Rationale: Atrial and brain natriuretic peptides (ANP and BNP, respectively) exert antihypertrophic effects in the heart via their common receptor, guanylyl cyclase (GC)-A, which catalyzes the synthesis of cGMP, leading to activation of protein kinase (PK)G. Still, much of the network of molecular mediators via which ANP/BNP-GC-A signaling inhibit cardiac hypertrophy remains to be characterized. Objective: We investigated the effect of ANP-GC-A signaling on transient receptor potential subfamily C (TRPC)6, a receptor-operated Ca 2؉ channel known to positively regulate prohypertrophic calcineurin-nuclear factor of activated T cells (NFAT) signaling. Methods and Results: In cardiac myocytes, ANP induced phosphorylation of TRPC6 at threonine 69, the PKG phosphorylation site, and significantly inhibited agonist-evoked NFAT activation and Ca 2؉ influx, whereas in HEK293 cells, it dramatically inhibited agonist-evoked TRPC6 channel activity. These inhibitory effects of ANP were abolished in the presence of specific PKG inhibitors or by substituting an alanine for threonine 69 in TRPC6. In model mice lacking GC-A, the calcineurin-NFAT pathway is constitutively activated, and BTP2, a selective TRPC channel blocker, significantly attenuated the cardiac hypertrophy otherwise seen. Conversely, overexpression of TRPC6 in mice lacking GC-A exacerbated cardiac hypertrophy. BTP2 also significantly inhibited angiotensin II-induced cardiac hypertrophy in mice. Conclusions: Collectively, these findings suggest that TRPC6 is a critical target of antihypertrophic effects elicited via the cardiac ANP/BNP-GC-A pathway and suggest TRPC6 blockade could be an effective therapeutic strategy for preventing pathological cardiac remodeling. (Circ Res. 2010;106:1849-1860.)Key Words: natriuretic peptides Ⅲ calcium Ⅲ ion channels Ⅲ hypertrophy I n response to pathological stimuli such as prolonged mechanical stress, massive tissue injury, or abnormal neurohumoral activation, hearts show hypertrophic growth and remodeling, which is characterized by an increase in myocyte cell size, assembly of sarcomere proteins, interstitial fibrosis, and reexpression of fetal cardiac genes. Although the hypertrophic response is initially compensatory, it ultimately causes heart failure, which is now a leading cause of morbidity and mortality around the world. Diverse intracellular signaling pathways exerting pro-or antihypertrophic effects have been shown to play important roles in the complex processes of cardiac remodeling, 1,2 but the details of the molecular mechanisms mediating the crosstalk among these signaling pathways remain uncertain. Unraveling those details should give us a better understanding of the molecular processes underlying the establishment of cardiac hypertro- phy and heart failure, which could ultimately lead to the discovery of novel therapeutic targets for prevention of pathological cardiac remodeling. The heart regulates cardiovascular homeostasis in part by secreting 2 peptide mediators, atrial natriuretic peptide (ANP) and brain n...

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Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1+/CXCR4+/VE-cadherin− (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

Background-Pharmacological interventions for prevention of sudden arrhythmic death in patients with chronic heart failure remain limited. Accumulating evidence suggests increased ventricular expression of T-type Ca 2ϩ channels contributes to the progression of heart failure. The ability of T-type Ca 2ϩ channel blockade to prevent lethal arrhythmias associated with heart failure has never been tested, however. Methods and Results-We compared the effects of efonidipine and mibefradil, dual T-and L-type Ca 2ϩ channel blockers, with those of nitrendipine, a selective L-type Ca 2ϩ channel blocker, on survival and arrhythmogenicity in a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor transgenic mice (dnNRSF-Tg), which is a useful mouse model of dilated cardiomyopathy leading to sudden death. Efonidipine, but not nitrendipine, substantially improved survival among dnNRSF-Tg mice. Arrhythmogenicity was dramatically reduced in dnNRSF-Tg mice treated with efonidipine or mibefradil. Efonidipine acted by reversing depolarization of the resting membrane potential otherwise seen in ventricular myocytes from dnNRSF-Tg mice and by correcting cardiac autonomic nervous system imbalance. Moreover, the R(Ϫ)-isomer of efonidipine, a recently identified, highly selective T-type Ca 2ϩ channel blocker, similarly improved survival among dnNRSF-Tg mice. Efonidipine also reduced the incidence of sudden death and arrhythmogenicity in mice with acute myocardial infarction. Conclusions-T-type Ca2ϩ channel blockade reduced arrhythmias in a mouse model of dilated cardiomyopathy by repolarizing the resting membrane potential and improving cardiac autonomic nervous system imbalance. T-type Ca 2ϩ channel blockade also prevented sudden death in mice with myocardial infarction. Our findings suggest T-type Ca 2ϩ channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure. Key Words: ion channels Ⅲ nervous system, autonomic Ⅲ heart failure Ⅲ calcium Ⅲ arrhythmia A s many as 50% of deaths among heart failure patients are sudden and unexpected, presumably the result of lethal arrhythmias. 1 Despite recent progress in nonpharmacological therapy, pharmacological interventions for the treatment and prevention of lethal arrhythmias associated with chronic heart failure remain limited. A prerequisite for the development of new pharmacological approaches is to identify potential targets based on knowledge of the molecular basis of arrhythmogenesis in failing hearts. Clinical Perspective on p 752Compelling evidence implicates T-type Ca 2ϩ channels in the progression of heart failure. 2,3 During development, T-type Ca 2ϩ channels are abundantly expressed in the embryonic ventricle, but their expression is suppressed in the adult ventricle, so that it is restricted to the conduction system. 4,5 However, T-type Ca 2ϩ channels are reexpressed in hypertrophied and failing ventricles, 4,6 -9 and the resultant T-type Ca 2ϩ currents (I Ca,T ) are thought to be involved in the pathologica...

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