Reduction of ischemia/reperfusion injury with bendavia, a mitochondria-targeting cytoprotective Peptide

Abstract

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.).

Keywords: cardioprotection; infarction; mitochondria; peptide.

Figure 1.

Left, Ischemic risk zone (% LV) and infarct size (% risk zone) in sheep exposed to in vivo ischemia/reperfusion (60 min/3 h). Bendavia (0.05 mg/kg per hour; black bars) was administered intravenously beginning 30 minutes before reperfusion (mean±SEM; P=0.02, t test). Right, Relationship between the extent of the ischemic risk zone (% LV) and the extent of necrosis (% LV) (P=0.03, ANCOVA).

Figure 2.

Left, Ischemic risk zone (fraction LV) and infarct size (fraction risk zone) in rabbits with risk zones >20% of the LV exposed to in vivo ischemia/reperfusion (30 min/3 h) in combined Bendavia treatment group and vehicle group (mean±SEM; P=0.09, t test). Note similarity to data observed in the sheep model (text, Figure 1). Right, Relationship between the extent of the ischemic risk zone (g) and the extent of necrosis (g) in combined Bendavia group and vehicle group. Regression lines: vehicle is indicated by dashed line; Bendavia, solid line.

Figure 3.

Infarct size in isolated guinea pig hearts exposed to global ischemia/reperfusion (20 min/2 h). Experimental compounds were either administered both before and after ischemia (whole time) or administered only at the onset of reperfusion (@ reperfusion) (mean±SEM; P<0.05 vs control, ANOVA followed by Newman-Keuls post hoc tests for between-group comparisons).

Figure 4.

Relationship between risk zone (g) and the extent of no-reflow (g). Lines of regression for vehicle-treated rabbits (dashed line) and Bendavia-treated rabbits (solid line) are shown. *P=0.0085 by ANCOVA testing for a treatment effect (Bendavia vs vehicle) on the relationship.

Figure 5.

Uptake of Bendavia by the myocardium before ischemia (Baseline) and during reperfusion. Data represent the percentage of Bendavia taken up by the heart over a 1-minute time course.

Figure 6.

Survival plot for myocytes in the study exposed to hypoxia and reoxygenation. Each cell death event is noted as a downward step in the survival curve. *P<0.02, log-rank (Mantel-Cox) test vs control for the reoxygenation period. Bendavia significantly lowered cell death rate during reoxygenation, but the extent of cell death during hypoxia was similar to control.

Figure 7.

Cellular ROS production during hypoxia/reoxygenation. A, Representative fluorescence images of cardiac ventricular myocytes loaded with the ROS sensor CM-DCF. ROS bursts during reoxygenation preceded cell death, and Bendavia treatment prevented ROS-induced cell death. B, Representative fluorescence intensity traces for cells in the study. CM-DCF fluorescence is normalized to the basal fluorescence (F_o) for each cell at the end of hypoxia. C, Contribution of ROS bursts to myocyte death during hypoxia/reoxygenation.

Figure 8.

Mitochondrial membrane potential (ΔΨ_m) in myocytes during cellular hypoxia/reoxygenation. A, Representative fluorescence images of myocytes loaded with the ΔΨ_m sensor TMRM. ΔΨ_m collapse often preceded cell death, and treatment with Bendavia improved the capacity to maintain ΔΨ_m. B, Representative fluorescence intensity traces for cells in the study.