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

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 microM MDL-28170, a membrane-permeant calpain inhibitor. Right trabeculae then were isolated from these hearts, skinned chemically, and pCa(2+)-force curves obtained. Samples of left ventricle were extracted subjected to SDS-PAGE, and Western analyzed for PKC epsilon and PKM epsilon.

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 approximately equal to 96 kDa species of PKC epsilon. I/R hearts contain the native PKC epsilon and a approximately equal to 25 kDa smaller species of PKC epsilon which corresponds to PKM epsilon, the calpain proteolyzed form of PKC epsilon. Pre-treatment of I/R hearts with MDL-28170 markedly diminishes PKM epsilon 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 PKM epsilon is present in R hearts.