Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice

Abstract

Objective: Autophagy is a critical cellular system for removal of aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in the development of heart failure, the role of autophagy in the development of diabetic cardiomyopathy has not been studied. We investigated whether chronic activation of the AMP-activated protein kinase (AMPK) by metformin restores cardiac function and cardiomyocyte autophagy in OVE26 diabetic mice.

Research design and methods: OVE26 mice and cardiac-specific AMPK dominant negative transgenic (DN)-AMPK diabetic mice were treated with metformin or vehicle for 4 months, and cardiac autophagy, cardiac functions, and cardiomyocyte apoptosis were monitored.

Results: Compared with control mice, diabetic OVE26 mice exhibited a significant reduction of AMPK activity in parallel with reduced cardiomyocyte autophagy and cardiac dysfunction in vivo and in isolated hearts. Furthermore, diabetic OVE26 mouse hearts exhibited aggregation of chaotically distributed mitochondria between poorly organized myofibrils and increased polyubiquitinated protein and apoptosis. Inhibition of AMPK by overexpression of a cardiac-specific DN-AMPK gene reduced cardiomyocyte autophagy, exacerbated cardiac dysfunctions, and increased mortality in diabetic mice. Finally, chronic metformin therapy significantly enhanced autophagic activity and preserved cardiac functions in diabetic OVE26 mice but not in DN-AMPK diabetic mice.

Conclusions: Decreased AMPK activity and subsequent reduction in cardiac autophagy are important events in the development of diabetic cardiomyopathy. Chronic AMPK activation by metformin prevents cardiomyopathy by upregulating autophagy activity in diabetic OVE26 mice. Thus, stimulation of AMPK may represent a novel approach to treat diabetic cardiomyopathy.

FIG. 1.

Inhibition of AMPK by overexpression of DN-AMPKα2 reduces autophagy in transgenic mice, aggravates diabetic cardiomyopathy, and increases mortality in STZ mice. A: AMPK expression and activity in cardiac tissues from WT and DN-AMPKα2 (DN) transgenic mice treated with/without STZ were detected as described in research design and methods. ♣P < 0.05 vs. WT control (Con; n = 5–6 in each group). B and C: Western blot analyses of heart homogenates with anti-LC3 antibody. Densitometric analysis of LC3-II levels is shown by the bar graph. Results shown are mean ± SEM. ♣P < 0.05 vs. FVB (n = 4 in each group). D: Langendorff perfusion analysis of the relationship between LV-developed pressure and volume in WT, DN-AMPKα2 (DN), WT STZ, DN STZ mice 6 months after induction of diabetes. ♣P < 0.05, WT vs. WT STZ, or DN STZ; *P < 0.05, DN vs. WT STZ or DN STZ; †P < 0.05 DN STZ vs. WT STZ (n = 4–5 in each group). E: Survival plots for WT STZ and DN STZ animals (n = 14 in each group).

FIG. 2.

Metformin (Met) restores autophagy in diabetic hearts. A: Immunoblot analysis of heart homogenates using an anti-LC3 antibody. B: Quantitative analysis of LC3-II levels (n = 6 in each group). ♣P < 0.05, FVB vs. OVE26; †P < 0.05, OVE26/Met vs. OVE26. C: Representative electron micrographs from cardiac tissues of FVB, OVE26, and metformin-treated OVE26 mice. The arrowheads indicate an autophagic vacuole (original magnification of ×1,000). D: Autophagic vacuoles were counted from five to six randomly selected fields. Values represent mean ± SEM (n = 6). ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26. Note: There were fewer autophagosomes in OVE26 mice. E: FVB and OVE26 mice were injected with metformin (200 mg/kg per day i.p.) for 24 h, and AMPK phosphorylation and LC3-II levels were detected by Western blotting and quantified to FVB control (Con). ♣P < 0.05 vs. FVB Con; †P < 0.05 vs. FVB Met; #P < 0.05 vs. OVE26 Con (n = 6 in each group). F: Western analyses of phosphorylation of AMPK and LC3-II levels in HL-1 cells treated with metformin (2 mmol/L) for 24 h. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Metformin (Met) increases the phosphorylation and activity of AMPK in diabetic hearts. A: AMPK activity in cardiac tissues from FVB, OVE26 mice, and metformin-treated OVE26 mice was detected as described in research design and methods. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26 (n = 5–6 in each group). B: Representative blots of phosphorylation of AMPK at threonine 172 and total AMPK. C: Densitometric analysis of expression of phospho-AMPK and total AMPK. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26 (n = 5–6 in each group).

FIG. 4.

Metformin (Met) prevents cardiac dysfunction in diabetic mice. A–F: Echocardiographic assessment of cardiac function as described in research design and methods. All measurements were determined in a short-axis view at the level of the papillary muscles. Representative images of M-mode echocardiography (A) and mitral valvular inflows show E wave and A wave (B). C: Ejection fraction. D: Percentage of fractional shortage as LV contractile function. E: LV end-systolic diameter. F: Diastolic filling as assessed by E/A ratio (E wave: LV early-filling wave; A wave: filling from atrial contraction). Values represent mean ± SEM. FVB, n = 11; OVE26, n = 8; metformin-treated OVE26, n = 6. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26. G: Analysis of LV-developed pressure vs. volume. The developed pressure was depressed in OVE26 mice, which was prevented by metformin. Metformin improved LV dp/dt_max (H) and dp/dt_min (I) in OVE26 mice. Results shown are mean ± SEM. ♣P < 0.05 vs. OVE26. FVB, n = 6; OVE26, n = 5; metformin-treated OVE26 mice, n = 5. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Metformin (Met) improves cardiac function and survival rate in an AMPK-dependent manner. A: AMPK expression and activity in cardiac tissues from the animals were detected as described in research design and methods. ♣P < 0.05 vs. WT STZ control (Con), †P < 0.05 vs. WT STZ Met (n = 5–6 in each group). B: Langendorff perfusion analysis of the relationship between LV-developed pressure and volume in WT STZ, DN STZ, WT STZ Met, and DN STZ Met mice 6 months after induction of diabetes. ♣P < 0.05, WT STZ Met vs. WT STZ, DN STZ Met, or DN STZ; †P < 0.05 DN STZ vs. WT STZ (n = 4–5 in each group). C: Survival plots for WT STZ, DN STZ, WT STZ Met, and DN STZ Met animals (n = 14–16 in each group).

FIG. 6.

Metformin (Met) increases the expression of Beclin1 and inhibits mTOR signaling in the heart. A: Immunohistochemical analysis of Beclin1 in the hearts from FVB, OVE26, and metformin-treated OVE26 mice. Nonspecific rabbit IgG was used as a negative control. B: Quantitative analysis of Beclin1-stained areas is shown by the bar graph. Results shown are mean ± SEM. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26 (n = 4 in each group). C: Western analysis of the mTOR signaling pathway in cardiac tissues from FVB, OVE26, and metformin-treated OVE26 mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Metformin (Met) protects cardiac ultrastructure from chronic diabetic damage. Representative transmission electron micrographs of cardiac tissues from LVs are shown from FVB mice (A), OVE26 mice (B), and OVE26 mice treated with metformin (C). A and C: FVB and metformin-treated OVE26 animals show normal myocardial structure, with myofibrils comprised of regular and continuous sarcomeres. Rows of moderately electron dense mitochondria (M) intervene between myofibrils. B: OVE26 diabetic myocardium shows randomly distributed mitochondria (M) between poorly organized myofibrils (original magnification of ×4,000; n = 6–7 in each group). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 8.

Metformin (Met) administration reduces apoptosis and ubiquitinated proteins in diabetic hearts. A: Immunoblot analysis of ubiquitin in the hearts from FVB, OVE26, and metformin-treated OVE26 mice. B: Densitometric analysis of ubiquitin. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26. C: Representative images of the TUNEL assay in the hearts from FVB, OVE26, and metformin-treated OVE26 mice. The number of TUNEL-positive cells is shown in the bar graph. Values represent mean ± SEM. ♣P < 0.05 vs. FVB; †P < 0.05 vs. OVE26 (n = 4 in each group). (A high-quality digital representation of this figure is available in the online issue.)