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(申请人提供)：家族性肥厚性心肌病(FHC)是一种常染色体显性遗传病，由包括心室肌球蛋白RLC在内的所有主要肌节蛋白突变引起。我们最近的研究表明，RLC与FHC连接的钙结合特性的改变可以被RLC的磷酸化逆转。此外，我们的结果表明，在肌肉收缩过程中，发生在磷酸化和与RLC结合的钙离子之间的功能偶联很可能被FHC突变改变。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，转基因E22K皮肤乳头肌纤维的最大ATPase活性和单位横截面上的力显著降低。我们的工作假设是，肌球蛋白RLC中的FHC突变改变了依赖钙和/或磷酸化的心肌收缩调节，并降低了力/ATPase水平，进而可能导致心力衰竭。为了验证这一假说，并探讨FHC与RLC相关的发病机制，我们将研究：特异性目标1：肌球蛋白RLC中FHC突变对依赖钙的心肌收缩调节的影响。根据我们最近用转基因E22K小鼠模型的结果，以及N47K和R58Q重组纤维系统中ATPase和FORCE的显著降低的结果，可以预测，与非转基因、转基因WT或A13T小鼠相比，N47K和/或R58Q转基因小鼠完整和剥皮的乳头肌纤维中的FORCE/ATPase的钙调节将发生更大的变化。具体来说，这些转基因小鼠将接受以下检测：a)钙敏感性及最大水平的力和肌动球蛋白ATPase；b)能量成本或跨桥解离速率的变化(ATPase/force)；c)力发展/松弛的动力学(Ktr和笼式钙络合剂)；d)缩短速度；e)舒张期和收缩期[钙]及力；f)[Ca2+]及力的持续时间；g)肌肉抵抗恒定后负荷的能力。特定目的2：FHC RLC突变对依赖磷酸化的心肌收缩调节的生理影响。利用不同动物模型的研究表明，RLC磷酸化水平与心脏功能之间存在相关性。我们假设，在肌肉收缩过程中，FHC突变干扰了RLC的磷酸化依赖的调节功能。我们将在这些转基因FHC RLC小鼠身上研究RLC磷酸化的作用及其在病理心脏中的生理意义。这些研究将把所提出的动物模型中观察到的RLC突变的影响与人类FHC的发病机制联系起来，并将破译RLC连锁FHC的关键机制。