Metformin promotes the survival of transplanted cardiosphere-derived cells thereby enhancing their therapeutic effect against myocardial infarction

Figures

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  Fig. 1. The culture and characterization of cardiosphere-derived cells. a Cells grew from the explant on top of a layer of stromal-like cells after 7 days. Cardiospheres formed after 4 days in suspension culture on poly-d-lysine-coated dishes. Cardiosphere-derived cells become confluent after 5 to 7 days. Scale bar = 100 μm. b Flow cytometric analysis showed that cardiosphere-derived cells were positive for CD29, CD105, and CD90, and negative for CD31, CD45, and CD34. GFP green fluorescent protein

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  Fig. 2. Combination of metformin (MET) treatment and cardiosphere-derived cell (CDC) transplantation improved cardiac function in myocardial infarction (MI) mice. a Representative echocardiographic images of hearts with different treatments at 4 weeks post-MI. Scale bar = 2 mm. b Heart rates were controlled to be similar in different groups. c,d Left ventricular ejection fraction and fraction shortening at 4 weeks post-MI. n = 8. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. sham; # P < 0.05 vs. MI + phosphate-buffered saline (PBS); &P < 0.05 vs. MI + MET; % P < 0.05 vs. MI + CDC. MET/CDC MET-pretreated CDCs

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  Fig. 3. Combination of metformin (MET) treatment and cardiosphere-derived cell (CDC) transplantation reduced infarct size in myocardial infarction (MI) mice. a1 Representative images of Masson’s trichrome staining for heart tissue obtained from hearts with different treatments at 4 weeks post-MI. Scale bar = 1 mm. a2 Graphic representation of the left ventricular (LV) infarct size calculated as the ratio of midline length of the infarcted LV wall to the midline length of total LV wall (n = 5). b Representative images (b1) and quantification (b2) of the fibrotic area in the infarct area 4 weeks post-MI. Scale bar = 200 μm. c Representative images (c1) and quantification (c2) of the fibrotic area at the infarct border zone 4 weeks post-MI. Scale bar = 100 μm. n = 5. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. MI + phosphate-buffered saline (PBS); # P < 0.05 vs. MI + MET; &P < 0.05 vs. MI + CDC. MET/CDC MET-pretreated CDC

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  Fig. 4. Metformin (MET) treatment increased cardiosphere-derived cell (CDC) survival after transplantation into post-myocardial infarction (MI) hearts. a Representative images of survived CDCs in hearts at 7 days after transplantation. Heart tissue was immunostained for tropomyosin (red), green fluorescent protein (GFP) (green) and DAPI (blue). Scale bar = 100 μm. b Post-MI hearts with CDC transplantation were enzymatically digested, and small cells from the heart (<30 μm diameter) were collected after depletion of cardiomyocytes. As indicated with the white arrows, GFP-positive cells represent surviving CDCs under fluorescence microscope. Scale bar = 50 μm. c The percentage of surviving CDCs out of the total transplanted CDCs at 7 days post-transplantation. n = 5. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. MI + CDC. MET/CDC MET-pretreated CDC

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  Fig. 5. The effect of metformin (MET) treatment on the inflammatory response in post-myocardial infarction (MI) hearts. Representative contour plots (a) and quantification (b) of infiltrating monocyte population (Ly6Chigh/CD11b+) in cells isolated from infarcted hearts at 48 hours post-MI analyzed by FACS analysis. n = 5. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. Sham; # P < 0.05 vs. MI + phosphate-buffered saline (PBS). MET/CDC MET-pretreated cardiosphere-derived cells

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  Fig. 6. Metformin (MET) treatment exerted an anti-apoptotic effect on CDCs subjected to oxidative stress by activating AMPK. a1 Annexin V-PI double staining assay of CDCs treated with H2O2, analyzed by flow cytometric analysis. a2 Average percentages of apoptotic CDCs. n = 9. b Representative images (b1) and quantification (b2) of TUNEL staining in CDCs subjected to H2O2 treatment. Cell nuclei were stained with DAPI (blue) and TUNEL+ nuclei were labeled with TMR (green). TUNEL positive rate = (TUNEL-positive nuclei/DAPI-positive nuclei) × 100%. n = 12. Scale bar = 30 μm. c Cell viability of CDCs detected with CCK8 analysis. The data were normalized to 100% of the control group. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. control; # P < 0.05 vs. H2O2. CC compound C

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  Fig. 7. Metformin (MET) treatment increased phosphorylation of AMP-activated protein kinase (AMPK)/endothelial nitric oxide synthase (eNOS) in CDCs. Representative blots (a1) and quantification (a2) of AMPK phosphorylation. AMPK phosphorylation was represented as the ratio of phosphorylated α-AMPK at Thr-172 to total AMPK of CDCs. Representative blots (b1) and quantification (b2) of eNOS phosphorylation. eNOS phosphorylation was represented as the ratio of phosphorylated eNOS at Ser-1177 to total eNOS of CDCs. Data were analyzed by one-way ANOVA with post-hoc comparisons by the Tukey’s test. *P < 0.05 vs. control; # P < 0.05 vs. H2O2. CC compound C