Molecular Mechanisms of Myosin Light Chain Mutation-Induced Cardiomyopathies

of a dissertation at the University of Miami. In this proposal, I have studied the HCM (hypertrophic cardiomyopathy) and DCM (dilated cardiomyopathy) disease causing mechanisms associated with mutations in the myosin regulatory (RLC) and essential (ELC) light chains. Specifically, four HCM mutations, RLC-A13T, RLC-K104E, ELC-A57G and ELC-M173V and one DCM mutation, RLC-D94A were studied. The RLC-A13T, RLC-K104E and ELC-A57G mutations were primarily investigated in transgenic (Tg) mice using in vitro and in vivo approaches, while RLC-D94A and ELC-M173V were studied in reconstituted system because of no Tg mice available in the laboratory (Specific Aims 1&2). In addition, the effects of RLC/ELC serine phosphorylation on the structure and function of the heart were examined (Specific Aim 3). Our studies indicated that RLC and ELC mutations lead to cardiomyopathy disease through different mechanisms and therefore resulted in different disease phenotypes. Specially, RLC mutations (exclude D94A) resulted in a classic HCM phenotype as left ventricular (LV) / septum hypertrophy and diastolic dysfunction. RLC mutations caused HCM or DCM through altering the secondary structure of the RLC, which further affected the structure (and function) of the lever arm domain imposing changes in the cross bridge cycling rate, myosin force generation ability and muscle relaxation. On the other hand, ELC mutations (e.g. A57G) caused a rare HCM phenotype as eccentric hypertrophy and systolic dysfunction. As for the RLC, ELC mutations also exert their detrimental effects through altering the structure (and function) of the ELC, especially its N-terminus. These changes further affect the N-ELC-actin interactions and the cross talk between the thin and thick filaments resulting in altered force generation and the calcium sensitivity of force (Special Aim 1&2). In Specific Aim 3, we have examined the rescue effects of Serine15 phosphorylation in the RLC and Serine 195 phosphorylation in the ELC. For RLC, we observed a myosin light chain kinase (MLCK)-induced phosphorylation was able to rescue the abnormally high IFS observed in the K104E fibers. However, compromised force generation observed in K104E myocardium was not restored upon K104E phosphorylation. The effects of phosphorylation on the A13T and D94A RLC mutants are still to be studied. For ELC, since no ELC specific kinase exists, Serine195 was mutated to Aspartic Acid (S195D) to mimic ELC phosphorylation. The S195D-ELC protein partially restored the reduced Vmax observed in M173V-ELC exchanged myosin and " corrected " the abnormally high calcium sensitivity of force in A57G-S195D and M173V-S195D exchanged porcine cardiac …