Exacerbation by knocking-out metallothionein gene of obesity-induced cardiac remodeling is associated with the activation of CARD9 signaling

Figures

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  Figure 1. The effect of MT knockout on HFD-induced obesity and metabolic disorders. A: Body weights during 26 weeks of ages and 18 weeks of HFD feeding (started at 8 weeks old). B-C: Weight of body and perirenal adipose tissue weight at the end of 18-week feeding. D: The changes of blood glucose levels over time after IPGTT in 26-week-old mice (Da, i.e., at the end of 18-wks HFD feeding) and the IPGTT results via calculation of the integrated area under the curves (AUC, Db). E-F: Plasma cholesterol and triglyceride. Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, HFD vs. ND in WT and MT-KO groups; #P<0.05, ##P<0.01, ####P<0.0001, MT-KO vs. WT in ND and HFD groups.

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  Figure 2. The effect of MT knockout on HFD-induced cardiac hypertrophy. A-B, Heart weight and heart weight to tibia length ratio. C, Cardiac MT protein expression evaluated by Western blot. D, WGA staining of cardiac tissue sections and the quantification of myocyte cross-sectional areas (scale bar = 50 μm). Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, HFD vs. ND in WT and MT-KO groups, ####P<0.0001, MT-KO vs. WT.

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  Figure 3. The effect of MT knockout on HFD-induced cardiac fibrosis. A, Picro-Sirius Red staining results of heart tissue sections. B, Quantitative analysis of Picro-Sirius Red staining for collagen accumulation. C-F: Cardiac pro-fibrotic mRNA expression of Fn, col1a1, col3a1, and TGF-β evaluated by qRT-PCR. G-I, Protein expression of COL1A1 and TGF-β1 detected by Western blot. Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, HFD vs. ND in WT and MT-KO groups, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, MT-KO vs. WT in ND and HFD groups.

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  Figure 4. The effect of MT knockout on HFD-induced macrophage infiltration into the heart. A, mRNA expression of CD68 detected by qRT-PCR.B, Cardiac protein expression of CD68 evaluated by Western blot. C, Immunohistochemistry staining of CD68 results in the heart and quantitative analysis of IHC staining (brown considered positive staining; scale bar = 50 μm). Data are presented as Mean ± SD (n=5). *P<0.05, ***P<0.001, HFD vs. ND in WT and MT-KO groups, #P<0.05, ###P<0.001, MT-KO vs. WT in ND and HFD groups.

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  Figure 5. The effect of MT knockout on HFD-induced secretion of adhesion molecules, proinflammation cytokines, and chemokines. A, mRNA expression of chemokines including CCL2 evaluated by qRCT-PCR. B-G, Cardiac protein expression of VCAM-1(C), ICAM-1(D), TNF-α(E), IL-6(F), IL-1β(G) detected by Western blot. Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, HFD vs. ND in WT and MT-KO groups, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, MT-KO vs. WT in ND and HFD groups.

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  Figure 6. The effect of MT knockout on HFD-induced activation of inflammation pathway. A-G, Cardiac protein expression of CARD9 (B), BCL10 (C), phosphorylation and total p38 MAPK (D), phosphorylation and total JNK (E), phosphorylation and total ERK1/2 (F), and phospho-NF-κB p65 (G) were detected by Western blot, with GAPDH used as the loading control. Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, HFD vs. ND in WT and MT-KO groups, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001, MT-KO vs. WT in ND and HFD groups.

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  Figure 7. The effect of MT knockout on HFD-induced oxidative stress damage in hearts. Cardiac expression of 4-HNE (A) and 3-NT (B), as markers of oxidative stress damage, was detected by Western blot, with GAPDH used as loading control. Data are presented as Mean ± SD (n=5). *P<0.05, **P<0.01, ***P<0.001, HFD vs. ND in WT and MT-KO groups, #P<0.05, ##P<0.01, MT-KO vs. WT in ND and HFD groups.

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  Figure 8. The effect of MT knockout on HFD-induced changes of the essential metal level in the heart. Zinc (Zn) (A), Iron (Fe) (B), Copper (Cu) (C) levels in the heart were measured in WT and MT-KO mice. The ratios of Fe/Zn (D), Cu/Zn (E), and Cu/Fe (F) in the heart were then calculated. Data are presented as Mean ± SD (n=5). *P<0.05, HFD vs. ND, #P<0.05, MT-KO vs. WT in ND and HFD groups.

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  Figure 9. An illustration outlining the potential protective mechanisms of MT against obesity-induced adverse impacts on the heart that we studied.