Protein Kinase C and Myocardial Calcium Handling during Ischemia and Reperfusion: Lessons Learned using Rhod-2 Spectrofluorometry

Abstract OBJECTIVE: We sought to assess myocardial Ca 2+ handling and excitation-contraction coupling in surgically relevant models of ischemia-reperfusion injury and to clarify the importance of protein kinase C (PKC) for cardioprotection. METHODS: Experimentally, surgical ischemia and reperfusion can only be mimicked in intact perfused heart models. We introduced the long-wavelength fluorescent Ca 2+ indicator Rhod-2 for real-time recording of cytosolic Ca 2+ transients in Langendorff-perfused rabbit, rat, and mouse hearts, and utilized it to study the impact of PKC on myocardial Ca 2+ handling during ischemia and reperfusion. RESULTS: We first established that the dissociation constant for Rhod-2 and Ca 2+ must be adjusted to account for changes in pH and temperature during ischemia and reperfusion. Based on this method, we determined the time-course and extent of cytosolic Ca 2+ accumulation during myocardial ischemia, which is associated with translocation of the PKC isoforms α and ε between the cytosolic and particulate compartments in cardiomyocytes. The PKC translocation is mediated by activation of phosphatidyl-inositol-specific phospholipase C (PI-PLC), and represents a cardioprotective mechanism. Finally, we studied the mechanism of action of PKC and found that it both limits the accumulation of cytosolic Ca 2+ during reperfusion and attenuates contractile protein Ca 2+ sensitivity via phosphorylation of troponin I. CONCLUSIONS: Rhod-2 spectrofluorometry is a valuable tool for assessment of cytosolic Ca 2+ in surgically relevant experimental models and can aid the development of more effective methods for myocardial protection.