Effects of dexmedetomidine postconditioning on myocardial ischemia and the role of the PI3K/Akt-dependent signaling pathway in reperfusion injury

Abstract

The present study aimed to determine whether post-ischemic treatment with dexmedetomidine (DEX) protected the heart against acute myocardial ischemia/reperfusion (I/R)‑induced injury in rats. The phosphatidylinositol‑3 kinase/protein kinase B(PI3K/Akt)‑dependent signaling pathway was also investigated. Male Sprague Dawley rats (n=64) were subjected to ligation of the left anterior descending artery (LAD), which produced ischemia for 25 min, followed by reperfusion. Following LAD ligation, rats were treated with DEX (5, 10 and 20 µg/kg) or underwent post‑ischemic conditioning, which included three cycles of ischemic insult. In order to determine the role of the PI3K/Akt signaling pathway, wortmannin (Wort), a PI3K inhibitor, was used to treat a group of rats that had also been treated with DEX (20 µg/kg). Post‑reperfusion, lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase isoenzymes (CK‑MB), superoxide dismutase (SOD) and malondialdehyde (MDA) serum levels were measured using an ultraviolet spectrophotometer. The protein expression levels of phosphorylated (p)‑Akt, Ser9‑p‑glycogen synthase kinase‑3β (p‑GSK‑3β) and cleaved caspase‑3 were detected in heart tissue by western blotting. The mRNA expression levels of B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax) were detected using reverse transcription‑polymerase chain reaction. At the end of the experiment, the hearts were removed and perfused in an isolated perfusion heart apparatus with Evans blue (1%) in order to determine the non‑ischemic areas. The risk and infarct areas of the heart were not dyed. As expected, I/R induced myocardial infarction, as determined by the increased serum levels of cTnI, CK‑MB and MDA, and the decreased levels of SOD. Post‑ischemic treatment with DEX increased the expression levels of p‑Akt and p‑GSK‑3β, whereas caspase‑3 expression was reduced following DEX treatment compared with in the I/R group. Compared with the I/R group, the ratio of Bcl‑2/Bax at the mRNA level was elevated in the DEX and ischemic post‑conditioning groups, whereas the expression levels of Bax were decreased. Conversely, the effects of DEX were attenuated by Wort. These results indicated that, similar to post‑ischemic conditioning, post‑ischemic treatment with DEX protects the heart against I/R via the PI3K/Akt‑dependent signaling pathway, possibly by activating GSK‑3β.

Figure 1

Experimental protocols used for in vivo experiments. I/R, ischemia/reperfusion group; IPO, ischemic post-conditioning group; D+Wort, DEX + Wort group; NS, normal saline; DEX, dexmedetomidine; Wort, wortmannin.

Figure 2

(A) LDH, (B) CK-MB, (C) cTnI, (D) SOD and (E) MDA levels in the serum of rats from each group. Data are presented as the mean ± standard error of the mean from at least six independent experiments. ^*P<0.05, ^**P<0.01 vs. S group, ^§P<0.05, ^§§P<0.01 vs. I/R group; ^#P<0.05, ^##P<0.01 vs. IPO group, ^&&P<0.01 vs. DEX5 group; ^{^^}P<0.01 vs. DEX10 group; ⁺P<0.05, ⁺⁺P<0.01 vs. DEX20 group. LDH, lactate dehydrogenase; cTnI, cardiac troponin I; CK-MB, creatine kinase isoenzymes; MDA, malondialdehyde; SOD, superoxide dismutase; S, sham; I/R, ischemia/reperfusion; IPO, ischemic post-conditioning; DEX, dexmedetomidine; Wort, wortmannin.

Figure 3

Comparison of infarct size in each experimental group in vivo. Data are presented as the mean ± standard error of the mean from at least four independent experiments. ^§§P<0.01 vs. I/R group; ⁺⁺P<0.01 vs. DEX20 group. I/R, ischemia/reperfusion; IPO, ischemic post-conditioning; DEX, dexmedeto-midine; Wort, wortmannin.

Figure 4

Expression levels of (A and B) p-Akt and (C and D) p-GSK-3β (Ser9) in heart tissue. Representive western blots of (A) p-Akt and (C) p-GSK-3β (Ser9) in the heart of each experimental group. β-actin was used as a loading control. (B and D) Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as the mean ± standard error of the mean from at least four independent experiments. ^§P<0.05, ^§§P<0.01 vs. I/R group; ⁺P<0.05, ⁺⁺P<0.01 vs. DEX20 group. p-, phosphorylated; Akt, protein kinase B; GSK-3β, glycogen synthase kinase-3β; I/R, ischemia/reperfusion; DEX, dexmedetomidine; Wort, wortmannin.

Figure 5

Expression levels of cleaved caspase-3 in heart tissue of each group. (A) Western blotting of cleaved caspase-3. β-actin was used as a loading control. (B) Average quantification obtained by densitometric analysis of the results of western blot analysis. Data are presented as the mean ± standard error of the mean from at least four independent experiments. ^§§P<0.01 vs. I/R group; ⁺P<0.05 vs. DEX20 group. I/R, ischemia/reperfusion; DEX, dexmedetomidine; Wort, wortmannin.

Figure 6

mRNA expression levels of myocardial Bcl-2 and Bax, and quantification of the Bcl-2/Bax ratio in the various groups. (A and B) Results of RT-PCR analysis in the heart tissue. β-actin was used as a loading control. (C) Quantification of the Bcl-2/Bax ratio obtained by densitometric analysis of RT-PCR. Data are presented as the mean ± standard error of the mean from at least six independent experiments. ^*P<0.05, ^**P<0.01 vs. S group; ^§§P<0.01 vs. I/R group; ⁺P<0.05, ⁺⁺P<0.01 vs. DEX20 group. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; RT-PCR, reverse transcription-polymerase chain reaction; S, sham; I/R, ischemia/reperfusion; IPO, ischemic post-conditioning; DEX, dexmedetomidine; Wort, wortmannin.