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在内的所有主要肌瘤蛋白突变引起。我们最近的研究表明，fhc相关的RLC Ca2+结合特性的改变可以通过RLC磷酸化逆转。此外，我们的研究结果表明，在肌肉收缩过程中，磷酸化和Ca2+结合到RLC之间发生的功能偶联很可能被FHC突变改变。我们对转基因E22K皮肤乳头状肌纤维的初步研究表明，与转基因WT小鼠纤维相比，最大atp酶活性和每横截面积的力大大降低。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，降低了力/ atp酶的水平，从而可能导致心力衰竭。为了验证这一假设并研究与RLC相关的FHC发病机制，我们将研究：特异性目标1：肌球蛋白RLC中FHC突变对心肌收缩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重组纤维系统中atp酶和力的显著降低，我们预测，与非转基因、转基因wt或A13T小鼠相比，N47K和/或R58Q转基因小鼠的完整和皮肤乳头状肌纤维中Ca2+对力/ atp酶的调节将会发生更大的改变。具体来说，这些转基因小鼠系将被检查：a) Ca2+敏感性和最大水平的力和肌动球蛋白atp酶；b)能量消耗或跨桥解离速率的变化（atp酶/力）c)力发展/弛缓动力学（ktr和笼型螯合剂）；D)缩短速度；e)舒张和收缩[Ca2+]和力；f) [Ca2+]和力瞬态持续时间；G)肌肉在恒定后负荷下完成工作的能力。具体目的2:FHC RLC突变对心肌收缩磷酸化依赖性调节的生理影响。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突变干扰了RLC在肌肉收缩过程中磷酸化依赖的调节功能。我们将在这些转基因FHC RLC小鼠中研究RLC磷酸化的作用以及磷酸化在病理心脏中的生理意义。这些研究将在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相关的FHC的关键机制。

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.