Modulation of Calcium Control of Cardiac Myofibrils

我们实验室研究的长期目标是了解肌动蛋白的功能意义 肌节水平钙流出改变对心功能下游的调节。现在 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 重点放在调节ITS的分子间和分子内的潜在协同和拮抗变化 钙对肌丝反应的影响。这一假设的基本原理与我们几乎完成的 对TM-P的功能意义缺乏了解，缺乏令人信服的试点数据演示新奇 TM-P的变化与心脏和肌节功能改变相关。目标是：目标1：做 CTnT和cTn1的修饰促进或减弱TM磷酸化的功能效应 (TM-P)对肌小球对钙的反应和/或强的协同激活肌丝 十字桥？目标2：内在应激，尤其是TM基因突变是否与心脏肌病有关？ 调节TM-P在肌瘤功能中的作用或改变其作为底物的分子内变化 用于激酶/磷酸酶吗？目的#3：做外部应激(高血压，p38 MAPK激活) 心肌引起TM-P的改变与功能改变有关？该方法采用了 表达TM、TM(S283D)和TM(S283A)突变型的转基因模型及病毒转移 不同形式的TM在心肌细胞中的表达及cTn1和cTnT在心肌细胞中的交换 带有和不带有TM(S283D)或TM(S283A)的结皮纤维。我们还研究了一种cTnL突变，这种突变会产生 与强大的交叉桥协同激活肌丝有关。 测量心肌细胞内钙离子和缩短，以及机械和生化活性。 以NEM-S1为探针的洗涤剂提取纤维及重组体系的研究 激活..应用磷酸化蛋白质组学方法测定改变的肌瘤蛋白 在这些实验中以及在心肌对p38MAPK和p38MAPK和 高血压。这些研究的结果将揭示TM磷酸化在脑内的功能意义 并为我们对心功能调节的理解增添了新的重要维度。 心脏细丝在生理和病理生理状态下的协同激活。

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.