Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes

Chaoyong He, Huaiping Zhu, Hongliang Li, Ming-Hui Zou, Zhonglin Xie, Chaoyong He, Huaiping Zhu, Hongliang Li, Ming-Hui Zou, Zhonglin Xie

Abstract

Diabetic cardiomyopathy is associated with suppression of cardiac autophagy, and activation of AMP-activated protein kinase (AMPK) restores cardiac autophagy and prevents cardiomyopathy in diabetic mice, albeit by an unknown mechanism. We hypothesized that AMPK-induced autophagy ameliorates diabetic cardiomyopathy by inhibiting cardiomyocyte apoptosis and examined the effects of AMPK on the interaction between Beclin1 and Bcl-2, a switch between autophagy and apoptosis, in diabetic mice and high glucose-treated H9c2 cardiac myoblast cells. Exposure of H9c2 cells to high glucose reduced AMPK activity, inhibited Jun NH2-terminal kinase 1 (JNK1)-B-cell lymphoma 2 (Bcl-2) signaling, and promoted Beclin1 binding to Bcl-2. Conversely, activation of AMPK by metformin stimulated JNK1-Bcl-2 signaling and disrupted the Beclin1-Bcl-2 complex. Activation of AMPK, which normalized cardiac autophagy, attenuated high glucose-induced apoptosis in cultured H9c2 cells. This effect was attenuated by inhibition of autophagy. Finally, chronic administration of metformin in diabetic mice restored cardiac autophagy by activating JNK1-Bcl-2 pathways and dissociating Beclin1 and Bcl-2. The induction of autophagy protected against cardiac apoptosis and improved cardiac structure and function in diabetic mice. We concluded that dissociation of Bcl-2 from Beclin1 may be an important mechanism for preventing diabetic cardiomyopathy via AMPK activation that restores autophagy and protects against cardiac apoptosis.

Figures

FIG. 1.
FIG. 1.
Activation of AMPK prevents autophagy inhibition by high glucose (HG) in H9c2 cells. A: H9c2 cells were treated with different concentrations of glucose for 48 h, and cell lysates were analyzed by Western blot using an antibody against LC3B (n = 6; *P < 0.05, †P < 0.01 vs. control [Con]). B: H9c2 cells were exposed to HG (30 mmol/L) for indicated times, and LC3-II protein levels in cell lysates were detected by Western blotting (n = 6; *P < 0.05 vs. Con). C: H9c2 cells were treated with HG for 48 h in the presence or absence of CQ (5 μmol/L), and expression of LC3-II in cell lysates was determined by Western blot (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. CQ). D and E: H9c2 cells were infected with an adenovirus encoding GFP-LC3 for 24 h. The cells were then treated with HG, CQ (5 μmol/L), or HG plus CQ for 48 h. GFP-LC3 was detected using an inverted fluorescence microscope. D: Representative microphotography of GFP-LC3 staining. E: Autophagy was quantified by counting the GFP-LC3 puncta in the cells (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. CQ). F: H9c2 cells were treated with HG, metformin (Met; 1 mmol/L), HG plus Met, or HG/Met/CQ for 48 h. Cell lysates were analyzed by Western blot using antibodies against P-AMPK (Thr172), P-ACC (Ser79), and LC3B (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG, ‡P < 0.05 vs. HG/Met). G and H: H9c2 cells transfected with GFP-LC3 were treated with HG, Met, HG plus Met, or HG/Met/CQ for 48 h. GFP-LC3 was detected using an inverted fluorescence microscope. G: Representative microphotography of GFP-LC3 staining. H: Autophagy was quantified by counting the GFP-LC3 puncta in the cells (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG, ‡P < 0.05 vs. HG/Met). I: H9c2 cells were transfected with control siRNA (C-siRNA) or AMPK siRNA (AMPK-si) for 48 h. The cells were incubated in HG medium and treated with or without Met (1 mmol/L) for 48 h. Cell lysates were analyzed by Western blot using antibodies against AMPK and LC3B (n = 3; *P < 0.05 vs. C-siRNA, †P < 0.05 vs. HG/C-siRNA, ‡P < 0.05 vs. HG/Met/C-siRNA). J: H9c2 cells infected with GFP or CA-AMPK adenovirus were incubated in HG medium for 48 h. Cell lysates were subjected to Western blot to determine the expression of LC3 (n = 3; *P < 0.05 vs. GFP, †P < 0.05 vs. GFP/HG, ‡P < 0.05 vs. CA-AMPK alone, ♣P < 0.05 vs. GFP/HG/Met, #P < 0.05 vs. CA-AMPK/HG).
FIG. 2.
FIG. 2.
AMPK activation inhibits high glucose (HG)–induced apoptosis in H9c2 cells. A and B: H9c2 cells were exposed to 5 mmol/L d-glucose (Control), 30 mmol/L d-glucose (HG), or 5.5 mmol/L d-glucose plus 24.5 mmol/L mannitol for 48 h. Then, TUNEL staining was performed (A) and the number of TUNEL-positive cells (B) were quantified (n = 6; *P < 0.05 vs. control or mannitol). C: Cleaved caspase 3 (C-Casp3) and cleaved PARP (C-PARP) were determined by immunoblot analysis (n = 5; *P < 0.05 vs. control). D and E: The apoptotic ratios of H9c2 cells in different groups were determined by flow cytometry using FITC-annexin V/PI double staining. D: Representative diagram of annexin V/PI staining. E: The apoptotic cells were calculated as the ratio of annexin V+/PI− cells to total cells (n = 5; *P < 0.05 vs. control or mannitol). F: H9c2 cells were pretreated with or without metformin (Met; 1 mmol/L) for 1 h and then treated with HG (30 mmol/L) for 48 h. AMPK activation in cell lysates was determined by Western analysis of phosphorylation of AMPK and ACC (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG). G: Expression of C-Casp3 and C-PARP in cell lysates was detected by Western blot analysis (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG). H: Apoptosis was assessed by flow cytometry using annexin V/PI double staining, and the apoptotic cells were calculated as the ratio of annexin V+/PI− cells to total cells (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG).
FIG. 3.
FIG. 3.
AMPK mitigates high glucose (HG)–induced apoptosis through induction of autophagy. A and B: H9c2 cells were pretreated with 3-MA (10 μmol/L) for 30 min and then treated with HG (30 mmol/L) in the presence or absence of metformin (Met; 1 mmol/L) for 48 h. Cell lysates were analyzed by Western blot to determine the expression of LC3 (A) and apoptosis markers (B) including cleaved caspase-3 (C-Casp3) and cleaved PARP (C-PARP) (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG, ‡P < 0.05 vs. HG/Met). C: H9c2 cells were transfected with control siRNA (C-siRNA) or Atg7 siRNA (Atg7-si) for 48 h. The cells were incubated in HG medium and treated with or without Met (1 mmol/L) for 48 h. Cell lysates were analyzed by Western blot using antibodies against C-Casp3, C-PARP, LC3B, and Atg7. D: Apoptosis was assessed by flow cytometry using annexin V/PI double staining, and the apoptotic cells were calculated as the ratio of annexin V+/PI− cells to total cells (n = 5; *P < 0.05 vs. control/C-siRNA, †P < 0.05 vs. C-siRNA/HG, ‡P < 0.05 vs. HG/Met/C-siRNA). E: H9c2 cells were infected with adenovirus encoding GFP or Atg7 for 48 h and then treated with or without CQ (5 μmol/L) for 16 h. Expression of LC3 in cell lysates was determined by Western blot (n = 5; *P < 0.05 vs. GFP, †P < 0.01 vs. CQ/GFP). F: H9c2 cells infected with GFP or Atg7 adenovirus were incubated in HG medium for 48 h. Cell lysates were subjected to Western blot to determine the expression of C-Casp3 and C-PARP (n = 5; *P < 0.05 vs. GFP, †P < 0.05 vs. GFP/HG). G: Apoptosis was assessed by flow cytometry using annexin V/PI double staining, and the apoptotic cells were calculated as the ratio of annexin V+/PI− cells to total cells (n = 5; *P < 0.05 vs. GFP, †P < 0.05 vs. GFP/HG).
FIG. 4.
FIG. 4.
AMPK activates JNK to regulate the association of Beclin1–Bcl-2, induce autophagy, and reduce apoptotic cell death. A and B: H9c2 cells were pretreated with or without metformin (Met; 1 mmol/L) for 1 h and then incubated in high glucose (HG; 30 mmol/L) medium for 48 h. Beclin1 (A) or Bcl-2 (B) was immunoprecipitated (IP) from cell lysates, and Bcl-2 or Beclin1 in the immunoprecipitates was detected by Western blot (IB) (n = 4; *P < 0.05 vs. control [Con], †P < 0.05 vs. HG). C: H9c2 cells were treated with HG (30 mmol/L) for 24 or 36 h in the presence or absence of metformin (Met; 1 mmol/L). Activation of JNK1 was determined by Western blot using antiphospho-JNK1 (Thr183/Tyr185) antibody (n = 5; *P < 0.05 vs. Con, †P < 0.05 vs. HG). C, right: H9c2 cells were treated with metformin (1 mmol/L) under normal or HG for 36 h. JNK1 phosphorylation was determined by Western blot (n = 3; *P < 0.05 vs. Con, †P < 0.05 vs. HG). D: Cell lysates were immunoprecipitated with anti–Bcl-2 antibody and then subjected to immunoblot using P-Bcl-2 (Ser70) and Bcl-2 antibodies (n = 5; *P < 0.05 vs. Con, †P < 0.05 vs. HG). D, right: H9c2 cells were treated with Met (1 mmol/L) under normal glucose or HG conditions for 36 h. Phosphorylation of Bcl-2 was determined by immunoprecipitation and Western blotting (n = 3; *P < 0.05 vs. Con, †P < 0.05 vs. HG). E: H9c2 cells were pretreated with SP600125 (SP; 50 μmol/L) for 30 min and then incubated in HG (30 mmol/L) medium in the presence or absence of Met (1 mmol/L) for 48 h. Total and phospho-JNK1 were detected by Western blot. In addition, cell lysates were subjected to immunoprecipitation with anti-Beclin1 antibody, and the immunoprecipitates were subjected to Western blotting with anti–Bcl-2 antibody (n = 5; *P < 0.05 vs. Con, †P < 0.05 vs. HG, ‡P < 0.01 vs. HG/Met). F: Expression of LC3 was analyzed by Western blot (n = 5; *P < 0.05 vs. Con, †P < 0.05 vs. HG, ‡P < 0.01 vs. HG/Met). G: H9c2 cells were transfected with LacZ or CA-JNK1 plasmid for 24 h and then incubated in HG medium for 36 h. Expression of JNK1 was detected by Western blot. Cell lysates were subjected to immunoprecipitation with anti-Beclin1 antibody, and then Bcl-2 was detected in the immunoprecipitates by Western blotting (n = 5; *P < 0.05 vs. LAZ/Con, †P < 0.05 vs. HG/LacZ). H: Expression of LC3 was analyzed by Western blot (n = 5; *P < 0.05 vs. LAZ/Con, †P < 0.05 vs. HG/LacZ). I: H9c2 cells were pretreated with SP (50 μmol/L) for 30 min followed by treatment with HG (30 mmol/L) in the presence or absence of Met (1 mmol/L) for 48 h. Cell lysates were subjected to Western blot using anti–C-Casp3 and anti–C-PARP antibodies (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG, ‡P < 0.05 vs. HG/Met). J: Apoptosis was assessed by flow cytometry using annexin V-PI double staining, and the apoptotic cells were calculated as the ratio of annexin V+/PI− cells to total cells (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. HG, ‡P < 0.05 vs. HG/Met).
FIG. 5.
FIG. 5.
AMPK phosphorylates JNK1. A: H9c2 cells were treated with indicated concentrations of metformin (Met) for 36 h. Cells lysates were subjected to Western analysis of P-AMPK (Thr172) and P-JNK1 (Thr183/Tyr185) (n = 3; *P < 0.05 vs. control [Con]). B: Recombinant JNK1 (500 ng) was incubated with indicated concentrations of recombinant AMPK. Phosphorylation of JNK1 was determined by Western blotting (n = 3; *P < 0.05 vs. control). C: H9c2 cells were treated with Met (1 mmol/L) for 36 h under normal glucose and HG. The interaction of AMPK and JNK1 was measured by immunoprecipitation (IP) and Western blotting (IB) (n = 3; *P < 0.05 vs. control, †P < 0.05 vs. HG). D: H9c2 cells were transfected with control siRNA (C-siRNA) or mTOR siRNA (mTOR-si) for 48 h, and then incubated in HG (30 mmol/L) medium for 48 h. The association of Beclin1 and Bcl-2 was determined by immunoprecipitation and Western blotting (n = 3; NS, not significant).
FIG. 6.
FIG. 6.
Activation of AMPK disrupts Bcl-2–Beclin1 association and restores autophagy in diabetic hearts. A: Phosphorylation of AMPK and ACC in cardiac tissues from FVB (control), metformin-treated FVB (Met), STZ-treated (STZ), and metformin-treated STZ (STZ/Met) mice was detected by Western blot using P-AMPK (Thr172) and P-ACC (Ser79) antibodies and quantified by densitometry (n = 6 per group; *P < 0.05 vs. Con, †P < 0.05 vs. STZ). B: Expression of total and phospho-JNK1 in cardiac tissues was analyzed by Western blotting. In addition, cardiac tissues were immunoprecipitated (IP) with an anti–Bcl-2 antibody, and then immunoprecipitates were probed by immunoblot (IB) using P-Bcl-2 (Ser70), Beclin1, and Bcl-2 antibodies. The result shown is representative of three independent experiments. C: Cardiac tissues were subjected to Western blotting to determine the expression of LC3 using a specific antibody (n = 5 in each group; *P < 0.05 vs. Con, †P < 0.05 vs. STZ). D: AMPK activity in cardiac tissues was assayed as described in research design and methods (n = 5; *P < 0.05 vs. WT/Con, †P < 0.05 vs. WT/STZ, ‡P < 0.05 vs. WT/STZ/Met). E: FVB (WT) and DN-AMPKα2 (DN) transgenic mice were treated with or without STZ. LC3-II protein levels in cardiac tissues were determined by Western blotting (n = 5; *P < 0.05 vs. WT). F: Diabetic WT and DN mice were treated with or without metformin (Met), and then heart homogenates from these mice were subjected to Western blot to determine the expression of LC3 (n = 5; *P < 0.05 vs. WT/STZ, †P < 0.05 vs. WT/STZ/Met). G: Phosphorylation of Akt at Ser437 in cardiac tissues from FVB control (Con), STZ, and STZ/Met mice was detected by Western blot and quantified by densitometry (n = 6 per group; *P < 0.05 vs. Con, †P < 0.05 vs. STZ).
FIG. 7.
FIG. 7.
Activation of AMPK protects against apoptosis in diabetic hearts. A: Representative images of TUNEL staining. Apoptotic cells in heart sections from control, metformin-treated FVB (Met), STZ-treated (STZ), and metformin-treated STZ (STZ/Met) mice were labeled by TUNEL staining. B: The number of TUNEL-positive cells is indicated in the bar graph (n = 6 per group; *P < 0.05 vs. control, †P < 0.05 vs. STZ). C and D: Western analysis of cleaved caspase-3 (C-Caspa3) and cleaved PARP (C-PARP) in cardiac homogenates (n = 5; *P < 0.05 vs. control, †P < 0.05 vs. STZ). E: FVB (WT) and DN-AMPKα2 (DN) transgenic mice were treated with or without STZ, and then cardiac tissues were subjected to Western blot analysis to determine the expression of C-Casp3 and C-PARP (n = 5 in each group; *P < 0.05 vs. WT, †P < 0.05 vs. DN). F: Diabetic WT and DN mice were treated with or without metformin (Met), and then heart homogenates were subjected to Western blot to determine the expression of C-Casp3 and C-PARP (n = 5; *P < 0.05 vs. WT/STZ, †P < 0.05 vs. WT/STZ/Met).
FIG. 8.
FIG. 8.
Metformin ameliorates cardiac fibrosis and improves cardiac function in diabetic hearts. A: Immunohistochemical staining for collagen I in cardiac tissues. Heart sections from control, metformin-treated FVB (Met), STZ-treated (STZ), and metformin-treated STZ (Met/STZ) mice were stained with antibody against collagen I, and the nuclei were counterstained with hemotoxylin. B: Quantitative analysis of collagen I–stained areas is shown by the bar graph (n = 6 in each group; *P < 0.05 vs. control, †P < 0.05 vs. STZ). C–E: The relationship between LV-developed pressure and volume in control, STZ, and STZ/Met mice was determined by Langendorff perfusion analysis. The developed pressure was depressed in STZ mice, and this depression was prevented by metformin treatment (C). Metformin improved LV dp/dtmax (D) and dp/dtmin (E) in STZ mice (n = 6 in each group; *P < 0.05 vs. control, †P < 0.05 vs. STZ).

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