Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity

Objective: Protein kinase C (PKC) activation impairs contractility in the n ormal heart but is protective during myocardial ischemia. We hypothesized t hat PKC remains activated post-ischemia and modulates myocardial excitation -contraction coupling during early reperfusion. Methods: Langendorff-perfus ed rabbit hearts where subjected to 25 min unmodified ischemia and 30 min r eperfusion. Total PKC activity was measured, and the intracellular transloc ation pattern of PKC-alpha, -delta, -epsilon, and -eta assessed by immunohi stochemistry and fractionated Western immunoblotting. The PKC-inhibitors ch elerythrine and GF109203X were added during reperfusion and also given to n on-ischemic hearts. Measurements included left ventricular function, intrac ellular calcium handling measured by Rhod-2 spectrofluorometry, myofibrilla r calcium responsiveness in beating and tetanized hearts, and metabolic par ameters. Results: Total PKC activity was increased at end-ischemia and rema ined elevated after 30 min of reperfusion. The translocation pattern indica ted PKC-epsilon as the main active isoform during reperfusion. Post-ischemi c PKC inhibition affected mainly diastolic relaxation, with lesser effect o n contractility. Both PKC inhibitors increased the Ca2+ responsiveness of t he myofilaments as indicated by a leftward shift of the calcium-to-force re lationship and increased maximum calcium activated tetanic pressure. Diasto lic Ca2+ removal was delayed and the post-ischemic [Ca2+](i) overload furth er exacerbated. Depressed systolic function was associated with a lower amp litude of [Ca2+](i) transients. Conclusion: PKC is activated during ischemi a and remains activated during early reperfusion. Inhibition of PKC activit y post-ischemia impairs functional recovery, delays diastolic [Ca2+](i) rem oval, and increases Ca2+ sensitivity of the contractile apparatus, resultin g in impaired diastolic relaxation. Thus. post-ischemic PKC activity may se rve to restore post-ischemic Ca2+ homeostasis and attenuate contractile pro tein calcium sensitivity during the period of post-ischemia rights reserved .