Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance

Abstract

Venable PW, Sciuto KJ, Warren M, Taylor TG, Garg V, Shibayama J, Zaitsev AV. Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance. Am J Physiol Heart Circ Physiol 308: H485-H499, 2015. First published December 31, 2014 doi:10.1152/ajpheart.00257.2014.-Mitochondrial membrane potential (⌬⌿m) 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 ⌬⌿m depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ⌬⌿m-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ⌬⌿m depolarization (t mito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ⌬⌿ m. The time of asystole (t asys) 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, t mito_depol and tasys were 24.4 Ϯ 6.0 and 26.0 Ϯ 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed t mito_depol and tasys (51.4 Ϯ 8.6 and 45.7 Ϯ 5.3 min, respectively; both P Ͻ 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (t mito_depol, 12.7 Ϯ 1.8 min; and t asys, 6.5 Ϯ 1.1 min; both P Ͻ 0.03 vs. control). In all groups pooled together, t asys was strongly correlated with tmito_depol (R 2 ϭ 0.845; P Ͻ 0.0001). These data may indicate a causal relationship between ⌬⌿ m 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. myocardial ischemia; mitochondrial depolarization; ATP-sensitive potassium channel; asystole; blebbistatin COLLAPSE OF MITOCHONDRIAL inner membrane potential (⌬⌿ m ) is a major adverse event in the course of global myocardial ischemia and/or reperfusion (I/R). It has been implicated in loss of excitability during ischemia and subsequent postreperfusion ventricular fibrillation (VF) (2), and in the development of proarrhythmic action potential alternans during ischemia (25). Despite the obvious importance of ⌬⌿ m loss for the outcomes of an ischemic insult, the timing and the determinants of this event are still poorly understood. One reason for that is the difficulty of monitoring ⌬⌿ m in realistic, whole heart models of ischemia. We addressed these issues in our previous publication (2...

Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step

“…We first analyzed the global dynamics of F TMRM and F YO-PRO1 during ischemia and reperfusion. During ischemia, we observed a global loss of ΔΨ m consistent with our previous publication [ 27 ], but did not observe SP (see S1 Text , S3 Fig and S1 Movie ). During reperfusion, the first event was recovery of ΔΨ m .…”

“…Confocal imaging was performed essentially as described in our previous publication [ 27 ], and only modifications will be described in detail. Briefly, the hearts were positioned in an imaging chamber mounted on the stage of an inverted Zeiss LSM 510 confocal microscope, and the posterior LV surface was imaged through a glass coverslip glued to the bottom of the chamber.…”

β-Adrenergic stimulation and rapid pacing mutually promote heterogeneous electrical failure and ventricular fibrillation in the globally ischemic heart

“…Use of BDM to immobilize the heart could distort the progressive electrophysiological changes during ischemia, but in two experiments using pacing at 200 ms and isoproterenol (30 nM) in the absence of uncouplers, the overall dynamics of electrical depression, emergence of inexcitable regions, and initiation of VF was similar to those observed in our 200ms_Iso group (not shown). In contrast, another uncoupler blebbistatin in three experiments postponed the adverse electrophysiological outcomes of ischemia (not shown) consistent with results from our recent study (56). Lack of mechanical load in the Langendorff-perfused hearts could also distort the ischemic changes (62) …”

A technical review of optical mapping of intracellular calcium within myocardial tissue

“…Blebbistatin is a popular uncoupling agent for optical mapping, especially when imaging Ca i 2ϩ because it specifically inhibits the myosin ATPase without altering sarcolemmal ion currents or SR Ca 2ϩ kinetics (28). However, myocyte ATP utilization is severely reduced by electromechanical uncoupling, potentially altering the time course of physiological changes, especially mitochondrial function, during experimental perturbations (47,105,114). Motion tracking algorithms (41,80,115) and ratiometry (42,44,93) are sometimes effective in removing motion artifact from optical action potentials, but there has been less work in the application of motion tracking algorithms to reduce artifact in optically mapped Ca i 2ϩ transients.…”