Introduction

In hypertrophic cardiomyopathy (HCM), the onset of septal hypertrophy is preceded by energetic deficits. In prior research, we demonstrated that increased energetic demand at the myofilaments of HCM animal models results in oxidation of mitochondrial NAD(P)H. Since its regeneration is not adequately matched by mitochondrial Ca2+ uptake, this results in increased reactive oxygen species (ROS) emission and arrhythmias. Here, we examined whether mitochondrial ROS further impair energy regeneration via the creatine kinase (CK) pathway, both at mitochondrial (Mt-CK) and myofilamental (M-CK) sites.

Methods & Results

Compared to non-failing donor controls (n=8-14), expression of M-CK in western blot analyses was reduced in human myocardium from patients with HCM (n=22-30), related primarily to a loss of the reduced form of CK to 58±3% of control. This correlated tightly to reduced CK activity (to 47±5% of control) as revealed by colorimetric CK activity assays. OxICAT labeling mass spectrometry revealed oxidation of M-CK at two distinct cysteines (Cys146 and Cys254) that are required for kinase activity. To recapitulate HCM conditions, mouse cardiomyocytes were exposed to the Ca²⁺ sensitizer EMD-57033 (EMD) or vehicle, paced at 5 Hz and stimulated with the β-adrenergic agonist isoproterenol. Compared to vehicle, EMD oxidized mitochondrial NAD(P)H, elevated H₂O₂ emission, and deactivated M-CK activity (by 28.1±2.1%). All these effects were prevented in myocytes from mice with mitochondrial overexpression of catalase. Mass spectrometry of human HCM myocardium further revealed oxidation of Mt-CK at Cys317, a critical site for enzyme function and protein assembly. Unlike dimeric M-CK, Mt-CK is an octamer that is thought to function also as a scaffold within the mitochondrial intermembrane space. Reduction of CK activity by the specific inhibitor DNFB (1 and 20µM) was associated with disassembly of the octameric Mt-CK supercomplexes into dimers and an increase of mitochondrial superoxide formation (+37%; determined by electroparamagnetic resonance) and H₂O₂ emission (detected by Amplex UltraRed), while ADP-induced O2 consumption in mitochondria respiring on pyruvate/malate was hardly affected. Additionally, native PAGE protein analysis revealed no alterations to the respirasome’s composition when mitochondria were subjected to 20 µM DNFB. To further exclude nonspecific effects of DNFB, cardiac mitochondria were compared to liver mitochondria, which express Mt-CK at a very low level. Intriguingly, formation of H₂O₂ and superoxide was essentially nonexistent in liver mitochondria exposed to DNFB.

Conclusion

Hypercontractility of myofilaments induces a Ca²⁺ mismatch in mitochondria, causing mitochondrial oxidation of NAD(P)H and ROS emission, which in turn oxidizes both M-CK and Mt-CK, which lowers their activities. In addition, disruption of Mt-CK octameres generates additional ROS, likely by electron leakage from the respiratory chain. These findings suggest a futile feed-forward mechanism of energetic and redox mismatch in HCM, which likely exacerbates diastolic dysfunction and – according to previous findings from our group – accounts for arrhythmias by providing a trigger (redox) and substrate (energy depletion) for arrhythmias.