Hypercontractility and Oxidative Stress Drive Creatine Kinase Dysfunction in Hypertrophic Cardiomyopathy.
Xu A., Weissman D., Ermer KJ., Bertero E., Federspiel JM., Stadler F., Grünler E., Tangos M., Zervou S., Waddingham MT., Pearson JT., Reil J-C., Scholtz S., Dudek J., Kohlhaas M., Nickel AG., Carrier L., Eschenhagen T., Michels M., Dos Remedios C., Lal S., Prates Roma L., Hamdani N., Kuster DWD., Falcão-Pires I., Johnson CN., Lygate CA., van der Velden J., Maack C., Sequeira V.
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a prevalent inherited cardiac disorder marked by left ventricular hypertrophy and hypercontractility. This excessive mechanical workload creates an energetic mismatch in which consumption exceeds production, leading to myocardial energy depletion. Although CK (creatine kinase) plays a key role in cardiac energy homeostasis, its involvement in HCM remains unclear. This study investigates how hypercontractility-driven mitochondrial stress and the resulting increase in mitochondrial H2O2 disrupt CK function in HCM. METHODS: CK function was analyzed using myocardial left ventricular tissue from 92 patients with HCM (with and without pathogenic sarcomere variants) and 30 non-failing human controls. Myofilament and mitochondrial CK isoforms were measured using mRNA analysis, protein immunoblotting, enzyme activity assays, mass spectrometry, and redox-sensitive proteomics. To explore links between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction, we used isolated cardiomyocytes from wild-type, mitochondrial-targeted catalase-overexpressing, CK knockout (myofilament and mitochondrial CK deletion), HCM-associated Mybpc3 knock-in, and mito-roGFP2-Orp1 mouse models. We also tested the effects of the Ca2+ sensitizer EMD-57033, the CK inhibitor 1-fluoro-2,4-dinitrobenzene (DNFB), and the myosin inhibitor MYK-581, a mavacamten derivative. RESULTS: Our analysis revealed significant reductions in myofilament and mitochondrial CK protein levels, as well as CK activity, in myocardium of patients with HCM, primarily because of oxidative modifications of CK. In isolated mouse cardiomyocytes from wild-type and CK knockouts, hypercontractility induced by EMD-57033 elevated mitochondrial H2O2, causing cellular arrhythmias and CK inactivation. Hypercontractility-induced oxidative stress, arrhythmias, and CK dysfunction were also observed in Mybpc3 knock-in cardiomyocytes. Mitochondrial-targeted catalase-overexpressing mice with enhanced H2O2 scavenging were protected against H2O2-induced (EMD-57033-mediated) arrhythmias and CK dysfunction. MYK-581 treatment in Mybpc3 knock-in cardiomyocytes reduced hypercontractility, lowered H2O2 production and arrhythmias, and preserved CK function. CK inhibition using DNFB in wild-type cardiomyocytes elevated mitochondrial H2O2 levels and triggered cellular arrhythmias. This mitochondrial oxidation was independently confirmed in mito-roGFP2-Orp1 cardiomyocytes exposed to DNFB. Mitochondrial-targeted catalase-overexpressing mice were protected from DNFB-induced oxidative stress and arrhythmogenic events. CONCLUSIONS: This study reveals a mechanistic link between hypercontractility, mitochondrial reactive oxygen species, and CK dysfunction in HCM, perpetuating a cycle of energetic dysfunction. Targeting hypercontractility and oxidative stress through myosin inhibition offers a strategy to restore energy balance and reduce arrhythmic risk in HCM.