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

Objective: Protein kinase C (PKC) activation impairs contractility in the normal heart but is protective during myocardial ischemia. We hypothesized that PKC remains activated post-ischemia and modulates myocardial excitation-contraction coupling during early reperfusion. Methods: Langendorff-perfused rabbit hearts where subjected to 25 min unmodified ischemia and 30 min reperfusion. Total PKC activity was measured, and the intracellular translocation pattern of PKC-alpha, -delta, -epsilon, and -eta assessed by immunohistochemistry and fractionated Western immunoblotting. The PKC-inhibitors chelerythrine and GF109203X were added during reperfusion and also given to non-ischemic hearts. Measurements included left ventricular function, intracellular calcium handling measured by Rhod-2 spectrofluorometry, myofibrillar calcium responsiveness in beating and tetanized hearts, and metabolic parameters. Results: Total PKC activity was increased at end-ischemia and remained elevated after 30 min of reperfusion. The translocation pattern indicated PKC-epsilon as the main active isoform during reperfusion. Post-ischemic PKC inhibition affected mainly diastolic relaxation, with lesser effect on contractility. Both PKC inhibitors increased the Ca2+ responsiveness of the myofilaments as indicated by a leftward shift of the calcium-to-force relationship and increased maximum calcium activated tetanic pressure. Diastolic Ca2+ removal was delayed and the post-ischemic [Ca2+](i) overload further exacerbated. Depressed systolic function was associated with a lower amplitude of [Ca2+](i) transients. Conclusion: PKC is activated during ischemia and remains activated during early reperfusion. Inhibition of PKC activity post-ischemia impairs functional recovery, delays diastolic [Ca2+](i) removal, and increases Ca2+ sensitivity of the contractile apparatus, resulting in impaired diastolic relaxation. Thus. post-ischemic PKC activity may serve to restore post-ischemic Ca2+ homeostasis and attenuate contractile protein calcium sensitivity during the period of post-ischemia rights reserved.