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

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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.