AMPK-mediated cardioprotection of atorvastatin relates to the reduction of apoptosis and activation of autophagy in infarcted rat hearts

Abstract

Atorvastatin (ATV) has an important pro-survival role in cardiomyocytes after acute myocardial infarction (AMI). The objectives of this study were to: 1) determine whether ATV could affect autophagy of cardiomyocytes via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, and 2) investigate the balance between autophagy and apoptosis pathways. Male Wistar rats (n = 100) were randomly divided into sham, control, ATV, Compound C, and ATV+ Compound C groups. In this AMI model, drug treatments were administered for 1 week before induction of MI by surgical ligation, and measurements were taken 1 and 4 weeks after AMI induction. Transthoracic echocardiography showed that the ejection fraction in the ATV group increased by 11.7% ± 6.83% over the control group 4 weeks after AMI. The fibrosis, infarcted area, and inflammatory level were determined by pathological and histological studies; these were found to be decreased substantially with ATV treatment (P<0.05). The expression of apoptotic, autophagic, and AMPK pathway proteins was detected by immunohistochemical staining and western blotting, while expression of their corresponding genes was measured with real-time polymerase chain reaction (PCR). ATV treatment increased AMPK/mTOR activity and the expression of autophagic protein LC3 in infarcted myocardium (P<0.05). The treatment also inhibited induction of pro-apoptotic protein Bax. AMPK inhibitor Compound C reversed these beneficial effects. In conclusion, ATV improves survival of cardiomyocytes and decreases alterations in morphology and function of infarcted hearts by inducing autophagy and inhibiting apoptosis through the activation of AMPK/mTOR pathway.

Keywords: AMPK; Myocardial infarction; apoptosis; atorvastatin; autophagy; mTOR.

Figure 1

Echocardiography assessment of cardiac function. A: M-mode echocardiograms are shown at 1 week after myocardial infarction (baseline) and 4 weeks after myocardial infarction (endpoint). B and C: Indices of cardiac function at baseline and endpoint. *P<0.05 vs. AMI; #P<0.05 vs. ATV; n = 8 for each group.

Figure 2

Results of hematoxylin & eosin and Masson’s trichome staining. (A) Representative Masson’s trichome staining images are shown each group. (B) Quantitative analysis of the fibrotic area. (C) Inflammatory cells infiltration in the peri-infarct area. The magnification for (A) is × 12.5, for (C) is × 400; n = 5 for each group; LV, left ventricle. *P<0.05 vs. AMI, #P<0.05 vs. ATV.

Figure 3

Inflammatory cytokines level in the peri-infarct myocardium. Inflammatory cytokines were analyzed by ELISA 4 weeks after AMI. n = 3 in each group. *P<0.05 vs. sham; #P<0.05 vs. AMI; $P<0.05 vs. ATV.

Figure 4

Detection of apoptosis in infarcted rat hearts. (A) Apoptotic cells detected by the TUNEL assay in infarcted hearts. (B) Quantitative analysis of Bax mRNA levels detected by real-time quantitative PCR. Expression values are reported relative to GAPDH and to the sham treatment group. (C) The expression of Bax, Bcl-2, and β-actin detected by western blot analysis. (D and E) Quantitative analysis of the expressions of Bax and Bcl-2 normalized to β-actin. Each column represents mean ± SD of three independent replicates. The magnification for (A) is × 400; *P<0.05 vs. AMI; #P<0.05 vs. ATV.

Figure 5

Detection of autophagy in infarcted rat hearts. (A) Autophagic cells detected by the immunohistochemistry with LC3B antibodies. (B) Quantitative analysis of LC3B mRNA levels measured by real-time quantitative PCR. Expression values were normalized to GAPDH and to sham. (C) The expression of Beclin, LC3I, LC3II, and β-actin detected by western blot analysis. (D and E) Quantitative analysis of the expression of Beclin/β-actin and LC3II/ LC3I was shown in the bar graphs. Each column represents mean ± SD of three independent experiments. The magnification for (A) is × 400; *P<0.05 vs. AMI; #P<0.05 vs. ATV.

Figure 6

AMPK/mTOR pathway proteins detected by western blot in infarcted rat hearts. (A) AMPK/mTOR pathway proteins, including p-AMPK, AMPK, p-p53, p53, p-mTOR, and mTOR, were examined by western blot analysis. (B) Quantitative analysis of the proportion of p-AMPK/AMPK, p-p53/p53, and p-mTOR/mTOR. p-AMPK, phosphorylation of AMP-activated protein kinase; p-p53, phosphorylation of p53; p-mTOR, phosphorylation of mammalian target of rapamycin. Each column represents mean ± SD of three independent experiments. The magnification for (A) is × 400; *P<0.05 vs. AMI; #P<0.05 vs. ATV.