Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

Abstract

Cardiac-specific myosin light chain kinase (cMLCK), encoded by <i>MYLK3</i>, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. We generated the knock-in mice (<i>Mylk3</i>^+/fs and <i>Mylk</i>3^fs/fs) with a familial dilated cardiomyopathy-associated <i>MYLK3</i> frameshift mutation (<i>MYLK3</i>^+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). Both mice (<i>Mylk3</i>^+/fs and <i>Mylk</i>3^fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_<i>MYLK3</i> vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3_+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_<i>MYLK3</i> vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the <i>V</i>_max for ventricular myosin regulatory light chain phosphorylation without affecting the <i>K</i>_m. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3_+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower <i>MYLK3</i>/<i>PPP1R12B</i> messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.