Molecular signaling in cardiac myofilaments

心肌丝中的分子信号传导

• 批准号: 6607095
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-06-15 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6607095
• 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.

（根据申请人摘要的改编）此处提出的实验检验了一种假设，即心脏TNI（CTNI）和CTNT的同工型切换和磷酸化是内在（Starling的定律）和外在（Neurohumoral）控制心脏输出的重要元素。长期目标是识别：1）CA2+ - 与心脏薄丝控制收缩和放松结合的独特分子机制，以及2）调制这些机制对激活和放松的影响。具体目的是：目标＃1：确定CTNT和CTNI相互作用以及CTNT与CTNC相互作用的功能效应。目的＃2：检验以下假设：蛋白激酶C（PKC）依赖性磷酸化的功能效应对CTNI和CTNT的磷酸化是特定的且协同作用。目的＃3：测试肌动蛋白，CTNT和CTNT和/或CTNI和CTNT以及用CTNC的CTNT的同工型切换的假设。目的＃2：检验以下假设：蛋白激酶C（PKC）依赖性磷酸化的功能效应对CTNI和CTNT的磷酸化是特定的且协同作用。目的＃3：测试肌动蛋白，CTNI和CTNT的同工型切换和/或CTNI和CTNT的磷酸化的假设调节肌膜长度和跨桥结合对肌膜激活的影响。目标＃4：用CTNI和CTNT确定CTNC的调节（N域）和结构（C-域）之间的稳态和稳态结合。该目标检验了以下假设：TN组分之间的关​​联/解离速率的调节会影响心脏收缩和/或放松的速度。使用诱变，重构和转基因模型与肌丝蛋白的结构 - 功能分析相结合，对此进行了假设。实验包括确定皮肤纤维束中的力学和力/ATPase速率，以及表面等离子体共振光谱，以确定TN组件之间的相互作用速率。这些实验的结果为了解正常事件的理解提供了信号收缩和心脏放松的信息至关重要。结果对于理解与缺血，心力衰竭以及涉及薄丝细丝蛋白的遗传连接的肥厚性肌病有关的薄丝信号传导机制变化的机制也至关重要。