Upregulated ATF6 contributes to chronic intermittent hypoxia-afforded protection against myocardial ischemia/reperfusion injury

Abstract

In the present study, we investigated the role of activating transcription factor 6 (ATF6) in the mechanism by which chronic intermittent hypoxia (CIH) increases tolerance to myocardial ischemia/reperfusion (I/R). Experiments were conducted using a rat model of I/R injury in vivo and isolated Langendorff-perfused rat hearts ex vivo. The role of Akt in this process was also investigated in vitro using rat myoblast H9c2 cells. Cell viability was measured using a cell counting kit-8 assay. Lactate dehydrogenase (LDH) and creatine kinase cardiac isoenzyme activity were also measured as markers of cellular damage. ATF6, Akt and phosphorylated (p)-Akt expression was analyzed by western blot analysis. RNA interference (RNAi) was used to suppress ATF6 expression. We noted that ATF6 expression in the ventricular myocardium was significantly increased in rats exposed to CIH. Furthermore, we noted that CIH preserved cardiac function after I/R in vivo and improved post-ischemic recovery of myocardial performance in isolated rat hearts. ATF6 and p-Akt expression was upregulated in cultured H9c2 cells exposed to chronic mild hypoxia compared with those cultured under normoxic conditions. Chronic mild hypoxia attenuated subsequent simulated I/R injury in H9c2 cells (48 h), as evidenced by increased cell viability and decreased LDH activity. By contrast, decreased cell viability and increased LDH activity were observed in siRNA-ATF6-transfected H9c2 cells, with a concomitant reduction in p-Akt levels. These results indicated that ATF6 upregulation is involved in the mechanism by which CIH attenuates myocardial I/R injury, possibly through upregulation of p-Akt, which is a key regulator of cardiomyocyte survival.

Figure 1

Chronic intermittent hypoxia (CIH) improves cardiac function recovery after ischemia/reperfusion (I/R) in isolated rat hearts. After 20 min of stabilization with Krebs-Henseleit (K–H) perfusate, the hearts were subjected to 30 min of global ischemia and 1 h of reperfusion. (A) Left ventricular developed pressure (LVDP) before ischemia. (B) LVDP recovery (percentage of pre-ischemic LVDP). (C) Representative sections for TTC staining (left) and the average infarct size vs. total area at risk (right). (D) CIH decreased I/R-induced necrosis, as shown by lactate dehydrogenase (LDH) release in the effluent after 60 min of reperfusion. Data are represented as the means ± SEM (n=6 rats/group). ^*P<0.05 and ^**P<0.01 vs. normoxia group.

Figure 2

In vivo effects of chronic intermittent hypoxia (CIH) on left ventricular function following myocardial ischemia/reperfusion. (A) Representative echocardiographic images taken pre-ischemia and post-reperfusion. (B) Pre-ischemic and post-reperfusion (24 h) cardiac function: left ventricular ejection fraction (LVEF) (left) and left ventricular fractional shortening (LVFS) (right). (C) Average infarct size. (D) Post-reperfusion (24 h) creatine kinase (CK)-MB activity. (E) Post-reperfusion (24 h) lactate dehydrogenase (LDH) activity. Data are represented as the means ± SEM (CH group, n=7; N group, n=6). P<0.05 vs. normoxia group.^*P<0.05 and ^**P<0.01.

Figure 3

Cleaved activating transcription factor 6 (ATF6) expression in rat hearts. (A) Representative images of western blot analysis of cleaved ATF6, binding immunoglobulin protein (BiP) and glyceraldehyde 3-phosphate dehydrogenase (GADPH) protein expression in rat myocardial tissue under normoxic or hypoxic conditions. (B) Quantification of cleaved ATF6 and BiP protein levels (normalized to GADPH protein levels). Representative data from five independent experiments are shown. ^**P<0.01 vs. normoxia group. (C) Immunofluorescence staining of the left ventricle (LV) and the right ventricle (RV) of rat hearts for ATF6 protein expression (green) under hypoxic or normoxic conditions; cell nuclei were counterstained with DAPI (blue). Scale bar, 25 mm.

Figure 4

Cleaved activating transcription factor 6 (ATF6) and Akt expression and effects on simulated ischemia/reperfusion (sI/R) injury induced in H9c2 cells subjected to mild hypoxia. (A) Representative expression of cleaved ATF6 and β-actin protein in H9c2 cells cultured under normoxic or hypoxic conditions for 0, 12, 24, 48 and 72 h (left); quantification of cleaved ATF6 and binding immunoglobulin protein (BiP) protein levels (normalized to β-actin protein levels) (right, ^*P<0.05 vs. 0 h; ^**P<0.01 vs. 0 h). (B) Representative expression of Akt, p-Akt and β-actin protein in H9c2 cells cultured under normoxic or hypoxic conditions for 0, 12, 24, 48 and 72 h (left); quantification of p-Akt protein levels (normalized to total Akt protein levels) (right, ^*P<0.05 vs. 0 h; ^**P<0.01 vs. 0 h). (C) Effect of mild hypoxia on the viability of H9c2 cells subjected to ischemia (1 h) followed by reperfusion (3 h). Representative data from five independent experiments are presented as the means ± SEM (^**P<0.01 vs. normoxia group). (D) Effect of chronic mild hypoxia on lactate dehydrogenase (LDH) activity in H9c2 cells subjected to ischemia (1 h) followed by reperfusion (3 h). Representative data from five independent experiments are presented as the means ± SEM (^**P<0.01 vs. normoxia group).

Figure 5

Cleaved activating transcription factor 6 (ATF6) and Akt expression and effects on simulated ischemia/reperfusion (sI/R) injury induced in siRNA-ATF6-transfected H9c2 cells subjected to mild hypoxia. (A) Representative expression of cleaved ATF6 and β-actin in siRNA-ATF6-transfected H9c2 cells cultured under hypoxic conditions for 48 h (left); quantification of cleaved ATF6 and binding immunoglobulin protein (BiP) protein levels (normalized to β-actin protein levels) (right, ^**P<0.01 vs. siRNA-control). (B) Representative expression of Akt, p-Akt and β-actin proteins in siRNA-ATF6-transfected H9c2 cells cultured under hypoxic conditions for 48 h (left); quantification of p-Akt protein levels (normalized to total Akt protein levels) (right, ^*P<0.05 vs. siRNA-control). (C) Effect of mild hypoxia on the viability of siRNA-ATF6-transfected H9c2 cells subjected to ischemia (1 h) followed by reperfusion (3 h). Representative data from five independent experiments are presented as the means ± SEM (^**P<0.01 vs. siRNA-control). (D) Effect of chronic mild hypoxia on lactate dehydrogenase (LDH) activity of siRNA-ATF6-transfected H9c2 cells subjected to ischemia (1 h) followed by reperfusion (3 h). Representative data from five independent experiments are presented as the means ± SEM (^**P<0.01 vs. siRNA-control).