Ablation of C-type natriuretic peptide/cGMP signaling in fibroblasts exacerbates adverse cardiac remodeling in mice.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

C-type natriuretic peptide moderates titin-based cardiomyocyte stiffness.

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase I (PKGI) increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signaling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte-restricted GC-B deletion (CM GC-B-KO mice). The impact on heart morphology and function, myocyte passive tension, and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC). Pressure overload increased left ventricular endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B-KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signaling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure overload.

Heart-Microcirculation Connection: Effects of ANP (Atrial Natriuretic Peptide) on Pericytes Participate in the Acute and Chronic Regulation of Arterial Blood Pressure.

Cardiac ANP (atrial natriuretic peptide) moderates arterial blood pressure. The mechanisms mediating its hypotensive effects are complex and involve inhibition of the renin-angiotensin-aldosterone system, increased natriuresis, endothelial permeability, and vasodilatation. The contribution of the direct vasodilating effects of ANP to blood pressure homeostasis is controversial because variable levels of the ANP receptor, GC-A (guanylyl cyclase-A), are expressed among vascular beds. Here, we show that ANP stimulates GC-A/cyclic GMP signaling in cultured microvascular pericytes and thereby the phosphorylation of the regulatory subunit of myosin phosphatase 1 by cGMP-dependent protein kinase I. Moreover, ANP prevents the calcium and contractile responses of pericytes to endothelin-1 as well as microvascular constrictions. In mice with conditional inactivation (knock-out) of GC-A in microcirculatory pericytes, such vasodilating effects of ANP on precapillary arterioles and capillaries were fully abolished. Concordantly, these mice have increased blood pressure despite preserved renal excretory function. Furthermore, acute intravascular volume expansion, which caused release of cardiac ANP, did not affect blood pressure of control mice but provoked hypertensive reactions in pericyte GC-A knock-out littermates. We conclude that GC-A/cGMP-dependent modulation of pericytes and microcirculatory tone contributes to the acute and chronic moderation of arterial blood pressure by ANP. Graphic Abstract A graphic abstract is available for this article.

Natriuretic Peptides Attenuate Retinal Pathological Neovascularization Via Cyclic Guanosine Monophosphate Signaling in Pericytes and Astrocytes.

OBJECTIVE: In proliferative retinopathies, complications derived from neovascularization cause blindness. During early disease, pericyte's apoptosis contributes to endothelial dysfunction and leakage. Hypoxia then drives VEGF (vascular endothelial growth factor) secretion and pathological neoangiogenesis. Cardiac ANP (atrial natriuretic peptide) contributes to systemic microcirculatory homeostasis. ANP is also formed in the retina, with unclear functions. Here, we characterized whether endogenously formed ANP regulates retinal (neo)angiogenesis. Approach and Results: Retinal vascular development and ischemia-driven neovascularization were studied in mice with global deletion of GC-A (guanylyl cyclase-A), the cGMP (cyclic guanosine monophosphate)-forming ANP receptor. Mice with a floxed GC-A gene were interbred with Tie2-Cre, GFAP-Cre, or PDGF-Rß-CreERT2 lines to dissect the endothelial, astrocyte versus pericyte-mediated actions of ANP in vivo. In neonates with global GC-A deletion (KO), vascular development was mildly delayed. Moreover, such KO mice showed augmented vascular regression and exacerbated ischemia-driven neovascularization in the model of oxygen-induced retinopathy. Notably, absence of GC-A in endothelial cells did not impact retinal vascular development or pathological neovascularization. In vitro ANP/GC-A/cGMP signaling, via activation of cGMP-dependent protein kinase I, inhibited hypoxia-driven astrocyte's VEGF secretion and TGF-ß (transforming growth factor beta)-induced pericyte apoptosis. In neonates lacking ANP/GC-A signaling in astrocytes, vascular development and hyperoxia-driven vascular regression were unaltered; ischemia-induced neovascularization was modestly increased. Remarkably, inactivation of GC-A in pericytes retarded physiological retinal vascularization and markedly enhanced cell apoptosis, vascular regression, and subsequent neovascularization in oxygen-induced retinopathy. CONCLUSIONS: Protective pericyte effects of the ANP/GC-A/cGMP pathway counterregulate the initiation and progression of experimental proliferative retinopathy. Our observations indicate augmentation of endogenous pericyte ANP signaling as target for treatment of retinopathies associated with neovascularization.