BackgroundManifestations of reperfusion injury include myocyte death leading to infarction, contractile dysfunction, and vascular injury characterized by the “no-reflow” phenomenon. Mitochondria-produced reactive oxygen species are believed to be centrally involved in each of these aspects of reperfusion injury, although currently no therapies reduce reperfusion injury by targeting mitochondria specifically.Methods and ResultsWe investigated the cardioprotective effects of a mitochondria-targeted peptide, Bendavia (Stealth Peptides), across a spectrum of experimental cardiac ischemia/reperfusion models. Postischemic administration of Bendavia reduced infarct size in an in vivo sheep model by 15% (P=0.02) and in an ex vivo guinea pig model by 38% to 42% (P<0.05). In an in vivo rabbit model, the extent of coronary no-reflow was assessed with Thioflavin S staining and was significantly smaller in the Bendavia group for any given ischemic risk area than in the control group (P=0.0085). Myocardial uptake of Bendavia was ≈25% per minute, and uptake remained consistent throughout reperfusion. Postischemic recovery of cardiac hemodynamics was not influenced by Bendavia in any of the models studied. Isolated myocytes exposed to hypoxia/reoxygenation showed improved survival when treated with Bendavia. This protection appeared to be mediated by lowered reactive oxygen species–mediated cell death during reoxygenation, associated with sustainment of mitochondrial membrane potential in Bendavia-treated myocytes.ConclusionsPostischemic administration of Bendavia protected against reperfusion injury in several distinct models of injury. These data suggest that Bendavia is a mitochondria-targeted therapy that reduces reperfusion injury by maintaining mitochondrial energetics and suppressing cellular reactive oxygen species levels. (J Am Heart Assoc. 2012;1:e001644 doi: 10.1161/JAHA.112.001644.)

The ability of exercise to protect the heart against ischemia-reperfusion (I/R) injury is well known in both human epidemiological studies and experimental animal models. In this review article, we describe what is currently known about the ability of exercise to precondition the heart against infarction. Just 1 day of exercise can protect the heart against ischemia/reperfusion damage, and this protection is upheld with months of exercise, making exercise one of the few sustainable preconditioning stimuli. Exercise preconditioning depends on the model and intensity of exercise, and appears to involve heightened oxidant buffering capacity, upregulated subunits of sarcolemmal ATP-sensitive potassium channels, and adaptations to cardiac mitochondria. We review the putative mechanisms involved in exercise preconditioning and point out many areas where future research is necessary to advance our understanding of how this stimulus confers resistance against I/R damage.

No abstract

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (ΔΨ m ) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high K ATP current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide ΔΨ m depolarization, through a reactive oxygen species (ROS) -induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of ΔΨ m with a ligand of the mitochondrial benzodiazepine receptor (4′-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200μM diamide for 35 minutes, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5 ± 0.5; n=6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64μM): arrhythmia scores with diamide + 4-ClDzp were 0.4± 0.2 (n=5; P < 0.05 vs. diamide alone). Imaging ΔΨ m in intact hearts revealed the heterogeneous collapse of ΔΨ m beginning 20 minutes into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of ΔΨ m was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia-reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.

We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia’s cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 min prior to reperfusion (0.05mg/kg/hr, i.v.) reduced myocardial infarct size by ~50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 min after the onset of reperfusion, the protection against infarction and no–reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1nM to 1mM, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at onset of reperfusion, but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia’s protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.