Modulation of Calcium Control of Cardiac Myofibrils

描述(申请人提供)：我们实验室的长期研究目标是在肌节水平上了解在改变的钙流的下游调节心脏功能的功能意义。目前的建议强调原肌球蛋白磷酸化(TM-P)在细丝激活的分子信号中的作用，重点关注潜在的协同和拮抗的分子间和分子内改变，这些改变调节了原肌球蛋白对钙的反应。这一假设的理论基础与我们几乎完全缺乏对TM-P的功能意义的了解有关，也与令人信服的试点数据显示TM-P与心脏和肌节功能改变相关的新变化有关。目标1：cTnT和cTn1的修饰效应是促进还是减弱TM磷酸化(TM-P)对肌节对钙的反应和/或由强大的交叉桥协同激活肌丝的功能效应？目的#2：内在应激，特别是与心肌病相关的TM突变，是否会导致分子内改变，从而调节TM-P对肌节功能的影响，或者改变它作为激酶/磷酸酶底物的作用？目的#3：心肌上的外源性应激(高血压，p38MAPK激活)是否会引起TM-P的改变，这与功能改变有关？该方法采用了表达TM、TM(S283D)和TM(S283A)突变形式的转基因模型，将表达各种形式TM的cDNA病毒转移到心肌细胞，以及在有或没有TM(S283D)或TM(S283A)的情况下，在皮肤纤维中交换各种形式的cTn1和cTnT。我们还研究了cTn1突变，它产生了“与强大的交叉桥协同激活肌丝有关的功能丧失”。用NEM-S1作为协同激活的探针，测定了心肌细胞内的钙离子和缩短，以及洗涤剂提取的纤维和重组系统的机械和生化活性。在这些实验和心肌对p38MAPK和高血压的长期反应中，应用磷酸化蛋白质组学方法来确定改变的肌节蛋白的磷酸化。这些研究结果将揭示TM磷酸化在心脏功能中的功能意义，并为我们理解心脏细丝在生理和病理生理状态下的协同激活调控提供新的重要维度。

DESCRIPTION (provided by applicant): The long term objective of research in our laboratory is to understand the functional significance of regulation of cardiac function downstream of altered Ca- fluxes at the level of the sarcomere. The present proposal emphasizes tropomyosin phosphorylation (Tm-P) in molecular signaling in thin filament activation with focus on potential synergistic and antagonistic inter- and intra-molecular alterations that modulate its effects on myofilament response to Ca. The rationale for this hypothesis is related to our nearly complete lack of understanding of the functional significance of Tm-P and to compelling pilot data demonstrating novel changes in Tm-P associated with altered cardiac and sarcomeric function. The objectives are: Aim #1: Do effects of modifications in cTnT and cTnl promote or diminish the functional effects of Tm phosphorylation (Tm-P) on sarcomeric response to Ca and/or cooperative activation of the myofilaments by strong crossbridges? Aim #2: Do intrinsic stresses, especially Tm mutations linked to cardio-myopathies, induce intra-molecular alterations that modulate the effect of Tm-P in sarcomeric function or alter it as a substrate for kinases/phosphatases? Aim #3: Do extrinsic stresses (hypertension, p38 MAPK activation) on the myocardium induce a change in Tm-P that correlates with altered function? The approach employs transgenic models expressing mutant forms of Tm, Tm (S283D) and Tm (S283A), viral transfer of cDNA expressing various forms of Tm into cardiac myocytes, and exchange of various forms of cTnl and cTnT in skinned fibers with and without Tm (S283D) or Tm (S283A). We also study a cTnl mutation that generates loss of function" with regard to cooperative activation of the myofilaments by strong crossbridges. Measurements are made of Ca2+ and shortening in myocytes, and mechanical and biochemical activity of detergent extracted fibers and reconstituted systems with the use of NEM-S1 as a probe of cooperative activation. Phospho-proteomic approaches are applied to determination of altered sarcomeric protein phosphorylation in these experiments and in long term responses of the myocardium to p38 MAPK and hypertension. Results of these studies will reveal the functional significance of Tm phosphorylation in cardiac function and add novel and important new dimensions to our understanding of modulation of cooperative activation of cardiac thin filaments in physiological and patho-physiological states.