Developmental biology
P46
Pericardial origin of proepicardial progenitor cells in the avian embryo

Abstract

The proepicardium (PE) is a cluster of mesothelial cells that develops at the venous pole of the heart. In many vertebrates including the chick, PE development is enhanced on the right side via a signalling pathway involving FGF8 and SNAI1 (Schlueter and Brand, 2009 PNAS 106:7485-7490), leading to the formation of a right-sided proepicardial tissue bridge via which PE cells colonize the heart and form the epicardium. In this study we have analysed the embryonic origin of proepicardial cells and uncovered a novel cellular contribution of pericardial mesothelium to the proepicardium. We performed labelling experiments of the lateral mesocardia and the pericardial mesoderm by DiI injection and electroporation of a GFP reporter gene and observed a cellular contribution of these tissues to the sinus venosus. Expression analysis of TWIST1, which represents a potential downstream target of the FGF8/SNAI1 pathway, revealed enhanced expression in the right somatopleura and subsequently in the right sinus horn and lateral mesocardium. Expression of TWIST1 terminated shortly before the onset of PE formation. We hypothesize that these proepicardial progenitor cells of somatopleural origin are asymmetrically mobilized by the expression of TWIST1. Indeed forced expression of TWIST1 on the left side lead to cell invasion of the sinus venosus and the heart by mesoderm cells of somatopleural origin. We are currently performing loss-of-function experiments to interfere with TWIST1 expression on the right side to study its effect on PE formation. These observations point to a similar origin of a subset of proepicardial cellsand the neighbouring pericardial mesoderm in the chick embryo.

P47
Conditional deletion of Scrib gene in the developing myocardium leads to congenital heart defects

Abstract

The Scrib gene is a component of the non-canonical Wnt/planar cell polarity (PCP) pathway which has been implicated in cardiogenesis. Mutation of Scrib, in the Circletail mouse mutant, results in cardiomyocyte disorganisation and disrupted cell-cell adhesion in the early heart tube. This is associated with a spectrum of cardiac septation and alignment defects and abnormalities of the ventricular myocardium that resemble certain types of human cardiomyopathy.

Utilising Cre-LoxPtechnology and Scribf/f mice, we generated embryos with the conditional deletion of Scrib in specific cell types within the developing heart, allowing us to determine the cardiac cell type that is critically dependent on Scrib function. Early deletion of Scrib in cardiac progenitors, utilising Nkx2.5Cre mice, confirmed the importance of Scrib at the earliest stages of myocardial formation, resulting in hearts with ventricular septal defects but a well-developed compact myocardium. Later deletion of Scrib in the myocardium, using Mlc2v-Cre, had only a mild affect on the ventricular myocardium.

We investigated how Scrib functions in the developing myocardium. Scrib localises to the adherens junctions of developing cardiomyocytes. Co-immunoprecipitation and immunofluorescence experiments indicated that Scrib forms a protein complex with Rac1 and its exchange factor, β-Pix, in both the H9C2 cardiomyocyte cell line and in hearts isolated from E10.5 embryos. Moreover, Rac1f/f;Nkx2.5Cre mice share a similar phenotype to Scrib1f/f;Nkx2.5Cre. To examine for a possible genetic interaction between Scrib and Rac1 we generated Scribf/ + ;Rac1f/ + ;Nkx2.5Cre mouse embryos. Double heterozygotes developed heart malformations that closely resembled those seen in Rac1f/f;Nkx2.5Cre embryos, confirming a genetic as well as physical interaction between Scrib and Rac1. These findings suggest that Scrib, together with Rac1, plays essential roles in cardiac progenitors, regulating adhesion and allowing the proper organisation of the myocardium that is essential for ventricular sepum integrity.

P48
Heart rate changes mediate the embryotoxic effects of antiarrhythmic drugs in the chick embryo

Abstract

Beta-blocking agents are frequently used cardiovascular drugs. With increasing average age of pregnant women we face the need of drug treatment duringpregnancy more frequently. As the heart rate is the most important determinant of cardiac output in the embryonic heart, we hypothesized that drug-induced bradycardia is the leading mechanism of heart failure.

ED4 and ED8 chick embryos were studied by video microscopy and ultrasound biomicroscopy ex ovo after intraamniotic injection of 200 μl of metoprolol or ivabradine or 200 μl of normal saline for a period of 30 minutes. Stroke volume was calculated by Simpson method from long parasternal short axis view in ED 8 embryos and prolate ellipsoid formula in video recordings (ED4). Cardiac output was then calculated from equation CO(μl/min) = SV(μl)*HR(BPM). Embryotoxicity was tested in ovo after administration of various doses of metoprolol or ivabradine compared to normal saline between ED3-ED8.

Metoprolol and ivabradine are drugs with strong negative chronotropic effect leading to 40% decrease of heart rate compared to normal saline within 30 minutes in ED 4 embryos. In more mature ED 8 embryos this effect was even more pronounced, with the heart rate decreased by 88% in metoprolol group and by 43% in ivabradine group. CO in ED 4 embryos decreased by 1% in the control group, by 11% in metoprolol group and by 43% in ivabradine group at 30 minutes. In ED 8 embryos the decrease in CO was 34% for normal saline group, 92% decrease for metoprolol group and 63% decrease for ivabradine group. There was no significant difference in stroke volume at either time point. A significant dose-dependent mortality (80%) was achieved in ED 4 embryos injected by 200 μl of ivabradine. In ED 8 embryos this effect was less pronounced with only 10% mortality at the same dose. No significant mortality was observed in ED 4 embryos injected by different doses of metoprolol but 39% mortality was achieved in ED 8 embryos injected by 200 ul of metoprolol.

Sensitivity to negative chronotropic effect of metoprolol and ivabradine increases with development. The embryonic heart has limited potential to vary stroke volume and significant bradycardia is followed by a significant decrease in cardiac output, likely leading to embryonic death. Metoprolol in usual doses appears to be relatively safe in pregnancy whereas ivabradine might have potential adverse effect on fetus.

P49
Functional deployment of ventricular conduction system in mouse during development

Abstract

Individual compartments of cardiac conduction system (CCS) became functional in order which correlates with cardiac morphogenesis. Ventricular CCS compartments mature with ventricular septation and it is accompanied by shift of activation pattern from primitive base to apex, which follows blood flow, to advanced apex to base. There are some important differences between avian and murine CCS. From the functional point of view, the main contrast is the appearance of mature activation sequence well before time of ventricular septation completion (embryonic day [ED] 13.5 in mouse) and also that there was never reported activation originating from the base of heart.

Function of CCS was studied by optical mapping and monitored parameters were speed of electrical impulse propagation, location first activation site on ventricular epicardial surface together with direction of action potential spread [evaluated as activation patterns, namely activation utilizing primary ring, left and right apical breakthroughs, only right or only left apical breakthrough - corresponding from ED14.5 with left and right bundle branches, respectively].

By measurement of time necessary for activation of the left ventricle from ED9.5 to ED18.5 we observed remarkable acceleration between ED9.5 and ED11.5 where activation time dropped to a half. This was due to decrease in frequency of primary ring activation pattern with a slow speed of action potential propagation compared to activation from apex to base. Primary ring is a primitive, temporary preferential activation pathway located in future interventricular septum and was the most common activation pattern for ED9.5 and 10.5; at ED11.5 one third of hearts was activated through primary ring and at ED12.5 it was recorded only sporadically. Activation from apex to base appeared from ED9.5, where it first originated from the primitive left ventricle; for mature activation originating from apex in the later stages was the most typical activation from left and right or only right apical breakthrough (since ED11.5). Appearance of these apical breakthrough sites correlated with expression of Cx40 in ventricular trabeculae. This analysis of normal development of CCS was useful for interpretation of changes observed in Cx40 deficient mice as well as in other transgenic strains with CCS phenotype.

P50
MicroRNA-130 regulates Fgf8 expression during early cardiac development

Abstract

At early gastrula stages of the chick embryo, cells with cardiogenic potential arise from a localized region of the primitive streak, just caudal to Hensen's node. These groups of cells migrate in an anterior-lateral direction to form the bilateral cardiogenic mesoderm, between the ectoderm and the endoderm, in the most anterior and lateral part of the embryo, constituting the primary cardiac field. At this moment, the precardiac mesoderm is surrounded by the adjacent endoderm, which comes from the more cranial part of the primitive streak, and which plays a crucial role during cardiac specification with the involvement of several molecular factors. Therefore, previous evidences suggest that Nkx2.5 expression is induced by Bmp2, emanating from the adjacent endoderm, which is also regulated by Fgf8.

To further analyze Bmp2-Fgf8 interaction during cardiac specification, we have analyzed the role of microRNA-130, which is expressed in the endoderm adjacent to precardiac mesoderm, in early cardiogenesis. For this purpose, we have electroporated Bmp2, Fgf8 and miR130 in precardiac cells as they pass the primitive streak. Our study has been complemented by experiments of ectopic administration of Bmp2, Fgf8 and SU5402 (an inhibitor of Fgfr1). We observe that Bmp2 induces the specific cardiac markers cNkx-2.5 and Gata4, and suppresses the endodermal endogenous expression of Fgf8. Moreover, Fgf8 suppresses Bmp2, cNkx-2.5 and Gata4 expression, while SU5402 administration suppresses the endogenous Fgf8 expression. Overexpression of miR130, a microRNA that binds to specific sequences in the 3′UTR of target gene Erk1/2 (Mapk1), induces the expression of Bmp2, cNkx-2.5 and Gata4, and suppresses the expression of Fgf8. The electroporation of antimiR130 shows the opposite results.

All our data supports the establishment of a negative feed-back between Bmp2 and Fgf8 through miR130, constituting a crucial element involved in the precise patterning and early development of the heart.

Funding: Spanish Ministry of Science and Innovation [BFU2007-66350], and the Junta de Extremadura with FEDER co-funding [CTS005].

Genetics, Epigenetics and Genomics
P51
Novel T(-4661)C polymorphic allele variant of ABCA1 gene

Abstract

ATP-binding cassette transporter A1 (ABCA1) is a main participant in reverse cholesterol transport (RTC) and can be implicated in atherosclerosis development. Liver X receptors (LXR) directly promote cholesterol efflux from macrophages, the first critical step of RTC, up-regulating ABCA1. There are sites for nuclear liver X receptors (LXR) in promote and 5′ flanking regions of ABCA1 gene. We suggest that polymorphic alleles in this region cay be associated with modulated level expression of ABCA1 gene. Purpose of our study was to search polymorphic alleles in 5′ flanking regions of ABCA1 gene and investigate their role in atherosclerosis development. Single-nucleotide polymorphism (SNP), which was not described previously, T(-4661)C of ABCA1 gene (NCBI, Gene Bank, AF275948) was found using SSCP analysis and direct sequencing of 5′ flanking region of ABCA1 gene among patients with extensive atherosclerosis and plasma HDL-C level lower than 0.9 mmol/l. For identification of T(-4661)C SNP a method, including PCR and restriction fragment length polymorphism analysis using endonuclease TaaI, was developed. We have showed that T(-4661)C SNP is widely spread in Russians. Genotypes TT(-4661), CT(-4661) и CC(-4661)frequencies were 81,32%, 17,58%, 0,00% и 78,35%, 21,64%, 0,75% among patients with angiographically verified atherosclerosis and control group, respectively. There were no differences in allele frequencies distribution of T(-4661)C SNP of ABCA1 gene between patients and control group. We can conclude that probably variants T(-4661)C of ABCA1 gene do not affect the atherosclerosis development. However further research is necessary to learn about influence of T(-4661)C variant on ABCA1 gene expression.

P52
Regulation of alternative splicing in the infarcted heart

Abstract

Purpose

Our knowledge of gene expression patterns in heart disease has increased considerably in the last few years. Unfortunately, this information is largely incomplete, as it relates to whole gene expression and does not take into account the variety of isoforms that are generated for each gene by alternative splicing. Alternative splicing (AS) affects 94% of multi-exonic genes and is carefully controlled by a collection of cis-regulatory motifs and trans-regulatory proteins that are not completely understood. In the heart, AS plays a main role during embryonic development. However its role in adult heart pathophysiology is only now beginning to be unveiled. In this work we aimed to gain deeper understanding of the AS events taking place in the infarcted heart and how these are regulated.

Methods and Results

Using unbiased microarray analysis we found that genes related to AS, in particular trans-regulatory factors like hnRNPs or SR proteins, are downregulated in the remote myocardium of infarcted hearts, both in mice and humans. To gain insight into the global regulation of AS in heart failure, we analysed exon expression in myocardial samples from uninjured and infarcted hearts 7 and 28 days after infarction using exon microarrays. We found that less than 20% of the genes alternatively spliced were also differentially expressed between infarcted and uninjured hearts. Gene ontology analysis showed association of alternatively spliced genes with ion transport, intracellular signalling and cell adhesion. In contrast, differentially expressed genes fell into extracellular matrix, cell adhesion and metabolism categories, suggesting that the cellular processes regulated by AS are largely different from those controlled by changes in gene expression. Interestingly, investigation of AS changes in a mouse model of cardiac pressure overload showed that similar biological processes were affected in the hypertrophic and the infarcted hearts, although only partial overlap was observed between the lists of alternatively spliced genes in both models. Using decision trees, we identified the major regulatory and structural elements controlling the AS changes observed in the infarcted heart. While several regulatory motifs seemed to be important to distinguish between cryptic and skipped exons, exon length was one of the main elements accounting for the presence of alternative splicing in a gene.

Conclusions

Our results suggest there is a specific pattern of As changes associated with heart failure and that AS affects biological processes that are different from those controlled by changes in gene expression.

P53
Polymorphisms in apolipoprotein AI and AIV genes and its combined effect on the occurrence of coronary artery disease

Abstract

Objectives

The aim of this study was to determine combined effect of Apolipoprotein AI(-75G/A) and AIV(T347S) gene polymorphisms and its role in coronary artery disease(CAD).

Background

A common polymorphism of aguanine (G) to adenine (A) substitution (G/A) at 75 bp(M1-) in the promoter region of the apoA1 gene has traditionally been associated with a significantly higher Lp(a) and HDL-c levels by numerous workers. A Thr347Ser polymorphism in exon 3 of Apo AIV gene has also been significantly associated with LDL-c and TG concentrations by many, but their combined effect in pathogenesis of CAD has seldom been investigated.

Methods

This was prospective study; the subjects were included from a tertiary care centre in Uttar Pradesh, the most populated province situated in northern India. The study population comprised of consecutive 200 proven CAD patients and 200 disease free controls. Genotyping technique used was polymerase chain reaction(PCR) followed by restriction fragment length polymorphism(RFLP).

Results

Baseline characteristics and risk factor distribution including incidence of diabetes, hypertension, smoking, family history of CAD along with mean levels of various lipid parameters were found to be significantly higher in patients than in controls (p < 0.001). No significant association with CAD was detected when the various genotypes/allele frequencies arising due to Apo AI(-75G/A) and AIV(T347S) gene polymorphisms were compared individually(p > 0.05). However when these two polymorphisms were clubbed together, we found significantly higher relative risk for CAD in patients carrying mutant alleles (Odds ratio = 1.33,p = 0.03) for both the polymorphisms. Other genotypic combinations did not reveal any significant association with the disease.

Conclusion

The results of our study indicate that apoAI(-75 G/A) and AIV(T347S) polymorphisms does not only affect the lipid levels of an individual (as reported in numerous studies all around the world) but also contribute in increasing the risk of CAD. The complex gene-gene interaction between the two studied genes could have resulted in an increased relative risk in patients carrying mutant combinations.

P54
Per3 VNTR polymorphism and expression in acute myocardial infarction with sT elevation

Abstract

Human autonomic processes such as the control of sleep, body temperature, hormone secretion, renal and cardiovascular functions are influenced by circadian variations. There is obvious diurnal variation in blood pressure, heart rate and cardiac output; moreover, it has been known for long time that the fatal cardiovascular events occur preferentially in certain time of the day. Molecular clocks present in individual cells consist of clock genes and their resultant proteins that generate self-sustained transcriptional feedback loops with a free-running period of 24h. One of the key components of the human clock system, Period 3 (Per3), has been recently demonstrated to affect circadian expression of various genes in different tissues including heart. Possible imbalance or impairment in clock mechanism within the cardiomyocytes may alter the cardiac metabolism and function and thus increase the susceptibility of cardiovascular diseases.

The aim of our study was to investigate the effect of variable number tandem repeat (VNTR) polymorphism in Per3 gene in acute myocardial infarction with ST elevation (STEMI). The study subjects (336 patients of Caucasian origin with STEMI, and 332 healthy controls) were genotyped for Per3 VNTR polymorphism using an allele-specific PCR. PER3 quantification of mRNA from circulation was performed using quantitative real-time polymerase chain reaction (qRT-PCR). The follow-up of PER3 expression in 10 patients within interval of 8 hours was carried out (6 samples per patient). Within the STEMI patients, the difference in allelic frequencies of Per3 VNTR polymorphism was observed when comparing the time of MI onset (pa = 0.01). The long variant (5/5) of Per3 VNTR polymorphism was more common within STEMI patients with pain onset during the night hours (p = 0.045). A trend of higher PER3 expression in short variant (4/4) was observed (p = 0.08). The lowest expression was found in time of MI onset with increasing PER3 expression in following hours. No significant differences in genotype and/or allele frequencies of Per3 VNTR polymorphism were observed when comparing STEMI cases and control group. In the multivariate regression modelling, no predictive function of VNTR Per3 polymorphism on ejection fraction, hyperlipidaemia or type II diabetes risk was observed.

Based on the results of the presented study, we consider a possible effect of the Per3 VNTR long variant on time of the pain onset during MI in STEMI patients.

P55
Next-generation sequencing used to discover novel genetic variants predisposing to heart disease

Abstract

Purpose

Cardiovascular disease is the leading cause of death worldwide. Most cardiomyopathies and channelopathies are genetic in origin and are characterized by their risk of premature death and chronic morbidity, affecting both the patient and his/her family. The detection of mutations in patients allows for therapeutic or preventive measures to be established as part of the genetic counseling. However, the large number of genes involved, makes it difficult to analyze using conventional techniques. The purpose of this study is the genetic characterization of heart disease patients in a fast, comprehensive, and cost-effective manner using an NGS approach, which includes 72 genes associated with different pathologies, coupled with a robust bioinformatics pipeline.

Methods

We developed a methodology for resequencing 72 genes (49 genes associated with cardiomyopathy, arrhythmogenic right ventricular dysplasia, Marfan syndrome, aortic aneurysm, and 23 genes associated with Brugada syndrome, long QT and short QT syndromes, familial atrial fibrillation and catecholaminergic polymorphic ventricular tachycardia). The design was done in our Bioinformatics Unit and includes 750Kb of exons, splicing regions and 5' and 3' unstranslated regions of the 72 selected genes. Exonic regions were thus captured (SureSelect, Agilent), and sequenced in the SOLiD v4 platform. The results were confirmed by Sanger sequencing. A set of 12 cases with known mutations was used for validation studies.

Results

To apply this methodology, we selected 26 patients (11 cases with aortic aneurism/Marfan syndrome, 2 cases with arrhythmogenic right ventricular dysplasia, 4 cases with Hypertrophic cardiomyopathy, 4 cases with long QT syndrome, 1 case with familial Arrhythmia, 1 case with Brugada syndrome and 3 cases with family history of sudden death). We detected 47 relevant nucleotide changes: 5 disease causing mutations and 42 unclassified variations, of which 36 are novel changes. Using in silico predictions for UVs, we classified 6 of this variants as likely pathogenic and 4 as unlikely pathogenic.

Conclusions

This NGS platform, which includes 72 genes associated with different cardiac pathologies, permits a quick and high throughput analysis of those genes. This study is important to explain the phenotypic variability of heart disease and to help establish new mutation-disease associations.

P56
miR-375: novel biomarker for early prognostic stratification of acute myocardial infarction

Abstract

Background

Despite great improvements in the diagnostic and therapeutic fields, morbidity and mortality in patients with acute myocardial infarction remains very high. Available tools for early prognostic stratification still have insufficient accuracy. microRNA (miR) are reported to be present in the blood and have been increasingly suggested as biomarkers of diseases.

miR-375 acts in the 3′-UTR of myotrophin mRNA, repressing its translation. Since myotrophin is a transcription factor that regulates several pathways required for cardiac-stress response, we sought to determine the potential of miR-375 to serve as biomarkers for early diagnosis and prognostic stratification of AMI patients.

Methods

Prospective observational study of consecutive ST-elevation AMI patients submitted to primary angioplasty. Blood samples were taken at the beginning of cardiac catheterization (T0) and after 8, 16 and 24 hours, and the serum levels of miR-375 were determined by real time-PCR. The accuracy for prognostic stratification was determined by Kaplan Meier survival and multivariate Cox regression analysis with stratification for left ventricular ejection fraction, considering the composite primary end point of death, re-infarction or hospitalization for cardiac causes and miR-375 tertiles distribution.

Results

Fourty-two ST-elevation AMI patients were evaluated (age 64 ± 11 years; 63% male). Initial miR-375 levels were very low (1.8x10−6 ± 4.6x10−6), only increasing 48 to 72h after angioplasty. Nevertheless, miR-375 at T0 were correlated with cardiac biomarkers, including troponin I (R = .46; p = .002), maximum troponin I (R = .46; p = .002), CPK-MB (R = .48; p = .001), maximum CPK-MB (R = .48; p = .001), CPK (R = .33; p = .03), maximum CPK (R = .33; p = .031) and NTproBNP (Rho = -337; p = .031). During follow-up (487 ± 261 days), 11 patients (29%) progressed unfavourably. High miR-375 levels both at T0 (≥7.5x10−7; 3th tertile) and T8 (≥5.6x10−7; 3th tertile) were identified as independent prognostic risks factors, significantly reducing the time-to-composite end-point survival [T0 Cox hazard ratio: 4.9 (95% CI 1.2-19.6), p = .048; T8 Cox hazard ratio: 5.7 (95% CI 1.4-24.0), p = .017].

Conclusions

Circulating miR-375 has the potential to become useful for long term prognostic stratification of ST-elevation AMI patients.

P57
Identification of hypertrophy and heart failure associated genes by in vitro and in vivo multi-model gene expression analysis

Abstract

Purpose

Cardiomyocyte growth (hypertrophy) is one of the hallmarks of heart failure (HF) development. Numerous studies have explored hypertrophy associated gene expression, but because of diversity in disease severity, etiologies and microarray platforms used the gene lists show only limited overlap. Here we performed a multi-model gene expression analysis to generate a concise, but robust list of genes that are differentially expressed both in in vivo HF and in vitro hypertrophy models.

Methods

Hypertensive Ren2 rats and animals with post-MI HF were used as in vivo HF models and neonatal rat cardiomyocytes treated with hypertrophy inducing hormones phenylephrine, endothelin-1 and isoproterenol were used as in vitro models. Whole genome gene expression analysis was performed using Illumina RatRef-12 Beadchips. Differentially expressed genes with at least 1.5 fold change as compared to the control groups (p < 0.05) were selected and gene lists were compared with each other.

Results

Analysis of differential gene expression in cardiomyocytes stimulated with hypertrophy inducing agents and two rat HF models generated large lists of differentially expressed genes. By combining these expression data sets we obtained a concise list of 62 differentially expressed genes. This list included most known HF associated genes, including NPPA (ANP), (NPPB) BNP and FHL1, indicating that this is a powerful approach to identify differentially expressed genes that are specifically linked to hypertrophy and HF development. In addition, novel genes not previously associated with hypertrophy/HF, like PTGIS, AKIP1 and Dhrs7c, were identified. These genes could constitute interesting targets for further investigations.

We also identified a number of in vivo specific genes and gene ontology (GO) analysis revealed a strong enrichment of these genes for fibrosis, wounding and stress responses. Therefore a number of novel genes within this in vivo specific list are related to fibroblasts or other non-cardiomyocytes present in the heart. Finally, interesting differences between the two HF rat models were observed. For example CA3 and WISP2 were strongly upregulated in post-MI HF, but not in Ren2 rats, indicative for etiology specific differences.

Conclusion

Together these results show that combining multiple models, both in vivo and in vitro, can provide a robust set of hypertrophy/HF associated genes. Moreover it provides insight into the differences between the different etiology models and neurohormonal effects.

P58
The relationship between angiotensinogen gene polymorphism and left ventricular remodeling in patients with myocardial infarction

Abstract

Purpose

The development of left ventricular (LV) remodeling after acute myocardial infarction (AMI) is a predictor of heart failure and mortality. The purpose of the present study was to assess whether the polymorphism of angiotensinogen (AGT) gene with threohine (T) instead of methionine (M) at amino acid 235 in exon 2 (M235T) had effects on cardiac remodeling after AMI.

Methods

141 patients (mean age 56.4 ± 11.1) with a first AMI were enrolled. Within 24-72 hours of the onset of AMI symptoms and at 4 months, 2-dimensional echocardiography was performed. LV remodeling was defined as ≥15% increase from the baseline in LV end diastolic volume. AGT M235T polymorphism was determined using polymerase chain reaction amplification.

Results

At follow-up, 49 patients (34.7%) were classified as having LV remodeling. Patients with LV remodeling were more frequently prone to anterior wall MI (p < 0.01), higher leukocyte count value at admission (p < 0.01), lower LV ejection fraction (p < 0.05) and increased LV end systolic volume (p < 0.05). Furthermore, AMI patients with LV remodeling significantly more often were carriers of TT genotype of AGT gene (28,6% vs.14.1%, p=0.038).

Conclusions

The TT genotype of the AGT gene may be related to the development of LV remodeling after acute myocardial infarction.

P59
Identification of A Kinase Interacting Protein 1 (AKIP1) as a novel cardiac hypertrophy gene

Abstract

Purpose

Heart failure (HF) is a complex disease and is accompanied by profound changes in gene expression patterns. We recently performed a multi-model gene expression analysis and identified A Kinase Interacting Protein 1 (AKIP1) as a HF associated gene. Here we confirm the up-regulation of this gene in different HF and hypertrophy models. We also show the basic characterization of AKIP1 and its potential role in hypertrophy development.

Methods

Using Real-time PCR (RT-PCR) AKIP1 mRNA expression was analysed in a post-MI rat HF model and in hypertensive Ren-2 transgenic rats that develop HF. In addition expression was analysed in primary isolated neonatal rat cardiomyocytes stimulated with hypertrophy inducing hormones (phenylephrine (PE), isoproterenol (Iso) and endothelin-1 (ET-1)). To investigate the function of AKIP1 overexpression and Silencing studies were performed in isolated cardiomyocytes and protein synthesis, cell growth (cell surface area) and RNA expression profiles were analysed. Western blot analysis was performed to investigate potential AKIP1 related signaling pathways.

Results

RT-PCR analyses showed that AKIP1 was significantly up-regulated in all hypertrophy and HF models. This confirms previous microarray data and shows that AKIP1 is a HF associated gene. AKIP1 overexpression in cardiomyocytes revealed more contracting cells and immunofluorescence microscopy showed that these cells have a more organized actin/sarcomere structure. Overexpressing cells also showed an increased cells size and protein synthesis analysis confirmed the AKIP1 hypertrophy inducing effect. Silencing of AKIP1 could, however, not prevent PE induced hypertrophy, suggesting that AKIP1 functions in a different pathway. Initial cell signaling pathway analysis has been performed suggesting a link between AKIP1 and the NFAT phosphorylation state.

Conclusion

AKIP1 is a novel HF associated gene and overexpression of AKIP1 in cardiomyocytes can induce hypertrophy, at least in vitro. Initial pathway analysis indicates that AKIP1 may operate via established hypertrophy inducing pathways.

Growth/death, Regeneration and Stem cells
P60
The PPARbeta agonist GW0742 protects cardiac myocytes from oxidative stress through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms

Abstract

Peroxisome proliferator-activated receptors (PPARs) (α, β and γ isoforms) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and are known to play a prominent role in the regulation of lipid metabolism and homeostasis. Emerging evidence indicates that their activation leads to cardioprotection after ischemia/reperfusion (I/R) and prevents cardiac remodelling and failure. However, the exact role of PPARs, in particular PPARβ isoform, in cardiac pathophysiology has not been defined yet. The aim of this study was to determine the effect of PPARβ activation during oxidative stress in cardiomyocytes and identify the underlying molecular mechanisms that lead to protection. Adult rat cardiomyocytes were subjected to oxidative stress (0.1 mM H2O2) in the presence or absence of PPARβ specific ligand GW0742. Cell viability, the extent of apoptosis as well as production of reactive oxygen species (ROS) were determined by flow cytometry. Activation of PPARβ inhibited ROS production and cardiac myocyte apoptosis and resulted in increased cell viability. Furthermore, heme oxygenase 1 (HMOX1), a stress-response protein was downregulated when GW0742 was present during oxidative stress. To explore further the underlying mechanisms, expression of the anti-apoptotic Bcl-2 protein and activation of metalloproteases (MMP) 2 and 9 was also determined. Decreased levels of Bcl-2, which were observed during exposure of cardiomyocytes to H2O2, were upregulated in the presence of GW0742, as determined by immunoblotting. In addition, PPARβ activation resulted in inhibition of MMP-2 and MMP-9 expression and enzymatic activity levels as determined by real time PCR and gelatin zymography, respectively. These data support the anti-oxidant, anti-apoptotic and anti-inflammatory role of PPARβ in cardiac myocytes. The underlying protective mechanisms include inhibition of ROS production, upregulation of the anti-apoptotic protein Bcl-2 and inhibition of MMP-2 and MMP-9.

P62
A novel CaMKII/ERK interaction in the heart sustains cardiac hypertrophy in spontaneously hypertensive rats

Abstract

The cardiac hypertrophy is an adaptive response to increased cardiac output that occurs in various cardiovascular diseases. The molecular mechanisms underlying it involve, among others, abnormal intracellular Ca + + dynamics. Ca + + /Calmodulin protein kinase II (CaMKII) activation has a role in the hypertrophic signaling transduction pathways. However, the mechanism by which CaMKII integrates with other pathways to develope cardiac hypertrophy is incompletely understood. We already showed that phenylephrine (PE) induced VSMC (vascular smooth muscle cell) proliferation results in CaMKII and ERK complex formation which promotes phosphorylation and nuclear localization of both proteins. We hypothesize that CaMKII, in association with ERK, promotes nuclear localization in hypertrophic cardiomyocytes. In H9c2 cardiomyoblasts PE induces both ERK and CaMKII activation, that were been affected by non-selective CaMKs inhibition with KN93 or by selective CaMKII inhibitor AntCaNtide, as well as MEK/ERK inhibitor UO126. Co-immunoprecipitating studies demonstrated that PE induces CaMKII/ERK interaction, which was prevented by pre-treatment with KN93, ANtCaNTide or UO126. Next, we tracked the intracellular localization of ERK and CaMKII. PE induces a time-dependent accumulation of both ERK and CaMKII into the nucleus. AnTCaNtide and UO126 pretreatment prevented not only ERK but also CaMKII nuclear localization, supporting the hypothesis that ERK activation is required for CaMKII nuclear localization. In order to determine the role of CaMKII in in vivo cardiac hypertrophy, Spontaneously Hypertensive Rats (SHR) were subjected to intracardiac injection of AnTCaNtide (ANTS,50mcg/kg, n = 8). Sham group of SHR (SS,n = 8) were subjected to intracardiac injection of saline. After 3 and 7 days from the treatment, we performed an ultrasound (US,VeVo-Visualsonics) cardiac evaluation and an invasive (Millar) arterial BP measurement in the rats. Mean arterial pressure and body weight were similar between SS and ANTS, but we observed in ANTS a significant reduction of cardiac size (ANTS vs SS:Heart weight/body ratio 3.961 ± 0.02586 vs 4.177 ± 0.02583,p < 0.05), of US left ventricular mass (ANTS vs SS:785.15 ± 47.4g vs 963.92 ± 51.6g,p < 0.05) and of US thickness of interventricular septum (ANTS vs SS:1.821 ± 0. 01063mm vs 1.918 ± 0.01797mm,p < 0.05). Finally in ANTS we observed a significant reduction of CaMKII and ERK phosphorylation levels. These results indicate that CaMKII promotes ERK activation in hypertrophic hearts and functional significance of CaMKII-ERK interaction offers a novel therapeutic approach to limit pathological cardiac hypertrophy.

P63
Constitutive Angiotensin II type 1 receptor activation in differentiated cardiomyocytes causes ventricular dysfunction in the absence of hypertrophy and fibrosis

Abstract

Ventricular dysfunction is commonly associated with elevated blood pressure and left ventricular hypertrophy (LVH) modulated by the renin-angiotensin system (RAS). Sustained LVH ultimately progresses to pathological remodelling and fibrosis resulting in heart failure. We have previously reported a conditional transgenic mouse model in which a mutant Human Angiotensin II type I Receptor Transgene (HART) can be activated exclusively in the differentiated cardiomyocyte. At 12 months of age HART mice exhibit a phenotype of relevance to clinical LVH, displaying cardiomyocyte hypertrophy, and subsequently interstitial fibrosis upon stimulation with the mutant receptor specific agonist, Ang IV, without alteration in blood pressure [1].

Here we have utilised the HART activation system in young adults to unravel cellular and molecular mechanisms involved in stimulating progression towards heart failure. At five months we find that HART mice show significant ventricular dysfunction (left ventricular dilatation and reduced ejection fraction) with no evidence of hypertrophy at either the individual cardiomyocyte or whole heart level. We also see no evidence of fibrosis or of increased cardiomyocyte death in these mice that may account for the observed dysfunction.

Stimulation of the mutant receptors with Ang IV for a period of 4 weeks induces cardiomyocyte (and whole heart) hypertrophy with worsening ventricular function, but only limited evidence of further pathological remodelling or modulation of pathological marker gene expression. However, TUNEL assay suggests an increase in number of dying cardiomyocytes in otherwise ‘healthy’ regions of the myocardium. Sporadic areas of fibrosis in some Ang IV treated hearts exhibit increased density of non-myocyte cells with evidence of increased cell turnover rate.

Our results demonstrate that activation of RAS in cardiomyocytes leads directly to dilated cardiomyopathy without causing cellular hypertrophy or death. Further sustained stimulation of RAS in adult cardiomyocytes appears to initiate sporadic remodelling through hypertrophy and death of individual cardiomyocytes, closely associated with infiltration and/or proliferation of non-myocyte cells (fibroblast and inflammatory). Further experiments are underway to dissect the mechanisms through which RAS activation in the cardiomyocyte first leads to ventricular dysfunction, and then progression to failure.

[1] Ainscough et al. (2009). Angiotensin II type-1 receptor activation in the adult heart causes blood pressure-independent hypertrophy and cardiac dysfunction. Cardiovasc Res 81(3):592-600

P64
The specific AMP-activated protein kinase activator A-769662 reduces cardiomyocyte hypertrophy by blocking multiple signalling pathways

Abstract

Purpose

The A-769662 compound is a specific activator of the AMP-activated protein kinase (AMPK). Several studies have shown that unspecific AMPK activators, like resveratrol and metformin, inhibit cardiac hypertrophy by preventing the increase in protein synthesis. This is accompanied by the modification of the phosphorylation state of two proteins involved in protein translation, namely p70 ribosomal S6 protein kinase (p70S6K) and elongation factor 2 (eEF2). The aim of this work was to evaluate the putative therapeutic effect of A-769662 for the treatment of cardiac hypertrophy.

Methods

Cultured neonatal rat cardiomyocytes were stimulated with phenylephrine (PE, 10μM) in the presence or not of A-769662 (100 μM) during 24h. Cardiomyocyte hypertrophy was visualized by immunostaining of α-actinin and by QPCR for ANP and BNP. Protein synthesis was measured by incorporation of radioactive methionine into proteins. Phosphorylation of AMPK, p70S6K, eEF2 and ERK was evaluated by immunoblot. Translocation of NFAT was studied by immunostaining.

Results

A-769662 was able to chronically stimulate AMPK signalling in cardiomyocytes. A-769662 treatment prevented the PE-induced cardiomyocyte hypertrophy. PE increased the mRNA level of both BNP and ANP and this was inhibited by A-769662 (Ctrl: 1 ± 0.03; PE: 2.49 ± 0.7; PE + A: 0.97 ± 0.21 in relative values for BNP and Ctrl: 1.07 ± 0.07; PE: 2.17 ± 0.27; PE + A: 0.82 ± 0.35 for ANP). This is associated with a decrease in PE-induced stimulation of protein synthesis (Ctrl: 4117 ± 621; A:4873 ± 204; PE: 7463 ± 854; PE + A: 4435 ± 777 incorporated dpm/dish, p < 0.05). A-769662 blocked the PE-mediated increase in p70S6K phosphorylation (Ctrl: 0.96 ± 0.11; A: 0.53 ± 0.10; PE: 2.54 ± 0.23; PE + A: 1.3 ± 0.20 arbitrary unit, p < 0.05). Similarly, A-769662 prevented the decrease in eEF2 phosphorylation induced by PE (Ctrl: 1 ± 0.07; A: 1.57 ± 0.09; PE: 0.49 ± 0.07; PE + A: 1.17 ± 0.11, arbitrary unit, p < 0.05). Moreover, PE promoted the nuclear translocation of NFAT, a transcription factor located downstream calcineurin and essential in the development of cardiac hypertrophy. This PE-induced translocation was reversed by the A-769662 treatment (Ctrl: 24 ± 2; A: 27 ± 2; PE: 85 ± 2; PE + A: 28 ± 1 % of NFAT-positive nuclei). Finally, the A-769662 compound blocked the increase in ERK phosphorylation caused by PE treatment (Ctrl: 1.00 ± 0.11; A: 0.71 ± 0.06; PE: 1.31 ± 0.08; PE + A: 0.61 ± 0.13 arbitrary unit, p < 0.05).

Conclusions

Activation of AMPK by A-769662 appears to be a potential therapeutic approach to treat cardiac hypertrophy by inhibiting protein synthesis and transcription factor signalling pathways.

P65
Adenosine A1-receptor agonism counteracts hypertrophy and fibrosis in response to alpha-adrenergic stimulation

Abstract

Introduction

Adenosine is an important signaling molecule in the cardiovascular system and has been proposed to exert anti-hypertrophic effects. The precise regulation and role of the different adenosine receptor subtypes in the heart and their effects on hypertrophic signaling, however, are largely unknown. Thus, the aim of our study was to characterize the expression and function of the Gαi-coupled adenosine A1-receptor in response to hypertrophic stimulation in vitro and in vivo.

Methods

Isolated rat cardiomyocytes were exposed to the α1-adrenergic receptor agonist phenylephrine (PE; 1-100 μM; 24h), to angiotensin II (AngII; 0.1-10 μM) or insulin-like growth factor (Igf-I; 0.2-20 ng/ml) and the expression of adenosine receptor subtypes was determined by PCR and Western blot analyses. The hypertrophic response was determined by cardiomyocyte cross sectional area (light microscopy), sarcomere organization (immunostaining of α–sarcomeric actinin), protein synthesis (3H-leucin-incorporation) and mRNA of the immediate early gene c-fos, in the absence and presence of the selective A1-receptor agonist CPA (N6-cyclopentyladenosine; 1 μM). C57Bl/6N mice were randomized to continuous PE-infusion (120 mg*kg*day) or vehicle (saline) for 21 days (osmotic mini-pumps) and cardiomyocyte cross sectional area, diameter and the amount of fibrosis analyzed by histology, respectively. Cardiac function in vivo was analyzed by echocardiography and MRI analyses.

Results

PE, but not AngII or Igf-I increased mRNA and protein expression of the A1-receptor in a concentration-dependent manner. A1-receptor stimulation reduced the PE-mediated increase in cardiomyocyte cross sectional area, the augmentation in sarcomeric organization, the rise in total protein expression as well as the increased expression of c-fos (p < 0.01, respectively). In contrast, CPA had no effects on the pro-hypertrophic effects of AngII or Igf-I. Accordingly, longterm PE-treatment in vivo upregulated A1-receptor protein expression. PE induced cardiomyocyte hypertrophy and fibrosis, which was blunted by co-treatment with CPA (PE: 14.1 ± 1.4%; PE + CPA: 3.3 ± 0.5%; p < 0.001).

Conclusion

α1-Adrenergic receptor stimulation leads to upregulation of the adenosine A1-receptor in vitro and in vivo, while stimulation of AngII- or Igf-I receptors has no such effect. Since A1-receptor stimulation counteracts PE-induced pro-hypertrophic and pro-fibrotic effects, this may resemble a distinct negative feedback mechanism regulating the myocardial maladaptive response induced by α-adrenergic receptor stimulation.

P66
Distinct intracellular signaling pathways in pressure overload and/or diabetes mellitus lead to different myocardial structural and functional phenotypes

Abstract

Diastolic heart failure (DHF) accounts for ∼50% of HF cases and represents a growing problem. Its pathophysiology is ascribed to impaired relaxation or increased myocardial stiffness. Often, chronic pressure-overload (PO) and diabetes mellitus (DM) lead to DHF. Moreover, hypertension is more prevalent among the diabetic population and exacerbates the extent of diabetic cardiomyopathy. Even so, functional and structural cardiac consequences of combined PO and DM are still unclear. The present study aimed to characterize myocardial morphological and functional changes induced by DM and/or PO as well as its underlying mechanisms.

PO was performed in Wistar-male rats by supra-renal aortic banding. After six-weeks, diabetes was induced by streptozotocin (65 mg/kg, ip) resulting in four groups: SHAM, banding (BA), diabetic (DM) and diabetic-banding (DB). Six weeks later, hemodynamic study was performed to evaluate cardiac performance. Samples were collected for histology, molecular studies and force measurement in isolated skinned cardiomyocytes.

Chronic PO increased LV hypertrophy (enlarged cardiomyocyte diameter) myofilament active force and Ca2+ sensitivity as well as phosphorylation of myofilamentary protein (MLC-2, MyBP-C) and hypertrophic and insulin signaling pathways proteins (Erk, Akt). At the extracellular matrix level, interstitial fibrosis and pro-MMP-2 e MMP-9 activity were increased. In vivo, contractility (LV peak systolic pressure (LV-ESP) and maximal LV wall stress (LV-Wstress) was increased and correlated positively with Factive, while relaxation was impaired. DM increased cardiomyocyte diameter, the expression of inflammatory (TNF-α) and apoptosis markers (Bax/Bcl-2). At the extracellular matrix level, diabetic animals displayed augmented myocardial fibrosis and AGEs deposition. In vivo, these abnormalities resulted in increased stiffness confirmed by the higher values of LV end-diastolic-pressure and end-diastolic pressure–volume relation. Furthermore, diabetic animals displayed lower contractility (end-systolic pressure–volume relation (ESPVR), expression of MHC-α/MHC-β and, as expected, lower activation of insulin signaling pathways (Akt phosphorylation levels).

DB animals combined overload-induced relaxation abnormalities and diabetes-induced stiffness. Additionally showed further pulmonary congestion. We conclude that DM and PO lead to distinct diastolic dysfunction phenotypes: while diabetes promoted myocardial stiffening, pressure overload impaired relaxation. The association of these damages accelerates the progression of DHF.

P67
Elevated expression of the myocardial sodium proton exchanger isoform 1 (NHE1) induces cardiac hypertrophy and the upregulation of osteopontin gene expression

Abstract

Cardiac hypertrophy(CH), a prominent feature that predisposes the heart to failure, is associated with the activation of multiple molecular and cellular changes in the circulation and heart. The Na+/H+ exchanger isoform 1 (NHE1) has been implicated in the development and progression of CH. NHE1 is an integral membrane protein that maintains intracellular pH by exchanging one intracellular H+ for one extracellular Na+. In various in vivo and in vitro models of cardiac dysfunction, NHE1 protein expression and activity have been shown to be upregulated. To better understand the involvement of NHE1, transgenic mice that express cardiac specific active NHE1 were studied (K-line mice). NHE1 activity of adult ventricular cardiomyocytes and protein expression were elevated by approximately 3-fold in the K-line mice vs. control. The K-line mice demonstrated significant global cardiac dysfunction. Left ventricular fractional cell shortening and ejection fraction were significantly decreased in the K-line mice (23.1 ± 3.8% and 45.2 ± 6.9% K-line vs. 36.5 ± 1.1% and 66.4 ± 1.5% control, respectively; P < 0.05). The K-line mice also exhibit myocardial remodeling. The heart weight to body weight ratio was significantly greater in the K-line mice (143 ± 10.0% of control; P < 0.05). Cross sectional area (K-line 195.6 ± 16.4% of control; P < 0.05) and interstitial fibrosis (K-line: 275.4 ± 11.6% of control; P < 0.05) were also elevated. Genechip analysis also revealed that expression of active NHE1 upregulated osteopontin (OPN) gene expression (>1,500 fold change) and its signaling pathways. OPN is a matricellular protein and a cytokine induced upon tissue injury and remodeling of various organs, including human heart failure. Our study shows that expression of activated NHE1 induces CH and elicits specific molecular changes that lead to CH.

P68
Investigation of effects of age on isolation and function of cardiac stem cells

Abstract

Cardiovascular disease remains the leading cause of morbility and mortality in many parts of the world. There are many risk factors for cardiovascular disease, and age is a major one. Recently, stem cell-based therapy has emerged as a promising approach to cardiac therapy with resident cardiac stem cells (CSCs) representing a desirable candidate because of their endogeneous origin and potential to develop into cardiac lineages. Furthermore, CSCs can be expanded in culture to give sufficient cells for therapy. However, increased age may result in a progressive decline in number and function of available cardiac stem cells. Therefore, the objectives of this study were to investigate the impact of age on CSC isolation and function in vitro.

CSCs were isolated and expanded, via the formation of cardiospheres, from the hearts of C57BL/10 mice at 1.5, 6, 18 and 24 months old. The number and function of explant-derived cells (EDCs) and cardiosphere-derived cells (CDCs) generated were examined. Young mice produced significantly more EDCs and CDCs per mg tissue, compared with 24 months old mice. CDCs derived from 1.5 months old mice exhibited higher expression of cardiac stem cell markers including c-kit and Sca-1 than those from 24 months old mice (c-kit: 13% vs 3%; Sca-1: 83% vs 50%, respectively). EDCs and CDCs from young animals showed increased migration, compared with those from aged ones. Higher proliferative capacity and clonogenic efficiency were also found in CDCs from younger mice. After induction of cardiomyogenic differentiation using either 5-azacytidine or DMSO, it was found that CDCs from young mice expressed a higher percentage of the cardiomyocyte-specific marker - cardiac troponin T.

In conclusion, aged animals generated a reduced number of cardiac stem cells, and the CDCs showed impaired proliferation, migration, clonogenicity and differentiation.

P69
IL-6 induces terminal differentiation of embryonic cardiomyocytes through phosphoinositide 3-kinase pathway

Abstract

Introduction

IL-6 is a pleiotropic cytokine expressed in a variety of cells mediating proliferation, growth, differentiation, survival and apoptosis signals. It can activate various cell types carrying the membrane bound IL-6R as well as IL-6R- gp130 + cells via the soluble IL-6R. IL-6 signaling involves the Jak/STAT-3, the Ras/Erk and PI-3K/Akt pathways. Increased circulating and intracardiac levels of IL-6 have been associated with chronic heart failure and myocardial infarction, and recent studies suggest that IL-6 signalling can be involved in the modulation of the compensatory hypertrophy by influencing remodelling processes and inducing Protein kinase C (PKC)-dependent apoptosis. Nevertheless, its role in cardiomyoblast differentiation remain unexplored.

Purpose

To analyze the possible role of IL-6 in inducing terminal differentiation of embryonic cardiomyoblasts, as well as its downstream intracellular signalling pathways.

Methods

H9C2 cells were cultured in medium supplemented with 1% FBS in presence of IL-6 (10 ng/ml) up to three days.

Results

In presence of IL-6 embryonic cardiomyoblasts underwent morphological modifications characterized by cells elongation and fusion into multinucleated tubes. This process was accompanied by the nuclear translocation of Nkx2.5 (a myocardial development transcription factor), an upregulation and spatial reorganization of α-myosin heavy chain (α-MHC; terminal cardiac differentiation marker) and a modulation and sub-cellular redistribution of gp130. The IL-6 signal was mainly mediated by a prompt involvement of PI3K that determined downstream a PKCzeta activation and a PTEN dependent Akt de-phosphorylation. These data were confirmed by the transient transfection of H9C2 cells with a catalytically active form of PKCzeta that induced cell fusion, Nkx2.5 nuclear translocation and upregulation of α-MHC expression. The IL-6 differentiating effect was abolished when the PKCzeta activity was blocked with a specific pseudo-substrate. No early modification in Erk1 and Erk2 expression or phosphorylation levels were detected in response to IL-6 treatment.

Conclusion

Our data evidence that IL-6 induces a terminal differentiation of embryonic cardiomyocytes through the activation of PI3K-PKCzeta pathway and that IL-6 may contribute to regenerative capacity of myocardium by inducing resident progenitor cells differentiation.

P70
Role of NF-Y in atherosclerotic disease as an essential effector of PDGF-BB-induced vascular smooth muscle cell proliferation

Abstract

Background

Excessive vascular smooth muscle cell (VSMC) proliferation is a hallmark of atherosclerosis and restenosis. The transcription factor NF-Y is essential for the expression of cyclin B1, a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. 

Results

By analyzing animal and human vascular specimens, we find co-expression of NF-Y and cyclin B1 in proliferative regions of atherosclerotic and restenotic lesions, which also show higher binding of NF-Y to target sequences in the CYCLINB1 gene promoter. Platelet-derived growth factor-BB (PDGF-BB), a key positive regulator of neointimal lesion development, induced in rat and human VSMCs the recruitment of NF-Y to the CYCLINB1 promoter and augmented both CYCLINB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt activation. We also demonstrate that NF-Y is essential for PDGF-BB-induced CYCLINB1 expression and VSMC proliferation. Moreover, we find that the T allelic variant of the single nucleotide polymorphism rs350099 in the human CYCLINB1 gene generates a functional NF-Y binding site in its promoter and is associated with increased CYCLINB1 expression in peripheral blood lymphocytes.

Conclusions

NF-Y is upregulated and activated in atherosclerosis and restenosis and is essential for PDGF-BB-Erk1/2-Akt-induced CYCLINB1 expression and VSMC proliferation. Polymorphic NF-Y-dependent regulation of CYCLINB1 transcription associated to the T allelic variant of rs350099 contributes to increased CYCLINB1 expression in human lymphocytes. These data identify NF-Y as an attractive target for the treatment of vasculoproliferative disease.

P71
Effect of lamin A/C mutations on differentiation properties of adipose derived stromal cells

Abstract

Purpose

Mutations in lamin A/C gene (LMNA) lead to development of severe disorders – laminopathies. Unlike most other types of intermediate filaments, where the pathological effect of mutations is tightly linked to alteration of mechanical and integrative function, the detailed mechanism of lamin mutations is still unclear and possibly involves the alteration of nuclear signaling and transcriptional process. Since in laminopathies mesenchymal lineage tissues are mostly affected such as myocardium, skeletal muscle, adipose and bone tissues, the role of lamin A/C in differentiation process of mesenchymal stem cells was assumed. The aim of the study was to estimate the effect of LMNA mutations on differentiation of mesenchymal stem cells into muscle, adipose and osteogenic lineages.

Methods

In vitro mutagenesis was perfomed on wild type LMNA gene incorporated in a lentiviral vector. Several previously described mutations in LMNA were studied, each associated with a certain phenotype: G232E –myodistrophies, R471C – cardiomyopathies, R482L –lypodistrophies, G465D – myodistrophies and lypodistrophies. Adipose-derived mesenchymal stem cells from healthy donors were transducted with lentiviruses bearing either wild-type or mutant LMNA. Cells were then induced to either muscle, adipose or osteogenic differentiation using standart techniques.

Results

Adipose differentiation was markedly decreased in all four mutations and showed 50% for LMNA G232E, 20% for G465D, 5,5% for R471C and 0% for R482L compared to wild type LMNA (100%). Osteogenic differentiation rate was 95,6%for G232E, 70,8% for R471C, 65,0% for R482L and 30,4% for G465D compared to wild type LMNA. Myogenic differentiation was severely reduced in the presence of mutant LMNA with more detailed mechanisms are currently under investigation.

Conclusions

Mutations of lamin A/C gene have strong effect on the process of mesenchymal stem cells differentiation into adipose, osteogenic as well as myogenic lineage with several mutations completely abolishing adipose differentiation. We conclude that lamin plays an important role in the process of mesenchymal stem cells differentiation and more detailed mechanisms of its actions will be investigated further.

P72
Krueppel-like factor 15 regulates Wnt/beta-catenin transcription and controls cardiac progenitor cell fate in the postnatal heart

Abstract

Wnt/β-catenin signaling controls cardiac progenitor cells (CPC) in early development and the postnatal heart. We identified and characterized a β-catenin interaction partner, the Krüppel like factor 15 (KLF15)concerning CPC biology. A reporter assay showed KLF15 to significant repress β-catenin/TCF-dependent transcription in a concentration-dependent manner. Mutagenesis and co-immunoprecipitation analysis showed both, the KLF15 N- and C-terminus to be necessary for transcription repression. KLF15 specifically interacted with TCF4, a β-catenin co-factor that activates target gene expression and target it to ubiquitination. Moreover, KLF15 interacted with the NEMO-like kinase (NLK), which phosphorylates TCF4 to target it for degradation. Thus, KLF15 may recruit NLK to the β-catenin/TCF-complex to prevent activation of transcription via targeting TCF to protein degradation. The analysis of Klf15 knock-out (KO) mice showed a cardiac de-repression of β-catenin transcription demonstrated by increase of its target genes TCF4 and c-Myc in KO mice. KLF15 KO mice showed impaired fractional shortening (n = 10). Flow cytometry analysis revealed a significant reduction of cardiogenic-committed CPC identified as Sca1/aMHC (WT 1.75 ± 0.08% vs KO 0.83 ± 0.19%) and Tbx5 (WT 5.23 ± 0.48% vs KO 3.47 ± 0.26%). In contrast, endothelial Sca1/CD31 cells were increased in KO mice (WT 8.60 ± 0.36% vs KO 11.25 ± 0.39% n≥9. Under the hypothesis that β-catenin transcriptional activation promotes an endothelial cell fate of CPCs, we analyzed the CPCs fraction in mice with a cardiac β-catenin inactivation, which indeed showed a reduction of CD31pos (WT 18.01 ± 0.84% vs KO 15.06 ± 0.76% n≥6) as well as CD31pos/Ki67pos CPCs in comparison to controls. In favour of an endothelial fate of CPCs in klf15 KO Sca-1 isolated cells showed increased RNA expression of endothelial markers: von Willebrand Factor and Flk-1 as well as upregulation of the β-catenin target genes c-myc and TCF4. Increased endothelial Flk1 cells and reduction of aMHC and Hand1 cardiogenic cells was observed in co-cultured KO CPCs at day 10 of culture (n = 6). Treating the co-cultures with Quercetin, an inhibitor of β-catenin transcription, resulted in abrogation of Flk1 cell increase and aMHC and Hand1 cell reduction. Our data allows us to conclude that KLF15-dependent β-catenin repression favors endothelial differentiation and antagonizes cardiogenic cell differentiation similar to what has been observed in embryonic development. This work may have identified a novel molecular switch to therapeutically modulate CPC fate and unlock the regenerative potential of the adult heart.

P73
Human cardiac biopsy-derived cells improve angiotensin II-induced heart failure

Abstract

Purpose

Cardiac fibrosis is a hallmark of left ventricular (LV) dysfunction. We isolated and identified novel cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). The aim of our study was to evaluate whether CAPs improve angiotensin II (Ang II)-induced heart failure.

Methods

CAPs were isolated from human cardiac biopsies. Ang II (1.8 mg/kg body weight/day) or saline were continuously delivered in C57BL/6 mice by a subcutaneously implanted osmotic pump. One week after the Ang II infusion, 2 x 105 CAPs or PBS were intramyocardially injected. Two weeks after CAPs or PBS application, LV function was determined with a conductance catheter and LVs were harvested for immunohistology, DNA, and RNA isolation. LV interleukin-10 (IL-10) mRNA expression and human Alu presence were quantified via real-time PCR. In vitro, (un)- or L-NAME-treated CAPs were added to un-, Dil- or CFSE-labeled cardiac fibroblasts in the presence or absence of 1 μM Ang II for 24 hours, with or without anti-human IL-10 neutralizing antibody. Reactive oxygen species (ROS), AT1R and alpha-smooth muscle actin (SMA) expression, and fibroblast proliferation were determined by FACS. Collagen production was analyzed via Sirius red staining and anti-collagen I and III immunohistology.

Results

CAPs presence in the murine myocardium was confirmed by human Alu detection. CAPs improved the impaired LV systolic and diastolic function in Ang II-treated mice as indicated by a 1.3-fold (p < 0.01) and 1.4-fold (p < 0.005) increase in LV pressure and dP/dt max, respectively, and a 2.5-fold (p < 0.05) and 1.3-fold (p < 0.05) decrease in LV end diastolic relaxation and dP/dt min, respectively, compared to Ang II-treated mice. In parallel, CAPs normalized the 1.8-fold (p < 0.001) Ang II-induced accumulation of collagen I to levels in PBS control mice. The 6.7-fold (p < 0.001 vs control) increase in collagen III in Ang II-treated mice was reduced by 2.4-fold (p < 0.01) in Ang II + CAPs mice. LV IL-10 mRNA expression was 4.1–fold (p < 0.05) upregulated in Ang II + CAPs versus Ang II + PBS mice. In vitro, addition of CAPs to cardiac fibroblasts decreased the AT1R expression by 2.3-fold and 2.4-fold under basal and Ang II conditions (p < 0.001), respectively, and decreased the Ang II-induced ROS production, alpha-SMA expression, and fibroblast proliferation by 3.2–fold, 2.1-fold and 1.8-fold, respectively (p < 0.05). Furthermore, CAPs reduced the Ang II-induced collagen production by 1.4–fold (p < 0.05). These anti-fibrotic features of CAPs were NO- and IL-10 dependent.

Conclusion

CAPs are promising tools to improve Ang II-induced heart failure.

P74
Paracrine effects of stem cells on cardiac remodelling

Abstract

Purpose of our study included assessment of paracrine effects of stem cell (SC) therapy on cardiac remodelling in patients with congestive heart failure (CHF) of different origin.

Methods

Overall 53 patients with CHF were enrolled in our study. Patients were divided into groups according to the SC/placebo (NaCl solution) delivery method: selectively percutaneously intracoronary or transendocardially into the regions of interest and transepicardially during open heart surgery based on the non-invasive/invasive methods of investigation. We applied autologous bone-marrow stem (BMS) cell progenitors CD133 + in the treatment of patients with CHF due to advanced coronary artery disease [CAD] (n = 27) and non-ischemic dilative cardiomyopathy patients (NICMP; n=26).

Results

Single isolated SC therapy with autologous CD133 + progenitors at average dosage 2mln, performed transendocardially, resulted in the significant reduction of left ventricular (LV) end-diastolic volume with moderate increase of ejection fraction in short-term follow-up (3-6 months) in patients with CHF due to CAD in comparison to placebo group. Alongside during this period was observed moderate reduction of perfusion defects in SC "treated" regions with viable myocardium according to single photon emission computed tomography. These positive changes eliminated in 1 year follow-up. Other main LV remodeling indexes such as myocardial mass and left atrial volume did not change in 3-6 months follow-up. In NICMP patients we observed no changes in any of LV remodeling indexes.

In order to evaluate biochemical processes and paracrine effects of stem cells we performed enzyme-linked immunoelectrodiffusion assay of patients plasma samples for VEGF, bFGF, angiogenin, angiopoetins-1,2, MMP-9, PlGF, endostatin, TNF-α, SDF-1α and NT-proBNP levels before and after elective SC therapy. In NICMP patients at 14 days after single isolated transendocardial SC delivery we noted significant increase of SC homing factor SDF-1α plasma concentration, whereas in patients with ischemic CHF - significant decrease of PlGF (placenta growth factor) concentration in comparison with placebo group. There were no changes in plasma NT-proBNP levels in both groups.

Conclusions

SC paracrine effects exerted transiently in ischemic scarred, but viable myocardium and did not exert in non-ischemic dilated myocardium. Thus isolated transendocardial delivery of BMS progenitors CD133 + at average dosage 2mln can have positive effects if it is repeated in 6 months after first delivery in patients with ischemic cardiomyopathy.

P75
Gap junctional coupling with cardiomyocytes is essential for cardiomyogenic differentiation of fetal human mesenchymal stem cells

Abstract

Purpose

Gap junctional coupling is important for functional integration of transplanted cells with host myocardium. However, the role of gap junctions in cardiomyogenic differentiation of transplanted cells has not been directly investigated. In this study, the role of connexin43 (Cx43) expression in cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) was investigated.

Methods

Knockdown of Cx43 gene expression was established in naturally Cx43-rich fetal amniotic membrane hMSCs (Cx43↓ fetal AM hMSCs), while Cx43 was overexpressed in inherently Cx43-poor adult adipose tissue hMSCs (Cx43↑ adult AT hMSCs). The hMSCs were exposed to cardiomyogenic stimuli by co-incubation with neonatal rat cardiomyocytes (nrCMCs) for 10 days. Differentiation was assessed by immunostaining and whole-cell current-clamping. To establish whether the effects of Cx43 knockdown could be rescued Cx45 was overexpressed in Cx43↓ fetal AM hMSCs.

Results

Ten days after co-incubation not a single Cx43↓ fetal AM hMSC or adult AT MsC expressed α-actinin, while control fetal AM hMSCs did (2.18 ± 0.4%, n=5,000). Moreover, functional cardiomyogenic differentiation, based on action potential recordings, occurred only in control fetal AM hMSCs. Of interest, Cx45 overexpression in Cx43↓ fetal AM hMSCs restored their ability to undergo cardiomyogenesis (1.57 ± 0.4%, n=2,500) in co-culture with nrCMCs.

Conclusion

Gap junctional coupling is required for differentiation of fetal AM hMSCs into functional cardiomyocytes after co-incubation with nrCMCs. Heterocellular gap junctional coupling thus plays an important role in the transfer of cardiomyogenic signals from nrCMCs to fetal hMSCs but is not sufficient to induce cardiomyogenic differentiation in adult AT hMSCs.

P76
Identification of the proteomes of c-kit and sca-1 expressing populations of mice cardiac stem cells

Abstract

Stem cell-based therapy using Cardiac Stem Cells (CSCs) is a promising approach for myocardial repair. As CSCs are a recent discovery, it is essential to identify the proteins involved in its mechanisms of mobilization, differentiation and proliferation. In this study, we have identified 122 proteins from CSCs isolated by Fluorescence Activated Cell Sorting, expressing the stem cell markers Sca-1 and c-Kit. Nuclei, membrane and whole cell fractions were isolated by differential centrifugation and proteins were identified by Matrix-Assisted Laser Desorption Ionization (MALDI)-Fourier Transform Ion Cyclotron (FT-ICR). From the proteins identified , uncharacterized proteins were found in both kinds of cells, along with proteins involved in the proliferation pathways common to both populations (Protein Chibby Homolog 1), specific of c-kit + population (Alpha Enolase) or of the Sca-1 + population (47 kDa heat shock protein). Immunity system specific proteins were also identified (Protein 100-A8), suggesting a possible hematopoietic relation of CSCs. Differentiation involved proteins as Sortilin, which were identified in the Sca-1 + population, which is the most committed CSC population. Also proteins involved in chemotactic reaction were identified, as is the case of Myristoylated Alanin-Rich C kinase substrate (c-kit + and Sca-1 + populations) and Calgranulin-A (c-kit + population). These results allow the characterization of cell autonomous and non-cell autonomous pathways involved in CSC proliferation and differentiation, as well as differences between CSC sub populations, which are the foundations for a pathway analysis of the processes involved in cardiac regeneration and organ homeostasis.

P77
Systems-based investigation of the effects of adenosine on endothelial progenitor cells

Abstract

Purpose

Whether adenosine may positively affect cardiac repair is still a matter of debate. We previously reported that adenosine beneficially regulates inflammation, extracellular matrix turnover and angiogenesis, all processes involved in cardiac repair. Here, using a combination of in silico, in vitro, and in vivo approaches, we investigated whether adenosine affects endothelial progenitor cells (EPC), other key players of cardiac repair.

Methods

In silico: gene expression data from adenosine-treated EPC were obtained by microarrays. Gene-gene functional similarity was estimated with Gene Ontology-based information. In vitro: early endothelial progenitor cells (EPC) were obtained from peripheral blood mononuclear cells of healthy volunteers. In vivo: 18 rats underwent permanent occlusion of the left anterior descending coronary artery (LAD) and were treated by NaCl (n = 6), CADO (stable analog of adenosine, n=6) and CADO with 8-SPT (pan-antagonist of adenosine receptors, n=6). Rats were injected ip twice daily for 2 months. 6 additional rats were sham-operated.

Results

Computational systems-based approaches allowed the implementation of a new integrative predictive model based on the combination of gene expression data and gene ontology-based similarity information. This model predicted that adenosine may regulate the expression of several members of the chemokine family in EPC (AUC = 0.92). This prediction was validated in cultured EPC, in which adenosine regulated the expression of multiple chemokines and chemokine receptors. Among these, CXCR4 was significantly up-regulated (3-fold increase, P < 0.001). Pharmacology and RNA interference experiments implicated the A2B adenosine receptor in this effect. Adenosine stimulated EPC migration towards stromal cell-derived factor-1α and conditioned medium from cardiac fibroblasts. This effect was blocked by anti-CXCR4 neutralizing antibodies. In rats, 2 months after induction of myocardial infarction, the amount of EPC recruited to the heart was enhanced by CADO treatment and inhibited by 8-SPT. This was accompanied by increased vascularization in the border zone.

Conclusion

Systems-based approaches identified adenosine as a major regulator of EPC. Adenosine up-regulates CXCR4 expression in EPC and stimulates their recruitment to the infarcted heart. Together with previous observations, these results suggest that adenosine has the potential to enhance cardiac repair.

P78
Activity status of resident adult cardiac stem cells determines their stem cell properties and growth kinetics

Abstract

Purpose

The identification of a small population of stem cells resident in the adult mammalian heart, with robust regenerative capacity, indicates the potential to develop new strategies using these cells for cardioprotective and regenerative purposes. Endogenous cardiac stem cells (eCSCs) positive for c-kit, isolated from the adult mammalian heart, are clonogenic, self-renewing and multipotent. Here we compared the properties and growth kinetics of quiescent eCSCs with activated eCSCs in vitro.

Methods

To induce tissue damage and resultant eCSC activation, 5mg kg-1 isoproterenol (ISO) was injected (s.c.) into 2-month-old adult male Wistar rats (∼250g). Saline was injected as control (CTRL; quiescent eCSC group). c-kitpos CD45neg eCSCs were isolated by retrograde coronary enzymatic perfusion, 24 hours post-ISO or saline injection. Quiescent and activated eCSCs were analysed for number, proliferative markers, multipotency genes, growth kinetics, clonogenicity and proliferation index using flow cytometry, immunocytochemistry, qRT-PCR, real time lapse cell culture imaging, and BrdU incorporation assay, respectively.

Results

ISO-induced injury significantly (P < 0.05) increased c-kitpos eCSC number (33 ± 7% of the CD45neg small cardiac cell population, compared to 8 ± 2% in controls). Most of these cells were actively proliferating, being 56 ± 7% Ki67 positive, compared to only 10 ± 1% Ki67 positive in the quiescent CTRL eCSCs. Activated c-kitpos eCSCs showed increased clonogenicity (24 ± 4% vs. 2 ± 4%), cardiosphere formation (563 ± 58 per 100,000 cells vs. 59 ± 39) and proliferation, measured by BrdU incorporation over 48 hours in culture (32 ± 8% vs. 5 ± 2%). Furthermore, activated and quiescent c-kitpos eCSCs showed differential multipotency gene expression and real time-lapse growth kinetics.

Conclusions

Activated c-kitpos CD45neg eCSCs show enhanced stem cell properties and growth kinetics, compared to quiescent c-kitpos CD45neg eCSCs, in vitro. These data imply that pre-activation of stem cells could improve their cardioprotective and regenerative ability. Identifying factors that influence the activation of eCSCs and the maintenance of this state is of paramount importance for the design and optimisation of cardiac regenerative therapies.

P79
Constrainment is crucial for the survival, alignment and differentation of cardiomyocyte progenitor cells in 3D tissue culture: relevance for cardiac regeneration

Abstract

Purpose

Stem cell therapy has emerged as a promising treatment for myocardial infarction. A population of stem cells that resides in the human heart (cardiomyocyte progenitor cells, CMPCs) can be isolated and differentiated into beating cardiomyocytes using biochemical factors in vitro. Upon injection in the heart, these cells will experience a three dimensional (3D) rather than a 2D environment and are exposed to mechanical forces. To investigate further the cardiac regeneration potential of CMPCs, we studied the behavior of these cells in a 3D constrained environment.

Methods

Human fetal CMPCs were encapsulated in either longitudinally constrained or free floating hydrogels composed of collagen-Matrigel (n = 3 each). Resulting constructs were cultured for 9 days in control or differentiation medium, and analyzed for compaction, cell survival, proliferation, alignment and cardiac differentiation using immunofluorescent stainings.

Results

Cell survival was higher in constrained constructs compared to free floating constructs. Furthermore, proliferation in constrained constructs was decreased, but 14% of the cells was still dividing. In contrast, in free floating gels cell proliferation was abolished, indicating that static strain stimulates proliferation of CMPCs in 3D. Moreover, cells cultured in constrained constructs aligned in the direction of the constraint, probably as a result of internal stress formation as demonstrated by the compaction of the constructs (20% after 1 day of culture, 27% after 9 days) and the formation of intracellular stress fibers. Construct compaction was greatly enhanced after culture in differentiation medium (51%). Lastly, cells cultured in constrained constructs showed expression of the cardiac markers Nkx2.5 and α-actinin after 9 days of culture and independent of culture medium. Expression of those cardiac markers was lost in free floating constructs, suggesting that static strain is necessary to preserve the cardiac differentiation potential of CMPCs in 3D. The early cardiac marker GATA4 was only expressed in constructs cultured in differentiated medium.

Conclusion

Constraining appeared to be a crucial factor for the survival, proliferation and cardiac differentiation of CMPCs cultured in 3D hydrogel-based constructs. Moreover, constraining resulted in cell alignment, which is important for proper tissue integration. Optimization of the culture system may be of great relevance for cardiac regeneration.

P80
Kinin-mediated recruitment of regenerative circulating cells promotes endothelial healing and is dysfunctional in CAD

Abstract

Background

Kinins derived from the vascular wall might initiate the recruitment of distinct circulating cell types which bear the B2 kinin receptor (B2R). We have investigated the role of this mechanism on endothelial healing in healthy subjects (HS) and in patients with coronary artery disease (CAD).

Methods

Expression of the B2R on peripheral blood mononuclear cell (PBMC) subsets of CAD patients and age- matched HS was assessed by flow cytometry. Adhesion to an endothelial monolayer and subsequent closure of a scratch gap, supported by paracrine effects of the adhering cells were studied in vitro. In vivo, recruitment of systemically injected cells to injured carotid endothelium, and re-endothelialization of the injured vessel were assessed in a mouse model. B2R blockade by icatibant, transplantation of B2R-deficient bone marrow cells (B2R-/- BMC), as well as adenoviral B2R overexpression were used to verify the relevance of the B2R.

Results

In HS PBMC, B2R was low expressed ( < 1000MFI) on CD14hi inflammatory monocytes and on lymphocytes, but high in angiogenic Tie2 + or KDR + monocytes and in KDR + or CXCR4 + angiogenic progenitor cells (PC) (2517 to 7516MFI; P < 0.05 vs. CD14hi). Recruitment of healthy CXCR4 + PC to endothelial cells or to the injured murine vascular wall was blocked by B2R inhibition in vitro (-67% vs. vehicle, P < 0.05) and in vivo (-58% vs. vehicle; P < 0.05), while adhesion of CD14hi monocytes was unchanged, indicating a critical role of B2R for vascular homing of CXCR4 + PCs, but not of CD14hi monocytes. In vivo endothelial healing was lower in mice receiving B2R-/- BMC than in mice receiving B2R + / + BMC (24.7% vs. 36.1%, P < 0.05). Kinin receptor expression on CXCR4 + PC (-75% vs. H; P < 0.05) and on angiogenic "early outgrowth cells" (EOC) (-68% vs. HS; P < 0.05) was reduced in CAD patients. Adhesion of CXCR4 + PC from CAD patients to endothelial cells was markedly reduced versus CXCR4 + PC of HS and not regulated by the B2R. Adenoviral B2R overexpression rescued the capacity of CAD EOC to support re-endothelialization in vivo, associated with enhanced vascular recruitment of CAD EOC after B2R overexpression.

Conclusions

We newly describe that vascular kinins do not only act via the endothelial B2R, but can also recruit endothelial-supportive circulating cells to the vessel wall via B2R on the circulating cell. The loss of B2R on angiogenic circulating cells might jeopardize the healing of endothelial injuries and thus promote the decay of endothelial function in CAD.

P81
Endogenous cardiac stem cell (eCSC) activation, myogenesis and angiogenesis contribute to cardiac remodelling following intensity-controlled exercise training

Abstract

Purpose

The adult mammalian heart was traditionally viewed as a post-mitotic organ. Increased cardiac mass and contractility following exercise training was thought to occur solely through physiological hypertrophy of existing myocytes. The adult mammalian heart harbors stem-progenitor cells which participate in adaptations to myocardial stress. Here, we sought to determine the role of the c-kitpos endogenous cardiac stem cells (eCSCs) and new cardiomyocyte formation in the physiological cardiac adaptation to exercise stress.

Methods

42 male Wistar rats (∼220g) were exercised on motorized treadmills for 30 min/day, 4 days/wk for up to 4 wks at a low (LI; 55-60% VO2 max) or high (HI; 85-90% VO2 max) intensity. 25 untrained rats acted as age-matched sedentary controls (CTRL). To track myocardial cell generation, BrdU was administered (i.p.) twice daily. New cardiomyocyte formation, c-kitpos eCSC number and differentiation were assessed by immunohistochemistry. Growth factor gene array was determined in isolated cardiomyocytes from exercised and CTRL hearts.

Results

Immunohistochemical and confocal microscopy analysis of the left ventricle (LV) showed an increase (P < 0.05) in average cardiomyocyte diameter and volume in exercised animals. In addition to larger (hypertrophied) cardiomyocytes, smaller BrdU (3.4 ± 0.2% LI, 7.4 ± 0.3% HI) and Ki67 (0.8 ± 0.1% LI; 1.0 ± 0.1% HI) positive cardiomyocytes were observed in exercised animals indicative of new cardiomyocyte formation. Capillary density in LI (2866 ± 48/mm2) and HI (4158 ± 47/mm2) animals was significantly (P < 0.05) greater than CTRL (2105 ± 51) and many of these capillaries were BrdU positive. c-kitpos eCSC number increased (P < 0.05) in exercising vs. CTRL rats and many eCSCs expressed transcription factors indicative of their commitment to the cardiomyocyte (Nkx2.5) or capillary (Ets-1) lineages. Gene array analyses revealed the up-regulation of IGF-1, TGF-β1, Neuregulin-1, BMP-10 and Periostin in cardiomyocytes isolated from exercised animals. In vitro, these factors differentially determined c-kitpos eCSC proliferation, clonogenicity and differentiation.

Conclusion

Treadmill exercise training induces myocardial remodeling initiated by increased growth factor expression, leading to activation and ensuing differentiation of c-kitpos eCSCs. These effects are followed by cardiomyocyte hypertrophy along with new capillary and cardiomyocyte formation, which were dependent on exercise training intensity. These findings highlight for the first time the role of the endogenous regenerative capacity of the adult heart in the physiological adaptation to exercise stress.

P82
Mutually exclusive expression of key cardiogenic transcription factors in single cardiac progenitor cells

Abstract

Fate-mapping studies in mice provide the strongest evidence to date that mammalian cardiac regeneration exists, occurring in newborns via myocyte proliferation but in adults primarily by differentiation of stem/progenitor cells. Characterization of these adult cardiac progenitors is still in its infancy, as existing work has largely relied on purified but potentially heterogeneous populations.

Here, we study the features that predict clonogenicity of mouse cardiac progenitor cells (CPCs) and define their molecular signature by analysing clonal derivatives and freshly isolated single cells. Clonal growth of adult heart-derived Lin-/Sca-1 + CPCs was identified prospectively by the "side population" (SP) phenotype. Cloned cardiac Lin-/Sca-1 + /SP + CPCs were propagated for > 10 months without senescence or loss of Sca-1 and the SP phenotype. A panel of 43 genes was analysed by QRT-PCR in 19 clones (ABI 7900HT) and > 60 freshly isolated single cells (Fluidigm). Both expressed a concordant molecular signature, including several stem cell-associated markers and key cardiac transcription factors; conversely, both lacked markers of pluripotency, primitive mesoderm, pre-cardiac mesoderm, hematopoietic lineages, and cardiomyocytes. The expression of most (but not all) cardiac transcription factors yet not their downstream targets suggests that CPCs resemble an incomplete but stable form of heart-forming mesoderm. Expression of cardiac transcription factor proteins was confirmed. Microarray analysis of 3 clones at passage 40 was compared to freshly isolated Lin-/Sca1 + /SP + CPCs, heart tissue and undifferentiated ESCs. By agglomerative clustering, clones largely retained the phenotype of freshly isolated progenitors, excepting an increase in proliferation-associated GeneOntology categories. Notably, clones and freshly isolated single CPCs expressed varying subsets of the three essential cardiogenic factors (Gata4, Mef2c, Tbx5) reported to reprogram fibroblasts to a cardiac phenotype; 62 of 63 single CPCs analysed showed incomplete expression of this triad. Nevertheless, cloned CPCs were capable of cardiogenic differentiation after grafting to injured hearts, shown by immunostaining for sarcomeric MyHC and sarcomeric α-actin.

In conclusion: Clonal and single-cell analyses demonstrate unexpected microheterogeneities in the phenotype of Lin-/Sca1 + /SP + CPCs which may represent different states of differentiation or lineage. Expression of key cardiogenic transcription factors in mutually exclusive patterns may represent a mechanism to maintain the CPC pool and prevent precocious differentiation.

P83
Transplantation of human embryonic stem cell-derived endothelial cells into rats: a new approach for vascular regeneration

Abstract

Stem cell therapy may transform the treatment of acute and chronic heart disease, with an anticipated impact rivalling the results of revascularization and device therapies. Human embryonic stem cells and their cardiac derivatives are currently being developed as sources of tissue-specific cells for organ regeneration. In this preclinical study, we tested the feasibility and efficacy of human embryonic stem cell-derived endothelial cells (hESC-EC) engraftment. To favour endothelial formation, undifferentiated H7 embryonic stem cells were treated with serum and endothelial growth factors and CD31 + cell population were sorted subsequently by FACS from differentiating cultures. A highly expandable population of hESC-EC was obtained thereby which express endothelial cell markers and form vascular networks in vitro. Using extracellular matrix Matrigel as scaffold, hESC-EC were implanted subcutaneously or intramyocardially into 3-months-old athymic nude rats. For studying angiogenesis, a small animal imaging system, a high-resolution NanoSPECT/CT was used to acquire whole-body SPECT/CT images. By radiolabelled albumin, a local significant increase in perfusion was detected at the grafted sites after 2 weeks, suggesting the functional incorporation of hESC-EC into the microvasculature. Post-mortem histology further showed that hESC-EC survive, engraft successfully and form capillary-like structures. As assessed by quantitative PCR, their expression of angiogenic factors such as angiopoietin-2 and apelin are induced after 2 weeks as compared with preimplanted cells, suggesting that hESC-EC may undergo an in vivo maturation. Cardiac cell replacement therapy by using hESC-EC may be a promising future approach to repair of ischemic tissues and induce the formation of blood vessel networks.

Signaling
P84
Phosphoregulation of the titin-cap protein telethonin in cardiac myocytes

Abstract

The titin-cap protein telethonin was previously identified by our group as an interaction partner for the protein kinase D (PKD) catalytic domain, through a yeast two-hybrid screen of a human cardiac cDNA library. In the present work, kinase assays confirmed that recombinant full-length telethonin is a novel PKD substrate in vitro, and tandem mass spectrometric analysis using electron transfer dissociation identified S157 and S161 as putative PKD target sites. Further in vitro kinase assays using recombinant mutated telethonin in which S157 and S161 were replaced (either individually or in combination) by non-phosphorylatable Ala (S157A, S161A or S157/161A) confirmed that both S157 and S161, but no other sites, are targeted by PKD. A novel method for simultaneously detecting multiple phospho-moieties of telethonin, based on Phos-tag phosphate affinity SDS-PAGE, was developed and used to reveal that endogenous telethonin exists predominantly in a dually-phosphorylated form in isolated adult rat ventricular myocytes (ARVM) and in ventricular tissue from rat and mouse hearts. Experiments with heterologous expression by adenoviral gene transfer of epitope-tagged telethonin in wild type (WT) or mutated (S157/161A) form in ARVM indicated that WT telethonin becomes fully-phosphorylated, S157/161A telethonin is completely non-phosphorylatable, and the phosphorylation status of telethonin does not regulate its Z-disk localisation, as detected by immunolabelling and confocal microscopy. In a mouse model of pressure overload-induced left ventricular hypertrophy, significant but inverse changes were observed in myocardial telethonin expression (increased) and phosphorylation (decreased), suggesting stress-induced regulation of these processes and a potential link between telethonin protein turnover and its phosphorylation status. Further work is required to identify the cellular mechanisms that regulate telethonin phosphorylation and to determine the functional importance of such phosphorylation in physiological and pathological settings.

P85
A proper characterization of myocardial iron load and homeostasis based on serum markers in advanced heart failure

Abstract

Correcting iron deficiency with the use of iv iron supplementation in patients with heart failure (HF) with/without anemia may result in clinical improvement, however iron deficiency diagnosis has been solely based on such serum markers as ferritin (FR) and transferrin (TR) saturation (TSAT), being the main criterion for introducing iron supplementation. A proper characterization of iron homeostasis seems important, as the additional iron supplementation could potentially exert a harmful effect related to production of intracellular reactive oxygen species. So the aim of this study was to characterize the relation between myocardial iron (Iron-M), FR (FR-M), transferrin receptor (sTfR-M) and serum iron markers and to compare HF population with relation to Iron-M. FR-M is the main iron storage protein, whereas sTfR-M takes part in iron acquisition.

Methods and Results

Study group 33 patients, left/right ventricle (LV/RV) (LVEDV 245 ± 84 ml; LVESV 189 ± 85 ml; LVEF 22 ± 11%; RVD 32 ± 10 mm), NTproBNP (5464 ± 4825 pg/ml). Serum iron homeostasis assessment: iron, FR, TR/TSAT, sTfR, sTfR/logFR TIBC, UIBC, EPO. Myocardial Iron-M (Instrumental Neutron Activation Analysis, μg/g), FR-M, sTfR-M (ELISA – ng/mg protein) in the explanted failing hearts (FH), compared to non-failing hearts (NFH n=11).

In multivariate regression analysis/Pearson correlation out of all serum iron markers the independent predictors of myocardial variables were In LV: for Iron-M – sTfR/logFR (R2=0.18 , p=0.04; r=-0.49, p=0.04, respectively); for sTfR-M – sTfR/logFR (R2=0.52, p < 0.0001; r=-0.77, p < 0.0001), for FR-M – FR (R2=0.17 , p=0.03; r=0.42, p=0.03); In RV: for Iron-M – sTfR(R2=0.29, p=0.03, r=-0.44, p=0.03, respectively); for sTfR-M –sTfR/logFR (R2=0.38 , p=0.0005; r=-0.62, p=0.0005); for FR-M – TR (R2=0.24, p=0.009, r=-0.48, p=0.009).

With regard to Iron-M, patients were divided into reduced (n = 14) and non-reduced Iron-M (n = 19) subgroups. Both subgroups had similar degree of LV/RV dysfunction, NT-proBNP levels. FR-M was lower in reduced than non-reduced Iron-M group (LV –178 ± 80 vs 199 ± 51; p=0.08) and (RV – 159 ± 46 vs 189 ± 39; p=0.024), without differences in sTfR-M.

Conclusions

In HF patients a proper characterization of myocardial iron load and homeostasis, based on serum markers, required TR, FR, sTfR assessment and sTfR/logFR calculation. In low myocardial iron group, decrease in myocardial storage protein FR-M was observed without differences in LV/RV dysfunction degree.

P86
Methylated arginines and nitric oxide in end-stage renal disease: relationship with inflammatory and oxidative status

Table 1

 CRP < 6.0 mg/L(n = 21) CRP > 6.0 mg/L(n = 26) MDA < 1.06 μmol/L(n = 21) MDA > 1.06 μmol/L(n = 26) 
 ADMA SDMA ADMA SDMA ADMA SDMA ADMA SDMA 
NOx r = 0.279 P = 0.22 r = 0.537 P = 0.012 r = 0.642 P < 0.001 r = 0.655 P < 0.001 r = 0.250 P = 0.28 r = 0.148 P = 0.52 r = 0.408 P = 0.038 r = 0.705 P < 0.001 
 CRP < 6.0 mg/L(n = 21) CRP > 6.0 mg/L(n = 26) MDA < 1.06 μmol/L(n = 21) MDA > 1.06 μmol/L(n = 26) 
 ADMA SDMA ADMA SDMA ADMA SDMA ADMA SDMA 
NOx r = 0.279 P = 0.22 r = 0.537 P = 0.012 r = 0.642 P < 0.001 r = 0.655 P < 0.001 r = 0.250 P = 0.28 r = 0.148 P = 0.52 r = 0.408 P = 0.038 r = 0.705 P < 0.001 

Pearson correlation coefficients and P values for NOx and ADMA or SDMA.

Abstract

Purpose

Plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine are increased in end-stage renal disease (ESRD), contributing to endothelial dysfunction through direct (ADMA) and indirect (SDMA) inhibition of nitric oxide (NO) synthesis. Little is known on the direct relationship between NO and methylated arginines in ESRD, however. We addressed this issue in ESRD patients undergoing haemodialysis.

Methods

Plasma concentrations of nitrite:nitrate (NOx, a marker of NO synthesis), L-arginine, ADMA, SDMA, and markers of inflammation (C-reactive protein, CRP) and oxidative stress (malondialdehyde, MDA) were measured in 47 ESRD patients (age 64.4 ± 13.4 years) on haemodialysis for 21 (3-126) months.

Results

There was a positive correlation between ADMA and NOx (r=0.404, P=0.005) and between SDMA and NOx (r=0.587, P < 0.001) in the whole population. ADMA correlated positively with MDA (r=0.426, P=0.007), SDMA (r=0.538, P < 0.001), and L-Arginine (r=0.434, P=0.002). L-Arginine, in turn, also correlated with MDA (r=0.367, P=0.022). When dividing patients based on median MDA (1.06 μmol/L) the ADMA-NOx and SDMA-NOx correlations were significant only in the higher MDA group (Table 1). When patients were divided based on median CRP (6.0 mg/L), the SDMA-NOx correlation was significant both in the low CRP and in the high CRP groups. The ADMA-NOx correlation was significant in the higher CRP group but not in the lower CRP group (Table 1).

Conclusions

We found positive correlations between plasma NOx, ADMA, and SDMA in ESRD, particularly in patients with higher CRP and MDA concentrations. These surprising findings, given the inhibitory effects of methylated arginines on NO synthesis, suggest that 1) higher NOx concentrations are secondary to excessive NO synthesis due to a pro-inflammatory state; and 2) the relationship between methylated arginines and NO depends on the existing inflammatory and oxidation status.

P87
Regulation of myocyte function by gch1 overexpression mediated increase in nitric oxide

Abstract

Purpose

By causing uncoupling of nitric oxide synthase (NOS) activity, myocardial depletion of the NOS co-factor BH4 has been associated with adverse LV remodelling and impaired relaxation in animal models of cardiac disease. Oral supplementation of BH4 has been shown to be beneficial under these conditions; however, whether this is due to a genuine increase in myocardial BH4 availability or to a systemic antioxidant effect of BH4 remains to be established. Here, we evaluated the effect of increasing myocardial BH4 availability by transgenic overexpression of GTP cyclohydrolase-1 (GCH) under the control of the MHC promoter (GCH-Tg) on cardiomyocyte function.

Methods & Results

There was no difference in body weight, cardiac mass or myocyte size between genotypes. As expected, BH4 and total biopterin concentrations (HPLC) in myocytes from GCH-Tg were significantly increased compared with controls. These increases resulted in a 2-fold increase in myocardial NOS activity, which was mostly accounted for by the neuronal NOs (nNOS) isoform. The speed of relaxation and the rate of decay of the [Ca2+]i transient were faster in mGCH-Tg myocytes and isolated hearts. These findings were associated with a reduction in total PLB and an increase in the PLB-Ser16 phosphorylated fraction. nNOS inhibition with SMTC (100nM) abolished the difference in the rate of relaxation and PLB-Ser16 phosphorylated fraction between genotypes. The Ca2+ load in the sarcoplasmic reticulum (SR) did not differ between genotypes, nor did myocyte contraction (3 Hz, 35C) or the amplitude of the [Ca2+]i transient (Fura-2, 3 Hz, 35C), despite a reduction in Ca2+ current density in the mGCH-Tg myocytes, which was reversed by specific nNOS inhibition with SMTC (100nM). Fractional release of Ca2+ from the SR was increased in mGCH-Tg myocytes.

Conclusions

Myocardial BH4 content regulates cardiac function through a nNOS-mediated effect on intracellular Ca2+ fluxes. The BH4-mediated increase in NOS-derived NO improves relaxation (by increasing the PLB phosphorylated fraction) and decreases ICa . However, contractility and [Ca2+]i transient amplitude are maintained in mGCH Tg myocytes by an increase in the fractional release of Ca2+ from the SR suggesting that nNOS-derived NO may increase the gain of EC coupling. Together, these findings indicate that strategies aimed at increasing myocardial BH4 availability may hold promise in the treatment of heart failure in the presence or absence of NOS uncoupling, suggesting that this therapeutic approach may have wider than expected applicability.

P88
NPR-A and NPR-B signal in different functional compartments in failing rat left ventricle

Abstract

Natriuretic peptides (NPs) are used as biomarkers in heart failure (HF) as they increase with severity of the disease. C-type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) activate NPR-B and NPR-A receptors, respectively. Earlier studies have shown that CNP elicits a direct negative inotropic response (NIR) and a positive lusitropic response (LR) through the cGMP - protein kinase G pathway. In this study we investigated cGMP increase and functional responses to CNP and BNP and the regulation by phosphodiesterases (PDEs) in isolated ventricular cardiomyocytes and muscle strips from Wistar rats with HF. CNP and BNP both increased cGMP levels, but only CNP caused functional responses and increased PLB and TnI phosporylation, the Ca2+ transient magnitude and the Ca2+ extrusion rate constant. Preincubation with BNP did not affect the CNP-induced NIR, however it increased the CNP-induced LR. cGMP measurements indicated that NPR-A and NPR-B stimulation involved different cGMP compartments. Both BNP- and CNP-induced cGMP increase is regulated by PDE2, 3 and 5 but a NIR to BNP was not revealed, even in an attempt to abolish compartmentation by the presence of combined PDE2, 3 and 5 inhibition. In conclusion, there is a strong functional compartmentation of the cGMP signal indicating different roles of BNP and CNP in the pathophysiology of HF.

P89
The negatively inotropic effect of adrenaline stress is not completely linked to the reduction of cAMP release mediated by the Gs/Gi switch

Abstract

At supra-physiological concentrations of adrenaline the beta-2 receptor (β2AR) undergoes ‘stimulus trafficking’ and binds Gi. This causes negatively inotropic effects as the dissociation of Gi results in its Gαi subunit inhibiting the production of cAMP via effects on adenyl cyclase. The Gβy subunit of Gi is proposed to cause negatively inotropic effects via the induction of cardio-protective kinases such as PI3K/AKT and p38-MAPK. We propose that a greater number of β2AR receptors at the apical myocardium coupled with this ability may result in the apical ballooning observed in the syndrome of stress cardiomyopathy. We investigated the effects of adrenergic overload on cells using measurements of contractility and real-time imaging of cAMP signals by FRET.

Adult rat ventricular myocytes (ARVM) were isolated from basal and apical regions of rat myocardium. A video microscopy technique (Ionoptix) was used to assess contraction of ARVM. Apical cells were pre-stimulated with 100nM adrenaline, then re-stimulated with 1μM isoproterenol (ISO) whilst inhibiting β1AR with 300nM CGP20712A (CGP). PTX (1.5ug/ml 3hrs) was used to assess the role of Gi. For FRET measurements apical and basal cells were transfected with a cAMP sensor and cultured for 48 hours. These cells were pre-stimulated with 100nM adrenaline and then re-stimulated with100nM ISO with 100nM CGP and then by forskolin analogue 5 μM NKH477. The contraction response of apical ARVMs to β2AR stimulation was larger than that of basal ARVMs (apical 2.28fold ± 0.12 n=10 vs. base 1.32 ± 0.06 n=8 p≤0.0001). After pre-stimulation of apical ARVMs with adrenaline no significant increase in contraction was observed upon β2AR stimulation (control 1.30 ± 0.13 N=9 vs. adrenaline 0.19 ± 0.1 N=10 p≤0.0001). This inhibition was restored to above the control level after removal of the effects of Gi by pretreatment with PTX (4.17 ± 1.2 N=6 p≤0.01).The cAMP response of apical and basal cells was not significantly different (apex 46.7% ± 5.0 n=14 vs. base 42.4% ± 4.5 n=16 p=NS: values are % maximal FRET response). Pre-stimulation of ARVMs with adrenaline resulted in a significant reduction in β2AR mediated cAMP response (control 4.23% ± 0.3 N=40 vs. adrenaline 2.44 ± 0.51 N=10 p≤0.01 values are %FRET). PTX treatment did not restore the β2AR response (3.37% ± 0.5 N=9 p=NS).

Neither the decrease in β2AR response after adrenaline nor the supernormal response after PTX is explained by cAMP changes. Non-cAMP factors are therefore implicated in the acute inhibition of β2AR by adrenaline pre-stimulation in ARVMs. These factors may have a role in the syndrome of stress cardiomyopathy.

P90
Compartmentation of cAMP generated by AC6 involves PDE4 but not PDE3 in adult mouse cardiomyocytes

Abstract

Background

AC5 and 6 are the two major cardiac adenylyl cyclase isoforms. Their involvement in the spatiotemporal regulation of intracellular cAMP ([cAMP]i) in cardiomyocytes has never been studied. Here, we used transgenic mice with a cardiac AC6 overexpression (AC6 Tg) to measure membrane [cAMP]i in response to β-AR stimulation and its regulation by cardiac phosphodiesterases (PDEs).

Material and Methods

3 months old AC6 Tg males or their wild type littermates (WT) underwent surgery to delive, by multiple injection sites, an adenovirus encoding for the membrane-bound cAMP sensor Lyn-Epac2-camps (2x109 pfu). Five to 7 days following surgery, cardiac myocytes were freshly isolated and used to measure real time subsarcolemmal [cAMP]I variations using FRET-technology. Membrane [cAMP]i was measured in response to isoproterenol (Iso, 15 s pulse application), in the presence and absence of inhibitors of the two main cardiac PDEs: cilostamide (Cil, 1 μM) for PDE3; Ro-201724 (Ro, 10 μM) for PDE4.

Results

Direct intramyocardial injection of Lyn-Epac2-camps adenovirus results in a reasonable percentage of infected cardiac myocytes. Dose-response curves for the effect of Iso on [cAMP]i have been established for each genotype. AC6 overexpression leads to an increase in membrane [cAMP]i at all tested Iso concentrations. At a submaximal concentration of 100 nM Iso, the percentage of maximal increase in FRET signal was 40% higher in AC6 Tg than in WT (p < 0.001) indicating that the AC6 transgene was functional and well coupled to the up-stream part of the β-AR signaling system. PDE4 inhibition with Ro produced a significant (p < 0.05) and similar relative increase of the effect of Iso on [cAMP]i in both WT (15.8 ± 1.1%, n=45 vs. 9.9 ± 0.5%, n=14, in Iso alone) and AC6 Tg mice (18.2 ± 0.7%, n=29 vs. 13.8 ± 0.9%, n=29 in Iso alone). However, PDE3 inhibition with Cil increased [cAMP]i concentration in WT (13.0 ± 0.6%, n=15 vs. 9.9 ± 0.5%, n=14, in Iso alone, p < 0.001) but had no effect in AC6 Tg mice (13.3 ± 0.4%, n=30 vs. 13.8 ± 0.9, n=29, in Iso alone).

Conclusion

When overexpressed, AC6 controls a pool of cAMP which is controlled by PDE4 but not by PDE3. This indicates a different subsarcolemmal organization of the cAMP compartments generated by the two cardiac AC isoforms.

P91
The beta-3 adrenoreceptor inhibits cardiac hypertrophy through nitric oxide synthase and activation of AMP-activated protein kinase

Abstract

Background

β3-adrenoreceptors (AR) are found in the human heart, & their activation couples to nitric oxide (NO) synthesis; however, their role in cardiac function & remodelling is unclear.

Methods

Wild type (WT) & heterozygote male mice with cardiomyocyte-specific overexpression of human β3AR (TG) were treated for 10 days with isoproterenol (Iso 30mg/kg/day), angiotensin II (AngII 2mg/kg/day),or saline by osmotic minipump, with/without L-NAME (2mg/mL) in drinking water. In vitro hypertrophic responses to phenylephrine (PE) & Iso were analysed in neonatal rat ventricular myocytes infected with an adenovirus expressing the human β3AR (hβ3-NRVMs). Subcellular localisation of the endogenous β3AR was studied in adult rat ventricular myocytes (ARVMs) using the proximity ligation assay (PLA).

Results

LV function was similar between WT & TG at baseline (DP/dt max (mmHg/sec): WT 5894 ± 614 vs TG 8036 ± 1332), & following acute Iso infusion (WT 9803 ± 119 vs TG 10099 ± 1416; n=12-15). Following 10 day Iso or AngII, cardiac myocyte hypertrophy & an upregulation of βMHC mRNA expression were observed in WT (μm2: 682 ± 18 Sal vs 914 ± 54 Iso; p < 0.001;n=9), but not TG mice (697 ± 16 Sal vs 783 ± 39 Iso; p=ns; n=9). L-NAME treatment abrogated the protection from Iso-induced hypertrophy in TG mice (728 ± 30 L-NAME vs 963 ± 60 L-NAME + Iso; p < 0.001; n = 9). Likewise, in hβ3-NRVMs PE or Iso did not induce an increase in cell size (μm2: 1187 ± 30 PBS vs 1232 ± 29 PE vs 1200 ± 35 Iso; p=ns; n=3 isolations, 40 cells/n) or protein synthesis, in contrast to GFP-NRVMs (1175 ± 31 PBS vs 1321 ± 30 PE vs 1392 ± 31 Iso: p < 0.001). Treatmentof hβ3-NRVMs with L-NAME or KT5823 (an inhibitor of cGMP-dependent protein kinase) abolished the protection from PE-induced hypertrophy (1180 ± 28 L-NAME vs 1425 ± 28 L-NAME + PE; p < 0.001; 1274 ± 42 KT vs 1567 ± 43 KT + PE; p < 0.05).  PE inhibited AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis & cardiac hypertrophy (activation measured as intensity of phospho-Thr172 AMPK), yet in hβ3-NRVMs AMPK phosphorylation was preserved after PE (1.27 ± 0.3 PBS vs 1.33 ± 0.4 PE; p=ns). In ARVMs, PLA showed colocalisation of the β3AR & eNOS with caveolin-3, which coimmunoprecipitated with AMPK & eNOS.

Conclusion

Cardiac-specific overexpression of β3ARs does not alter LV function but inhibits hypertrophy following neurohormone stimulation in vivo and in vitro, partly through a NO-dependent mechanism. This involves a caveolar signalosome including the β3AR, eNOS & AMPK. Cardiac β3ARs may be a therapeutic target in the treatment of hypertrophic cardiomyopathies associated with elevated sympathetic tone.

P92
Functional characterization of phosphorylation-deficient endothelin receptor A

Abstract

Purpose

Endothelins are potent vasoconstrictor peptides which mediate their activity through the endothelin receptor A (ETA) and B (ETB) belonging to the superfamily of G-protein-coupled receptors (GPCRs). In the myocardium endothelin-1 (ET1) has growth promoting and positive chronotropic as well as positive inotropic effects. Multiple phosphorylation sites have been reported for the ETA receptor. However, their functional roles for signaling and receptor desensitization are widely unknown.

Methods

To analyze the impact of serine/threonine receptor phosphorylation on ET1-signaling we generated different phosphorylation-deficient alanine mutants of the ETA receptor: 1) ETA4PD with substitutions of the four distal C terminal amino acids T417, S420, S421 and S425 by alanine; 2) ETAPDZPD with substitutions of six amino acids belonging to the C-terminal PDZ-motif S391, S393, T396, S397, T403 and S404 and 3) ETA10PD with the combined substitutions of ETA4PD and ETAPDZPD. We analysed the ET1-induced inositol-trisphosphate (IP3) production in living cells transfected with ETA and mutant receptors by homogenous time resolved fluorescence (HTRF).

Results

Stimulation of cells transfected either with ETA4PD and ETAPDZPD, showed the same second messenger production as the wild type form of ETA after 2 hours of ET1-treatment. By contrast, ETA10PD resulted in a significant (p < 0,001) increase of IP3 production, indicating that multiple phosphorylation of the C-terminal domain of the receptor modifies signal transduction of ETA.

Conclusions

GRK phosphorylation of ETA receptor might play an important role for receptor desensitization after prolonged ET1 stimulus. We suggest that the desensitization process of the phosphorylation-deficient receptor mutant ETA10PD lacking 10 distal serine/threonine residues is significantly hampered, resulting in increased second messenger accumulation during permanent ET1 stimulation. Of note, the mutations in both receptors variants ETAPDZ-PD and ETA4PD each containing a sub-fraction of these 10 alanine substitutions cause no alteration in signaling. In summary, we suppose a cumulative phosphorylation effect at specific C-terminal sites for ETA-receptor desensitization.

P93
Hypoxia/reoxygenation induces TGFbeta/SMAD/GATA signalling in endothelial cells

Abstract

Under different pathophysiological conditions, as after myocardial infarction, the growth factor TGFβ is increased in hearts and contributes to cardiac remodelling. Since there are various cell types in the heart that can serve as a source of TGFβ, we now analyzed if cardiac endothelial cells can release bioactive TGFβ under hypoxic (Hx) and reoxygenated (Rx) conditions, and which signalling pathways are induced by TGFβ.

Microvascular endothelial cells were isolated from rat hearts and exposed to hypoxia (Hx) for 2 hours and followed by reoxygenation (Rx) up to 24 hours. The TGFβ precursor protein expression increased significantly after 1h and 24 h Rx (n = 21, p < 0.05). In order to analyse, if this increase of TGFβ precursor protein results in enhancement of bioactive TGFβ, we analysed activation of SMAD transcription factors, as classical signalling molecules of TGFβ. An increase in phosphorylation of SMAD2 to 130 ± 15 % or 113 ± 4% (n = 13, p < 0.05) was found after 2 h Hx followed by 2 h or 24 h Rx, respectively. This effect was abolished by incubation of endothelial cells with TGFβ type I receptor blocker SB431542 (1μM). Simultaneously with the induction of P-SMAD2 after 2 h Rx, expression of transcription factor GATA2 increased to 126 ± 10 % (n = 11, p < 0.05), whereas after 24 h Rx, enhancement of GATA2 was not present. This indicates a TGFβ induced gene regulation in endothelial cells via GATA2/SMAD2 interaction at early time points of reoxygenation, whereas after 24 h Rx SMAD2 interacts probably with different binding partners to regulate different target genes. To analyse, if TGFβ released from endothelial cells can also influence other cell types in the heart, supernatants of endothelial cells that underwent 2 h Hx followed by 1h Rx were added to ventricular cardiomyocytes of rat. These supernatants of hypoxia/reoxygenated endothelial cells enhanced phosphorylation of SMAD2 within 1.5 h in cardiomyocytes to 112 ± 1% (n = 4, p < 0.05).

In conclusion, after hypoxia endothelial cells release TGFβ at early and late time points of reperfusion. Then TGFβ acts in an autocrine manner on endothelial cells to regulate gene expression via the transcription factors SMAD2 and GATA2. Furthermore, TGFβ acts in a paracrine manner on cardiomyocytes and activates SMAD2 signalling in theses cells as well.

P94
PPAR agonists attenuated leptin induces endothelin-1-Rho/Rho-kinase-interleukin-18 pathway in cardiomyocytes

Abstract

Purpose

Leptin is known to be an adipocyte-derived hormone and regulates weight control and energy metabolism. Recent studies indicate that leptin may contribute to heart failure. Interleukin-18 (IL-18), a member of the IL-1 family, is a proinflammatory cytokine with multiple biological functions. IL-18 induces myocardial hypertrophy, loss of contractility of cardiomyocytes and apoptosis leading myocardial dysfunction. Increased levels of circulating IL-18 are thought to be one of risk factors for heart failure. However, the effect and mechanism by which leptin induces heart failure with inflammatory cytokine were still unclear. Therefore, in the present study, we examined how leptin induces heart failure with increased IL-18.

Methods

We used cultured rat neonatal cardiomyocytes stimulated with leptin in order to measure IL-18 mRNA and protein expression, and Rho-kinase and NF-kB activity. We also investigated the effects of peroxisome proliferator-activated receptors (PPAR) agonists on these actions.

Results

Leptin increased IL-18 mRNA and protein expression with dose- and time-dependent manner. BQ123, an endothelin A receptor (ETAR) antagonist inhibited leptin-induced IL-18 expression. Moreover, leptin induced endothelin-1(ET-1) production in cultured media and ET-1 increased IL-18 expression. These results indicate leptin induces IL-18 expression intermediates ET-1 via ETAR. Furthermore, C3 toxin, RhoA inhibitor, fasudil, Rho-kinase inhibitor, simvastatin, an HMG-CoA reductase inhibitor, and PPAR agonists, pioglitazone and bezafibrate led to a significant reduction in leptin-induced IL-18 expression. On the other hand, leptin up-regulated the activities of Rho-kinase and NF-kB. Western blots showed PPAR agonists attenuated the leptin-induced IL-18 expression and NF-kB activity but not the Rho-kinase activity. These results indicate that leptin induced the IL-18 expression through a mechanism that involves, at a minimum, ET-1 acting via the Rho/Rho-kinase and PPAR/NF-kB pathway and PPAR agonists attenuate the leptin-induced IL-18 expression at a point downstream from Rho/Rho-kinase.

Conclusions

The induction of IL-18 in cardiomyocytes by leptin and ET-1 might, therefore, cause a deterioration of the cardiac function in an autocrine and paracrine fashion. The inhibition of the IL-18 expression by PPAR agonists might be one of the mechanisms whereby the beneficial cardiovascular effects are exerted.

P95
Dipyridamole reduces the expression of metalloproteinase-9 in human monocytes by reducing nf-kappab signaling: an anti-inflammatory effect with a possible role in stroke prevention

Abstract

Matrix metalloproteinase (MMP)-9 putatively plays an important role in stroke by accelerating matrix degradation, disrupting the blood-brain barrier, and increasing infarct size. Therefore, the inhibition of MMP-9 activity is a potential target for stroke prevention. Dipyridamole (DP) is an antiplatelet agent with recognized benefits in stroke prevention. In addition to its antiplatelet properties, recent studies have reported that DP also features anti-inflammatory and anti-oxidant properties. We here investigated whether DP regulates MMP-9 expression in human monocytes and explored underlying mechanisms.

Methods

Human peripheral blood mononuclear cells (PBMC) and the U937 monocytoid cells were treated with 1-10 μmol/L DP for 1 h before stimulation with 10 ng/mL tumor necrosis factor (TNF)α or 10 ng/mL phorbol myristate acetate (PMA) for 24 h. Conditioned media were then tested for the release of MMP-9 and tissue inhibitors of MMP (TIMPs) by ELISA, while MMP-9 activity was assessed by gelatin zymography. In parallel, MMP-9 mRNA expression was investigated by RT-PCR, the activation of NF-κB by EMSA and Western analysis and finally activation status of p38 mitogen-activated protein (MAP) kinase was assessed by Western analysis using an antibody that recognize the dual phosphorylated (activated) form of the enzyme.

Results

MMP-9 antigen and activity, in both PBMC and U937, increased significantly after stimulation with TNFα or PMA for 24 h (P < 0.01). DP reduced TNFα and PMA-induced MMP-9 activity in a concentration.dependent manner, with an half maximal inhibitory concentration (IC50), for both stimuli, at 5 μmol/L (P < 0.01). At the same concentrations DP also reduced MMP-9 protein release by 60% (P < 0.01 versus TNFα) and MMP-9 mRNA expression by 40% (P < 0.05 versus TNFα), without significantly affecting the release of TIMP-1. At 5 and 10 μmol/L DP also significantly inhibited (P < 0.05) PMA- and TNFα -induced activation of NF-κB (at EMSA) as well as the nuclear translocation of p65 (at Western analysis), through a mechanism involving inhibition of the degradation of the NF-κB inhibitor IkBα and the activation of p38 MAP kinase, thus yielding a plausible mechanism for the reduction of MMP-9 expression.

Conclusions

DP, at therapeutically achievable concentrations, reduces the expression and release of MMP-9 through a mechanism involving NF-κB inhibition. These results indicate that DP exerts anti-inflammatory properties that may favorably contribute to its actions in the secondary prevention of stroke, independent of its antiplatelet properties.

P96
Expression of the regulatory receptor CD200R on macrophages is regulated by polarization signals in atherosclerosis

Abstract

Background

Macrophage activation and polarization are key steps in host defense and chronic inflammatory diseases, including atherosclerosis. The glycoprotein receptor CD200R1 belongs to a family of four isoforms and signals by binding to its counterpart ligand CD200. The receptor is expressed on cells of the myeloid lineage and inhibits inflammatory signaling by blocking pro-inflammatory mediators.

Aims

Our work aims to determine the signals that regulate CD200R expression during macrophage polarization and the development of atherosclerosis.

Methods

Bone marrow-derived macrophages from WT mice were cultured in the presence of combinations of Interferon gamma (IFN-g), Lipopolysaccharide (LPS) (pro-inflammatory M1 macrophages) or IL-4 and IL-13 (M2 macrophages). The expression of CD200R1 and polarization markers was evaluated via Q-PCR. Using the hypercholesterolaemic ApoE-/- model which spontaneously develops atherosclerotic lesions, we assessed the kinetics of CD200R1 expression (at 8, 12-15 and 35 weeks of age) during disease progression in the aorta and in secondary lymphoid organs with immunohistochemistry and Q-PCR.

Results

CD200R1 gene expression is significantly reduced after exposure to M1 signals IFN-g and LPS alone (respectively 3 ± 0.09 fold change; p < 0.001 and 2.38 ± 0.05; p < 0.01 vs. un-stimulated) or in combination (6.02 ± 0.039, p < 0.001). A similar pattern is followed by the M2 marker CD206 (1.78 ± 0.11, p < 0.05). Conversely, IL-4 and IL-13 increased expression of CD200R1 using IL-4 alone (2.83 ± 0.41, p < 0.05) or IL-4 together with IL-13 (2.3 ± 0.38, p < 0.05). This correlated with the increased expression of CD206 p < 0.01. Treatment with oxidized low-density lipoprotein (oxLDL) did not induce significant changes. Finally, CD200R1 expression in ApoE-/- mice is reduced at late stages of atherosclerosis compared to healthy age matched controls (p < 0.05)

Conclusion

The expression of CD200R1 is negatively regulated by pro-inflammatory M1 signals and positively regulated by anti-inflammatory M2 signals. CD200R1 is also reduced at late stages of atherosclerosis. The CD200R-CD200 axis could be failing to regulate inflammation in atherosclerosis.

P97
MicroRNA-155 mediates cardiac inflammation and injury in acute viral myocarditis

Abstract

Backgroud

Viral myocarditis (VM) is the most prevalent cause of heart failure in young adults. The pathogenesis is based on an adverse immune response inflicting irreversible damage to the myocardium. In this study, we examined the role of inflammatory microRNAs – and microRNA-155 in particular – in VM.

Methods and Results

Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible to non-susceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b and -21 were consistently and strongly up-regulated during acute myocarditis in both humans and susceptible mice. In situ hybridization revealed that microRNA-155 expression during myocarditis was localized primarily in infiltrating inflammatory cells. Inhibition of miR-155 in C3H mice by a systemically delivered LNA-antimiR suppressed CVB3-induction of miR-155 in the heart (75% reduction, p < 0.001) and attenuated cardiac inflammation and necrosis (LV necrotic area: LNA-control: 35.3% versus LNA-antimiR-155 19.7%, p < 0.01, n=11 and 12, respectively) during acute myocarditis. MicroRNA-155 inhibition did not affect viral load before onset of inflammation, but decreased viral copy numbers during the inflammatory phase 7 days after infection (4-fold, p < 0.01), indicating that microRNA-155 loss-of-function did not compromise viral clearance while preventing adverse cardiac inflammation.

Conclusions

MicroRNA-155 is selectively upregulated in inflammatory cells during acute viral myocarditis and is a mediator of adverse cardiac inflammation after CVB3 infection in mice. Its knockdown attenuates myocardial inflammation and necrosis without compromising viral clearance. Our data identify microRNA-155 as a potential therapeutic target in the treatment of viral myocarditis. 

Ion channels and Electrophysiology
P98
The possible proarrhythmic effects of diclofenac

Abstract

Introduction

Sudden cardiac death among athletes is very rare but 2-4 times more frequent than in the age- matched control population. Non-steroidal anti-inflammatory drugs (NSAIDs) like diclofenac are widely used in the treatment of sports injuries, however, their effects on the cardiac electrophysiological parameters are not properly understood. It is possible that the NSAID diclofenac might cause ventricular repolarisation abnormalities contributing to the increased arrhythmic risk of young athletes. Therefore the aim of our study was to characterize the cellular electrophysiological effect of diclofenac on dog right ventricular preparations.

Methods

Action potential measurements were carried out by applying the standard intracellular microelectrode technique in right ventricular papillary muscle preparations isolated from mongrel dogs of either sex weighing 12–20 kg. Ionic currents were recorded using the whole-cell configuration of the patch-clamp technique in single ventricular myocytes isolated from dog hearts. The experimental temperature was 37°C.

Results

Diclofenac slightly lengthened the action potential duration at 90% repolarisation (APD90) without affecting the maximum upstroke of AP (Vmax). In the presence of 100 nM IKr blocker dofetilide, 20 μM diclofenac caused significant additional APD90 lengthening in a reverse frequency dependent manner. The drug induced a marked further increasing relative to the APD values measured after the administration of 100 nM Dofetilide and 30 μM BaCl2, ie. the APD lengthening effect of diclofenac was significantly augmented in preparations where the “repolarisation reserve” was attenuated by previous application of dofetilide and BaCl2. In some experiments early afterdepolarisations (EADs) developed in this setting. During the experiments transmembran ionic currents were also measured. In dog ventricular myocytes the amplitude of IKr was concentration dependently decreased by diclofenac. IKs was also depressed by 30 μM diclofenac.

Conclusions

At therapeutic concentration diclofenac alone does not influence ventricular repolarisation significantly. However, in the case of impaired "repolarisation reserve" such as organic heart disease or in athlete's heart, diclofenac may enhance the arrhythmic risk and sudden cardiac death by increasing the APD and developing EADs.

P99
PI3Kgamma protects against catecholamine-induced ventricular arrhythmia through PKA-mediated regulation of distinct phosphodiesterases

Abstract

Purpose

Phosphoinositide 3-kinase γ (PI3Kγ) signaling downstream β-adrenergic receptors (β-ARs) plays a pivotal role in the regulation of myocardial contractility and maladaptive remodelling. However, the function of PI3Kγ in catecholamine-induced arrhythmia is presently unknown.

Methods and Results

Adrenaline stimulation in mice lacking PI3Kγ triggered runs of premature ventricular contractions thus uncovering PI3Kγ as a crucial mediator of arrhythmia. Consistently, FRET probes revealed abnormal cAMP accumulation following β2-AR activation that depended on PI3Kγ scaffold activity. Downstream β2-ARs, PI3Kγ was found to participate in multi-protein complexes linking PKA to the activation of PDE3A, PDE4A and PDE4B, but not PDE4D. The ensuing negative feedback loop lowered cAMP and limited PKA-mediated phosphorylation of L-type calcium channel and phospholamban, thus preventing arrhythmogenesis.

Conclusions

PI3Kγ protects against catecholamine-induced ventricular arrhythmia by coordinating the coincident signaling of the major cardiac PDE3 and PDE4 isoforms in distinct cellular compartments.

P100
Mice with conditional knockout of Galphai2 in the cardiac pacemaker system exhibit tachycardia with loss of HF power on HRV analysis

Abstract

Purpose

We have previously shown that inhibitory G protein Galphai2 (Gαi2) is important in mediating vagal tone to the murine heart with global Gαi2 knockout (KO) mice developing tachycardia with loss of high frequency (HF) power and resistant to the effects of the muscarinic agonist Carbachol. However, it is unclear whether the effect is mediated entirely via Gαi2 signaling at the end-organ level or whether there is a central component. We sought to investigate this by studying mice with conditional KO of Gαi2 in the cardiac pacemaker tissue.

Methods

Using the Cre/loxP approach, Gαi2 was deleted by Tamoxifen inducible Cre-recombinase driven by the promoter HCN-4, which is selectively expressed in cardiac pacemaker cells.  Littermates without either the Cre and/or loxP alleles acted as controls. 48 hour ECG tracings in freely moving mice were obtained using implantable telemetry probes. The mice were injected with Tamoxifen i/p (1mg/25g body weight for 5 days) and the ECG telemetry recordings were repeated 10 days after the last dose. Heart rate (HR) and heart rate variability (HRV) was then derived from the ECG tracings.

Results

6 KO and 7 control mice aged between 3-4 months were studied. There was no significant difference in mean HR between the groups pre-Tamoxifen (508 ± 104 v 536 ± 96 bpm, p=0.12). Post Tamoxifen, KO mice were more tachycardic (538 ± 91 v 496 ± 79 bpm, p=0.06), with a significant increase in daytime HR (530 ± 85 v 457 ± 63 bpm, p < 0.001) (Figure). On HRV analysis, there was a reduction in daytime HF power in the KO mice (41 ± 21 v 24 ± 12 ms2, p=0.03).

Conclusion

Our findings suggest that Gαi2 signaling at the level of the pacemaker system is important in mediating parasympathetic effect on murine hearts in vivo.

P101
Chronotropic pharmacology of cardiomyocytes derived from of human iPS-cells

Abstract P101 figure

Abstract P101 figure

Abstract

Highly homogenous and pure cell preparations with a cardiomyocyte phenotype can be obtained from human pluripotent stem cells. When seeded on fibronectin-coated surfaces in a 37°C CO2 incubator, these cells form spontaneously-contracting syncytia after 2-4 days, which, after 7-10 days, exhibit a stable and "human-like" spontaneous rhythm (SR = 40-60 bpm). SR is highly sensitive to temperature and gas exchange conditions such that special care must be taken to ensure homogenous conditions across the preparations. We have characterized the chronotropic response of this preparation to the application of compounds known to modify heart rate in humans. The adrenergic receptor agonists norepinephrine, isoproterenol and salbutamol increase SR (Δ, EC50 (nM): + 73%, 30; + 102%, 354; + 73%, 5.8). Histamine also increases SR (Δ, EC50 (nM): + 72%, 285). In contrast, the muscarinic receptor agonist carbachol, the S1P receptor agonists S1P, p-FTY720 and BAF-312, the selective adenosine A1 receptor agonist N6-cyclopentyladenosine, and the If blocker ivabradine all reduce SR (Δ, EC50 (nM): −27%, 462; −13%, 7.2; −16%, 0.11; −26%, 0.11; −25%, 21; −53%, 80). The slowing of rhythm induced by the GPCR agonists is prevented by inhibiting Gi-protein coupling with pertussis toxin (figure) and it is reduced by blocking GIRK channels with tertiapin Q. We also examined the effect of the IKr blockers E-4031 and quinidine: they induce arrhythmic beating in the preparation. Therefore the human iPS-cardiomyocyte represents a new tool to predict the pharmacological response of human hearts in regard to their chronotropic behavior. It can be used as well to dissect the cellular mechanisms of chronotropic control in human cardiac tissue and compare them to what has been extensively described in animal models.1

P102
Long QT3 mice have disrupted sympathovagal balance and in vivo ventricular stimulation does not determine risk of sudden cardiac death, suggesting that a second perturbation may be required

Abstract

Purpose

Long QT 3 (LQT 3) is a cause of sudden cardiac death (SCD) by Torsades de pointes (TdP). SCD often occurs during sleep, rest and bradycardia, suggesting that heightened parasympathetic tone provokes TdP in LQT 3. It is challenging to ascertain the risk of SCD in these patients. We performed in vivo electrophysiological studies (EPS), ventricular tachycardia (VT) stimulation and telemetry in LQT 3 (ΔKPQ) and wild type (WT) mice, without and with provocation with the muscarinic agonist, carbachol.

Methods

EPS were performed in young (8-week) and old (≥6 months of age) anaesthetised mice with a 1.1F catheter inserted into the right ventricle via the internal jugular vein. ECG and EP parameters were recorded. VT stimulation was attempted with 1 to 5 extrastimuli, coupled at 75 to 10ms following a train of 8 beats at 100ms. This was repeated after injection with 0.5mg/kg carbachol. Telemetry probes were inserted intra-abdominally into young (12-week old) mice and old (≥6 months of age) mice. After a 2 week recovery period, ECGs were recorded in conscious mice for 48 hours and studied for VT and ventricular ectopics (VE). An ECG post carbachol was analysed. Heart rate variability (HRV) was measured from 12 to 2pm, when murine vagal tone is highest.

Results

Young ΔKPQ had a prolonged PR interval (41.2 ± 0.5 vs 39.2 ± 0.6ms, P=0.003), QRS duration (11.0 ± 0.6 vs 9.6 ± 0.3ms, P=0.03), as well as a prolonged corrected QT interval (74.2 ± 3.6 vs 57.2 ± 1.4, P < 0.0001), compared to WT. EP parameters were comparable between young and old cohorts and respective WT controls. One young WT developed VT. Of the older mice, 1 WT had a VE pre and post carbachol and 1 ΔKPQ had a VE post carbachol. ΔKPQ mice had disrupted sympathovagal balance shown by normalised low (9.8 ± 3.6 vs 53.2 ± 5.6nu, P=0.004) and high (12.2 ± 3.1 vs 46.7 ± 5.6nu, P=0.004) frequency power. On examination of telemetry ECGs, 1 young ΔKPQ had 2 pauses (435 and 394ms) and 1 old ΔKPQ had 3 pauses (316, 308 and 378ms), but no pauses were seen in WT. No VT was found.

Conclusions

To our knowledge, this is the first report of in vivo EPS in the ΔKPQ mouse. Our results concur with human studies where VT stimulation is unhelpful in predicting risk of SCD.

HRV assessment shows disrupted symapthovagal balance, but an increase in vagal tone with carbachol did not induce VT. However, the effects of carbachol are short. It is feasible that a second perturbation sustaining increased vagal tone, for example, through increased inhibitory G-protein activity, is responsible for TdP and predisposition to SCD in LQT 3 patients.

P103
Cellular mechanisms involved in antiarrhythmic effects of omega-3 fatty acids in young and old SHR

Abstract

Background and Purpose

Hypertension-induced myocardial remodeling is known to be associated with increased propensity to malignant arrhythmias and sudden death partially due to alterations in cell-to-cell coupling protein, connexin-43 (Cx43) at the gap junctions. We investigated whether omega-3 fatty acids diet can protect from malignant arrhythmias via modulation of intercellular Cx43-mediated signaling and intracellular signaling mediated by protein kinase C (PKC) at early and late stage of structural remodeling.

Design and Methods

Untreated male 3 and 12-month-old spontaneously hypertensive rats (SHR) and age-matched healthy Wistar rats were compared with animals supplemented by omega-3 FA (EPA + DHA 30 mg/day) for two month. Body weight, blood pressure, glucose and plasma lipids profile were monitored. Left ventricular tissues were taken for immunoblotting of Cx43 and PKC-isoforms. In situ immunostaining of Cx43 and electron microscopy were performed to examine distribution and subcellular alterations of gap junctions. Langendorff-heart preparation was used to test VF inducibility.

Key results

Immunobloting of all rat hearts revealed conventional patterns of Cx43 expression, i.e. two phosphorylated forms Cx43 (P-Cx43) that are needed for the function of Cx43 channels and one non-phosphorylated (noP-Cx43) form. Neither total Cx43 expression nor P-Cx43 was significantly altered in young unlike old SHR heart, which exhibited marked decrease of both parameters. However, the ratio of P-Cx43 to total Cx43 was reduced (P < 0.05) in young SHR hearts while markedly increased due to omega-3 FA diet. Moreover, omega-3 FA enhanced significantly both P-Cx43 and total Cx43 levels in old SHR. In addition, abnormal distribution (lateralization and inernalization) of Cx43-positive gap junctions was attenuated by athe treatment. Expression of PKC-epsilon, which phosphorylates Cx43, was significantly decreased in both young and old SHR, while augmented due to omega-3 FA diet. On the other hand, the expression of PKC-delta (which promotes fibrosis) was increased in the hearts of SHR and suppressed by the treatment. Furthermore, omega-3 FA diet significantly reduced incidence of electrically-inducible VF in both young and old SHR.

Conclusions

Results indicate that both young and old hypertensive rats benefit from omega-3 fatty acids diet due to alleviation of myocardial abnormalities in connexin-43 and protein kinase-C signaling that was associated with suppression of malignant arrhythmias.

This work was supported by VEGA 2/0046/12 grant.

P104
Preferential re-entrant excitations for atrial fibrillation/flutter arising from the coronary sinus through the left atrial roof in rat hearts, a combined analysis by optical mapping and histology

Abstract

Re-entrant excitations are accepted as being responsible for genesis of atrial fibrillation and flutter (AF/AFL); however, detailed activation patterns of these arrhythmias are not fully elucidated with reference to the morphological correlations. We hypothesized that intrinsic regional differences in atrial structure, especially those in muscle content, can be a substrate for abnormal impulse propagation leading to generation of AF/AFL. To address this issue, we evaluated the possible re-entrant pathways on the whole atrial tissue in excised rat hearts and explored their correlations of histologic background.

By using Langendorff-perfused hearts with bilateral lungs at 32°C (11-13-week-old males, n=19), spatiotemporal patterns of excitation on the posterior surface of the di-4ANEPPS-stained whole atria (15.9x13.6mm, 188x160 pixels) were optically visualized by a high-speed CMOS camera (MiCAM02; Brainvision) at 500 frames/s. Atrial arrhythmias were induced by a premature stimulus after 5-Hz consecutive pacing at the right atrium. Following the optical imaging of atrial arrhythmias, histological analyses (n = 9) were conducted on the posterior surface of the whole atria sectioned on the short axis plane by using a digital slide scanner system (ScanScope, Aperio).

Of 19 hearts examined AF/AFL was induced in 15 hearts, most of which initially showed re-entrant excitations through the roof region of the left atrium and the coronary sinus with subsequent development of complex patterns of re-entrant propagations, i.e., meandering. The conduction velocity at the roof of the left atrium was significantly slower than that of the coronary sinus (42.4 ± 16.6 vs 53.3 ± 9.2 cm/s, p < 0.05) just before the initiation of re-entry, suggesting the existence of slow and fast conduction pathways. Quantitative histologic evaluation by Masson's trichrome staining of atrial tissues, divided into seven different areas, i.e., anterior region of the right atrium, roof region, anterior region, lateral region, and posterior region of the left atrium, septum, and coronary sinus, revealed that the myocardial density in the roof region of the left atrium was significantly lower than that in the coronary sinus (76.3 ± 2.5 vs 82.3 ± 4.7 %, p < 0.01) and the remaining atrial areas.

In summary, the combined functional and histological analyses of the rat atria enabled us to elucidate a possible morphological substrate for arrrhythmias. The observed regional differences in myocardial density between the left atrial roof and the coronary sinus may become an important substrate for AF/AFL under certain diseased conditions.

P105
Circulating heat shock protein 70 and anti-heat shock protein 70 antibodies in atrial fibrillation: relation with atrial fibrillation type and response to catheter ablation

Abstract

Background

This study investigated the association between heat shock protein 70 (HSP70) and anti-HSP70 antibodies as well their changes and rhythm outcome after atrial fibrillation (AF) catheter ablation.

Methods

We studied 67 patients with AF (59 ± 11 years, 66 % male, 66 % lone AF) undergoing catheter ablation. Circulating HSP70 and anti-HSP70 antibody levels were quantified using commercially available assays before and 6 months after catheter ablation. Serial 7-day Holter ECGs were used to detect AF recurrences.

Results

At baseline, HSP70 was detectable in 14 patients (21 %), but there was no correlation between clinical or echocardiographic variables and the presence or the level of HSP70. In contrast, patients with paroxysmal AF (n = 39) showed lower anti-HSP70 antibodies (median 43, IQR 28 – 62 μg/ml) than patients with persistent AF (n = 28; 53, 41 – 85 μg/ml, p=0.035). Using multivariable regression analysis, AF type was the only variable associated with anti-HSP70 antibodies (Beta=0.342, p=0.008). At 6 months, HSP70 was present in 27 patients (41 %, p < 0.001 vs. baseline) with an overall increase (median 0, IQR 0 – 0 vs. 0, 0 – 0.09 ng/ml, p=0.029). Similarly, there was an increase of anti-HSP70 antibodies (48, 36 – 72 vs. 57, 43 – 87 μg/ml, p < 0.001). AF recurrence rates were higher in patients with HSP70 increase ≥0.025 ng/ml (32 vs. 11 %, p=0.038) or anti-HSP70 increase ≥2.5 μg/ml (26 vs. 4 %, p=0.033).

Conclusion

HSP70 and anti-HSP70 antibodies may be involved in the progression of AF and AF recurrence after catheter ablation.

P106
Computational modeling of the cellular mechanisms of cardiac pacemaking

Abstract

The cellular basis of heart's pacemaking, and specifically the degree of contribution of the different mechanisms involved, is still debated. Reliable computational models of the sinoatrial (SAN) action potential (AP) may help gain a deeper understandingof the phenomenon. Recently, novel models incorporating a detailed calcium-handling dynamics have been proposed, but they fail in reproducing experimental effects of "funny" current (If) reduction. We therefore developed a SAN AP model, based on available experimental data, to reproduce and investigate autonomic and drug-induced rate modulation.

Cellcompartmentalization and all the intracellular Ca2+ handling mechanisms were formulated as in the Maltsev-Lakatta model. Membrane current equations were revised on the basis of published experimental data. Autonomic modulation and drug effects (Acetylcholine, Isoprenaline, Ivabradine, Cesium, BAPTA, Ryanodine, CPA) were simulated by modifying the affected currents.

The model generates AP waveforms typical of rabbit SAN cells, whose parameters fall within the experimental ranges:176ms AP duration, 329ms cycle length, 73mV AP amplitude, -56mV maximum diastolicpotential and 6.23V/s maximum upstroke velocity. More importantly, the model reproduces the autonomic and drug-induced rate modulationntal findings. In particular, 18% Cesium-induced (5mM) and and 20% Ivabradine-induced (3μM) rate reductions were reproduced. Model testing of Ryanodine, CPA and BAPTA effects showed slowing of rate without cessation of beating. Our up-to-date model describes satisfactorily experimental data concerning autonomic stimulation, funny-channel blockade and inhibition of the Ca-related system by specific drugs, making it a useful tool for further investigations. Simulation results suggest that a detailed description of the intracellular calcium fluxes is fully compatible with the observation that If is a major component of pacemaking and rate modulation.

P107
Electrical properties of human right atrial fibroblasts from patients in sinus rhythm and atrial fibrillation

Abstract

Introduction

Cardiac fibroblasts can couple to cardiomyocytes via gap junctions and thus alter the electrical properties of the latter. The interaction between the 2 cell types might be of particular importance in the context of atrial fibrillation which is characterized by increased fibrosis. In order to understand the potential role of fibroblasts in atrial electric dysfunction, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (SR) and chronic atrial fibrillation (cAF).

Methods

Fibroblasts were isolated by outgrowth culture from right atrial biopsies and maintained in culture for up to 12 weeks. We used whole-cell patch clamp techniques to investigate ion currents and membrane potential.

Results

SR and cAF fibroblasts showed similar capacitance (SR: 43.6 ± 4.6 pF, n=33; cAF: 54.7 ± 5.1 pF, n = 17) and membrane potential (SR: -21.0 ± 4.3 mV, n = 14; cAF: -27.4 ± 4.8 mV, n = 16). In both groups, we observed fast activating outward currents with a mean threshold at -20 mV. Interestingly, current amplitude was significantly larger in SR than cAF cells at early stages of culture (SR: 23.8 ± 4.2 pA/pF, n = 15; cAF: 6.1 ± 1.0, n = 6; p < 0.05). After 3-5 weeks in culture, cells from both groups developed Na+ currents. The number of cells showing such currents was larger in cAF (SR: 15%; cAF: 38%), and increased with culture time. After 11-12 weeks of culture, Na+ currents were present in 87% of SR cells and 63% of cAF cells. Similarly, current amplitude was larger in cAF fibroblasts at early stages of culture (SR: 6.1 ± 2.0 pA/pF, n = 5; cAF: 17.4 ± 4.4 pA/pF, n = 6; p < 0.05) but comparable after 11-12 weeks of culture (SR: 25.0 ± 7.8 pA/pF, n = 13; cAF: 20.5 ± 4.2 pA/pF, n = 10, p = 0.6). Na+ currents were not altered by 100 nM Tetrodotoxin (TTX), but 10 μM TTX reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac TTX-resistant isoform Nav1.5. Some cells showing large Na+ currents at late stages of culture became excitable and could be stimulated to generate action potentials. AP parameters were similar in both SR and cAF.

Conclusions

The potential of atrial fibroblasts to become excitable is of significance for our understanding of their role in the pathophysiology of cAF. Since culture time diminishes the electrophysiological differences in properties of atrial fibroblasts derived from patients in SR and cAF, characterization of these cells as early as possible after isolation may provide an estimate close to their in vivo phenotype.

P108
Deficiency of cardiac cytidine monophospho-N-acetylneuraminic acid synthetase (CMAS) determines arrhythmias in the murine heart

Abstract

Purpose

N-acetylneuraminic acid (NeuNAc) is a terminal sugar residue on cell surface glycoproteins and -lipids and plays a pivotal role in many biological processes including in the functionality of voltage gated potassium and sodium channels. The enzyme cytidine monophospho-N-acetylneuraminic acid synthetase (CMAS) is localized in the nucleus and catalyzes the activation of NeuNAc to Cytidine 5-prime-monophosphate N-acetylneuraminic acid (CMP-NeuNAc), a substrate required for its addition to target glycans. CMAS is ubiquitinously expressed and its systemic knockout is embryonic lethal. Here we investigate the role of cardiomyocytes-specific CMAS in the postnatal heart.

Methods and Results

Mice with a cardiomyocyte-specific deletion of CMAS were generated using the Cre/LoxP system with the Cre recombinase driven by the aMHC-promoter, a floxed CMAS allele (flox) and a deleted (delta) CMAS allele (aMHC-Cretg/ + ; CMASfl/delta: CMAS-KO). CMAS-KO mice were born according to Mendelian inheritance ratios and survived into adulthood. At 3 months of age male CMAS-KO mice revealed normal LV morphology (determined with Sirius red and H&E staining), no inflammatory infiltrates (CD45 staining) and normal left ventricular function (fractional shortening assessed by echocardiography: CMAS-KO: 26 ± 9%; WT: 30 ± 8%, n.s.). However, HOLTER analysis of CMAS-KO mice displayed marked ECG abnormalities including atrioventricular (AV)-blocks from 1st to 3rd degree and slow heart rate  (beats per minute, bmp: CMAS-KO: 283 ± 29; WT: 523 ± 67, p < 0.01 assessed by HOLTER). Programmed electrical stimulation (PES) did not induce additional arrhythmias and challenge with atropine or orciprenaline did not overcome the AV-block in CMAS-KO mice. Acetylcholin-esterase staining of heart sections displayed no morphological changes in the formation of the AV node in CMAS-KO hearts.

At 6 months of age CMAS-KO mice developed dilated cardiomyopathy and heart failure (FS: CMAS-KO: 18 ± 14%; WT: 30 ± 14%, p < 0.05).

In cultivated neonatal rat cardiomyocytes, CMAS expression was reduced after ischemia (4 h) and reperfusion (3 h, -38 ± 4 %, p < 0.005 and 16 h, -60 ± 6 %, p < 0.05).

Conclusion

Cardiomyocyte-specific CMAS deficiency causes AV-blocks in the murine heart before onset of heart failure. CMAS is down-regulated in cardiomyocytes exposed to I/R suggesting that reduced CMAS expression and subsequent alterations in NeuNAc content after I/R may contribute to a higher risk of arrhythmias after myocardial infarction.

P109
Cardioprotective effect of dipeptidyl peptidase-4 inhibitor during ischemia-reperfusion injury is via prevention of cardiac mitochondrial dysfunction

Abstract

Dipeptidyl peptidase-4 (DPP-4) inhibitor has been used to treat type-2 diabetes mellitus. Despite its benefits on glycemic control, the effects of DPP-4 inhibitor on the heart during ischemia-reperfusion (I/R) are not known. We tested the hypothesis that during I/R, 1) DPP-4 inhibitor attenuates cardiac arrhythmias and reduces the infarct size, and 2) the cardioprotective effects of DPP-4 inhibitor is via its prevention of cardiac mitochondrial dysfunction.

Methods

Fourteen pigs were randomized to receive either DPP-4 inhibitor (vildagliptin, Vil) 50 mg or normal saline (NSS) intravenously prior to a 90-min left anterior descending artery occlusion, followed by a 120-min reperfusion. The hemodynamics, cardiac electrophysiological parameters, and the infarct size were determined. Since I/R is known to cause severe oxidative stress and cardiac mitochondrial dysfunction, rat cardiac mitochondria were used to elucidate the cardioprotective mechanisms of DPP-4 inhibitor.

Results

Compared to the NSS group, DPP-4 inhibitor attenuated effective refractory period (ERP) shortening, decreased the number of premature ventricular contractions and arrhythmia score, increased the ventricular fibrillation threshold (VFT) during I/R, and also decreased the infarct size (Figure). In cardiac mitochondria, DPP-4 inhibitor significantly decreased the reactive oxygen species production and prevented mitochondrial depolarization caused by severe oxidative stress (Figure).

Conclusions

During I/R, DPP-4 inhibitor stabilizes cardiac electrophysiology by preventing the ERP shortening, decreasing arrhythmias, increasing the VFT, and decreasing the infarct size. This cardioprotective effect could be due to its prevention of cardiac mitochondrial dysfunction caused by severe oxidative stress during I/R.1

P110
A unique CaMKII betaisoform and anchoring protein alphaKAP assemble with the Tyrosine kinase signaling complex in myocardium

Abstract

aKAP is a Kinase Anchoring Protein derived from the CaMKIIa gene. aKAP is expressed in various tissue including muscle and brain. Cardiac tissue is shown to have a predominance of CaMKII dB and dC isoforms while a 73 kDa CaMKIIb4 isoform that is generated by alternative splicing of the CaMKIIb gene is also expressed in myocardium and muscle. The CaMKIIb4 isoform differs from the ubiquitous CaMKIIb isoform by the presence of three exons encoding three proline rich repeats in the C-terminal association domain. The proline rich motifs in CaMKIIb4  were found to directly bind SH3 domains and immunoprecipitation assays indicated the presence of Grb-2 and c-Src in the CaMKIIb4 complex. A direct interaction of aKAP with CaMKIIb4 could target the enzyme to subcellular membranes including the sarcoplasmic reticulum (SR). Furthermore, aKAP can associate with SERCA2a and target CaMKIIb4 and promote phosphorylation of phospholamban and the ryanodine receptor. These data suggest that aKAP via it's interactions with the unique CaMKIIb4 may serve to modulate calcium transport and integrate calcium signals with SH3 domain containing proteins at the SR.

P111
R-L3 enantiomers have adverse modulating effects on IKs in rabbit ventricular myocytes

Abstract

Background and Aim

Activators of the slow delayed rectifier K+ current (IKs) have been suggested as promising tools to suppress ventricular arrhythmias due to prolongation of repolarization. A recently synthesized compound, L-364,373 (R-L3), was nominated to activate IKs in ventricular cells isolated from guinea pigs, rabbits and dogs, however, in some studies it failed to activate IKs (N-S Arch Pharmacol., 373:85-89, 2006). One later study suggested a possible explanation for this discrepancy: the two enantiomers of the racemic R-L3 have different activities, namely that the d enantiomer activates, while the l enantiomer potently blocks IKs. This mixed activating and blocking effect might be a plausible answer why the racemic substance failed to activate IKs in dogs previously. Therefore, the aim of the present study was to analyse the effect of the enantiomers on IKs current in isolated ventricular rabbit myocytes, by applying the whole-cell patch clamp technique at 37 C.

Results

We have synthesised two substances, ZS 1271B_R (right) and Zs 1271B_L (left) the two enantiomers of R-L3. In rabbit myocytes, ZS 1271B_R (1 μM) enhanced the IKs tail current by about 30% (at 40 mV, IKs tail current amplitude increased from 45.9 ± 4.97 pA to 66.09 ± 4.54 pA, after drug superfusion, n=6), while the left enantiomer ZS 1271B_L (1 μM) reduced IKs tail current by approximately 45% (at 40 mV IKs tail current amplitude decreased from 81.9 ± 10.9 pA to 39.8 ± .9 pA after drug superfusion, n=5).

Conclusion

These results indicate that the two enantiomers of R-L3 indeed have adverse modulating effects on IKs in the same concentration range, which may explain why the racemic drug R-L3 failed to activate IKs in previous studies. ZS 1271B_R is a potent activator of IKs, therefore, this substance is adequate to test whether IKs activators are indeed ideal tools to suppress ventricular arrhythmias originating from prolongation of action potentials.

P112
Both two-pore channels and Kir2.x channels contributes for altered IK1 current in cardiomyopathy

Abstract

Two-pore channels and inward rectifier potassium current (IK1) determine the resting membrane potential and contributes to the final repolarization in cardiac muscle but its molecular background is still uncertain. Although they are structurally very different, it is thought that both Kir2.x are pore-forming alfa-subunit genes and TWIK1 such as TASK1 two-pore forming alfa-subunit genes may underline the structural base of IK1. Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation that is associated with systolic dysfunction and low resting membrane potential with prolonged action potential duration. Here we examined the contribution of Kir2.x, TWIK1 and TASK1 to IK1 and its possible contribution to electrophysiological remodeling during dilated cardiomyopathy in human ventricular muscle by applying the real-time qPCR, immunofluorescence and Western blot methods. In cardiac left ventricular tissue samples obtained from 17 hearts of patients with dilated cardiomyopathy and from 17 undiseased donors we observed that Kir2.1 and Kir2.3 mRNA and the corresponding protein densities were upregulated while the TWIK1, TASK1 and Kir2.2 mRNA and protein densities with different distribution were down-regulated in dilated cardiomyopathy compared to undiseased control hearts. In addition, the expression of the DLG1 gene coding for the synapse-associated protein 97 (SAP97), a Kir2.x anchoring molecule which exhibits 2.0-2.5 fold reduction was robustly down-regulated in young DCM patients. The results of the present study suggest that both Kir2.x and two-pore channels contribute to IK1 and they can be differently altered in diseased states Such as dilated cardiomyopathy. Such an opportunity may have important new aspect for the explanation of the generally observed physiological alterations and possible therapeutical implications in the future.

This work was supported by grants OTKA NI-6190, DFG, EU FP6 LSHM-CT-2005-018833, EUGeneHeart and TAMOP-4.2.2.-08/1-2008-0013.

Excitation-contraction coupling, Cardiomyopathy
P113
Cellular signalling in the diseased left ventricle influences cellular signalling in the non-diseased right ventricle of patients with aortic stenosis or ischaemic disease

GENE DESCRIPTION LVAVS/LVCABG RVAVS/RVCABG ROLE 
ATP2A2 sarcoplasmic reticulum Ca2+ ATPase 0.4* 0.7 E C coupling 
c-Kit c-kit (tyrosine kinase receptor) 3.6* 4.2* CPC marker 
IGF1 Insulin growth factor 1 2.7 1.9 Growth/Survival 
IGF1R Insulin growth factor 1 receptor 2.2 4.8 Growth/survival 
HRAS Small GTPase protein 2.1 3.5 Growth/Survival 
CTNNB1 β-catenin 0.4* 0.6 Apoptosis 
TNF Inflammatory factor 2.9* 2.7* Inflammation 
IL6 Inflammatory factor 3.7* Inflammation 
GENE DESCRIPTION LVAVS/LVCABG RVAVS/RVCABG ROLE 
ATP2A2 sarcoplasmic reticulum Ca2+ ATPase 0.4* 0.7 E C coupling 
c-Kit c-kit (tyrosine kinase receptor) 3.6* 4.2* CPC marker 
IGF1 Insulin growth factor 1 2.7 1.9 Growth/Survival 
IGF1R Insulin growth factor 1 receptor 2.2 4.8 Growth/survival 
HRAS Small GTPase protein 2.1 3.5 Growth/Survival 
CTNNB1 β-catenin 0.4* 0.6 Apoptosis 
TNF Inflammatory factor 2.9* 2.7* Inflammation 
IL6 Inflammatory factor 3.7* Inflammation 

Gene expression changes when the left and right ventricles of AVS patients are compared to the corresponding ventricle in CABG patients. The numbers indicate fold difference. * p < 0.05 using Mann-Whitney U test.

Abstract

Cardiac pathologies including hypertrophy and coronary artery disease are associated with changes in signalling pathways. It is currently unknown whether the changes in the diseased left ventricle (LV) also alter signalling pathways in the clinically "normal" right ventricle (RV). This is important as different maladaptation in both sides of the heart can result in different vulnerability to ischemia and reperfusion injury during cardioplegic arrest. This study aims to identify if cellular signalling occurring in the diseased LV of hypertrophic and CAD patients influences cellular signalling in the clinically normal RV.

Left and right ventricular biopsies were taken from patients undergoing either aortic valve Surgery (AVS, n=5) or coronary artery bypass surgery (CABG, n=4). RNA from biopsies was extracted using trizol. Markers of inflammatory response, growth/survival signalling, apoptosis and E-C coupling were quantified by TaqMan PCR within the left and the right ventricle of each patient and normalised using 18S ribosomal RNA. The data was expressed as fold differences for LV(AVS)/ LV(CABG) and RV(AVS)/RV(CABG).

Differences in the expression of key genes between AVS and CABG were observed between both the LV and RV. This observation suggests that signalling due to disease is altering signalling in the clinically "normal" RV. Quantification of protein will be undertaken to further understand functional changes occurring in the right ventricle.

This work was approved by the local ethics committee with all patients giving consent.1

P114
Altered location of peripheral couplings point to a decreased E-C coupling efficiency in intrauterine growth restriction

Abstract

Purpose

Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with increased risk of cardiovascular mortality in adulthood. IUGR fetuses and newborns show signs of cardiovascular remodelling and dysfunction that persist postnatally, resulting in dilated and less efficient hearts. However, the precise underlying mechanism remains elusive. In mammalian heart, specialized junctional domains of the Sarcoplasmic reticulum (jSR) are associated with the plasmalemma and T-tubules to form peripheral couplings or dyads, respectively. They are collectively called Calcium Release Units (CRUs), and constitute the structural and functional units for E−C coupling. Because during fetal stages T-tubule system is either absent or poorly developed, CRUs are mostly in the form of peripheral couplings. It is known that a fixed coupling distance between the plasmalemma and jSR is required to ensure a proper E-C coupling, and alterations in this distance could decrease its efficiency affecting cardiac muscle contractility. Therefore, the purpose of the present study was to assess a reduced myocardial performance in an animal model of IUGR and to evaluate a potential association with the peripheral CRUs couplings.

Methods

IUGR was induced in 6 New Zealand pregnant rabbits by a surgical standard protocol. At 30 days of gestation a caesarean section was performed and cardiac function was evaluated by echocardiography. Morphometric analysis of peripheral CRUs was performed in 3 different left ventricular areas from 3 paired control and IUGR rabbit fetuses, using transmission electron microscopy imaging.

Results

Fetal echocardiography showed an increased ductus venosus and aortic isthmus pulsatility index (p < 0.01), together with a lower systolic ring displacement (p < 0.01) and annular velocity under IUGR (p < 0.05). Morphometric analysis showed a significatively increased distance between jSR and plasmalemma under IUGR (in nm: 32,25 ± 0,8914 vs. 35,59 ± 1,215 in control and IUGR, respectively. p < 0.05).

Conclusions

Results reported here show a reduced cardiac performance and an abnormal systolic function in fetal IUGR rabbits, reproducing the same features observed in human growth restriction. This may be, at least in part, due to a less efficient E-C coupling, since the distance between the plasmalemma and jSR was increased in fetal IUGR myocardium, as previously reported in models of heart failure. Thus, this finding may partly explain the abnormal cardiac contractility observed in IUGR, contributing to explain a proportion of cardiomyopathies with origin in the fetal life.

P115
CRH regulates cardiac function in normal conditions and infection

Abstract

Myocardial dysfunction leading to heart failure is a major cause of death due to endotoxemia and sepsis. Mammalian adaptation to stressors is mediated primarily by Corticotropin Releasing Hormone or Factor (CRH/CRF) and the corresponding activation of the hypothalamic-pituitary-adrenal (HPA) axis. In this study, we demonstrate for the first time that mice genetically deficient in CRH (Crh-/-), exhibit defective responses following systemic exposure to lipopolysaccharide (LPS). Intriguingly, Crh-/- mice showed unexpected elevated levels of mortality (90-100% 16-28h post-LPS treatment), compared to no mortality observed among their wild-type (Crh + / + ) littermates. Crh-/- mice have significantly reduced EF and FS (60%) values, histological abnormalities, including perivascular fibrosis, increased vasculature, vascular thickness and hyperplastic intima, and rate of cardiomyocytes' apoptosis after LPS administration compared to Crh + / + mice. LPS treatment induces MMP-8 and MMP-13 expression in Crh-/- mice, suggestive of myofibrillar dysfunction. Corticosterone administration to correct the glucocorticoid insufficiency of Crh-/- mice did not rescue them from LPS-induced mortality or ameliorate the cardiac function indices. We found that CRH is expressed in both atria and ventricles. In further support of the impact of Crh deficiency in cardiac function, Crh-/- mice have lower FS and EF values in basal state. Crh + /- exhibit intermediate phenotype. Comparison of microarray results of Crh + / + and Crh-/- cardiac tissue revealed differences in genes involved in lipid metabolism, cell proliferation and extracellular matrix organization processes. Our preliminary real time PCR results show significantly lower levels of PPARα, PPARgamma, AMPKα, and Cpt1b in Crh-/- heart suggestive of their impaired metabolic activity. As known, the heart relies mostly on fatty acids for its energy demands, a process altered in states of increased stress such as ischemia and heart failure. Based on the above findings, our working hypothesis is that CRH may play a fundamental role in cardiac function and its adaptation to increased metabolic demands, which is unmasked in states of increased stress such as infections. On-going studies in mice and human cells aim to elucidate the exact role of CRH in normal cardiac development and function and its putative use in cardioprotection in states of altered metabolic demands.

P116
Glucose-6-phosphate dehydrogenase deficiency exacerbates LV dilation but does not affect function after myocardial infarction

Abstract

Purpose

Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects 400 million people worldwide and is the most common enzyme deficiency. Heart failure increases myocardial [NADPH] and NADPH-dependent reactive oxygen species (ROS). G6PD activity controls cytoplasmic [NADPH], thus suppression of G6PD activity may decrease NADPH-dependent ROS. On the other hand, NADPH also fuels the antioxidant glutathione system. Therefore we determined the effect of G6PD deficiency on heart failure after myocardial infarction.

Methods

WT and G6PD deficient (G6PDX) mice were subjected to Sham (n = 10/group) surgery or permanent LAD occlusion (n = 19 WT and n=28 G6PDX surviving the duration of the study) for12 weeks.

Results

sham G6PDX mice had a 53% reduction in myocardial G6PD activity. No differences were observed in Survival or body mass. Infarction increased heart mass and LV dilation in G6PDX mice compared to WT mice (Figure). Infarction also decreased ejection fraction and dp/dt min, and increased LV end diastolic pressure and MPI, indicative of systolic and diastolic dysfunction, but there was no effect of G6PD deficiency on any functional parameter. Further, G6PD activity was increased by infarction in WT mice. Mitochondrial oxidative enzyme activities (citrate synthase, medium chain acyl-CoA dehydrogenase & aconitase) were decreased after infarction in both WT & G6PDX mice, with no differences between WT & G6PDX mice.

Conclusions

The results indicate that G6PD deficiency worsened LV dilation in response to myocardial infarction, but did not affect systolic or diastolic function or oxidative enzyme capacity. The effects of G6PD deficiency on development and progression of heart failure in patients should be assessed.1

P117
The HCM-associated cardiac Troponin T mutation K280N accelerates tension generation and relaxation in human cardiac myofibrils

Abstract

In spite of extensive work on the functional sequelae of HCM-associated mutations in sarcomeric proteins, the mechanisms by which the mutant proteins cause the disease have not been definitely identified. Here we use the single myofibril technique (Tesi et al., Biophys. J., 2002, 83, 2142-2151) to compare the kinetics of contraction and relaxation of myofibrils isolated from frozen left ventricular samples of one homozygous HCM patient carrying the K280N cTnT mutation (underwent heart transplantation) to those from "control" hearts. Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (< 10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in the myofibrils from the K280N patient (1.7- 2 s-1) compared to controls (0.7-1 s−1). Force relaxation kinetics upon Ca2+ removal were also faster in K280N myofibrils; the rate constant of isometric relaxation (slow kREL) was 2-3 times faster in K280N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the HCM preparations. Replacement of the mutant protein by exchange with wild-type recombinant human Tn reduced both kACT and slow kREL of HCM myofibrils close to control values. The results indicate that the HCM-associated cTnT mutation K280N alters apparent cross-bridge kinetics. This can lead to increased energy cost of tension generation that may be central to the HCM disease process. Supported by the 7th Framework Program of the European Union, "BIG-HEART" grant agreement 241577, & Telethon-Italy GGP07133.

P118
The HCM-associated cardiac Troponin T mutation K280N causes reduced responsiveness to protein kinase A

Abstract

Background

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Mutations in cardiac Troponin T (cTnT) account for ∼15% of known HCM-causing mutations and are associated with a high incidence of sudden cardiac death at young age. In the present study we investigated myofilament properties in failing human myocardium with a homozygous cTnT charge mutation (K280N), obtained during transplant surgery. Troponin exchange experiments were performed in single cells to study the specific effect of the K280N mutation.

Methods

Force was measured in cardiomyocytes before and after exchange of endogenous mutant troponin with recombinant (healthy) human troponin at various calcium concentrations. To investigate myofilament responsiveness to β-adrenergic stimulation force measurements were repeated after incubation with protein kinase A (PKA).

Results

Maximal and passive force were significantly lower in K280N cells (21 ± 2 and 1.9 ± 0.1 kN/m2, respectively) compared to non-failing cardiomyocytes (33 ± 4 and 2.9 ± 0.4 kN/m2, respectively), while Ca2+-sensitivity was significantly higher in K280N (pCa50 = 5.58 ± 0.02) compared to control (pCa50 = 5.52 ± 0.02). Myofilament response to PKA was smaller in K280N (ΔpCa50 = 0.03 ± 0.02) compared to non-failing (ΔpCa50 = 0.06 ± 0.01) cells. Partial exchange (∼70%) of endogenous K280N in HCM cells with healthy troponin did not correct the high myofilament Ca2+-sensitivity to control values, while myofilament responsiveness to PKA was significantly enhanced (ΔpCa50 = 0.17 ± 0.02). Protein analysis revealed lower cTnT phosphorylation in K280N compared to non-failing myocardium and previously studied end-stage failing hearts. Phosphorylation of the PKA target proteins, cardiac myosin-binding protein-C was similar as found in non-failing myocardium, while troponin I phosphorylation was slightly lower in K280N in comparison with non-failing tissue. Phosphorylation of myosin light chain 2 was significantly higher in K280N compared to non-failing heart muscle.

Conclusions

Our data reveal reduced maximal force generating capacity and increased Ca2+- sensitivity of human myofilaments harbouring the HCM-associated cTnT mutation K280N. The lower force generating capacity and absence of myofilament Ca2+-desensitization upon PKA may impair cardiac function in human HCM with mutant cTnT at baseline and during exercise. Funding: Seventh Framework Program of the European Union "BIG-HEART," grant agreement 241577.

P119
Beta-2 adrenergic receptor signaling in adult rat ventricular myocytes at 4, 8 and 16 weeks after myocardial infarction

Abstract

We have previously shown that β2AR-induced cAMP signal localisation is disrupted at 16 weeks post-myocardial infarction in a rat model of heart failure. In this study we aimed to investigate the changes in β2AR signaling as failure develops. Chronic myocardial infarction (MI) was induced by permanent coronary arterial ligation in adult rats. Adult rat ventricular myocytes (ARVM) were isolated at 4, 8 and 16 weeks after MI. A video-microscope technique (IonOptix) was used to assess contraction of ARVM derived from the ventricular apex. Selective β2AR stimulation was achieved with 1μM Isoproterenol whilst inhibiting β1AR with 300nM CGP20712A. PTX was used to assess the contribution of Gi in the signalling. Cells were transfected with a cAMP sensor construct EPAC2 and cultured for 48 hours for FRET. These were stimulated with 100nM ISO in the presence of 100nM CGP and then further stimulated with Forskolin analogue NKH477 at 5 μM.

ARVMs were hypertrophied at 4, 8 and 16 weeks post-MI. Contraction amplitude (%shortening) was increased compared to control at all 3 time points (Control 3.91 ± 0.5 % n=21; 4 Week 6.70 ± 0.60 n=20 p≤0.001; 8 Week 7.95 ± 0.70 N=9 p≤0.001; 7.89 ± 0.39 N=48 p≤0.001).

In control cells both contractility and cAMP FRET response increased appropriately following β2AR stimulation (Contraction: CGP Baseline 3.34% ± 0.51 N=11 vs. CGP + ISO 7.09% ± 1.13 N=11 p≤0.001). At 4 weeks, contraction was unresponsive to β2AR stimulation but FRET response was normal (expressed as a percentage of maximal AC stimulation after 5 μM NKH477: Control 39.3 ± 2.2% N=42 vs. 35.3% ± 1.5 N=12 p=NS) . ARVMs at 8 weeks had an increased ability of β2AR stimulation to promote cAMP production (Control 39.3% ± 2.2% N=42 vs. 8 Week 53.2 ± 5.0% N=9 p≤0.01). However, contraction was again unresponsive to β2AR stimulation. At 16 Weeks post MI cells did not increase cAMP production upon β2AR stimulation but did increase contraction amplitude (CGP Baseline 8.1 ± 0.75 N=17 vs. CGP + ISO 10.42 ± 1.1 N=17 p≤0.001). PTX pre-treatment produced only modest increases in β2AR-mediated contraction at 8 and 16 weeks, without complete rescue.

Increased basal contraction of surviving myocytes from the infarcted ventricle is consistent with a compensatory hypertrophic phase. Disconnect between cAMP responses and contractile effects suggest that both non-cAMP dependent positive inotropic and concurrent negative inotropic signalling are activated through β2ARs. The lack of rescue by PTX indicates a minor role for Gi coupling in the negative responses. β2AR coupling is dynamically regulated through the development of heart failure.

P120
Altered unfolded protein response in Mybpc3-targeted mice with cardiac hypertrophy

Abstract

Purpose

The unfolded protein response (UPR) is initiated to maintain endoplasmic reticulum (ER) homeostasis upon diverse stresses. It involves the activation of different signal transduction pathways that cope with the accumulation of unfolded proteins by induction of ER resident chaperones such as glucose-regulated protein 78 (GRP78), reduction of protein synthesis and/or activated degradation of unfolded proteins. When ER stress is excessive and/or prolonged, it can lead to apoptotic cell death by induction of C/EBP homologous protein (CHOP). Recent findings have shown that UPR is activated in several cardiovascular diseases, including heart failure. In the present study, we evaluated the UPR in two mouse models of hypertrophic cardiomyopathy (HCM), which were targeted in the Mybpc3 gene encoding cardiac myosin-binding protein C (cMyBP-C), and developed a similar degree of left ventricular hypertrophy.

Methods

Homozygous cMyBP-C knock-in (KI) mice mimic human HCM by carrying a point mutation, which results in a low level of mutant cMyBPC proteins. The other model is a homozygous cMyBP-C knock-out (KO), which does not express any cMyBP-C. Each group was compared to wild-type (WT) littermates (n = 7 per group). Transcript and protein levels of major UPR markers were determined by RT-qPCR and Western blot, respectively.

Results

In KO, inositol-requiring kinase 1 alpha (IRE1α) protein level was ∼70% higher and associated with 90% higher levels of spliced X-box-binding protein 1 (XBP1) mRNA than in WT hearts. On the other hand, levels of ER chaperones (GRP78, GRP94, calreticulin, calnexin) and CHoP mRNA did not differ between Ko and WT. Interestingly in KI, IRE1α protein levels did not differ from WT and spliced XBP1 and GRP78 mRNA levels were ∼50% and ∼35% lower, respectively. Levels of other ER chaperones (GRP94, calreticulin, calnexin) and CHOP mRNA did not differ between KI and WT mice.

Conclusions

These data suggest i) activation of the UPR without apoptosis in KO mice, and ii) impairment of the UPR in KI mice. Thus, our data provide evidence of differently regulated UPR depending on the absence/presence of mutant cMyBP-C in Mybpc3-targeted mice with cardiac hypertrophy.

P121
Stabilin-1 protects against adverse inflammation and injury during viral myocarditis

Abstract

Background

Viral myocarditis (VM) is an important cause of heart failure (HF) in young patients and there are no current therapies. In response to cardiac infection, inflammation has long been considered a double edge sword, therefore understanding the cellular and molecular steps of cardiac inflammation is essential in order to develop new therapies.

Hypothesis

We hypothesize that Stabilin-1 has an immunoregulatory role and protects against adverse cardiac inflammation during VM.

Results

In the coxsackie B3 (CVB3) murine model of viral myocarditis, stabilin-1 expression increased 3-fold in cardiac tissue 9 days post infection and stabilin-1 was primarily expressed in macrophages. This was similarly observed in endomyocardial biopsies from patients with fulminant myocarditis, where stabilin-1 was expressed by CD-68 + macrophages, forming a protective barrier around the remaining healthy tissue. When challenged with the CVB3 virus, stabilin-1 null mice showed increased cardiac related mortality as compared to wild type mice (p < 0,001, n=19). Subsequent histological analysis showed that the stabilin-1 null mice had increased cardiac necrosis due to CD3 + T-lymphocyte infiltration, and low F4/80 + macrophages as compared to their wildtype littermates, suggesting that stabilin-1 may be important for monocytes recruitment. We induced peritonitis in stabilin-1 WT and null mice, and using fluorescence-activated cell sorting, found fewer monocytes in the peritoneal lavages of the stablin-1 null mice, 18hrs after injection. Finally, we found that depletion of circulating monocytes with clodronate liposomes induced a similar phenotype after CVB3 infection to the stabilin-1 null mice, suggesting that should there be impaired monocytes recruitment during myocarditis, the subsequent T-lymphocyte response can be fatal.

Conclusion

Here we demonstrate for the first time that during myocarditis impaired monocytes recruitment leads to uncontrolled T-lymphocytic infiltration, with fatal consequences. We identify stabilin-1 as an important up-regulated protein during VM in both mice and humans and that absence of stabilin-1 results in increased cardiac inflammation, injury and mortality, demonstrating a novel cardio-protective effect of stabilin-1.

P122
Sarcomere permanent morphometric changes underlie cardiac programming in intrauterine growth restriction

Abstract

Purpose

Intrauterine growth restriction (IUGR) is associated with cardiovascular remodelling and dysfunction persisting into adulthood, resulting in dilated and less efficient hearts. However, evidence of direct fetal programming of cardiac cytoarchitecture is scarce, and the underlying molecular mechanisms remain to be elucidated. Here we show that IUGR is associated with direct changes of Sarcomere morphometry that persist in adulthood and altered fetal gene expression of sarcomere components that are in line with the reported dilation of the heart. These results identify an important mechanism within the process of fetal programming of cardiac disease at the subcellular scale.

Methods

IUGR was induced in 8 New Zealand pregnant rabbits. Puppies were assigned to: i) fetal (30 days of gestation) or ii) young adult (70 days postnatally) groups. Fetal cardiac function was assessed by echocardiography. IUGR-gene expression profile was analyzed by a bioinformatic gene set analytic tool. Sarcomere quantitative morphometric changes were assessed by multiphoton microscopy based on the SHG signal, called second harmonic generation microscopy (SHGM).

Results

Fetal echocardiography showed that ductus venosus and aortic isthmus pulsatility index were increased in IUGR rabbits, revealing a reduced cardiac performance. Additionally, both systolic and diastolic cardiac function was compromised by IUGR, illustrated by a lower systolic ring displacement and annular velocity, and an increased isovolumetric relaxation time. Gene set analysis suggested that the sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR hearts (p < 0.001). Results provided by SHGM showed that resting sarcomere length, defined by the distances between the two Z-discs, was shorter in IUGR fetuses (p < 0.01). Distances between intrasarcomeric A-bands was also shorter under IUGR (p < 0.03). Additionally, thick-thin filaments overlap was also shorter in IUGR fetuses (p < 0.05). We assessed the postnatal persistence of all sarcomere morphometric changes in adult myocardium (p < 0.05).

Conclusion

Results reported here suggest that IUGR induces permanent changes of cardiac sarcomeres in fetal life to cope with the adverse intrauterine environment. Importantly, these changes persist postnatally and might explain the stiffer and less contractile hearts of adult IUGRs, resembling hallmarks of high-pressure cardiac disease models. Since sarcomere changes persist in adulthood, this could be one of the molecular mechanisms leading to abnormal cardiac function in IUGR that may explain a proportion of cardiomyopathies with fetal origin.

P123
Myocardial nNOS-derived NO regulates excitation-contraction coupling via differential and cGMP-independent effects on protein phosphorylation

Abstract

Background

Nitric oxide (NO) has long been known to regulate protein phosphorylation in discreet myocardial subcellular domains via a cyclic GMP-mediated regulation of PKG and phosphodiesterase activity. More recently, nNOS-derived NO has been shown to hasten basal myocardial relaxation by enhancing PKA- phosphorylation of phospholamban via a cGMP-independent inhibition of protein phosphatase activity. However, the mechanism underlying the effect of myocardial nNOS-derived NO on inotropy and the sarcolemmal Ca2+ current (ICa) remains to be explored.

Methods and Results

PKA inhibition (PKI, 1 μmol/L) did not affect basal contraction in voltage-clamped LV myocytes from WT mice but caused a significant reduction in shortening in nNOS-/- myocytes (from 9.15 ± 1.17% to 6.27 ± 0.8% with PKI, n=9-16 cells, P < 0.05). In agreement with these findings, the phosphorylated fraction α1.2 subunit of the L-type Ca channel was significantly larger in nNOS-/- myocytes (0.93 ± 0.15 v. 0.68 ± 0.17 in WT, n=12, P < 0.05) and in WT myocytes after nNOS inhibition with SMTC (n = 3, P < 0.05).

Total protein phosphatase activity in α1.2 immunoprecipitates did not differ between genotypes (RFU: 2.75 ± 0.02 in WT v. 2.81 ± 0.02 in nNOS-/-; n=15, P=NS) but the PP2A contribution, i.e., the okadaic acid (OA, 10 nmol/L)-inhibitable fraction of phosphatase activity, was significantly reduced in nNOS-/- α1.2 immunoprecipitates (12 ± 1.2% v. 21 ± 1.0% in WT, n=8 hearts per genotype, P=0.03) in the absence of changes in PP2A protein. Consistent with this result, PP2A inhibition by intracellular perfusion of OA significantly increased cell shortening and ICa in WT mice but not in nNOS-/- (pA/pF: 5.85 ± 0.6 v. 7.97 ± 0.58 after OA in WT, n=9-10, P=0.03 and 7.92 ± 1.05 v. 7.54 ± 0.49 in nNOS-/-, n=9-10, P=NS) whereas PP1 inhibition had no effect in either group. Similarly, OA enhanced α1.2 phosphorylation in WT but not nNOS-/- myocytes. Soluble guanylate cyclase inhibition with ODQ (10 μM) did not affect the difference α1.2 phosphorylation or PP2A activity between genotypes.

Conclusions

Myocardial NO production by nNOS targets phosphorylation of proteins involved in EC coupling and regulates myocardial function via differential cGMP-independent effects on protein phosphatase activity in discreet subcellular domains.

P124
Initiation of relaxation is delayed in fetal ventricular myocytes from rabbits suffering intrauterine growth restriction and worsens postnatally

Abstract

Purpose

Intrauterine growth restriction (IUGR) is associated with cardiovascular remodelling and dysfunction persisting into adulthood, resulting in dilated and less efficient hearts. However, the underlying molecular mechanisms remain to be elucidated. Here we test the hypothesis that unbalanced calcium (Ca2+) homeostasis is a key contributor to cardiac fetal programming.

Methods

IUGR was induced in 6 pregnant New Zealand rabbits by a surgical standard protocol. Puppies were assigned to: i) fetal (30 days of gestation) or ii) young adult (70 days postnatally) groups. Fetal myocardial performance was assessed by echocardiography on day 30 of gestation. Ventricular myocytes were isolated by a multistep enzymatic digestion of myocardial fragments of the fetal heart, or by enzymatic Langendorff perfusion of the adult heart. Then, intracellular Ca2+ concentration was monitored with the calcium indicator fluo-3 and the responses to electrical field stimulation at different pacing rates were recorded and analyzed.

Results

Fetal echocardiography showed that ductus venosus, aortic isthmus pulsatility index and isovolumetric relaxation time were increased in IUGR hearts (p < 0.01). When subjected to rapid pacing, fetal IUGR cardiomyocytes also needed more time in order to reach 10% relaxation of the peak calcium transient (RT10) 0.121 ± 0.104s vs. 0.068 ± 0.079s in control (p < 0.05). The increased RT10 under IUGR persisted postnatally (0.141 ± 0.015s vs. 0.115 ± 0.016s in control, p=0.01) and was accompanied by an increased RT50 (0.529 ± 0.05s vs. 0.435 ± 0.055s in control, p < 0.001).

Conclusion

IUGR induces a permanent impairment in the initial relaxation phase that could be interpreted as a delayed onset of relaxation. Importantly, this phenomenon worsens postnatally as evidenced by an overall slowing of the relaxation process. This, suggests that IUGR-induced impairment of Ca2+ removal from the cytosol may contribute to the development of inefficient hearts with diastolic dysfunction, observed in both fetal humans and rabbits suffering IUGR, and thus provide an explaination for some cardiomyopathies with origin in the fetal life.

P125
Transthyretin aggregates affect viability and functional properties of cardiomyocytes

Abstract

Purpose

Senile systemic amyloidosis (SSA), is a sporadic disease whose main symptom is a severe cardiomyopathy associated with arrhythmias. SSA is characterized by the presence of extracellular amyloid fibrillar aggregates of transthyretin (TTR), a plasma protein carrying the thyroid hormone and the retinol binding protein. The aggregates are deposited in several tissues and are responsible for tissue functional impairment. To date, liver and heart transplantation are the only medical treatments; so, a thorough investigation of the molecular basis functional and viability impairment induced by TTR aggregates is expected to identify new pharmacological targets and to develop novel therapeutic strategies.

Methods

Cardiomyocytes cell line (HL-1) and ventricular cardiac myocytes isolated from mouse heart were exposed to prefibrillar and fibrillar aggregates of TTR. The TTR aggregates internalization into HL-1 is assessed by immunofluorescence detection whereas cell citotoxicity is determined by JC-1 assay. Modifications of intracellular calcium levels were studied by fluorescence microscopy on HL-1 and the effect of TTR aggregates on action potential profile is determined by single cell patch-clamp technique on mouse ventricular cardiomyocytes.

Results

Only prefibrillar aggregates were able to interact with cell membrane and were internalized. This resulted in a moderate impairment of cell viability at concentration of 10 μM. In the same cells exposed to TTR prefibrillar aggregates, the cytosolic calcium content showed a progressive rise over time; it did not reach a steady state level and came back to its basal levels upon TTR removal from bath solution. By patch-clamp technique we investigated the effect of the enhanced intracellular calcium on the electrical properties of mouse ventricular myocytes. Action potential recordings were performed at increasing rate of stimulation (0.5, 1 and 2 Hz) before and after application of TTR. The results showed a progressive prolongation of the action potential that was associated with a marked increase of the duration of the plateau phase. These effects were seen at any frequency of stimulation.

Conclusions

TTR internalization is associated with rise of cytoplasmic calcium content and electrical abnormalities in exposed cells. These data demonstrate a proarrhythmic effect of TTR aggregates in cardiomyocytes, a possible cause of SSA cardiomyopathy.

P126
Chronic changes in cardiac load dynamically alter T-tubule morphology and local Ca2+-induced Ca2+ release in rat ventricular cardiomyocytes

Abstract

Purpose

Transverse (t) tubules enable tight coupling between L-type Ca2+ channels and ryanodine receptors throughout the depth of cardiomyocytes, and are disrupted in several cardiac diseases. We have previously reported that mechanical unloading can influence t-tubule structure and Calcium-induced Calcium-release (CICR). In this study, we used graded mechanical overloading and mechanical unloading to comprehensively assess the effect of load on t-tubule structure and CICR. 

Methods

To test this hypothesis, rat hearts were unloaded for 4 or 8 weeks using heterotopic abdominal heart-lung transplantation, or overloaded for 6 or 10 weeks using thoracic aortic constriction. Left ventricular cardiomyocytes were isolated enzymatically and studied using confocal microscopy. Results: 10 weeks (TAC10) but not 6 weeks (TAC6) of chronic mechanical overload increased cell size (Control 45,348 ± 2373μm3, n=47 vs. TAC6 weeks 48,785 ± 2237μm3, n=60 vs. TAC10 weeks 56,066 ± 3091μm3, n=42; p < 0.05) and disrupted t-tubule regularity, which was measured as the power of the dominant frequency of the Fourier transform of Di-8-ANEPPS images (Control 1.69 ± 0.1x10^7, n=44 vs. OV 10 weeks 9.45 ± 1.0x10^6, n=33; p < 0.001), despite preserved Ca2+ transients. Unloading decreased cardiomyocyte size and induced time-dependent Ca2+ transient changes. Ca2+ transient synchronicity (measured by the variance of the time-to-peak of the Ca2+ transient) was disrupted at 8 but not 4 weeks (Control 269.6 ± 17.13ms2, n=106 vs. UN 4 weeks 331.6 ± 23.67ms2, n=46 vs. UN 8 weeks 403.5 ± 35.4ms2, n=50; p < 0.01), as was the t-tubule regularity (Control 1.59 ± 0.01x10^7, n=107 vs. UN 4 weeks 1.75 ± 0.1x10^7, n=53 vs. UN 8 weeks 1.1 ± 0.0007x10^7, n=82; p < 0.01).

Conclusions

We demonstrate that changes in load can dynamically influence t-tubule structure and CICR. Deviations from normal cardiac load are initially not associated with changes at the level of Calcium cycling but chronic load variation can lead to pathological deterioration of the t-tubules which may have functional consequences. The mechanisms by which changes in load influence t-tubule structure require further studies.

Metabolism, Oxygen, Ischaemia and Protection
P127
Energetic depletion in intact papillary muscles from ACTC E99K transgenic mouse

Abstract

It was proposed that higher Ca2+-sensitivitywill cause an inefficient use of ATP which leads to energy depletion in thecell that could be a trigger for hypertrophy. We directly measured the contractile efficiency as ratio of work/(work + heat) of intact papillary muscle from ACTCE99K transgenic mice and NTG littermates and confirmed the energy depletionis associated with HCM.

The cardiac actin mutation E99K was reported to causepredominantly apical hypertrophic cardiomyopathy (HCM) in human patients. The ACTC E99K transgenic mouse model,expressing 50% mutant actin in the heart, recapitulates many symptoms includingsudden cardiac death, apical hypertrophy, fibrosis, myocyte disarray and highermyofibrillar Ca2+-sensitivity. Intact papillary muscle of left ventricle from 10 wks old ACTC E99K mouse and NTG littermates wasused in the experiments.

To mimic the cardiac cycle, each stimulus was followed by anisometric period (isovolumic contraction) of 0.12s, shortening by 10%L0,an isometric period (isovolumic relaxation), and lengthening by 10% L0. Four movement velocities, 0.5, 0.67, 1 and 2 L0/s were tested. The isovolumic relaxation periods were adjusted accordingly. The force and the heat production weremeasured during a set of 40 runs with no stimulation as control. The efficiency during contractions was measured with a protocol of 40 twitches in20s (27oC). Net work wascalculated as the integral of active force and length change. Heatproduction, an index of metabolic cost, was calculated from temperature changemeasured with a thermopile of constantan-chromel thermocouples.

Muscle of ACTCE99K mouse (n = 12) did 74% more work/mg muscle than muscles from NTG (n = 11) (33 ± 1 vs 19 ± 4, mJ/g), with disproportionately 131% more total energy than NTG (263 ± 10 vs 114 ± 18, mJ/g). ACTC E99Kmouse muscle was 23% less efficient than NTG muscle (0.127 ± 0.005 vs 0.166 ± 0.015).

Thus ACTCE99K mouse muscle was thus found to have a hyper-contractile phenotype with a lowerefficiency. Although reduced efficiencymay be due to increased ATP consumption to pump ions or a reduced efficiency toresynthesize ATP, since the mutation affects contractile properties directly, thereduced efficiency is most likely related to the increased Ca2+-sensitivityin HCM. Improving energy utilization is therefore a method worth investigatingto manage the progress of the HCM.

P128
NADPH oxidase (NOX2) activation by hyperglycemia in cardiomyocytes is independent of glucose metabolism but requires SGLT-1 and a caveolae-associated signalosome

Abstract

Purpose

Prolonged exposure to hyperglycemia results in toxic effects in the heart inducing cell death by an oxidative stress-dependent mechanism. Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production through NOX2 activation. This activation depends on Rac1. The underlying mechanism responsible for NOX2 activation remains elusive. In this study, we have dissected the link between glucose transport, its metabolism, and NOX2 activation under hyperglycemic conditions.

Methods

Primary cultured rat cardiomyocytes were incubated with high (HG, 21 mM) or low (LG, 5 mM) glucose concentration for 24 h. Cell mortality was evaluated using propidium iodide. ROS production was measured using H2DCFDA. p47phox translocation was quantified by immunofluorescence and Rac1 activity by a pull down assay method. Protein co-localization was assessed by in situ Proximity Ligation Assay.

Results

HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane resulting in NOX2 activation, increased ROS production, insulin resistance and eventually cell death. Inhibition of the pentose phosphate pathway (PPP) by 6-amino-nicotinamide (6AN) counteracted ROS production in response to HG but did not prevent Rac-1 activation and p47phox translocation. Modulation of facilitated glucose uptake through GLUTs by insulin (stimulation) or phloretin (inhibition) barely affected oxidative stress and toxicity induced by HG. Interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and alpha-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of sodium/glucose cotransporter SGLT1 by phloridzin counteracted HG-induced NOX2 activation and ROS production. p47phox and SGLT1 co-localised with Caveolin-3 (Cav3). Caveolae was required for glucotoxicity since caveolar disruption with methyl-beta-cyclodextrin completely blocked ROS production under HG. Phosphorylation of ERM proteins can stimulate Rac1. Under HG, ERM proteins were phosphorylated and also located closed to cav-3.

Conclusions

Increased glucose metabolism by itself does not trigger NOX2 activation, although PPP is necessary to provide NOX2 with NADPH and to produce ROS. The major finding of our study is that glucose transport through GLUT and metabolism are not required to induce glucotoxicity. NOX2 activation results from glucose transport through a sodium-glucose co-transporter, SGLT1 thus transducing a metabolic signal into an intracellular ionic signal. SGLT1 activates ERM-dependent signalling cascade located in the caveolar structure leading to NOX2 activation.

P129
Mechanical efficiency of hypertrophied rat papillary muscle related to phosphatidylglycerol/cardiolipin and cytosolic cytochrome c

Abstract

Hypertrophied hearts are inefficient, i.e. consume more oxygen per unit of myocardial work than normal. The inefficiency may increase myocardial oxygen demand several fold and can be a major contributor to the development of hypoxia in hypertrophied myocytes and reduced cardiac output. We hypothesize that the inefficiency is due to mitochondrial dysfunction.

Papillary muscles were dissected from the right ventricle of control (n = 5) and monocrotaline-induced (60mg/kg sc) pulmonary hypertensive Wistar rats (n = 7). Mechanical efficiency was determined in a 0.038 ml glass chamber from work loops and oxygen uptake during 5 Hz sinusoidal length changes, peak-to-peak amplitude 15% of optimal length, at 37C. The efficiency was 35 (SD 5) % in control and 14 (SD 14) % in hypertrophied preparations (P=0.006). Experiments using blebbistatin to inhibit cross-bridge interaction demonstrated that the inefficiency is not due to sarcomere dysfunction. The right ventricular free wall was used to determine the phosphatidylglycerol/cardiolipin ratio using HPLC mass spectroscopy and cytosolic cytochrome c by quantitative immunohistochemistry. Mechanical efficiency of papillary muscles correlated with phosphatidylglycerol/cardiolipin (r = -0.82, P < 0.01) and cytosolic cytochrome c concentration (r = -0.78, P=0.01).

Cardiolipin is an essential component of the mitochondrial innermembrane - regulating its permeability, respiratory chain complexes and cytochrome c release - and is synthesized from phosphatidylglycerol. Our results suggest that the mechanical inefficiency of hypertrophied myocardium is caused by mitochondrial dysfunction due to a disturbance of cardiolipin metabolism.

P130
The effects of a non-obesogenic high-fat diet on cardiac mitochondrial morphology and function

Abstract

High-fat diet can trigger cardiomyopathies that are associated with obesity-induced co-morbidities (e.g. diabetes, hypertension). High-fat diet can also directly trigger cardiac changes without obesity (e.g. altering cardiac metabolism) and has been shown to increase vulnerability to ischemia-reperfusion (I/R) injury. The underlying mechanism for the latter could be due to changes in mitochondria as it has been reported that larger, fused mitochondria are better able to survive I/R. The aim of this work was to investigate the effects of high-fat diet, without obesity and associated co-morbidities, on cardiac mitochondrial morphology and function.

Male C57BL/6 mice were fed either a normal diet (13% kcal from fat) or a high-fat diet (45% kcal from fat) for 20 weeks. Isolated hearts and isolated mitochondria were used to study mitochondrial morphology and function, respectively. Hearts were fixed and sliced in longitudinal plane for electron microscopic imaging. Isolated mitochondria were supplied with either pyruvate and malate (P/M) or palmitoyl-carnitine and malate (Pal-Car) as substrates. Isolated mitochondria were assessed for oxygen consumption and H2O2 production. Data are presented as mean ± SEM and analysed using unpaired student's t-test.

C57BL/6 mice fed a high-fat diet had elevated blood cholesterol and triglycerides but no evidence of hypertrophy or change in body weight and insulin sensitivity.

Following high-fat feeding interfibrillar mitochondria were smaller, shorter and less dense (n = 4 hearts/group and ∼1000 mitochondria/heart from >10 electron micrographs). The mitochondrial size decreased from 0.682 ± 0.06μm2 in the normal diet group to 0.427 ± 0.021μm2 in the high-fat group (P>0.01). The amount of longer mitochondria (>2.1μm in length) in the high-fat group was lower compared to the normal diet (6.7 ± 0.1 vs. 11.8 ± 1.6% of total mitochondria, P>0.05). The density of interfibrillar mitochondria was higher, 31.4 ± 0.8%, in the normal diet group compared to 26.6 ± 1.6% in the high-fat group (P>0.05).

The oxygen consumption rates of isolated mitochondria were not different between normal diet and high-fat diet in state 3, 4 and 3.5. H2O2 production in state 3.5 was not different when using P/M as a substrate. With Pal-Car H2O2 production in state 3.5 was higher in mitochondria isolated from high-fat fed mice compared to normal diet, (46.0 ± 3.4 vs. 30.9 ± 0.9 pmol/min/mg protein, P>0.05, n=3 isolations/group using 2 mice per isolation).

This works shows changes in mitochondrial morphology and increased H2O2 production in high-fat diet hearts, which could contribute to the increased vulnerability during I/R.

P131
The myocardial postconditioning preserve the bioenergetics systems during ischemia-reperfusion

Abstract

Purpose

This study investigated the effect of myocardial ischemic postconditioning (IPostC) and pharmacologic postconditioning by cyclosporine A on the changes in capacity of oxidative phosphorylation, and coupling between adenine nucleotide translocase (ANT) and mitochondrial creatine kinase (miCK).

Methods

Isolated perfused hearts from C57BL/6J mice were subjected to 40-min of ischemia followed by 20-min of reperfusion (Control group). Hearts were divided into 4 groups: 1) Control; 2) IPostC: elicited by ten cycles of 5-s ischemia/5-s reperfusion after the 40-min ischemia; 3) cyclosporine A : administered during the first min of reperfusion; 4) Sham: perfusion with normoxic condition (n = 6 in each group). Parameters of mitochondrial function were assessed on saponin-skinned fibers from isolated hearts. The protocol of the respiration rate determination included a cytochrome c test to check the intactness of the outer mitochondrial membrane. The apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km) in the presence and absence of creatine were also evaluated to test the ANT-miCK functional coupling.

Results

Our results show that the capacity of oxidative phosphorylation was significantly reduced in control and cyclosporine A groups, and equivalent in IPostC group in comparison to Sham group. The cytochrome c test was negative in IPostC and cyclosporine A groups, suggesting intact outer mitochondrial membrane ; and positive in control group, suggesting some alteration of the outer mitochondrial membrane. In Sham and IPostC groups, Km is high and addition of creatine significantly decreases Km, suggesting a high efficiency of the ANT-miCK functional coupling. Alterations in mitochondrial function in control and cyclosporine A groups were characterized by a significant decrease in Km and partially loss of the stimulatory effect of creatine.

Conclusions

IPostC and cyclosporine A prevent the loss of integrity of the outer mitochondrial membrane after prolonged ischemia, moreover IPostC contribute to the preservation of the ANT-miCK functional coupling.

P132
Endothelial progenitor cells mobilization and increased levels in the injured myocardium after sevoflurane preconditioning

Abstract

Introduction

Exposure to volatile anesthetics triggers an adaptive response similar to that achieved by ischemic preconditioning, in both temporal and mechanistic patterns. Current experimental data suggests a possible link between cardioprotection provided by anesthetic preconditioning and myocardial tissue regeneration through endothelial progenitor cells.

Objectives

This study aims at demonstrating that sevoflurane exposure enhances endothelial progenitor cell mobilization and recruitment in the infarcted myocardium, an event that accounts for part of the cardioprotective effect of delayed anesthetic preconditioning.

Methods

The experiments were performed on Wistar rats (n = 36) randomly assigned to a control and a preconditioned group. The rodents were intubated and ventilated with room air for the control group and with 1 MAC sevoflurane for the preconditioned group. Luminex was used to evaluate the plasma levels of VEGF and G-CSF, whereas the mobilization of CD 34 + / flk-1 + progenitor cells in the peripheral blood was estimated by flow cytometry and immunofluorescence. The level of progenitor cells (c-kit + ) present in the injured myocardium was measured through immunofluorescence one day after the ischemia-reperfusion lesion in similar experimental groups (n = 18). The cardioprotection was assessed through apoptosis assays (one day after ischemia/ reperfusion injury) and by measuring the area of fibrosis (2 weeks after ischemia/ reperfusion injury).

Results

Increased levels of VEGF and G-CSF were recorded at 3 and 6 h respectively after sevoflurane exposure, followed by a rise in CD34 + / flk-1 + cells beginning at 12 hours after preconditioning, compared to the control group, with a maximum level at 24 hours after exposure to sevoflurane (p < 0.05). The number of c-kit + cells in the myocardial area damaged after the ischemia/ reperfusion procedure was significantly increased (p=0.001), the degree of apoptosis was reduced (p=0.007) and the area of fibrosis was diminished (p < 0.05) in the late preconditioned group compared to the control group.

Conclusions

Our results indicate for the first time the mobilization of a population of mononuclear cells, with endothelial progenitor cell markers (CD 34, flk-1) in the late phase of anesthetic preconditioning. These cells are a potential source of endothelial repair and myocardial regeneration in the context of perioperative or periprocedural ischemia in patients with coronary artery disease.

P134
On the role of NOX4 in different forms of ischemia/reperfusion injury

Abstract

Aim

Reactive oxygen species (ROS) are thought to be key players in ischemia-reperfusion-injury (IRI). However, clinical trials using antioxidants to scavenge ROS failed to show a benefit. Thus, inhibition of ROS-production may be a superior therapeutic strategy. We recently identified NOX4 as a major source of ROS in ischemic stroke in mice. Therefore, we hypothesized that NOX4 may also play a detrimental role in IRI of other organs such as heart and lung.

Methods

We performed ischemia-reperfusion (I/R) of the heart in male and female NOX4 KO and matched WT mice by ligating the left descending coronary artery (LAD) for 45 min, followed by a 24 h reperfusion and observation period. Basal and 24 h post-reperfusion ultrasounds were performed. After 24 h, area at risk and infarct size (Evans' Blue-TTC double-staining) and left ventricular haemodynamic function were determined. In another set of experiments, isolated perfused mouse lungs were subjected to 30 min of ischemia and 90 min of reperfusion. Post-ischemic vascular leakage and lung capillary filtration coefficient were measured as parameters of tissue damage.

Results

In the hearts of NOX4 KO mice infarct size and area at risk were unchanged compared to WT mice. Functional parameters 24h post-reperfusion were impaired after IRI, but not different between KO and WT mice. Also, the changes in left ventricular haemodynamic function were not different between the genotypes in male mice (female not determined). Data on 4 weeks observation period post-MI and further read-outs will be presented and discussed. In the isolated perfused lung, I/R caused edema formation with an increase in lung weight and increased capillary filtration coefficients, but again no differences between KO and WT mice.

Conclusions

After I/R of the heart no significant differences in infarct size, area at risk or functionality were found between NOX4 KO and matched WT mice. Similarly, in lung IRI, no effect of NOX4 was observed. These results are contrasting with those found in ischemic stroke, where NOX4 KO mice showed a smaller infarct size and better neurological outcome. Thus, NOX4 appears not to play a general, but rather an organ- or even cell-specific role in IRI. A complicating factor in the vascular phenotype of NOX4 may be a possible beneficial role NOX4 in angiogenesis and remodeling. Therefore, we are currently investigating the role of NOX4 in the chronic phase after I/R of the heart and in the chronic ischemia model of the hindlimb. The possibility that neuronal NOX4 is the key source in ischemic stroke will be addressed using neuron-specific NOX4 KO mice.

P135
The early markers of ischemic heart disease in cardiac remodeling in patients with metabolic syndrome and impaired glucose metabolism

Abstract

Material and methods

345 patients were examined: 95 patients with arterial hypertension (AH) and dyslipidemia (DLP) without abdominal obesity (AO) – group 1 (control group; 50 women, 45 men, 49,85 ± 10,6 years), 103 patients with AH, DLP and AO (waist girth > 80 sm in women, > 94 sm in men) – group 2 (67 women, 36 men, 46,99 ± 10,97 years), 34 patients with AH, DLP, AO and impaired glucose metabolism – group 3 (26 women, 12 men, 53,29 ± 7,82 years), 113 patients with ischemic heart disease (IHD) (comparison group, 64 women, 49 men, 62,12 ± 9,93 years). All patients went through transthoracic echocardiography with remodeling indexes calculation, lipid and glucose testing.

Results

groups 1 and 2 showed increase of linear dimensions of LV and myocardial mass (LVMM/hight2,7 47,21 (41,07; 53,56) g/m2,7 in group 1, 58,07 (49,56; 68,13) g/m2,7 in group 2, p=0,03). In group 3 along with increasing of MM and it's indexes (p < 0,001), we mentioned rise of systolic (0,46 (0,42; 0,49) ea. in group 1, 0,49 (0,46; 0,56) ea. in group 3, p < 0,01) and diastolic (0,64 (0,61; 0,69) ea. in group 1, 0,69 (0,64; 0,73) ea. in group 3, p < 0,01) indexes of spherisity and worsening of systolic function: growth of systolic myocardial stress (127,48 (114,25; 140,75) din/m2 in group 1, 139,85 (129,62; 148,39) din/m2 in group 3, p < 0,01) and reduction of systolic remodeling index (105,9 (98,17; 116,32) ea. in group 1, 97,95 (86,62; 100,55) ea. in group 3, p < 0,01) and deterioration of functional parameters of heart remodeling (increase of the indexes, which show the contribution of dilatation in heart function compensation: MSs/ESVind (6,94 (6,02; 8,1) ea. in group 1, 8,5 (6,88; 10,35) ea. in group 3, p < 0,001) and MSd/EDVind (2,8 (2,48; 3,08) ea. in group 1, 3,19 (2,89; 3,79) ea. in group 3, p=0,03), and decrease of the parameter, which shows adequacy of systolic function to the myocardial stress: EF/MSs (0,53 (0,47; 0,63) ea. in group 1, 0,48 (0,43; 0,53) ea. in group 3, p=0,02). Distinction of groups 3 and 4 was described only by the value of MS (139,85 (129,62; 148,39) and 147,51 (134,2; 184,61) ea., p=0,04) and MSd/EDVind (3,19 (2,89; 3,79) and 2,86 (2,31; 3,24) ea., p < 0,01), thus impaired glucose metabolism conduces to early cardiac remodeling, same as in CAD. Using correlation analysis we fixed out that the value of remodeling index is associated with plasma glucose concentration (r=-0,43, p=0,04).

Conclusion

Hyperglycemia introduces a significant contribution in structural and functional disorder of LV in patients with MS, causing changes similar to CAD.

P136
Farnesol treatment decreases infarct size in rat hearts

Abstract

Purpose

We have previously shown that the beneficial effect of preconditioning on ischemic myocardial function was recaptured by farnesol (a key metabolite of the mevalonate pathway) in the heart of cholesterol fed rats. Farnesol is also known as an antioxidant. Our aim was to examine whether farnesol treatment decreases infarct size in rats fed with normal rat chow and modulates the activity of the mevalonate pathway and/or cardiac oxidative/nitrosative stress.

Methods

Male Wistar rats were treated with oral administration of 0.2, 1, 5, and 50 mg/kg/day farnesol or its solvent for 12 days, respectively. On day 13, isolated perfused hearts were subjected to 30 min coronary occlusion followed by 120 min reperfusion. At the end of reperfusion, infarct size was determined. In separate experiments, basal cardiac level of mevalonate pathway derivates (Q9, Q10, cholesterol, prenylated proteins), and 3-nitrotyrosine (marker of oxidative/nitrosative stress) were measured following the 12-day farnesol pretreatment.

Results

We found that 1 mg/kg/day farnesol pretreatment significantly decreased infarct size (22.3 ± 3.9% vs. 40.0 ± 4.1%, p < 0.05). However, 0.2, 5, and 50 mg/kg/day farnesol remained ineffective (37.3 ± 5.1%, 31.9 ± 3.1%, 43.5 ± 5.1% vs. 40.0 ± 4.1%, respectively). The effective dose of farnesol significantly increased cardiac N-acetyl-S-geranylgeranyl-L-cysteine level (96.8 ± 11.1 vs. 56.0 ± 9.0 ng/mg tissue, p < 0.05), but did not influence cardiac N-acetyl-S-farnesyl-L-cysteine level (21.4 ± 1.5 vs. 30.7 ± 5.9 ng/mg tissue). One mg/kg/day farnesol significantly increased cardiac cholesterol level (0.73 ± 0.02 vs. 0.56 ± 0.04 μg/g tissue p < 0.05), and caused a non-significant increase in cardiac coenzyme Q9 and Q10 levels (94.79 ± 6.14 vs. 68.91 ± 16.48 μg/g tissue, 6.46 ± 0.52 vs. 5.46 ± 1.02 μg/g tissue, respectively). Cardiac 3-nitrotyrosine level was modified significantly only by the highest dose 50 mg/kg/day farnesol (1.16 ± 0.18 vs. 2.44 ± 0.47 ng/mg protein, p < 0.05).

Conclusion

Farnesol dose-dependently decreased infarct size and 1 mg/kg/day farnesol was the most effective dose. We conclude that the infarct size limiting effect of farnesol likely involves increased protein geranylgeranylation and seems to be independent of the antioxidant effect of farnesol.

P137
Cyclophilin-D ablation offers long-term protection against lethal myocardial reperfusion injury

Abstract

Purpose

Lethal myocardial reperfusion injury attenuates the benefits of myocardial reperfusion following an acute myocardial infarction in terms of myocardial salvage. The opening of the mitochondrial permeability transition pore (mPTP) at the onset of reperfusion is a major contributor of lethal myocardial reperfusion injury. Genetic ablation of cyclophilin-D (CypD, a component of the mPTP) has been reported to reduce myocardial infarct size (IS) after 2hrs reperfusion. However, the role of CypD ablation in long-term cardioprotection after 72hrs reperfusion has not been examined. Whether lethal myocardial reperfusion injury progresses with increasing periods of reperfusion is controversial, and is also investigated here.

Methods

Using B6sv129 male mice, our first objective was to establish for the first time in our laboratory an in vivo recovery model of acute myocardial ischaemia-reperfusion injury (IRI) comprising 30min occlusion of the left anterior descending (LAD) artery followed by extended reperfusion for 2, 6, 24 and 72hrs. Infarct size was expressed as a % of the area-at-risk (IS/AAR%). Ischaemic preconditioning (IPC, comprising one-5 min cycle of LAD occlusion and reflow prior to IRI) was used as a positive control. Mice deficient in CypD (CypD-/-) and wild-type littermates (CypD + / + ) were subjected to IRI.

Results

There was no increase in IS/AAR% as the reperfusion time was prolonged over the 72hr period (38.4 ± 3.4% at 2hrs, 37.4 ± 1.4% at 6hrs, 31.4 ± 3.5% at 24hrs, 32.0 ± 3.7% at 72hrs:P > 0.05:N > 7/group). As expected, IPC significantly reduced IS/AAR% after 72hrs reperfusion (32.0 ± 3.7% in control versus 16.2 ± 2.7% with IPC:P < 0.05:N=6/group). Male mice deficient in CypD sustained smaller IS/AAR% (35.4 ± 4.3% in male CypD + / + versus 22.6 ± 2.4% in male CypD -/-:P < 0.05:N=9/group). Similar results were also observed in female mice deficient in CypD (39.7 ± 5.4% in female CypD + / + versus 24.7 ± 3.5% in female CypD -/-:P < 0.05:N=7/group).

Conclusions

Myocardial infarct size did not enlarge with increasing duration of reperfusion suggesting that in the murine model lethal myocardial reperfusion injury does not progress over time. Genetic ablation of CypD confers long-term protection against IRI in both male and female mice.

P138
S-nitrosylation-mediated inhibition of protein tyrosine phosphatases attenuates ischemia-induced cardiac injury

Abstract

Ischemia-induced injury contributes to the progression of coronary artery disease. To date it is not known what governs ischemia-mediated perturbation of signaling in heart, or what strategy might be used to counteract such perturbation thus protecting heart against ischemic insults. It has been proposed that dysfunctions of protien kinases or phosphatases might lead to injury in heart suffering from ischemia. However, it remains elusive how ischemia-induced perturbations of intrinsic phosphorylation signaling contribute to cardiac damage. In the present study, using surgical ligation of the left anterior descending coronary artery (LAD) as a model, we have shown for the first time that the levels of phosphotyrosine (pTyr) signaling were decreased significantly in the left ventricular tissue of mouce heart, concomitant with a burst of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) released to the circulation. To gain insights into this disaese process at the molecular level, we examined signaling events in rat myocardial H9c2 cells. Interestingly, H9c2 cells underwent a drastic decrease of pTyr signaling in response to hypoxia. Such results recapitulated the ischemia-induced perturbation of signaling in heart. This change was associated with the activation of protein tyrosine phosphatases (PTPs) and a loss of cytoskeletal integrity. Employing a pan-PTP inhibitor phenyl vinyl sulfone in H9c2 cells, we showed a critical role of PTP activation in hypoxia-induced adverse effects. Furthermore, we identified that the hypoxic condition drives reduction of the active-site Cys residue of PTPs, leading to a significant increase of their activity. These observations led to a hypothesis that the intervention by nitric oxide (NO) might prevent PTP activation via S-nitrosylation on the active-site Cys residue, thus suppressing the hypoxic injury. Importantly, treatment with S-nitrosoglutathione (GSNO) in H9c2 cells not only sustained pTyr levels but also reversed hypoxia-induced cytoskeletal damages. We further showed that four PTPs, which control the function of cytoskeletal regulators, were indeed S-nitrosylated and inactivated by the GSNO treatment under the hypoxic condition. The application of GSNO therapy was tested in mice with LAD ligation. Intriguingly, ischemia-induced cardiac injury as evidenced by release of CPK and LDH was markedly attenuated by GSNO treatment, along with a significant rebound of pTyr signaling. Taken together, our data suggest a protective role of NO via inactivation of PTPs, thus providing a novel therapeutic opportunity for heart suffering from ischemic insult.

P139
PPAR-gamma agonist facilitated fatal arrhythmia in ischemic-reperfusion rat hearts by decreased cardiac connexin43 phosphorylation

Abstract

Background

Although PPAR-gamma agonist rosiglitazone has been used to improve insulin sensitizing in type II diabetes patients, growing evidence in both basic and clinical studies indicated that it could decrease cardiac function, have proarrhythmic effect and increased cardiac mortality. However, how rosiglitazone facilitates fatal arrhythmias including ventricular fibrillation (VF) during ischemia and reperfusion (I/R) is unknown. In the present study, we tested the hypothesis that rosiglitazone facilitates VF occurrence during I/R by decreasing the level of phosphorylated connexin43.

Methods

Sixteen male Wistar rats were used. In each rat, either rosiglitazone (1mg/kg) or normal saline solution was administered intravenously. Then, the left anterior descending coronary artery was ligated for 30 minutes and released to promote reperfusion for 120 minutes. Cardiac function before ischemia and during I/R was determined using the pressure-volume recording system. The level of phosphorylated connexin43 at serine368 residues, Bax and Bcl-2, and the infarct size were also determined.

Results

Rosiglitazone increased both the arrhythmia score and VF incidence in I/R rats (Figure), without preventing cardiac dysfunction. The phosphorylated connexin43 level in rosiglitazone-treated hearts was markedly decreased in both ischemic and non-ischemic myocardium, compared to the vehicle-treated rats (Figure). The infarct size was also markedly decreased in rosiglitazone-treated rats. However, the Bax/Bcl-2 ratio was not different between the two groups.

Conclusions

Rosiglitazone facilitated the occurrence of VF during I/R by decreasing the phosphorylation of connexin43 in the heart. This proarrhythmic effect of rosiglitazone could be responsible for increased mortality reported previously.1

P140
Deficiency in Tissue Kallikrein abolishes the cardio protective effects of CsA during post-conditioning in mice

Abstract

Cardioprotective strategies, called postconditionning (PostC), that limit lethal reperfusion injury have a major clinical potential in acute myocardial infarction. Indeed, bolus injection of calcineurin inhibitor Cyclosporine A (CsA) at reperfusion reportedly reduces myocardial infarct size (IS) in mouse as well as in human. Bradykinin (BK) is equally protective in mouse and both molecules CsA and BK are able to modulate the opening of the mitochondrial Permeability Transition Pore (mPTP) to trigger their cardioprotection effects during ischemia-reperfusion (IR). We investigate for the first time whether in mouse BK is involved in the cardioprotection afforded against IR by CsA using Tissue Kallikrein deficient mice (TK-/-) lacking BK and their wild-type littermate (TK+/ + ).

Methods and Results

A branch of the left main coronary artery was reversibly ligated in mice to produce 30-min-ischemia followed by reperfusion (120 min), after which the degree of myocardial necrosis (IS as a percent of area at risk (AAR)) was assessed by measuring the AAR and IS. Two-year-old TK+/ + and TK-/- male mice were divided in two groups receiving 5 min before the reperfusion either the vehicule or 10 mg/kg of CsA. As expected, CsA (10mg/kg) reduced infarct size from IS/AAR= 35.4 ± 3.4 % of the ischemic zone in Ct-TK+/ + hearts to IS/AAR =13.6 ± 2.5% (p=0.001) in Csa-TK+/ + hearts. Interestingly, CsA is unable to reduce the Is in TK-/- mice (Ct-TK-/- IS/AAR= 31.8 ± 6.8%, vs CsA-TK-/- IS/AR= 29.8 ± 6.1%) suggesting that the protective effets of CsA are abolished in absence of the TK. Investigating the mitochondria functions, we demonstrated that TK presence does not impact on mitochondrial membrane potential or oxidative phosphorylation. However, we demonstrated that CsA treatment is able to inhibit the mPTP opening in TK+/ + mice while the CsA administration is unable to do so in TK-/- mice.

Conclusions

While still investigating the molecular mechanisms of CsA lack of efficacy in TK-/- mice, the present study demonstrate for the first time that null expression of TK abrogates the cardioprotective effects of a Pharmacological PostC afforded by a CsA bolus prior myocardial reperfusion in TK+/ + mice. Furthermore, we demonstrate that this loss of reactivity to CsA injection is due to mPTP loss of sensitivity to CsA.

P141
LRP1 mediates the hypoxia induced uptake of VLDL-cholesteryl esters in cardiomyocytes

Abstract

Little is known about the contribution of lipoprotein receptors to intracellular lipid accumulation in the ischemic myocardium.

The aims of this study were 1) to analyze the effect of hypoxia on low density lipoprotein receptor-related protein 1 (LRP1), very low density lipoprotein receptor (VLDLR) and low density lipoprotein receptor (LDLR) expression in vitro in cultured cardiomyocytes and in vivo in a pig model of acute myocardial ischemia and 2) the role of LRP1 on very low density lipoprotein (VLDL) uptake and intracellular lipid accumulation in hypoxic cardiomyocytes. Real time PCR and western blot analysis demonstrated that hypoxia upregulated LRP1 and VLDLR expression in cultured cardiomyocytes and in the ischemic myocardium of infarcted pigs. Thin layer chromatography after lipid extraction showed that in in vitro cardiomyocytes, hypoxia increased VLDL-derived intracellular cholesteryl ester (CE) and triglyceride (TG) accumulation and that in in vivo model, ischemia increased myocardial lipid accumulation. To know the role of LRP1 on VLDL-cholesterol ester and triglyceride uptake, cardiomyocytes were transduced with miR RNAi lentivirus designed to knockdown LRP1 expression. Hypoxia induced uptake and accumulation of VLDL-[3H]-cholesterol ester but not of VLDL-[14C]-triglyceride was prevented in LRP1-deficient cardiomyocytes. In conclusion, LRP1 is upregulated in hypoxic cardiomyocytes and ischemic myocardium and LRP1 overexpression plays a major role in the hypoxia-induced cholesterol but not triglyceride uptake and accumulation in cardiomyocytes.

P142
Activation of Oxytocin receptors protects the myocardium via recruitment of PI3K-AKT intracellular signalling pathway during reperfusion

Abstract

Recent studies have emphasized an important role for Oxytocin in protecting against myocardial ischemia-reperfusion injury. In this study, we assessed whether phosphatidylinositol-3-kinase (PI3K-AKT) pathway was involved in Oxytocin mediated cardioprotection. Isolated perfused rat hearts were subjected to 35minutes of ischaemia and 120minutes of reperfusion. Hearts underwent triphenyl-tetrazolium staining for infarct size assessment, or frozen for Western blot analysis. Oxytocin (1nM, 10 nM, 100nM) was administered throughout reperfusion in the presence and absence of the Oxytocin receptor antagonist of L371,527 (10nM), PI3K inhibitor Wortmannin (100nM). Data was analysed using one way ANOVA followed by Tukey's test (n = 4-6). Oxytocin (1nM, 10nM, 100nM) when administered during reperfusion significantly reduced infarct size when compared to control (22.7 ± 1% (1nM); 30.2 ± 71% (10nM); 37.3 ± 1.5% (100nM); vs. 56.1 ± 2.2% (control), p < 0.05). The protective effect of Oxytocin (1nM) was abrogated by administration of either L371,527 (10nM) (49.2 ± 3.1%, p < 0.001), Wortmannin (100 nM), (59.1 ± 4.0%, p < 0.001). Western blot analysis further demonstrated that Oxytocin receptor activation during reperfusion induced a significant increase in p-Akt(Ser473) compared to non-treated control hearts. Oxytocin dependent phosphorylation of Akt was abrogated by Wortmannin. This is the first study to show that Oxytocin receptor activation can protect the ischaemic-reperfused myocardium via recruitment of the PI3K-AKT cell Survival pathway.

P143
Role of NO in apelin-induced protection against myocardial ischemia and reperfusion injury

Abstract

Background

Cardioprotective activity of the adipocytokine apelin is attributed to upregulation of endothelial nitric oxide synthase (NOS). This study was designed to examine effects of a synthesized 12 C-terminal residue of apelin (A-12) and NG-nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, in ex vivo and in vivo models of ischemia/reperfusion injury.

Methods

Isolated working rat hearts were subjected to 35-min global ischemia followed by 30-min reperfusion. 140 μM A-12 and 100 μM L-NAME were administered separately or in combination before global ischemia. Metabolic state of reperfused hearts and lactate dehydrogenase (LDH) leakage in perfusate were assessed by enzymatic methods. Anaesthetized open-chest rats were subjected to 40-min regional ischemia and 60-min coronary reperfusion. L-NAME (37 μmol/kg 10 min prior to reperfusion) and A-12 (0.35 μmol/kg at the onset of reperfusion) were injected intravenously; control rats received saline. Myocardial injury was evaluated by MB-creatine kinase (MB-CK) and LDH activities in plasma. Infarct size was determined by the Evans Blue/2,3,5-triphenyl tetrazolium chloride staining method.

Results

Preischemic infusion of A-12 increased recovery of cardiac function during reperfusion compared with control and resulted in enhanced restoration of myocardial ATP, adenine nucleotide pool, phosphocreatine and reduction of myocardial lactate and lactate/pyruvate ratio. Coadministration of A-12 and L-NAME aggravated recovery of coronary flow and cardiac function compared with these indices after A-12 treatment. Cardiac dysfunction was associated with increase in LDH release in myocardial effluent, reduction of glucose oxidation and abolishment of augmented restoration of high energy phosphates. A-12 administration after regional ischemia in anesthetized rats led to a transient reduction of mean arterial blood pressure (MABP), significant limitation of infarct size expressed as a percent of area at risk (MI/AR, %) and decreased LDH and MB-CK activities in plasma at the end of reperfusion compared with control. Subsequent administration of L-NAME and A-12 resulted in less reduction of MABP and substantially increased MI/AR ratio and MB-CK and LDH activities in plasma compared to the values in the A-12 group.

Conclusions

The results demonstrate the principal role of NO as a mediator of overall cardioprotection afforded by apelin.

P144
The role of prolyl-hydroxylase inhibition in rat aorta during ischemia/reperfusion

Abstract

Purpose

Although, vascular grafts are frequently applicated in the cardiac surgery, the storage protocols need further improvement against ischemia/reperfusion (IR) injury. Endothelial dysfunction may result in postoperative graft failure and promote late graft vasculopathy. Low oxygen tension elicits a variety of complex cellular responses by altering the activity of many signaling pathways, such as the oxygen dependent prolyl-hyroxylase-domain containing enzymes (PHD). Reduction of PHD-activity during hypoxia leads to stabilisation and accumulation of hypoxia inducible factor 1-α, a transcription factor which regulates the expression of target genes in response to hypoxia. We investigated the effects of PHD-inhibitor dimethyloxalylglycine (DMOG) on the vasomotor responses of isolated rat aorta and aortic vascular smooth muscle cells (VSMC) in a model of cold ischemia/warm reperfusion.

Methods

Isolated rat aortic rings underwent a 24h cold ischemic preservation in NaCl or DMOG (10-5M) supplemented saline solution. In organ bath experiments we investigated endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) on phenylephrine-precontracted isolated aortic rings with an additional external oxidant (hypochlorite, 200μM, 30min). VSMC under normoxic conditions are plated and 24h later the medium is changed to NaCl or DMOG (1mM) solution. The cells are incubated (4°C, 24h-cold ischemia) and after the medium is changed for a supplied standard medium (37°C, 6h-warm reperfusion). Different gene expression analysis was performed by quantitative real-time PCR.

Results

Compared with the control the NaCl-group showed a prominent endothelial dysfunction induced by hypoxia/reoxygenation, which was significantly improved by DMOG supplementation (maximal relaxation to ACh, control:90 ± 1% vs. NaCl:30 ± 6% vs. DMOG:62 ± 7%, p < 0.05). Following hypoxia/reoxygenation, concentration-response curves to SNP were significantly shifted to the right and DMOG has no effect (pD2, control:8.3 ± 0.1 vs. NaCl:8.0 ± 0.1 vs. DMOG:8.0 ± 0.1). Decreased mRNA-expression of heme oxygenase-1 in the NaCl-group as compared to control group was increased after DMOG-pretreatment (control:1.62 ± 0.11 vs. NaCl:0.50 ± 0.03 vs. DMOG:1.52 ± 0.40). The changes in the expression of vascular endothelial growth factor expression showed similar tendency (control:1.62 ± 0.14 vs. NaCl:0.37 ± 0.04 vs. DMOG:0.98 ± 0.23).

Conclusion

Inhibition of prolyl hyroxylase could be a new therapeutic avenue for protecting endothelium and vascular muscle cells against IR injury.

P145
Myocardial protection conferred by high density lipoproteins implicates connexin43 gap junction channels

Abstract

Purpose

High-density lipoprotein (HDL) is known for its cardioprotective properties independent from its cholesterol transport activity. These properties are mediated by activation of protein kinases, such as PKC. Connexin43 (Cx43) is a gap junction protein present in ventricular cardiomyocytes. PKC-dependent phosphorylation modifies Cx43 gap junction channel properties and is involved in cardioprotection. We hypothesized that cardioprotective properties of HDL may be mediated in part by affecting Cx43 gap junction channels.

Methods and Results

Neonatal rat cardiomyocytes were treated with HDL and Cx43 phosphorylation was evaluated by Western blotting and immunofluorescence. We found that HDL promoted phosphorylation of Cx43 with a maximal induction at 5 min, which was inhibited by pre-treatment with various PKC inhibitors. Sphingosine-1-phosphate (S1P), a component of HDL, induced similar effects as HDL. The effect of S1P on Cx43 phosphorylation was mediated by S1P2 and S1P3 receptors. HDL or S1P significantly reduced diffusion of fluorescent dye between cardiomyocytes (∼50%), which could be prevented by PKC inhibition. As observed during optical recordings of transmembrane voltage, HDL and S1P depressed impulse conduction only minimally ( < 5%). Moreover, 5 min of HDL and S1P treatment at the onset of reperfusion significantly reduced infarct size in response to 30 min of ischemia in ex vivo experiments (Control: 23 ± 3 %, HDL: 13 ± 2 % and S1P: 11 ± 2 %, p < 0.05). Moreover, Left Ventricular Developed Pressure was also significantly improved by 5 min treatment with HDL or S1P at the onset of reperfusion (23 ± 6 mmHg, 32 ± 4 mmHg, respectively, vs. controls: 8 ± 2 mmHg, p < 0.05).

Conclusions

Short-term treatment with HDL or S1P induces phosphorylation of Cx43 by a PKC-dependent pathway. HDL-induced phosphorylation of Cx43 reduced the diffusion of large tracer molecules between cells, whereas impulse conduction was maintained. Moreover, 5 min treatment with HDL confers cardioprotection against ischemia/reperfusion injury. These results link Cx43 for the first time to the short-term cardioprotective effects of HDL.

P146
The endothelial gap junction protein connexin40 limits myocardial infarct size after ischemia-reperfusion in mice

Abstract

Purpose

Intercellular channels formed by connexins (Cx) have been shown to play a critical role in cardiovascular disease. For instance, vascular Cx43 influences atherosclerosis development and plaque stability, and cardiac Cx43 may determine the development of arrhythmias after myocardial infarction. In the heart, Cx40 is expressed in atrial cardiomyocytes, the conduction system and in endothelial cells, whereas Cx37 is only found in the endothelium. Here, we study the implication of the endothelial connexins during ischemia and reperfusion in mice.

Methods and Results

We used the Cre-loxP system to create a mouse line in which Cx40 is deleted from the endothelium only. Immunostainings on Tie2Cre + Cx40fl/fl ApoE-/- mice confirmed the absence of Cx40 in the endothelium, whereas the protein was normally expressed in the atria and cardiac conduction system. Moreover, Cx40 was normally expressed in the endothelium, atria and conduction system of Tie2Cre + Cx40wt/wt ApoE-/- and Tie2Cre- Cx40fl/fl ApoE-/- control mice. Sixteen-week-old mice were subjected to in vivo left coronary artery occlusion for 30 minutes and sacrificed 24-hours after reperfusion for analysis of infarct size. Myocardial surfaces areas at risk and infarcted areas were measured from computed images using NIH Image software. Areas at risk, normalized to total left ventricle surfaces areas, were similar between the experimental groups, i.e Tie2Cre + Cx40wt/wt ApoE-/-: 29.2 ± 1.2% (N=11), Tie2Cre- Cx40fl/fl ApoE-/-: 33.7 ± 3.7% (N=9), and Tie2Cre + Cx40fl/fl ApoE-/- 31.7 ± 2.2% (N=11, n.s.). Interestingly, the infarct area, normalized to areas at risk, was significantly increased in Tie2Cre + Cx40fl/fl ApoE-/- (20.2 ± 3.1%, P < 0.05) mice as compared to controls (10.1 ± 2.0% and 11.3 ± 1.8%). To investigate the possible implications of another endothelial connexin in myocardial infarction, Cx37-/- ApoE-/- mice were submitted to the same protocol of in vivo ischemia-reperfusion. Areas at risks were similar between Cx37-/- ApoE-/- and control ApoE-/- mice (44.9 ± 3.3% (N=10), 38.4 ± 3.8% (N=12), n.s.), and the infarcted area appeared not affected by the deletion of Cx37 (Cx37-/- ApoE-/-: 13.8 ± 1.3%, ApoE-/-: 14.7 ± 2.7% n.s.).

Conclusions

We conclude that endothelial Cx40, but not Cx37, is implicated in resistance of the heart to ischemia-reperfusion injury. These findings may point towards a specific novel therapeutic target to limit the cardiac injury after coronary interventions.

Circulation, Vascular biology
P147
Fibrinolytic status in acute coronary syndromes and factors of its regulation

Abstract

Background

Thrombosis-related perturbations and demand for fibrinolysis in patients with acute coronary syndrome (ACS) may persist even after successful thrombus recanalization. Experimental data also suggest important ‘non-fibrinolytic’ effects of the fibrinolytic factors including myocardial remodelling and angiogenesis. Scarce data are available on the comparative status of fibrinolytic system in ST-elevation myocardial infarction (STEMI) and non-STEMI. The aim of this study was to address these questions.

Methods

Fibrinolytic status (tissue-type-[tPA] and the urokinase type-[uPA] plasminogen activators, plasminogen activator inhibitor [PAI-1] antigen and activity and thrombin activable fibrinolysis inhibitor [TAFI]) were determined in 97 ACS patients (50 with STEMI and 47 with NSTEMI), 40 patients with stable coronary artery disease (CAD) and 39 healthy volunteers. In ACS the parameters were measured within 24 hours of admission and at days 3, 7 and 30. Counts of monocyte subsets, their activity (intracellular IKKβ levels), numbers of monocyte-platelet aggregates, and plasma levels of inflammatory cytokines were measured by flow cytometry at hospital admission.

Results

Significant changes were evident in all fibrinolytic factors amongst ACS patients. On regression analysis, NSTEMI (vs. STEMI) status at admission was significantly and independently associated with TAFI concentrations, PAI-1 antigen (both higher in NSTEMI) and PAI-1 activity (higher in STEMI). In STEMI,uPA activity was reduced at admission but its values increased significantly by day 30. TAFI levels were elevated in NSTEMI at admission, whilst in STEMI they only peaked at day 7 following the disease onset and dropped significantly by day 30. There were obvious discrepancies between PAI-1 antigen levels (increased in NSTEMI) and its fibrinolytic activity (increased in STEMI).

On regression analysis the CD14 + + CD16 + monocyte subset was independently associated with PAI-1 activity in ACS. Monocyte aggregates with platelets (involving only CD14 + + CD16− and/or CD14 + + CD16 + subsets) rather than platelet-free monocytes were independent predictors of tPA, PAI-1 and TAFI levels.

Conclusions

There are significant differences in the status of the fibrinolytic system between patients with STEMI and NSTEMI at admission and follow-up. These changes could reflect the role of these factors in post-MI cardiac remodelling and recovery.Monocyte interactions with platelet are implicated in the regulation of fibrinolytic status in ACS.

P148
The activity and concentration of angiotensin converting enzyme (ACE) and ACE 2 in the plasma and platelets of rats developing stasis induced venous thrombosis

Abstract

Recent data indicate that ACE 2 activity may change after myocardial infarction. In patients with the presence of venous or arterial thrombosis the activity of ACE may depend on the endothelial dysfunction. The aim of our study was to assess the activity of ACE and ACE 2 in platelets and plasma of rats with venous thrombosis (VT) induced by ligation of the inferior vena cava.

Shame operated (SO) male Wistar rats (250-350g) served as a control group. 60% of the VT animals developed venous occlusion and formed thrombus (0,58 ± 0,33mg), whereas none of the SO animals developed thrombosis. VT induction caused a significant change in the activity of ACE and ACE 2 (measured by HPLC) in plasma and platelets. Both plasma ACE and ACE 2 activity significantly increased (3.062 ± 0.210 in VT vs. 1.540 ± 0.259 in SO; p < 0.001 and 0.115 ± 0.016 in VT vs. 0.063 ± 0.012 in SO; p < 0.05, respectively). The specificity of enzymes was confirmed by the use of the specific inhibitors: captopril (CAP) (ACE) and DX600 (ACE 2). There was no significant change of ACE activity in homogenates of platelets isolated from VT and SO rats (0,0015 ± 0,0007 vs. 0,0002 ± 0,0002; p=0,55). In contrast after VT platelets ACE 2 activity was significantly decreased (0.032 ± 0.008 in VT vs. 0.078 ± 0.014 in SO; p < 0.05). Additionally, the concentration of ACE and ACE 2 (measured by ELIsA) was significantly increased in plasma (101.49 ± 1.80 ng/ml in VT vs. 23.44 ± 2.89 ng/ml in SO; p < 0.001 and 92.39 ± 1.82 ng/ml in VT vs. 26.40 ± 1.55 ng/ml in SO; p < 0.001 for ACE and ACE 2 respectively), whereas decreased in platelets (26.64 ± 1.59 ng/ml in VT vs. 34.88 ± 2.09 ng/ml in SO; p < 0.05 and 57.65 ± 2.08 ng/ml in VT vs. 77.84 ± 5.86 ng/ml in SO; p < 0.01 for ACE and ACE 2, respectively).

In conclusion our study showed that stasis induced venous thrombosis leads to the increase of the plasma activity and concentration of ACE and ACE 2. The platelets activated during thrombosis development might be a source of these enzymes, proving that platelets are an important component of the renin-angiotensin system.

P149
Involvement of KCNQ K channels in hypoxic vasodilatation in porcine coronary arteries

Abstract

Purpose

The aim was to investigate the mechanisms behind the vasodilation seen in response to acute hypoxia in porcine arteries. We hypothesized that hypoxia results in K+ channel opening in porcine large coronary arteries thereby leading to vasorelaxation.

Experimental Approach

Porcine left anterior descendent coronary artery segments without endothelium were mounted in myographs for isometric tension recording. Functional studies examining the influence of K+ channels were performed and the presence of K+ channels was examined by PCR and immunoblotting.

Key Results

In prostaglandin F2α (PGF2α)-contracted arteries relaxations induced by gradually reducing oxygen from 95% to 1% were associaed with lowering of smooth msucle calcium and more pronounced than in 30 mM K+-contracted arteries. The hypoxic relaxation in PGF2α-contracted arteries were inhibited by TEA, a non-specific potassium channel blocker, by iberiotoxin, a blocker of large-conductance calcium-activated K+ channels, by 4-aminopyridine, a blocker of voltage-dependent K+ channels, by glibenclamide, a blocker of ATP-sensitive K+ channels, but the largest effect was seen by XE991 and linopirdine, blockers of the Kv7.1-7.5 channels, while no effect was seen by chromanol 293B a blocker of Kv7.1. KV7.1, KV7.4, KV7.5 and the large-conductance calcium-activated BKCa channels were expressed in porcine coronary arteries.

Conclusion

Our findings suggest that hypoxia induces K+ channel opening in isolated porcine coronary arteries. The effect is mainly mediated through Kv7 channels, which for the first time has been identified in porcine coronary arteries.

P150
Hypercoagulable phenotype of the arterial wall of spontaneously hypertensive rats: involvement of vascular smooth muscle cells

Abstract

The anticoagulant role of the vascular wall suggests that there are connections between coagulation and the phenotype of vascular smooth muscle cells (VSMCs). Our objective was to determine whether hypertension confers a hypercoagulable state. We used the model of spontaneously hypertensive rats (SHR) compared with Wistar rats. The reactivity of the coagulation system was studied in vitro by monitoring the kinetics of thrombin generation (Thrombography) in two experimental conditions: (i) the plasma in the presence or absence of thoracic aortic rings and (ii) on the surface of cultured VSMCs from aortas of rats in the presence of platelet-poor plasma of Wistar rats.

At baseline, plasma from SHR has a rate of thrombin generation significantly lower (p < 0.05) than the control. The addition of aortic rings of SHR to a pool of plasma from Wistar rats results in a significant increase in thrombin generation compared to the addition of rings of Wistar rats (699 ± 23 vs. 637 ± 8 nM.min, p < 0.05) to the same pool control. At the surface of cultured VSMC from SHR, thrombin generation was significantly higher than at the surface of VSMCs from Wistar rats (636 ± 10 versus 490 ± 12 nM.min, p < 0.05).

These results show that the wall of the SHR is more thrombotic than the vessel wall of Wistar rat. This phenotype is partly due to the ability of VSMCs to serve as a cellular support for the generation of thrombin. These results, obtained in vitro without hemodynamic stresses suggest a specific structural effect of the arterial wall producing an increased stiffness.

P151
Different potassium channels are involved in relaxation of arterial graft induced by nicorandil

Abstract

The drug nicorandil is a vasodilator approved for treatment of angina. In addition to its well-known effect on the opening of ATP-sensitive K+ (KATP) channels, nicorandil-induced vasorelaxation also involves the opening of Ca2+-activated K+ (KCa) channels.

Purpose

The aim of this study was to investigate the effects of nicorandil, K+ channel opener, on the isolated human internal mammary artery (HIMA) and to define the contribution of different K+ channel subtypes in nicorandil action on this blood vessel.

Methods

The HIMA segments were collected from male patients suffering from coronary artery disease who were undergoing coronary artery bypass surgery and studied in organ bath. HIMA rings were pre-contracted with phenylephrine (10 μM). Endothelium was removed mechanically.

Results

Our results show that nicorandil (0.001 μM – 300 μM) induced a concentration-dependent relaxation of HIMA rings pre-contracted by phenylephrine. Glibenclamide (10 μM), a selective KATP channels inhibitor, as well as iberiotoxin (100 nM), a most selective blocker of large-conductance KCa (BKCa) channels, partly antagonized relaxation of HIMA induced by nicorandil. In contrast, a non-selective blocker of voltage-gated K+ (KV) channels, 4-aminopyridine (4 AP, 0.5 mM), as well as margatoxin (10 nM), a potent inhibitor of KV1.3 channels, did not abolish the nicorandil-induced relaxation of HIMA.

Conclusions

Our results showed that nicorandil induced strong endothelium-independent relaxation of HIMA. It seems that KATP and BKCa channels located in the smooth muscle of HIMA mediated relaxation induced by nicorandil.

P152
Galectin-3 is a potential mediator of aldosterone effects in vascular remodeling

Abstract

Background

Aldosterone (Aldo) is involved in extracellular matrix (ECM) remodeling and inflammation leading to heart failure (HF), but its mechanisms remains unknown. Galectin-3 (Gal-3), a β-galactosidase-binding lectin, plays an important role in inflammation and HF. We have investigated whether Gal-3 mediates Aldo-induced ECM remodeling in vascular smooth muscle cells (VSMCs) in vitro and in vivo.

Methods

In vitro, primary cultured VSMCs were stimulated with Aldo (10-8M) for 24h, with or without mineralocorticoid receptor (MR) antagonists (eplerenone, RU28318) and Gal-3 inhibitors (modified citrus pectin, N-acetyllactosamine, lactose). Gal-3 was over-expressed (transfection) and knocked-down (siRNA). In vivo, Wistar rats were treated with Aldo (1mg/kg/day) + salt or Aldo + salt + spironolactone (200mg/kg/day) for 3 weeks. Gal-3 expression, ECM production (collagen type I and III, fibronectin and elastin) and degradation (MMP activities) were evaluated by RT-PCR, Western blot, zymography and immunohistochemistry in VSMCs and aorta.

Results

Gal-3 was spontaneously expressed in cultured VSMCs. Its over-expression enhanced collagen type I production. Aldo up-regulated Gal-3 levels in a dose- and time-dependent manner via the mineralocorticoid receptor. Gal-3 chemical inhibitors blocked Aldo-induced ECM protein production. In addition, Gal-3 silencing abolished Aldo-induced collagen type I synthesis. In Aldo-salt hypertensive rats, aortic Gal-3 expression, ECM proteins and MMP activities were enhanced. Spironolactone treatment reversed all the above effects. Aortic Gal-3 expression was positively correlated with vascular collagen type I, elastin, MMP-2 and MMP-13 activities.

Conclusions

Aldo up-regulates Gal-3 expression via its mineralocorticoid receptor in VSMCs in vitro and in vivo. Gal-3 over-expression induces collagen type I synthesis. Moreover, Gal-3 is required for the fibrotic response to Aldo. Our data suggest a key role for Gal-3 in Aldo-induced vascular collagen accumulation.

P153
Role of fibronectin extra domain-a (FN-EDA) in neo-intima formation and arterial remodelling in mouse models

Abstract

Purpose

Fibronectins (FN) are multifunctional glycoproteins present in the plasma and in the extracellular matrix. The primary transcript undergoes alternative-splicing to generate isoforms namely fibronectin extra domain-a (FN-EDA), fibronectin extra domain-b (FN-EDB) and type-III connecting segments (III-CS). Previous evidences indicated that  FN-EDA levels are increased during series of pathophysiological conditions including thrombosis, lung fibrosis, inflammation. The aim of the work is investigate the role of FN-EDA in neo-intimal hyperplasia in murine perivascular collar models.

Methods

Specific gene targeting allowed the generation of mice expressing fibronectin constitutively containing extra domain-a (FN-EDA) or lacking  extra domain-a (EDA) both in plasma and pheripheral tissues. In order to study the neo-intima formation an non-obstructive collar was placed on the right carotid artery of FN-EDA + / + , FN-EDA-/- and FN-EDAwt/wt mice for a period of 9 weeks. Formalin fixed paraffin embedded carotid and sham operated sections were stained for haematoxylin and eosin and morphometrically evaluated by OPTIMAS 6.2 image software. Data were analyzed for ANOVA followed by tukey's multiple comparision tests. The vascular smooth muscle cells were isolated from respective genotypes analyzed for proliferation by MTT assay. The migration of vascular smooth muscle cells were observed in vitro by scratch wound assay.

Results

At 4 months of age, male mice from corresponding groups were used for the study. Morphometric analysis showed that neo-intimal thickening (measured as intima to media ratio, IMT) was greater in mice lacking fibronectin extra domain-a (FN-EDA-/-) compared to mice with fibronectin extra domain-a (FN-EDA + / + ) and controls (FN-EDAwt/wt) (IMT 1.42 ± 0.21 vs 0.8 ± 0.11 and 1.00 ± 0.35 respectively). The  remodelling index (RI- measured as the slope of external elastic lamina area versus IMT curve) was found to be lower in both FN-EDA + / + and FN-EDA-/- compared to the controls (FN-EDAwt/wt) (RI: 0.83 ± 0.11, 0.56 ± 0.20 vs 1.20 ± 0.27 respectively). The preliminary data on MTT assay showed that vascular smooth muscle cells (VSMC's) from mice lacking fibronectin extra domain-a (FN-EDA-/-) showed and higher proliferation rate with respective to the VSMC's from mice containing extradomain-a (FN-EDA-/-) and control animals (FN-EDA wt/wt).

Conclusion

These data suggest a possible role of fibronectin containing extra domain-a (FN-EDA) in the process of intimal hyperplasia. Further studies are warrented to elucidate the molecular mechanisms and pathways involved.

P154
Proteomic study of atheroma-derived smooth muscle cells reveals signatures of increased oxidative stress and mitochondrial dysfunction

Abstract

Background

Atherosclerosis is a chronic disease with acute clinical manifestations due to plaque rupture and inflammation. Smooth muscle cells (SMC) contribute to the development and fate of atherosclerotic lesions. The study of their proteome may suggest molecular mechanisms through which SMC contribute to disease pathogenesis.

Methodology

SMC were isolated and cultured in vitro from intimal segments of carotid endarterectomies from 6 patients that experienced acute cerebrovascular symptoms and from 4 that did not. Commercially available aortic SMC (n = 6) were used as controls. Cells at an early passage (P3) were consistently used. Using 2 dimensional gel electrophoresis (2DE) and tandem mass spectrometry we compared protein expression of intimal SMC from human atherosclerotic lesions (AthSMC, n = 10) to that of control aortic medial SMC (AoSMC, n = 6). Differences were validated via immunoblotting, quantitative-PCR, functional assays and gene expression measurements in the Tampere Vascular Study cohort (n = 72).

Results

AthSMC exhibited protein signatures of increased oxidative stress and mitochondrial damage or dysfunction when compared to AoSMC, e.g. increased oxidized peroxiredoxin-4 (PRDX4, p = 0.019) and decreased mitochondrial proteins ATP Synthase subunit-beta (ATP5B, p = 0.0004) and Aldehyde dehydrogenase 2 (ALDH2; p = 0.011). Differences in the gene expression of the genes encoding for these proteins were also confirmed in the Tampere Vascular Study (PRDX4, p < 0.0001; ATP5B, p = 0.13; ALDH2, p < 0.0001). Accordingly, a decrease in mitochondrial mass was observed in AthSMC vs. AoSMC determined by citrate synthase assay (p = 0.0001). Surprisingly, a comparison between AthSMC isolated from patients with or without recent acute cerebrovascular symptoms revealed further differences, including an increase in Annexin 1 in the asymptomatic group (p = 0.030). This difference was also confirmed in the Tampere Vascular Study (p < 0.0001). Inhibition of Annexin 1 function was associated with increased cytokine production both at baseline (p < 0.01), and after stimulation with the proinflammatory cytokine TNFα (p < 0.01).

Conclusions

Our study shows that atheroma-derived SMC display signatures of increased oxidative stress and mitochondrial damage/dysfunction. Moreover, we found an increased expression of the anti-inflammatory protein Annexin 1 by SMC in asymptomatic plaques, that may represent an additional mechanism through which SMC promote plaque stability through the modulation of inflammation.

P155
Targeting myocardial edema: The depletion of extracellular RNA reduces vascular permeability and myocardial infarction size

Abstract

Cell injury resulting from myocardial infarction (MI) leads to exposureof intracellular material and is associated with increased permeability ofvessels in the vicinity of the damage, a process that contributes to tissueinjury throughout the ventricle. As previously demonstrated by our group,released, natural extracellular RNA significantly increases the permeability acrossmicrovascular endothelial cells through a vascular endothelial growth factor(VEGF)-dependent mechanism. In the present study we thereforeevaluated changes of vascular permeability, edema formation andmyocardial infarction size following a depletion ofextracellular RNA after in vivoinduction of MI .

The leftcoronary artery (LAD) of C57/Bl 6 mice was ligated and RNase (100μg) or controlbuffer was administered intravenously 30 min, 3 and 6 hours following LADligation. Wet and dry weight of heart slices were measuerd for analysis ofmyocardial edema, and evans blue dye and tetrazolium were used to delineate thearea at risk and infarction size within the myocardium 24h after ligation.Cardiac function as measured by fractional shortening was assessed byechocardiography. Water content of myocardial tissue and myocardial perfusionwas calculated by wet/dry-ratio or micro-CT-Imaging respectively.

RNAsetreatment had no effect on blood pressure, total plasma protein or albuminlevels, peripheral blood cell counts or glucose levels. However, edemaformation was significantly decreased in RNAse treated mice as measured bywet/dry ratio (3.38 ± 0.19 vs. 3.93 + - 0.27; P < 0.05). Assessment of myocardial perfusion by micro-CTangiography revealed a significantly increased perfusion of the peri-infarct zone.Since risk zone sizes were similar between groups, the percentageinfarction of the risk zone was significantly smaller in RNAsetreated mice (P < 0.05). Left ventricular function as assessed by echocardiography and fractionalshortening analysis was significantly enhanced in RNAse treated mice (25.3 ± 2.6% vs. 13.8 ± 2.6 %; P < 0.05). Consequentelythe application of RNAse led to a significant increase of survival of mice following MI hier noch zahlen, Ns und P einfügen …

These results identify extracellular RNA as a novel natural permeabilityfactor which augments ischemia-induced edema formation and myocardialinfarction. Moreover, RNase treatment serving as a novel vessel-protectivemodality, prevents MI-induced edema formation and tissue injury and thussignificantly improves survival following MI.

P157
Nitroso-redox imbalance contribute to vascular dysfunction and elevated blood pressure in ACE2-deficient mice

Fig.1 Endothelial function in ACE2 mice

Fig.1 Endothelial function in ACE2 mice

Abstract

Purpose

Angiotensin-converting enzyme 2 (ACE2) is the main enzyme responsible for the degradation of Ang II and generation of Ang-(1-7), peptides which play critical roles in the regulation of blood pressure (BP) and endothelial function. The aim of the present study was to evaluate the effects of chronic Ang-(1-7) treatment and AT1 receptor blockage by losartan on BP in ACE2-deficient (ACE2-/-) mice and to assess the endothelial function and the vascular redox balance in these animals.

Methods and Results

The study was conducted in 20 week-old ACE2-/- male mice on C57Bl/6 background. Telemetric blood pressure measurements confirmed an increase in mean arterial pressure (MAP) in these mice (ACE2-/-: 112.5 ± 3.3 vs C57Bl/6: 105.6 ± 2.3 mmHg, p < 0.01). Chronic Ang-(1-7) infusion led to a 2 mmHg decrease in BP in both ACE2-/- and C57Bl/6 animals, whereas the BP lowering effect of losartan was more pronounced in ACE2-/- than in control animals (MAP post-losartan: ACE2-/-: 101.8 ± 1.9 vs C57Bl/6: 101.7 ± 2.6). Endothelial function was evaluated by measuring changes in MAP in response to bolus intra-aortic acetylcholine (ACh) and sodium nitroprusside (SNP) administration. The endothelium-dependent vascular reactivity was impaired in ACE2-/- mice compared to C57Bl/6 animals (Fig. 1, p < 0.001). ACE2-/- mice presented a lowered plasma and urine nitrite concentration, and reduced aorta NO levels. Lipid peroxidation was significantly increased and superoxide dismutase activity was decreased in the aorta homogenate of ACE2-/- mice in comparison to controls, indicating impaired antioxidant capacity in these animals.

Conclusion

These data demonstrate that oxidative stress and NO imbalance, induced by increased local Ang II levels may cause elevated blood pressure and endothelial dysfunction in ACE2-/- mice.1

P158
EDN1 Lys198Asn is associated with microvascular angina or cardiac syndrome X in women

Abstract

Relevance

In women with a diagnosis of ischemic heart disease (IHD) besides atherosclerosis of coronary artery unchanged coronary vessels are revealed by angiography. The one reason of smooth coronary artery is microvascular angina or "cardiac syndrome X". Leading role in the development of this disease is belongs to endothelial dysfunction, which may be because of genetic polymorphism of the endothelin-1 and NO synthase.

Purpose

To study the distribution of allele and genotype frequencies of polymorphisms Lys198Asn gene endothelin-1 and 4a/4b gene ecNOS in women of young and middle age with IHD compared with women of similar age without clinical manifestations of coronary artery disease and family history of cardiovascular disease.

Materials and Methods

222 women were included. 115 women had coronary atherosclerosis (stenosis of coronary artery more than 70%) (average age 52,7 ± 0,5 years), 36 women had diagnosis "cardiac syndrome X" (mean age 55.6 ± 1,0 years) and 71 women were without clinical evidence of coronary heart disease (average age 51,3 ± 1,0 years). Detection of polymorphisms Lys198Asn EDN1 and 4a/4b ecNOS was conducted by PCR followed by restriction analysis.

Results

In group of women with coronary atherosclerosis was found the following distribution of genotypes - 54%, 42% and 4% for the 4b4b, and 4a4a, a group of women with "cardiac syndrome X"- 58%, 36% and 6% for the 4b4b, 4a4b and 4a4a, and a group of women with no history of coronary heart disease - 66%, 32% and 2% for genotypes 4b4b, 4a4b and 4a4a, respectively (p=0.06). Thus, for ecNOS 4a4b polymorphism statistically significant differences were not found.

In the analysis of gene EDN1 genotype distribution the following data were found. In group of women with coronary atherosclerosis - 60%, 31% and 9%, with cardiac syndrome X - 3%, 89% and 8% in women without coronary heart disease - 37%, 62% and 1% for genotypes LysLys, LysAsn and AsnAsn, respectively were revealed. It was found statistically significant increase in frequency of mutant allele Asn gene EDN1 (in hetero-and homozygous state) in women with cardiac syndrome X in comparison with a group of women with coronary atherosclerosis (p = 0.0003) and a group of healthy individuals ( p = 0.00001).

Conclusions

The polymorphic markers 4a/4b ecNOS is not involved in the development of coronary heart disease among women of young and middle age, but it is quite obvious that the polymorphic marker Lys198Asn EDN1 may be associated with the development of women "cardiac syndrome X".

P159
The nature and mediation of flow-induced responses of cerebral arteries depends on the origin of vessels

Abstract

Purpose

In most, if not all peripheral arteries increases in intraluminal flow elicit dilations. Much less is known regarding the nature of flow-induced responses in cerebral vessels. Previous studies reported both dilations and constrictions to flow in vessels isolated from different regions of the brain. We hypothesized that the nature of the response depends on the origin of cerebral vessels.

Methods

Isolated middle cerebral arteries (MCA) and basilar arteries (BA) of rats were studied in a pressure/flow chamber. Changes of inner diameter to stepwise increases in intraluminal flow (at a constant intraluminal pressure of 80 mmHg) were measured by a microangiometer. Intraluminal flow was established by increasing the pressure difference throughout the vessels (ΔP = from 0 to 60 mmHg). Inhibitors of known mechanisms of action were used: Nω-nitro-L-arginine methyl ester (L-NAME) and indomethacin (INDO) to inhibit the synthesis of nitric oxide and prostaglandins, respectively. At the end of experiments the passive diameters (PD) of vessels (in Ca2+ free solution) were measured.

Results

In the presence of 80 mmHg intraluminal pressure the basal diameter of MCA and BA were (181 ± 6μm and 328 ± 19μm, which were ∼58% and ∼78 % of PD, respectively). Increases in flow elicited significant constrictions in MCA (from 61 ± 1.2 to 50 ± 1.3% of PD, p < 0.05) and dilations in BA (from 281 ± 36 to 371 ± 21% of PD, p < 0.05). L-NAME, which reduced the dilation to acetylcholine, did not affect flow-induced constriction of MCA and dilation of BA. Indomethacin inhibited the constriction of MCA, but did not affect the dilation of BA to increases in flow, confirming the findings of previous studies.

Conclusions

We propose that the nature and the mediation of responses of cerebral arteries to increases in flow are depending on the location of vessels in the brain. Furthermore, the opposite responses may have important physiological roles: dilations of basilar arteries could be important to amplify the increases in blood flow to the brain in case of increased demand, whereas constrictions of middle cerebral arteries could be important to limit increases of intracranial blood volume.

Support

AHA-FA 0855910D, Hungarian Sci. Res. Funds (OTKA) K71591 and K67984.

P161
Contribution of phospholipids to thrombus resolution

Abstract

Purpose

Besides the fibrinolytic system, leukocyte recruitment and angiogenesis are two major determinants of thrombus resolution. Previous studies in a murine model of stagnant flow venous thrombosis have shown that splenectomy effectively delays thrombus resolution, and is associated with accumulation of anionic phospholipids. Here we propose that thrombus angiogenesis is inhibited in the presence of anionic, procoagulant phospholipids, e.g. phosphatidylserine (PS), as compared to neutral phospholipids such as phosphatidylcholine (PC) or phosphatidylethanolamine (PE).

Methods

Phospholipid vesicles were prepared, containing (1) 100% PC, (2) 60% PC / 40% PS, (3) 60% PC / 40% PE, (4) 60% PC / 20% PS / 20% PE. Biological activity of the mixtures was confirmed by thrombin generation assay. For analysing effects on endothelial cells, a BrdU cell proliferation assay and a lactate dehydrogenase cytotoxicity assay were performed with human umbilical vein endothelial cells (HUVEC).

Results

In the BrdU cell proliferation assay, exposure of HUVEC towards 50 μM or 100 μM phospholipids containing PS for 24 h and 48 h inhibited DNA synthesis rates significantly, whereas neutral phospholipids had hardly any effect. The lactate dehydrogenase cytotoxicity assay confirmed a lack of cytotoxicity in all cases.

Conclusions

Together with their procoagulant function, anionic phospholipids such as phosphatidylserine attenuate proliferation of endothelial cells and may thus reduce or inhibit thrombus-associated angiogenesis, a factor relevant for thrombus resolution.

P162
Aquaporin-1 induces actin-remodeling via activation of beta-catenin in human induced pluripotent stem cells following exposure to glucose-induced hyperosmolarity

Abstract

Background and Objective

Diabetic hyperglycemia increases plasma osmolarity, leading to adaptive cell reponses. Aquaporin-1 (AQP1) is induced by hyperosmolarity and plays a role in the vascular permeability. Induced pluripotent stem (iPS) cells offer a disease cellular model for the interplay of risk factors in the vascular complications of diabetes. We tested the hypothesis that  glucose induced hyperosmolarity promotes angiogenesis in human iPS cells through activation of AQP1 and downstream osmosignaling pathway, thus orchestrating cell sprouting and migration.

Methods and Results

Human iPS cells were generated from skin fibroblasts by lentiviral transduction of 3 weeks compact refractive embryonic stem cell-like colonies emerged. All the iPS cell colonies expressed OCT4, Nanog, c-Myc and SOX2, characteristic of successful dedifferentiation. After reprogramming, iPS cells were transfected with siRNA-AQP1 or scrambled controls, and exposed to 5.5 mmol/L glucose (normoglycemia), high glucose (HG) at 12.5, 25 and 45 mmol/L, or with high mannitol (HM) at 12.5, 25 and 45 mmol/L for 24-72 hours. Exposure to either HG or HM increased expression of AQP1 and tonicity enhancer binding protein (TonEBP). In iPS cells, AQP1 could be co-immunoprecipitated with β-catenin. HG and HM strongly induced the expression of β-catenin (n = 3, p < 0.001 vs normal glucose by ANOVA). Under these conditions, proteins co-immunoprecipitated with anti-AQP1 and β-catenin showed increased ratio of F-actin vs G-actin (n = 3, p < 0.01 vs normal glucose by ANOVA). iPS cells formed bundles in methylcellulose matrix and tubing networks in matrigel, especially when they were exposed to HG and HM (HG 2.8 ± 0.2 fold; HM 3.3 ± 0.5 fold; n=3, p < 0.01 by ANOVA). SiRNA to AQP1 or to TonEBP/NFAT5 all reverted the inducing effects of HG and HM on β-catenin expression, actin polymerization and angiogenic activities.

Conclusions

The hyperosmolarity serves as a biophysical factor that promotes angiogenesis in human iPS cells. This effect may occur through an AQP1-associated cytoskeleton remodeling. Targeting the osmosignaling pathway offers a novel strategy to reduce vascular complications of diabetes.

P163
Angiopoietin-2: mediator of septic hypercirculation

Abstract

Background

Angiopoietins play a central role in the quiescence of the endothelium. Pathological stimuli, such as inflammation, lead to a destabilization of the endothelial cell layer via  Angiopoietin-2 and its receptor Tie-2. In clinical studies, elevated Angiopoietin-2 levels in sera of patients suffering severe sepsis have been described (Kümpers 2008). Recently, it has been demonstrated, that treatment with an Ang-1 adenovirus in septic mice resulted in improved cardiac function as well as enhanced survival (Witzenbichler 2005). Here, we assessed the protective effect of Ang-2 antibodies (Ang2-ab) in a murine model of lipopolysaccharide (LPS)-induced sepsis.

Methods

To induce Sepsis, LPS [20mg/kg] was injected intraperitoneally into C75BL/6 mice. 24 hours before sepsis induction, groups were pre-treated with Ang2-ab or an unspecific antibody (control-ab) as a control. After sepsis induction a sepsis-severity-score was assessed after 6 and 12 hours, followed by invasive and non-invasive hemodynamic measurements or observation of further survival. The sepsis-severity-score includes five different parameters (behaviour, pain, ascites, dispnea, weight loss), according to the severity points between 0 and 20 are given. Blood samples and organs were harvested for histological and molecular analysis. 

Results and Conclusion

Whereas in control-ab treated mice, hemodynamic function was severely depressed 12 hoursafter LPS injection, as seen in decreased left ventricular developed bloodpressure (74 ± 9 mmHg) and severely reduced systemic blood pressure (45 ± 3 mmHg), in Ang2-ab treated mice left ventricular developed blood pressure fell only to 92 ± 6 mmHg. Similarly, the drop in systemic blood pressure was less pronounced (77 ± 10 mmHg). This resistance to LPS-induced hemodynamic changes was reflected by reduced sepsis-severity-score results after 6 (5 ± 1 points vs.7 ± 1 points) and 12 hours (10 ± 1 points vs.14 ± 1 points) and at least by trend improved survival rate of the Ang2-ab treated mice. Histological analysis revealed that the dropout in pericytes seen in septic mice is abolished by Ang2-ab treatment. These findings highlight the pivotal role of Ang-2 in the onset and progression of sepsis and identify Ang-2 as a potential target for treatment of Sepsis.

P164
Adenosine up-regulates thrombospondin -1 production by macrophages

Abstract

Background

Increase of blood capillary density at the interface between normal and ischemic tissue after acute myocardial infarction reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component thrombospondin-1 (TSP-1) and adenosine. Adenosine is thought to be involved in cardiac repair and is known to stimulate angiogenesis. The role of TSP-1 in angiogenesis is less clear, since both anti- and pro-angiogenic activities have been reported. We hypothesized that adenosine controls angiogenesis through modulation of TSP-1 production.

Methods

Primary human macrophages were obtained by differentiation of peripheral blood monocytes from healthy volunteers, and were treated with adenosine (0.1-50 μmol/L) under ischemic conditions (hypoxia and starvation) or stimulation by cytokines (IL-1β and TNF-α). A rat aortic ring assay was implemented to evaluate angiogenesis.

Results

Adenosine dose-dependently increased the production of TSP-1 by macrophages, reaching a 4-fold increase at 10 μmol/L (n = 11, P < 0.001). A 13-fold induction of TSP-1 mRNA expression was measured. These effects were observed both under basal conditions, during ischemia, and after stimulation with cytokines. Use of agonists and antagonist of adenosine receptors, coupled to RNA interference experiments suggested that the A2A and A2B receptors mediate the effect of adenosine on TSP-1. This effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator), and blocked by the PKA inhibitor H89. Low doses of purified TSP-1 ( < 5μg/mL) increased microvessel outgrowth from rat aortic rings, whereas high doses ( > 5μg/mL) decreased this outgrowth. Conditioned medium from adenosine-treated macrophages (containing approximately 0.2ng/mL of TSP-1) enhanced microvessel outgrowth. Addition of anti-TSP-1 antibodies to conditioned medium blocked angiogenesis in this model.

Conclusions

We show for the first time that adenosine up-regulates TSP-1 production by macrophages. This effect involves the A2-type adenosine receptors and is mediated through the cAMP/PKA pathway. In our model, adenosine induced low levels of TSP-1 which were pro-angiogenic. However, higher levels of adenosine and TSP-1, which are anti-angiogenic, may be reached in the ischemic myocardium. This is an important information for the design of adenosine-based cardiac therapies.

P165
MicroRNA-15a and mir-16 regulate sdf-1 migration of endothelial progenitor cells in diabetic patients with limb ischemia and healthy controls

Abstract

Purpose

Circulating endothelial progenitor cells (EPCs) are reduced and functionally impaired in diabetic patients, possibly contributing to the higher number and severity of ischemic events observed in this population. The molecular mechanisms underlying diabetes-associated EPC impairment are incompletely understood. microRNAs (miRs) post-transcriptionally inhibit the expression of their target genes. We aim to obtain mechanistic insights of diabetic EPCs impairment and to provide the first characterization of the EPC-associated miRNA with special respect to those potentially involved in the control of angiogenesis. Here we report results on miR-15a/-16.

Methods

EPCs were prepared from the peripheral blood of type-2 diabetic patients undergoing revascularization for limb ischemia (LI) and from healthy individuals. The migratory activity of EPCs was measured in a transwell migration assay using SDF-1 (100ng/ml) as a chemo-attractant. A miR screening was performed (TaqMan PCR) in diabetic LI EPCs (n = 55) (controls: healthy donors n=17) to selected some differentially expressed miRs to be further analyzed. miR expression in EPCs was regulated using pre-miR, anti-miR and scramble control (all from Ambion).

Results

As expected, EPC migration (modified Boyden chamber) to SDF was reduced by disease (migration index – ratio of SDF-1 to BSA migrated EPCs -: 1.08 + 0.02 vs 1.87 + 0.09 for healthy EPCs, P < 0.01, n=6 patients and n=7 controls). The miR-15a/-16 expression (relative to snRU6) was increased in "diseased" EPCs in comparison to controls (miR-15a: 5.19 + 0.85 vs 1.4 + 0.15, p < 0.05; miR-16: 4.91 + 0.91 vs 1.13 + 0.13, p < 0.05). To understand the effect of miR-15a/-16 upregulation on EPC capacities, we transfected pre-miR-15a and pre-miR-16 in healthy EPCs. miR-15a/-16 overexpression inhibited EPC migration to SDF-1 (migration index: 1.24 + 0.74 vs. 1.62 + 0.04 in scramble, p < 0.05). Next, to understand if increased endogenous miR-15a/-16 contributes to impaired EPC migration observed in patients, we transfected "diseased" EPCs with anti-miR-15a and anti-miR-16. This corrected the migratory capacity of patient-derived EPCs (migration index: 1.51 + 0.14 vs 1.1 + 0.09 in scramble, p < 0.05 and P=NS vs. healthy EPCs given scramble). Bioinformatic analyses predict BCL2, VEGFA, and AKT3 to be target genes of both miRNA-15a and miR-16, which additionally suggest anti-angiogenic and pro-apoptotic roles of these miRs.

Conclusions

miR-15a/-16 are involved in SDF-1 migratory impairment of EPCs from diabetic patients with LI.

P166
Tissue factor induces endothelial neovessel formation through Akt signaling

Abstract

Prone to rupture atherosclerotic plaques (AT) show significant angiogenesis. The mechanism of neovessel formation in the growing AT is still unknown. We have previously shown that tissue factor (TF) and its signaling mediators have a significant role in mature neovessel formation.

Objective

To define the signaling pathways involved in TF-induced angiogenesis.

Methods and Results

We analyzed the Akt, a downstream effector of the TF-induced signaling via activation of PI3K. The following results were obtained: a) TF-siRNA inhibition of TF in the human endothelial cells (HMEC-1) resulted in a highly significant inhibition of endothelial-tube-likeformation (ETub) with stable phenotype in 3D cultures (2500 ± 25 μm random-siRNA versus 260 ± 45 μm TF-siRNA). The inhibition in ETub was associated with a down-regulation of Akt expression (2x) and with increased Raf-phosphorylation at Ser259 (1x up) resulting in a reduced Raf kinase expression and a reduction of ERK1/2 phosphorylation (1x down) and Ets-1 transcription factor inhibition (2x down); b) Overexpression of TF resulted in an increase in ETub (3250 ± 15 μm versus 2500 ± 25 μm) and in the up-regulation of Akt protein; c) Immunoprecipitation of Akt revealed that TF is directly associated with Akt; d) The effect of silencing TF was only reversed by PAR2 agonist (H-5042) to a 65% of control ETub (1630 ± 25 μm versus 260 ± 45 μm); e) Enforced expression of Akt by pcDNA3-MyrHA-Akt1 plasmid in TF-silenced endothelial cells rescued ETub formation (2250 ± 45 μm versus 260 ± 45 μm) and induced Ets-1 phosphorylation.

Conclusion

TF and Akt1 form a complex that regulates Etub formation signaling through Raf/ERK/Ets-1. The triggering effect of TF on the formation of endothelial-tube-like formation may have a significant impact not only in atherosclerotic plaque neovessel formation but in cancer metastasis and other processes cursing with neovascularization.

P167
Dynamic course of serum-induced endothelial cell apoptosis in ST-segment elevation myocardial infarction patients treated with primary angioplasty: implications for a deregulation of immune response

Abstract

Purpose

Acute loss of endothelial cells may play a role in the ischemia-reperfusion injury process. The aim of this study was to evaluate the pro-apoptotic effect of blood serum on endothelial cells in reperfused ST-segment elevation myocardial infarction (STEMI) patients.

Methods

Human umbilical vein endothelial cells (HUVEC) were incubated with serum of 20 patients with a first STEMI treated with primary angioplasty drawn before reperfusion and 24h, 96h and 30 days afterwards. Apoptosis, necrosis and viability percentages were evaluated by flow cytometry. Cytokine levels and lymphocyte subtypes were evaluated by multiplexed immunoassay and flow cytometry respectively. Values were compared with serum of 12 age- and sex- matched control subjects with normal coronary arteries. 

Results

In comparison with controls, serum of STEMI patients induced a loss of HUVEC viability mainly due to apoptosis but not necrosis. In patients, the pro-apoptotic effect of serum was maximum at 96h post-reperfusion (Figure). A pro-inflammatory response paralleled the pro-apoptotic effect of serum. In comparison with controls, at 96 hours anti-inflammatory cytokines IL-4 and IL-10 did not vary but pro-inflammatory cytokines IL-6 (p < 0.001) and IL-1β (p < 0.001) and pro-apoptotic cytokines TNF-α (p < 0.01) and TGF-β (p < 0.05) increased. Similarly a pro-inflammatory response in adaptative immune cells occurred: CD4 + cells count and Th1/Th2 ratio increased but FOXP3 + T regulatory cells count diminished (p < 0.05 in all cases). 

Conclusion

Serum of STEMI patients induces apoptosis on endothelial cells. This effect progressively increases in the days following reperfusion and it is acompanied by an acute pro-inflammatory deregulation of the adpatative immune system.1

Lipids, Atherosclerosis
P168
Metabolic hormone levels in patients undergoing on pump coronary artery bypass grafting

Abstract

Purpose

To explore the response pattern of plasma adipokine and ghrelin levels to coronary artery bypass graft (CABG) surgery in patients receiving cardiopulmonary bypass (CPB) with glucose-insulin-potassium (GIK) infusion.

Methods

16 consecutive patients (age: 62 ± 10 years, male: 10) with obstructive coronary artery disease (CAD) who underwent elective CABG surgery with CPB and intraoperative GIK infusion were selected. Blood samples were taken before, during and after surgery. Intraoperative samples were withdrawn simultaneously for peripheral vein and sinus coronarius (SC). Plasma adipokine concentrations were measured by ELISA, those of ghrelin by RIA kits.

Results

The baseline values of insulin and leptin were higher (p < 0.01), those of resistin and ghrelin were lower (p < 0.01) in patients undergoing CABG with CPB than the respective values of controls. In response to surgical intervention there was an early, transient fall in plasma levels of leptin (p < 0.06), adiponectin (p < 0.001) and resistin (p < 0.002) followed by an increase to approach their initial values. Plasma ghrelin also increased (p < 0.045), this increase, however, was confined to the period of GIK supported CPB. Moreover, it was demonstrated that plasma leptin, adiponectin and ghrelin in peripheral vein did not differ from those in SC. By contrast, plasma insulin (p < 0.003) and resistin (p < 0.009) was significantly higher in the periphery than in SC.

Conclusions

Adipose tissue-derived factors may mediate the metabolic and vascular effects of insulin in patients with CABG surgery. Epicardial adipose tissue is unlikely to have major contribution to the development of CAD as adipokines are not elevated in SC.

P169
High dose statines reduce carotid artery intima-media thickness (IMT) and decrease the risk for general cardiovascular disease

Abstract

Purpose

To use High dose Statins in non symptomatic patients with increased Carotid Intima-Media Thickness (IMT) to reduce the incidence of Cardiac Arrhythmias and of fatal or non-fatal Coronary Artery Disease (CAD) among patients at increased risk for cardiovascular disease

Methods

We investigated 170 patients who had increased ITM 1.1-1.4mm, had low-density lipoprotein (LDL) cholesterol levels of 100 to 190 mg per deciliter (2.6 to 4.9 mmol per liter), and had not known Cardiac Arrhythmias and CAD. The patients were divided in two groups. One group (69 patient) was treated with 80 mg Simvastatin per day and second group (101patient) with 20mg simvastatin per day. ACI and Aspirin 100mg were prescribed to both groups

Results

The mean LDL cholesterol level during the trial was 73 mg per deciliter (1.9 mmol per liter) among patients receiving simvastatin 80mg per day and 117 mg per deciliter (2.9 mmol per liter) among patients receiving 20mg simvastatin per day. IMT in patient receiving 80mg simvastatin preserved or diminished 0.1-0.89mm, and in patients receiving 20mg simvastatin preserved or increased after 1,9 years. During a median follow-up of 1,9 year, 15 patients (21,7 %) receiving simvastatin 80mg per day and 30 patients (29,75%) receiving 20mg had a fatal or nonfatal CAD and atrial and ventricular arrhythmias.

The group received 80mg simvastatin had 3 atrial fibrilacion,1 ventrikular arrhytmias;7 NONSTEMI and 4 STEMI, whereas the group, received 20mg simvastatine had 8 atrial fibrilacion and other atrial arrhythmias,3 ventricular arrhythmias;12 NONSTEMI and 7 STEMI.

4 (5,7%) patients from 80mg simvastatine group and 9 (8,9%) from 20mg simvastatin group died from cardiac and cardio-cerebral event.

The five-year absolute reduction in the risk of major cardiovascular events was 3.5 percent.

The overall mortality rate was low in patient received high dose simvastatin.

IMT also that is one of them marker of cardiovascular disease, did not changed or improved. Elevated liver enzyme values were more common in patients taking simvastatin in high dose

Conclusions

In patients with increased Carotid IMT and increased risk for cardiovascular disease and without known cardiac arrhythmias and coronary heart disease, 80 mg of simvastatin per day reduced the overall incidence of Cardiac Aarrhythmias and ACS and of other cardiovascular events, and improves Carotid IMT values as a marker of general cardiovascular disease.

P170
Comparison of atherogenic porcine and rabbit models in restenosis studies

Abstract P170 figure Elastic Stain of WHHL and swine arteries

Abstract P170 figure Elastic Stain of WHHL and swine arteries

Abstract

Background

Animal models are needed to preclinically evaluate in-stent restenosis (ISR) reducing therapies. Currently used models vary in large measure between different research groups and separate studies. Our aim was to compare lesion characteristics and feasibility of the models for ISR studies.

Methods

We compared experimental atherosclerotic lesions in landrace porcine coronary arteries (n = 33) and rabbit aortas (n = 12) by combining endothelial injury by balloon dilation with high serum lipid levels. Dyslipidemia was diet induced, except for WHHL rabbits which have naturally high serum cholesterol levels.

Results

Rabbit aortas developed uniform lesions rich in lipids (Fig. A). Due to different anatomy of the arteries, porcine coronary artery denudation and balloon over dilatation lesions (Fig. B) had high variation in size and structure inside the groups. Porcine coronary stenosis differed from 25 to 70 % compared to WHHL stenosis of 55.1 to 85.1%. The porcine coronary lesions had more connective tissue and SMCs than lesions in rabbit aorta. Landrace porcine did not develop high serum lipid levels with feasible lipid diets. Serum cholesterol levels were 4.2 + /- 1.2 mmol/L in porcine high lipid diet group compared to 18.7 + /- 4.6 mmol/L in NZW lipid diet group and 17.3 + /- 1.3 mmol/L in WHHL group.

Conclusions

The variation in the degree of stenosis and lesion characteristics was high within the porcine denudation groups. We suggest the atherogenic rabbit aorta models lipid and inflammatory profile of atherosclerotic human coronary better than the atherogenic porcine model. Intact porcine coronary is more comparable to human coronaries for its structure and heamodynamics. Hence it can be used to assess restenosis unaffected by atherosclerosis.1

P171
P. gingivalis-induced aggregation and ros production in whole blood is dependent on gingipains

Abstract

A large body of data accumulated over the past several years suggests that the periodontal pathogen Porphyromonas gingivalis is associated with cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. We have previously demonstrated that P. gingivalis induces aggregation and ROS production in whole blood, and that the anti-inflammatory mediator lipoxin A4 (LXA4) inhibits these responses by modulating platelet-neutrophil interaction through a down-regulation of the bacterium-induced surface expression of CD11b/CD18 on neutrophils, likely by inhibiting Rac2 and Cdc42 signaling pathways. Furthermore, P. gingivalis, unlike other periodontopathic bacteria, has been shown to trigger platelet aggregation, mainly through the interaction between bacterial gingipains and protease-activating receptors (PARs) on the platelets. Since platelet aggregation precedes thromboembolic events, this is an important pathogenic feature of the bacterium. The aim of this study was to investigate the effect of gingipains on P. gingivalis-induced cell activation in whole blood. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry. This study shows that leupeptin, a protease inhibitor of gingipains, inhibits P. gingivalis-induced aggregation and ROS production in whole blood. Supernatants of bacteria suspensions induced no ROS-production, but an aggregatory response that was also inhibited by leupeptin. In conclusion, P. gingivalis-induced aggregation and ROS production in whole blood is mainly dependent on gingipains. However, since bacterial supernatants (containing soluble gingipains) stimulate only aggregation, this suggests that a gingipain/PAR-mediated mechanism in combination with phagocytosis of whole bacterium is a prerequisite for inducing a respiratory burst and an inflammatory response. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.

P172
HSPA1A phenotypes and risk of cardiovascular disease

Abstract

Purpose

HSPA1A is a serum and intracellular heat shock protein with antiapoptotic / antithrombotic properties. Possible polymorphisms of the regulatory region of HSPA1A could affect HSPA1A protein synthesis, determining diminished, normal or increased HSPA1A-producing phenotypes. The hypo-producing phenotype would entail a greater risk of developing atherosclerotic disease. The present study was made to identify SNPs in the HSPA1A regulatory region and evaluate whether any of them could affect HSPA1A synthesis in a randomly selected population which was later stratified into different groups according to the degree of vascular risk.

Methods

Serum and intragranulocytic HSPA1A was quantified by ELISA and direct Sanger sequencing performed in all subjects. An analysis was made of the association of two SNP (db rs1008438 -110A/C and db rs1043618 + 190G/C) with circulating and intragranulocytic HSPA1A and the risk of atherosclerosis. Data were analyzed using a nonparametric Mann-Whitney rank sum test or a Kruskal-Wallis test. A Chi-square test or a Fisher's exact test was used to assess the goodness-of-fit between the observed allele frequencies and the expected counterparts by Hardy-Weinberg equilibrium, and to evaluate differences in allele distributions between groups.

Results

The study population consisted of 452 randomly selected subjects, 234 females (49.95 ± 6.89 years), and 218 males (48.86 ± 7.27 years). They were stratified into three groups according to Task Force Chart Criteria: no vascular risk or risk < 5%, n=239; moderate vascular risk (10-20%) without clinical atherosclerosis, n=161; and overt atherosclerosis n=52. The greatest percentage of intragranulocytic HSPA1A hypo-producers corresponded to the subjects with the CC genotypes of -110A/C and + 190G/C SNPs. The assignment to a given vascular risk group revealed differences in the percentages of hyper-, normal or hypo-producing individuals. The CC genotype entails a risk of being a low intracytoplasmic HSPA1A producer with respect to normal production of 1.673 (95% CI 1.191-2.351, p=0.004) versus the AC genotype, and of 1.419 (95% CI 1.034-1.947, p=0.033) versus the AA genotype.

Conclusions

Based on our results, -110A/C and + 190G/C homozygous carrier status entails a risk of presenting moderate vascular risk or declared atherosclerosis – probably as a result of diminished intracellular HSPA1A synthesis and a consequent partial loss of its antiinflammatory and antithrombotic properties. In coincidence with other authors, this leads us to postulate the -110 A and + 190 G alleles as possible genetic markers of less severe clinical phenotypes

P173
Interleukin-33 induces urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 in human endothelial cells in vitro

Abstract

Background

Urokinase-type plasminogen activator (u-PA) and its inhibitor, PA inhibitor type-1 (PAI-1) by regulating extracellular proteolysis are involved among other pathophysiological events in vascular remodeling and in plaque angiogenesis and stability in atherosclerosis. IL-33 is a novel member of the IL-1 cytokine family and is a ligand of the ST2 receptor. IL-33 has recently been implicated in the pathogenesis of atherosclerosis. It was shown to induce vascular permeability and angiogenesis and the production of inflammatory cytokines in endothelial cells. Here we aimed to study a possible regulation of u-PA and PAI-1 by IL-33 in human endothelial cells (EC).

Methods

Human umbilical vein EC (HUVEC) and human coronary artery EC (HCAEC) were treated with IL-33 alone or together with soluble ST2 fusion-protein (sST2 Fc) or together with simvastatin. Specific mRNA levels for u-PA and PAI-1 were determined by RT-PCR and u-PA and PAI-1 antigen and activity were measured by specific ELISAs.

Results

u-PA mRNA was up-regulated up to 5-fold in HUVEC and up to 2.4-fold in HCAEC when these cells were treated with 100 ng/ml IL-33 for 9h whereas PAI-1 mRNA increased up to 2.5-fold and up to 2-fold, respectively. IL-1 receptor antagonist had no effect on IL-33-induced increase in u-PA and PAI-1, which suggests that these effects are IL-1-independent. u-PA antigen increased up to 30-fold and PAI-1 antigen increased up to 2-fold after 48h of incubation with 100 ng/ml IL-33 in HUVEC. In HCAEC u-PA increased up to 10-fold after 4h and PAI-1 increased up to 3.5 fold after 24h of incubation with 100 ng/ml IL-33. PAI-1 activity increased up to 4.5-fold after 4h of incubation with 100 ng/ml IL-33 in HUVEC and up to 5-fold after 6h of incubation in HCAEC. The increase in u-PA and PAI-1 antigen was concentration-dependent when the cells were incubated with IL-33 at concentrations ranging from 1 to 100 ng/ml. sST2 Fc abrogated the IL-33-induced increase in u-PA and PAI-1 antigen suggesting that these effects of IL-33 are ST2 receptor-mediated. Simvastatin at concentrations ranging from 0.5-2.5 μM also abrogated IL-33-induced increase of u-PA and PAI-1 antigen, thus providing further evidence that statins have effects beyond reduction of cholesterol.

Conclusion

Via induction of u-PA and PAI-1 in endothelial cells, IL-33 could contribute to the modulation of endothelial cell-mediated extravascular proteolysis in processes such as neovascularization and vascular remodeling. By modulating these processes IL-33 could affect plaque angiogenesis thereby impacting on the stability of these vascular lesions in atherosclerosis.

P174
The influence of the IL-18 + 183 A/G polymorphism on gene- and protein expression in stable coronary artery disease patients

Abstract

Purpose

The pro-inflammatory cytokine interleukin (IL)-18 is shown to be a potential predictor of cardiovascular events. Increased IL-18 serum levels have been associated with diabetes type 2, metabolic syndrome and the severity of atherosclerosis.

In this study we investigated the presence of the 3'UTR + 183 A/G polymorphism and its influence on gene- and protein expression in stable coronary artery disease (CAD) patients and in subgroups of CAD. Additionally, levels of IL-18 BP were measured.

Methods

The frequency of the + 183 A/G (rs 5744292) was analyzed in 1001 patients with angiographically verified CAD, all enrolled in the ASCET study (ASpirin non-responsiveness and Clopidogrel Endpoint Trial)*, of which 20% presented with diabetes type 2, 24% with metabolic syndrome, 56% with hypertension, 22% were women, mean age 62 years. IL-18 gene-expression was measured in circulating leukocytes (PAXGene tubes) in 240 randomly selected patients.

Results

The proportion of the + 183 A/G polymorphism was: AA (55%), AG (39%) and GG (6%).

The AA genotype was more frequent in patients with hypertension (p=0.04, adjusted for age, body mass index and gender), but no further differences in frequencies between subgroups of CAD were observed.

The G-allele associated significantly with lower serum levels of IL-18 (p=0.002, adjusted for age, glucose, body mass index and gender) without any changes in IL-18 BP levels according to genotypes. Serum IL-18 levels were significantly higher in men as compared to women, in patients with diabetes type 2 and metabolic syndrome compared to those without (p < 0.01, all). The G-allele induced reduction in IL-18 levels was 3-4 fold more pronounced in diabetes and metabolic syndrome as compared to unaffected patients.

Unexpectedly, the G-allele associated with a 1.13 fold higher IL-18 gene-expression level (p=0.01).

Conclusion

The difference in serum IL-18 levels across numbers of G-alleles were especially apparent in patient with diabetes type 2 and metabolic syndrome, suggesting a non-preferable AA genotype in relation to cardiovascular outcome in these patients.

* Pettersen A-Å  Scand Cardiovasc J. 2004 Dec;38(6):353-6.

P175
Low-density lipoprotein receptor-related protein 5 is differentially expressed in CD14/CD16 macrophage subpopulations and regulates migration

Abstract

Background

Previous results from our lab have shown the involvement of a member of the LDL receptor family, low-density lipoprotein receptor-related protein 5 (LRP5), in the macrophage reaction to lipid infiltration in atherosclerosis. LRP5 participates in lipid uptake and macrophage migration, critical steps in atherosclerosis progression. We have recently shown that in human coronary artery plaques only a subpopulation of infiltrated macrophages (stained with HAM56) express LRP5. Macrophages are tissue-differentiated monocytes, mononuclear cells that circulate several days until they extravasate and populate healthy and diseased tissues. At least two principal monocyte subsets have been identified that can be distinguished by the expression of surface markers: CD14 + CD16- and CD14 + CD16 + . These two cell subsets are morphologically and functionally different. CD16 + monocytes are known to participate in atherosclerotic progression by the release inflammatory cytokines, adherence to endothelial cells and induction of cellular migration to the vascular wall.

Objective

The aim of this work was to investigate LRP5 expression and function in the two monocyte subsets.

Methods

Magnetic cell sorting (MACS) with CD16 monoclonal antibodies was used to fractionate human monocytes from healthy individuals yielding highly purified populations of CD16 + and CD16- monocytes. Monocytes were differentiated to macrophages and LRP5 levels were measured, LRP5-gene was Over-expressed or silenced and macrophage migration assays performed.

Results

A significantly higher LRP5-mRNA expression level was observed in CD16 + compared to CD16- macrophages. Furthermore, CD16 + macrophages had higher migration rates than CD16-. over-expressing LRP5 in CD16- macrophages rescued their migration capacity. Also, silencing LRP5 in CD16 + macrophages induced reduction of migration.

Conclusions

Here we show for the first time that CD16 + macrophages have a high expression of LRP5. This increased expression results in an increased macrophage migration, a process involved in the infiltration of inflammatory cells in the atherosclerotic lesion.

P176
Relationship between natural killer cells and platelet reactivity in patients with acute coronary syndrome

NK cells by platelet reactivity in ACS

NK cells by platelet reactivity in ACS

Abstract

Purpose

Preliminary studies show that patients with Coronary Artery Disease (CAD) present reduced NK cell compartment and low cytotoxic NK activity. NK cell functions may be affected by inflammatory processes with consequent impairment of immunological response leading to progression of CAD. High levels of inflammation associated with enhanced platelet aggregation are typical of acute phases of coronary disease. Thus, we analyzed the relationship between thrombogenicity and NK cell distribution in patients with non-ST-elevation acute coronary syndrome (NSTEACS).

Methods

Peripheral blood samples were taken from 50 statin-naïve patients with NSTEACS and analyzed for the distribution of NK cells by Flow Cytometry (using anti-CD3, -CD8, -CD16 + 56, -CD57 monoclonal antibodies) and for platelet reactivity by the VerifyNow P2Y12 assay. Platelet reactivity was measured as residual ADP induced aggregation (PRU) after P2Y12 receptor blockade and as thrombin induced aggregation (Base PRU) which provides an estimation of total platelet reactivity.

Results

Overall, there were a significant positive correlation between PRU and Base PRU values (r = 0.38, p=.02) and inverse correlations between the absolute number of NK cells (CD3-CD16 + 56 + and CD8-CD57 + ) and PRU (p < 0.05) and Base PRU (p < 0.01). We divided the patient population into two groups: Low Reactivity (Base PRU < 305) and High Reactivity (Base PRU ≥ 305). NK cell frequencies were significantly lower in the High Reactivity than the Low Reactivity group (Figure 1).

Conclusions

This study shows that in patients with NSTEACS there is a link between platelet reactivity and NK cell distribution. As PRU values correlate with clinical outcome, our results may suggest a possible link between NK cell frequencies and prognosis in patients with ACS.

P177
Apoptosis pathways are deregulated in CD4 + CD28null T cells from patients with acute coronary syndrome

Abstract

Purpose

T lymphocytes, the main effectors of adaptive immunity, have key roles in the development and progression of atherosclerosis. One subset of T lymphocytes that is altered in patients with acute coronary syndrome (ACS) is the CD4 + CD28null T cells. These cells expand significantly in ACS patients compared to stable angina and healthy controls and have been suggested to mediate plaque instability and recurrence of acute coronary events. CD4 + CD28null T cells characteristically do not express the CD28 receptor which has pivotal roles for the activation and survival of T lymphocytes. Apoptosis is a homeostatic process that regulates the size of the T cell compartment. Very little is known about the mechanisms that lead to the accumulation of CD4 + CD28null T cells in ACS. Our aim was to characterise apoptosis pathways in CD4 + CD28null T cells in ACS patients to identify alterations that could explain the persistence of this cell subset.

Methods

We analysed the expression of death receptors (Fas) and ligands (FasL), as well as the levels of anti-apoptotic (Bcl-2, Bcl-xL, survivin) and pro-apoptotic (Bax, Bim) proteins in CD4 + CD28null T cells in patients with ACS using flow cytometry. In addition, we have quantified apoptosis sensitivity of in vitro activated CD4 + CD28null T cells using Annexin V and 7-AAD staining and detection of activated caspase-3.

Results

We found that CD4 + CD28null T cells express significantly lower levels of the death receptor Fas compared to conventional CD4 + CD28 + T cells. Furthermore, the pro-apoptotic protein Bim was significantly decreased in CD4 + CD28null T cells compared to their CD28 + counterparts. Interestingly, CD4 + CD28null T cells failed to upregulate Bim following activation, in stark contrast to CD4 + CD28 + T cells. No differences were found in the levels of anti-apoptotic proteins between the two CD4 + T cell subsets.

Conclusion

We identified defects in the death receptor Fas and the pro-apoptotic protein Bim in CD4 + CD28null T cells from ACS patients which suggest that these cells are resistant to apoptosis. These findings could open the way for novel therapies aimed at targeted induction of apoptosis in CD4 + CD28null T cells to stabilise atherosclerotic lesions.

P178
Cardiac expression and activation of NF-kB in association with enhanced spleen inflammation in mice with obesity and diabetes

Lipids in the heart of db/db mice

Lipids in the heart of db/db mice

Abstract

Inflammation serves as a risk factor for atherosclerosis. In diabetes, abnormal production of inflammatory factors contribute to cardiovascular dysfunction. The nuclear factor NF-kB plays an important role in inflammatory signal transduction.

Objective

To test the association of spleen inflammatory responses and activation of CD1d-restricted NKT cells, with expression and activation of NF-kB in the hearts of obese hyperinsulinemic diabetic mice.

Db/db and CD1d-knockout mice of both genders, in comparison with their respective background wild-type C57BL/6 and Balb/C were examined. The spleen tissues of db/db mice displayed increased mRNA levels of TNF-α and TNF-α receptor type 1, and higher ratio of CD1 + NK-T cells vs. CD3 + cells compared with controls. In the heart of db/db mice, although no obvious infiltration of inflammatory cells was found, mRNA levels of TNF-α receptor type 1, NF-kB DNA-binding activity, nuclear expression of p65 and activation of p52 subunit were significantly higher compared with  controls. Histological examinations showed marked accumulation of lipid droplets within cardiac myocytes, but no hypertrophy or increased deposition of collagen between myocytes in db/db compared with controls. On echocardiography, the ratio of E to A transmitral flow velocities (an indicator of diastolic function) was significantly decreased in db/db mice, while indexes of systolic function were marginally affected. These changes were paralleled by increased mRNA expression of brain natriuretic peptide.

Conclusions

These results provide evidence for the presence of NF-kB activation and diastolic dysfunction in the heart of db/db mice. The abnormal immune responses and TNF-α production in the spleen tissue can contribute to NF-kB activation and cardiac dysfunction in type 2 diabetes.1

P179
Lp(a), apo-b and ldl levels in premature coronary heart diseases in pakistani adults

Abstract

Background

Premature coronary heart disease(CHD) is a highly complex disorder with multiple risk factors.

Objective

To identify the risk factors responsible for premature (CHD) in local population.

Method

A case control study of 100 angiographically diagnosed patients and 100 healthy subjects was conducted. The risk factors assessed in this study were Serum Lipids, Lipoprotein and Apolipoprotein-A1,Apolipoprotein-B, Lp(a), Diabetes Mellitus (DM), smoking, hypertension,family history of CAD, obesity and sedentary lifestyle. Descriptive,uni-variate and multivariable analyses were conducted using statistical package for social sciences (SPSS). 

Results

 Sedentary life style (OR= 12.57, 95% CI: 3.68, 42.98), serum Triglyceride (TAG) levels(OR= 2.45, 95% CI: 1.27, 4.72), HDL-c (OR= 4.95, 95% CI: 2.35, 10.42), APO-A(OR= 1.86, 95% CI: 4.40, 24.76), APO-B (OR= 10.49, 95% CI: 4.40, 24.76) andlipoprotein (a) (OR= 2.15, 95% CI: 1.20, 3.84) were found to be significant factors.TAG levels, HDL-c, APO-A, LDL-c and HDL-c ratio, total cholesterol and HDL-cratio were found to be significant among patients of age ≥40 years, while LDL-capolipoproten B and LP(a) was highly significant among age < 40 years.

Conclusion

 Raised level of Lp(a), apo-B and LDL-c among premature CAD patients have identified as risk factors in Pakistani adults < 40years.

P180
The influence of beta1-adrenergic receptor gene polymorphism on lipids metabolism in patients with heart failure and systolic dysfunction

Abstract

Purpose

To investigate the influence of Ser49Gly and Arg389Gly β1-adrenergic receptor (β1-AR) gene polymorphism on lipids metabolism in patients with severe chronic heart failure.

Methods

Ninety nine patients (78 males and 21 females; mean age 61,7 ± 0,96 years) with chronic heart failure and systolic dysfunction were examined. Geno-typing was performed to identify the individual β1-AR Arg389Gly and Ser49Gly polymorphism by the restriction fragment length analysis of polymerase chain reaction products. The parameters of lipid metabolism were determined enzymatically by using kits of reagents "HUMAN" (Germany), according to manufacturer's instructions. Taking into account the abnormality of distribution non-parametric tests were performed. Results were present as Median (low : upper quintiles). For nonparametric comparisons Mann-Whitney U test was used. All statistical tests were 2-tailed, and p < 0.05 was considered statistically significant.

Results

Serum total cholesterol ((5,61 (5,00 : 6,37) versus 4,60 (3,81 : 5,58) mmol/l), triglycerides ((2,13 (1,64 : 2,47) versus 1,64 (1,14 : 1,98) mmol/l), low-density lipoprotein (LDL) cholesterol ((3,63 (3,14 : 4,33) versus 2,88 (2,18 : 3,58) mmol/l) and very low-density lipoprotein (VLDL) cholesterol ((0,96 (0,74 : 1,11) versus 0,74 (0,51 : 0,89) mmol/l) were significantly higher in the group of Ser49Gly carriers than in the homozygous carriers of Ser49 allele of β1-AR gene polymorphism, (p < 0,01). The concentration of serum total cholesterol ((5,00 (4,20 :5,96) versus 4,17 (3,91 : 4,60) mmol/l), triglycerides ((1,78 (1,37 : 2,12) versus 1,50 (0,9 :2,00) mmol/l), LDL cholesterol ((3,13 (2,34 : 4,09)) versus 2,60 (2,42 :3,04) mmol/l) and VLDL cholesterol ((0,80 (0,62 : 0,95)) versus 0,68 (0,44 6 0,90) mmol/l) were higher in the group of Arg389Arg carriers than in the homozygous carriers of Gly389 allele of β1-AR gene polymorphism but these differences didn't reach a significance, (p > 0,05)

Conclusions

The presents of Ser49Gly β1-AR gene polymorphism was associated with significantly greater lipids metabolism abnormalities comparing with the Gly49Gly variants. The Arg389Gly polymorphism wasn't influence on lipids metabolism in the patients with severe chronic heart failure.

P181
Apolipoprotein(a) impairs vascular smooth muscle cell motility via alpha V beta 3 integrin and RhoA activation

Abstract

Purpose

Lipoprotein(a) (Lp(a)) is an independent risk factor for cardiovascular disease (CVD). Although structurally similar to LDL, it contains a unique glycoprotein, apolipoprotein(a) (apo(a)), which is the active part of the Lp(a) molecule. Plasma Lp(a) levels remain constant throughout an individual's lifetime and are resistant to lipid-lowering strategies. Remodelling of vascular smooth muscle cells (VSMC) plays a key role in the pathogenesis of CVD through phenotypic and functional alterations. The aim of this study was to determine the role of apo(a) on VSMC migration and explore the underlying mechanisms.

Methods

VSMC were cultured by explant technique from human aortic root and saphenous vein. Local ethical committee and informed patient consent were obtained. Migration was analysed using modified Boyden chambers in response to recombinant apo(a) in the presence and absence of a recognised chemoattractant (PDGF; 10ng/ml). Morphology of the cytoskeleton was detected by immunocytochemistry. RhoA activity was measured using G-LISA. Where apo(a) was seen to mediate effects the underlying mechanism was examined using gene silencing (RhoA), tyrosine kinase inhibition and neutralising antibody against αVβ3.

Results

Apo(a) potently repelled the migration of human VSMC over 6h in a concentration-dependent manner (p < 0.001, n=6) whereas prolonged exposure (48h, 25nM) induced actin bundling and increased spread cell area (125%, n=200 cells, p < 0.001). Apo(a) rapidly activated RhoA (150%, n=8, p < 0.05). The chemorepellent effect of apo(a) was negated when cells were treated with RhoA siRNA (p < 0.001, n=6) or fluvastatin (p < 0.001, n=5). Blocking αVβ3 using a neutralising antibody significantly reduced the chemorepellent effect of apo(a) (p < 0.001, n=6), as did tyrosine kinase inhibition using genistein (p < 0.001, n=5).

Conclusions

Our data reveal a unique mechanism by which apo(a) may modulate VSMC behaviour in a detrimental manner. Apo(a) has been shown to accumulate in atheromatous regions, and we demonstrate here that it acts as a potent chemorepellent to VSMC. The underlying mechanism is likely the activation of RhoA and actin bundling, mediated via αVβ3 integrin and tyrosine kinase signalling. As circulating levels of Lp(a) are resistant to current lipid-lowering strategies, our work identifies the integrin / RhoA signalling pathways as potential therapeutic targets for modulating VSMC function.

P182
Does platelet dysfunction account for thrombolysis-related upper gastrointestinal bleeding (UGB) in STEMI patients? A 4year prospective study in Constanta County, Romania

Abstract

Background

In Constanţa County, Romania, coronary reperfusion in acute STEMI is achieved only by pharmacological therapy. UGB represents a major and potentially fatal haemorrhagic complication of thrombolysis.