Cardiac Sarcoplasmic Reticulum Calcium Cycling Proteins

描述（由申请方提供）：导致人类和实验性心力衰竭收缩性能受损的常见临床标志和特征是肌浆网（SR）Ca循环抑制。主要的重点放在纠正无论是抑制SR钙摄取或受损SR钙释放，以恢复细胞钙稳态和收缩性能在心力衰竭。SR钙摄取在阿托洛尔是介导的SERCA 2a和其可逆的调节剂受磷蛋白（PLN）;钙储存的钙螯合蛋白;和钙通过兰尼碱受体及其锚定蛋白连接蛋白和三聚体在心脏收缩期间释放。我们的中心假设是，SR钙摄取，储存和释放之间的精细串扰的改变在心力衰竭进展中起着至关重要的作用。我们以前的研究表明，去磷酸化受磷蛋白抑制SERCA 2钙亲和力，它代表了SR钙循环和心肌收缩力的基本“刹车”机制。事实上，通过激活其抑制剂-1来减弱蛋白磷酸酶1活性，从而增加PLN的磷酸化，可能会改善SR Ca循环并停止衰竭心脏的重塑。抑制剂-1的活化通过其在Thr 35处的PKA磷酸化发生。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：Ser 67和Thr 75。这两个位点的磷酸化增加蛋白磷酸酶1活性，导致SR Ca转运减弱和心肌收缩力降低。重要的是，在衰竭的心脏中，Ser 67和Thr 75的磷酸化增加。因此，我们在此提出进一步的研究，以阐明新发现的心脏抑制剂-1磷酸化位点的功能作用，其表达水平在生理和压力（压力超负荷和心肌梗死）条件下的成人心脏的时间调节。此外，我们发现SR Ca-释放可能受连接蛋白调节，其表达的消融与延迟后除极和室性心律失常有关。值得注意的是，在人类衰竭心脏中几乎检测不到连接蛋白水平，这可能是导致SR Ca循环受损的因素。因此，我们建议进一步阐明连接蛋白在体内的功能作用和其对兰尼碱受体钙释放通道的调节作用的机制。我们将采用一种综合方法，将分子、生物化学和生理学水平的研究整合在一起。我们的初步结果是非常令人兴奋的，他们支持抑制剂-1和junctin作为两个关键的调节SR钙循环和节点在心肌细胞钙稳态。总的来说，拟议的研究将推进我们的理解，并提供进一步的基本见解SR钙处理在心脏生理学和病理生理学的调节机制。 公共卫生相关性：衰竭心脏的普遍特征是通过肌浆网的钙循环受到抑制，这反映了心脏功能的恶化。本研究的重点是阐明肌浆网钙循环中两种蛋白质的作用：抑制剂-1，它调节蛋白磷酸酶1的活性和钙向肌浆网的转运;和连接蛋白，它存在于肌浆网腔中，调节SR的钙释放。我们的研究将清楚地推进我们对这两种参与心脏调控的蛋白质的认识。功能和功能障碍，这可能会导致更好的治疗心力衰竭的途径。

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.