Metformin Protects against Diabetic Cardiomyopathy: An Association between Desmin-Sarcomere Injury and the iNOS/mTOR/TIMP-1 Fibrosis Axis

Abstract

The intermediate filament protein desmin is essential for maintaining the structural integrity of sarcomeres, the fundamental unit of cardiac muscle. Diabetes mellitus (DM) can cause desmin to become dysregulated, following episodes of nitrosative stress, through the activation of the iNOS/mTOR/TIMP-1 pathway, thereby stimulating collagen deposition in the myocardium. In this study, type 2 diabetes mellitus (T2DM) was induced in rats. One group of animals was pre-treated with metformin (200 mg/kg) prior to diabetes induction and subsequently kept on metformin until sacrifice at week 12. Cardiac injuries developed in the diabetic rats as demonstrated by a significant (p < 0.0001) inhibition of desmin immunostaining, profound sarcomere ultrastructural alterations, substantial damage to the left ventricular tissue, collagen deposition, and abnormal ECG recordings. DM also significantly induced the cardiac expression of inducible nitric oxide synthase (iNOS), mammalian target of rapamycin (mTOR), and the profibrogenic biomarker tissue inhibitor of metalloproteinase-1 (TIMP-1). The expression of all these markers was significantly inhibited by metformin. In addition, a significant (p < 0.0001) correlation between desmin tissue levels/sarcomere damage and glycated hemoglobin, heart rate, iNOS, mTOR, and fibrosis was observed. These findings demonstrate an association between damage of the cardiac contractile unit—desmin and sarcomere—and the iNOS/mTOR/TIMP-1/collagen axis of fibrosis in T2DM-induced cardiomyopathy, with metformin exhibiting beneficial cardiovascular pleiotropic effects.

Keywords: cardiomyopathy; desmin; diabetes; fibrosis; metformin; sarcomere.

Figure 1

Metformin (Met) protects the cardiac architecture against injuries secondary to T2DM. H&E-stained images (400×) of left ventricles obtained 10 weeks post diabetic induction from the control untreated (A), T2DM (B), and Met + T2DM (C) groups are illustrated (Scale = 20 μm). Note that the arrowhead in (A) points to the intercalated disc, and the wavy arrow in (B) points to dilated cardiac myocytes. V: vesicular nuclei; S: spaces between cardiac cells; D: darkly stained nuclei; H&E: hematoxylin and eosin; T2DM: type 2 diabetes mellitus. Histograms in (D) represent a quantitative analysis of cardiomyocytes injury assessed on the basis of the cardiomyocyte diameter in μm in the groups described above. (E,F) Blood levels of glucose (E) and glycated hemoglobin (F) were measured at the end of the experiment, at week 12. The presented p values are all significant. * p < 0.05 versus control, ** p < 0.01 versus T2DM.

Figure 2

Metformin (Met) protects the cardiac ultrastructure against alterations secondary to T2DM. TEM images (10,000×) of left ventricles obtained 10 weeks post diabetic induction of untreated control (A,B), T2DM (C,D), and Met + T2DM (E,F) groups are illustrated. Note that the asterisk in (C) points to degenerated fragmented myofibrils, and the arrows in (D) point to the widely separated festooned sarcolemma. N: nucleus; nu: nucleolus; chr: chromatin; ne: nuclear envelope; V: vacuoles; m: mitochondria; m1: damaged mitochondria; H: H band; Z: Z discs; T2DM: type 2 diabetes mellitus. The histogram in (G) represents a quantitative analysis of % sarcomere damage in cardiac sections from the groups above. Presented p values are all significant. * p < 0.05 versus control, ** p < 0.0001 versus T2DM.

Figure 3

Metformin (Met) protects against the inhibition of cardiac desmin expression and ECG abnormalities caused by T2DM. Immunohistochemistry of desmin (400×) in left ventricle sections obtained 10 weeks post diabetic induction from the untreated control (A), T2DM (B), and Met + T2DM (C) groups are depicted (Scale = 20 μm). Note that the arrows in (A,C) point to desmin immunostaining in the intercalated discs, and the arrowheads in (A,C) point to the organized desmin distribution in the cardiomyocytes. Histograms in (D) represent a quantitative analysis of desmin-immunostained area % in cardiac sections from the above groups. Representative ECG recordings from the untreated control (E), T2DM (F), and Met + T2DM (G) groups are depicted. The heart rate of the groups was recorded at the end of the experiment (H). (I,J) Correlation between desmin and heart rate (I) and sarcomere damage (J). T2DM: type 2 diabetes mellitus; ECG: electrocardiographs. Presented p values are all significant. * p < 0.0001 versus control, ** p < 0.0001 versus T2DM.

Figure 4

Metformin (Met) protects against cardiac fibrosis secondary to T2DM. Masson’s trichrome-stained images (400×) of left ventricles obtained 10 weeks post diabetic induction from the untreated control (A), T2DM (B), and Met + T2DM (C) groups are visualized using light microscopy (Scale = 20 μm). Note that the arrows point to collagen deposition. Histograms in (D) represent a quantitative analysis of collagen area % in cardiac sections from the above groups. (E–H) Correlation between collagen deposition (fibrosis) score and glucose levels (E), desmin (F), sarcomere injury (G), and heart rate (H). The presented p values are all significant. * p < 0.01 versus control, ** p < 0.0001 versus T2DM. T2DM: type 2 diabetes mellitus.

Figure 5

Metformin (Met) protects against the inhibition of cardiac desmin expression and ECG abnormalities caused by T2DM. iNOS immunohistochemistry of left ventricle sections (400×) obtained 10 weeks post diabetic induction from the untreated control (A), T2DM (B), and Met + T2DM (C) groups are depicted (Scale = 20 μm). Histograms in (D) represent a quantitative analysis of desmin immunostaining area % in cardiac sections from the above groups. Cardiac lysates prepared from the above groups were immunoblotted with antibodies against p-AMPK, p-mTOR, and β-actin as a loading control (E). The relative expression of these signaling proteins is shown (F,G). Histograms in (H) display the relative mRNA expression of TIMP-1 in all animal groups. In E, --, represent the Control group; -+, represent T2DM group; and ++, represent the treated group. The presented p values are all significant. * p < 0.01 versus control, ** p < 0.0001 versus T2DM. (I–L) Correlation between either the desmin score versus iNOS (I) and p-AMP (J) or the score of sarcomere damage versus p-mTOR (K) and TIMP-1 (L). iNOS: inducible nitric oxide synthase; T2DM: type 2 diabetes mellitus; AMPK: AMP-activated protein kinase; mTOR: mammalian target of rapamycin; TIMP-1: tissue inhibitor of metalloproteinase-1.

Figure 6

The proposed model for diabetes-induced cardiomyopathy (left side) appears inhibited by metformin (right side). ↑ = increased; ↓ = decresed. iNOS: inducible nitric oxide synthase; T2DM: type 2 diabetes mellitus; Met: metformin.