Metformin Inhibits Cardiac Fibroblast Differentiation by Promoting Fatty Acid β-Oxidation: Implications for Age-Associated Cardiac Fibrosis

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  Figure 1. Metformin promotes fatty acid β-oxidation in adult mouse cardiac fibroblasts. (A–C) Adult mouse cardiac fibroblast cells were pretreated with or without metformin (2 mM) for 12 h followed by TGF-β (10 ng/mL) for 48 h, and CPT1 transcript levels (A), protein levels (B), and enzyme activity (C) were measured. (D) Same as A, except that transcript levels of FABP3, ACSL1, ACADM, and ACADS were measured by RT-qPCR analysis. (E) Same as A, except that transcript levels of HIF-1α, GLUT1, and LDHA were measured by RT-qPCR analysis. (F) Adult mouse cardiac fibroblast cells were pretreated with metformin (2 mM) followed by treatment with TGF-β (10 ng/mL) in the presence or absence of etomoxir (40 µM) for 48 h; lipid accumulation was measured by Nile Red staining, and images were taken in a fluorescence microscope equipped with a rhodamine filter, scale bar = 175 µm. (G,H) Same as A, except that acetyl-CoA levels (G) and malonyl-CoA levels (H) were measured in cell lysates. (I) Same as A, except that the indicated protein levels were measured by immunoblot analysis. (J,K) Quantification of (I). GraphPad Prism 10 was used for statistical analysis. Data are presented as mean ± SD from at…

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  Figure 2. Inhibition of AMPK activation abolishes metformin-mediated enhancement of fatty acid β-oxidation in cardiac fibroblasts. (A,B) Adult mouse cardiac fibroblasts were treated with Compound C (10 μM) in the presence or absence of metformin (2 mM) for 12 h, followed by TGF-β (10 ng/mL) stimulation for 48 h, and CPT1 transcript levels (A) and enzyme activity (B) were measured. (C–F) Same as A, except that transcript levels of FABP3, ACSL1, ACADM, and ACADS were measured by RT-qPCR analysis. (G,H) Same as A, except that acetyl-CoA levels (G) and malonyl-CoA levels (H) were measured in cell lysates. (I) Same as A, except that transcript levels of TGF-β (I) and the indicated protein levels (J) were assessed by RT-qPCR and Western blot analysis, respectively. (K,L) Quantification of (J). GraphPad Prism 10 was used for statistical analysis. Data are presented as mean ± SD from at least three independent experiments. ns is non-significant, *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001. #, p ≤ 0.05; ##, p ≤ 0.005; ###, p ≤ 0.0005; ####, p ≤ 0.0001 significantly different from TGF-β. Welch’s ANOVA followed by Dunnett’s T3 post hoc test for unequal variances and one-way ANOVA…

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  Figure 3. Etomoxir reverses metformin-mediated increase in mitochondrial oxygen consumption rate (OCR) in adult mouse cardiac fibroblast cells. (A) Adult mouse cardiac fibroblast cells were pretreated with or without metformin (2 mM) for 12 h followed by TGF-β (10 ng/mL) for 48 h, and the oxygen consumption rates (OCR) were measured by sequentially adding (indicated by arrows) Etomoxir (40 µM), Oligomycin (1 µM), FCCP (1 µM), Rotenone (1 µM), and Antimycin (1 µM) using a Seahorse XF 24 Extracellular Flux Analyzer. (B–E) Same as A, except that basal respiration (B), maximal respiration (C), ATP production (D), and coupling efficiency (E) were measured from the data obtained in A. GraphPad prism 10 was used to perform the statistics. The data shown in the graphs represent the mean ± SD of at least three different experiments. ns is non-significant, *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001 by two-way ANOVA followed by Tukey’s multiple-comparison test.

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  Figure 4. Metformin-mediated increase in mitochondrial oxygen consumption rate (OCR) is due to enhanced FAO in cardiac fibroblasts. (A) Adult mouse cardiac fibroblast cells were pretreated with or without metformin (2 mM) for 12 h followed by TGF-β (10 ng/mL) for 48 h and palmitate-BSA fatty acid oxidation substrate assay was performed to measure the mitochondrial OCR as described in methods. (B–D) Same as A, except that basal respiration (B), maximal respiration (C), ATP production (D) and coupling efficiency (E) were measured from the data obtained in A. GraphPad prism 10 was used to perform the statistics. The data shown in the graphs represent the mean ± SD of at least three different experiments. ns is non-significant, *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001 by two-way ANOVA followed by Tukey’s multiple-comparison test.

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  Figure 5. Metformin mitigates cardiac fibrosis markers and cellular senescence by promoting fatty acid β-oxidation. Adult mouse cardiac fibroblast cells were pretreated with metformin (2 mM) followed by treatment with TGF-β (10 ng/mL) in the presence or absence of etomoxir (40 µM) for 48 h. (A) Cells were labelled with PKH26 lipophilic dye and images were acquired using a laser scanning confocal microscope (scale bar = 50 µm). (B–D) Same as A, except that transcript levels of the indicated fibrosis markers were measured by RT-qPCR analysis. (E) Same as A, except that α-SMA protein levels were measured by immunoblotting. (F,G) Same as A, except that cellular senescence (F) was measured by SA-β-Gal staining (scale bar = 175 µm) and its quantification (G). (H) Same as A, except that senescence marker proteins like p21 and p27 were measured by immunoblot analysis. (I,J) Quantification of H. GraphPad Prism 10 was used for statistical analysis. Data are presented as mean ± SD from at least three independent experiments. ns is non-significant, *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001. #, p ≤ 0.05; ##, p ≤ 0.005; ###, p ≤ 0.0005; ####, p ≤ 0.0001 significantly different from…

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  Figure 6. Metformin administration enhances CPT1 levels and fatty acid β-oxidation (FAO) pathway in the hearts of aged Apoe−/− mice. (A–D) CPT1 Transcript (A), protein (B,C), and enzyme activity (D) were measured in Apoe−/− mice heart (n = 3 per group). (E–H) Transcript levels of FABP3, ACSL1, ACADM, and ACADS were measured in Apoe−/− mice heart tissue by RT-qPCR analysis (n = 3 per group). (I,J) Acetyl-CoA (I) and Malonyl-CoA (J) levels were measured in Apoe−/− mice heart tissue (n = 3 per group). (K) Lipid content was measured by Nile Red staining in Apoe−/− mice heart sections and images were taken in a fluorescence microscope equipped with a rhodamine filter (n = 3 per group; scale bar = 85 µm). (L) Indicated protein levels were measured in Apoe−/− mice heart tissue homogenates (n = 3 per group) by immunoblot analysis. (M,N) Quantification of L. GraphPad Prism 10 was used for statistical analysis. Data are presented as mean ± SD from at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001. Welch’s ANOVA followed by Dunnett’s T3 post hoc test for unequal variances and one-way ANOVA followed by Tukey’s multiple-comparison test for equal…

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  Figure 7. Metformin administration attenuates cardiac fibrosis markers in Apoe−/− mice. (A) TGF-β levels were measured in Apoe−/− mice heart tissue homogenates by ELISA (n = 3 per group). (B–D) Transcript levels of the indicated fibrosis markers were measured in Apoe−/− mice heart tissue by RT-qPCR analysis (n = 3 per group). (E) α-SMA protein levels were measured in Apoe−/− mice heart tissue homogenates by immunoblotting (n = 3 per group). (F) Quantification of E. (G) Masson’s trichome staining for collagen accumulation in the heart sections (n = 3 per group; scale bar = 85 µm). (H) Quantification of G. (I) Sirius Red/Fast Green staining for collagen accumulation in the heart sections (n = 3 per group; scale bar = 85 µm). (J) Quantification of I. (K) Total collagen content in heart tissues by hydroxyproline assay (n = 3 per group). GraphPad Prism 10 was used for statistical analysis. Data are presented as mean ± SD from at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005; ****, p ≤ 0.0001. Welch’s ANOVA followed by Dunnett’s T3 post hoc test for unequal variances and one-way ANOVA followed by Tukey’s multiple-comparison test for equal variances, depending…