My-BP-C Modulation of Cardiac Contraction

描述（由申请人提供）：本研究的总体目标是阐明Ca 2+、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。本研究旨在阐明肌球蛋白结合蛋白C（myosin binding protein-C，cMyBP-C）在心肌中的作用，特别强调cMyBP-C磷酸化对心肌收缩的调节作用以及对心肌收缩力和心肌收缩力发展动力学的影响。(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，从而物理控制肌动蛋白交叉桥的可用性。这一想法将通过评估旨在破坏小鼠皮肤心肌中cMyBP-C/S2相互作用的干预措施的功能效应以及使用X射线衍射和电子显微镜评估这些干预措施的结构效应来进行测试。(2)我们假设，我们在cMyBP-C敲除小鼠中观察到的收缩功能改变是由于cMyBP-C缺失导致的跨桥动力学和牵张激活加速所致。我们将通过测量无效心肌中力和牵张激活的上升速率的激活依赖性，并通过用cMyBP-C重建无效心肌来评估这些效应的可逆性，来测试这些想法。(3)我们假设α-肾上腺素能激动剂诱导的正性肌力至少有一部分是由于蛋白激酶A介导的cMyBP-C磷酸化。我们将通过评估PKA对力和力发展动力学的影响来测试这一想法：（i）在表达不能被PKA磷酸化的突变型心脏Tnl的小鼠心肌中，（ii）在表达不可磷酸化的cTnl的cMyBP-C无效心肌中，和（iii）在表达cMyBP-C的突变体的心肌中，其中可磷酸化的丝氨酸被丙氨酸或谷氨酸盐取代。认为磷酸化的影响是由于肌动蛋白的跨桥可用性的改变的想法将通过X射线衍射进行评估。基因敲除和转基因小鼠的收缩表型部分是由于补偿机制的可能性将通过用野生型和突变型蛋白质重建无效心肌以及无效和突变等位基因的条件表达来研究。结果应提供新的信息机制的收缩状态调制在健康心肌和新的见解的基础上，在患病的心脏功能缺陷。

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.