Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (
asystole) during global
ischemia, which is relevant for the success of defibrillation and
resuscitation after
cardiac arrest. However, the relationship between ΔΨm depolarization and
asystole during no-flow
ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive
dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of
asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms),
myosin II ATPase inhibitor
blebbistatin (heart immobilizer), and the combination of
blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of
blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic
solution and after the contraction failed between 7 and 15 min of
ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter.
Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with
blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and
asystole or a similar dependence of the two events on energy depletion during
ischemia. Our results urge caution against the use of
blebbistatin in studies addressing pathophysiology of
myocardial ischemia.