Introduction

In a mouse model of hypertrophic cardiomyopathy (HCM) we previously established the link between cellular hypercontractility and mitochondrial redox alterations with synergistic alterations to the energetic buffering system. In particular, oxidized and hypoactive myofilamental creatine kinase (M-CK) was observed in human HCM biopsies. Because of the complementary involvement between M-CK and the mitochondrial CK (mt-CK) in cardiomyocytes, we investigated mt-CK´s function to mitochondrial anti-oxidative capacity and Ca²⁺ load.

Methods & Results

Oxidation of mt-CK Cys317 in human HCM samples was identified via mass spectrometry ICAT labelling, a known site essential for enzyme activity and protein assembly. To investigate the implications of mt-CK inactivation to mitochondrial oxidative stress and Ca²⁺ dynamics we used the CK selective inhibitor DNFB in isolated cardiac mouse mitochondria. Mt-CK comprises of a cluster of 8 identical monomers, which is assumed to bridge the inner-to-outer mitochondrial membrane. Using gel electrophoresis we identified that alongside reducing mt-CK´s activity, DNFB caused the loss of the octameric assembly, which is reminiscent of the disruption caused by oxidation of mt-CK Cys317. Mitochondrial O₂ respirometry with simultaneous H₂O₂ recording (Amplex UltraRed) was performed with pyruvate/malate (i.e., “state 2”), saturating ADP levels  (1mM, “state 3”) and DNFB (1 and 20µM) (Oroboros Instr). O₂ consumption was not compromised and native PAGE protein analysis revealed no alterations to the composition of the respirasome, in the presence of DNFB. Most important, DNFB stimulated a large H₂O₂ generation in cardiac mitochondria. To validate upstream formation of superoxide, potentially caused by membrane electron leakage, we measured the effects of DNFB using electroparamagnetic resonance and observed substantial increases of superoxide. To rule out unspecific off-target effects of DNFB, cardiac mitochondria was compared against liver mitochondria, which have very low expression of mt-CK. H₂O₂ and superoxide formation was almost negligible in liver mitochondria with DNFB. Finally, to investigate the impact of mt-CK inactivation/disruption to mitochondrial Ca²⁺ dynamics, extramitochondrial Ca²⁺ was recorded using Ca²⁺ green. Mitochondria were pre-incubated with 5mM K-glutamate/2.5mM malate and the assay initiated by sequential 10µM free Ca²⁺ additions. In the presence of DNFB, mitochondria showed early opening of the mitochondria permeability transition pore (mPTP) with Ca²⁺ extrusion. Notably, liver mitochondrial Ca²⁺ was unaffected by DNFB.

Conclusion

Here we show, in cardiac mitochondria, that inhibition of mt-CK causes both H₂O₂ and superoxide formation and associates with higher mitochondrial Ca²⁺ extrusion. These, we speculate, likely occur due to loss of the stabilizing function of octameric mt-CK structure, leading to electron leakage and destabilization of the mPTP. Mt-CK inhibition however did not alter respirasome´s formation and O₂ consumption. Mt-CK hypoactivity is known in heart failure and we suspect those alterations are likely occurring in human HCM, where the hypercontractility provokes mitochondrial redox alterations and likely feed-forwards to mt-CK inactivation. We are currently evaluating the state of mt-CK´s octameric assembly in human HCM biopsies.