Dilated cardiomyopathy (DCM) is a major cause of heart failure, characterized by ventricular dilatation and systolic dysfunction. Familial DCM is reportedly caused by mutations in more than 50 genes, requiring precise disease stratification based on genetic information. However, the underlying genetic causes of 60 to 80% of familial DCM cases remain unknown. Here, we identified that homozygous truncating mutations in the gene encoding Bcl-2–associated athanogene (BAG) co-chaperone 5 ( BAG5 ) caused inherited DCM in five patients among four unrelated families with complete penetrance. BAG5 acts as a nucleotide exchange factor for heat shock cognate 71 kDa protein (HSC70), promoting adenosine diphosphate release and activating HSC70-mediated protein folding. Bag5 mutant knock-in mice exhibited ventricular dilatation, arrhythmogenicity, and poor prognosis under catecholamine stimulation, recapitulating the human DCM phenotype, and administration of an adeno-associated virus 9 vector carrying the wild-type BAG5 gene could fully ameliorate these DCM phenotypes. Immunocytochemical analysis revealed that BAG5 localized to junctional membrane complexes (JMCs), critical microdomains for calcium handling. Bag5 -mutant mouse cardiomyocytes exhibited decreased abundance of functional JMC proteins under catecholamine stimulation, disrupted JMC structure, and calcium handling abnormalities. We also identified heterozygous truncating mutations in three patients with tachycardia-induced cardiomyopathy, a reversible DCM subtype associated with abnormal calcium homeostasis. Our study suggests that loss-of-function mutations in BAG5 can cause DCM, that BAG5 may be a target for genetic testing in cases of DCM, and that gene therapy may potentially be a treatment for this disease.

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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.

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