Conditional Knock-Out of Cardiac Myosin Light Chain Kinase Ameliorates Hypertrophic Cardiomyopathy Phenotype in a Murine Model

Abstract

Hypertrophic cardiomyopathy (HCM) is the most commonly inherited cardiac disorder estimated to affect 1 in 200 individuals. It is strongly linked to sarcomeric protein mutations, including cardiac troponin C (cTnC). Within the cardiac sarcomeric thick filament, each myosin heavy chain subunit contains one cardiac regulatory light chain (cRLC). Phosphorylation of the cRLC has been found to increase cardiac muscle force production, myofilament Ca2+-sensitivity, and rate of force redevelopment. Our group has previously established a successful HCM knock-in mouse model through a cTnC A8V (Alanine => Valine) point mutation. This mouse model exhibited decreased ventricular dimensions, diastolic dysfunction, and enhanced systolic function changes that were consistent with the HCM phenotype. The goal of this study was to establish the efficacy of treating an HCM mutation exhibiting enhanced systolic function with a decrease of cRLC phosphorylation through the conditional knock-out of cardiac myosin light chain kinase (cMLCK). Echocardiographic, heart weight/tibia length, histopathology, fibrosis quantification, pressure-volume loop, protein quantification, and RNA sequencing measurements were performed before and after cMLCK KO in cTnC-A8V mouse hearts. This study resulted in overall improvement of cardiac morphology and systolic function as well as a trend in less fibrosis over time in cTnC-A8V mouse hearts with reduced levels of cMLCK. Histopathological analysis concurred with the echocardiography findings displaying a thinning of the previously hypertrophic walls and larger inner left ventricular chambers post-cMLCK KO. Next, western blot analysis will determine potential alterations/corrections in Ca2+ handling protein expression levels. RNA sequencing will assist to determine which genes show significant changes after cMLCK reduction levels in cTnC-A8V mouse hearts. In conclusion, our data strongly support cMLCK inhibition and/or reduction in cRLC phosphorylation levels as a unique treatment opportunity for the reversal of the HCM phenotype.