Cardiac Sarcoplasmic Reticulum Calcium Cycling Proteins

描述(由申请人提供)：导致人类和实验性心力衰竭收缩性能受损的一个共同的临床标志和特征是肌浆网(SR)钙循环受抑。在心力衰竭中，主要的重点是纠正降低的肌浆网钙摄取或受损的肌浆网钙释放，以恢复细胞钙稳态和收缩性能。舒张期肌浆网钙摄取由SERCA2a及其可逆调节蛋白磷脂蛋白(PLN)介导，钙由钙调素储存，收缩期钙通过兰尼定受体及其锚定蛋白连接素和Triadin释放。我们的中心假设是，SR钙摄取、储存和释放之间的细微串扰的变化在心力衰竭的进展中起着关键作用。我们以前的研究表明，去磷酸化的磷蛋白抑制SERCA2的钙亲和力，它代表了SR钙循环和心肌收缩的一个基本的“刹车”机制。事实上，通过激活蛋白磷酸酶1的抑制物-1来减弱蛋白磷酸酶1的活性，增加PLN的磷酸化，可能会改善SR钙循环，阻止衰竭心脏的重塑。抑制物-1的激活是通过其Thr35处的PKA磷酸化来实现的。然而，我们最近发现心脏抑制因子-1有两个额外的磷酸化位点：Ser67和Thr75。这两个位点的磷酸化增加了蛋白磷酸酶1的活性，导致肌浆网钙转运减弱，心脏收缩能力下降。重要的是，Ser67和Thr75的磷酸化在衰竭的心脏中增加。因此，我们建议在此进行进一步的研究，以阐明新发现的心脏抑制物-1磷酸化位点在生理和应激(压力超负荷和心肌梗死)条件下的表达水平的时间调节的功能作用。此外，我们发现SR钙释放可能受连接素的调节，其表达的阻断与延迟后除极和室性心律失常有关。值得注意的是，在衰竭的心脏中几乎检测不到连接素水平，这可能是导致SR钙循环受损的一个因素。因此，我们建议进一步阐明连接素在体内的功能作用及其对兰尼定受体钙释放通道的调节机制。我们将采用一种综合的方法，综合分子、生物化学和生理水平的研究。我们的初步结果是非常令人兴奋的，它们支持抑制物-1和连接素作为肌浆网钙循环的两个关键调节因子和心肌细胞钙稳态的结点。总体而言，拟议的研究将促进我们的理解，并为心脏生理学和病理生理学中SR钙处理的潜在调节机制提供进一步的基本见解。

DESCRIPTION (provided by applicant): A common clinical hallmark and characteristic, contributing to the impaired contractile performance in human and experimental heart failure, is the depressed sarcoplasmic reticulum (SR) Ca-cycling. Major emphasis has been placed on correcting either the depressed SR Ca-uptake or impaired SR Ca-release to restore cellular Ca-homeostasis and contractile performance in heart failure. SR Ca-uptake during diastole is mediated by SERCA2a and its reversible regulator phospholamban (PLN); Ca is stored by calsequestrin; and Ca is released during systole through the ryanodine receptor and its anchoring proteins junctin and triadin. Our central hypothesis is that alterations in the fine cross-talk between SR Ca-uptake, storage and release play a critical role in heart failure progression. Our previous studies showed that dephosphorylated phospholamban inhibits the SERCA2 Ca-affinity and it represents a fundamental "brake" mechanism in SR Ca-cycling and cardiac contractility. Indeed, increased phosphorylation of PLN by attenuated protein phosphatase 1 activity through activation of its inhibitor-1 may improve SR Ca-cycling and halt remodeling in the failing heart. Activation of inhibitor-1 occurs by its PKA-phosphorylation at Thr35. However, we have recently shown that cardiac inhibitor-1 has two additional phosphorylation sites: Ser67 and Thr75. Phosphorylation of these two sites increases protein phosphatase 1 activity, resulting in attenuated SR Ca-transport and depressed cardiac contractility. Importantly, phosphorylation of Ser67 and Thr75 is increased in failing hearts. Thus, we propose herein further studies to elucidate the functional role of the newly identified cardiac inhibitor-1 phosphorylation sites by temporal regulation of their expression levels in the adult heart under physiological and stress (pressure-overload and myocardial infarction) conditions. In addition, we uncovered that SR Ca-release may be regulated by junctin and ablation of its expression is associated with delayed after-depolarizations and ventricular arrhythmias. Notably, the junctin levels are almost non-detectable in human failing hearts and this may be a contributing factor to the impaired SR Ca-cycling. Thus, we propose to further elucidate the functional role of junctin in vivo and the mechanisms underlying its regulatory effects on the ryanodine receptor Ca-release channel. We will employ a comprehensive approach integrating studies at the molecular, biochemical and physiological levels. Our preliminary results are very exciting and they support inhibitor-1 and junctin as two key regulators of SR Ca-cycling and nodal points in cardiomyocyte Ca-homeostasis. Overall, the proposed studies will advance our understanding and provide further fundamental insights into the mechanisms underlying regulation of SR Ca-handling in cardiac physiology and pathophysiology.