Molecular signaling in cardiac myofilaments

（改编自申请人的摘要）这里提出的实验检验了心脏TnI（cTnI）和cTnT的同种型转换和磷酸化是心输出量的内在（Starling定律）和外在（神经体液）控制中的重要因素的假设。长期目标是确定：1）与心脏细丝结合的Ca 2+和交叉桥控制收缩和舒张的独特分子机制，以及2）这些机制的调节对激活和舒张的影响。具体目的是：目的#1：确定cTnT和cTnI以及cTnT和cTnC相互作用的亚型特异性区域的功能效应。目标二：验证cTnI和cTnT的蛋白激酶C（PKC）依赖性磷酸化的功能效应具有位点特异性和协同性的假设。目的#3：检验肌动蛋白、cTnT和cTnT的亚型转换和/或cTnI和cTnT的磷酸化以及cTnT与cTnC的磷酸化的假设。目标二：验证cTnI和cTnT的蛋白激酶C（PKC）依赖性磷酸化的功能效应具有位点特异性和协同性的假设。目标3：检验肌动蛋白、cTnI和cTnT的亚型转换和/或cTnI和cTnT的磷酸化调节肌节长度和跨桥结合对肌丝激活的影响的假设。目的#4：确定cTnC的调节（N结构域）和结构（C结构域）与cTnI和cTnT之间的稳态和稳态前结合。该目的检验Tn组分之间的缔合/解离速率的调节影响心脏收缩和/或舒张速率的假设。这一假设是接近使用诱变，重建和转基因模型结合肌丝蛋白的结构-功能分析。实验包括确定力学和力/ATP酶速率在剥皮的纤维束，和表面等离子体共振光谱，以确定Tn组件之间的相互作用的速率。这些实验的结果为理解心脏收缩和舒张信号的正常事件提供了至关重要的信息。这些结果对于理解与缺血、心力衰竭和涉及细丝蛋白的遗传相关的肥大性肌病相关的细丝信号机制的变化机制也是必不可少的。

(Adapted from the Applicant's Abstract) Experiments proposed here test the hypothesis that isoform switching and phosphorylation of cardiac TnI (cTnI) and cTnT are important elements in the intrinsic (Starling's Law) and extrinsic (neurohumoral) control of cardiac output. The long term objective is to identify: 1) unique molecular mechanisms by which Ca2+- and cross-bridge binding to cardiac thin filaments control contraction and relaxation, and 2) the impact of modulation of these mechanisms on activation and relaxation. The specific aims are: Aim #1: To identify functional effects of isoform specific regions of interaction of cTnT and cTnI and of cTnT with cTnC. Aim #2: To test the hypothesis that functional effects of protein kinase C (PKC) dependent phosphorylation of cTnI and cTnT are site specific and synergistic. Aim #3: To test the hypothesis that isoform switching of actin, cTnT and cTnT and/or phosphorylation of cTnI and cTnT and of cTnT with cTnC. Aim #2: To test the hypothesis that functional effects of protein kinase C (PKC) dependent phosphorylation of cTnI and cTnT are site specific and synergistic. Aim #3: To test the hypothesis that isoform switching of actin, cTnI and cTnT and/or phosphorylation of cTnI and cTnT modulate effects of sarcomere length and cross-bridge binding on myofilament activation. Aim #4: To determine steady- and pre-steady state binding between the regulatory (N domain) and structural (C- domain) of cTnC with cTnI and cTnT. This objective tests the hypothesis that modulation of association/dissociation rates between Tn components affects the rates of cardiac contraction and/or relaxation. This hypotheses are approached using mutagenesis, reconstitution, and transgenic models combined with structure-function analysis of myofilament proteins. The experiments include determination of mechanics and force/ATPase rate in skinned fiber bundles, and surface plasmon resonance spectroscopy to determine rates of interaction between Tn components. Results of these experiments provide information crucial to the understanding of normal events that signal contraction and relaxation of the heart. The results are also essential in understanding the mechanism for changes in the thin filament signaling mechanism associated with ischemia, heart failure, and genetically linked hypertrophic myopathies involving the thin filament proteins.