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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL071778-01A1
Application #
6686520
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Program Officer
Reinlib, Leslie
Project Start
2003-07-01
Project End
2007-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
1
Fiscal Year
2003
Total Cost
$367,420
Indirect Cost
Name
University of Miami School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
052780918
City
Miami
State
FL
Country
United States
Zip Code
33146
Yadav, Sunil; Kazmierczak, Katarzyna; Liang, Jingsheng et al. (2018) Phosphomimetic-mediated in vitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain. FEBS J :
Wang, Li; Kazmierczak, Katarzyna; Yuan, Chen-Ching et al. (2017) Cardiac contractility, motor function, and cross-bridge kinetics in N47K-RLC mutant mice. FEBS J 284:1897-1913
Yuan, Chen-Ching; Muthu, Priya; Kazmierczak, Katarzyna et al. (2015) Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice. Proc Natl Acad Sci U S A 112:E4138-46
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; 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
Karabina, Anastasia; Kazmierczak, Katarzyna; Szczesna-Cordary, Danuta et al. (2015) Myosin regulatory light chain phosphorylation enhances cardiac ?-myosin in vitro motility under load. Arch Biochem Biophys 580:14-21
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
Farman, Gerrie P; Muthu, Priya; Kazmierczak, Katarzyna et al. (2014) Impact of familial hypertrophic cardiomyopathy-linked mutations in the NH2 terminus of the RLC on ?-myosin cross-bridge mechanics. J Appl Physiol (1985) 117:1471-7
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
Nagwekar, J; Duggal, D; Rich, R et al. (2014) The spatial distribution of actin and mechanical cycle of myosin are different in right and left ventricles of healthy mouse hearts. Biochemistry 53:7641-9

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