sdc4/Sdc4 expression profile analyses between zebrafish and mice after heart injury. A The zebrafish ventricles were collected at different time points after cryoinjury for microarray analysis: 0, 6, 24, 48, 72 and 120 hpci. (NCBI GEO accession number: GSE94617) (hpci: hours post cryoinjury). B The zebrafish ventricles were collected at different time points after ventricular resection for microarray analysis: 0, 0.25, 1, 3, 6, 10, 15, 21 and 28 dpa. (NCBI GEO accession number: GSE72348) (dpa: days post amputation). C The mice ventricles were collected at different time points after injury for microarray analysis: 0, 0.25, 1, 4, 12, 24 and 48 hpi. (NCBI GEO accession number: GDS2331) (hpi: hours post injury). D The zebrafish ventricles were collected at different time points after ventricular cryoinjury for real-time quantitative PCR (RT-qPCR): 0, 0.25, 1, 3, 5 and 7 dpci. (*p < 0.05 vs. 0 dpci; n = 6 per group) (dpci: days post cryoinjury)

Knockdown of sdc4 expression decreased the scar deposition of zebrafish hearts. Acid fuchsin orange G (AFOG) staining of heart sections was used to visualize fibrin (red), myocardium (light orange), and collagen (blue). Dashed line: post-cryoinjury area. Data showed scar deposition both in the negative control group and sdc4 siRNA group at 3 dpci (A) and 7 dpci (C). The hearts of the negative control group contained abundant collagen at the injury area at 7 dpci. However, in the sdc4 siRNA group, the scar was nearly completely resolved and replaced with new muscle. The statistical results showed that the scar deposition percentage of zebrafish hearts after cryoinjury did not change at 3 dpci (B) but significantly reduced in the sdc4 siRNA group compared to the negative control group at 7 dpci (D). Scale bar: 200 μm (****p < 0.001 vs. negative control; n = 3–5 per group) (dpci: days post cryoinjury)

Inhibition of sdc4 reduced the recruitment of innate immune cells to the injury site after cryoinjury. A–H The whole mount view of post-cryoinjury ventricles at 12, 24, 36 and 96 hpci. White: endothelial cells (kdr:EGFP); Red: myeloid cells (lyz:DsRed). I Quantitative analysis showed the cell numbers of myeloid cells at the injury site at each time point. The silence of sdc4 slightly decreased the aggregation of myeloid cells at the injury site at 12 and 24 hpci and significantly reduced it at 96 hpci. Scale bar: 100 μm (*p < 0.05 vs. negative control) (hpci: hours post cryoinjury)

sdc4 inhibition resulted in a decrease in cell proliferation at the injury site. Expression patterns of PCNA (red) and Hoechst (blue) immunostaining were analyzed after cryoinjury. PCNA (proliferating cell nuclear antigen) was used as a marker for cell proliferation, and Hoechst was used to label nuclei. Dashed line: injury site. Double-headed arrow: compact myocardium. A–F The PCNA signals of the negative control group were significantly higher than the sdc4 siRNA group at 3 dpci. G–L At 7 dpci, the expression levels of PCNA were the same at the injury site in both groups. Moreover, the Hoechst signals of the sdc4 siRNA group were weaker at the injury site than the negative control group. M The statistics indicated the cell numbers of PCNA-positive cells at the injury site. Knockdown of sdc4 significantly reduced the cell proliferation at the injury site at 3 dpci, but no significant difference compared to the negative control at 7 dpci. Scale bar: 100 μm (*p < 0.05 vs. negative control; n = 2–4 per group) (dpci: days post cryoinjury)

sdc4 expression affected ECM-associated marker genes during the cardiac repair. Extracellular matrix (ECM)-associated marker genes: tgfb1a, col1a1a, mmp2, and mmp9 were examined by real-time quantitative PCR with or without sdc4 siRNA treatment at 3 and 7 dpci. A After treating sdc4 siRNA, the sdc4 gene expression was down-regulated as expected. Besides, tgfb1a and col1a1a were significantly down-regulated after sdc4 siRNA treatment at 3 dpci. B At 7 dpci, expression of sdc4, tgfb1a, and col1a1a were all significantly reduced in the sdc4 siRNA group. However, mRNA expression of mmp2 was significantly up-regulated after the sdc4 knockdown (*p < 0.05 vs. negative control; ***p < 0.005 vs. negative control; n = 8 per group) (dpci: days post cryoinjury)

sdc4 knockdown inhibited Fibronectin expression at the injury site. Immunofluorescence staining of heart sections after cryoinjury was performed to visualize MF-20 (red), Fibronectin (green), and Hoechst (blue). MF-20 is a myosin heavy chain labeled myocardium. Hoechst labeled nuclei. Dashed line: injury site. A–D In negative control group at 3 dpci, MF-20 signals at the injury site were absence. Fibronectin signals were observed in the margin of the infarct area and partially expressed at the injury site. The large DNA fragments were detected at the injury site labeled by Hoechst. (*labeled the strong signals of Hoechst). E–H In the sdc4 siRNA group at 3 dpci, the signals of Fibronectin were nearly undetectable. Also, the Hoechst signals were absent at the injury area. I–L In the negative control group at 7 dpci, the Fibronectin signals were more highly expressed than 3 dpci of the negative control group at the injury area. M–P In the sdc4 siRNA group at 7 dpci, the reduction of Fibronectin signals was observed compared to the negative control group. Scale bar: 100 μm (dpci: days post cryoinjury)

Pathological Q wave and ST elevation were observed after sdc4 siRNA treatment. The zebrafish electrocardiography (ECG) with or without sdc4 siRNA treatment was analyzed at control, 1, 3, and 7 dpci after cryoinjury. Two ECG signs of myocardial infarction (MI) were analyzed: pathological Q wave and ST elevation. A, Bsdc4 knockdown group showed significantly augmented pathological Q wave and slight ST elevation compared to the negative control group, which suggested sustained cardiac conduction abnormalities. C, D Representative electrocardiogram figures of the pathological Q wave and ST elevation at 1 and 3 dpci in the negative control group and sdc4 siRNA group. (*p < 0.05 vs. negative control; **p < 0.01 vs. negative control; n = 3–7 per group) (dpci: days post cryoinjury)