Functional Consequences of FHC-linked RLC Mutations.

描述（由申请人提供）：家族性肥厚性心肌病（FHC）是一种发现于心脏的病理性代偿表现，其原因是心脏不能充分泵血，从而导致肥厚并常导致心脏性过早死亡。在过去的4年里，我们的实验室一直在研究转基因小鼠中肌球蛋白调控轻链（RLC）中几种FHC突变的功能后果。我们假设，通过改变RLC Ca2+- mg2 +结合位点的特性，FHC突变干扰了RLC作为暂时延迟Ca2+缓冲液的细胞内功能，并导致肌肉松弛动力学的增加或减少。另一种假说涉及突变控制RLC的Ca2+-Mg2+结合位点的金属占用，以及Ca2+或Mg2+结合RLC可能影响肌凝蛋白与肌动蛋白相互作用和张力产生的机制。我们进一步假设FHC诱导的病理心脏表型可以通过Ca2+钙调蛋白激活的rlc突变心肌的MLCK磷酸化来挽救。该应用程序将继续使用从单分子、细胞到器官水平的综合多学科方法和新型转基因小鼠模型来解决以下问题：具体目标1:FHC诱导RLC Ca2+-Mg2+结合位点的特性变化是否抑制或促进RLC作为临时细胞内钙缓冲剂的功能？在肌肉收缩期间，FHC突变是否会改变RLC Ca2+-Mg2+结合位点的金属占用？特异性目标2:Ca2+钙调蛋白（CaM）激活的肌球蛋白轻链激酶（MLCK）的RLC磷酸化是否受到fhc相关RLC突变的影响？MLCK磷酸化能否挽救突变引起的病理性心脏表型？特异性目标3:RLC中fhc相关突变是否会改变RLC与肌凝蛋白重链（HC）之间的分子间相互作用，最终改变肌凝蛋白和肌动蛋白之间的相互作用？这些改变会导致肌丝紊乱、心肌肥厚和突变心肌功能障碍吗？这一建议的成功实施将带来关于RLC在健康和疾病中心肌收缩作用的新的机械、生理和组织学信息。

DESCRIPTION (provided by applicant): Familial hypertrophic cardiomyopathy (FHC) is one of the pathological compensatory manifestations found in the heart resulting from its inability to adequately pump blood, thus leading to hypertrophy and often to premature cardiac death. Over the past 4 years our laboratory has been studying the functional consequences of several FHC mutations in the regulatory light chain (RLC) of myosin expressed in transgenic mice. We hypothesize that by changing the properties of the RLC Ca2+-Mg2+ binding site, the FHC mutations interfere with the intracellular function of RLC as a temporary delayed Ca2+- buffer and lead to increased or decreased kinetics of muscle relaxation. Another hypothesis pertains to the mutation controlled metal occupancy of the Ca2+-Mg2+ binding site of RLC and the mechanism by which Ca2+ or Mg2+ binding to RLC may influence the interaction of myosin with actin and tension generation. We further hypothesize that an FHC induced pathological cardiac phenotype can be rescued by Ca2+-calmodulin activated MLCK phosphorylation of the RLC-mutated myocardium. This application will continue the use of integrated multidisciplinary approaches from single molecule, cell to organ levels and novel transgenic mouse models to address the following questions: SPECIFIC AIM 1: Do FHC induced changes in the properties of the RLC Ca2+-Mg2+ binding site inhibit or facilitate the function of RLC as a temporary intracellular calcium buffer? Do FHC mutations shift the metal occupancy of the RLC Ca2+-Mg2+ binding site during muscle contraction? SPECIFIC AIM 2: Is RLC phosphorylation by Ca2+-calmodulin (CaM) activated myosin light chain kinase (MLCK) affected by FHC-linked RLC mutations? Can MLCK phosphorylation rescue a mutation induced pathological cardiac phenotype? SPECIFIC AIM 3: Do FHC-associated mutations in RLC alter intermolecular interactions between RLC and myosin heavy chain (HC) and ultimately myosin and actin? Do these changes lead to myofilament disarray, cardiac hypertrophy and dysfunction of the mutated myocardium? Successful execution of this proposal will result in new mechanical, physiological and histological information regarding the role of the RLC in cardiac muscle contraction in health and disease. Relevance: Cardiovascular diseases are the number one cause of mortality worldwide with heart failure being highly prevalent in most affluent parts of the world. There is an urgent need for a better understanding of the mechanisms underlying Familial Hypertrophic Cardiomyopathy (FHC) that often leads to premature sudden cardiac death (SCD). This proposal addresses the mechanisms by which the mutations in myosin regulatory light chain (RLC) cause FHC and lead to SCD. Determining the mechanisms of the RLC- mediated regulation of contraction in the healthy and hypertrophic heart will provide insight and be instrumental in developing specific therapeutic strategies that can be employed to reverse or prevent FHC RLC pathology.