Metformin induces apoptosis in mesenchymal stromal cells and dampens their therapeutic efficacy in infarcted myocardium

Figures

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  Fig. 1. Metformin dampens MSC therapeutic efficacy in MI. a Representative images of TTC-stained heart sections obtained from Sham, MI + PBS, MI + metformin, MI + MSCs, and MI + metformin + MSCs groups at 4 weeks after MI. b LV infarct sizes expressed as the ratio of the area of the infarct band to the total area of LV (n = 23). c Representative M-mode images of hearts with sham surgery or MI at 4 weeks after PBS, metformin, MSCs, and metformin + MSCs treatment. Scale bar: X axis: 0.1 s; Y axis: 0.2 cm. d Heart rates were controlled to be similar in different groups (n = 23). e–g LV fraction shortening (e), LV ejection fraction (f), and diastolic left ventricle internal diameter (LVIDd) (g) at 4 weeks after treatment (n = 23). *p < 0.05 vs. sham; #p < 0.05 vs. MI + metformin; &p < 0.05 vs. MI + MSCs, by one-way ANOVA

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  Fig. 2. Metformin induces MSC apoptosis. a Diabetic mice were administered with PBS or metformin by gavage for 4 weeks, and then all mice were sacrificed to isolate mouse bone marrow mesenchymal stem cells (mBMSCs) for flow cytometry assay. b, c In vivo effects of metformin on mBMSCs. Compared with the PBS group, metformin decreased the number of mBMSCs (CD45−/CD105+/CD29+/Sca-1+). Lines in c represent the mean. **p < 0.01, by t test, n = 10. mBMSC, mouse bone marrow mesenchymal stromal cell. d, e Human umbilical cord MSC apoptosis ratio (annexin V+) induced by different concentrations of metformin at 24 h, 48 h, and 72 h. f, g Comparison of cell cycle distribution between the metformin group and control group by flow cytometry. In e, c, and g, **p < 0.01 by one-way ANOVA (n = 3 per group in d and f)

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  Fig. 3. Metformin induces the activation of AMP-activated protein kinase (AMPK)-mediated apoptosis. a AMPK, mTOR, S6K1, their phosphorylated forms, and cleaved-caspase 3 were assessed by western blot analysis after treatment with or without metformin (2 mM). β-actin was used as loading control. b, c Treatment with an AMPK inhibitor (compound C) (1 μM) and siAMPK significantly blocked metformin-induced hUC-MSC apoptosis. Bars in c represent the mean ± SD (n = 3 per group). **p < 0.01, by one-way ANOVA. d Real-time cellular analysis (RTCA) of compound C and siAMPK on metformin-induced hUC-MSC apoptosis. e Compound C and siAMPK significantly inhibited metformin-induced AMPK phosphorylation but have no effect on LC3B, Akt, and Rag B. β-actin was used as loading control. f, g Expression of AMPK in hUC-MSCs 24 h after transient transfection of siAMPK at the transcriptional (qRT-PCR, **p < 0.01, by t test, n = 3) and translational (western blot) level. h hUC-MSCs were treated with metformin (2 mM) with or without compound C (1 μM) and siAMPK for 36 h. After treatment, cell lysates were immunoprecipitated with an anti-Bcl-xL antibody and immunoblotted with an anti-Bad antibody. The presence of…

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  Fig. 4. Counteraction of AMPK partially restored MSC cardioprotective effects in diabetic mice with MI. a Representative images of TTC-stained heart sections obtained from sham, MI + PBS, MI + metformin, MI + MSCs, MI + metformin + MSCs, and MI + metformin + MSCs + compound C groups at 4 weeks after MI. b LV infarct sizes expressed as the ratio of the area of the infarct band to the total area of LV (n = 28). c Representative M-mode images of hearts with sham surgery or MI at 4 weeks after PBS, metformin, MSCs, metformin + MSCs, and metformin + MSCs + compound C treatment. Scale bar: X axis: 0.1 s; Y axis: 0.2 cm. d Heart rates were controlled to be similar in different groups. e–g LV fraction shortening (e), LV ejection fraction (f), and diastolic left ventricle internal diameter (LVIDd, g) at 4 weeks after treatment (n = 28). *p < 0.05 vs. sham; #p < 0.05 vs. MI + metformin; &p < 0.05 vs. MI + MSCs; ɸp < 0.05 vs. MI + metformin + MSCs, by one-way ANOVA

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  Fig. 5. Counteraction of AMPK attenuated metformin-induced MSC apoptosis in vivo. a Diabetic mice were administered with PBS, metformin (250 mg/kg/day, i.g.), or metformin + compound C (0.1 mg/kg/day, i.g.) by gavage for 4 weeks, and then all mice were sacrificed to isolate mBMSCs for flow cytometry assay. b Metformin treatment induced a significant decrease in mBMSCs compared with PBS treatment. Compared with metformin, compound C reduced the metformin-induced mBMSC decrease (CD45−/CD105+/CD90+/Sca-1+). *p < 0.01 vs. PBS, #p < 0.01 vs. Met, by one-way ANOVA, n = 5 per group. c Post-MI hearts with CM-DiI-labeled MSC transplantation were enzymatically digested, and small cells from the heart (< 30 μm diameter) were collected after the depletion of cardiomyocytes. As indicated with a yellow arrow, CM-DiI-labeled cells represent surviving MSCs under fluorescence microscopy. Scale bar = 100 μm. d Representative flow cytometric plots of surviving CM-DiI+ MSCs counted by FCM. Gate R4 indicates the CM-DiI+ cells out of all the isolated cells from the heart. e The percentage of surviving MSCs out of the total transplanted MSCs at different time points. *p < 0.05 vs. MSCs, #p < 0.05 vs. MSCs +…