Functional Consequences of FHC-linked RLC Mutations

FHC 相关 RLC 突变的功能后果

基本信息

批准号：
6765918
负责人：
Danuta Szczesna-Cordary
金额：
$ 37.62万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
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/6765918
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

项目摘要

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）是一种常染色体显性遗传疾病，由所有主要肌节蛋白（包括心室肌球蛋白 RLC）突变引起。我们最近的研究表明，RLC 的 Ca2+ 结合特性中 FHC 相关的改变可以通过 RLC 磷酸化来逆转。此外，我们的结果表明，肌肉收缩过程中磷酸化和 Ca2+ 与 RLC 结合之间发生的功能耦合很可能因 FHC 突变而改变。我们对转基因 E22K 去皮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 ATP 酶活性和单位横截面积的力大幅下降。我们的工作假设是，肌球蛋白 RLC 中的 FHC 突变改变了心肌收缩的 Ca2+ 和/或磷酸化依赖性调节，并降低了力/ATP 酶水平，进而可能导致心力衰竭。为了检验这一假设并研究 FHC 与 RLC 相关的发病机制，我们将研究：具体目标 1：肌球蛋白 RLC 中的 FHC 突变对心肌收缩的 Ca2+ 依赖性调节的影响。根据我们最近对转基因 E22K 小鼠模型的研究结果以及 N47K 和 R58Q 重建纤维系统中 ATP 酶和力显着降低的结果，预测完整和去皮乳头肌纤维中的 Ca2+ 对力/ATP 酶的调节与非转基因、转基因WT或A13T小鼠相比，N47K和/或R58Q转基因小鼠的改变甚至更大。具体而言，将检查这些转基因小鼠系的： a) Ca2+敏感性以及力和肌动球蛋白 ATP 酶的最大水平； b) 能量消耗或跨桥解离速率的变化（ATP 酶/力） c) 力发展/松弛动力学（ktr 和笼状 Ca 螯合剂）； d) 缩短速度； e) 舒张压和收缩压 [Ca2+] 和力； f) [Ca2+] 和力瞬变的持续时间； g) 肌肉在恒定后负荷下做功的能力。具体目标 2：FHC RLC 突变对磷酸化依赖性心肌收缩调节的生理影响。利用各种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 FHC 突变会干扰肌肉收缩过程中 RLC 的磷酸化依赖性调节功能。我们将研究 RLC 磷酸化的影响以及磷酸化在这些转基因 FHC RLC 小鼠病理心脏中的生理意义。这些研究将在所提出的动物模型中观察到的 RLC 突变的影响与人类 FHC 的发病机制相关联，并将破译 RLC 相关 FHC 的关键机制。