Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling

Abstract

Dexmedetomidine (DEX) has been reported to attenuate the ischemia and reperfusion (I/R) induced cardiomyocyte apoptosis. However, mechanisms underlying these protective effect remain to be fully elucidated. Cardiomyocyte apoptosis is associated with ischemic heart disease. Here we investigated the role of DEX in I/R -induced cardiomyocyte apoptosis. Mice and H9c2 cardiomyocyte cells were subjected to cardiomyocyte I/R injury and hypoxia/reoxygenation (H/R) injury, respectively. The roles and mechanisms of DEX on H9c2 cardiomyocyte cells and mice cardiomyocyte cells exposured to H/R or I/R injury were explored. The results showed that DEX attenuates H/R injury-induced H9c2 cell apoptosis and alleviated mitochondrial oxidative stress; it also reduced myocardial infarct size and protected the cardiac function following cardiomyocyte I/R injury. In addition, H/R and I/R injury increased p53 expression and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling in H9c2 cardiomyocyte cells and cardiomyocytes. Targeting p53 expression or FOXO3a/PUMA signaling inhibited cell apoptosis and protected against H/R injury in H9c2 cardiomyocyte cells and cardiomyocytes. Pretreatment with DEX reduced the H/R or I/R injury-induced activation of p53 expression and FOXO3a/PUMA signaling, and alleviated H/R or I/R injury-induced apoptosis and mitochondrial oxidative stress. Therefore, DEX could alleviate H/R- or I/R-induced cardiomyocytes injury by reducing cell apoptosis and blocking p53 expression and FOXO3a/PUMA signaling. Targeting p53 or/and FOXO3a/PUMA signaling could alleviate cardiomyocyte I/R injury.

Keywords: Dexmedetomidine; apoptosis; forkhead box o3a; ischemia and reperfusion; p53; p53-upregulated modulator of apoptosis.

Figure 1.

Effect of H/R on apoptosis in H9c2 cardiomyocytes. Control and H9c2 cardiomyocytes transfected with p53 siRNA or treated with DEX were subjected to 18 h of hypoxia followed by 24 h of reoxygenation. (a) CCK-8 assay was used to determine cell viability. (b) Hoechst 33258 staining was used to determine cell apoptosis. (c) Reverse transcription-quantitative PCR and (d) Western blotting was used to determine p53 mRNA and protein expression levels, respectively. *P < 0.05, **P < 0.01 and ***P < 0.001. H/R, hypoxia/reoxygenation; siRNA, small-interfering RNA; DEX, dexmedetomidine; OD, optical density; NC, negative control; LOG, logarithmic.

Figure 2.

Effect of DEX on p53 protein expression in H9c2 cardiomyocytes subjected to H/R. (a) H9c2 cardiomyocytes were transfected with p53 siRNA or treated with DEX followed by H/R treatment, and the p53 protein expression was detected by Western blot analysis. (b) H9c2 cardiomyocytes were transfected with FOXO3a siRNA followed by H/R treatment, and the FOXO3a, PUMA and Bim protein expression was detected by Western blot analysis. (c) H9c2 cardiomyocytes were transfected with FOXO3a, PUMA or Bim siRNA, followed by H/R treatment, and the cleaved-caspase-3/9 protein expression was detected by Western blot analysis. (d) H9c2 cardiomyocytes were transfected with FOXO3a, PUMA or Bim siRNA, followed by H/R treatment, and cell apoptosis was detected by Hoechst 33258 staining. **P < 0.01. DEX, dexmedetomidine; H/R, hypoxia/reoxygenation; siRNA, small-interfering RNA; FOXO3a, forkhead box O3a; PUMA, p53-upregulated modulator of apoptosis; Bim, Bcl-2-interacting mediator of cell death; MCL-1, myeloid-cell leukemia 1; Noxa, NADPH oxidase activator.

Figure 3.

DEX inhibits cell apoptosis and oxidative stress following myocardial I/R injury. (a) Representative photomicrographs of TUNEL-stained heart sections. The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total cells. (b) Effect of DEX on LDH release. (c) Effect of DEX on CK-MB release. (d) Cytotoxicity was measured by analyzing the MDA level. (e) Western blot analysis of the levels of apoptotic proteins.**P < 0.01. DEX, dexmedetomidine; I/R, ischemia/reperfusion; LDH, lactate dehydrogenase; CK-MB, creatine kinase myocardial band; MDA, malondialdehyde; SOD, superoxide dismutase; MCL-1, myeloid-cell leukemia 1; FOXO3a, forkhead box O3a; PUMA, p53-upregulated modulator of apoptosis.

Figure 4.

Effect of DEX on cardiac function and infarct size following myocardial I/R. (a) Evans blue/TTC double staining. (b) Echocardiography following DEX treatment during I/R. *P < 0.05. DEX, dexmedetomidine; TTC, triphenyltetrazolium chloride; I/R, ischemia/reperfusion; LVES, large vessel endothelial supplement.