Metformin alleviates diastolic dysfunction in mice with experimental diabetic cardiomyopathy

Figures

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  Fig. 1. Metformin treatment improves glucose tolerance in mice with T2D. (A) Schematic representation of the experimental design used for the in vivo animal studies. (B) Body weight, (C) % fat mass and (D) % lean mass in lean mice and mice with T2D before and after 7.5-wks of treatment with either vehicle control (VC) or metformin (n = 8–20 mice/group). (E) Ad-libitum blood glucose measured bi-weekly in lean mice and mice with T2D treated with VC or metformin for 7.5-wks (n = 8–20 mice/group). (F) Intraperitoneal insulin tolerance test, (G) intraperitoneal glucose tolerance test and (H) the associated integrated area under the curve (iAUC) in lean mice and mice with T2D at 4–5 weeks post-VC or metformin treatment (n = 8–20 mice/group). The iAUC was calculated using the fasting glycemia value as the baseline. Values represent mean ± SEM. Differences were determined using a repeated measures two-way ANOVA (B–G) or a two-way ANOVA (H) followed by a Bonferroni post-hoc analysis. *P < 0.05, significantly different versus respective VC counterpart. #P < 0.05, significantly different versus respective lean counterpart. δP < 0.05, significantly different versus respective pre-treatment…

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  Fig. 2. Metformin alleviates left ventricular diastolic dysfunction without affecting systolic function in mice with T2D. (A) Representative images of pulse-wave Doppler, (B) representative images of tissue Doppler, (C) mitral E/A ratio, (D) percent change in mitral E/A ratio after treatment, (E) mitral e′/a′ ratio, (F) percent change in mitral e′/a′ ratio after treatment, (G) E/e′ ratio, (H) percent change in mitral E/e′ ratio after treatment, (I) Representative M-mode images, (J) percent left ventricular (LV) ejection fraction (LVEF), (K) percent fractional shortening (LVFS) and (L) cardiac output (CO) assessed using ultrasound echocardiography in lean mice and mice with T2D mice after a 7.5-wk treatment with either vehicle control (VC) or metformin (n = 8–20 mice/group). Values represent mean ± SEM. Differences were determined using a two-way ANOVA (C, E, G, J–L) or a repeated measures two-way ANOVA (D, F, H) followed by a Bonferroni post-hoc analysis. *P < 0.05, significantly different versus respective VC counterpart. #P < 0.05, significantly different versus respective lean counterpart.

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  Fig. 3. Metformin treatment decreased cardiomyocyte size but not wall thickness in mice with T2D. (A) Left ventricular (LV) anterior wall thickness during diastole (LVAWd) and LV posterior wall thickness during diastole (LVPWd), (B) LV internal diameter during diastole (LVIDd), (C) LV mass and (D) LV mass normalized to lean body mass (LBM) as assessed by ultrasound echocardiography in lean mice and mice with T2D 7.5-wks after treatment with vehicle control (VC) or metformin (n = 8–20 mice/group). (E) Representative images of wheat germ agglutinin staining of LV sections and (F) LV cardiomyocyte (CM) cross-sectional area (CSA) in lean mice and mice with T2D following treatment with either VC or metformin for 7.5-wks (n = average of 15 cardiomyocytes from 4 to 5 mice/group). Scale bar, 50 μm. (G) Relative mRNA expression of biomarkers for cardiac hypertrophy (Nppa, Nppb, Myh7 and Acta2) normalized to Ppia mRNA expression in myocardial extracts of lean mice and mice with T2D at 7.5-wks post-treatment (n = average of 3 technical replicates from 5 to 11 mice/group). Values represent mean ± SEM. Differences were determined using a two-way ANOVA (A–D, F–G) followed by a Bonferroni post-hoc…

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  Fig. 4. Myocardial fibrosis was unchanged with metformin treatment in mice with T2D. (A) Representative images of picrosirius red staining of the left ventricular (LV) myocardium, (B) percent LV interstitial fibrotic area and (C) percent LV perivascular fibrotic area in lean mice and mice with T2D following a 7.5-wk treatment with vehicle control (VC) or metformin (n = average of 3 LV sections from 4 to 5 animals/group). Scale bar, 50 μm. (D) Relative myocardial mRNA expression of key regulators of fibrosis (Col1a1, Col1a2, and Ctgf) normalized to Ppia mRNA expression in lean mice and mice with T2D treated with VC or metformin for 7.5-wks (n = average of 3 technical replicates from 5 to 11 mice/group). Values represent mean ± SEM. Differences were determined using a two-way ANOVA (B–D) followed by a Bonferroni post-hoc analysis. *P < 0.05, significantly different versus respective VC counterpart. #P < 0.05, significantly different versus respective lean counterpart. Col1a1, collagen type 1 alpha 1; Col1a2, collagen type 1 alpha 2; Ctgf, connective tissue growth factor; Ppia, peptidylprolyl isomerase A.

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  Fig. 5. Metformin treatment did not alter myocardial substrate metabolism in mice with T2D. (A) Relative myocardial mRNA expression of key enzymes involved in glucose metabolism (Pdk4, Hk2, Fbp2, Glut4 and Pfkm) normalized to Ppia mRNA expression in lean mice and mice with T2D treated with vehicle control (VC) or metformin for 7.5 weeks (n = average of 3 technical replicates from 5 to 11 mice/group). (B) Protein expression of PDK4, (C) PDH phosphorylation at serine 232 and (D) serine 300 in myocardial extracts from lean mice and mice with T2D at 7.5-wks post-treatment (n = 4–5 samples/group). (E) Relative myocardial mRNA expression of key regulators of fatty acid metabolism (Acc2, Acox1, Acadm, Cd36 and Ppara) normalized to Ppia mRNA expression in lean mice and mice with T2D after 7.5-wks treatment with VC or metformin (n = average of 3 technical replications from 5 to 11 mice/group). (F) TAG content in myocardial extracts from mice with T2D at 7.5-wks post-treatment (n = 7–8 samples/group). (G) Relative myocardial mRNA expression of key enzymes involved in ketone metabolism (Oxct1, Bdh1 and Acat1) normalized to Ppia mRNA expression. (H, I) Representative western blot images and…

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  Fig. 6. Metformin treatment does not attenuate inflammation in mice with T2D or H9C2 cardiac myoblasts. (A) Relative mRNA expression of pro-inflammatory cytokines (Il6, Il1b and Tgfb) and chemokines (Ccl2 and Ccl5) normalized to Ppia mRNA expression in myocardial extracts of lean mice and mice with T2D following a 7.5-wk treatment with vehicle control (VC) or metformin (n = average of 3 technical replicates from 5 to 11 mice/group). (B) Representative immunofluorescence images of H9C2 cells stained with nuclear factor κ B (NFκB) and 4′,6-diamidino-2-phenylindole (DAPI) (C) Integrated density of NFκB (n = average of 10 myoblasts from 3 biological replicates), (D) percent nuclear area of NFκB expression (n = average of 10 myoblasts from 3 biological replicates), (E) cell viability determined via MTT assay (n = average of 6 technical replicates from 3 biological replicates/group), (F) representative immunofluorescence images of H9C2 cells stained with WGA and DAPI and (G) relative cardiomyocyte (CM) cross-sectional area (CSA) (n = average of 35–60 cardiac myoblasts from 3 biological replicates/group), following a 1-hr incubation with either vehicle (sterile PBS) or lipopolysaccharide (LPS;…