Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry.
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 alpha and epsilon 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.
Stamm
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Klinik und Poliklinik für Herzchirurgie, Universität Rostock, Rostock, Germany. christoff.stamm@med.uni-rostock.de
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D000818:Animals; D002118:Calcium; D004734:Energy Metabolism; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000378:metabolism; D017202:Myocardial Ischemia / Q000378:metabolism*; D015425:Myocardial Reperfusion; D009206:Myocardium / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D019210:Troponin I / Q000378:metabolism
D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D009124:Muscle Proteins; D019210:Troponin I; C068483:rhod-2; D011493:Protein Kinase C; D002118:Calcium