Protein kinase C and myocardial calcium handling during ischemia and reperfusion: Lessons learned using Rhod-2 spectrofluorometry

objective: We sought to assess myocardial Ca2+ handling and excitation-contraction coupling in surgically relevant models of ischemia-reperfusion injury and to clarify the importance of protein kinase C (PI(C) for cardioprotection. Methods: Experimentally, surgical ischemia and reperfusion can only be mimicked in intact perfused heart models. We introduced the long-wavelength fluorescent Ca2+ indicator Rhod-2 for real-time recording of cytosolic Ca2+ transients in Langendorff-perfused rabbit, rat, and mouse hearts, and utilized it to study the impact of PKC on myocardial Ca2+ handling during ischemia and reperfusion. Results: We first established that the dissociation constant for Rhod-2 and Ca2+ 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 Ca2+ accumulation during myocardial ischemia, which is associated with translocation of the PI(C isoforms alpha. and c between the cytosolic and particulate compartments in cardiomyocytes. The PI(C 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 PI(C and found that it both limits the accumulation of cytosolic Ca2+ during reperfusion and attenuates contractile protein Ca2+ sensitivity via phosphorylation of troponin 1. Conclusions: Rhod-2 spectrofluorometry is a valuable tool for assessment of cytosolic Ca2+ in surgically relevant experimental models and can aid the development of more effective methods for myocardial protection.