Emulsified isoflurane postconditioning produces cardioprotection against myocardial ischemia-reperfusion injury in rats

Abstract

Emulsified isoflurane (EIso) preconditioning can induce cardioprotection. We investigated whether EIso application after ischemia protects hearts against reperfusion injury and whether it is mediated by the inhibition of apoptosis. Rats were subjected to 30-min coronary occlusion followed by 180-min reperfusion. At the onset of reperfusion, rats were intravenously administered saline (sham, control group), 30 % intralipid (IL group) or 2 ml kg(-1) EIso (EIso group) for 30 min. After reperfusion, infarct sizes, myocardial apoptosis and expression of Bcl-2, Bax and caspase-3 proteins were determined. Hemodynamic parameters were not different among groups. Compared with control and intralipid group, EIso limited infarct size, inhibited apoptosis, increased the expression of Bcl-2, decreased the expression of Bax, cleaved caspase-3, and enhanced Bcl-2/Bax ratio. EIso protects hearts against reperfusion injury when administered at the onset of reperfusion, which may be mediated by the inhibition of apoptosis via modulation of the expression of pro- and anti-apoptotic proteins.

Fig. 1

Myocardial infarct size expressed as a percentage of left ventricular area at risk in rats receiving no postconditioning stimuli (CON), 30 % intralipid (IL) or 2 ml kg⁻¹ emulsified isoflurane (EIso). The upper and lower edges of each box plot represent the 25th percentile and the 75th percentile, respectively. The solid horizontal line within the box indicates the median value for the variable. Data are expressed as mean ± SD (n = 8 per group). **Significantly (P < 0.01) different from CON. ^†Significantly (P < 0.05) different from IL group

Fig. 2

Apoptosis determined by measurement of terminal transferase dUTP nick end labeling (TUNEL) positive cardiomyocyte nuclei in the area at risk of myocardium obtained from rats receiving saline (sham, CON), 30 % intralipid (IL) or 2 ml kg⁻¹ emulsified isoflurane (EIso) during early reperfusion after coronary artery occlusion. Arrows denote TUNEL positive nucleus. Positive cells were not detected in non-ischemic zone after ischemia and reperfusion. Magnification 400×. Sham, Sham operated group; CON, control group; IL, 30 % intralipid group; EIso, emulsified isoflurane group of 2 ml kg⁻¹. Box-plot graph illustrates the average percentage of TUNEL-positive ventricular myocytes in the ischemic regions of LVs. Each group n = 8. Data are expressed as mean ± SD. **P < 0.01 compared with CON

Fig. 3

Caspase-3 protein expression and cleavage in myocardium of rats in each group. a Cleaved caspase-3 immunoreactivity in the cytoplasm of myocytes of hearts. Magnifications, 400×. b Western blot analysis of total and cleaved caspase-3 protein expression in hearts subjected to ischemia/reperfusion and receiving different treatments. Representative blots of total and cleaved caspase-3 (left). Data are quantified as percentage of cleaved caspase-3 of total caspase-3 and displayed using a box-plot. Data are presented as mean ± SD. Sham, Sham operated group; CON, control group; IL, 30 % intralipid group; EIso, emulsified isoflurane group of 2 ml kg⁻¹. Each group n = 8. PEI, positive expressive index. *P < 0.05, **P < 0.01 vs sham; ^† P < 0.05, ^†† P < 0.01 vs CON; ^‡‡ P < 0.01 vs IL

Fig. 4

Myocardial Bcl-2 and Bax protein immunostaining. Rats were subjected to 3 h of sham ischemia and reperfusion or 30 min of myocardial ischemia followed by 3 h of reperfusion. Sham, Sham operated group; CON, control group; IL, 30 % intralipid group; EIso, emulsified isoflurane group of 2 ml kg⁻¹. Magnification 400×. Data are presented as mean ± SD. Each group n = 8. PEI, positive expressive index; CI, 95 % confidence interval. *P < 0.05, **P < 0.01 vs sham; ^† P < 0.05, ^†† P < 0.01 vs CON; ^‡ P < 0.05, ^‡‡ P < 0.01 vs IL

Fig. 5

Effect of emulsified isoflurane on expression of Bcl-2 and Bax proteins visualized by Western blot analysis. Representative Western blot bands of Bcl-2 (26 kD), Bax (21kD) expression in the ischemic myocardium 3 h after reperfusion are shown. Corresponding GAPDH band as loading controls is shown in the upper panels. The box-plot graphs in the lower panel show quantification of the Western blot analysis for the respective proteins of Bcl-2 (a), Bax (b) and Bcl-2/Bax ratio (c) compared with GAPDH, respectively. Data are given as mean ± SD (n = 8, each group). *P < 0.05, **P < 0.01 vs sham; ^† P < 0.05, ^†† P < 0.01 vs CON; ^‡ P < 0.05, ^‡‡ P < 0.01 vs IL