Molecular signaling in cardiac myofilaments

(改编自申请人的摘要)这里提出的实验验证了心脏TnI(CTnI)和cTnT的异构体转换和磷酸化是心输出量的内在(斯塔林定律)和外部(神经体液)控制的重要因素的假设。长期目标是确定：1)钙离子和跨桥结合到心脏细丝控制收缩和松弛的独特分子机制，以及2)这些机制的调制对激活和松弛的影响。具体目的是：目的1：确定cTnT和cTnI相互作用的异构体特异区以及cTnT与cTNC相互作用的功能效应。目的#2：验证蛋白激酶C(PKC)依赖的cTnI和cTnT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：验证肌动蛋白、cTnT和cTnT的异构体转换和/或cTnI和cTnT的磷酸化以及cTnT与cTNC相互作用的假说。目的#2：验证蛋白激酶C(PKC)依赖的cTnI和cTnT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：验证肌动蛋白、cTnI和cTnT的异构体转换和/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说。目的#4：确定cTnC调节域(N结构域)和结构域(C结构域)与cTnI和cTnT的稳态和稳态前结合。这一目标验证了TN组分之间的结合/解离率的调节影响心脏收缩和/或松弛速率的假说。这些假说是通过突变、重组和转基因模型结合肌丝蛋白的结构-功能分析来探讨的。这些实验包括测定去皮纤维束中的力学和力/ATPase比率，以及确定TN组分之间相互作用的速度的表面等离子体共振光谱。这些实验的结果提供了对理解正常事件至关重要的信息，这些事件是心脏收缩和放松的信号。这些结果对于理解涉及细丝蛋白的与缺血、心力衰竭和遗传相关的肥厚性肌病相关的细丝信号机制的变化机制也是至关重要的。

(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.