CTRP9 knockout (on a C57BL/6J background) mice, were generated by Shanghai Biomodel Organism Science & Technology Development Co., Ltd. STZ (MCE, USA, 55 mg/KG) in citrate buffer was injected intraperitoneally for five consecutive days to induce type 1 diabetes mellitus (T1DM) (Meng et al., 2023). Fasting glucose values above 16.7 mM were considered to be diabetes. After the mice were anesthetized with Pentobarbital Sodium (70 mg/kg, IP), animal tissues were retained for subsequent experiments. The Ethics Committee of Qiluhospital of Shandong University reviewed (KYLL-2022(ZM)-1300) and authorized all animal procedures performed with the Guide for the Care and Use of Laboratory Animals.

Mice were anesthetized with the inhalational anesthetic isoflurane and underwent echocardiography. The induction anesthetic dose was 2%, 0.2 mg/10 g, 400 mg/kg, and the maintenance anesthetic dose was 1.2%, 0.2 mg/10 g, 240 mg/kg. The operator is unaware of the animal grouping. The left ventricle function was evaluated using a Vevo2100 imaging system (VisualSonics, Toronto, Canada). The echocardiography parameters included left ventricular end-diastolic internal diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), fraction shortening (FS), early-to-late diastolic mitral flow velocities (E/A), and the ratio of early diastolic mitral inflow to mitral annular velocity (E/e’).

Hearts were prepared as 4 μm paraffin sections for subsequent staining procedures, including hematoxylin and eosin (H&E) staining as well as Masson’s trichrome staining. Anti-Collagen I (Abcam, ab34710, 1:200), Anti-Collagen III (Abcam, ab7778, 1:200), Anti-α-SMA (HUABIO, ET1607-53, 1:5000), and Anti-YAP (ABclonal, A19134, 1:200), Anti-CTRP9 (NOVUSBIO, NBP2-46834, 1:200) were applied to the paraffin sections at 4°C. Subsequent treatment included exposure to secondary antibodies (Gene Tech, GK600505) and hematoxylin staining. Three fields of view were selected for each sample, and the mean optical density was measured and then averaged. The resulting data represents one biological replicate. The results were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics Inc., USA).

Primary cardiac fibroblasts were isolated from the hearts of 3- to 5-day-old C57BL/6J mice. The neonatal mouse hearts were removed and sliced into tissue fragments in pre-cooled D-Hank’s solution. The fragments were then transferred into conical flasks containing type II collagenase (Solarbio) and incubated overnight at 4°C on a shaker. The next day, the fragmented heart tissue was digested in a 37°C water bath using an EDTA-free trypsin (Solarbio) digestion solution. The collected supernatant was inoculated into flasks for cell culture. After an incubation period of 2 h at 37°C with 5% CO₂, the fibroblasts attached to the flasks and the complete medium was subsequently substituted.

During the experiment, only cardiac fibroblasts of one to three generations were used. Primary cardiac fibroblasts were grown in complete DMEM (Gibco). The cells were preincubated with recombinant gCTRP9 (1 μg/mL) (Lei et al., 2021) for 2 h before they were exposed to a high-glucose environment (33.3 mM). Following a 48-h incubation period, the cells were harvested for analysis. CQ (1 mM) was administered 12 h before the end of the experiment to inhibit autophagy.

Transfection of cells with the YAP overexpression plasmid (Shandong Gene & Bio Co., Ltd.) (1,000 ng/mL) and the control plasmid was conducted using Lipofectamine™ 3000 reagent (Invitrogen) in Opti-MEM™ reduced serum medium (Gibco). Complete medium was added to replace the medium 8 hours after transfection.

To knock down CTRP9, small interfering RNA (siRNA) (shandong Gene&Bio) was transfected into CFS using Lipofectamine™3000 reagent (Invitrogen) in Opti-MEM™ reduced serum medium (Gibco). The medium was replaced with complete medium 6–8 h later, and small interference was screened by detecting the mRNA expression of CTRP9 24 h later.

Extracted protein samples were underwent separation through SDS-PAGE, followed by transfer onto PVDF membrane (Millipore). Then, the membranes underwent overnight with antibodies against Collagen I (Proteintech, 66761-Ig, 1:1000), Collagen III (Abcam, ab184993, 1:1000), α-SMA (HUABIO, ET1607-53, 1:5000), GAPDH (Proteintech, 60004-1-Ig, 1:10,000), p62 (Abcam, ab109012, 1:1000), LC3B (CST, 3868S, 1:1000), and YAP (ABclonal, A19134, 1:1000), CTRP9 (NOVUSBIO, NBP2-46834, 1:500). Subsequently, secondary antibodies (HUABIO, HA1006 and HA1001) were applied to the membranes, followed by visualization using an Amersham Imager 680.

Cardiac fibroblasts were sequentially treated with methanol on ice and 5% BSA. Subsequently, the cells were exposed to anti-α-SMA (HUABIO, ET1607-53,1:500) or anti-LC3B (CST, 3868S, 1:200) primary antibodies and left to incubate overnight. The coverslips were subjected to secondary antibody incubation, followed by staining with DAPI. Images were captured using a fluorescence microscope (Nikon Eclipse TE2000-S) or a Zeiss confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany). Three fields of view were selected for each sample, and the fluorescence intensity was measured and averaged. The results obtained represented a biological replication. The ImageJ software was used for analysis.

FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Biotech Co., Ltd.) extracted total RNA from fibroblasts and determined its concentration, followed by HiScript II Q RT SuperMix (Vazyme Biotech Co., Ltd.) and ChamQ universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) for reverse transcription and PCR quantification. The primer sequences used are as follows: Collagen I-F, CCC​TGG​TCC​CTC​TGG​AAA​TG, Collagen I-R, GGA​CCt​ttg​ccc​cCT​TCT​TCT​TT; Collagen III-F, TGA​CTG​TCC​CAC​GTA​AGC​AC, Collagen III-R, GAG​GGC​CAT​AGC​TGA​ACT​GA; α-SMA-F, TTC​GTG​ACT​ACT​GCC​GAG​C, α-SMA-R, GTC​AGG​CAG​TTC​GTA​GCT​CT; p62-F, CCT​CAG​CCC​TCT​AGG​CAT​TG, p62-R, TTC​TGG​GGT​AGT​GGG​TGT​CA; LC3B-F, AGA​GCG​ATA​CAA​GGG​GGA​GA, LC3B-R, TGC​AAG​CGC​CGT​CTG​ATT​A; ATG-7-F, CCT​TCT​GGA​GCA​GTC​AGC​AA, ATG-7-R, AGG​AGC​ATG​GGG​TTT​TCG​AG; YAP1-F, TCC​AAC​CAG​CAG​CAG​CAA​AT, YAP1-R, CCT​GTT​GTT​TCA​ACC​GCA​GTC; CTRP9-F, GTG​CCC​AAG​AGT​GCT​TTC​AC, CTRP9-R, AAC​TTC​CCC​GTC​GCT​ACA​TT; GAPDH-F, TGT​CTC​CTG​CGA​CTT​CAA​CA, GAPDH-R, GGT​GGT​CCA​GGG​TTT​CTT​ACT.

The proliferation of cardiac fibroblasts was detected using Cell-Light EdU DNA Cell Proliferation Apollo567 Kit (RiBoBio). The treated cardiac fibroblasts were incubated with 10 nm EdU (5-ethynyl-2’-deoxyuridine) for 16 h, followed by subsequent experimental steps according to the instructions. Images were captured using a Zeiss fluorescence microscope, and the percentage of EdU-positive cells was analyzed using the ImageJ software.

The data are presented as mean ± SEM. Shapiro-Wilk test was used to evaluate the normality of the data distribution before data analysis. Statistical analyses involved Student’s t-test for comparing two groups and one-way ANOVA for assessing differences among three or more groups. Statistical analysis was performed using GraphPad Prism version 8, with significance defined as p < 0.05.

To demonstrate the involvement of CTRP9 in cardiac fibrosis, immunohistochemistry and Western blot analysis were conducted. The findings demonstrated a marked decrease in CTRP9 expression within the hearts of diabetic mice (Figures 1A–D). In vitro, CTRP9 expression was identified by Western blot following the stimulation of primary cardiac fibroblasts with high glucose (HG). Fibroblast CTRP9 expression was significantly lower in the HG group compared with the group without added high glucose (Figure 1E, F).

To elucidate the influence of CTRP9 deficiency on cardiac function, we performed echocardiograms prior to anesthesia (Figure 2A). In contrast to the NC group, the DM group demonstrated marked impairment in cardiac function, with a notable reduction in LVEF, LVFS, and E/A. These impairments were further accompanied by elevated E/e’, increased LVEDD and LVESD (Figure 2B–G). Notably, CTRP9 knockout diabetic mice exhibited greater deterioration in LVEF, LVFS, and E/A. Additionally, they exhibited increased E/e', LVEDD, and LVESD values (Figure 2B–G). Furthermore, diabetic mice in the CTRP9 deficiency group displayed significantly higher blood glucose levels and decreased body weights than those in the DM group (Figure 2H, I). In addition, in the absence of diabetes, there were no notable disparities observed in cardiac function, body weight, or blood glucose levels between the CTRP9 knockout mice and the control mice. These findings validated the successful establishment of the T1DM mouse model and demonstrated that CTRP9 deletion aggravated cardiac dysfunction in diabetic mice.

To elucidate the impact of CTRP9 knockout on fibrosis within the myocardium of diabetic mice, we performed pathological staining and immunohistochemistry. Hematoxylin and eosin (H&E) staining showed significant myocardial disarray within the DM group relative to the NC group. The disruption was more severe in CTRP9 knockout mice (Figure 3A). Moreover, Masson’s trichrome staining further displayed augmented collagen accumulation within the DM group, which intensified with CTRP9 gene deletion (Figure 3A, B). Immunohistochemical analysis revealed that collagen I, collagen III, and α-SMA was significantly elevated within the DM group. This increase was even more pronounced in the absence of CTRP9, indicating a heightened fibrotic response (Figure 3C–F). Western blot analysis provided additional support for these findings, showing similar trends in protein expression (Figure 3G–J). Collectively, these results suggested that CTRP9 deletion exacerbates myocardial fibrosis in diabetic mice.

Previous studies have shown a strong link between suppressed autophagy, activated YAP and the advancement of myocardial fibrosis in DCM (Ikeda et al., 2019; Wang et al., 2022). Building upon this foundation, our study examined the expression levels of p62 and LC3-II, alongside YAP, recognized for its critical involvement in organ fibrosis. Western blot analysis of cardiac tissues demonstrated a dramatic increase of p62 and LC3-II with the DM group relative to the NC group, with CTRP9 deletion exacerbating this trend further (Figure 4A–C). In parallel, YAP protein expression exhibited a significant upregulation in the DM group in comparison to the NC group, a disparity that was further amplified by CTRP9 deletion (Figure 4A, D). Immunohistochemical analysis confirmed the upregulation of YAP in the DM group, especially in the absence of CTRP9 (Figure 4E, F). These findings demonstrated that CTRP9 knockout exacerbated autophagy inhibition and upregulated YAP protein expression in diabetic mice.

Examining the influence of CTRP9 on fibroblasts activation triggered by HG, we pretreated primary cardiac fibroblasts isolated from mice with exogenous CTRP9, followed by stimulation with a 33.3 mM HG solution. Western blot analysis revealed that after 48 h of HG stimulation, myofibroblast marker α-SMA, as well as collagen level, significantly elevated within the HG group in contrast to both the NC and HO (high osmotic control) groups, whereas treatment with CTRP9 notably reduced the expression levels of these fibrosis markers (Figure 5A–D). Similar results were obtained in the RT-PCR experiment (Supplementary Figures S1A–C). Immunofluorescence staining for α-SMA further confirmed the inhibitory effect of CTRP9 on fibroblast activation (Figure 5E, F). Meanwhile, the results of the cell proliferation assay also showed that CTRP9 treatment caused a significant reduction in the level of HG-induced fibroblast proliferation (Figure 5G, H). These results suggested that CTRP9 treatment significantly inhibited myofibroblasts activation and the extracellular matrix accumulation induced by HG.

This research sought to investigate the influence of autophagy on myofibroblast activation. Western blot analysis revealed significant upregulation of p62 and LC3-II in the HG group, suggesting possible impairment of autophagy. Notably, exogenous CTRP9 administration reduced the expression of these markers, indicating the restoration of autophagic activity (Figure 6A–C). The results of RT-PCR were consistent with the above results (Supplementary Figures S1F–G). Confocal microscopy further revealed a decrease in the number of LC3B puncta upon CTRP9 treatment, indicating a partial reversal of autophagy inhibition under HG conditions (Figure 6D, E). To investigate whether the CTRP9-mediated inhibition of cardiac fibroblast activation was autophagy dependent, cardiac fibroblasts were exposed to CQ, an autophagy inhibitor. Western blot analysis demonstrated a notable elevation of α-SMA, collagen I, and collagen III in the HG + CTRP9+CQ group compared to the HG + CTRP9 group (Figure 6F–I). The same results were obtained in the RT-PCR experiment (Supplementary Figures S2A–C). Immunofluorescence staining also revealed similar results (Figure 6J, K). In addition, the cell proliferation level in HG + CTRP9+CQ group was significantly higher than that in HG + CTRP9 group (Figure 6L, M). These combined results suggest that CTRP9 can suppress fibroblast activation and extracellular matrix secretion through restoring autophagy disrupted by high-glucose conditions.

Given that YAP is closely associated with fibrosis (Francisco et al., 2020), we elucidated its role in cardiac fibroblast activation. Western blot revealed notable YAP increase within the high glucose (HG) group relative to both the NC and HO groups, and the addition of exogenous CTRP9 led to a significant suppression of YAP expression (Figure 7A, B). The mRNA change level of YAP was consistent with the protein level (Supplementary Figure S1D). To determine the impact of YAP on autophagy and its potential role in mediating the regulatory effect of CTRP9 on cardiac fibroblast activation, we overexpressed YAP in primary mouse cardiac fibroblasts via plasmid-mediated transfection (Figure 7C–E). We observed that overexpression of YAP eliminated CTRP9-mediated autophagy recovery (Figure 7F–J). The experimental results of RT-PCR also support the above results (Supplementary Figures S3E, F). Additionally, overexpressing YAP abolished the inhibitory effect exerted by CTRP9 toward myofibroblast activation. In the group overexpressing YAP, the levels of fibrosis markers and collagen were significantly elevated compared to the group treated with HG + CTRP9+NT/pcDNA3.1(Figure 7K–N). The results of RT-PCR were consistent with the above results (Supplementary Figures S3A–C). Immunofluorescence staining for α-SMA provided additional confirmation of these effects (Figure 7O–P). Overexpression of YAP significantly increased the proliferation of cardiac fibroblasts treated with high glucose and CTRP9 (Figure 7Q–R). Based on the observations, CTRP9 suppresses high glucose-induced cardiac fibroblast activation and extracellular matrix deposition through YAP-mediated autophagy.