Critical Events in Myocardial Ischemia/Reperfusion: Mitochondrial Depolarization Versus Sarcolemmal Permeability

The critical events determining cell death in the aftermath of myocardial ischemia/reperfusion (I/R) remain poorly understood. Here we investigated the temporal relationship between the time of apparently complete mitochondrial inner membrane potential (ΔΨm) depolarization and the onset of sarcolemmal permeabilization (SP) during I/R using confocal imaging of the mitochondrial fluorophore TMRM and the SP indicator YO-PRO1. Whole rabbit hearts (n=6) were subjected to 60-minutes global ischemia and 180-minutes reperfusion (37oC). In some hearts the electromechanical uncoupler 2,3-Butanedione monoxime (20 mM) or temporary perfusion with high [K+] (20 mM) was used to abolish motion during imaging. Two or three day-old cultured neonatal rat ventricular cardiomyocyte monolayers (NRVCM, n=3) were subjected to 30-minutes simulated ischemia (including near-anoxia, pH=6.5, [K+]o = 8.8 mM and [deoxyglucose] = 11 mM, 37oC) and 60-minutes reperfusion. In hearts and NRVCMs the relatively uniform ΔΨm loss occurred at 31.2±5.6 and 18.3±0.6 minutes of ischemia, respectively, amid absence of SP events. ΔΨm recovered to a variable degree upon reperfusion in both models, followed by a secondary and heterogeneous ΔΨm loss in individual myocytes. Total of 7 myocytes from whole hearts and 6 myocytes from NRVCMs were tracked throughout the entire period of reperfusion to accurately determine the delay between ΔΨm loss and the earliest detectable SP (Δt). Δt varied between −5.3 and +14.5 minutes in the heart myocytes and between −3.0 and +19.0 minutes in NRVCM myocytes. Excluding outlier cells with exceedingly large positive Δts, the mean ± standard deviation of Δt became 0.8±4.0 minutes in hearts and 0.2±3.2 minutes in NRVCMs. Conclusions: (1) critical ΔΨm loss upon reperfusion typically overlaps in time with the emergence of sarcolemmal pores of at least 1 nm diameter; (2) 2-3 day-old NRVCM qualitatively recapitulate the critical I/R events observed in whole hearts.