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。该项目的目标是阐明钙离子、分子间协同作用和蛋白质磷酸化调节健康和疾病心肌收缩的机制。这个建议的目的是确定肌球蛋白结合蛋白-C(cMyBP-C)的作用，重点是通过cMyBP-C的磷酸化来调节收缩。我们提出：假设1，cMyBP-C调节 通过与肌球蛋白亚片段2(S2)结合收缩，从而控制对肌动蛋白的交叉桥的可用性；假设2，我们的cMyBP-C缺失小鼠的收缩功能降低是由于cMyBP-C缺失导致跨桥动力学加速；以及假设3，B-肾上腺素能激动剂诱导的正性肌力作用部分是由于PKA介导的cMyBP-C的磷酸化。在当前的授权期内，在检验这些主要假说方面已经取得了相当大的进展，包括以下结果：1)cMyBP-C是肌原纤维收缩动力学的主要调节因子，这是由于在B-肾上腺素能刺激心肌过程中PKA对收缩蛋白的磷酸化，2)cMyBP-C沿着粗丝的长轴与肌球蛋白结合，从而驳斥了广泛存在的cMyBP-C结合的“项圈模型”，3)cMyBP-C的磷酸化或消融导致交叉桥向细丝移动，以及4)CaMKII的磷酸化 CMyBP-C介导心肌的正性力频反应。这些和其他结果为研究这些发现的分子机制奠定了基础。我们将建立表达cMyBP-C磷酸化突变体的新小鼠品系，以确定cMyBP-C中的PKA和CaMKII位点，这些磷酸化的顺序，以及它们对心肌功能的影响。我们将确定cMyBP-C的作用是由于它与肌球蛋白的相互作用，还是像一些人提出的那样，它与肌动蛋白的假定结合是 也牵涉其中。进一步的研究将通过研究cMyBP-C在活体和离体肌中对收缩的影响来研究cMyBP-C在活体肌肉中的功能和结构作用。CMyBP-C基因敲除和磷酸化突变小鼠的疾病表型部分归因于代偿机制的可能性将通过用野生型和突变蛋白重建无效心肌，通过有条件地表达无效和突变等位基因，以及通过重新表达野生型等位基因来研究。 这些结果有望为了解健康心肌收缩状态的调节机制，以及疾病心脏功能缺陷的基础提供见解。

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 Ca2+, 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 B-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 )B-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.