LMK235 ameliorates inflammation and fibrosis after myocardial infarction by inhibiting LSD1-related pathway

Fig. 1. HDAC5 was gradually upregulated and underwent phosphorylation-dependent nuclear export post-MI. A-D, Western blot results and statistical analysis of LAP-TGF-β1, HDAC5 and p-HDAC5 (S498) in noninfarcted hearts and infarcted left ventricles 7, 14, and 21 days post-MI (n = 6 for each group). The band of LAP-TGF-β1, HDAC5 and GAPDH were from the same gel, and the band of p-HDAC5 (S498) were from the other gel. E, Immunofluorescence analysis of HDAC5 in noninfarcted heart tissue and the border zone of infarcted heart tissue. The data are presented as the mean ± SD, ☨P<0.05 vs. sham group, *P < 0.01 vs. sham group. The samples derive from the same experiment and that blots were processed in parallel. The original blots are presented in Supplementary Figs. 5–9.

Fig. 2. LMK235 improved left ventricular dysfunction 21 days after MI. A-D, Representative images of M-mode echocardiography of rats in each group. E-G, Statistical analysis of LVESd, LVEDd and LVEF (n = 8 for sham group and sham + LMK235 group, while n = 10 for MI group and MI + LMK235 group). The data are presented as the mean ± SD, *P < 0.001 vs. the sham group, #P < 0.01 vs. the MI group.

Fig. 3. LMK235 attenuated chronic inflammation in the border zone 21 days after MI. A-B, Representative images of HE staining in each group at a magnification of 40×, and statistical analysis of inflammatory infiltration area (n = 3 for each group). C-G, Western blot results and statistical analysis of TNF-α, IL-6, cleaved-IL-1β and pro-IL-1β expression (n = 5 for sham group and sham + LMK235 group, while n = 7 for MI group and MI + LMK235 group). The band of TNF-α, cleaved-IL-1β and GAPDH were from the same gel, and the band of IL-6 and pro-IL-1β were from the other two gels. The data are presented as the mean ± SD, ☨P < 0.05 vs. the sham group, *P < 0.001 vs. the sham group, #P < 0.001 vs. the MI group. The samples derive from the same experiment and that blots were processed in parallel. The original blots are presented in Supplementary Figs. 10–16.

Fig. 4. LMK235 attenuated interstitial fibrosis in the border zone 21 days after MI, and LMK235 inhibited upregulation of LSD1 in the border zone 21 days after MI. A-D & F, Representative images of Masson’s trichrome staining in each group at magnifications of 40× and 100× and statistical analysis of the fibrotic area (n = 3 for each group). E & G-K, Western blot results and statistical analysis of collagen I, collagen III, LAP-TGF-β1 and LSD1 expression (n = 5 for sham group and sham + LMK235 group, while n = 7 for MI group and MI + LMK235 group). The band of collagen I, LAP-TGF-β1 and GAPDH (Fig. 4E) were from the same gel, the band of LSD1 and GAPDH (Fig. 4J) were from the same gel, and the band of collagen III were from the other gel. The data are presented as the mean ± SD, *P < 0.001 vs. the sham group, #P < 0.001 vs. the MI group. The samples derive from the same experiment and that blots were processed in parallel. The original blots are presented in Supplementary Figs. 17–23.

Fig. 5. LMK235 suppressed LPS-induced inflammatory macrophage polarization and inflammatory cytokine expression by inhibiting NF-κB pathway in RAW264.7 cells, while overexpression of LSD1 abrogated the anti-inflammatory effect of LMK235. A-B, Flow cytometry results and statistical analysis of the percentage of CD86+ macrophages (n = 4 for each group). C-J, Western blot results and statistical analysis of IL-6, TNF-α, LSD1, p-p65 and p65 expression in RAW264.7 cells in each group. n = 4 for each group. The band of IL-6 and GAPDH (Fig. 5C) were from the same gel, the band of TNF-α were from the other gel. The band of LSD1, t-p65 and GAPDH (Fig. 5F) were from the same gel, and the band of p-p65 were from the other gel. The data are presented as the mean ± SD, *P < 0.001 vs. the NC group, #P < 0.01 vs. the LPS group, ☨P < 0.01 vs. the LPS + LMK235 group. The samples derive from the same experiment and that blots were processed in parallel. The original blots are presented in Supplementary Figs. 24–32.

Fig. 6. LMK235 suppressed TGF-β1-induced fibroblast-myofibroblast transformation by inhibiting Smad2/3 pathway in MCFs, while overexpression of LSD1 abrogated the antifibrotic effect of LMK235. A‒H, Western blot results and statistical analysis of collagen I, collagen III, α-SMA, LSD1, p-Smad2, Smad2, p-Smad3 and Smad3 expression in MCFs in each group. n = 4 for each group. The band of collagen I, α-SMA and α-tublin (Fig. 6A) were from the same gel, the band of collagen III were from the other gel. The band of LSD1, t-Smad2 and α-tublin (Fig. 4J) were from the same gel, and the band of p-Smad2, p-Smad3 and t-Smad3 were from the other three gels. The data are presented as the mean ± SD, *P < 0.001 vs. the NC group, #P < 0.01 vs. the TGF-β1 group, ☨P < 0.01 vs. the TGF-β1 + LMK235 group. The samples derive from the same experiment and that blots were processed in parallel. The original blots are presented in Supplementary Figs. 33–46.