Modulation of Calcium Control of Cardiac Myofibrils

我们实验室研究的长期目标是了解 在肌节水平改变钙流下游的心脏功能调节。本 一项提案强调了原肌球蛋白磷酸化（Tm-P）在细丝激活中的分子信号传导 重点是潜在的协同和拮抗分子间和分子内的改变， 影响肌丝对Ca.这一假设的基本原理与我们几乎完全的 缺乏对Tm-P功能意义的了解，以及缺乏令人信服的试验数据， 与心脏和肌节功能改变相关的Tm-P变化。目标#1：做 cTnT和cTnI修饰的作用促进或减弱Tm磷酸化的功能作用 （Tm-P）对肌节对Ca的反应和/或肌丝的协同激活的影响 天桥？目的#2：内在应激，特别是与心肌病相关的Tm突变，是否诱导 调节Tm-P在肌节功能中的作用或改变其作为底物的分子内改变 对于激酶/磷酸酶？目的#3：外源性应激（高血压，p38 MAPK激活）对 心肌诱导Tm-P变化与功能改变相关？该方法采用 表达Tm、Tm（S283 D）和Tm（S283 A）突变形式的转基因模型，cDNA的病毒转移 将各种形式的Tm表达到心肌细胞中，并在心肌细胞中交换各种形式的cTnI和cTnT。 具有和不具有Tm（S283 D）或Tm（S283 A）的结皮纤维。我们还研究了一种cTnl突变， 关于通过强的横桥协同激活肌丝的“功能丧失”。 测量心肌细胞中的Ca 2+和缩短，以及心肌细胞的机械和生物化学活性。 洗涤剂提取的纤维和重组系统与使用NEM-S1作为探针的合作 激活..磷酸化蛋白质组学方法用于测定改变的肌节蛋白 在这些实验中以及在心肌对p38 MAPK的长期反应中， 高血压这些研究的结果将揭示Tm磷酸化的功能意义， 心功能和增加新的和重要的新的层面，我们的理解调制的 在生理和病理生理状态下心脏细丝的协同激活。

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.