Sulodexide attenuates endoplasmic reticulum stress induced by myocardial ischaemia/reperfusion by activating the PI3K/Akt pathway

2.1 Animals and materials

Male C57/B6 mice aged 8-12 weeks were supplied by the Animal Center of the Wenzhou medical university. The animal use and care protocols were in line with the National Institutes of Health Guide of the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wenzhou medical University (Number: 2017-010). Rat cardiomyocyte H9C2 cells were purchased from the American Type Culture Collection. DMEM and foetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Anti-p-PI3K (ab182651) was purchased from Abcam (Cambridge, MA). Anti-PI3K (C73F8), anti-AKT (C67E7), p-AKT (Ser473), anti-BAX (D3R2M), Bcl-2 (D17C4), anti-CHOP (D46F1), GRP-78 (C50B12), ATF-6 (D4Z8V), GAPDH (D16H11), β-actin (13E5) antibodies and goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc (Danvers, MA). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA). Tert-butyl hydroperoxide (TBHP) and PI3K/AKT inhibitor (LY294002) were purchased from Sigma-Aldrich.

2.2 Cardiac I/R model in mice and treatment

The MI/R in mice was induced by transient myocardial ischaemia for 30 minutes followed by reperfusion for 2 hours. Specifically, mice were anaesthetized by inhaling isoflurane. The heart was completely exposed in the intercostal space by a lateral cut along the third or fourth rib. Then the left anterior descending coronary artery (LAD) was found and was ligatured. The heart grew grey quickly after using a 7-0 silk suture at the distal 1/3 of the LAD.

The ligation was released after coronary artery occlusion for 30 minutes and reperfusion for 2 hours. The Sham group mice were also anaesthetized and the heart was exposed, a silk suture was passed under the LAD but not ligated. The MI/R mice were administered with 60 µg SDX/mouse through gastric administration at 30 minutes before ischaemia.27 The PI3K inhibitor LY294002 was administered into mouse through intramyocardial injection at 30 minutes before ischaemia and the dose is 0.2 mg/mouse.

2.3 Myocardial infarct size measurement

The myocardial infarct size was assessed by Evan's Blue and triphenyltetrazolium chloride (TTC) double staining method. At the end of reperfusion, the LAD was reoccluded and 0.2 mL of a 2% Evan's Blue dye was injected into the inferior vena cava. When the right side of the heart turned blue, the heart was quickly removed, rinsed in NS and frozen at −20°C. Five 1-mm thick heart sections were made and incubated in 1% TTC for 20 minutes at 37°C. The viable tissue which was stained red by TTC was defined as area at risk (AAR). The non-ischaemic myocardium was stained deep blue by Evans Blue. Infarct area (INF) appeared pale after staining. A percent of infarcted area over total AAR (% INF/AAR) was calculated.

2.4 Myocardial TUNEL staining

After 2 hours of reperfusion, the heart was removed. The heart tissues were then embedded in optimal cutting temperature compound and approximately 5-µm thick sections were cut. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) (Roche, Mannheim, Germany) staining was used to detect cardiomyocytes apoptosis according to the kit's manual. The section images were taken by a Nikon ECLIPSE Ti microscope (Olympus BX51, Japan).

2.5 Determination of superoxide dismutase activity and malondialdehyde levels

The superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were measured in mice plasm, which were conducted using commercial kit reagents (JianCheng Bioengineering Institute, Nanjing, China) according to the manufacturers' instructions.

2.6 Cell viability

H9C2 cells were cultured in high-glucose DMEM with 10% (v/v) FBS without penicillin/streptomycin in a humidified atmosphere (5% CO₂, 21% O₂) at 37°C. H9C2 cells were seeded into 96-well plates at a concentration of 5000 cells per well. The cells were pre-treated with SDX at different concentration for 30 minutes before being exposure to TBHP for 12 hours. The PI3K inhibitor LY294002 was added to the cell at the concentration of 15 µmol/L for 30 minutes before adding TBHP. The number of viable cells was evaluated by CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS). Briefly, 20% MTS dye solution was added to each well and incubated for 2.5 hours. The number of viable cells was measured by evaluating absorbance at 480 nm. The MTS assay was repeated three times for consistency.

2.7 Cell apoptosis assays

H9C2 cells (2 × 10⁵) were plated in 6-well plates for 24 hours. After different treatments (note: LY294002 and SDX added at the same time), cells of each well were collected, washed twice with PBS and resuspended with 200 µL PBS. Then cells (100 µL) were incubated with 5 µL annexin V-fluorescein isothiocyanate for 5 minutes, followed by 3 µL propidium iodide (PI) for 10 minutes in the dark at room temperature. The fluorescence intensities of annexin V/PI-stained cells were analysed by a flow cytometry. The apoptosis ratio was quantified by BD FACS software.

2.8 Western blot

Total proteins in the heart tissue and H9C2 cells were extracted and denatured using protein extraction reagents. Fifty microgram of protein was then loaded in lanes of 10% gels, separated by electrophoresis and then transferred onto a polyvinylidene fluoride membrane. After blocking with 5% fat-free milk, the membranes were incubated with the relevant primary antibodies overnight on a refrigerator shaker. The membranes were washed three times with TBS Tween and incubated with the relevant secondary antibodies at room temperature for 2 hours. The signals were visualized using ECL reagents. The amounts of the proteins were analysed using Image Pro Plus and normalized to their respective controls.

2.9 Statistical analysis

The data in this study are shown as the mean ± SEM Differences among groups were analysed using one-away ANOVA accompanied with Turkey multiple-comparisons test (IBM spss statistical 21). Two-tailed Student's t test was used for comparison between two groups; P < 0.05 was considered significant.

3.1 SDX pre-treatment decreased MI/R injury

To determine the role of SDX in cardiac protection, SDX was administered to mice 30 minutes before ischaemia. After 30 minutes of ischaemia and 2 hours of reperfusion, the size of myocardial infarcts was measured by TTC staining. Compared with the MI/R group, the infarct size was greatly reduced in the SDX pre-treatment group (Figure 1A). Myocardial ischaemia/reperfusion injury affects ER integrity and promotes ER stress.28, 29 The level of MDA in plasma was markedly increased by MI/R and this effect was significantly reduced by SDX pre-treatment. In contrast, the level of SOD was decreased by MI/R and this effect was reversed by SDX pre-treatment (Figure 1B).

3.2 SDX pre-treatment inhibited MI/R-induced apoptosis

Loss of cardiac cells in MI/R injury is caused by ER stress-induced apoptosis.30 We investigated the effect of SDX on apoptosis during MI/R A TUNEL assay and showed that MI/R significantly increased apoptosis of cardiac cells, whereas SDX pre-treatment ameliorated this effect (Figure 2A). The B-cell lymphoma 2 (BCL2) protein family is associated with ER stress-induced apoptosis31; within this family, Bcl-2 plays an anti-apoptotic and Bax a pro-apoptotic role. As expected, MI/R increased Bax and decreased Bcl-2 protein levels in cardiomyocytes and these effects were alleviated by SDX pre-treatment (Figure 2B).

3.3 SDX exerted the cardioprotective effect by reducing ER stress and activating the PI3K/AKT pathway

To assess the role of SDX in ER stress induced by MI/R, we assayed the expression of several ER stress-related proteins and the activation state of the PI3K/Akt signalling pathway. Pro-apoptotic transcription factor C/EBP homologous protein (CHOP), ER-targeted cytoprotective chaperones glucose-regulated protein 78 (GRP78) and activating transcription factor 6 (ATF-6) were up-regulated in the heart of mice with MI/R (Figure 3A); however, this effect was reversed by SDX pre-treatment. Importantly, administration of SDX to sham mice did not affect the levels of these proteins. The PI3K/Akt pathway is an important survival pathway in a variety of cell types and is involved in the regulation of ER stress.32, 33 We examined the activity of the PI3K/Akt pathway in the hearts of mice with MI/R As expected, MI/R activated the PI3K/Akt pathway and the magnitude of this activation was enhanced by SDX pre-treatment, as determined by Western blotting (Figure 3B).

3.4 SDX reduced oxidative stress-induced apoptosis in H9C2 cells

We next evaluated the cardioprotective effect of SDX in vitro. Tert-butyl hydroperoxide induces oxidative stress and apoptosis in H9C2 cells.34 Dose-response experiments showed that the optimal concentrations of TBHP and SDX in H9C2 cells were 100 µmol/L and 0.25 µg/mL respectively (Figure 4A). Flow cytometry indicated that TBHP induced the apoptosis of H9C2 cells and this effect was reduced by SDX pre-treatment (Figure 4B). Moreover, the Bax protein level was significantly increased by TBHP treatment but reduced by SDX pre-treatment. In contrast, the Bcl-2 level was decreased by TBHP treatment and increased by SDX pre-treatment (Figure 4C).

3.5 SDX attenuated ER stress and activated the PI3K/AKT pathway in H9C2 cells

Based on the above results, we hypothesized that ER stress and the PI3K/Akt pathway are involved in the SDX-mediated inhibition of apoptosis of H9C2 cells. Compared with the control group, the CHOP, GRP78 and ATF-6 levels were significantly increased in the TBHP group and decreased in the SDX pre-treatment group (Figure 5A). In addition, compared with the control group, the phosphorylated p-PI3K and p-Akt levels were increased in the TBHP-treated group and the magnitudes of these increases were enhanced by SDX pre-treatment (Figure 5B).

3.6 Inhibition of the PI3K/AKT pathway reversed the protective effect of SDX

To determine whether the SDX-mediated suppression of ER stress-induced apoptosis was related to the activation of the PI3K/Akt pathway, the PI3K inhibitor LY294002 was used. LY294002 significantly increased apoptosis compared with the MI/R + SDX group (Figure 6A). Moreover, LY294002 increased the Bax protein level and decreased that of Bcl-2 compared with the SDX group in mice with MI/R injury (Figure 6B). Compared with the SDX pre-treatment group, the levels of CHOP, GRP78 and ATF-6 were increased, whereas those of p-PI3K and p-Akt were decreased, by LY294002 treatment (Figure 7A,B). Furthermore, LY294002 reversed the protective effect of SDX in H9C2 cells. Compared with the SDX pre-treatment group, LY294002 significantly increased the apoptosis of H9C2 cells (Figure 6C). Similarly, the Bax protein level was increased and that of Bcl-2 decreased in the LY294002 group compared with the control group (Figure 6D). Moreover, the LY294002-induced changes in the CHOP, GRP78, ATF-6 and p-Akt protein levels in H9C2 cells were consistent with those in mice with MI/R (Figure 8A,B).