Abstract-Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) ] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(1-7)/Mas axis in NO generation and Ca 2ϩ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca 2ϩ imaging, and confocal microscopy. Acute Ang-(1-7) treatment (10 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(1-7)-dependent NO raise was abolished by pretreatment with A-779 (1 mol/L). To confirm that Ang-(1-7) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(1-7) failed to increase NO levels. Moreover, Mas-ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthase activity, indicating that endothelial NO synthase and its binding partners are important effectors of the Mas-mediated pathway in cardiomyocytes. We then investigated the role of the Ang- (1-7)

Abstract-The renin-angiotensin (Ang) system plays a pivotal role in the pathogenesis of cardiovascular disease, with Ang II being the major effector of this system. Multiple lines of evidence have shown that Ang-(1-7) exerts cardioprotective effects in the heart by counterregulating Ang II actions. The questions that remain are how and where Ang-(1-7) exerts its effects. By using a combination of molecular biology, confocal microscopy, and a transgenic rat model with increased levels of circulating Ang-(1-7) (TGR[A1-7]3292), we evaluated the signaling pathways involved in Ang-(1-7) cardioprotection against Ang II-induced pathological remodeling in ventricular cardiomyocytes. Rats were infused with Ang II for 2 weeks. We found that ventricular myocytes from TGR(A1-7)3292 rats are protected from Ang II pathological remodeling characterized by Ca 2ϩ signaling dysfunction, hypertrophic fetal gene expression, glycogen synthase kinase 3␤ inactivation, and nuclear factor of activated T-cells nuclear accumulation. Moreover, cardiomyocytes from TGR(A1-7)3292 rats infused with Ang II presented increased expression levels of neuronal NO synthase. To provide a signaling pathway involved in the beneficial effects of Ang-(1-7), we treated neonatal cardiomyocytes with Ang-(1-7) and Ang II for 36 hours. Treatment of cardiomyocytes with Ang-(1-7) prevented Ang II-induced hypertrophy by modulating calcineurin/nuclear factor of activated T-cell signaling cascade. Importantly, antihypertrophic effects of Ang-(1-7) on Ang II-treated cardiomyocytes were prevented by N G -nitro-L-arginine methyl ester and 1H-1,2,4oxadiazolo4,2-aquinoxalin-1-one, suggesting that these effects are mediated by NO/cGMP. Taken together, these data reveal a key role for NO/cGMP as a mediator of Ang-(1-7) beneficial effects in cardiac cells. (Hypertension. 2010; 55:153-160.)

Recent work has provided compelling evidence that increased levels of acetylcholine (ACh) can be protective in heart failure, whereas reduced levels of ACh secretion can cause heart malfunction. Previous data show that cardiomyocytes themselves can actively secrete ACh, raising the question of whether this cardiomyocyte derived ACh may contribute to the protective effects of ACh in the heart. To address the functionality of this non-neuronal ACh machinery, we used cholinesterase inhibitors and a siRNA targeted to AChE (acetylcholinesterase) as a way to increase the availability of ACh secreted by cardiac cells. By using nitric oxide (NO) formation as a biological sensor for released ACh, we showed that cholinesterase inhibition increased NO levels in freshly isolated ventricular myocytes and that this effect was prevented by atropine, a muscarinic receptor antagonist, and by inhibition of ACh synthesis or vesicular storage. Functionally, cholinesterase inhibition prevented the hypertrophic effect as well as molecular changes and calcium transient alterations induced by adrenergic overstimulation in cardiomyocytes. Moreover, inhibition of ACh storage or atropine blunted the anti-hypertrophic action of cholinesterase inhibition. Altogether, our results show that cardiomyocytes possess functional cholinergic machinery that offsets deleterious effects of hyperadrenergic stimulation. In addition, we show that adrenergic stimulation upregulates expression levels of cholinergic components. We propose that this cardiomyocyte cholinergic signaling could amplify the protective effects of the parasympathetic nervous system in the heart and may counter-act or partially neutralize hypertrophic adrenergic effects.

Overwhelming evidence supports the importance of the sympathetic nervous system in heart failure. In contrast, much less is known about the role of failing cholinergic neurotransmission in cardiac disease. By using a unique genetically modified mouse line with reduced expression of the vesicular acetylcholine transporter (VAChT) and consequently decreased release of acetylcholine, we investigated the consequences of altered cholinergic tone for cardiac function. M-mode echocardiography, hemodynamic experiments, analysis of isolated perfused hearts, and measurements of cardiomyocyte contraction indicated that VAChT mutant mice have decreased left ventricle function associated with altered calcium handling. Gene expression was analyzed by quantitative reverse transcriptase PCR and Western blotting, and the results indicated that VAChT mutant mice have profound cardiac remodeling and reactivation of the fetal gene program. This phenotype was attributable to reduced cholinergic tone, since administration of the cholinesterase inhibitor pyridostigmine for 2 weeks reversed the cardiac phenotype in mutant mice. Our findings provide direct evidence that decreased cholinergic neurotransmission and underlying autonomic imbalance cause plastic alterations that contribute to heart dysfunction.