Fig 2: BMSCs osteogenesis analysis in vitro. A The ALP staining and B activityon the 4th and the 7th day (n = 3); C the Alizarin red S staining was carried out on the 14th day; D the relative mRNA expressions of Runx2 and Alp in BMSCs cultured on different hydrogels for 4 and 7 days, while Ocn and Opn for 7 and 14 days (n = 3); E the Runx2 immunofluorescent staining of BMSCs cultured on different hydrogels for 4 days and 7 days and the Opn for 7 days and 14 days (n = 3). The scale bar is 200 µm. ***p < 0.001, **p < 0.01, *p < 0.05

Fig 3: The SUV39H1/SPP1 axis modulates cardiac function and myocardial function in rats. (a) Protein levels of SUV39H1 and SPP1 in rat myocardial tissues examined by the IHC assay (n = 8). (b) LVEF and LVFS values of MI rats as manifestations of myocardial function (n = 8). (c) The cardiac remodeling in rats examined by HW/TL and LV+RV/TL values (n = 8). (d) Infarct area in the cross section of LV examined by Masson's trichrome staining (n = 8). (e) Percentage of the perimeter of infarcted endocardium in total endocardium (n = 8). (f) Pathological changes in rat myocardium determined by HE staining (n = 8). The si-NC (small interfering RNA-negative control) and the si-SUV39H1 groups refer to the H9C2 cells transfected with si-NC or si-SUV39H1. Differences were analyzed by the one-way ANOVA. *p < 0.05 vs. the si-NC group; #p < 0.05 vs. the si-SUV39H1 group.

Fig 4: Time course and quantitative analysis of osteogenic gene expression. (A,B) Time course of pooled RNA samples from four different donors were screened by a NanoString® kit for 66 (mainly osteogenic) genes after 1, 3 or 5 weeks of control or osteogenic condition. Week timepoints are shown as W1 (red), W3 (yellow), W5 (purple). Meanings: 2D = adherent cell cultures, 3D = Col I gel cultures, U = Undifferentiated cells in control medium, and O = differentiated cells in osteogenic medium. (A) Time course of the three most upregulated out of sixty-six tested genes: SPP1, MMP13, COL10A1. (B) Time course of the three most downregulated out of sixty-six tested genes: COL5A1, COL14A1, FGF2. (C) Multivariate analysis of the sixty-six tested genes, after 5 weeks of differentiation, displayed as a volcano plot (created with nSolver® software) with the differentiation set as covariate. The plot shows each gene’s -log10 (p-value) and log2 fold change with the differentiation set as covariate. Highly statistically significant genes fall at the top of the plot above the indicated p-value lines, and highly differentially expressed genes fall to either side (right = upregulated, left = downregulated). The most significantly (p-value < 0.01) upregulated genes are highlighted in the green circle (COL10A, COL7A1, SPP1, MMP13), and the most significantly (p-value < 0.01) downregulated genes are highlighted in the blue circle (COL15A1, COL14A1, COL12A1, FGF2).

Fig 5: Lcz696 regulates the SUV39H1/SPP1 axis in MI rats. (a) Volcano plots for DEGs in MI rats in the GSE47495 dataset. (b) Biofunctional processes the DEGs-enriched analyzed by GO functional enrichment analysis. (c) mRNA expression of SUV39H1, SPP1, and THBS1 in rat cardiac tissues examined by RT-qPCR (n = 8). (d) Histone modification peaks at the SPP1 promoter in LV obtained from the ENSEMBL system. (e) Protein levels of SUV39H1, H3K9me3, and SPP1 in H9C2 cells determined by Western blot analysis (n = 3). (f) Enrichment of SUV39H1 and H3K9me3 fragments in SPP1 promoter examined by the ChIP-qPCR (n = 3). Differences were analyzed by the unpaired t-test and one-way ANOVA or two-way ANOVA. The sham group refers to rats underwent sham operation without LAD (left anterior descending) artery ligation; the MI (myocardial infarction) group refers to the model group where rats induced with MI via LAD artery ligation; the lcz69l group refers to rats with MI treated with lcz696; and the si-NC (small interfering RNA-negative control) and the si-SUV39H1 groups refer to the H9C2 cells transfected with si-NC or si-SUV39H1. *p < 0.05 vs. the sham group/control group/si-NC group; #p < 0.05 vs. the MI group.