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.

Keywords: Heart failure; Cardiomyocyte; Endothelium; Ischaemia/reperfusion injury; Natriuretic peptide; Natriuretic peptide receptor.

Figure 1

Cardiomyocyte-specific ablation of CNP has modest effects on basal cardiac function. 24 hr and mean radiotelemetry evaluation of (A and B) MABP, (C and D) heart rate and (E and F) QA interval in WT and cmCNP^−/− mice. (G) Echocardiographic analyses of left ventricular internal diameter at systole (LVIDs), left ventricular posterior wall diameter at systole (LVPWs), ejection fraction, left ventricle to body weight ratio (LV/BW), intraventricular septum diameter at systole (IVSs) and RR interval in WT and cmCNP^−/− animals. Data are presented as mean ± SD and analysed using two-way ANOVA with Šídák post-hoc test (A, C and E) or Student's t-test (B, D, F and G). Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Figure 2

Cardiomyocyte-specific deletion of CNP worsens the cardiac response to pressure-overload. Ejection fraction (A), left ventricular internal diameter at systole (LVIDs; B), left ventricle to body weight ratio (LV/BW; C), mean arterial blood pressure (MABP; D), fibrotic burden (collagen fraction; E and G; scale bar = 50 μm) and cardiomyocyte size (F and H) in WT and cmCNP^−/− animals exposed to 6 weeks abdominal aortic constriction (AAC). Data are presented as mean ± SD and analysed using one-way ANOVA with Šídák post-hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Figure 3

Cardiomyocyte-specific deletion of CNP worsens the cardiac response to sympathetic hyperactivation. Ejection fraction (A), left ventricular internal diameter at systole (LVIDs; B), left ventricle to body weight ratio (LV/BW; C), heart rate (HR; D), fibrotic burden (collagen fraction; E and G; scale bar = 50 μm) and cardiomyocyte size (F and H) in WT and cmCNP^−/− animals exposed to 7 days isoprenaline (ISO; 20mg/kg/day). Data are presented as mean ± SD and analysed using one-way ANOVA with Šídák post-hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Figure 4

Global deletion of NPR-C worsens the cardiac response to pressure-overload. Ejection fraction (A), left ventricular internal diameter at systole (LVIDs; B), left ventricle to body weight ratio (LV/BW; C), mean arterial blood pressure (MABP; D), fibrotic burden (collagen fraction; E and G; scale bar = 50 μm) and cardiomyocyte size (F and H) in WT and NPR-C^−/− animals exposed to 6 weeks abdominal aortic constriction (AAC). Data are presented as mean ± SD and analysed using one-way ANOVA with Šídák post-hoc test. Each statistical comparison undertaken has an assigned i value (adjusted for multiplicity).

Figure 5

Pharmacological administration of CNP rescues the detrimental cardiac phenotype in response to pressure-overload in wild type, but not NPR-C^−/−, mice. Ejection fraction (A), mean arterial blood pressure (MABP; B), and fibrotic burden (collagen fraction; C and D; scale bar = 50 μm) in WT or NPR-C^−/− animals exposed to 6 weeks abdominal aortic constriction (AAC) in the absence and presence of CNP (0.2 mg/kg/day; s.c. by osmotic minipump, initiated 3 weeks following AAC surgery and maintained throughout the study). Intrinsic hypertrophic response to Angiotensin (Ang) II and the effect of CNP (100nM) on cardiomyocytes isolated from WT and cmCNP^−/− mice (E and F). Data are presented as mean ± SD and analysed using one-way ANOVA with Šídák post-hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Figure 6

Expression and co-localization of CNP and NPR-C are altered in human heart failure. CNP, NPR-B and NPR-C mRNA (and protein) expression in murine pressure overload -induced (6 weeks abdominal aortic constriction, AAC) heart failure (A) and in human non-failing (NF) and failing (HF) hearts (B). NPR-C is highly expressed on cardiomyocytes in both non-failing and failing hearts but co-localizes with cardiac fibroblasts in heart failure patients (C; cardiomyocyte marker troponin T; fibroblast marker vimentin; scale bars, 50x magnification; white triangles highlight NPR-C co-localization in fibroblasts in HF). Data are presented as mean ± SD and analysed using Student's t-test.

Figure 7

Fibroblast-specific deletion of CNP worsens the cardiac response to pressure-overload. Ejection fraction (A), left ventricular internal diameter at systole (LVIDs; B), left ventricle to body weight ratio (LV/BW; C), mean arterial blood pressure (MABP; D), fibrotic burden (collagen fraction; E and G; scale bar = 50 μm) and cardiomyocyte size (F and H) in WT and fbCNP^−/− animals exposed to 6 weeks abdominal aortic constriction (AAC). Data are presented as mean ± SD and analysed using one-way ANOVA with Šídák post-hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

Figure 8

Endothelial CNP regulates coronary vascular reactivity and ischaemia/reperfusion injury via NPR-C. (A and B) Bradykinin (10 nmol), (C and D) acetylcholine (0.1–1 nmol), and (E and F) flow-mediated dilatation (zero flow for 20-80 s followed by reperfusion at 2 mL/min)—dependent decreases in coronary perfusion pressure (CPP) in isolated Langendorff hearts from WT, ecCNP^−/− and NPR-C^−/− mice. (G and H) Infarct size and (I and J) left ventricular developed pressure (LVDP) in isolated Langendorff hearts from WT, cmCNP^−/− and NPR-C^−/− mice subjected to 35 mins global ischaemia (zero flow) followed by 60 mins reperfusion (2 mL/min constant flow). Data are presented as mean ± SD and analysed using two-way ANOVA with Šídák post-hoc test (C, D, E, F, I and J) or Student's t-test (A, B, G and H).

Take home figure

C-type natriuretic peptide (CNP) produced by multiple cell types within the heart acts in concert to reduce cardiac hypertrophy, cardiac fibrosis and improve coronary blood flow.