Cardiac fibrosis and dysfunction in experimental diabetic cardiomyopathy are ameliorated by alpha-lipoic acid

Abstract

Background: Alpha-lipoic acid (ALA), a naturally occurring compound, exerts powerful protective effects in various cardiovascular disease models. However, its role in protecting against diabetic cardiomyopathy (DCM) has not been elucidated. In this study, we have investigated the effects of ALA on cardiac dysfunction, mitochondrial oxidative stress (MOS), extracellular matrix (ECM) remodeling and interrelated signaling pathways in a diabetic rat model.

Methods: Diabetes was induced in rats by I.V. injection of streptozotocin (STZ) at 45 mg/kg. The animals were randomly divided into 4 groups: normal groups with or without ALA treatment, and diabetes groups with or without ALA treatment. All studies were carried out 11 weeks after induction of diabetes. Cardiac catheterization was performed to evaluate cardiac function. Mitochondrial oxidative biochemical parameters were measured by spectophotometeric assays. Extracellular matrix content (total collagen, type I and III collagen) was assessed by staining with Sirius Red. Gelatinolytic activity of Pro- and active matrix metalloproteinase-2 (MMP-2) levels were analyzed by a zymogram. Cardiac fibroblasts differentiation to myofibroblasts was evaluated by Western blot measuring smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β). Key components of underlying signaling pathways including the phosphorylation of c-Jun N-terminal kinase (JNK), p38 MAPK and ERK were also assayed by Western blot.

Results: DCM was successfully induced by the injection of STZ as evidenced by abnormal heart mass and cardiac function, as well as the imbalance of ECM homeostasis. After administration of ALA, left ventricular dysfunction greatly improved; interstitial fibrosis also notably ameliorated indicated by decreased collagen deposition, ECM synthesis as well as enhanced ECM degradation. To further assess the underlying mechanism of improved DCM by ALA, redox status and cardiac remodeling associated signaling pathway components were evaluated. It was shown that redox homeostasis was disturbed and MAPK signaling pathway components activated in STZ-induced DCM animals. While ALA treatment favorably shifted redox homeostasis and suppressed JNK and p38 MAPK activation.

Conclusions: These results, coupled with the excellent safety and tolerability profile of ALA in humans, demonstrate that ALA may have therapeutic potential in the treatment of DCM by attenuating MOS, ECM remodeling and JNK, p38 MAPK activation.

Figure 1

Diabetes-induced left ventricular dysfunction were improved by ALA treatment. Cardiac dysfunction were evaluated by measuring (A) heart rate (HR), (B) left ventricular end-diastolic pressure (LVEDP), (C) maximum rate of fall of left ventricle pressure (-dP/dtmax), (D) left ventricular end-systolic pressure (LVSP), (E) maximum rate of rise of left ventricle pressure (+dP/dtmax). Eleven weeks of STZ-induced diabetes was associated with decrease in HR, LVSP and ± dp/dtmax and an increase in LVEDP, which were significantly improved by ALA treatment for 11 weeks. Results are presented as mean values ± standard deviation. *p < 0.05 vs Control group, #p < 0.05 vs. STZ group, n = 8 per group.

Figure 2

Diabetes induced cardiac fibrosis. (A) The red color of Sirius red staining under the common microscope indicates total collagen deposition, representative images are from NC, NC + ALA, STZ and STZ + ALA (200 × magnification). (B) Type I and II collagen deposition were shown by orange and green color under the polarized light respectively, representative images are from NC, NC + ALA, STZ and STZ + ALA. (C) The protein level of type I and II collagen were determined by western blot, β-Actin was used as loading control. Bar graph, density analysis results from 8 rats per group. Results are mean values ± standard deviation. *p < 0.05 vs Control group, #p < 0.05 vs. STZ group.

Figure 3

ALA treatment ameliorated diabetes-induced myocardial mitochondrial oxidative stress. Myocardial MOS were determined by measuring (A) malondialdehyde (MDA) (B) endogenous antioxidants reduced glutathione (GSH) (C) oxidized glutathione (GSSG) (D) the ratio of GSH and GSSG (E) superoxide dismutase (SOD) activity. Results are mean values ± standard deviation. *p < 0.05 vs Control group, #p < 0.05 vs. STZ group.

Figure 4

ALA treatment decreased the TGF-β,α-SMA, and TIMP-2 expression and increased cardiac active- and pro- MMP-2 levels. The protein expression of TGF-β (A), α-SMA (B), and TIMP-2 (C) were determined by Western blot with specific antibodies as indicated, β-Actin was used as loading control. Cardiac active- and pro- MMP-2 (D/E) levels were assayed by zymogram. Representative image, bar graph, density analysis results from 8 hearts per group. Data represent mean ± standard deviation. *p < 0.05 vs Control group, #p < 0.05 vs. STZ group.

Figure 5

ALA Reduces diabetes-induced myocardial activation of JNK and P38. Total protein was obtained from the hearts of STZ-induced diabetic rats and vehicle rats. Phosphorylated and total JNK, P38 and ERK were determined by Western blot with specific antibodies as indicated. The extent of JNK (A), P38 (B) and ERK (C) phosphorylation was quantified by phosphorylated protein/total protein. Representative image, bar graph, density analysis results from 8 hearts per group. Data represent mean ± standard deviation. *p < 0.05 vs Control group, #p < 0.05 vs. STZ group.