SR Ca2+ ATPase, a determinant of cardiac contractility

描述（由申请人提供）：SERCA泵表达和活性降低与舒张功能障碍和心力衰竭有关。为了更好地定义降低的SERCA泵表达的作用，我们开发了SERCA 2基因敲除（-/+）模型。然而，杂合子小鼠（出生时仅具有SERCA 2基因的单个功能等位基因）诱导其他Ca 2+处理蛋白的表达和活性的几种补偿性改变，以弥补SERCA泵功能的降低。因此，SERCA 2活性的慢性降低导致用于调节心肌细胞Ca 2+稳态的新的平衡设定点。为了克服这个问题，我们将开发一种条件性敲除（cKO）小鼠模型，以在成年阶段以心脏特异性和可诱导的方式消融SERCA 2基因。研究还表明，SERCA 2a和2b在心脏中共表达，SERCA 2b可以替代SERCA 2a的功能。与SERCA 2a相比，SERCA 2b的C-末端多了49个氨基酸，并具有更高的钙离子亲和力和更低的周转率。我们假设SERCA 2b在SR Ca 2+摄取中起着独特的作用（因为其对Ca 2+离子的高表观亲和力），并且对于维持低细胞溶质Ca 2+含量很重要。为了确定SERCA 2b的作用，我们将使用腺病毒介导的SERCA基因转移到成年大鼠心肌细胞中。此外，我们有初步的数据表明，SERCA蛋白要么形成一个复合物，要么与其调节分子（PLB，sarcolipin，PKA，CaMKII，磷酸酶1和2a，通过锚定分子栓系）共定位，以有效地调节SR Ca 2+摄取功能。在这个建议中，我们试图确定使用免疫沉淀和二维凝胶分析和质谱法的SERCA大分子复合物。总的来说，这些研究将使我们能够更好地了解SERCA 2a和2b泵在心肌搏动功能中的作用。因此，这些研究很可能提出新的临床策略，可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.