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We previously reported the secretion of C-type natriuretic peptide (CNP) from vascular endothelial cells and proposed the existence of a vascular natriuretic peptide system composed of endothelial CNP and smooth muscle guanylyl cyclase-B (GC-B), the CNP receptor, and involved in the regulation of vascular tone, remodeling, and regeneration. In this study, we assessed the functional significance of this system in the regulation of blood pressure in vivo using vascular endothelial cell–specific CNP knockout and vascular smooth muscle cell–specific GC-B knockout mice. These mice showed neither the skeletal abnormality nor the early mortality observed in systemic CNP or GC-B knockout mice. Endothelial cell–specific CNP knockout mice exhibited significantly increased blood pressures and an enhanced acute hypertensive response to nitric oxide synthetase inhibition. Acetylcholine-induced, endothelium-dependent vasorelaxation was impaired in rings of mesenteric artery isolated from endothelial cell–specific CNP knockout mice. In addition, endothelin-1 gene expression was enhanced in pulmonary vascular endothelial cells from endothelial cell–specific CNP knockout mice, which also showed significantly higher plasma endothelin-1 concentrations and a greater reduction in blood pressure in response to an endothelin receptor antagonist than their control littermates. By contrast, vascular smooth muscle cell–specific GC-B knockout mice exhibited blood pressures similar to control mice, and acetylcholine-induced vasorelaxation was preserved in their isolated mesenteric arteries. Nonetheless, CNP-induced acute vasorelaxation was nearly completely abolished in mesenteric arteries from vascular smooth muscle cell–specific GC-B knockout mice. These results demonstrate that endothelium-derived CNP contributes to the chronic regulation of vascular tone and systemic blood pressure by maintaining endothelial function independently of vascular smooth muscle GC-B.

Background: It has been reported that biological inactive pro-brain natriuretic peptide(proBNP), precursor peptide of bioactive BNP, is increased in patients with heart failure, as well as mature BNP-32. However, underlying mechanisms of increased plasma proBNP levels in heart failure remain unclear. Methods: We used the conventional BNP assay system, which detects both proBNP and BNP-32, and our recently developed specific human proBNP assay system. We assessed cardiac production of proBNP and BNP(=proBNP+BNP-32) in patients with heart failure. To elucidate the molecular mechanisms underlying the processing and secretion of proBNP, we generated lentivirus vectors carrying several mutations in glycosylation sites of human proBNP. We evaluated the proBNP/BNP ratio, as an efficacy of processing of proBNP to BNP-32. Results: In coronary sinus, plasma proBNP/BNP ratios were significantly elevated in heart failure patients in accordance with the disease severity. In conditioned medium of cultured ventricular myocytes expressing wild type human proBNP, proBNP/BNP ratio was 37%, while in those of myocytes expressing glycosylation-resistant mutant of proBNP, proBNP/BNP ratio was only 3%, demonstrating the essential contribution of glycosylation to the secretion of proBNP. Among 7 glycosylation sites, we found that glycosylation of Thr48 and Thr71 in proBNP is critical for the secretion of proBNP from ventricular myocytes by attenuating proBNP processing. GalNAc-transferase(GALNT) is the largest glycosyltransferase enyme family, which controll O-glycosylasion. We identified GALNT 1 and 2 mediated the glycosylation-regulated increase in cardiac proBNP secretion by screening with siRNAs. Indeed, GALNT1 and 2-expression was increased in FCS-treated ventricular myocytes and failing hearts. We further found that GALNT1 and 2 expression is suppressed by miR-30 family, and that miR-30s expression is decreased in FCS-treated ventricular myocytes and failing hearts. Conclusions: We have elucidated novel molecular pathways that miR-30-GALNTs axis-mediated O -glycosylation in proBNP contributes to increased proportion of proBNP among secreted BNP in the pathological myocardium.

Background The efficacy of pharmacological interventions to prevent sudden arrhythmic death in patients with chronic heart failure remains limited. Evidence now suggests increased ventricular expression of hyperpolarization‐activated cation ( HCN ) channels in hypertrophied and failing hearts contributes to their arrythmicity. Still, the role of induced HCN channel expression in the enhanced arrhythmicity associated with heart failure and the capacity of HCN channel blockade to prevent lethal arrhythmias remains undetermined. Methods and Results We examined the effects of ivabradine, a specific HCN channel blocker, on survival and arrhythmicity in transgenic mice (dn NRSF ‐Tg) expressing a cardiac‐specific dominant‐negative form of neuron‐restrictive silencer factor, a useful mouse model of dilated cardiomyopathy leading to sudden death. Ivabradine (7 mg/kg per day orally) significantly reduced ventricular tachyarrhythmias and improved survival among dn NRSF ‐Tg mice while having no significant effect on heart rate or cardiac structure or function. Ivabradine most likely prevented the increase in automaticity otherwise seen in dn NRSF ‐Tg ventricular myocytes. Moreover, cardiac‐specific overexpression of HCN 2 in mice ( HCN 2‐Tg) made hearts highly susceptible to arrhythmias induced by chronic β‐adrenergic stimulation. Indeed, ventricular myocytes isolated from HCN 2‐Tg mice were highly susceptible to β‐adrenergic stimulation‐induced abnormal automaticity, which was inhibited by ivabradine. Conclusions HCN channel blockade by ivabradine reduces lethal arrhythmias associated with dilated cardiomyopathy in mice. Conversely, cardiac‐specific overexpression of HCN 2 channels increases arrhythmogenicity of β‐adrenergic stimulation. Our findings demonstrate the contribution of HCN channels to the increased arrhythmicity seen in failing hearts and suggest HCN channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

Accumulating evidence suggests increased ventricular expression of hyperpolarization-activated cation channels (HCNs) in hypertrophied and failing hearts contributes to the induction of arrhythmias. In this study, we addressed the capacity of HCNs blockade to prevent lethal arrhythmias associated with heart failure. Transgenic mice expressing a dominant-negative mutant of neuron-restrictive silencer factor in a cardiac-specific manner (dnNRSF-Tg) exhibited dilated cardiomyopathy and sudden arrythmic death with an increase in ventricular HCNs expression, which are potentially responsible for the observed lethal arrhythmias. Ivabradine (Iva), a specific HCN channel inhibitor, significantly improved the survival among dnNRSF-Tg mice. Though echocardiographic, hemodynamic, and histological analyses showed no significant difference between Iva and control, ECG telemetric monitoring showed the significant reduction of arrhythmias in dnNRSF-Tg mice treated with Iva (VT; Iva 19/h, control 92/h ; p We also found that the transgenic mice overexpressing HCN2 specifically in the heart (HCN2-Tg) are susceptible to ventricular arrhythmias induced by chronic isoproterenol infusion. In isolated ventricular myocytes from HCN2-Tg, but not in those from wild type mice, isoproterenol induced abnormal spontaneous action potentials, which were suppressed with Iva. Our findings suggest that increased ventricular expression of HCN channels possibly contributes to the ventricular arrhythmias, and HCN channels blockade may represent a new and effective means of preventing sudden arrhythmic death in patients with heart failure.

Myocardin-related transcription factor (MRTF)-A is a Rho signaling-responsive co-activator of serum response factor (SRF). Here we show that induction of MRTF-A expression is key to pathological vascular remodeling in mouse models of vascular disease. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE -/- mice than in healthy control tissues, whereas myocardin expression was significantly lower. In addition, neointima formation in wire-injured femoral arteries in MRTF-A knockout ( Mkl1 -/- ) mice and atherosclerotic lesions in Mkl1 -/- ;ApoE -/- mice were both significantly attenuated. Expression of vinculin, MMP-9 and integrin β1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1 -/- mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), a cellular model of dedifferentiated VSMCs, knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1 concomitant with a decrease in myocardin expression. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.

Objective: Rho-ROCK pathway is involved in the progression of pathological vascular remodeling through the regulation of cell proliferation and migration by controlling gene transcription. While a transcriptional co-activator Myocardin-related transcription factor(MRTF)-A transduces Rho-actin signaling to SRF activation in the nucleus, roles of MRTF-A in the process underlying vascular diseases remain unknown. Methods and Results: We found that MRTF-A mRNA and protein expression in femoral arteries 2weeks after wire injury was significantly higher than that in sham-operated arteries of mice. In contrast, the expression of myocardin or MRTF-B was significantly decreased in injured arteries. We then evaluated the role played by MRTF-A in pathological vascular remodeling by creating several vascular disease models of mice lacking MRTF-A (MRTF-A -/- ). Neointima formation induced by ligation in carotid arteries of MRTF-A -/- was significantly smaller than that of MRTF-A +/- . Neointima formation induced by wire injury in femoral arteries of MRTFA -/- was significantly smaller than that of MRTF-A +/+ . Atherosclerotic lesions of MRTF-A -/- ;ApoE -/- was also significantly smaller than those of MRTF-A +/+ ;ApoE -/ . The expression of SRF-target genes involved in cellular migration, such as MMP9 and vinculin,within injured arteries of MRTF-A -/- was significantly attenuated compared to that of MRTF-A +/+ . In cultured, de-differentiated rat aortic vascular smooth muscle cells (RAVSMC), the expression of these genes was decreased by knocking down MRTF-A. Indeed, the promoter activity of vinculin gene was controlled by MRTF-A in RAVSMC. In addition, knocking down MRTF-A in RAVSMC resulted in a significant impairment in migration capacity. Furthermore, treatment with a small molecule CCG1423 inhibiting MRTF-A significantly reduced neointima formation induced by wire injury in femoral arteries of mice. Conclusions: Our study revealed that in de-differentiated vascular smooth muscle cells, in which myocardin and MRTTF-B expression is decreased, increased expression of MRTF-A contributes to the acquisition of migration capacity by maintaining SRF-target genes transcription, thereby promoting stress-induced vascular remodeling.