Autophagy Plays a Protective Role in Advanced Glycation End Product-Induced Apoptosis in Cardiomyocytes

Advanced glycation end products (AGEs) arise from a non-enzymatically glycated, irreversible process called the Maillard reaction [1]. The accumulation of AGEs has been implicated in the accelerated process of diabetic cardiovascular complications through interactions with the receptor for advanced glycation end products (RAGE) [2]. AGEs play an important role in the development and progression of diabetic cardiomyopathy through endothelial dysfunction, myocardial apoptosis, oxidative stress, increased inflammation, and enhanced extracellular matrix accumulation in diabetes [3,4,5].

Autophagy is a degradation process that delivers intracellular components to lysosomes through autophagosomes in all eukaryotic cells [6]. There are multiple stages, structures, and proteins involved in the process of autophagy. During autophagy, to begin with, LC3-I is recruited to the autophagosome in the cytoplasmic form [7]. Moreover, by the activating enzyme Atg7 and the conjugating enzyme Atg3, phosphatidylethanolamine-conjugated LC3-II is localized in the membrane of autophagosome [8]. Finally, LC3-II is degraded by autophagy. Another autophagy marker, p62, also called sequestosome 1 (SQSTM1), binds LC3 and recruits proteins into autophagosomes for degradation [9]. Thus, LC3-II and SQSTM1 levels indicate autophagic activity. Autophagic flux is a method that is used to measure autophagy, which is always assessed using MAP1LC3B/LC3 and SQSTM1/p62 western blotting [10]. PI3K/AKT/mTOR is the most important intracellular signal pathway to negatively regulate autophagy [11,12,13]. In addition, MAPKs, a family of serine/threonine protein kinases, are also involved in the process of autophagy [14,15].

The crosstalk between autophagy and apoptosis is complex [16]. Autophagy and apoptosis are under the control of multiple common upstream signals, including the PI3K/AKT/mTOR signalling pathway. Generally, autophagy can promote cell survival by blocking apoptosis [17]. However, under certain special conditions, autophagy or autophagy-related proteins can culminate in apoptosis or autophagic cell death. Our previous research shows that AGEs can induce autophagy in a time- and dose-dependent manner, which is involved in AGE-induced proliferation of rat vascular smooth muscle cells [18]. It is known that AGEs increase the rate of apoptosis and ROS generation of cardiomyocytes through the ERK1/2 and p38 MAPK signalling pathways but do not affect the distribution of the cell cycle [19,20]. Another recent study indicates that advanced glycation end products trigger autophagy via the PI3K/AKT/mTOR signalling pathway in cardiomyocytes [21]. However, the relationship between AGE-RAGE mediated autophagy and apoptosis in cardiomyocytes has not been elucidated. In this study, we sought to explore the signalling pathway involved in AGE-induced autophagy in cardiomyocytes and the crosstalk between AGE-induced autophagy and apoptosis in cardiomyocytes.

A polyclonal rabbit anti-LC3B antibody, MTT, BSA, 3-MA, and MAPK inhibitors, including PD98059, SP600125 and SB203580, were obtained from Sigma (St. Louis, MO, USA). Monoclonal rabbit antibodies, includinganti-SQSTM1/p62, anti-P38, anti-phospho-P38, anti-JNK, anti-phospho-JNK, anti-ERK, anti-phospho-ERK, anti-AKT, anti-mTOR, anti-phospho-mTOR and anti-phospho-AKT, were obtained from Cell Signalling Technology (MA, USA). The Vybrant apoptosis assay kit (488) was obtained from Invitrogen (CA, USA). An HRP-marked anti-GAPDH antibody was purchased from Kangchen (Shanghai, China). Rat IGF-1 was obtained from R&D (Minneapolis, MN, USA).

Cardiomyocytes were obtained from 1-2 day old Sprague-Dawley rats (provided by the experimental animal centre of Zhejiang Chinese Medicine University). Heart tissue was cut into small pieces of ∼1 mm³ and digested with PBS containing 0.1% trypsin and 0.1% type II collagenase five to six times. The suspension was centrifuged and the supernatant was collected, centrifuged and resuspended in DMEM cell culture medium with 10% FBS. Fibroblasts in the cell suspension were reduced by pre-plating for 1 h according to differential cell adhesion [22]. 5ʹ-BrdU (0.01 mM) was also added to inhibit the growth of fibroblast during the first three days. The medium was replaced every 48 h until the cardiomyocytes reached >80% confluence for use.

AGEs were prepared as described previously [23]. Briefly, BSA was incubated with 0.5 M glucose in phosphate-buffered saline (PBS) in dark and sterile conditions for 16 weeks at 37°C. Unincorporated sugars were removed by dialysis with PBS (pH 7.4). Control non-glycated BSA was incubated in the absence of glucose under the same conditions. Preparations were tested for endotoxins using an endotoxin testing kit (Chromogenic TAL Endpoint Assay Kit, China), and the AGE-BSA solutions were confirmed to be endotoxin free (<2.5U/ml of endotoxin).

Cells were solubilized in a lysis buffer containing RIPA and a mixture of protease and phosphatase inhibitors. The total protein concentrations were determined by a BCA Protein Assay Kit (Applygen Technologies, Inc., China). After the samples were heat-denatured, they were analysed on a 10 or 15% tris-glycine gradient gel, transferred to nitrocellulose membranes and blocked with 5% non-fat dry milk in tris-buffered solution (TBS) for 1 h at room temperature. The membranes were incubated with primary antibody overnight at 4ºC. After being washed three times, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. The immune complexes were visualized by ultrachemiluminescence (ECL) reagents and imaged using an Image Quant LAS-4000 (Fujifilm, Tokyo, Japan). The band densities were determined using Multi-Gauge Software (Fujifilm, Tokyo, Japan).

The ultrastructural analysis was performed to examine autophagy. Rat neonatal cardiomyocytes were grown in 6-well plates treated with 100 μg/ml AGEs for 6 h, fixed with a solution containing 3% glutaraldehyde and then sent to Zhejiang University for electron microscopic analysis.

Cardiomyocytes were plated in a 96-well plate. After 48 hours, the medium was changed, and the cells were incubated with fresh medium containing AGEs (100 μg/ml) or 3-MA (2 mM) for another 48 hours. Then, 20 μl of MTT was added to each well for a final concentration of 0.5 mg/mL for 4 hours at 37°C. The medium was then discarded, and 100μl of DMSO was added to each well. The absorbance was measured at 490 nm.

After treatment, cells (5 × 10⁵ cells/ml) were collected and suspended in 100μl of 1× annexin-binding buffer, and then, 5μl of Alexa Fluor 488 and 5μl of propidium iodide (PI) were added and then incubated for 15 min at room temperature in the dark. After incubation, 400 μl of 1× binding buffer was added for FCM detection. Flow cytometry was performed using a Zeiss fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

All data were obtained from at least 3 individual experiments. The values are expressed as the mean ± SEM. Statistical analysis between groups was performed by one-way ANOVA. The statistical significance was set at p<0.05.

To determine whether AGEs can affect the level of autophagy in cardiomyocytes, we treated cells with AGEs at various concentrations (0, 4, 20, 100 and 500 μg/ml) for 6 h. The expression of LC3-II and the ratio of LC3- II to LC3-I were notably increased, whereas SQSTM1/p62 was decreased in a dose-dependent manner in AGE-treated cells (Fig. 1A). Cells were also treated with AGEs (100μg/ml) for various times (0, 1, 2, 6, 12 and 24 h). The expression of LC3- II and the ratio of LC3- II to LC3-I were significantly increased after treatment with AGEs, peaking at 6 h. However, the expression of SQSTM1/p62 was decreased under the same conditions (Fig. 1B).

To directly visualize autophagy, we used transmission electron microscopy to examine autophagicvacuoles (autophagosomes). We treated cells with 100μg/ml AGEs for 6h. In the control group, autophagic vacuoles were rarely detected. However, we found that autophagic vacuoles containing cellular material or membranous structures were increased in cardiomyocytes (bold arrows in Fig. 1C).

To determine whether AGEs induced apoptosis, we used MTT and FCM analysis with Alexa Fluor 488 Annexin V/PI. Cardiomyocytes were treated with AGEs at various concentrations (0, 20, 100 and 500 μg/ml) for 48 h. As shown in Fig. 2, the apoptotic rate of cardiomyocytes increased and the cardiomyocyte viability was decreased in a dose-dependent manner in the AGE-treated group.

It has been well documented that the MAPK signalling pathway is important for cells to respond to many extracellular signals. To investigate the mechanisms involved in AGE-induced autophagy and apoptosis, we examined the phosphorylation of the MAPKs (ERK1/2, p38, and JNK) cascade in cardiomyocytes by western blotting. Cells were treated with 100 μg/ml AGEs for 0, 7.5,15,30, 60 and 120 min. As is shown in Fig. 3, AGEs stimulated the phosphorylation of ERK1/2 and p38 in cardiomyocytes in a time-dependent manner, but not activated the phosphorylation of JNK (Fig. 3A). ERK1/2 and p38 Phosphorylation peaked at 15-30 min and then declined.

Then, to delineate the pathways involved in AGE-induced autophagy and apoptosis, we assessed the effects of a panel of pharmacologic inhibitors, including those of ERK1/2(PD98059, 20μM), JNK (SP600125, 20μM) and p38 (SB203580, 10μM). In our experiments, the ERK1/2 inhibitor PD98059, but not the p38 inhibitor SB203580 or JNK inhibitor SP600125, suppressed the expression of LC3-II (Fig. 3B). The results indicate that the ERK1/2 signal transduction pathway is involved in AGE-induced autophagy. By contrast, the p38 inhibitor SB203580, but not the ERK1/2 inhibitor PD98059 or JNK inhibitor SP600125, suppressed AGE-induced apoptosis (Fig. 3C). These results demonstrate that AGEs induced apoptosis via the p38 MAPK signalling pathway in cardiomyocytes.

The PI3K/Akt/mTOR signalling pathway is a major pathway that negatively regulates autophagy. In cardiomyocytes, phosphorylation of Akt and mTOR decreased 30 min to 2 h after treatment with AGEs (100μg/ml) (Fig. 4A). To examine the role of the PI3K/Akt/mTOR pathway in AGE-induced autophagy, we used insulin-like growth factor 1 (IGF-1) to activate this signalling pathway [24].

In addition, pretreatment with IGF-1 (200ng/ml) suppressed AGE-induced LC3-II expression in cardiomyocytes. This result suggests that the PI3K/Akt/mTOR signalling pathway is involved in AGE-induced autophagy in cardiomyocytes (Fig. 4B). Similarly, compared with the control group, pretreatment with IGF-1 (200ng/ml) also reduced the apoptotic rate in the AGE group in cardiomyocytes (Fig. 4C). These results demonstrate that the PI3K/Akt/mTOR signalling pathway is involved in AGE-induced apoptosis (Fig. 4B).

To understand the role of autophagy in AGE-induced apoptosis in cardiomyocytes, we used 3-MA, an inhibitor that blocks early stages of the autophagy pathway. Compared with the control group, cells treated with 100μg/ml AGEs for 48 h showed an increased apoptotic rate and decreased viability of cardiomyocytes by FCM analysis and MTT assays (Fig. 5). These results indicate that autophagy plays a protective role in AGE-induced apoptosis in cardiomyocytes.

Diabetic cardiomyopathy is characterized by cardiac dysfunction that develops in many cases of type 2 diabetes mellitus in the absence of coronary artery disease or hypertension [25]. Advanced glycation end products (AGEs) play an important role in the development and progression of diabetic cardiomyopathy [3]. However, the underlying mechanisms are not completely understood. It has been reported that increased oxidative injury in diabetic hearts may trigger the activation of stress signalling pathways, facilitating cardiomyocyte cell death [26,27]. However, recent evidence also suggests that, in addition to apoptosis, other processes, such as autophagy, may also be involved in controlling cell death in the pathogenesis of diabetic cardiomyopathy [28].

Autophagy is a cellular housekeeping process that is important for the removal of misfolded proteins, damaged organelles and intracellular pathogens [29]. In general, the decreased levels of SQSTM1/p62 and conversion of LC3-I to LC3-II are commonly used as markers for autophagic flux [30]. In our study, we found that the level of LC3-II was up-regulated and SQSTM1/p62 was down-regulated after cultured cardiomyocytes were treated with AGEs. The number of autophagosomes was also detected under the same condition by transmission electron microscopy. These demonstrated that autophagic flux was enhanced by AGEs in cardiomyocytes.

There is a complex crosstalk between autophagy and apoptosis. Nevertheless, studies have yielded conflicting results. Under certain cellular conditions, autophagy may be utilized as an adaptive response for survival and to avert apoptosis [31]. Under other conditions, autophagy appears to promote apoptosis or apoptotic programmed cell death. Autophagy is a double-edged sword in cardiomyocyte demise and survival. Yutaka et al. previously reported that autophagy plays a protective role during ischemia, whereas it may be detrimental during reperfusion [32]. In this study, the autophagy inhibitor 3-MA could attenuate the effects of AGE-induced apoptotic rate and increase the viability of cardiomyocytes. This result indicates that AGE-induced autophagy plays a protective role in the AGE-stimulated apoptosis of cardiomyocytes. This may provide a new therapeutic strategy for preventing diabetic cardiomyopathy in diabetic patients.

In our study, we found that AGEs increased the phosphorylation of ERK and p38 and decreased the phosphorylation of AKT and mTOR in a time-dependent manner. Furthermore, autophagy in cardiomyocytes treated with AGEs was reduced when cells were pretreated with the ERK inhibitor PD98059 but not the p38 inhibitor SB203580. Interestingly, the apoptotic rate of cardiomyocytes was reduced when cells were pretreated with the p38 inhibitor SB203580 but not the ERK inhibitor PD98059. Activation of the AKT pathway using IGF-1 inhibited AGE-induced autophagy and apoptosis. These results imply that the ERK and AKT pathways are involved in AGE-induced autophagy, whereas the P38 and AKT pathways are involved in AGE-induced apoptosis. Depending on the experimental conditions, the members of the MAPK and AKT signalling pathways may have different roles in the regulation of autophagy and apoptosis. Zhang et al. reported that the PI3K/AKT/mTOR and JNK signalling pathways were involved in cathepsin S inhibition-induced autophagy and apoptosis in human glioblastoma cells [14]. However, because we could not exclude autophagy-regulating proteins in the modulation of apoptosis, further investigation is needed.

In summary, our studies demonstrate that AGEs induced autophagy through the PI3K/AKT/mTOR and ERK signalling pathways and induced apoptosis through the PI3K/AKT/mTOR and P38signalling pathways. Autophagy plays a protective role in AGE-induced apoptosis in cardiomyocytes. This suggests that regulating the AGE-autophagy pathway can attenuate apoptosis of cardiomyocytes and therefore may reduce the development of diabetic cardiomyopathy in diabetic patients. Further studies are needed to dissect the relationship between autophagy and apoptosis in animal models and to explore possible drug-targeting methods to regulate this pathway.

This work was supported by grants from the Natural Science Foundation of Zhejiang Province (Y13H020030, S.W. Huang) and the foundation of Zhejiang education department (Y201225030, P.F. Hu). All research was performed in the Biomedical Research Centre, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China.

The authors have no conflict of interest.

P. Hu and H. Zhou contributed equally to this work.