Functional Consequences of FHC-linked RLC Mutations

FHC 相关 RLC 突变的功能后果

• 批准号: 6897942
• 负责人: Danuta Szczesna-Cordary
• 金额: $ 37.75万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6897942
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; autosomal dominant trait; binding sites; calcium; gene mutation; genetically modified animals; heart ventricle; histochemistry /cytochemistry; hypertrophic myocardiopathy; laboratory mouse; muscle contraction; myocardium; myosins; phosphorylation; polymerase chain reaction; sarcomeres

DESCRIPTION (provided by applicant): Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease caused by mutations in all of the major sarcomeric proteins, including ventricular myosin RLC. Our recent studies have demonstrated that FHC-linked alterations in the Ca2+ binding properties of RLC could be reversed by RLC phosphorylation. Furthermore, our results suggest that a functional coupling that occurs between phosphorylation and Ca2+ binding to RLC during muscle contraction is most likely altered by the FHC mutations. Our preliminary studies on transgenic E22K skinned papillary muscle fibers demonstrated a large decrease in maximal ATPase activity and force per cross-sectional area compared with transgenic WT mouse fibers. Our working hypothesis is that FHC mutations in myosin RLC alter the Ca2+- and or phosphorylation-dependent regulation of cardiac muscle contraction and decrease the level of force/ATPase that in turn may lead to heart failure. To test this hypothesis and to investigate the mechanisms involved in the RLC-linked pathogenesis of FHC, we will study: SPECIFIC AIM 1: EFFECTS OF THE FHC MUTATIONS IN MYOSIN RLC ON THE Ca2+-DEPENDENT REGULATION OF CARDIAC MUSCLE CONTRACTION. Based on our recent results with transgenic E22K mouse model and the results of profoundly decreased ATPase and force in the N47K- and R58Q-reconstituted fiber systems, it is predicted that the Ca2+ regulation of force/ATPase in intact and skinned papillary muscle fibers derived from N47K and/or R58Q transgenic mice will be even more altered compared to non-transgenic, transgenic-WT or A13T mice. Specifically these transgenic mouse lines will be examined for: a) Ca2+-sensitivity and maximal levels of force and actomyosin ATPase; b) alterations in energy cost or rate of cross-bridge dissociation (ATPase/force) c) kinetics of force development/relaxation (ktr and caged Ca-chelator); d) velocity of shortening; e) diastolic and systolic [Ca2+] and force; f) duration of [Ca2+] and force transients; g) the ability of the muscle to do the work against a constant afterload. SPECIFIC AIM 2: PHYSIOLOGICAL CONSEQUENCES OF THE FHC RLC MUTATIONS ON THE PHOSPHORYLATION-DEPENDENT REGULATION OF CARDIAC MUSCLE CONTRACTION. Studies utilizing various animal models have shown a correlation between the level of RLC phosphorylation and cardiac performance. We hypothesize that FHC mutations interfere with the phosphorylation-dependent regulatory function of the RLC during muscle contraction. We will study the effects of RLC phosphorylation and the physiological significance of phosphorylation in the pathological heart in these transgenic FHC RLC mice. These studies will correlate the observed effects of the RLC mutations in the proposed animal models with the pathogenesis of FHC in humans and will decipher the key mechanisms of the RLC-linked FHC.

描述（由申请人提供）：家族性肥厚性心肌病（FHC）是一种由所有主要肉瘤蛋白（包括心室肌球蛋白RLC）突变引起的常染色体显性疾病。我们最近的研究表明，RLC磷酸化可以逆转RLC的Ca2+结合特性中FHC连接的变化。此外，我们的结果表明，FHC突变很可能会改变肌肉收缩期间磷酸化和与RLC的Ca2+结合之间发生的功能耦合。我们对转基因E22K皮肤乳头肌纤维的初步研究表明，与转基因WT小鼠纤维相比，最大ATPase活性和每个横截面面积的最大ATPase活性和力降低。我们的工作假设是，肌球蛋白RLC中的FHC突变改变了心脏肌肉收缩的Ca2+ - 和/或磷酸化依赖性调节，并降低了力量/ATPase的水平，而力量/ATPase的水平又可能导致心力衰竭。为了检验该假设并研究FHC RLC连接发病机理所涉及的机制，我们将研究：特定目的1：FHC突变在肌球蛋白RLC中对CA2+依赖性心脏肌肉收缩调节的影响。根据我们最近使用转基因E22K小鼠模型的结果以及N47K和R58Q重新定位的纤维系统的ATPase和ATPase和力的结果，可以预测，Intact和Intacted和Skined Papillary肌肉的Ca2+调节力/ATPase的ca2+调节来自N47K的N47K和/或R58Q Tressen-ny-or-R58Q Trresten cy vern-Or-R58Q Trresteric My-cons ca转基因-WT或A13T小鼠。具体而言，将检查这些转基因小鼠系：a）ca2+ - 敏感性和最大力量的力和肌动蛋白ATPase； b）改变跨桥解离的能量成本或速率（ATPase/force）c）力开发/松弛动力学（KTR和笼子Ca-Chelator）； d）缩短速度； e）舒张期和收缩期[Ca2+]和力； f）[Ca2+]和力瞬变的持续时间； g）肌肉能够抵制恒定后负载进行工作的能力。具体目标2：FHC RLC突变对心肌收缩的磷酸化依赖性调节的生理后果。利用各种动物模型的研究表明，RLC磷酸化水平与心脏性能之间的相关性。我们假设FHC突变会干扰肌肉收缩期间RLC的磷酸化依赖性调节功能。我们将研究这些转基因FHC RLC小鼠在病理心脏中RLC磷酸化的作用和磷酸化的生理意义。这些研究将将RLC突变在提出的动物模型中观察到的影响与人类中FHC的发病机理相关联，并破译RLC链接的FHC的关键机制。