Oxidants are produced endogenously and can react with proteins causing post-translational modification of target proteins. They are involved in the redox regulation of signal transduction pathways for cell protection. However, upon chronical increase of oxidative stress, cell damage occurs due to protein oxidation and aggregation. Here we investigated oxidative modifications of myofilament proteins and its role in cardiomyocyte function modulation in hypertrophic cardiomyopathy patients (HCM).

Permeabilized cardiomyocytes from LV human HCM hearts compared with those of non-failing hearts (donor group). Myofilament oxidation was assayed by measuring in vivo redox state of cardiac proteins in HCM patients and compared to donors. We used the OxICAT method coupled with mass spectrometry (MS). OxICAT, which allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It uses cysteine-specific isotope-coded affinity tag (ICAT) reagent to differentially label oxidized and reduced cysteines, which can then be detected with MS. By systematically labeling all reduced cysteines in the tissue sample first and then labeling the remaining cysteines that were in an oxidized state in the tissue. We also used Western blots with specific monoclonal anti‐glutathione antibody that recognizes S‐glutathionylated proteins. We were able to detect and quantify many peptides that were oxidized in HCM compared to donors. We detected many peptides from different cardiac proteins to be highly oxidized and myofilament proteins titin, myosin binding protein C, troponin I, and actin were abundantly oxidized. Ca²⁺ sensitivity of force production was significantly increased in cardiomyocytes in HCM than in non-failing. Subsequently, it was significantly reduced upon treatment with reduced glutathione enzyme suggesting protein oxidation as a cause of the changes in Ca²⁺-sensitivity. Maximal tension generated by a single cardiomyocyte was lower in HCM compared to non-failing cardiomyocytes and was recovered upon treatment with reduced glutathione enzyme.  Furthermore, impairment of Ca²⁺ sensitivities and maximal tension correlated strongly not only with myofilament oxidation levels but also with oxidative stress parameters measured in all samples. Many oxidative stress and inflammation parameters were significantly increased in HCM hearts compared to non-failing hearts.

Hypertrophic cardiomyopathy involves increased myofibrillar protein oxidation, which contributes to increased Ca²⁺ sensitivity of force production and maximal tension, leading potentially to contractile dysfunction and development of heart disease.