MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKC proteolysis in reperfused ferret hearts

. .Objectives: This paper tests the hypothesis that calpains are activated in the ischemic I rreperfused R heart and contribute to myocardial stunning. Methods: Isolated ferret hearts were Langendorff perfused isovolumically, and subjected to 20 min of global I followed by 30 min of R in the presence or absence of 0.2 mM MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa2q-force curves obtained. Samples of left ventricle were extracted, subjected to SDS-PAGE, and Western analyzed for PKC and PKM. Results: Perfused ferret hearts exhibit a 43 % decline in left ventricular developed pressure during R. Pre-treatment of hearts with MDL-28170 prior to I significantly improves function during R. Trabecular myofilaments from normal hearts have a K for Ca2q of 6.27"0.06; IrR decreased the K to 6.09"0.04; trabeculae fromD D IrR hearts pre-treated with MDL-28170 have a K of 6.28"0.04. Western analysis shows ferret hearts to contain a single f96 kDaD species of PKC. IrR hearts contain the native PKC and a f25 kDa smaller species of PKC, which corresponds to PKM, the calpain proteolyzed form of PKC. Pre-treatment of IrR hearts with MDL-28170 markedly diminishes PKM in reperfused hearts. Conclusions: Mechanical stunning during R is sensitive to MDL-28170. Depressed mechanical function is reflected in a hyposensitization of trabecular myofilaments to Ca2q. Western analysis shows that PKM is present in R hearts