Introduction

Hypertrophic cardiomyopathy (HCM) is characterized by abnormal hypertrophy of the septum and it is commonly caused by mutations of genes that encode sarcomeric proteins. Many of the mutations increase the Ca²⁺ 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. Here, we present evidence for additional disease drivers, including the uncoupling of the energetic buffering system (i.e., creatine kinase) and cellular redox state.

Methods

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. Human tissue collected from HCM patients (n=22-30) was compared against non-failing controls (n=8-14). To investigate if myofilaments sensitized to Ca²⁺ are able cause ROS generation, and deactivate M-CK, isolated mouse cardiomyocytes (N=17) were paced at rest (0.5Hz) and during stress (with isoproterenol and 5Hz pacing), in the presence of the Ca²⁺ sensitizer EMD (3µM). Simultaneous monitoring of H₂O₂ was conducted with DCF. M-CK was thereafter extracted from cells and kinase activity measured via the colorimetric assay. Proof-of-concept experiments were performed in isolated mouse cardiomyocytes exposed to 20µM of the M-CK inhibitor DNFB at rest and during stress (N=19) to evaluate the involvement between M-CK and the cellular 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). The plotting of M-CK´s activity (acquired from the individual samples) against the corresponding protein oxidation showed strong correlation between kinase hypoactivity and higher oxidation of M-CK (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 M-CK´s proper activity, validating that oxidation of M-CK is causative for kinase hypoactivity in HCM. Mouse cardiomyocytes perfused with the Ca²⁺ sensitizer EMD exhibited large elevations of H₂O₂, which coincided with a deactivation of M-CK activity at rest (16.4±0.8% reduction) that became more pronounced following stress conditions (28.1±2.1% reduction). Finally, cardiomyocytes exposed to the stress protocol, in the presence of the M-CK inhibitor DNFB, showed a 45±7% reduction of kinase´s activity in concert with substantial increases of H₂O₂ generation.

Discussion

M-CK expression and activity are diminished in human HCM, and correlate with high oxidation of M-CK. Single cell experiments confirmed the mechano-energetic (un)coupling between hypercontractility, higher ROS, oxidation of M-CK and kinase hypoactivity. More, the present study additionally demonstrates that direct inactivation of M-CK directly contributes to ROS generation, irrespective of contractility alterations. We speculate the changes to M-CK are early contributors to impaired cardiac energetics and redox alterations in HCM.