Redefining The Role Of Myosin Essential Light Chain In Cardiac Muscle

摘要 本应用的科学前提是认为长ELC的心脏特异性表达包含 N-末端ELC延伸(N-ELC)及其在肌球蛋白运动功能调节中的重要生理作用 并推动肌肉的生产。我们假设心脏N-ELC作为分子连接物和/或 肌动蛋白-肌球蛋白相互作用的能量开关--通过调节肌球蛋白跨桥的转变 松弛(SRX)到无序松弛(DRX)状态，以及可用于 与肌动蛋白的相互作用。在DRX中，肌球蛋白头部伸入细丝间隙，但受到限制 与肌动蛋白结合，而在SRX状态下，头部沿着粗丝轴整齐排列，但以 高度抑制ATP周转率。我们进一步假设肥厚型心肌病基因突变 肌球蛋白ELC促进SRX到DRX的转变，N-ELC的缺失抵消了这一转变，并 稳定节能SRX状态。我们将使用两个小鼠模型来检验这些假设 肥厚型HCM-A57G(丙氨酸57甘氨酸)和限制性RCM-E143K(谷氨酸143赖氨酸) 心肌病。我们还将使用表达43-aa截短ELC的转基因Δ43小鼠，以及双转基因 小鼠：A57GxΔ43和E143KxΔ43，在HCM或RCM突变的背景下表达Δ43-ELC。数据 将与表达人心室ELC(MYL3基因)的年龄和性别匹配的WT-ELC小鼠进行比较。 特异性目的1：确定肌球蛋白ELC及其N末端(N-ELC)在调节中的作用 肌球蛋白运动功能和心肌收缩。这个目标将集中在分子上 从肌球蛋白分子、肌纤维和整体水平研究ELC诱导的心脏重构的触发因素 心。N-ELC依赖的肌动蛋白-肌球蛋白相互作用的调节将使用：1)MANT-ATP-Chase SRX和DRX状态下肌球蛋白头的测定和比例；2)量子点标记的肌动蛋白±TM-TN运动 3)剥离和完整的肌纤维和力-PCA以及力和钙的瞬变测量； 4)超声心动图和有创血流动力学评价活体心脏的收缩反应。 特异性目标2：肌球蛋白ELC在小鼠心脏能量重塑中的作用 心肌病的症状。A57G、E143K、Δ43、A57GxΔ43和A57Gx基因的代谢图谱分析 将执行E143KxΔ43与WT心脏对比，以确定管理能源利用的特定机制 在所有ELC突变小鼠与WT小鼠中。线粒体功能将通过测定线粒体来评估 呼吸作用、呼吸复合体的酶活性以及ATP的产生/消耗。稳态 与能量产生有关的候选蛋白的水平，如己糖激酶I和己糖激酶II，各种线粒体 呼吸链亚单位(复合体I至V)以及过氧化物酶体增殖物激活受体共激活物α (pGC-1α)将在所有模型中进行评估，以表征心肌疾病的更广泛影响 由ELC链接的修改产生。

ABSTRACT The scientific premise of this application regards the cardiac specific expression of the long ELC containing the N-terminal ELC extension (N-ELC) and its physiologically important role in regulating myosin motor function and force production in muscle. We hypothesize that cardiac N-ELC works as a molecular linker and/or energetic switch of the actin-myosin interaction by regulating the transition of myosin cross-bridges from a super- relaxed (SRX) to a disordered-relaxed (DRX) state, and the proportion of myosin heads that are available for the interaction with actin. In DRX, the myosin heads protrude into the interfilament space but are restricted from binding to actin, while in the SRX state, the heads are neatly ordered along the thick filament axis but cycle at a highly inhibited ATP turnover rate. We further hypothesize that hypertrophic cardiomyopathy mutations in myosin ELC promote the SRX-to-DRX transition and the deletion of the N-ELC counteracts this transition and stabilizes the energy conservation SRX state. We will test these hypotheses using two mouse models of hypertrophic HCM-A57G (Alanine57Glycine) and restrictive RCM-E143K (Glutamate143Lysine) cardiomyopathy. We will also use transgenic Δ43 mice expressing a 43-aa truncated ELC, and double transgenic mice: A57GxΔ43 and E143KxΔ43, expressing Δ43-ELC in the background of HCM or RCM mutations. The data will be compared to age and sex matched WT-ELC mice expressing the human ventricular ELC (MYL3 gene). SPECIFIC AIM 1: DEFINE THE ROLE OF MYOSIN ELC AND ITS N-TERMINUS (N-ELC) IN THE REGULATION OF MYOSIN MOTOR FUNCTION AND CARDIAC MUSCLE CONTRACTION. This aim will focus on the molecular triggers of ELC-induced heart remodeling studied at the level of myosin molecules, myofibers and the whole heart. The N-ELC-dependent regulation of actin-myosin interactions will be studied using: 1) mant-ATP-chase assays and the proportion of myosin heads in SRX vs. DRX states; 2) Quantum dot-labeled actin±Tm-Tn motility assays; 3) Skinned and intact muscle fibers and force-pCa as well as force and calcium transient measurements; and 4) echocardiography and invasive hemodynamics to assess contractile responses of the heart in vivo. SPECIFIC AIM 2: THE ROLE MYOSIN ELC IN ENERGETIC REMODELING OF THE HEART IN MOUSE MODELS OF CARDIOMYOPATHY. The analysis of the metabolic landscape in A57G, E143K, Δ43, A57GxΔ43 and E143KxΔ43 vs. WT hearts will be performed to identify the specific mechanisms that govern energy utilization in all ELC-mutant vs. WT mice. Mitochondrial function will be assessed by determination of mitochondrial respiration, enzymatic activity of respiratory complexes as well as ATP production/consumption. Steady-state levels of candidate proteins implicated in energy production such as Hexokinases I and II, various mitochondrial respiratory chain subunits (Complexes I to V) as well as peroxisome proliferator-activated receptor coactivator α (PGC-1α) will be assessed in all models to characterize the broader implications of cardiac muscle disease resulting from ELC-linked modifications.