Reactive oxygen species trigger ischemic and pharmacological postconditioning: in vivo and in vitro characterization

Abstract

Reactive oxygen species (ROS) generated by ischemic and pharmacological preconditioning are known to act as triggers of cardiac protection; however, the involvement of ROS in ischemic and pharmacological postconditioning (PostC) in vivo and in vitro is unknown. We tested the hypothesis that ROS are involved in PostC in the mouse heart in vivo and in the isolated adult cardiac myocyte (ACM). Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion with or without ischemic or pharmacologic PostC (three cycles of 20 s reperfusion/ischemia; 1.4% isoflurane; 10 mg/kg SNC-121). Additional groups were treated with 2-mercaptopropionyl glycine (MPG), a ROS scavenger, 10 min before or after the PostC stimuli. Ischemia-, isoflurane-, and SNC-121- induced PostC reduced infarct size (24.1+/-3.2, 15.7+/-2.6, 24.9+/-2.6%, p<0.05, respectively) compared to the control group (43.4+/-3.3%). These cardiac protective effects were abolished by MPG when administered before (40.0+/-3.6, 39.3+/-3.1, 38.5+/-1.6%, respectively), but not after the PostC stimuli (26.6+/-2.3, 17.0+/-2.2, 23.9+/-1.7%, respectively). Additionally, ACM were subjected to a simulated ischemia/reperfusion protocol with isoflurane and SNC PostC. Isoflurane- and SNC-induced PostC in vitro were abolished by prior treatment with MPG. These data indicate that ROS signaling is an essential trigger of ischemic and pharmacological PostC and this is occurring at the level of the cardiac myocyte.

Figure 1

Schematic illustration of the experimental protocol. Mice were subjected to 30 min coronary artery occlusion and 2 h of reperfusion. No intervention was performed in Control animals (n = 9). Hearts underwent postconditioning (PostC) in early reperfusion periods. Ischemic PostC (R/I) was elicited by three cycles of 20 s reperfusion and ischemia before 2 h of reperfusion (n = 8). Isoflurane (ISO, n = 8) or SNC-121 (SNC, n = 8) was administered for 5 min, beginning at 3 min before and ending 2 min after reperfusion. Groups were treated with 2-mercaptopropionyl glycine (MPG) before reperfusion (Pre MPG, MPG + R/I, MPG + ISO, and MPG + SNC, n = 8), or after reperfusion (Post MPG, R/I + MPG, ISO + MPG, and SNC + MPG, n = 8).

Figure 2

Myocardial infarct size expressed as percentage of the left ventricular area at risk. Data are means ± SEM. *p < 0.05 vs. Control; **p < 0.05 vs. Post MPG; #p < 0.05 vs. R/I; §p < 0.05 vs. ISO; †p < 0.05 vs. SNC.

Figure 3

Percent cell death as determined by trypan blue staining. Data are means ± SEM. *p < 0.01 vs. Basal Ischemia.