Redefining The Role Of Myosin Essential Light Chain In Cardiac Muscle

重新定义肌球蛋白必需轻链在心肌中的作用

基本信息

批准号：
10589886
负责人：
Danuta Szczesna-Cordary
金额：
$ 38.38万
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：
2020
资助国家：
美国
起止时间：
2020-04-05 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10589886
关键词：
Actins Age Alanine Alkalies Amino Acids Binding Biological Assay Calcium Cardiac Cardiac Muscle Contraction Cardiomyopathies Cells Complex Consumption Cytoskeleton Data Disease Echocardiography Etiology Event Filament Fluorescence Fluorescent Probes Frequencies Genes Glycine Health Heart Heart Diseases Homeostasis Human Hypertrophic Cardiomyopathy Hypertrophy In Vitro Investigation Label Light Link Lysine Measurement Metabolic Mitochondria Modeling Modification Molecular Motor Mus Muscle Muscle Contraction Muscle Fibers Mutate Mutation Myocardium Myosin ATPase Myosin Alkali Light Chains N-terminal Organ Organelles Output Oxidative Phosphorylation Pathologic Peroxisome Proliferator-Activated Receptors Phenotype Physiological Play Production Proteins Quantum Dots RNA Splicing Regulation Relaxation Respiration Respiratory Chain Role Skeletal Muscle Skin Smooth Muscle Myosins System Testing Thick Filament Transgenic Mice Transgenic Organisms Variant Ventricular Work cardiac muscle disease cell motility hemodynamics hexokinase in vivo insight mouse model mutant novel novel therapeutics papillary muscle respiratory response sex

项目摘要

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.

抽象的该应用的科学前提涉及含有长ELC的心脏特异性表达 N末端ELC扩展（N-ELC）及其在调节肌球蛋白运动功能中的物理重要作用并在肌肉中产生武器。我们假设心脏N-ELC充当分子接头和/或通过从超级 - 放松（SRX）到无序 - 浮肿（DRX）状态，以及可用于该状态的肌球蛋白头的比例与肌动蛋白的相互作用。在DRX中，肌球蛋白头突出到丝间空间，但受到限制与肌动蛋白结合，而在SRX状态下，头部沿着厚的细丝轴整齐地排序，但在A 高度抑制的ATP周转率。我们进一步假设肥厚的心肌病突变肌球蛋白ELC促进SRX-to-DRX过渡和N-ELC的删除，以抵消此过渡和稳定节能SRX状态。我们将使用两种鼠标模型来检验这些假设肥厚的HCM-A57G（丙氨酸57-甘氨酸）和限制性RCM-E143K（谷氨酸盐143期）心肌病。我们还将使用表达43-AA截短的ELC和双转基因的转基因Δ43小鼠小鼠：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。特定目的1：定义肌球蛋白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。这个目标将集中于分子肌球蛋白分子，肌纤维和整个水平的ELC诱导的心脏重塑研究的触发器心。肌动蛋白 - 肌球蛋白相互作用的N-ELC依赖性调节将使用：1）MANT-ATP-CHASE SRX与DRX状态中的肌球蛋白头的比例和肌球蛋白头的比例； 2）量子点标记的肌动蛋白±TM-TN运动性测定； 3）皮肤和完整的肌肉纤维以及力-PCA以及力和钙瞬态测量； 4）超声心动图和侵入性血流动力学评估体内心脏的收缩反应。特定目标2：肌球蛋白ELC在小鼠模型中心脏的能量重塑中的作用心肌病。 A57G，E143K，Δ43，A57GXΔ43和将执行E143KXΔ43与WT心脏，以识别控制能量利用的特定机制在所有ELC突变与WT小鼠中。线粒体功能将通过测定线粒体评估呼吸，呼吸复合物的酶活性以及ATP的生产/消费。稳定状态在能源生产中实施的候选蛋白水平，例如己糖酶I和II，各种线粒体呼吸链亚基（复合物I至V）以及过氧化物组增殖物激活的受体共激活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含义由ELC连接的修改产生。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.

DOI：
10.3389/fcvm.2022.988066
发表时间：
2022
期刊：
FRONTIERS IN CARDIOVASCULAR MEDICINE
影响因子：
3.6
作者：
Kazmierczak, Katarzyna;Liang, Jingsheng;Gomez-Guevara, Michelle;Szczesna-Cordary, Danuta
通讯作者：
Szczesna-Cordary, Danuta

Phosphorylation Mimetic of Myosin Regulatory Light Chain Mitigates Cardiomyopathy-Induced Myofilament Impairment in Mouse Models of RCM and DCM.