Selective dysregulation of nitric oxide synthase type 3 in cardiac myocytes but not coronary microvascular endothelial cells of spontaneously hypertensive rat

Abstract

Objective: Recent studies indicate that endothelial type nitric oxide synthase (NOS3) modulates cardiac systolic and diastolic function and the inotropic responsiveness to β-adrenergic agonists, and may affect myocardial oxygen consumption. Although NOS3 is a constitutive protein, its levels of expression can be modified by various physiological and pathophysiological stimuli. We investigated whether the cell-specific expression of NOS3 mRNA and protein are altered in cardiac hypertrophy. Methods: Left ventricular cardiac myocytes and coronary microvascular endothelial cells were freshly isolated from 12 week old male spontaneously hypertensive rat (SHR) and matched normotensive Wistar rat hearts. NOS3 protein levels were assessed by Western analysis, and mRNA levels by RT-PCR and Southern blotting. Results: Left ventricular/body weight ratios were significantly increased in SHR compared to Wistar controls, indicating significant hypertrophy. The levels of NOS3 protein were markedly decreased in SHR compared to Wistar cardiac myocytes (by ∼85%). By contrast, the expression of NOS3 mRNA normalized for GAPDH was increased ∼3 fold in SHR cardiac myocytes relative to Wistar controls. In freshly isolated microvascular endothelial cells, however, levels of NOS3 protein and NOS3 mRNA were similar between the two groups. Conclusions: The expression of NOS3 is selectively altered in cardiac myocytes but not coronary microvascular endothelial cells of young SHR hearts, with a marked decrease in NOS3 protein but an increase in NOS3 mRNA. This dysregulation of NOS3 could contribute to contractile dysfunction in left ventricular hypertrophy.

Experimental, Heart, Molecular biology, Hypertrophy, Myocytes, Endothelial factors, Hypertension, Nitric oxide, Rat

Time for primary review 21 days.

1 Introduction

Nitric oxide (NO) has a key role in many physiological processes including the regulation of cardiovascular function through effects on blood pressure, flow, vascular proliferation, platelet function, and the peripheral and central nervous systems (for recent reviews, see Refs. [1, 2]). Three main isoforms of NO synthase (NOS) have been described. The constitutive isoforms NOS1 and NOS3, originally found in neurones and endothelial cells respectively, are also expressed in several other tissues, e.g., NOS3 in cardiac myocytes [3]. The inducible isoform NOS2 is usually expressed only in response to certain stimuli, e.g., cytokines. Although NOS3 is “constitutively” expressed, significant changes in gene expression have been reported in many tissues in response to various physiological and pathophysiological stimuli. An increase in NOS3 expression in endothelial cells in vitro occurs in response to chronic fluid shear stress [4], exposure to transforming growth factor β (TGF β) [5], or during cell proliferation [6], while chronic exercise increases NOS3 mRNA in endothelial cells in vivo [7]. NOS3 is also upregulated in vascular and non-vascular tissues during pregnancy [8]. On the other hand, endothelial NOS3 expression is decreased by tumour necrosis factor-α (which induces NOS2 in many tissues) [9], oxidized low-density lipoprotein [10], and hypoxia in some studies [11], while elevation of cAMP is reported to reduce the expression of NOS3 in rat cardiac myocytes both in vitro and in vivo [12].

Recent studies in isolated preparations as well as human subjects indicate a physiological role for NO in the regulation of cardiac contractile function (for recent reviews, see Refs. [13, 14]). A number of different effects of NO on contractile function have been reported, including (i) an enhancement of myocardial relaxation and diastolic function [13, 15–18], (ii) modulation of β-adrenergic inotropic responses [3, 19, 20], (iii) interaction with the Frank-Starling response [21], and (iv) modulation of the force-frequency relationship [22, 23]. NO may also have anti-hypertrophic activity [24], and has been shown to reduce oxygen consumption [25]. The main physiological source of NO in the heart with respect to contractile function is probably NOS3 in coronary microvascular endothelial cells (CMEC) and in cardiac myocytes, although NOS3 in endocardial endothelial cells and NOS1 in neurones could also play a role [13, 14]. There is very little published data on the molecular regulation of NOS3 in the heart in pathological states. The focus of the present study was hypertensive cardiac hypertrophy. Previous studies have suggested abnormalities of coronary endothelial function in hypertensive hypertrophy. NO-dependent regulation of coronary vascular resistance is abnormal in spontaneously hypertensive rats (SHR) [26]. In human subjects with hypertension and LV hypertrophy, the coronary flow response to acetylcholine is impaired, probably reflecting reduced release or activity of NO [27]. On the other hand, a recent study reported that Ca²⁺-dependent NOS biochemical activity was increased in cardiac tissue of SHR [28]. In the present study, we sought to examine whether the cell-specific expression of NOS3 was altered in the hypertrophied heart of SHR. We chose to study young (12 week old) SHR, that is, at a stage when there is significant LV hypertrophy [29, 30]and evidence of contractile (especially diastolic) dysfunction [31, 32], but as yet no marked degree of extracellular fibrosis or features of decompensation [29].

2 Methods

The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985). Male SHR and age- and sex-matched normotensive Wistar rats were obtained from Charles River Breeding Laboratories (UK), and housed under identical conditions. All animals were studied at 12 weeks age. They were sacrificed by intraperitoneal overdose of sodium pentobarbitone and hearts rapidly excised into oxygenated physiological saline solution. A total of 29 SHR and 28 Wistar rats were used in these studies.

2.1 Isolation of cardiac myocytes

LV myocytes were isolated by collagenase digestion of hearts, as described previously [33]. Briefly, hearts were mounted on a Langendorff apparatus and perfused at 9 ml/min and 37°C with a series of solutions based on an “isolation solution” of the following composition (in mM): NaCl 130, KCl 5.4, MgCl₂ 1.4, NaH₂PO₄ 0.4, HEPES 5, glucose 10, taurine 20, creatine 10, pH 7.3. The first solution, containing CaCl₂ 750 μM, was perfused for 4 min. Next, the heart was perfused for 4 min with Ca²⁺-free isolation solution containing Na₂EGTA 100 μM. Finally, perfusion was switched to isolation solution containing CaCl₂ 200 μM, collagenase (Worthington type 2; 0.1 mg/ml) and protease (Sigma type 14; 0.01 mg/ml) for 9–12 min. The LV was dissected and finely chopped in an enzyme-containing solution with bovine serum albumin (BSA) 1%, and gently agitated in a water bath at 37°C. Aliquots of the cell suspension were examined every 5 min until a >80% yield of rod-shaped cells with a clear striation pattern was obtained. Myocytes were collected by filtration through nylon gauze and gentle centrifugation, followed by passage through two 5% BSA density gradients, and re-suspension in isolation solution containing CaCl₂ 500 μM. The final purity of myocyte preparations was >95% rod-shaped cells with a clear striation pattern.

2.2 Isolation of coronary microvascular endothelial cells

CMEC were isolated as described by Piper et al. [34]. Cells were separately pooled from 4–6 SHR hearts or Wistar hearts for each isolation. Briefly, hearts mounted on a Langendorff apparatus were perfused at 37°C with a solution of the following composition (in mM): NaCl 118, KCl 4.7, NaH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, glucose 11, pH 7.4 (gassed with 95% O₂/5% CO₂) – Buffer 1. Epicardial mesothelial cells were devitalised with 70% (v/v) ethanol. After flushing out blood from the coronary circulation, perfusion was changed to Buffer 1 with added CaCl₂ 0.25 μM and collagenase 0.04% (Sigma type II), which was recirculated for 30 min. Ventricles – excluding any visible large vessels – were then chopped into 15 ml of recirculating solution containing BSA (200 mg, Sigma fraction V), and triturated gently during a 10 min incubation period at 37°C. The suspension was filtered through nylon gauze and centrifuged (150 g, 3 min) to sediment myocytes. The supernatant, including added BSA (100 mg), trypsin 0.01%, and CaCl₂ 50 μM, was incubated at 37°C for 15 min with stirring. The CMEC pellet was obtained by centrifugation (1000 g, 10 min), washed twice in Buffer 1 with CaCl₂ 250 μM and 500 μM respectively, and resuspended in 40 ml pre-warmed Medium 199 (Gibco) with added 10% newborn calf serum, 10% fetal calf serum, benzylpenicillin 250 U/ml, streptomycin 250 μg/ml, amphotericin B 12.5 μg/ml, and gentamycin 50 μg/ml. Cell suspensions were plated in 75 cm² tissue culture flasks and incubated at 37°C. After 1 h, unattached cells and debris were washed off with 0.9% saline. Cells were either used for RNA isolation at this stage, or following a period of culture for 7–14 days. Cultured cells formed confluent monolayers with a “cobblestone” morphology within about 5–7 days. For more prolonged culture, cells were trypsinised and subcultured in fresh flasks.

Cells were characterised as endothelial by the uptake of fluorescently labelled acetylated LDL [34]. The endothelial cell population obtained with this method may include some endocardial endothelial cells and some endothelial cells from larger coronary vessels, but the statistical average represents predominantly CMEC since the vast majority of all cardiac endothelial cells are found in small vessels and the capillary bed [34]. In keeping with this, cells stained positively with the microvascular-specific lectin Lycopersicon esculentum lectin [35], negatively for α-smooth muscle actin, and rapidly formed capillary-like tubes on the basement membrane preparation Matrigel [36]. No cardiac myocytes (easily identifiable by their size and characteristic morphology) were observed in endothelial cell isolates.

2.3 Western blotting

Cells were washed twice in ice-cold PBS prior to lysis in 1 ml of boiling lysis solution containing 1% SDS and 10 mM Tris pH 7.4. After scraping the cells into this solution and further boiling for 5 min, insoluble material was removed by centrifugation. Protein concentration was measured in an aliquot using a micro BCA kit (Pierce). Equal amounts of protein were run on 8% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membrane. Equal transfer among lanes was confirmed by reversible staining with Ponceau S (Sigma). The membrane was incubated with a mouse monoclonal antibody raised against a polypeptide (amino acid residues 1030–1209) of the human NOS3 protein (Transduction Labs.). The secondary antibody was a sheep horseradish peroxidase-linked anti-mouse antibody (Amersham). Blots were assessed by densitometry of bands, with subtraction of the background counts measured outside loaded lanes. The values for SHR samples were normalized by the mean value obtained with control Wistar rat samples.

2.4 RNA isolation, semi-quantitative RT-PCR and Southern blotting

Total RNA was isolated from cardiac myocytes or CMEC by extraction with guanidinium isothiocyanate [37]. In view of the relatively low level of NOS3 expression in cardiac myocytes [3]and the requirement to pool freshly isolated CMEC from at least 4–6 hearts per experiment, RT-PCR and Southern blotting were used in these studies. First strand cDNA synthesis was performed using 5 μg total RNA, quantified spectrophotometrically, in the presence of random hexamers (500 ng, Promega), dNTPs (10 mM each, Boehringer), Tris–HCl 10 mM (pH 8.4), KCl 50 mM, MgCl₂ 2.5 mM, and RNase inhibitor 33 U (Promega). The reaction mixture was preincubated at 70°C for 3 min followed by cooling on ice prior to addition of Moloney murine leukemia virus reverse transcriptase (200 U, Gibco-BRL). The RT reaction was performed at 42°C for 90 min. Primers for amplification of a 324-bp rat NOS3 PCR product were based on published sequences [3]: 5′-GGGCCAGGGTGATGAGCTCTG-3′ and 5′-CCCTCCTGGCTTCCAGTGTCC-3′ (sense and antisense respectively). A 996-bp rat GAPDH cDNA PCR product, as a reference cellular transcript, was amplified using the following primers: 5′-GTGAAGGTCGGTGTCAAC-3′ and 5′-CTCCTTGGAGGCCATGT-3′ (sense and antisense respectively) [38]. PCR reactions were carried out in 100 μl final volumes containing RT reaction (2 μl), sense and antisense primers (48 ng each), dNTPs (200 μmol), Mg²⁺ 1.5 mM, and Taq Polymerase (2.5 U, Promega). Amplification cycles (n=35 for NOS3; n=25 for GAPDH) comprised 1 min at 94°C, 1 min at 65°C, and 2 min at 72°C, followed by a 10 min extension reaction at 72°C. The same preparation of RNA was reverse transcribed and amplified two to three times to confirm reproducibility of the analysis.

PCR products were separated on 1.2% agarose gels (Bio-Rad) and transferred to Hybond^N filters (Amersham) by Southern blotting [37]. Filters were pre-hybridized at 65°C in 6×SSC/1% SDS and 0.5 mg/ml Heparin for 2-3 h and then hybridized overnight with dCTP-random-prime-labelled NOS3 or GAPDH cDNA probes (10 mCi []dCTP ml⁻¹ 3000 Ci mmol⁻¹; Amersham). These probes consisted of the PCR fragments described above, amplified from rat heart tissue and sequenced. Filters were washed to a final stringency of 0.1×SSC/0.1% SDS at 65°C, and exposed to KODAK X-100 XAR film with intensifying screens at −70°C. Quantification of autoradiograms was performed by densitometry using an Image Quant Densitomer (Molecular Dynamics), and values for NOS3 were normalized with respect to GAPDH [39, 40].

2.5 Statistics

Data are presented as mean±SEM. Comparisons were performed by a two-tailed Student's unpaired t-test, and P<0.05 considered statistically significant.

3 Results

The LV/body weight ratios were significantly greater in 12 week old SHR compared to normotensive Wistar rats (3.84±0.20 mg/g compared to 2.82±0.06 mg/g; n=9 each; P<0.002), indicating significant LV hypertrophy and consistent with other published data in this model (e.g., Ref. [30]).

Fig. 1 shows the results of Western analysis for NOS3 protein, using a NOS3-specific monoclonal antibody, in SHR and Wistar cardiac myocytes. A 140 kDa protein band compatible with NOS3 [1]was identified in the whole cell lysate of both groups. The levels of NOS3 protein were significantly lower in SHR relative to Wistar cardiac myocytes (SHR 14.5±0.11% of the level in Wistar myocytes; n=6 SHR and 6 Wistar hearts; P<0.001). Fig. 2 indicates that the levels of NOS3 protein in freshly isolated CMEC were similar in SHR and Wistar rats, in contrast to the downregulation of NOS3 in SHR cardiac myocytes. The mean level of NOS3 protein in SHR CMEC was 100±1.4% of the level in Wistar CMEC (n=8 SHR and 8 Wistar hearts each; P=NS).

Fig. 2

Western blot showing NOS3 protein levels in CMEC freshly isolated from 4 SHR hearts (lane 1) and 4 Wistar rat hearts (lane 2). Similar results were obtained in 2 experiments.

Fig. 1

Western blot showing NOS3 protein levels in cardiac myocytes isolated from individual SHR (lanes 1–3) and Wistar rats (lanes 4–6). Equal amounts of protein (100 μg) were used for each lane. Similar results were obtained in 2 experiments.

In order to investigate the underlying cause of this downregulation of myocyte NOS3 protein, we studied the expression of NOS3 mRNA. Using species-specific oligonucleotide primers [3], a 324-bp NOS3 product was amplified by RT-PCR from both cardiac myocytes (1 Wistar heart) and freshly isolated CMEC (4 Wistar hearts) (Fig. 3). No PCR products were detected in parallel-processed RNA samples subjected to PCR without prior RT, indicating an absence of contaminating genomic DNA. The authenticity of the NOS3 PCR product was confirmed by (a) partial restriction digestion using ECOR V, which resulted in expected size fragments of 191-bp and 133-bp as well as uncut 324-bp product (Fig. 3b), and (b) automated sequencing on an ABI Prism 377 DNA sequencer (Perkin Elmer), following ligation into a TA cloning vector (Invitrogen) and transformation into E. coli INVαF′ competent cells. Interestingly, we observed that the level of expression of NOS3 mRNA was significantly reduced in cultured compared to freshly isolated CMEC (Fig. 3a), so that experiments were restricted to freshly isolated CMEC.

Fig. 3

(a) Southern blots of PCR products showing NOS3 transcripts amplified from freshly isolated cardiac myocytes (lane 1), freshly isolated CMEC (lane 2), and CMEC cultured for 7 days (lane 3) and 14 days (lane 4) respectively, all from Wistar rat hearts. (b) Ethidium bromide-stained gel showing 324-bp NOS3 PCR product amplified from cardiac myocyte cDNA (lane 1), and ECOR V restriction digest resulting in the production of expected 191-bp and 133-bp fragments (lane 2). Hae III restricted φX174 DNA was used as a size marker.

The expression of NOS3 mRNA, normalised for GAPDH mRNA, was significantly increased in cardiac myocytes isolated from SHR compared to Wistar rats by RT-PCR–Southern blotting (Fig. 4). The level of expression of GAPDH was unaltered. This difference was also confirmed by examining PCR products after varying degrees of amplification. As shown in Fig. 5, a NOS3 transcript was easily apparent in SHR myocytes after 30 cycles of amplification, whereas in Wistar myocytes this was only the case after 35 cycles of amplification.

Fig. 5

Gel showing NOS3 PCR products amplified from Wistar cardiac myocyte (lanes 1, 3, 5, 7) and SHR cardiac myocyte cDNA (lanes 2, 4, 6, 8). The number of PCR cycles was 20 for lanes 1 and 2, 25 for lanes 3 and 4, 30 for lanes 5 and 6, and 35 for lanes 7 and 8.

Fig. 4

(a) Representative Southern blots of NOS3 transcripts (top) and GAPDH transcripts (bottom) amplified from normotensive Wistar rat myocytes (lane 1) and SHR myocytes (lanes 2–4). (b) Histogram showing changes in NOS3 mRNA in SHR myocytes (n=9 hearts) relative to control myocytes (n=3 hearts), after normalization of Southern blot autoradiograms by the levels of GAPDH amplification products. ***P<0.0001

In contrast to the findings in cardiac myocytes, the level of expression of NOS3 mRNA, normalized for GAPDH, was similar in freshly isolated CMEC from SHR and Wistar rat hearts (SHR 1.25±0.085 fold that in Wistar CMEC; n=6 hearts each; P=NS).

4 Discussion

The “constitutive” endothelial-type isoform of NO synthase, NOS3, is expressed in a number of cell types in the mammalian heart, notably the endothelial cells of the coronary vasculature and cardiac myocytes. Recent studies from a number of laboratories including our own indicate that NO can modulate cardiac contractile function. It has become clear that both CMEC-derived NO and myocyte-derived NO, acting in a paracrine and autocrine/intracrine fashion respectively, may be involved in the physiological regulation of contractile and other functions [13, 14]. It seems likely that NO derived from these different cellular sources may have differing effects on cardiac myocytes in situ because of spatial factors (e.g., diffusion distance), varying levels of expression (greater in CMEC than in cardiac myocytes), and potential differences in the stimuli that evoke release of NO. Possible changes in the expression of NOS3 in the heart in pathological states and the consequences of such alterations, particularly for contractile function, have not so far been characterised. In the present study, we show that the cardiac expression of NOS3 protein is significantly reduced in a cell-specific manner (i.e., in cardiac myocytes but not in CMEC) in a genetic model of hypertensive left ventricular hypertrophy, namely the SHR. At the stage that the SHR animals were studied (i.e., 12-week-old), they are known to have significant hypertension and compensated LV hypertrophy but as yet no evidence of marked extracellular fibrosis nor of decompensation into heart failure [29, 30]. Contractile dysfunction at this stage is reported to be characterised by a significant rightward shift of the Frank-Starling curve, particularly at lower filling pressures, compatible with diastolic dysfunction and increased myocardial diastolic stiffness [31, 32, 41]. The fact that the changes in NOS3 protein and mRNA in SHR compared to Wistar control rats were selective for cardiac myocytes, whereas levels of NOS3 in CMEC were similar between the two groups, suggests that this difference is indeed related to the myocardial abnormalities rather than being simply due to other possible differences between these animal strains. This contention is also supported by previous studies which have demonstrated specific cardiac abnormalities in SHR compared to normotensive Wistar rats and related these to the hypertrophy of SHR hearts [42–44].

Decreased expression of NOS3 protein in cardiac myocytes could have a number of functional effects. Although hypertensive LV hypertrophy is initially an adaptive response that normalises wall stress and preserves baseline contractile function, it leads to contractile dysfunction (e.g., abnormal myocardial relaxation) and other abnormalities such as energy imbalance. A reduction of NOS3 protein in cardiac myocytes in the hypertrophied heart could (a) contribute to the abnormalities of myocardial relaxation and diastolic function [15–18, 21], (b) alter responsiveness to β-adrenergic stimulation [3, 19, 20], and (c) increase myocardial oxygen consumption, thereby worsening oxygen supply-demand balance [25]. It is interesting that the main contractile abnormalities in young SHR are a significant rightward shift of the Frank-Starling curve and an increase in myocardial diastolic stiffness [31, 32, 41], given that NOS3 is known to enhance myocardial relaxation and diastolic function [13, 15–18]and to augment the Frank-Starling response [21]. Although no change in NOS3 was observed in CMEC, the amount of NO from this source reaching cardiac myocytes may also conceivably be reduced in the hypertrophied heart as a consequence of the increased diffusion distances between EC and cardiac myocytes.

Unexpectedly, the reduction in NOS3 protein in SHR cardiac myocytes was not accompanied by a reduction in NOS3 mRNA, but rather an increase. This finding clearly indicates that the reduction in NOS3 protein levels does not result from reduced transcription. Possible explanations for the reduction in NOS3 protein despite abundant message include alterations in translational efficiency, a deficiency in essential co-factors for protein synthesis and stability (e.g., haem, FAD, FMN, calmodulin), abnormal post-translational processing, and/or other changes in NOS3 protein stability. In this regard, Belhassen and coworkers [45]have recently reported that the intracellular post-translational processing (in particular, palmitoylation) of NOS3 in rat cardiac myocytes, and its subsequent targeting to sarcolemmal caveolae, are both influenced by changes in intracellular cAMP – effects which may be applicable to the hypertrophied heart. There are several other examples in the literature of a similar discrepancy between mRNA and protein expression; for instance, with respect to expression of cytochrome b558 in Epstein–Barr virus-immortalized B-lymphocytes [46].

The basis for the increase in NOS3 mRNA in SHR myocytes was not defined in this study. Several recent studies indicate that the expression of NOS3, a “constitutive” protein, is subject to significant transcriptional regulation by various physiological and pathophysiological stimuli. The promoter of the NOS3 gene contains shear stress response elements as well as a number of putative binding domains that may be regulated by various transcription factors. In particular, an AP-1 and a nuclear factor-1 (NF-1)-like binding site have been identified. It has been suggested that protein kinase C-mediated signalling pathways may regulate NOS3 expression through the AP-1 site, whereas cAMP and TGF β may act through the NF-1 binding site [47]. In this regard, it may be relevant that protein kinase C-mediated signalling pathways are implicated in the development of cardiac hypertrophy [48].

We observed no significant differences in the level of NOS3 mRNA and protein in SHR CMEC relative to Wistar controls in this study. At first sight, this might suggest that alterations in NOS3 do not contribute to coronary vascular abnormalities in SHR, and would seem to be in conflict with previous studies that have reported reduced endothelium-dependent coronary vascular relaxation in hypertension and LV hypertrophy [26, 27, 49]. However, the biological actions of NO may be reduced even with normal levels of protein if (a) there is increased inactivation of NO, e.g., by reactive oxygen species, and/or (b) NOS3 protein activity is impaired, e.g., because of a deficiency of substrate (l-arginine) or essential co-factors such as tetrahydrobiopterin. Thus, the findings of the present study with respect to NOS3 in CMEC are not inconsistent with reports of reduced endothelium-dependent coronary vascular relaxation in hypertension and LV hypertrophy. On the other hand, Nava et al. [28]recently reported an increased biochemical activity of Ca²⁺-dependent NOS in cardiac endothelium of SHR. With respect to biochemical assays of NOS activity, it is important to remember that these are performed in the presence of excess substrate and co-factors [28]. Thus, these in vitro assays may not necessarily reflect the true level of NOS3 activity in vivo, especially if there is a deficiency of substrate and/or co-factors or an increased inactivation of NO.

The NO pathway appears to be involved in many aspects of physiological blood pressure control and possibly in hypertension [1, 2]. Transgenic mice that lack the NOS3 gene develop significant systemic hypertension [50]. Abnormalities of NO-dependent regulation of vascular tone have been described both in peripheral vessels and the coronary circulation in hypertension and LV hypertrophy [26, 27, 49, 51]. In the present study, we have documented a selective dysregulation of NOS3 in the cardiac myocytes (but not CMEC) of SHR, which may be relevant to the pathophysiology of myocardial dysfunction associated with LV hypertrophy. It should be noted that the effects of NO on contractile (and other) function will also be influenced by other factors such as substrate and co-factor availability, redox state, the presence or absence of reactive oxygen species, and the activities of downstream signalling pathways such as the cGMP system. Furthermore, the findings of this study cannot necessarily be extrapolated to other types of LV hypertrophy. Nevertheless, this study suggests that dysregulation of cardiac myocyte NOS3 may be relevant in the pathophysiology of hypertensive LV hypertrophy and further investigation of the functional effects of NO on myocardium in this condition are indicated.

Acknowledgements

This work was supported by the UK Medical Research Council and the British Heart Foundation. We thank Angela Walters for isolation of CMEC, and Dr. D. Lang for technical advice relating to this procedure.