SR Ca2+ ATPase, a determinant of cardiac contractility

描述(由申请人提供)：SERCA泵的表达和活性降低与舒张期功能障碍和心力衰竭有关。为了更好地确定SERCA泵表达降低的作用，我们建立了SERCA2基因敲除(-/+)模型。然而，杂合子小鼠(出生时只有一个SERCA2基因的功能等位基因)会导致其他钙处理蛋白的表达和活性发生代偿性变化，以弥补SERCA泵功能的下降。因此，SERCA2活性的慢性降低导致了心肌细胞钙稳态调节的新的平衡设定点。为了克服这个问题，我们将开发一个条件基因敲除(CKO)小鼠模型，在成年阶段以心脏特异的、可诱导的方式去除SERCA2基因。研究还表明，SERCA2a和SERCA2b在心脏中共表达，SERCA2b可以替代SERCA2a的功能。与SERCA2a相比，SERCA2b多了49个C端氨基酸，具有更高的钙亲和力和更低的周转速率。我们推测，SERCA2b在肌浆网钙离子摄取中发挥着独特的作用(因为它对钙离子具有很高的表观亲和力)，并且对于维持细胞内低钙含量是重要的。为了明确SERCA2b的作用，我们将使用腺病毒介导的SERCA基因转移到成年大鼠的心肌细胞。此外，我们有初步的数据表明，SERCA蛋白要么与其调控分子(PLB、肌磷脂、PKA、CaMKII、磷酸酶1和2a，通过锚定分子连接)形成复合体，要么与其共定位，从而有效地调节SR钙摄取功能。在这项提案中，我们试图使用免疫沉淀、二维凝胶分析和质谱分析来鉴定SERCA大分子复合体。总的来说，这些研究将使我们能够更好地了解SERCA2a和2b泵在心肌搏动功能中的作用。因此，这些研究可能会提出新的临床策略，可能被用于人类慢性心力衰竭的管理。

DESCRIPTION (provided by applicant): Decreased SERCA pump expression and activity have been implicated in diastolic dysfunction and heart failure. To better define the role of decreased SERCA pump expression we developed a SERCA2 gene knockout (-/+) model. However, heterozygous mice (born with only a single functional allele of the SERCA2 gene) induce several compensatory alterations in expression and activity of other Ca2+ handling proteins to make up a decrease in SERCA pump function. Thus, a chronic reduction in SERCA2 activity results in a new equilibrium set point for the regulation of cardiomyocyte Ca2+ homeostasis. To overcome this problem we will develop a Conditional knockout (cKO) mouse model to ablate the SERCA2 gene in a heart specific and inducible manner in adult stages. Studies also indicate that SERCA2a and 2b are co-expressed in the heart and SERCA2b can substitute for SERCA2a function. Compared with SERCA2a, SERCA2b has 49 extra C-terminal amino acids and has a higher Ca2+ affinity and lower turnover rate. We hypothesize that SERCA2b plays a unique role in SR Ca2+ uptake (because of its high apparent affinity for Ca2+ ion) and is important for maintaining low cytosolic Ca2+ content. To define the role of SERCA2b we will use adenoviral-mediated SERCA gene transfer into adult rat myocytes. In addition, we have preliminary data to suggest that SERCA protein either forms a complex or co-localizes with its regulatory molecules (PLB, sarcolipin, PKA, CaMKII, Phosphatase 1 and 2a, tethered via anchoring molecules) for efficient regulation of SR Ca2+ uptake function. In this proposal we seek to identify the SERCA macromolecular complex using immuno-precipitations and 2D gel analyses and MASS spectrometry. Collectively, these studies will allow us to better understand the role of SERCA2a and 2b pumps in the beat-to-beat function of the myocardium. As such, these studies are likely to suggest novel clinical strategies that might be pursued for the management of chronic heart failure in humans.