My-BP-C Modulation of Cardiac Contraction

本申请是一项请求，要求将PI的功绩奖延长为 再提供5年的支持。这个项目的目标是阐明分子间钙离子的作用机制 协同性和蛋白质磷酸化调节健康和疾病中的心肌收缩。这个 这项建议的目的是确定肌球蛋白结合蛋白-C(cMyBP-C)的作用，重点是 CMyBP-C磷酸化对收缩的调节。我们提出：假设1，cMyBP-C调节 通过与肌球蛋白亚片段2(S2)结合而收缩，从而控制交叉桥的可用性 肌动蛋白；假设2，我们的cMyBP-C基因缺失小鼠的收缩功能降低是由于加速的交叉刺激引起的。 CMyBP-C缺失所致的桥接动力学；和假设3，肾上腺素能诱发的正性肌力 激动剂的部分原因是PKA介导的cMyBP-C的磷酸化。已经取得了相当大的进展 在本授权期内检验上述每一项主要假设，包括以下结果：1) CMyBP-C是由PKA磷酸化引起的肌原纤维收缩动力学的主要调节因子 2)cMyBP-C沿长轴与肌球蛋白结合。 粗丝，从而驳斥了广泛持有的cMyBP-C结合的“项圈模型”，3)磷酸化或 CMyBP-C的消融导致跨桥向细丝移动，以及4)CaMKII的磷酸化 CMyBP-C介导心肌的正性力频反应。这些结果和其他结果设置为 研究这些发现的分子机制的阶段。我们将开发新的鼠系，表达 CMyBP-C的磷酸化突变体用于鉴定cMyBP-C中的PKA和CaMKII位点，顺序为 磷酸化，以及每种作用对心肌功能的影响。我们将确定是否会产生 CMyBP-C是由于它与肌球蛋白的相互作用，或者如一些人所建议的那样，它与肌动蛋白的假定结合是 也牵涉其中。进一步的研究将集中于cMyBP-C在活体中的功能和结构作用 研究其消融或磷酸化对活体和离体肌收缩的影响。这个 CMyBP-C基因敲除和磷酸化突变小鼠的疾病表型可能是由于 部分代偿机制将通过野生型和野生型零心肌的重建来研究 突变蛋白，通过零和突变等位基因的条件表达，以及通过野生型等位基因的重新表达。 这些结果有望提供对收缩状态调节机制的深入了解。 健康的心肌，也是疾病心脏功能缺陷的基础。

The present application is a request, to extend the Pi's MERIT Award for a further 5 years of support. The goal of this project is to elucidate mechanisms by which Ca^*, intermolecular cooperativity, and protein phosphorylations regulate myocardial contraction in health and disease. The objective of this proposal is to determine the roles of myosin binding protein-C (cMyBP-C), with emphasis on the regulation of contraction by cMyBP-C phosphorylation. We propose: Hypothesis 1, cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby controlling the availability of cross-bridges to actin; Hypothesis 2, reduced systolic function in our cMyBP-C null mouse results from accelerated cross- bridge kinetics due to deletion of cMyBP-C; and Hypothesis 3, the positive inotropy induced by ^ff-adrenergic agonists is due in part to PKA-mediated phosphorylation of cMyBP-C. Considerable progress has been made in the current grant period in testing each of these principal hypotheses, including the following results: 1) cMyBP-C is the primary regulator of myofibrillar contractile kinetics due to PKA phosphorylation of contractile proteins during )?-adrenergic stimulation of myocardium, 2) cMyBP-C binds to myosin along the long axis of the thick filament, thereby refuting the widely held "collar model" of cMyBP-C binding, 3) phosphorylation or ablation of cMyBP-C causes cross-bridges to move toward the thin filament, and 4) CAMKII phosphorylation of cMyBP-C mediates the positive force-frequency response in myo-cardium. These and other results set the stage for studies of the molecular mechanisms of these findings. We will develop new mouse lines expressing phosphorylation mutants of cMyBP-C to identify the PKA and CAMKII sites in cMyBP-C, the order of these phosphorylations, and the effects of each on myocardial function. We will determine whether the effects of cMyBP-C are due to its interactions with myosin or if, as proposed by some, its putative binding to actin is also involved. Further studies will focus on the functional and structural roles of cMyBP-C in living nnuscle by studying the effects of its ablation or phosphorylation on contraction in vivo and in isolated muscle. The possibility that the disease phenotypes of cMyBP-C knock-out and phosphorylation mutant mice are due in part to compensatory mechanisms will be studied by reconstitution of null myocardium with wild-type and mutant proteins, by conditional expression of null and mutant alleles, and by re-expression of wild-type alleles. These results promise to provide insights into the mechanisms by which contractile state is modulated in healthy myocardium and also the basis for functional deficits in diseased hearts.