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

Abstract

Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , 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. Methods: We generated the knock-in mice ( Mylk3 +/fs and Mylk 3 fs/fs ) with a familial dilated cardiomyopathy–associated MYLK3 frameshift mutation ( MYLK3 +/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). Results: Both mice ( Mylk3 +/fs and Mylk 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_ MYLK3 vector. MYLK3 +/fs induced pluripotent stem cell–derived cardiomyocytes 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_ MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V max for ventricular myosin regulatory light chain phosphorylation without affecting the K m . LEUO-1154 treatment of human MYLK3 +/fs induced pluripotent stem cell–derived cardiomyocytes 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 MYLK3 / PPP1R12B 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. Conclusions: 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.

Letter by Okamoto et al Regarding Article, “Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin”

“…We read with great interest the article by Hitsumoto et al showing that cardiac myosin light chain kinase (cMLCK) knock-in mice and cells with the human dilated cardiomyopathy gene mutation developed systolic dysfunction and could be rescued by CRISPR-mediated gene correction and adenoviral vector expression of cMLCK. 1 They used C57BL/6N mice to develop cMLCK knock-in mice. However, we and another group independently discovered that C57BL/6N mice are a natural strain with cMLCK( MYLK3 ) deficiency due to a point mutation that produces an out-of-frame start codon 5 nucleotides upstream from a normal codon compared with C57BL/6J mice.…”

“…The fact that the cMLCK mutant can cause dilated cardiomyopathy both in humans 4 and rodents in their natural courses 1,3 indicates that the regulation of the MLC phosphorylation level plays a very important role in the development of dilated cardiomyopathy and can be a crucial therapeutic target in the heart, as they proposed. 1 We would like to encourage every researcher to use C57BL/6J mice, and not C57BL/6N mice, when they study the regulation of MLC phosphorylation by the balance between cMLCK and MLC phosphatase signaling. 5…”

Response by Tsukamoto and Takashima to Letter Regarding Article, “Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Super-Relaxed Myosin”

“…We thank Okamoto et al for their thoughtful comments regarding our article. 1 They highlighted the importance of considering mouse strains in studying the regulation of myosin light chain phosphorylation via the balance between cMLCK (cardiac-specific myosin regulatory light chain kinase) and myosin regulatory light chain phosphatase signaling. 2 First, we apologize for the error in describing the method of creating knock-in (KI) mice models with a familial dilated cardiomyopathyassociated Mylk3 [myosin light chain kinse 3] frameshift (fs) mutation.…”

“…Indeed, wild-type (Mylk3 +/+ ) and heterozygous mice (MYLK3 +/fs ) express the wild-type cMLCK protein, consistent with the approximately 90-kDa band detected by the cMLCK antibody on western blotting. 1 In addition, the knock-in allele does not have an single nucleotide polymorphism located at nucleotide 146 of exon 1 of Mylk3, a T to A mutation that creates a new initiation codon and causes frameshift and elimination of cMLCK protein expression. 2 Therefore, we concluded that the systolic dysfunction observed in our knock-in mice was caused by cMLCK protein deficiency.…”

Letter by Komamura Regarding Article “Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin”

“…The myocardial contractile mechanisms involving cMLCK (cardiac-specific myosin light chain kinase) discovered by Hitsumoto et al 1 appear to be related to the major causes of human heart failure. Doxorubicin cardiomyopathy due to mitochondrial disorders, ischemic cardiomyopathy due to inflammation and fibrosis after myocardial ischemia, dilated and hypertrophic cardiomyopathy due to sarcomeric protein abnormalities, whatever the cause of contractile dysfunction, the authors found that the ratio of MYLK3/PPP1R12B mRNA was significantly decreased in the myocardium.…”