AUTHOR=Kazmierczak Katarzyna , Liang Jingsheng , Gomez-Guevara Michelle , Szczesna-Cordary Danuta 

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

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=Volume 9 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2022.988066

DOI=10.3389/fcvm.2022.988066

ISSN=2297-055X

ABSTRACT=In this study, we focused on the effects of myosin regulatory light chain (RLC) pseudo-phosphorylation on the function of myosin motors in cardiac myosin and left ventricular papillary muscle (LVPM) preparations reconstituted with two phosphomimetic mutants of the human ventricular RLC, S15D and T160D RLCs. The S15D mimics the phosphorylation of the established serine-15 site and the T160D, the phosphorylation of threonine-160 identified by computational analysis as a high-score phosphorylation site of the RLC. Myosin and LVPM fibers were isolated from the hearts of transgenic (Tg) wild-type (WT) and hypertrophic cardiomyopathy (HCM) Tg-R58Q mice. They were then subjected to depletion of endogenous RLC followed by reconstitution with recombinant S15D, T160D phosphomimetic RLCs, and WT and R58Q-RLC controls. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D or WT-RLC-reconstituted myosin preparations. Both S15D and T160D phosphomimetic RLCs rescued Vmax of Tg-R58Q myosin reconstituted with R58Q-RLC, but the effect of S15D-RLC was significantly larger than T160D-RLC. Likewise, low tension observed for R58Q-reconstituted LVPM was rescued by both phosphomimetic RLCs. In the HCM-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to disordered relaxed (DRX) increasing the number of heads readily available to interact with actin and produce force, while T160D-RLC stabilized the SRX state at the level similar to R58Q-reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC versus the C-terminal T160-RLC with S15D-RLC showing therapeutic potential in mitigating the hypocontractile behavior of HCM-R58Q myocardium.