Cardiac Sarcoplasmic Reticulum Calcium Cycling Proteins

描述（由申请人提供）：一个共同的临床标志和特征，导致人类和实验性心力衰竭的收缩性能受损，是肌浆网（SR）钙循环抑制。主要的重点放在纠正被抑制的SR钙摄取或受损的SR钙释放，以恢复心力衰竭时细胞钙稳态和收缩性能。舒张期SR钙摄取由SERCA2a及其可逆调节因子磷蛋白（PLN）介导；钙被钙螯合蛋白储存；Ca在收缩期通过ryanodine受体及其锚定蛋白连接蛋白和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改变在心力衰竭的进展中起关键作用。我们之前的研究表明，去磷酸化的磷蛋白抑制SERCA2的ca亲和力，它代表了SR ca循环和心脏收缩性的基本“制动”机制。事实上，通过激活蛋白磷酸酶1的抑制剂-1来减弱蛋白磷酸酶1的活性，从而增加PLN的磷酸化，可能会改善SR钙循环，并阻止衰竭心脏的重塑。抑制剂-1的激活是通过其在Thr35位点的pka磷酸化发生的。然而，我们最近发现心脏抑制剂-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SR - ca转运减弱和心脏收缩力下降。重要的是，Ser67和Thr75的磷酸化在衰竭的心脏中增加。因此，我们建议进一步研究阐明新发现的心脏抑制剂-1磷酸化位点在生理和应激（压力过载和心肌梗死）条件下通过时间调节其在成人心脏中的表达水平的功能作用。此外，我们发现SR - ca的释放可能受连接蛋白的调节，其表达的消融与去极化后延迟和室性心律失常有关。值得注意的是，在人类衰竭的心脏中，连接蛋白水平几乎无法检测到，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蛋白在体内的功能作用及其调控ryanodine受体ca释放通道的机制。我们将采用综合的方法，在分子、生化和生理水平上进行研究。我们的初步结果非常令人兴奋，它们支持抑制剂-1和连接蛋白作为SR钙循环和心肌细胞钙稳态节点的两个关键调节因子。总的来说，拟议的研究将促进我们对SR - ca处理在心脏生理学和病理生理学中的调节机制的理解，并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. PUBLIC HEALTH RELEVANCE: A universal characteristic of the failing heart is depressed calcium cycling through the sarcoplasmic reticulum, which reflects deteriorated heart function. This proposal concentrates on elucidating the role of two proteins involved in sarcoplasmic reticulum (SR) calcium cycling: inhibitor-1, which has been known to regulate protein phosphatase 1 activity and calcium transport into the SR; and junctin, which is present in the sarcoplasmic reticulum lumen and regulates calcium release from the SR. Our studies will clearly advance our knowledge on these two protein players involved in cardiac function and dysfunction, which may lead to better therapeutic avenues in heart failure.