Blebbistatin Delays Mitochondrial Depolarization and Asystole during Myocardial Ischemia, and Prevents Cell Death Upon Reperfusion

Abstract

Myosin II inhibitor Blebbistatin is frequently used for heart immobilization during imaging experiments involving ischemia/reperfusion (I/R). Here we investigated the extent to which Blebbistatin modulates critical events during I/R, such as mitochondrial membrane potential depolarization (ΔΨm), asystole, and infarction. Langendorff-perfused rabbit hearts were subjected to 60-90 minutes of ischemia and 180 minutes of reperfusion in the absence (control) or in the presence of Blebbistatin (5.7 µM). We monitored ΔΨm depolarization using spectral analysis of confocal images yielded by cationic fluorescent dye TMRM. We detected the onset of sarcolemmal permeabilization (SP) in individual myocytes as a massive uptake of anionic dye DiBac4(3) or cell-impermeable nucleic acid dye YO-PRO1. Control hearts were briefly perfused with 20 mM potassium at 35 minutes prior to ischemia, and at 30 and 120 minutes of reperfusion to achieve immobilization necessary for confocal imaging. In control hearts, cessation of contraction permitted confocal imaging after 10-15 minutes of ischemia. Global and local bipolar electrograms monitored electrical activity. During ischemia, Blebbistatin significantly (p<0.01) and proportionally delayed both ΔΨm depolarization (50.5 minutes versus 24.0 minutes in control) and asystole (40.2 minutes versus 22.3 minutes in control). Upon reperfusion, a heterogeneous ΔΨm recovery was observed in both groups, but Blebbistatin largely prevented SP events observed on a massive scale in control hearts. Additionally, Blebbistatin almost completely prevented infarction in this model (1.9±3.2% versus 75.0±7.8% in control by tetrazolium chloride staining). Another electromechanical uncoupler, 2,3-Butanedione monoxime (20 mM), did not prevent SP and infarction despite efficient immobilization. Conclusions: (1) great caution has to be exercised with regard to interpretation of I/R events observed in the presence of Blebbistatin; (2) the mechanism(s) of prominent cardioprotection by Blebbistatin may not be fully explained by immobilization and deserve further study.