Prolonged oxidative stress inverts the cardiac force-frequency relation: Role of altered calcium handling and myofilament calcium responsiveness

The normally positive force- and Ca²⁺-frequency responses (FFR and CaFR) are inverted in heart failure (HF); whether oxidative stress contributes to these abnormalities is unknown. We evaluated the impact of acute and prolonged oxidative stress on contraction and Ca²⁺ handling in adult rat cardiomyocytes. Acute (30:min) exposure to H₂O₂ (100 μM) induced a twofold increase (P < 0.025) in intracellular oxyradicals together with contractile depression despite preservation of the Ca²⁺ transient and the FFR and CaFR to 3:Hz, indicating reduced myofilament Ca²⁺ responsiveness. In contrast, prolonged (24:h) exposure to the copper-zinc superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC, 1 μM) similarly augmented oxyradicals but also increased cell size, and contraction and Ca²⁺ transient duration (P < 0.025). DDC-treated myocytes displayed inverted FFRs and attenuated (though still positive) CaFRs as compared to control, indicating reduced myofilament Ca ²⁺ responsiveness coupled with altered Ca²⁺ handling. Protein levels of the Na⁺-Ca²⁺ exchanger (NCX), sarcoplasmic reticular (SR) Ca²⁺ ATPase (SERCA2), and serine-16 phosphorylated phospholamban (pSer16-PLB) were increased (P < 0.025), whereas dihydropyridine receptor abundance was decreased. Total PLB and ryanodine receptor protein expression were unchanged. Caffeine-induced Ca²⁺ release showed increased NCX activity (P < 0.025) without changes in total releasable SR Ca²⁺, suggesting compensatory changes in SERCA2 and pSer16-PLB to maintain SR Ca²⁺ load. The superoxide scavenger Tiron attenuated these effects. Thus, acute oxyradical exposure rapidly depresses myofibrillar Ca²⁺ responsiveness. Prolonged oxidative stress further induces alterations in Ca²⁺ handling that combined with extant reductions in myofibrillar responsiveness invert the FFR. With regard to Ca ²⁺ handling, reduced transsarcolemmal Ca²⁺ flux rather than reduced SR Ca²⁺ uptake was the primary determinant of a negative FFR. Analogous changes may be operative in HF, a state characterized by both oxidative stress and Ca²⁺ dysregulation.