Chad R. Frasier

Reduction of Ischemia/Reperfusion Injury With Bendavia, a Mitochondria-Targeting Cytoprotective Peptide

Background Manifestations 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 Results We 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. Conclusions Postischemic 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.)

Exercise-induced cardiac preconditioning: how exercise protects your achy-breaky heart

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.

Mitochondrial permeability transition in the diabetic heart: Contributions of thiol redox state and mitochondrial calcium to augmented reperfusion injury

Mitochondria from diabetic hearts are sensitized to mitochondrial permeability transition pore (PTP) opening, which may be responsible for the increased propensity for cardiac injury in diabetic hearts. The purpose of this study was to determine if redox-dependent PTP opening contributes to augmented injury in diabetic hearts, and if compounds targeted at mitochondrial PTP, ROS, and calcium influx protected diabetic hearts from injury. Hearts from control or streptozotocin-induced diabetic rats were excised for either whole-heart or isolated mitochondria experiments. Myocardial glutathione content was oxidized in diabetic hearts when compared to control, and this translated to increased oxidation of the adenine nucleotide translocase in diabetic hearts. Diabetic mitochondria displayed significantly greater sensitivity to PTP opening than non-diabetic counterparts, which was reversed with the thiol-reducing agent dithiothreitol. The thiol-oxidant diamide increased calcium sensitivity in control, but not diabetic mitochondria. Diabetic animals treated with the mitochondria-targeted ROS suppressing peptide MTP-131 also showed improved resistance to PTP opening. In separate experiments hearts underwent ex vivo ischemia/reperfusion (IR). Diabetic hearts were more susceptible to IR injury, with infarct sizes of 60 ± 4% of the area-at-risk (vs. 46 ± 2% in non-diabetics; P<0.05). Administration of the PTP blocker NIM811 (5 μM), MTP-131 (1 nM) or the mitochondrial calcium uniporter blocker minocycline (1 μM) at the onset of reperfusion reduced infarct sizes in both control and diabetic hearts. These findings suggest that augmented susceptibility to injury in the diabetic heart is mediated by redox-dependent shifts in PTP opening, and that three novel mitochondria-targeted agents administered at reperfusion may be suitable adjuvant reperfusion therapies to attenuate injury in diabetic patients.

Reduction of early reperfusion injury with the mitochondria-targeting peptide Bendavia

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 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes 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 1 nmol/L to 1 mmol/L, 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 the 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.

Redox-dependent increases in glutathione reductase and exercise preconditioning: role of NADPH oxidase and mitochondria

AIMS We have previously shown that exercise leads to sustainable cardioprotection through a mechanism involving improved glutathione replenishment. This study was conducted to determine if redox-dependent modifications in glutathione reductase (GR) were involved in exercise cardioprotection. Furthermore, we sought to determine if reactive oxygen species generated by NADPH oxidase and/or mitochondria during exercise were triggering events for GR modulations. METHODS AND RESULTS Rats were exercised for 10 consecutive days, after which isolated hearts were exposed to ischaemia/reperfusion (25 min/120 min). Exercise protected against infarction and arrhythmia, and preserved coronary flow. The GR inhibitor BCNU abolished the beneficial effects. GR activity was increased following exercise in a redox-dependent manner, with no change in GR protein levels. Because fluorescent labelling of GR protein thiols showed lower amounts of reduced thiols after exercise, we sought to determine the source of intracellular reactive oxygen species that may be activating GR. Subsets of animals were exercised immediately after treatment with either NADPH-oxidase inhibitors apocynin or Vas2870, or with mitoTEMPO or Bendavia, which reduce mitochondrial reactive oxygen species levels. The cardioprotective effects of exercise were abolished if animals exercised in the presence of NADPH oxidase inhibitors, in clear contrast to the mitochondrial reagents. These changes correlated with thiol-dependent modifications of GR. CONCLUSION Adaptive cardioprotective signalling is triggered by reactive oxygen species from NADPH oxidase, and leads to improved glutathione replenishment through redox-dependent modifications in GR.

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 μM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.

Pulmonary instillation of multi-walled carbon nanotubes promotes coronary vasoconstriction and exacerbates injury in isolated hearts

Abstract The growing use of multi-walled carbon nanotubes (MWCNTs) across industry has increased human exposures. We tested the hypothesis that pulmonary instillation of MWCNTs would exacerbate cardiac ischaemia/reperfusion (I/R) injury. One day following intratracheal instillation of 1, 10 or 100 μg MWCNT in Sprague-Dawley rats, we used a Langendorff isolated heart model to examine cardiac I/R injury. In the 100 μg MWCNT group we report increased premature ventricular contractions at baseline and increased myocardial infarction. This was associated with increased endothelin-1 (ET-1) release and depression of coronary flow during early reperfusion. We also tested if isolated coronary vascular responses were affected by MWCNT instillation and found trends for enhanced coronary tone, which were dependent on ET-1, cyclooxygenase, thromboxane and Rho-kinase. We concluded that instillation of MWCNTs promoted cardiac injury and depressed coronary flow by invoking vasoconstrictive mechanisms involving ET-1, cyclooxygenase, thromboxane and Rho-kinase.