Molecular signaling in cardiac myofilaments

心肌丝中的分子信号传导

• 批准号: 6340113
• 负责人: R John Solaro
• 金额: $ 28.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6340113
• 关键词: actins; genetically modified animals; heart contraction; laboratory mouse; microfilaments; muscle contraction; myocardium; myosins; protein isoforms; protein kinase C; protein protein interaction; protein reconstitution; protein structure function; sarcomeres; site directed mutagenesis; surface plasmon resonance; troponin

(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.

(改编自申请人的摘要)本文提出的实验测试了以下假设：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)和外在(神经体液)控制中的重要元素。长期目标是确定：1​​) Ca2+- 和跨桥与心脏细丝结合的独特分子机制控制收缩和舒张，以及 2) 这些机制的调节对激活和舒张的影响。具体目标是： 目标#1：确定 cTnT 和 cTnI 以及 cTnT 与 cTnC 相互作用的异构体特定区域的功能效应。目标 #2：检验以下假设：蛋白激酶 C (PKC) 依赖性 cTnI 和 cTnT 磷酸化的功能效应具有位点特异性和协同作用。目标#3：检验肌动蛋白、cTnT 和 cTnT 异构体转换和/或 cTnI 和 cTnT 以及 cTnT 与 cTnC 磷酸化的假设。目标 #2：检验以下假设：蛋白激酶 C (PKC) 依赖性 cTnI 和 cTnT 磷酸化的功能效应具有位点特异性和协同作用。目标#3：检验以下假设：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。目标#4：确定 cTnC 的调节（N 结构域）和结构（C 结构域）与 cTnI 和 cTnT 之间的稳态和预稳态结合。该目的测试了以下假设：Tn 成分之间的结合/解离速率的调节会影响心脏收缩和/或舒张的速率。该假设是通过使用诱变、重构和转基因模型并结合肌丝蛋白的结构功能分析来实现的。实验包括测定带皮纤维束中的力学和力/ATP 酶速率，以及确定 Tn 组分之间相互作用速率的表面等离子体共振光谱。这些实验的结果提供了对于理解心脏收缩和舒张信号的正常事件至关重要的信息。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肥厚性肌病相关的细丝信号传导机制的变化机制也至关重要。