C-type natriuretic peptide co-ordinates cardiac structure and function

Abstract Aims C-type natriuretic peptide (CNP) is an essential endothelium-derived signalling species that governs vascular homoeostasis; CNP is also expressed in the heart but an intrinsic role for the peptide in cardiac function is not established. Herein, we employ unique transgenic strains with cell-specific deletion of CNP to define a central (patho)physiological capacity of CNP in maintaining heart morphology and contractility. Methods and results Cardiac structure and function were explored in wild type (WT), cardiomyocyte (cmCNP−/−), endothelium (ecCNP−/−), and fibroblast (fbCNP−/−)—specific CNP knockout mice, and global natriuretic peptide receptor (NPR)-B−/−, and NPR-C−/− animals at baseline and in experimental models of myocardial infarction and heart failure (HF). Endothelium-specific deletion of CNP resulted in impaired coronary responsiveness to endothelium-dependent- and flow-mediated-dilatation; changes mirrored in NPR-C−/− mice. Ex vivo, global ischaemia resulted in larger infarcts and diminished functional recovery in cmCNP−/− and NPR-C−/−, but not ecCNP−/−, vs. WT. The cardiac phenotype of cmCNP−/−, fbCNP−/−, and NPR-C−/− (but not ecCNP−/− or NPR-B−/−) mice was more severe in pressure overload- and sympathetic hyperactivation-induced HF compared with WT; these adverse effects were rescued by pharmacological CNP administration in WT, but not NPR-C−/−, mice. At a molecular level, CNP/NPR-C signalling is impaired in human HF but attenuates activation of well-validated pro-hypertrophic and pro-fibrotic pathways. Conclusion C-type natriuretic peptide of cardiomyocyte, endothelial and fibroblast origins co-ordinates and preserves cardiac structure, function, and coronary vasoreactivity via activation of NPR-C. Targeting NPR-C may prove an innovative approach to treating HF and ischaemic cardiovascular disorders.

To confirm the selective deletion of CNP from cardiomyocytes, neonatal cardiomyocytes from cmCNP -/-and WT littermates were isolated and cultured using the Pierce Primary Cardiomyocyte Isolation Kit (Thermo Scientific,USA). Cardiomyocytes in each well were collected with 350µL of buffer RLT (Qiagen RNeasy Mini Kit, Germany) containing 1% β-mercaptoethanol into a 1.5mL tube. The collected cells were snap-frozen immediately with liquid nitrogen and stored at -80°C for RNA extraction. Total RNA was extracted from cardiomyocytes and organs using a RNeasy Fibrous Tissue Mini Kit (Qiagen, Germany) according to manufacturer instructions. 250ng RNA from each cell sample and 1000ng from each tissue sample was converted to cDNA using the QuantiTect® Reverse Transcription Kit (Qiagen) according to manufacturer instructions. The cDNA products were stored at -20°C for qPCR analysis. Quantitative real-time PCR was performed using the Quantitect SYBR green kit (Qiagen). The cardiomyocyte cDNA samples were diluted 1:2 and the samples from organ tissue were diluted 1:20 resulting in cDNA concentrations of 6.25ng/μL and 50ng/μL, respectively. 2µL of the cDNA sample was then added to 8µL of the qPCR master mix.
Results were analyzed using the ABI Prism 7900HT software package SDS 2.4 (Advanced Biotechnologies Ltd). CNP mRNA levels were normalized to β-actin and RPL-19 (internal controls) for each sample. The relative quantification of CNP expression between cmCNP -/and WT were determined by the 2 -ΔΔCT method, where ΔΔCT is the fold change relative to WT levels.
Expression of CNP mRNA was significantly reduced (~60%) in cardiomyocytes isolated from cmCNP -/-mice compared to WT littermates (Supplementary Figure 1). Further analysis indicated that the isolated cardiomyocyte population was approximately 80% pure (ratio of Troponin T-positive to DAPI-positive cells), resulting in ~80% efficiency in CNP deletion. In addition, CNP mRNA expression was significantly reduced in whole hearts from cmCNP -/-mice, but equivalent in all other tissues/organs examined (Supplementary Figure   1). Deletion of the LoxP sites was confirmed by PCR in which an excised DNA band was only observed in cmCNP -/-hearts, and not in WT or cmCNP -/-liver or lung (Supplementary Figure   1). Deletion of CNP from (cardiac) fibroblasts was confirmed by isolating and culturing these cells from hearts of WT and fbCNP -/-animals and determining CNP mRNA expression levels (Supplementary Figure 1). These studies revealed an approximate 75% deletion of CNP from cardiac fibroblasts, but without a significant reduction in expression in whole organs 3 (Supplementary Figure 1). In concert, these data confirm efficient, exclusive removal of the Nppc gene from the cardiomyocyte or (cardiac) fibroblast.

Ex-vivo functional assessment of coronary vascular reactivity
Coronary reactivity was evaluated in murine hearts set-up in Langendorff mode.
Animals were injected with heparin (100 µL; i.p.) prior to anesthesia with isoflurane (3 % in O2). Following opening of the chest cavity, the heart was quickly excised and placed into ice- nitro-L-arginine methylester (L-NAME; 300µM). Hearts that did not show a >50mmHg increase in CPP in the presence of L-NAME (indicative of a functional endothelium) were discarded. Acute changes in CPP were recorded in response to bolus injections of the endothelium-dependent vasodilators bradykinin (BK; 10 nmol) and acetylcholine (ACh; 0.1-1 nmol), CNP (10 nmol) and the NO-donor sodium nitroprusside (SNP; 1 nmol). 10 μL of each drug was administered with a Hamilton syringe into the aortic cannula and each injection was separated by at least 5 min in order for the CPP to return to baseline before adding the next dose or drug. Hearts were also exposed to three periods of zero flow (20, 40 and 80 seconds) and the resulting response to restoration of flow (flow-mediated dilatation; shear stressinduced release of endothelium-borne vasodilators 5 ) was assessed using area under the curve (AUC). Occlusion periods were at least 5mins apart, allowing the CPP to return to baseline. The extent of vasodilatation was quantified by percentage change in CPP.
Additionally, following administration of 1 nmol ACh, 10ml (5 min of perfusion) of coronary effluent was collected from the apex of the hearts and immediately frozen in liquid nitrogen for CNP bioassay. 4

Ischemia-reperfusion injury
In some studies, cardiac function was investigated following ischemia-reperfusion (I/R) injury. This was induced by global cessation of flow for 35 min then reperfusion for 60 min.
After reperfusion, the hearts were frozen at -20°C for 10 min and then immediately sliced with a scalpel into 1mm thick sections perpendicular to the long-axis of the heart. The slices were subsequently incubated with 1% triphenyl tetrazolium chloride (TTC) made in PBS at 37°C for 15 min. The heart sections were photographed with a scanner on both sides and the infarcted area (white) was measured in Image J and expressed as a percentage of total ventricular area. arteries. This produces a 30% constriction of the luminal diameter 6 . For sham operations, the 4-0 surgical thread was passed under the aorta and removed without tying it against the needle. In some studies, WT mice undergoing AAC were administered (a) CNP (0.2mg/kg/day; s.c. by osmotic minipump, initiated 3 weeks following AAC surgery and maintained throughout the study) and/or (b) the NOS inhibitor L-N G -nitroarginine methylester (L-NAME; 100 mg/kg/day via the drinking water, initiated 3 days prior to AAC surgery and maintained throughout the study). Plasma natriuretic peptide concentrations were 5 determined by specific enzyme immunoassay (Phoenix Pharmaceuticals Inc., Karlsruhe, Germany) as previously described 1 .
In vivo cardiac functional assessments

Histology, staining and imaging
For murine studies, the isolated left ventricles were cut transversely below the mitral valves, fixed in 10% formalin for 24h, then stored in 70% ethanol before embedding in paraffin wax and sectioning.

Wheat germ agglutinin fluorescent staining
In order to measure the ventricular myocyte size, the tissue slides were stained with a fluorescent cell membrane antibody, wheat germ agglutinin alexafluor 647 (1:500; Molecular Probes, Invitrogen, UK) and mounted with Prolong gold DAPI mountant as per standard immunohistochemistry protocols. Images were taken on a Zeiss 710 confocal microscope and the cardiomyocyte size analyzed with Image J (National Institutes of Health). 6 Cardiomyocyte size was estimated with investigator blinded to treatment type and as an average of >1000 cells per heart.

Picrosirius red staining
Tissue slides were dewaxed, rehydrated and stained using a Picrosirius Red Stain kit following the manufacturer's instructions (Polysciences, Inc. Warrington, PA, USA. A Nikon Eclipse TS100 microscope (Nikon UK Limited, Surrey, UK) was used to capture images of the stained slides. Images were analyzed by threshold analysis using Image J (National Institutes of Health) with investigator blinded to treatment type.

Immunofluorescence analysis
Immunofluorescence was used to localize NPR-C (anti-NPR-C, ab37617, 1:100; This was then incubated in a 37°C incubator for 7 days to allow fibroblasts to grow out from the tissue. Once the cells approached confluency, the old media was removed and tissues were washed with sterile PBS twice. 3 mL trypsin was added into the flask for 5 min to detach the cells. An equal amount of FBS was then added and centrifuged at 1000 rpm for 5 min.  WT, ecCNP -/-, cmCNP -/-and NPR-C -/-animals. Data are presented as mean ± SD and analysed using Student' t-test (A-E; unadjusted for multiplicity) or two-way ANOVA with Šídák post-hoc test (F; adjusted for multiplicity; n=12). Each statistical comparison undertaken has an assigned P value.