Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy

Abstract

We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM.

Figure 1

Nrf2-KO mice showed increased diabetes-induced cardiac hypertrophy and reduced SFN-induced cardiac protection. A: Nrf2 mRNA level was detected by qRT-PCR. B: Nuclear Nrf2 protein expression was detected by Western blot. C: Heart size. D and E: Cardiac tissue FITC-conjugated WGA staining and quantification of myocyte cross-sectional areas (scale bar = 25 μm). F and G: qRT-PCR analysis of hypertrophic markers ANP and β-MHC to determine mRNA expression. Data were presented as means ± SD (n = 6). *P < 0.05 vs. C57 Ctrl; #P < 0.05 vs. C57 DM; §P < 0.05 vs. C57 DM/SFN.

Figure 2

Nrf2-KO mice showed increased diabetes-induced cardiac fibrosis and reduced SFN-mediated cardiac protection. A: Cardiac fibrosis, determined by Sirius Red staining of collagen accumulation (collagen is red; scale bar = 50 μm). B: Quantitative analysis of Sirius Red staining for collagen accumulation. C–E: Protein expression of CTGF and TGF-β1 by Western blot. F and G: Cardiac inflammation, measured by mRNA expression of IL-6 and MCP-1 by qRT-PCR. Data were presented as means ± SD (n = 6). *P < 0.05 vs. C57; #P < 0.05 vs. C57 DM; §P < 0.05 vs. C57 DM/SFN.

Figure 3

SFN completely abolished induced Nrf2 expression and function as well as MT expression. Cardiac oxidative damage, tested by IHC staining for 8-OHdG (A; brown considered positive staining; scale bar = 50 μm) followed by a quantitative analysis of the IHC stains (B) and lipid peroxidation with MDA assay (C). Activation of Nrf2, reflected by its nucleus accumulation (indicated by white arrows) determined by immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope (D) (scale bar = 25 μm) and mRNA expression of Nrf2 downstream genes HO-1 and NQO1 (E). F: MT expression measured by quantitative PCR and Western blots for its mRNA and protein levels, respectively. Data were presented as means ± SD (n = 6). *P < 0.05 vs. C57; #P < 0.05 vs. C57 DM; §P < 0.05 vs. C57 DM/SFN.

Figure 3

SFN completely abolished induced Nrf2 expression and function as well as MT expression. Cardiac oxidative damage, tested by IHC staining for 8-OHdG (A; brown considered positive staining; scale bar = 50 μm) followed by a quantitative analysis of the IHC stains (B) and lipid peroxidation with MDA assay (C). Activation of Nrf2, reflected by its nucleus accumulation (indicated by white arrows) determined by immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope (D) (scale bar = 25 μm) and mRNA expression of Nrf2 downstream genes HO-1 and NQO1 (E). F: MT expression measured by quantitative PCR and Western blots for its mRNA and protein levels, respectively. Data were presented as means ± SD (n = 6). *P < 0.05 vs. C57; #P < 0.05 vs. C57 DM; §P < 0.05 vs. C57 DM/SFN.

Figure 4

MT-KO mice showed increased diabetes-induced cardiac hypertrophy and preserved SFN-induced cardiac protection. A and B: MT mRNA and protein expression, evaluated by quantitative PCR and Western blot. C: Heart size. D: WGA-FITC staining in cardiac tissue sections and the quantification of myocyte cross-sectional areas (scale bar = 25 μm). E and F: Cardiac hypertrophy, detected with hypertrophic markers ANP and β-MHC with qRT-PCR. *P < 0.05 vs. 129S; #P < 0.05 vs. 129S DM; §P < 0.05 vs. 129S DM/SFN; +P < 0.05 vs. MT-KO DM.

Figure 5

MT-KO mice showed increased diabetes-induced cardiac fibrosis and preserved SFN-induced cardiac protection. A and B: Cardiac fibrotic response, examined by Sirius Red staining of collagen (collagen is red; scale bar = 50 μm) and quantitative analysis. C–E: Protein expression of CTGF and TGF-β1 with Western blot. F and G: qRT-PCR analysis for the inflammatory factors IL-6 and MCP-1 to determine mRNA expression. Data were presented as means ± SD (n = 6). *P < 0.05 vs. 129S; #P < 0.05 vs. 129S DM; §P < 0.05 vs. 129S DM/SFN; +P < 0.05 vs. MT-KO DM.

Figure 6

SFN-induced Nrf2 expression was MT independent. Cardiac oxidative damage, detected by IHC staining with an anti–8-OHdG antibody (A; brown considered positive staining; scale bar = 50 μm) and quantitative analysis of IHC staining (B) and lipid peroxidation with MDA assay (C). D: Immunofluorescent staining with Nrf2 antibody (red) and nuclear staining with DAPI (blue) on cardiac tissue sections by fluorescence microscope (scale bar = 25 μm). White arrows indicate Nrf2 localized in the nucleus. Data were presented as means ± SD (n = 6). *P < 0.05 vs. 129S; #P < 0.05 vs. 129S DM; §P < 0.05 vs. 129S DM/SFN; +P < 0.05 vs. MT-KO DM.

Figure 7

SFN-induced Nrf2 downstream gene expression was MT independent. A: The mRNA levels of Nrf2 were detected by qRT-PCR. B: The activation of Nrf2, which was reflected by its nuclear accumulation, was quantified by Western blot of the cardiac nucleus protein. C and D: HO-1 and NQO1 mRNA expression. Data were presented as means ± SD (n = 6). E: Schematic illustration for SFN protection against type 2 DCM. T2DM induces cardiac oxidative stress, inflammation, and fibrosis, leading from myocardial remodeling and dysfunction to development of DCM. SFN-induced cardiac protection from diabetes via upregulating Nrf2 expression and function, including Nrf2 downstream MT, NQO1, and HO-1 expression. MT, as one of the major Nrf2 downstream targets, plays an important role in the protection by SFN-induced Nrf2 pathway against type 2 DCM. *P < 0.05 vs. 129S; #P < 0.05 vs. 129S DM; §P < 0.05 vs. 129S DM/SFN; %P < 0.05 vs. MT-KO; +P < 0.05 vs. MT-KO DM; &P < 0.05 vs. 129S SFN.