Myofilament carbonylation modulates contractility in human cardiomyocytes

This study investigated the effects of myofilament carbonylation in permeabilized human left ventricular cardiomyocytes. Protein carbonylation was monitored by the oxyblot method following the in vitro application of the Fenton reaction reagents [iron(II), ascorbic acid and hydrogen peroxide (H ₂O₂)], known to produce hydroxyl radicals. Sulfhydryl group oxidation was assessed in parallel by the Ellman assay. During force measurements, the Ca²⁺-activated active force, the Ca ²⁺-independent passive force and the Ca²⁺ sensitivity of force production (pCa₅₀) were measured in permeabilized cardiomyocytes before and after in vitro carbonylation. Carbonylation at the levels of several myofilament proteins (e.g. myosin heavy chain, α-actinin, actin, myosin binding protein C, desmin and myosin light chain 1) was enhanced by increasing concentrations of H₂O₂ (0-10⁵ μM) in the Fenton solution. Carbonylation suspended active force generation following aggressive Fenton treatment (10⁵ μM H₂O₂), whereas the application of 3*10³ μM H₂O₂ decreased pCa₅₀ (from 5.74±0.01 to 5.65±0.01; mean±SEM, P<0.05) and increased the passive force (from 1.72±0.21 kN/m² to 2.33±0.22 kN/m²). None of these changes was influenced by sulfhydryl group reduction. Thus, myofilament carbonylation dysregulates the contractile function in human cardiomyocytes, and may therefore mediate the contractile dysfunction during oxidative stress.