My-BP-C Modulation of Cardiac Contraction

描述(申请人提供)：本研究的总体目标是阐明钙离子、协同作用和蛋白质磷酸化调节哺乳动物心肌收缩的分子机制。本研究的目的是阐明肌球蛋白结合蛋白-C(cMyBP-C)在心肌中的作用，特别是收缩的调节和cMyBP-C的磷酸化对力的影响和力发展的动力学。(1)我们假设cMyBP-C通过与肌球蛋白亚片段2(S2)结合来调节收缩，从而物理上控制肌动蛋白的交叉桥的可用性。这一想法将通过评估旨在破坏小鼠去皮心肌cMyBP-C/S2相互作用的干预措施的功能效果，并使用X射线衍射和电子显微镜来评估这些干预措施的结构效果来检验。(2)我们假设我们在cMyBP-C缺失小鼠中观察到的收缩功能改变是由于cMyBP-C缺失导致跨桥动力学和拉伸激活加速的结果。我们将通过测量无效心肌中力上升速率和拉伸激活的激活依赖来检验这些想法，并通过用cMyBP-C重建无效心肌来评估这些影响的可逆性。(3)我们推测，α-肾上腺素能激动剂诱导的正性肌力作用至少有一部分是由蛋白激酶A介导的cMyBP-C磷酸化所致。我们将通过评估PKA对以下力量和动力学的影响来检验这一想法：(I)在表达PKA不能磷酸化的突变心脏TNL的小鼠心肌中，(Ii)在表达非磷酸化cTnL的cMyBP-C缺失心肌中，以及(Iii)在表达cMyBP-C突变体的心肌中，可磷酸化的丝氨酸被丙氨酸或天冬氨酸取代。认为磷酸化的影响是由于对肌动蛋白的跨桥可获得性的改变，这一想法将通过X射线衍射进行评估。敲除和转基因小鼠的收缩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零心肌以及通过零和突变等位基因的条件表达来研究。研究结果将为健康心肌收缩状态的调节机制提供新的信息，并为疾病心脏功能缺陷的基础提供新的见解。

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate molecular mechanisms by which Ca2+, cooperation, and protein phosphorylations regulate contraction of mammalian myocardium. The objective of this proposal is to elucidate the roles of myosin binding protein-C (cMyBP-C) in myocardium, with particular emphasis on the regulation of contraction and the effects of cMyBP-C phosphorylation on force and the kinetics of force development. (1) We hypothesize that cMyBP-C modulates contraction by binding to myosin subfragment 2 (S2), thereby physically controlling the availability of cross-bridges to actin. This idea will be tested by assessing the functional effects of interventions designed to disrupt cMyBP-C/S2 interactions in mouse skinned myocardium and using X-ray diffraction and electron microscopy to assess the structural effects of these interventions. (2) We hypothesize that the altered systolic function we have observed in our cMyBP-C null mouse results from accelerated cross-bridge kinetics and stretch activation due to deletion of cMyBP-C. We will test these ideas by measuring the activation dependence of the rate of rise of force and stretch activation in null myocardium and assessing the reversibility of these effects by reconstituting null myocardium with cMyBP-C. (3) We hypothesize that at least some of the positive inotropy induced by (alpha-adrenergic agonists is due to protein kinase A-mediated phosphorylation of cMyBP-C. We will test this idea by assessing the effects of PKA on the force and kinetics of force development (i) in mouse myocardium expressing mutant cardiac Tnl that cannot be phosphorylated by PKA, (ii) in cMyBP-C null myocardium expressing non-phosphorylatable cTnl, and (iii) in myocardium expressing mutants of cMyBP-C in which phosphorylatable serines are replaced with alanines or with aspartates. The idea that the effects of phosphorylation are due to alterations in cross-bridge availability to actin will be assessed by X-ray diffraction. The possibility that the contractile phenotypes of knock-out and transgenic mice are due in part to compensatory mechanisms will be studied both by reconstitution of null myocardium with wild-type and mutant proteins and by conditional expression of null and mutant alleles. Results should provide new information about mechanisms by which contractile state is modulated in healthy myocardium and new insights as to the basis for functional deficits in diseased hearts.