CARDIAC SARCOPLASMIC RETICULIM CALCIUM CYCLING PROTEINS

在心脏兴奋-收缩偶联中，肌浆网（SR）在调节胞浆游离Ca ~（2+）浓度中起着重要作用。 SR有三个主要功能：a）Ca 2+从胞质溶胶摄取到SR腔中，导致肌肉松弛; B）Ca 2+储存在SR腔中;和c）Ca 2+从SR释放到胞质溶胶中，导致肌肉收缩。 负责这些功能的主要SR蛋白分别是：Ca 2+转运ATP酶（SERCA）、Ca 2+储存蛋白钙螯合蛋白和Ca 2+释放通道或ryanodine受体。 Phospholamban是另一种SR蛋白，其在Ca 2 +-ATP酶活性和心肌收缩性的调节中起关键作用。 在这个项目中，我们提出进一步的研究阐明PLB在哺乳动物心脏的调节作用，并定义PLB和钙泵之间的化学计量耦合比，这似乎是心脏收缩参数的一个关键决定因素。 我们还建议阐明PLB磷酸化状态的作用，通过调节其磷酸酶活性的抑制剂1，在控制收缩基础和β-激动剂条件下。 此外，由于PLB水平或PLB磷酸化程度的改变反映了SR Ca 2+负荷和收缩性的改变，我们建议通过钙螯合蛋白（SR腔中的主要Ca 2+储存蛋白）来阐明SR Ca 2+负荷的功能作用。 将生成该蛋白表达水平改变（过表达和敲除）的动物模型，并在亚细胞、细胞、器官和完整动物水平分析其心脏表型。 这些研究将为钙螯合蛋白在体内的生理作用提供重要信息。 总体而言，我们提出的研究将推进我们的知识的机制的钙离子稳态的SR功能的调节。 他们还将提供有价值的见解之间的串扰的各种SR钙处理蛋白质和它们对心肌收缩力的调节作用。

In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR) plays an essential role in the regulation of the cytosolic free Ca2+ concentration. There are three major functions of the SR: a) Ca2+-uptake from the cytosol into the SR lumen resulting in muscle relaxation; b) Ca2+ storage in the SR lumen; and c) Ca2+-release from the SR into the cytosol resulting in muscle contraction. The main SR proteins responsible for these functions are: the Ca2+-transport ATPase (SERCA), the Ca2+ storage protein calsequestrin, and the Ca2+ release channel or ryanodine receptor, respectively. Phospholamban is another SR protein, which plays a crucial role in the regulation of the Ca2+-ATPase activity and myocardial contractility. In this project, we propose further studies on elucidating the regulatory role of PLB in the mammalian heart and defining the stoichiometric coupling ratio between PLB and the Ca2+-pump, which appears to be a key determinant of cardiac contractile parameters. We also propose to elucidate the role of the PLB phosphorylation status, through regulation of its phosphatase activity by inhibitor 1, in the control of contractility under basal and beta-agonist conditions. Furthermore, since alterations in the levels of PLB or in the degree of PLB phosphorylation reflect alterations in SR Ca2+ load and contractility, we propose to elucidate the functional role of SR Ca2+ load through calsequestrin, the major Ca2+ storage protein in the SR lumen. Animal models with alterations in the expression levels of this protein (overexpression and knockouts) will be generated and their cardiac phenotype will be analyzed at the subcellular, cellular, organ and intact animal levels. These studies will provide important information on the physiological role of calsequestrin in vivo. Overall, our proposed studies will advance our knowledge on the mechanisms underlying regulation of Ca2+ homeostasis by the SR function. They will also provide valuable insights into the crosstalk between the various SR Ca2+ handling proteins and their regulatory effects on cardiac contractility.