Myocardial Ischemic Postconditioning Promotes Autophagy against Ischemia Reperfusion Injury via the Activation of the nNOS/AMPK/mTOR Pathway

Abstract

Autophagy participates in the progression of many diseases, comprising ischemia/ reperfusion (I/R). It is reported that it is involved in the protective mechanism of ischemic postconditioning (IPostC). According to research, neuronal nitric oxide synthase (nNOS) is also involved in the condition of I/R and IPostC. However, the relationship between nNOS, autophagy and IPostC has not been previously investigated. We hypothesize that IPostC promotes autophagy activity against I/R injury partially through nNOS-mediated pathways. Mouse hearts were subjected to I/R injury through the ligation of the left anterior descending coronary artery. H9c2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro. IPostC, compared with I/R, restored nNOS activity, increased the formation of autophagosome and restored the impaired autophagic flux, thus autophagic activity was raised markedly. IPostC increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and suppressed mammalian target of rapamycin (mTOR), but a selective nNOS inhibitor abolished those effects. Similar effects of IPostC were demonstrated in H9c2 cells in vitro. IPostC decreased infarct size and preserved most of the normal structure. The level of reactive oxygen species (ROS) and cell apoptosis were reduced by IPostC with improved cell viability and mitochondrial membrane potential. However, an autophagy inhibitor suppressed the protective effects. These results suggest that IPostC promoted autophagy against I/R injury at least partially via the activation of nNOS/AMPK/mTOR pathway.

Keywords: adenosine monophosphate-activated protein kinase (AMPK); autophagy; ischemia/reperfusion; ischemic postconditioning (IPostC); mammalian target of rapamycin (mTOR); neuronal nitric oxide synthase (nNOS).

Figure 1

Activity of neuronal nitric oxide synthase (nNOS) was restored by ischemic postconditioning (IPostC) in the myocardium and H9c2 cells. (A) Expression of nNOS and nNOS^Ser852 in the myocardium at 30 min of reperfusion; (B) The level of nNOS and nNOS^Ser852 in H9c2 cells at 30 min of reoxygenation. ^& p < 0.05 versus control, * p < 0.05 versus ischemia/reperfusion (I/R). The mean values ± SEM, n = 6.

Figure 2

Autophagy activity was promoted by IPostC via nNOS in myocardium. (A) The protein expression of LC3 in the myocardium at 30 min of reperfusion; (B) The level of p62 was detected in the myocardium; (C) Autophagosomes (AVs) which were marked with red arrows were confirmed using electron microscopy in the control, I/R, IPostC group and IPostC treated with nNOS inhibitor L-VNIO. ^& p < 0.05 versus control, * p < 0.05 versus I/R, ^# p < 0.05 versus IPostC. The mean values ± SEM, n = 6.

Figure 3

Hypoxic postconditioning (HPostC) enhanced autophagic activity via nNOS in H9c2 cells; (A) H9c2 cells were infected with GFP-LC3 adenovirus. The nuclei were labeled with DAPI (blue); (B) The statistics of GFP-LC3 dots; (C) Expression of LC3 in H9c2 cells at 30 min of reoxygenation; (D) Expression of p62 at 30 min of reoxygenation. ^& p < 0.05 versus control, * p < 0.05 versus hypoxia/reoxygenation (H/R), ^# p < 0.05 versus HPostC. The mean values ± SEM, n = 6.

Figure 4

IPostC activated the nNOS/AMPK/mTOR signaling pathway during early reperfusion. (A) The protein expression and optical density analysis of p-AMPK (Thr172)/AMPK, p-mTOR(Ser2448)/mTOR in the myocardium at 30 min of reperfusion; (B) Expression of p-AMPK, p-mTOR in H9c2 cells at 30 min of reoxygenation. ^& p < 0.05 versus control, * p < 0.05 versus I/R, ^# p < 0.05 versus IPostC. The mean values ± SEM, n = 6.

Figure 5

The protective effect of IPostC in the myocardium was inhibited by an autophagy inhibitor. (A) Infarct size was measured at 120 min of reperfusion; (B) Representative histological images of hearts at 120 min of reperfusion. ^& p < 0.05 versus control, * p < 0.05 versus I/R, ^# p < 0.05 versus IPostC. The mean values ± SEM, n = 6.

Figure 6

HPostC protected H9c2 cells by promoting autophagy in vitro. (A,B) ROS production was detected in H9c2 cells at 30 min of reoxygenation; (C) H9c2 cells were collected and stained with annexin V-FITC/propidium iodide and detected by flow cytometry; (D) A bar diagram of the cell apoptosis; (E) Cell viability was assessed by CCK-8 kits. ^& p < 0.05 versus control, * p < 0.05 versus H/R, ^# p < 0.05 versus HPostC. The mean values ± SEM, n = 6.

Figure 7

Determination of mitochondrial function. (A) Mitochondrial morphology was observed under electron microscopy in the myocardium (mitochondrial vacuoles were indicated by red arrows); (B) Measurement of mitochondria membrane potential after 30 min of reoxygenation. ^& p < 0.05 versus control, * p < 0.05 versus H/R, ^# p < 0.05 versus HPostC. The mean values ± SEM, n = 6.