Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Oxidation of mitochondrial creatine kinase in hypertrophic cardiomyopathy facilitates ROS formation and premature permeability transition pore opening through loss of its chaperone function
A. Xu1, F. Stadler1, C. Lygate2, D. Kuster3, J. van der Velden3, J. Dudek1, A. Nickel1, N. Hamdani4, C. Maack1, V. Sequeira1
1Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg; 2Dept. of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, UK; 3Dept. of physiology, Amsterdam University Medical Center, Amsterdam, NL; 4Forschungslabor Molekulare Kardiologie, Kath. Klinikum Bochum, Bochum;

Introduction

In mouse models of hypertrophic cardiomyopathy (HCM), we previously revealed that increased myofilament Ca2+ sensitivity exaggerates energetic demand that oxidizes NAD(P)H in mitochondria, resulting in reactive oxygen species (ROS) emission and arrhythmias. Furthermore, we found that these ROS oxidize and thereby inactivate myofilament creatine kinase (M-CK), and similar M-CK oxidation correlates with decreased M-CK activity in human HCM myocardium. Since phosphocreatine (PCr) flux depends on both M-CK and mitochondrial CK operating as a functional unit, we addressed the role of mt-CK for mitochondrial respiration, ROS formation and Ca2+ retention.

Methods & Results

Using comprehensive mass spectrometry in human myocardium from patients with HCM and non-failing donors, we observed protein downregulation and oxidation of mt-CK at Cys317 in HCM, a site known to be essential for enzyme activity and protein assembly. Functional Mt-CK exists as an octamer, which (besides its enzymatic activity) is thought to behave as a scaffold that bridges the inner and outer mitochondrial membrane in the intermembrane space. The chemical compound 2,4-dinitro-1-fluorobenzene (DNFB) is a known inhibitor of CK activity and led to a disassembly of the octameric structure of mt-CK, visualized by gel electrophoresis, at similar concentrations as its inhibitory effect on CK activity (i.e., between 1 and 20 µM). Similar disassembly of octameric mt-CK was previously shown to be induced by oxidation at Cys317 in reconstituted cell systems, the site we found oxidized in human HCM myocardium. Therefore, we used DNFB to mimic Cys317-oxidation-induced mt-CK disassembly in isolated murine cardiac mitochondria respiring on pyruvate/malate. Here, DNFB led to a concentration-dependent increase in mitochondrial superoxide formation (determined by electroparamagnetic resonance, EPR) as well as H2O2 emission (detected by Amplex UltraRed) in both the absence (state 2) or presence of 1 mM ADP (state 3), without reducing the rates of respiration (i.e., O2 consumption) per se or affecting the assembly of respiratory chain complexes, as determined by native PAGE protein analysis. Furthermore, DNFB led to premature opening of the mitochondrial permeability transition pore (mPTP) in response to repetitive extramitochondrial Ca2+ pulses in cardiac mitochondria. To rule out that these effects of DNFB were related to non-specific effects, we performed similar experiments in liver mitochondria, which have very low mt-CK expression. Intriguingly, DNFB-induced .O2- and H2O2 formation as well as premature mPTP opening (as observed in cardiac mitochondria) were all absent in liver mitochondria, and respiration was also unafffected.

Conclusion

In human HCM, mt-CK is oxidized at Cys317, a modification previously shown to destabilize the mt-CK octamer. Similar mt-CK disassembly with DNFB increases mitochondrial ROS formation and emission and sensitizes the mPTP to premature opening in cardiac, but not liver mitochondria, without affecting respiratory chain assembly or respiration per se. Therefore, mt-CK oxidation and subsequent disassembly may sustain a feed-forward loop in which pathological energy demand at the myofilaments aggravates deficient ATP delivery to myofilaments through redox-dependent inactivation of mt-CK, which further increases mitochondrial ROS formation by losing its chaperon function in the intermembrane space.


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