The cardiac ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) comprises a macromolecular complex that includes a kinase and two phosphatases that are bound to the channel via targeting proteins. We previously found that the RyR2 is protein kinase A (PKA)-hyperphosphorylated in end-stage human heart failure. Because heart failure is a progressive disease that often evolves from hypertrophy, we analyzed the RyR2 macromolecular complex in several animal models of cardiomyopathy that lead to heart failure, including hypertrophy, and at different stages of disease progression. We now show that RyR2 is PKA-hyperphosphorylated in diverse models of heart failure and that the degree of RyR2 PKA phosphorylation correlates with the degree of cardiac dysfunction. Interestingly, we show that RyR2 PKA hyperphosphorylation can be lost during perfusion of isolated hearts due to the activity of the endogenous phosphatases in the RyR2 macromolecular complex. Moreover, infusion of isoproterenol resulted in PKA phosphorylation of RyR2 in rat, indicating that systemic catecholamines can activate phosphorylation of RyR2 in vivo. These studies extend our previous analyses of the RyR2 macromolecular complex, show that both the kinase and phosphatase activities in the macromolecular complex are regulated physiologicallyin vivo, and suggest that RyR2 PKA hyperphosphorylation is likely a general feature of heart failure.

Background — Heart failure (HF) frequently follows the occurrence of myocardial infarction (MI). Questions about how HF develops and what cellular defects contribute to this dysfunction led to this study. Methods and Results — MI was induced in rats by coronary artery ligation. Clinical examination of the post-MI (PMI) surviving animals indicated that they were in overt HF by all measures. Cellular examination of the cardiomyocytes by patch-clamp and confocal [Ca ²⁺ ] _i imaging methods indicated that cellular function was significantly compromised. At the single-cell level, [Ca ²⁺ ] _i transient amplitudes were reduced and contractions were decreased and slowed, although Ca ²⁺ current ( I _Ca ) remained unchanged. The excitation-contraction coupling (ECC) gain function measured as Δ[Ca ²⁺ ] _i / I _Ca was significantly decreased. Ouabain, a cardiotonic steroid that blocks the Na ⁺ ,K ⁺ -ATPase and activates Ca ²⁺ entry via cardiac Na ⁺ channels, largely alleviated this defect. Conclusions — After MI, I _Ca becomes less able to trigger release of Ca ²⁺ from the sarcoplasmic reticulum. This failure of ECC is a major factor contributing to the development of contractile dysfunction and HF in PMI animals. The improved ECC gain, enhanced Ca ²⁺ entry, and augmented Ca ²⁺ signaling due to cardiotonic steroids contribute to the beneficial effects of these agents.

Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) [Ang-(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 ²⁺ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca ²⁺ 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 μmol/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)/Mas axis on Ca ²⁺ signaling. Cardiomyocytes treated with 10 nmol/L of Ang-(1-7) did not show changes in Ca ²⁺ -transient parameters such as peak Ca ²⁺ transients and kinetics of decay. Nevertheless, cardiomyocytes from Mas-deficient mice presented reduced peak and slower [Ca ²⁺ ] _i transients when compared with wild-type cardiomyocytes. Lower Ca ²⁺ ATPase of the sarcoplasmic reticulum expression levels accompanied the reduced Ca ²⁺ transient in Mas-deficient cardiomyocytes. Therefore, chronic Mas -deficiency leads to impaired Ca ²⁺ handling in cardiomyocytes. Collectively, these observations reveal a key role for the Ang-(1-7)/Mas axis as a modulator of cardiomyocyte function.