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
Kazmierczak, Katarzyna; Yuan, Chen-Ching; Liang, Jingsheng et al. (2014) Remodeling of the heart in hypertrophy in animal models with myosin essential light chain mutations. Front Physiol 5:353
Muthu, Priya; Liang, Jingsheng; Schmidt, William et al. (2014) In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain. Arch Biochem Biophys 552-553:29-39
Huang, Wenrui; Liang, Jingsheng; Kazmierczak, Katarzyna et al. (2014) Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice. J Mol Cell Cardiol 74:318-29
Michael, John Jeshurun; Gollapudi, Sampath K; Ford, Steven J et al. (2013) Deletion of 1-43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca(2+) sensitivity and cross-bridge detachment kinetics. Am J Physiol Heart Circ Physiol 304:H253-9
Wang, Li; Muthu, Priya; Szczesna-Cordary, Danuta et al. (2013) Diversity and similarity of motor function and cross-bridge kinetics in papillary muscles of transgenic mice carrying myosin regulatory light chain mutations D166V and R58Q. J Mol Cell Cardiol 62:153-63
Kazmierczak, Katarzyna; Paulino, Ellena C; Huang, Wenrui et al. (2013) Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol 305:H575-89