Transforming growth factor-β (TGF-β, 763104) was purchased from Biolegend (San Diego, CA, USA). Isoprenaline (ISO, I5627) was obtained from Sigma-Aldrich and prepared in DD water. The SMOC2 (SC-376104, 1:500 for western blot and 1:100 for satining) and ILK (SC-20019, 1:500) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies against collagen type I (Col I, 1:1000), collagen type III (Col III, 1:1000), phospho-JNK (Tyr185,1:1000), JNK (1:2000), phospho-p38 MAPK (Thr180/Tyr182,1:1000), t-p38 (1:1000), phospho-AKT (1:5000), t-AKT (1:5000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH,1:4000) were obtained from Proteintech (Wuhan, China). Anti-α smooth muscle actin (a-SMA, 1:100 for staining) antibody was purchased from Abcam (Cambridge, UK).

All animal experimental procedures were carried out under the supervision of the Animal Care and Use Committee of Renmin Hospital of Wuhan University and in compliance with established guidelines published by the National Institutes of Health of United States. Eight-week-old male C57BL/6 mice (body weight 23.5 ± 2 g) were provided by the Experimental Animal Center of the Three Gorges University (Hubei, China) for this study, and were acclimated to the experimental scenarios for 5–7 days before they were used. Under standard environmental conditions, all animals were fed a pellet diet and had access to water without restrictions. Mice were subcutaneously injected with ISO (10 mg/kg for 3 days and 5 mg/kg for 11 days) to induce cardiac fibrosis (25–27). For the control group, an equal volume of normal saline solution was administered. Four weeks before ISO injection, mice received a single intravenous injection of adeno-associated virus 9 (AAV9) carrying small hairpin RNA against SMOC2 (sh-SMOC2) and its corresponding negative control (shRNA) generated by Obio technology (Shanghai, China) to verify its role in vivo. Animals were randomly assigned to four groups: Control + sh-RNA, Control + sh-SMOC2, ISO + sh-RNA, and ISO + sh-SMOC2. Animals were sacrificed after an excessive inhalation of CO₂, having their hearts and tibias harvested and measured to calculate the heart weight (HW)/body weight (BW) and HW/tibia length (TL).

Following modeling, echocardiographic evaluations were conducted under light anesthesia with a Vinno 6th ultrasound device with Doppler imaging (VINNO6, Vinno Corporation, China). At the level of the left ventricular short-axis papillary muscle, M-mode tracking was used to measure the following parameters: left ventricular end-systolic diameter (LVEDs), left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall thickness (LVPW), interventricular septal thickness (IST), ejection fraction (EF), and left fractional ventricular shortening (FS).

A mixture of trypsin and collagenase II was used to extract CFs from the hearts of 1–3 days old mice. CFs were cultured in a medium with 10% FBS (fetal bovine serum) at 37°C with 5% CO2 after removing non-adherent cells after 1 h. Non-adherent cells were removed after 1 h, and the adherent CFs were cultured in DMEM/F12 medium containing 10% FBS (fetal bovine serum) at 37°C with 5% CO2. The following experiments were conducted with 2–3 passages of CFs. Following 8 h of serum-free DMEM/F12 culture, cells were stimulated with TGFβ. In order to knock down the SMOC2, CFs were transfected with short hairpin (sh)RNA adenovirus against SMOC2 performed by Shanghai Jikai Gene Chemical Technology Co., Ltd. As a negative control, adenovirus expressing short hairpin (sh)GFP was used. To confirm the role of ILK, CFs were transfected with adenovirus carrying ILK (Ad-ILK) or green fluorescent protein (Ad-GFP).

Commercial ELISA kits (collagen I and collagen III ELISA kits from Uscnlife, Wuhan, China) were used to measure collagen I and III content in the cell culture supernatant after cardiac fibroblasts were incubated with or without TGFβ.

Heart tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin before being cut into 5-micron sections. Heart tissue morphology and cardiac fibrosis were assessed with hematoxylin and eosin (HE) and Masson staining, respectively. Quantitative analysis was performed using Image J software.

CFs were stained with immunofluorescence in a well-cultured condition. Briefly, cell permeabilization was enhanced after fixation with 4% paraformaldehyde for 20 min, followed by incubation with 50–100 μl of disruption buffer for 10 min at room temperature. Cell coverslips were incubated with anti-α-SMA and anti-SMOC2 overnight at 4°C. The next day, cells were incubated with goat anti-mouse (SMOC2) pre-adsorbed or goat anti-rabbit (α-SMA) pre-adsorbed secondary antibodies for 1 h. Nuclei were subsequently stained with 4′,6-diamino-2-phenylindole (DAPI) and images were taken with a fluorescence microscope.

The heart tissues or cells were prepared to extract total proteins with RIPA lysis buffer (Servicebio, Wuhan, China). The extracts were resolved via 8 and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore Corp. Bedford, MA, USA) using standard protocols. And then, the membranes were incubated with skim milk at room temperature for 1 h. Membranes were incubated with specific primary antibodies at 4°C overnight, followed by bio-tinylated secondary antibodies (goat anti-mouse HRP and goat anti-rabbit HRP) for 2 h at room temperature. The membranes were washed with TBST three times between each step mentioned above. Finally, the blots were detected by a hypersensitive ECL reagent (Biosharp, Beijing, China).

Total RNA was extracted using TRIzol reagent (Servicebio, Wuhan, China), and a cDNA synthesis kit was carried out to synthesize cDNA following the manufacturer’s protocol. Real-time PCR was carried out using EnTurboTM SYBR Green PCR SuperMix Kit (ELK Biotechnology). The mRNA levels were normalized to GAPDH. Molecular biological tests were conducted by individuals who were unaware of the treatment conditions of the animals and cells.

SPSS 26.0 software was used for analysis. All results are presented as mean ± standard error of the mean, and t-test was used for comparison between two groups, and one-way ANOVA was used for three or more groups, followed by post L-S-D test. Differences were considered statistically significant with p < 0.05.

We first investigated whether SMOC2 expression was altered in ISO-induced mouse model. Mice were treated daily with ISO or an equal volume of saline as control for 21 days as described. It was found that SMOC2 protein levels increased markedly in the ISO group, as was the accumulation of collagen types I and III (Figures 1A–D). Notably, RT-PCR further revealed increased expression level of SMOC2 in the ISO group (Figure 1E). The above observations strongly implicate that SMOC2 may play a critical role in regulating ISO-induced myocardial remodeling.

We next sought to explore the specific effects of SMOC2 in ISO-induced cardiac fibrosis. Considering the up-regulation of SMOC2 expression in the ISO-induced mouse model, we constructed a SMOC2-knockdown AAV9 system to infect mice as previously described.

As shown in Figures 2A–C, hearts of animals administered sh-SMOC2 showed a significant decrease in SMOC2 mRNA and protein expression after 4 weeks compared to hearts of mice treated with sh-RNA. Mice infected with sh-RNA subjected to ISO injection for 3 weeks developed cardiac hypofunction, manifested as a decrease in EF, FS, and elevation in LVEDd, LVEDs, HW/TL, and HW/BW (Figures 2D–J). Notably, cardiac function was partially reversed in mice injected with sh-SMOC2 (Figures 2D–J). Neither ISO administration nor infection with sh-SMOC2 affected ventricular wall thickness, however (Figures 2K, L).

In addition, morphological examination revealed that mice experienced more severe myofibrillar disarrangement and fibrosis after 3 weeks of ISO injection (Figures 3A, B). Moreover, these histological changes weakened after SMOC2 deficiency (Figures 3A, B). To substantiate this further, we detected biomarkers of myocardial fibrosis. As expected, collagen I and collagen III protein and mRNA levels were down-regulated post-sh-SMOC2 injection (Figures 3C–G). These findings implied an underlying physiopathological impact of SMOC2 in modulating cardiac fibrosis, and SMOC2 knockdown in mice can partially reverse the myocardial function reduction and myocardial fibrosis in response to ISO stimulation in vivo.

Next, we went on to achieve further insight into the potential mechanisms accounting for our previous findings. Pre-existing research has identified that SMOC2 could activate downstream ILK (20, 28), which was reported to be involved in the fibrotic pathological process (21). Consequently, we focused on the expression of ILK. It was revealed that, compared with the control group, the ISO + shRNA injection group had a significantly higher ILK level, and, importantly, the effect of ILK production can be relieved by SMOC2 knockdown (Figures 4A, B). Given that the AKT and AMPK pathways have been implicated in the pathophysiology of fibrosis, we examined whether these pathways are involved in the process of SMOC2-medicated myocardial fibrosis. It was found that p-AKT, p-p38-MAPK, and p-JNK-MAPK were significantly elevated in the ISO + sh-RNA group, while the p-p38 was decreased in the ISO + sh-SMOC2 group (Figures 4A, D). Strikingly, knocking down SMOC2 did not affect the expression of p-JNK and p-AKT (Figures 4C, E and Supplementary Figures 1A–F). Collectively, these findings demonstrate that the ILK and p38 pathway is likely to participate in the ISO-induced cardiac fibrotic process in vivo.

To gain further insight into the regulation of SMOC2 in cardiac fibrosis, we conducted in vitro experiments subsequently. Neonatal myocardial fibroblasts were separated by a differential adherent as described to explore the role of SMOC2 in TGFβ-induced fibroblast activation. The results showed TGFβ induced fibroblasts activation in a dose-dependent and time-dependent manner. After being treated with TGF β (10 ng/ml) for 24 h, the protein concentrations of SMOC2 reached the highest (Figures 5A–F). Consequently, the cells were treated with TGFβ at 10 ng/ml for 24 h before being harvested in subsequent analysis.

In order to discuss the potential effects and mechanisms underlying the pro-fibrotic effect of SMOC2, we infected CFs with sh-SMOC2 or sh-RNA as control. As shown in Figures 6A–C, after being treated with TGFβ for 24 h, SMOC2 protein and mRNA levels increased. However, these changes could be relieved post sh-SMOC2 infection. Furthermore, these findings were confirmed by an immunofluorescence stain for SMOC2 (Figures 6D, F).

The up-regulation of α-SMA expression, as a hallmark marker for differentiated myofibroblasts, was examined using immunofluorescence staining. As anticipated, the level of α-SMA was elevated in TGFβ + shRNA group, while SMOC2 down-regulation reduced the level of α-SMA (Figures 6E, G). Further analysis revealed that knocking SMOC2 could decrease the proliferation of TGFβ-induced CFs, as indicated by wound-healing scratch experiments (Figures 6H, I). Further analysis implicated that the protein and mRNA levels of collagen I and collagen III increased in the TGFβ + shRNA group, while knockdown of SMOC2 expression significantly inhibited type I and type III collagen production (Figures 6J–N).

Following that, we measured the collagen production of fibroblasts in the media. In agreement with the results above, the protein levels of fibrotic biomarkers such as type I and Type III collagen in the medium were elevated in the TGFβ group, whereas SMOC2 knockdown could relieve these changes (Supplementary Figures 2A, B).

In order to explore the mechanisms of anti-fibrotic effects of sh-SMOC2, we also detected ILK, AKT, MAPK pathways. Consistent with the findings in vivo, the ILK level was elevated after being treated with TGFβ, which could be down-regulated by sh-SMOC2 administration (Figures 7A, B). In-depth studies also found that p-AKT, P-JNK, and p-P38 were significantly up-regulated, while the sh-SMOC2 infection significantly reduced p-p38 without affecting the phosphorylation levels of p-AKT and p-JNK (Figures 7A, C–E and Supplementary Figures 3A–F).

Next, we also verified whether ILK participated in the activation of p-p38 in CFs in vitro, and we infected CFs with Ad-ILK to overexpress ILK. In line with the above findings, the anti-fibrotic effect of sh-SMOC2 was knocked over after ILK overexpression, and the scratch experiment also suggested that ILK further promoted the migration of CFS and activated p-p38 (Figure 7F). To further confirm this notion, the expression of α-SMA by IHC staining was examined. The results also identified that Ad-ILK worsened the transformation of CFs (Figure 7G). Results of in vitro studies matched those of in vivo studies. The level of SMOC2 did not change due to overexpression of ILK (Figures 7H, I), but ILK, p-p38, collagen I and collagen III were significantly up-regulated (Figures 7H, J, K–M and Supplementary Figures 4A–D).