The myosin essential light chain (ELC) is a structural component of the actomyosin cross-bridge, but little is known about its function or the significance of the cardiac specific N-terminal ELC extension on the interaction of myosin with actin, development of force and heart performance. The functional importance of ELC is highlighted by the recent identification of several missense mutations shown to cause Familial Hypertrophic Cardiomyopathy (FHC), a genetic disorder manifested by ventricular enlargement, myofilament disarray and sudden cardiac death (SCD). This proposal will elucidate the mechanisms of ELC-mediated FHC and the role of the direct N-terminal ELC-actin interaction in the normal and FHC heart.
SPECIFIC AIM 1 : DETERMINE THE MECHANISM FOR THE ELC-MUTATION INDUCED DEVELOPMENT OF FHC. We hypothesize that ELC mutations may lead to FHC by affecting the ability of the ELC to form stable structures with the MHC, thus altering the mechanical (stiffness, force) and/or kinetic properties of the myosin cross-bridge during the contractile cycle and development of force. This study is focused on two FHC- mutations localized in two different ELC domains and demonstrating different clinical phenotypes. The N- terminal A57G mutation was shown to cause a classic asymmetric hypertrophy and SCD, and the C-terminal E143K mutation, which presented with mid-cavity hypertrophy, obstruction in systole and restrictive physiology. We propose to determine: (1A) The effect of FHC mutations on the binding of ELC to the 2-MHC, myosin lever arm stiffness and myosin cross-bridge kinetics. (1B) The contractile properties of the mutated myocardium in skinned and intact papillary muscle fibers. (1C) The age-dependent morphology and function of mutated transgenic mouse hearts by histopathology, echocardiography, invasive hemodynamics and MRI.
SPECIFIC AIM 2 : EXPLORE THE MECHANISMS BY WHICH THE N-TERMINUS OF ELC REGULATES MYOSIN FORCE GENERATING CAPABILITY AND CARDIAC MUSCLE CONTRACTION. We hypothesize that the N- terminus of ELC interacts with the actin-tropomyosin-troponin filaments to promote strong cross-bridge formation~ thus it serves as a mechanism to modulate the actin-myosin interaction and myofilament cooperativity, thereby regulating contractile force. Using cardiac muscle preparations from Tg- 43 mice expressing the N-terminal truncated ELC and Tg-WT expressing the full length ELC wild-type, we will study: (2A) the importance of the N-terminus of ELC for myosin force generating capability determined at the molecular and muscle fiber levels. (2B) the effect of the N-terminal ELC extension on myosin cross-bridge kinetics.(2C) The mechanism of the N-terminus ELC-dependent development of cardiac hypertrophy. These studies will help to determine the role(s) of the ELC and the N-terminal cardiac specific ELC extension in the regulation of contractile force in healthy and FHC-diseased hearts.

Public Health Relevance

The goal of this proposal is to understand the mechanism by which the cardiac myosin essential light chains (ELC) regulate the interaction of myosin with actin, force generation and cardiac muscle contraction. In addition, the proposal will unravel the mechanisms, by which genetic mutations in myosin ELC lead to Familial Hypertrophic Cardiomyopathy (FHC). Our developed transgenic animal models and the integration of in vitro approaches with a physiological assessment of cardiac function in vivo will enable us to address the fundamental questions regarding ELC-mediated physiology of the heart and the impact of FHC-linked ELC mutations. Successful execution of this proposal may lay a foundation for the development of novel pharmacological strategies aimed at improving ELC - induced cardiac disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL108343-03
Application #
8586553
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Krull, Holly
Project Start
2011-12-01
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
3
Fiscal Year
2014
Total Cost
$346,687
Indirect Cost
$107,325
Name
University of Miami School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
Wang, Yihua; Ajtai, Katalin; Kazmierczak, Katarzyna et al. (2016) N-Terminus of Cardiac Myosin Essential Light Chain Modulates Myosin Step-Size. Biochemistry 55:186-98
Zhou, Zhiqun; Huang, Wenrui; Liang, Jingsheng et al. (2016) Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants. Front Physiol 7:240
Huang, Wenrui; Liang, Jingsheng; Yuan, Chen-Ching et al. (2015) Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain. FEBS J 282:2379-93
Gomes, Aldrin V; Kazmierczak, Katarzyna; Cheah, Jenice X et al. (2015) Proteomic analysis of physiological versus pathological cardiac remodeling in animal models expressing mutations in myosin essential light chains. J Muscle Res Cell Motil 36:447-61
Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta et al. (2015) Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load. Arch Biochem Biophys 580:14-21
Nagwekar, Janhavi; Duggal, Divya; Midde, Krishna et al. (2015) A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein. Front Cardiovasc Med 2:35
Liang, Jingsheng; Kazmierczak, Katarzyna; Rojas, Ana I et al. (2015) The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice. Biomed Res Int 2015:742536
Matusovsky, Oleg S; Mayans, Olga; Szczesna-Cordary, Danuta (2015) Molecular mechanism of muscle contraction: new perspectives and ideas. Biomed Res Int 2015:694345
Duggal, D; Nagwekar, J; Rich, R et al. (2015) Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions. Am J Physiol Heart Circ Physiol 308:H1248-57
Huang, Wenrui; Szczesna-Cordary, Danuta (2015) Molecular mechanisms of cardiomyopathy phenotypes associated with myosin light chain mutations. J Muscle Res Cell Motil 36:433-45

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