CARDIAC SARCOPLASMIC RETICULIM CALCIUM CYCLING PROTEINS

在心脏兴奋-收缩耦合中，肌浆网（SR）在调节胞浆内游离Ca2+浓度中起重要作用。SR有三个主要功能：a)钙离子从细胞质吸收到SR管腔，导致肌肉松弛；b) SR腔内Ca2+蓄积；c) Ca2+从SR释放到胞质中导致肌肉收缩。负责这些功能的主要SR蛋白分别是：Ca2+运输atp酶（SERCA）， Ca2+储存蛋白calsequestrin和Ca2+释放通道或ryanodine受体。磷蛋白是另一种SR蛋白，在Ca2+- atp酶活性和心肌收缩性的调节中起着至关重要的作用。在本项目中，我们建议进一步研究阐明PLB在哺乳动物心脏中的调节作用，并确定PLB与Ca2+泵之间的化学计量偶联比，这似乎是心脏收缩参数的关键决定因素。我们还建议阐明PLB磷酸化状态的作用，通过抑制剂1调节其磷酸酶活性，在基础和β激动剂条件下控制收缩性。此外，由于PLB水平或PLB磷酸化程度的变化反映了SR Ca2+负荷和收缩性的变化，我们建议通过钙调蛋白（SR管腔中主要的Ca2+储存蛋白）来阐明SR Ca2+负荷的功能作用。将生成具有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上分析其心脏表型。这些研究将为钙甲肾上腺素在体内的生理作用提供重要信息。总的来说，我们提出的研究将推进我们对SR功能调控Ca2+稳态的机制的认识。他们还将为各种SR Ca2+处理蛋白之间的串扰及其对心脏收缩性的调节作用提供有价值的见解。

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.