A DUSP6 inhibitor suppresses inflammatory cardiac remodeling and improves heart function after myocardial infarction

Figures

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  Fig. 1.. BCI ameliorates cardiac injury in post-MI rats. (A) Experimental scheme showing the timing of (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) injections, myocardial infarction (MI) and echocardiography (ECHO) of rats. i.v., intravenous. (B) Representative M-mode tracings from ECHO of sham-operated and vehicle- or BCI-treated groups at 4 weeks post-MI. (C) The BCI treatment group showed improved ejection fraction and fractional shortening compared with the vehicle treatment group, and partially rescued heart function compared with the sham group (n=9–10). (D) BCI treatment improved left ventricle (LV) volumes and LV end-diastolic diameter (LVEDD) compared with vehicle treatment (n=9–10). (E) Masson's trichrome staining showing less fibrosis in BCI-treated hearts than in control hearts at 28 days post-MI (scale bar: 3 mm, n=9–10). (F) High-magnification images of Masson's trichrome and H&E staining showing decreased fibrosis in BCI-treated hearts at 28 days post-MI (scale bar: 300 μm, n=9–10). (G) Quantification of the Masson's trichrome-stained fibrotic area in sham-operated, vehicle-treated and BCI-treated hearts at 28 days post-MI (n=9–10 per group). (H)…

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  Fig. 2.. BCI has no effect on CM proliferation, H2O2-induced CM death, CM hypertrophy and coronary vessel regeneration. (A) Released lactate dehydrogenase (LDH) was comparable in neonatal rat ventricular myocytes (NRVMs) treated with DMSO or BCI for 24 h in the presence or absence of H2O2 (n=3 per group). (B) Western blots and quantification of Bcl-2 (α-tubulin was used to normalize protein) and p-AKT (AKT was used to normalize protein) in NRVMs treated with DMSO or BCI for 24 h (n=3 per group). (C) Western blots and quantification of H2O2-induced cleaved PARP (α-tubulin was used to normalize protein) and cleaved caspase 3 (total caspase 3 was used to normalize protein) in NRVMs treated with DMSO or BCI (n=3 per group). (D) Immunofluorescent staining showing cTnT+ and pH3+ CMs of sham-operated, vehicle-treated or BCI-treated LV tissues at 7 days after MI (scale bar: 100 μm). (E) Immunofluorescent staining showed that the numbers of either α-actinin+ Ki67+ or α-actinin+ pH3+ CMs were comparable in DMSO- and BCI-treated NRVMs (scale bars: 50 μm; n=3 per group). HPF, high-power field. (F) Immunofluorescent staining and statistics for CD31+ and α-SMA+ vessels of sham-operated, vehicle-treated…

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  Fig. 3.. BCI inhibits macrophage recruitment in infarcted rat hearts and LPS-induced mouse abdominal macrophages. (A) Experimental scheme showing the timing of BCI injections and MI in rats. (B,C) Percentages of His36+ macrophages in the LV of rats at 7 days post-MI by flow cytometry (B) and statistics of His36+ macrophages (C) (n=3 per group; two hearts per sample). (D) Immunofluorescent staining showing decreased numbers of His36+ macrophages (red) in the LV of rats after BCI treatment at 7 days post-MI compared with vehicle treatment (scale bar: 50 μm). (E) The percentage of His36+ macrophages to DAPI+ cells in each group (n=5 per group). (F) The expression levels of Il1b, Il6, Il12b, Retnla, Arg1 and Ym1 mRNAs were determined in sham and MI hearts (day 7) with or without BCI treatment (n=3–4 per group). (G,H) Percentages of lipopolysaccharide (LPS)-induced CD11b+ (also known as ITGAM+) F4/80+ macrophages in mouse abdominal cells were measured by flow cytometry (G), and statistics of CD11b+ F4/80+ cells are shown (H) (n=3 per group; two mice per sample). (I) LPS-induced IL-1β, IL-6 and IL-12 proteins in mouse serum detected by ELISA (n=5 mice per group). One-way ANOVA followed by…

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  Fig. 4.. BCI inhibits differentiation of BMCs into macrophages. Bone-marrow cells (BMCs) were isolated and cultured from the femurs and tibias of 6-week-old wild-type Sprague-Dawley rats. (A) Bright-field images of BMC-derived macrophages, and the numbers of adherent macrophages in each HPF after 7-day induction by M-CSF in the presence of DMSO or BCI (scale bar: 100 μm; n=3 per group). (B) Numbers of adherent macrophages in each HPF at different time points of BCI treatment (n=3 per group). (C) Expression levels of Egr1, Grb2 and Mnda mRNAs in BMCs at day 3 after M-CSF induction with DMSO or BCI treatment (n=3 per group). (D) Western blots showing DUSP6 expression in M-CSF-induced bone-marrow-derived macrophages (BMDMs) from wild-type (WT) or Dusp6 mutant rats (n=3 per group). (E) Bright-field images of BMC-derived macrophages and the numbers of adherent macrophages in each HPF after 7-day induction by M-CSF from WT or Dusp6 mutant rats (scale bar: 100 μm; n=3 per group). (F) Expression levels of Egr1, Grb2 and Mnda mRNAs in BMDMs at day 3 after M-CSF induction from WT or Dusp6 mutant rats (n=3 per group). (G) RNA-seq showing differential gene expression in myeloid cells at 3 days after…

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  Fig. 5.. BCI attenuates inflammation through the p38–NF-κB signaling pathway. BMDMs were stimulated by LPS in the presence of DMSO or BCI, and the treated cells were used for qRT-PCR, immunostaining and western blotting. (A) qRT-PCR showing expression levels of LPS-induced Inos, Il1b, Il6, Il12b and Cd14 mRNAs in BMDMs with DMSO or BCI (n=3 per group). (B) qRT-PCR showing expression levels of LPS-induced Mcp1, Ccl4, Ccr2 and Cxcl9 mRNAs in BMDMs with DMSO or BCI (n=3 per group). (C) Western blots and quantification of LPS-induced p-p65 and p65 in BMDMs with DMSO or BCI treatment (n=3 per group). (D) Immunofluorescent staining and quantification of LPS-induced p-p65 (green) in BMDMs with DMSO or BCI treatment (n=3 per group; scale bars: 20 μm). (E) Immunofluorescent staining and quantification of LPS-induced reactive oxygen species measured by dihydrorhodamine 123 (DHR123) (green) in BMDMs with DMSO or BCI treatment (n=3 per group; scale bars: 20 μm). (F-H) Western blots and quantification of LPS-induced p-ERK (F), p-p38 (G) and p-JNK (H) in BMDMs with DMSO or BCI treatment (n=3 per group). One-way ANOVA followed by Dunnett's multiple comparison test; mean±s.e.m.; *P<0.05, **P<0.01,…

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  Fig. 6.. Intramyocardial injection of PLGA-encapsulated BCI improves cardiac function post-MI in rats. (A) Poly (D, L-lactic-co-glycolic acid) (PLGA) microspheres under scanning electron microscopy (scale bar: 5 μm). (B) In vivo fluorescence imaging of rats 4 weeks after intramyocardial injection of normal saline (control), 1,1'-dioctadecyl-3,3,3′,3'-tetramethylindotricarbocyanine iodide (DiR) or PLGA-encapsulated DiR (n=3 per group). (C) Imaging of hearts 4 weeks after intramyocardial injection of normal saline (control), DiR or PLGA-encapsulated DiR (n=3 per group). (D) Representative M-mode tracings from ECHO of control, PLGA-treated and PLGA+BCI treated rat hearts at 4 weeks after MI. (E) Statistical analysis of ejection fraction and fractional shortening of control, PLGA-treated and BCI+PLGA-treated rat hearts 1 day before MI, and on days 1, 7, 14 and 28 after MI (n=8–9). (F) Masson's trichrome staining of control, PLGA-treated and BCI+PLGA-treated hearts at 28 days post-MI. Scale bar: 3 mm. (G) Quantification of Masson's trichrome-stained fibrotic area in F (n=8–9). (H) Masson's trichrome staining and H&E staining of control, PLGA-treated and BCI+PLGA-treated hearts at 28 days…