Hypertrophic cardiomyopathy (HCM) is characterized by abnormal hypertrophy of the septum and commonly caused by mutations of genes that encode sarcomeric proteins, many of which increase the Ca affinity of myofilaments. While this readily explains the functional phenotype of diastolic dysfunction and often hypercontractility, it is largely unclear how these mutations impair cardiac energetics and cause maladaptive cardiac remodeling. In previous work, we revealed that increased energetic demand at the myofilaments in HCM mouse models oxidizes mitochondrial NAD(P)H, since its regeneration is not sufficiently matched by mitochondrial Ca uptake, which results in elevated emission of reactive oxygen species (ROS) and arrhythmias. Here, we investigated whether mitochondrial ROS further compromise energetic regeneration through the creatine kinease (CK) system.

Methods

Human tissue collected from HCM patients (n=22-30) was compared against non-failing controls (n=8-14). Myofilament creatine kinase (M-CK) protein expression and oxidation were evaluated by Western Blot and OxICAT mass spectrometry (MS)-labeling, while M-CK activity was assessed via a colorimetric assay. To investigate whether myofilamental Ca sensitization per se deactivates M-CK through ROS, isolated mouse cardiomyocytes (n=17) were paced at 0.5Hz and 5 Hz in the presence of isoproterenol (10 nM) and the Ca sensitizer EMD (3µM), and ROS were monitored with DCF. M-CK was thereafter extracted from cells and kinase activity measured via a colorimetric assay. Similar experiments were performed in the presence of the M-CK inhibitor DNFB (20 µM; n=19) to evaluate the role of M-CK for the regulation of the mitochondrial redox state.

Results

Protein analysis revealed total M-CK levels were lower in human HCM biopsies (58±3% of total) and coincided with greater oxidation of M-CK (22% more oxidized vs controls). M-CK activity was reduced in human HCM (47±5% of total), with a close correlation between reduced M-CK activity and oxidation (R²=0.96). OxICAT labeling identified two oxidized cysteine sites in M-CK from human HCM at positions 146 and 254. The latter residues are known sites responsible for proper M-CK activity, validating that oxidation of M-CK is causative for kinase hypoactivity in HCM. Mouse cardiomyocytes (at 5 Hz and isoproterenol) treated with the Ca sensitizer EMD exhibited oxidation of mitochondrial NAD(P)H, elevated H₂O₂ emission, and deactivated M-CK activity (by 28.1±2.1%) compared to vehicle. All these effects were prevented by co-treatment with CGP-37157 (10 µM), an inhibitor of the mitochondrial Na/Ca exchanger. Finally, pretreatment of M-CK using the inhibitor DNFB (20 µM) reduced M-CK activity by 45±7%, oxidized NAD(P)H redox state and provoked a substantial increase in mitochondrial ROS emission.

Discussion

Increased Ca sensitivity of myofilaments induces a Ca mismatch in mitochondria, causing mitochondrial oxidation of NADPH and ROS emission, which in turn oxidize M-CK and thereby reduce its activity. Inactivation of M-CK per se aggravates mitochondrial oxidation and ROS emission through a mechanism that is currently addressed in ongoing studies. Together, these results suggest a futile feed-forward mechanism of energetic- and redox mismatch in HCM that likely aggravates diastolic dysfunction and – according to previous results of our group – account for arrhythmias through providing a trigger (redox) and substrate (energy depletion) for arrhythmias.