The MEK inhibitor U0126 ameliorates diabetic cardiomyopathy by restricting XBP1's phosphorylation dependent SUMOylation

Figures

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  Figure 1. Establishment of diabetic cardiomyopathy mouse model. (A) schematic illustration of interventional studies in mice with STZ-induced hyperglycemia. (B) Line graphs reflecting blood glucose levels in mice with STZ-induced hyperglycemia. Blood glucose was measured at indicated time points. Histological analyses of the H&E staining and WGA staining of Con and DCM mice 26 weeks after STZ injection. (C) Representative images of H&E staining in the hearts of Con and DCM mice (n ≥ 8; scale bar, 100 μm). (D) Statistical results of the myocyte cross-sectional areas (n ≥ 6, at least 100 cells per mouse were analyzed). (E) Representative images of WGA staining in the hearts of Con and DCM mice (n ≥ 8; scale bar, 100 μm). (F) Quantitative analysis of myocyte cross-sectional areas (n ≥ 6, at least 100 cells per mouse were analyzed). (G-H) Representative immunoblots of Atrial natriuretic peptide (ANP), B type natriuretic peptide (BNP) and β-myosin heavy chain (MHC) expression and quantitative analysis are shown. (I-K) Echocardiographic measurements of ventricular functional parameters includes: Ejection fraction (J) and fractional shortening (K). Representative echocardiographic M-mode images…

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  Figure 2. XBP1s' nuclear translocation was significantly reduced and negatively correlated with the level of its SUMOylation in STZ-induced diabetic mouse heart. XBP1s nuclear translocation is impaired in DCM mice and in H9C2 cardiomyocytes treated with HG (25mM). (A-D) Representative immunoblots (A) and bar graph (B) showing nuclear and cytoplasm levels of XBP1s in heart samples of wild-type control (Con) and STZ-induced diabetic cardiomyopathy mice (DCM). As loading controls, Lamin A/C was used for nuclear extracts and GAPDH for cytoplasm extracts (n=6 mice per group). (C-D) Representative immunoblots showing cytoplasmic (C, top panel) and nuclear levels (C, lower panel) of XBP1s in H9C2 cardiomyocytes at indicated time points after treatment with HG (25mM). Line graph (D) reflecting the Mean ± SEM of three independent experiments. (E) XBP1s' SUMOylation was increased in DCM group. Heart samples from Con and DCM mice were immunoprecipitated with anti-XBP1s antibody, and Western blot analysis was performed with anti-SUMO1 (top panel) and anti-SUMO2/3 (lower panel) antibodies. A significant increase in XBP1s' SUMOylation was observed in DCM group. Con, control mice without diabetes, black…

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  Figure 3. SUMOylation of XBP1s at K276 residue at the C-terminal transactivation domain reduced its nuclear localization under HG condition. (A-B) Immunoblots and bar graph of Flag-XBP1s from Nuclear and cytoplasmic protein extracts in H9C2 cells upon transfection with different Flag-XBP1s constructs. As loading controls, Lamin A/C was used for nuclear extracts and GAPDH for cytoplasm extracts. (C) Fluorescent microscopic images of XBP1s-GFP fusion constructs upon 24 h transfection into H9C2 cells following HG intervention for 6 h. Nuclei were counter-stained with DAPI. Scar bar: 25μm. (D) Heat map summarizes enrichment of condition-specific concordant UPR genes. (E) And their impact on regulation of UPR-gene expression (E; n=3; Mean ± SEM, wild type vs K276R mutant).

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  Figure 4. Differential regulation of MAPK kinase in STZ-induced diabetic mouse heart. (A) Western blot analysis of protein extracts from hearts in different mice as indicated using specific antibodies against the phosphorylated and the non-phosphorylated form of the MAPKs ERK1/2, p38, JNK and (B) scatter blot summarized the results. We could show an increase in the phosphorylated form of the MAPKs ERK1/2 but not in p38 and JNK. Con, control mice without diabetes, black dots; DCM, diabetic cardiomyopathy, red squares; Data represent ratios of densitometric measurements of either p-JNK or JNK, p-ERK and ERK1/2 or p-p38 and p38 and are presented as Mean ± SEM. **P < 0.01 vs Con group; NS, non-significant (t-test). Representative immunoblots of at least three independent experiments (A).

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  Figure 5. Predicting and validating ERK1/2 Serine phosphorylation sites on XBP1s. (A) Potential phosphorylated Serine sites on XBP1s by ERK1/2 were predicted by the phosphorylation prediction software GPS 3.0. (B) Plasmid wild type XBP1s (WT) and serine-to-alanine phospho-mutants of XBP1s (S61A, S169A and S348A) were expressed in HEK293T cells and lysates were immunoblotted using the indicated antibodies. (C)Additionally, lysates were immunoprecipitated (IP) using Flag-agarose and immunoblotted using the indicated antibodies. (D) H9C2 cells were infected with wild type and XBP1s S348A mutant carrying adenoviruses, lysates were immunoblotted using the indicated antibodies. (E) Subsequently, lysates were immunoprecipitated (IP) using Flag-agarose and immunoblotted using the indicated antibodies. Results are representative of three independent experiments (B-E).

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  Figure 6. U0126 increased XBP1s' nuclear translocation through inhibiting its SUMOylation in HG-treated H9C2 cells in vitro. (A) Expression of total-ERK1/2 (t-ERK1/2) and phospho-ERK1/2 (p-ERK1/2) was detected by Western blot after H9C2 cells pretreated with U0126 (10 μM) for 1 h and HG (25mM) for additional 6 h. Representative immunoblots (top panel) and scatter blot (lower panel) summarizing results. (B) Extracted total proteins were immunoprecipitated with anti-XBP1s antibody, and Western blot analysis was performed with anti-SUMO1 (top panel) and anti-SUMO2/3 (lower panel) antibodies. A significant increase in XBP1s-SUMOylation was observed in HG group. (C) Representative immunoblots (top panel) and scatter blot (lower panel) showing cytoplasm and nuclear levels of XBP1s in the extracted total proteins of Con, HG and HG + U0126 group. As loading controls, Lamin A/C was used for nuclear extracts and GAPDH for cytoplasm extracts. (D) In the same experimental setting immunofluorescence staining was conducted, XBP1s was stained with green color, while nuclei were counter stained with DAPI (blue color). Scar bar: 25μm.Con, control without HG, black dots; HG: high glucose, squares; U0126,…

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  Figure 7. U0126 improved cardiac function and ameliorated cardiac hypertrophy in STZ-induced diabetic mice. (A) Schematic illustration of interventional studies in mice with STZ-induced hyperglycemia. Treatment with U0126 for 8 weeks was initiated after manifestation of abnormal cardiac function at week 18. (B) Line graphs reflecting blood glucose levels in mice with STZ-induced hyperglycemia. Blood glucose was measured at indicated time points. Histological analyses of the H&E staining and WGA staining of hearts from different mice as indicated 26 weeks after STZ injection. (C) Representative images of H&E staining in the hearts of each group mice (n ≥ 8; scale bar, 100 μm). (D) Statistical results of the myocyte cross-sectional areas (n ≥ 6, at least 100 cells per mouse were analyzed). (E) Representative images of WGA staining in the hearts of each group mice (n ≥ 8; scale bar, 100 μm). (F) Quantitative analysis of myocyte cross-sectional areas (n ≥ 6, at least 100 cells per mouse were analyzed). Representative immunoblots (G) and scatter blots summarizing results (H) for cardiac hypertrophy-related proteins expression in hearts from different mice as indicated. (I) Representative…

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  Figure 8. U0126 increased XBP1s' nuclear translocation through inhibiting its SUMOylation in STZ-induced diabetic mouse heart in vivo. Heart samples of each group were lysed and the extracted total proteins were processed for the detection of p-ERK1/2 and t-ERK1/2 using Western blot. (A) Representative immunoblots (top panel) and scatter blots (lower panel) summarizing results. (B) The extracted total proteins of heart samples were also immunoprecipitated with anti-XBP1s antibody, and Western blot analysis was performed with anti-SUMO1 (top panel) and anti-SUMO2/3 (lower panel) antibodies. (C) Representative immunoblots and (D) scatter blots showing nuclear and cytoplasm levels of XBP1s in the extracted proteins of each group mice. As loading controls, Lamin A/C was used for nuclear extracts and GAPDH for cytoplasm extracts. Con, control without HG, black dots; DCM: Diabetic cardiomyopathy, red squares; U0126 1mg/kg, blue triangles; Data are shown as mean ± SEM; *p < 0.05 vs Con group; #p < 0.05 vs DCM group. IB, immunoblot; IP, immunoprecipitation. Representative IBs of at least three independent experiments (A, B and C).

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  Figure 9. Schematic model of the signaling pathway for XBP1s' nuclear translocation regulated by U0126 through restricting its phosphorylation and SUMOylation. In healthy subjects the XBP1s' nuclear translocation maintains normal level. In contrast, in diabetic individuals, hyperglycemia activates Ras/MEK/ERK cascades, these activation signals lead to XBP1s Ser 348 phosphorylation, resulting in XBP1s K276 SUMOylation and subsequent impairing its nuclear translocation. U0126 could ameliorates diabetic cardiomyopathy by inhibiting Ras/MEK/ERK cascades to maintain XBP1s' nuclear translocation.