Ischemia-reperfusion induced calcium overload results in activation of calpain-1 in the heart. Calpain-dependent proteolysis contributes to myocardial dysfunction and cell death. Previously, preischemic treatment with low doses of H₂O₂ was shown to improve postischemic function and reduce myocardial infarct size. Our aim was to determine the mechanism by whichH₂O₂ protects the heart. We hypothesized that H₂O₂ causes the activation of p38 MAPK which initiates translocation of heat shock protein 25/27 (HSP25/27) to the myofilament Z-disk. We further hypothesized HSP25/27 shields structural proteins, particularly desmin, from calpain induced proteolysis. To address this hypothesis we first determined that an ischemia-reperfusion induced decrease in desmin content could be blocked by H₂O₂ pretreatment of hearts from rats. Next, we determined that ventricular myocytes which underwent Ca²⁺ overload also demonstrated a calpain-dependent disruption of desmin that could be reduced by H₂O₂-p38 MAPK activation. Further, myocytes acutely treated with H₂O₂ prior to demembranation exhibited a decrease in cleavage of desmin upon exposure to exogenous calpain-1 as compared to myocytes not pretreated with H₂O₂. The H₂O₂ induced attenuation of desmin degradation by calpain-1 was blocked by inhibition of p38 MAPK. In a final series of experiments we demonstrated that cardiac myofilaments exposed to recombinant phosphorylated HSP27, but not non-phosphorylated HSP27, had a significant reduction in the calpain induced degradation of desmin as compared to non-HSP27 treated myofilaments. These findings are consistent with the hypothesis that H₂O₂ induced activation of p38 MAPK and subsequent HSP25/27 translocation attenuates desmin degradation brought about by calpain-1 activation in ischemia-reperfused hearts.