Inhibiting the Histone Demethylase Kdm4a Restrains Cardiac Fibrosis After Myocardial Infarction by Promoting Autophagy in Premature Senescent Fibroblasts

Figure 1. Premature senescent fibroblasts are elevated and persistent in the heart post‐MI A. A tSNE projection of cardiac cells analyzed by scRNA‐seq. Cells are colored according to the 8 major cell types. B. Heatmap illustrating the AUC scores of the SIPS gene set across different cell types, as projected on the tSNE plot. C. Violin plot illustrating the AUC scores of the SIPS gene set across various time points. D. Western blot analysis of the protein levels p16 and p21 in adult hearts after MI and in normal adult hearts. (n = 6/group) E‐F. Representative immunohistochemical staining and densitometric analysis of p16 and p21 in MI heart tissue compared to normal heart tissue. (n = 6 /group; scale bars = 100 µm) G. Representative images of Masson's trichrome staining and SA‐β‐gal staining in adult hearts harvested at 14 days post‐MI and in normal adult hearts. (n = 4 /group; scale bars = 100 µm) H. Box plot depicting the AUC scores of the SIPS gene set across various cell types. I. Bar plots showing the number of the eight‐cell types within the total SIPS cells at different time points. J. Line plot depicting the count of stress‐induced premature senescent cells and fibroblasts over…

Figure 2. Kdm4a is upregulated in premature senescent fibroblasts after MI and H2O2‐induced fibroblasts A. Box plot depicting the AUC scores of the histone methylation and histone demethylation across various cell types. B. Dot plots showing the expression of Kdm family members in different fibroblast cell types. C. Heatmap showing the differential expression of Kdm family members between sham and MI (GSE183272). D‐G. Western blotting was used to detect the cardiac expression of Kdm4a and the relative levels of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were quantified in different groups. (n = 6/group) H. Representative images of Masson's trichrome staining and immunohistochemical staining of Kdm4a in different regions after MI. (n = 6 /group; scale bars = 100 µm) I‐J. Western blot analysis of Kdm4a protein levels in cardiomyocytes (CMs), fibroblasts (FBs) and endothelial cells (ECs). The cells were isolated from the hearts of mice with MI. (n = 6/group) K‐L. Western blot analysis of Kdm4a protein levels in isolated fibroblasts from the hearts of mice with MI and in control fibroblasts. (n = 6/group) M. Immunofluorescence staining analysis for CD31, vimentin and cTnT costaining to…

Figure 3. Knockdown of Kdm4a improved cardiac function and cardiac fibrosis after MI in adult mice A‐B. Cardiac function was assessed by echocardiography at pre‐MI and 1, 14, and 28 days after MI. (n = 10 /group) C. Percentage of cardiac rupture between mice injected with AAV9‐NC or AAV9‐shKdm4a post‐MI. (n = 20/group) D. The myocardium of three different zones in mice was harvested on day 14 post‐MI for the following study. Representative images of heamatoxylin and eosin (H&E), picrosirius red staining, and Masson's trichrome staining in the mouse heart. (n = 6 /group; scale bars = 100 µm) E. Western blot analysis of α‐SMA, periostin, collagenI, and collagenIII and GAPDH expression in heart tissues of mice and quantification for the indicated proteins. (n = 6 /group) F. Representative immunohistochemical staining of α‐SMA, periostin, collagen I, and collagen III in transverse sections from adult mouse MI model hearts at 14 days after MI. (n = 6 /group; scale bars = 100 µm) Data are expressed as the means SD for each group. B was analyzed by the two‐way ANOVA followed by Tukey's test. C was analyzed by the log‐rank (Mantel‐Cox) test. E and F were analyzed by one‐way ANOVA followed by…

Figure 4. Deficiency of Kdm4a inhibits premature senescence of fibroblasts both in vitro and in vivo A‐B. Western blot analysis of p16, p21, p53 and γH2AX in cardiac tissue from mice harvested at 14 days post‐MI in each group. (n = 6 /group) C‐D. Representative images of SA‐β‐gal assay in the left ventricle of hearts harvested at 14 days post‐MI in different groups. (n = 6 /group; scale bars = 100 µm). E‐G. Representative densitometric analysis and immunostaining micrographs of senescence‐associated markers p16 and p21 in MI model hearts at 14 days post‐MI. (n = 6 /group; scale bars = 100 µm) H‐I. The mouse cardiac fibroblasts were pre‐treated with si‐Kdm4a and then exposed to H2O2 (100 µM) for an additional 96 h, and then the cells were analyzed. Western blotting and densitometric analysis of the protein levels of γH2AX, p16, p21 and p53 in each group. (n = 6 /group) J‐K. Representative immunofluorescence images for p16 in fibroblasts. Fibroblasts are stained with vimentin, nuclei are labelled with DAPI. (scale bars = 50 µm; n = 6/group) L‐M. Cytochemical evaluation of the SA‐β‐gal activity in fibroblasts and the percentage of positive fibroblasts was quantified. (scale bars = 200 µm; n =…

Figure 5. Kdm4a deficiency inhibits H2O2‐induced cardiac fibroblast activation and extracellular matrix (ECM) production A. Schematic diagram of transwell support coculture experiments to assess the effect of H2O2‐induced senescent fibroblasts on normal fibroblasts. B. Western blotting of the protein levels of fibrotic markers α‐SMA, periostin, collagen I, and collagen IIII in cultured fibroblasts transfected with siRNA against Kdm4a (si‐Kdm4a). (n = 6 /group) C–E. Representative immunofluorescence images for α‐SMA (red) (C), collagen I (red) (D), and collagen III (red) (E) in fibroblasts. Fibroblasts are stained with vimentin, nuclei are labeled with DAPI. (scale bars = 20 µm) F‐G. Representative images showing the scratch wound assay in fibroblasts transfected with si‐Kdm4a. Images of the wounds were captured at 0 and 24 h after the assay. The area between the wound edges was measured and compared between the groups. (scale bars = 200 µm; n = 6 /group) H‐I. Collagen gels containing fibroblasts were photographed at 0, 24, and 48 h. The sizes of the gels were measured and analyzed to assess changes in gel. Data are expressed as the means SD for each group. B and G were analyzed by one‐way…

Figure 6. Kdm4a knockdown suppresses Trim44 expression by directly enhancing H3K9me3 deposition A. Volcano plot illustrating differentially expressed genes in adult fibroblasts incubated with H2O2+si‐NC or H2O2+si‐Kdm4a. A |log2FC|>1 and a p value <0.05 were considered indicative of significant differential expression. B‐C. GO and KEGG pathway enrichment analysis of differentially expressed genes after Kdm4a knockdown. The x‐axis indicates the gene count, and the y‐axis specifies the GO or KEGG terms. D. Western blot analysis was performed to show the global changes of histone methylation modifications induced by Kdm4a knockdown in fibroblasts. (n = 6 /group) E. Schematic diagram to illustrate the workflow of H3K9me3 ChIP‐seq experiments. F. Bar chart showing the genomic distribution of H3K9me3 binding peaks in H2O2‐treated fibroblasts transfected with either si‐NC or si‐Kdm4a, including promoters, 5′ UTR, 3′ UTR, exons, introns, and intergenic regions. G. Normalized ChIP‐seq density was assessed in H2O2‐treated fibroblasts transfected with either si‐NC or si‐Kdm4a. The signal intensities were ranked according to H3K9me3 read density within ±3 kilobases of the peaks relative to the…

Figure 7. Kdm4a knockdown attenuates fibroblast senescence by regulating Trim44‐mediated autophagy A. Western blot analysis and quantification of the expression of LC3B‐II, Beclin1, and p62 protein expression in H2O2‐treated fibroblasts after si‐Trim44 and inhibition of autophagy with 3‐MA. GAPDH was used as a loading control. (n = 4/group) B. H2O2‐treated fibroblasts were incubated with si‐Trim44 and 3‐MA and then transfected with mCherry‐GFP‐LC3 adenovirus. LC3 spots were captured by confocal microscopy. (scale bars = 20 µm) C. Western blot analysis and quantification of the expression of p16, p21, and p53 protein levels in H2O2‐treated fibroblasts after si‐Trim44 and inhibition of autophagy with 3‐MA. GAPDH was used as a loading control. (n = 4/group) D. Immunofluorescence staining and quantification of p16‐positive fibroblasts to show the effects of Trim44 knockdown on the regulatory role of 3‐MA in fibroblast senescence. (scale bars = 50 µm) E. Western blot analysis and quantification of the expression of p16, p21, and p53 protein levels in H2O2‐treated fibroblasts after OE‐Trim44 and induction of autophagy with rapamycin. GAPDH was used as a loading control. (n = 4/group) F.…

Figure 8. Kdm4a knockdown prevents fibroblast activation by inhibiting SASP secretion A. TGF‐β1 was detected by enzyme‐linked immunosorbent assay (ELISA). ELISAs were performed on conditioned medium collected from fibroblasts incubated in OE‐NC + si‐NC, OE‐Kdm4a+ si‐NC, OE‐NC + si‐Trim44 or OE‐Kdm4a+ si‐Trim44. (n = 6/group) B‐C. Western blot analysis and quantification of α‐SMA, periostin, collagen I and collagen III protein levels in fibroblasts with or without exogenous rTGF‐β1 treatment. (n = 4/group) D‐E. Western blot analysis and quantification of α‐SMA, periostin, collagen I and collagen III protein levels in fibroblasts cocultured with H2O2‐induced fibroblasts with vehicle or TGF‐β1 neutralising antibody. (n = 4/group) F‐G. Representative images of α‐SMA and collagen I immunostaining in fibroblasts which were cocultured with H2O2‐induced fibroblasts incubated with vehicle or TGF‐β1 neutralising antibody. (scale bars = 20 µm) H‐I. Western blot analysis and quantification of α‐SMA, periostin, collagen I and collagen III protein levels in fibroblasts cocultured with H2O2‐induced fibroblasts with TGF‐β1 neutralising antibody+OE‐NC or TGF‐β1 neutralising antibody+OE‐Kdm4a. (n = 4/group)…