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

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Abstract
Objectives: This paper tests the hypothesis that calpains are activated in the ischemic (I)/reperfused (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 μM MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa2+-force curves obtained. Samples of left ventricle were extracted, subjected to SDS-PAGE, and Western analyzed for PKCε and PKMε. 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 KD for Ca2+ of 6.27±0.06; I/R decreased the KD to 6.09±0.04; trabeculae from I/R hearts pre-treated with MDL-28170 have a KD of 6.28±0.04. Western analysis shows ferret hearts to contain a single ≈96 kDa species of PKCε. I/R hearts contain the native PKCε and a ≈25 kDa smaller species of PKCε, which corresponds to PKMε, the calpain proteolyzed form of PKCε. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKMε 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 Ca2+. Western analysis shows that PKMε is present in R hearts.

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