MDL-28170, a membrane-permeant calpain inhibitor, attenuates stunning and PKC epsilon proteolysis in reperfused ferret hearts.
OBJECTIVE
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.
Urthaler
F
F
Department of Medicine, University of Alabama at Birmingham 35294, USA.
Wolkowicz
P E
PE
Digerness
S B
SB
Harris
K D
KD
Walker
A A
AA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors*; D002463:Cell Membrane Permeability; D004151:Dipeptides / Q000494:pharmacology*; D005289:Ferrets; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism; D017682:Myocardial Stunning / Q000378:metabolism / Q000517:prevention & control*; D009206:Myocardium / Q000378:metabolism*; D010477:Perfusion; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon
D004151:Dipeptides; D007527:Isoenzymes; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D002154:Calpain; D002118:Calcium; C058076:calpain inhibitor III