Hydrogen peroxide‐induced oxidative stress to the mammalian heart‐muscle cell (cardiomyocyte): Lethal peroxidative membrane injury

Abstract

Oxidative stress induced by hydrogen peroxide (H₂O₂) may contribute to the pathogenesis of ischemic‐reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H₂O₂ on heart muscle, a cellular model of H₂O₂‐induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal‐rat cardiomy‐ocytes and discrete concentrations of reagent H₂O₂ in defined, supplement‐free culture medium. Cardiomyocytes challenged with H₂O₂ readily metabolized it such that the culture content of H₂O₂ diminished over time, but was not depleted. The consequent H₂O₂‐induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain‐breaking membrane antiperoxidant (α‐tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H₂O₂‐induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H₂O₂. Development of lethal cardiomyocyte injury during H₂O₂‐induced oxidative stress did not require the presence of H₂O₂ itself; a brief “pulse” exposure of the cardiomyocytes to H₂O₂ was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox‐active iron and the iron‐dependent transformation of internalized H₂O₂ into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl‐radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte‐membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of α‐tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H₂O₂‐induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H₂O₂‐induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H₂O₂‐induced oxidative stress, a nonperoxidative route of H₂O₂ cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte‐membrane peroxidation. The latter mode of H₂O₂‐induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de‐energization on the other could be completely dissociated. The cellular pathophysiology of H₂O₂ as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mechanisms of oxidative injury in the postischemic heart.