Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

TL;DR

This study shows that combining CHIR99021 and FGF2 reduces heart scarring in mice by regulating Serpine1, offering a potential new treatment for myocardial fibrosis.

Contribution

The study identifies Serpine1 as a key target through which CHIR99021 and FGF2 inhibit myocardial fibrosis.

Findings

CHIR99021 and FGF2 together inhibit cardiac fibroblast activation and collagen scar formation in mice.

Serpine1 expression is reduced via TGF-β and FAK pathways by the drug combination.

Serpine1 overexpression or knockdown confirms its role in fibrotic regulation.

Abstract

Cardiac fibrosis is a pathological phenomenon caused by tissue remodeling and excessive matrix proliferation under stress conditions. CHIR99021 is a selective glycogen synthase kinase-3 inhibitor that has shown potential in cardiovascular regeneration therapy. Fibroblast growth factor 2 (FGF2) has a protective effect on ischemic myocardium. However, the effect and underlying mechanism of the combined use of CHIR99021 and FGF2 on myocardial fibrosis remains unclear. In this study, we found that the combination of CHIR99021 and FGF2 could significantly inhibit the activation of cardiac fibroblasts (CFs) and alleviate the formation of collagen scars in mouse myocardium. By analyzing the expression levels of fibrotic proteins, such as ColI, ColIII and alpha smooth muscle actin (α-SMA) in fibroblasts in vitro and in vivo, we confirmed the inhibitory effect of CHIR99021 combined with FGF2 on the activation of fibroblasts. Transcriptome sequencing showed that CHIR99021 and FGF2 inhibited the expression level of Serpine1 through the transforming growth factor-β (TGF-β) and Focal Adhesion Kinase (FAK) signaling pathways. By analyzing the regulatory effect of overexpressed and knocked-down Serpine1 on fibrotic pathway-related proteins in CFs, we verified that Serpine1 is a key target for inhibiting fibrosis. In conclusion, this study provides evidence that Serpine1 may be a potential mechanism that enables CHIR99021 combined with FGF2 to improve myocardial fibrosis.

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Full text

1. Introduction

Cardiac fibrosis is a common pathological feature of chronic heart failure, for which no specific therapy is currently available. Persistent and excessive proliferation and activation of cardiac fibroblasts (CFs) following myocardial infarction can lead to scar formation, reduced tissue compliance, and widespread fibrotic remodeling—major contributors to myocardial dysfunction and eventual heart failure [1,2,3,4]. Targeting fibroblasts within the infarcted area to suppress their excessive proliferation and promote the degradation of existing fibrotic scars may effectively reduce fibrosis and support the recovery of myocardial function [5]. Although several approaches identified through basic research have shown promise in controlling and inhibiting pathological myocardial fibrosis, safe and effective clinical treatments remain lacking. Therefore, it is imperative to identify novel modulators and potential therapeutic targets to facilitate clinical intervention.

Multiple cardioprotective signaling cascades converge on glycogen synthase kinase-3 (GSK-3) [6,7] (Figure 1). Inhibiting GSK-3 can reduce cardiomyocyte apoptosis, decrease infarct size, and improve cardiac function, thereby offering cardioprotective effects [8,9,10,11]. CHIR99021, a selective GSK-3 inhibitor, has been shown to enhance myocardial reprogramming and holds therapeutic potential for cardiovascular regeneration [12]. However, the specific role and mechanism of CHIR99021 in cardiac fibrosis remain incompletely understood. Recent research suggests that GSK-3α may promote fibrosis in the injured heart via the RAF-MEK-ERK pathway [13,14], indicating a complex and nuanced role for GSK-3 in myocardial repair. Our previous research demonstrated that CHIR99021 does not only promote the differentiation of CFs into cardiomyocytes, but also inhibits the proliferation of fibroblasts, degrades the extracellular matrix and reduces collagen secretion function [15]. These findings suggest that CHIR99021 may serve as a therapeutic agent for myocardial fibrosis by not only protecting the myocardium but also through promoting its regeneration after ischemic injury.

Growth factor 2 (FGF2) is widely expressed in various tissues and regulates key cellular processes such as proliferation, differentiation, migration, and apoptosis [16]. FGF2 has been shown to inhibit the activation and secretion of CFs, suppress matrix protein and collagen production, and maintain the stem-like characteristics of CFs [17,18]. These findings highlight FGF2 as a promising therapeutic target for improving myocardial fibrosis.

In our preliminary study, it was confirmed that the small molecule compound CHIR99021, in combination with FGF2, has a synergistic regulatory effect on the proliferation and activation of cardiac fibroblasts. In the fibrotic repair stage after myocardial infarction in mice, the combined intervention of CHIR99021 and FGF2 can reduce the infarct size by inhibiting myocardial cell death and improve the degree of ischemic myocardial fibrosis by reducing collagen fiber deposition. Further analysis showed that Serpine1 was involved in the antifibrosis effect and regulation process of CHIR99021/FGF2, but the underlying mechanism is still unclear. This study aims to demonstrate the synergistic effect of CHIR99021 and FGF2 in antifibrosis and the mechanism of its downstream key gene Serpine1 in regulating fibrosis, providing a new therapeutic strategy for reducing myocardial fibrosis.

2. Results

2.1. The Effects of CHIR99021 and FGF2 on the Viability of Cardiac Fibroblasts

To verify the purity of the cultured cells, we used CF-specific markers Vimentin and discoidin domain receptor tyrosine kinase 2 (DDR2). The expression rates of DDR2 and Vimentin in the isolated cells were 98.47 ± 0.11% and 99.19 ± 0.41%, respectively, confirming that the cultured cells were indeed CFs (Figure 2A,B). To determine the optimal working concentration of CHIR99021, we assessed its effects on CF viability using a CCK-8 assay. The results indicated that 10 μM CHIR99021 was the most effective concentration for promoting cell viability (Figure 2C). Additionally, a Calcein AM assay was performed to further evaluate cell viability. Both CHIR99021 and FGF2 significantly enhanced the viability of CFs. Notably, the combined treatment with CHIR99021 and FGF2 produced a more pronounced effect compared to either treatment alone (Figure 2D,E).

2.2. CHIR99021 in Combination with FGF2 Inhibits the Activation of Cardiac Fibroblasts

Fibroblast activation, characterized by the expression of alpha smooth muscle actin (α-SMA) and the synthesis of abundant extracellular matrix (ECM) proteins, is a critical event in the progression of cardiac fibrosis. Consequently, we analyzed the expression of ColI, ColIII and α-SMA, markers for activated fibroblasts. It is obvious that the fibroblasts in the control group were cruciform or stellate shaped, while the cells in the CHIR99021 combined with the FGF2 treatment group stayed spindle-shaped since they were quiescent fibroblasts (Figure 3A). Immunofluorescence staining showed that the expression of α-SMA, ColI and ColIII in CFs was significantly inhibited after treatment with CHIR99021 and FGF2, either alone or in combination (Figure 3B,C). Western blotting confirmed the expression levels of α-SMA was significantly inhibited by CHIR99021 with or without FGF2, suggesting the inhibitory effect of the combined treatment on fibroblast activation (Figure 3D).

In addition, collagen secretion capacity was assessed through hydroxyproline assay, which reveals that CHIR99021 alone or in combination with FGF2 significantly reduced collagen secretion. However, in contrast, the inhibitory effect of FGF2 alone on collagen secretion was not readily discernible (Figure 3E). Collectively, these findings highlight the strong inhibitory effect of CHIR99021 on fibroblast activation. While FGF2 alone has limited impact, it may enhance the antifibrotic effect of CHIR99021 when used in combination.

2.3. Gene Expression Profiles of CFs Treated with CHIR99021 and FGF2 Reveal Suppression of Extracellular Matrix Activation

Cell viability assay and immunofluorescence staining confirmed that CHIR99021 in combination with FGF2 could significantly inhibit the expression of extracellular matrix components (collagen and SMA) and maintain cell viability. To explore the underlying mechanisms, we analyzed the differentially expressed genes (DEGs) in CFs treated with CHIR99021/FGF2 using RNA-seq. Based on defined criteria (absolute log_2_ fold change ≥1 and adjusted p value ≤ 0.01), a total of 1549 DEGs were identified, including 557 upregulated and 992 downregulated genes (Figure 4A,B).

Gene ontology (GO) enrichment analysis further supported these findings. Downregulated molecular functions were primarily associated with integrin binding, GTP binding, growth factor activity, actin binding, and extracellular matrix structural constituents. In terms of cellular components, DEGs were enriched in receptor complexes, actin cytoskeleton, cell–substrate junctions, and the extracellular matrix. The most significantly enriched biological processes included cell–substrate adhesion, ECM organization, actin filament organization, fibroblast activation, and response to hypoxia (Figure 4C), supporting the notion that cardiac fibroblast activation was broadly suppressed.

Gene set enrichment analysis (GSEA) revealed distinct transcriptional regulatory patterns influenced by CHIR99021/FGF2. Upregulated genes were significantly enriched in biological processes related to cell growth and RNA regulation, such as “ribosome biogenesis” and “RNA processing.” In contrast, downregulated genes were predominantly associated with processes related to cytoskeletal dynamics (e.g., “actin filament-based process”), cellular movement (e.g., “cell adhesion” and “cell migration”), and fibrogenesis (e.g., “extracellular matrix organization”) (Figure 4D).

To further identify key regulatory targets, we prioritized specific DEGs associated with fibroblast activation. Notably, several fibroblast activation markers, including Acta2, Pdgfrb, Cygb, and Akap12, were significantly downregulated. Additionally, genes involved in integrin-mediated signaling pathways, such as Itga1, Itgb1, Itgb3, Ptk2b, and Emp2, were also suppressed, aligning with their established roles in fibrogenesis. Importantly, several genes related to the TGF-β signaling pathway and ECM remodeling, including Tgfb1, Fn1, Tgfb2, Smad7, Smad6, Tgfb3, Ltbp2, and Eln, were enriched among the downregulated group (Figure 4E). The heatmap of differentially expressed genes in the control group and the CHIR99021/FGF treatment group further revealed a marked downregulation of key fibrogenic genes and TGF-β signaling components, including Acta2, Eln, Tgfb3, Tgfb1, Fbn1, Ltbp1, and Serpine1 (Figure 4F). Together, our results suggest that CHIR99021 and FGF2 cooperatively suppress extracellular matrix production and fibroblast activation at the transcriptional level in cardiac fibroblasts.

2.4. CHIR99021 Combined with FGF2 Inhibits AngII-Induced Activation of Cardiac Fibroblasts and the Expression of Serpine1

According to RNA-seq results, several genes associated with fibroblast activation, such as Tgfβ1, Acta2, and Col3a1 were significantly downregulated in CFs following treatment with CHIR99021 and FGF2. Among them, Serpine1 was identified as a key gene that potentially undergoes great changes in CFs via RNA-seq analysis and qRT-PCR. To further investigate the inhibitory effects of this combination on CFs activation, we used Angiotensin II (AngII) to stimulate CFs activation. Moreover, the combination therapy effectively suppressed the upregulation of these genes induced by AngII stimulation (Figure 5A). Immunofluorescence staining demonstrated that AngII significantly enhanced the expression of both α-SMA and ColIII in CFs, whereas treatment with CHIR99021 and FGF2 substantially reduced their expression levels (Figure 5B). Since hydroxyproline content is a key indicator of collagen turnover and fibrosis severity, we further assessed collagen secretion. The results showed a marked reduction in hydroxyproline levels in the CHIR99021/FGF2 group, confirming the inhibitory effect of this combination on CFs activation (Figure 5C). Western blot analysis corroborated these findings, showing significantly decreased protein expression levels of α-SMA and Col III in CFs treated with CHIR99021/FGF2 (Figure 5D,E). Notably, Serpine1 expression at both mRNA and protein levels was significantly elevated in AngII-activated fibroblasts and was markedly suppressed following treatment with CHIR99021/FGF2. These results suggest that Serpine1 is likely involved in fibroblast activation and collagen deposition and may serve as a downstream target of the CHIR99021/FGF2 combination therapy.

2.5. CHIR99021 Combined with FGF2 Inhibits Serpine1 Expression in Myocardial Fibrotic Regions and Attenuates Fibrosis in Mice

Fibroblast proliferation and collagen deposition are hallmark pathological features following myocardial infarction. To assess whether Serpine1 is involved in the fibrotic process in vivo, and to evaluate the effects of CHIR99021 combined with FGF2 on cardiac remodeling, morphological and histological analyses were conducted two weeks after treatment (Figure 6A). Masson’s staining revealed extensive myocardial fibrosis in the MI group, whereas treatment with CHIR99021 and FGF2 significantly reduced collagen fiber content in the infarct area (Figure 6B,C). ImageJ analysis demonstrated that collagen deposition was markedly decreased by the combined treatment, from an average of 5.008 ± 0.406% to 2.668 ± 0.129% (Supplementary Figure S1). Immunofluorescence staining for α-actinin and ColIII, which directly impact cardiomyocyte contractile and diastolic functions, showed that the control group retained well-organized myocardial sarcomeres and normal interstitial architecture. In contrast, the MI model group exhibited disorganized and fragmented sarcomeres, with fibrotic tissue replacing cardiomyocytes in the infarcted area. Notably, the CHIR99021 plus FGF2 treatment group displayed a reduction in myocardial structural disruption within the infarcted area (Figure 6D). CHIR99021 and FGF2 may also have increased survival of the native cardiac cells and contributed to functional improvement. Moreover, this combined treatment significantly inhibited the expression of Serpine1 in infarcted area (Figure 6E). These findings suggest that the upregulation of ColIII and Serpine1 contributes to the cardiac fibrotic response post-MI, and that CHIR99021 with FGF2 can substantially reduce collagen deposition and Serpine1 expression in the infarct region. However, whether this antifibrotic effect is directly mediated through Serpine1 inhibition warrants further investigation.

2.6. Knockdown of Serpine1 Inhibited the Activation of Cardiac Fibroblasts

To investigate the role of Serpine1 in CFs activation, we performed siRNA-mediated knockdown and overexpression of Serpine1 in CFs. Morphological analysis revealed that CFs with Serpine1 knockdown adopted a spindle-shaped morphology typical of quiescent fibroblasts, while Serpine1-overexpressing cells displayed a triangular or polygonal morphology characteristic of activated fibroblasts (Figure 7A). Successful knockdown of Serpine1 protein expression was confirmed by Western blotting. Compared with the negative control (lentivirus harboring empty vector), the protein level of α-SMA was significantly decreased by Serpine1 knockdown (Figure 7B). Furthermore, we detected the expression of fibroblast activation-related proteins in fibroblasts after Serpine1 knockdown and overexpression by using immunofluorescence. Markers of fibroblast activation, including α-SMA, ColIII and Cryab, were significantly downregulated in Serpine1-siRNA-treated cells compared to untransfected controls in the same field of view. Conversely, Serpine1 overexpression led to increased α-SMA, ColIII and Cryab expression (Figure 7C). Taken together, these results suggest that Serpine1 knockdown inhibits the activation of cardiac fibroblasts, indicating that Serpine1 may play a critical role in promoting collagen synthesis and fibroblast activation in the context of cardiac fibrosis.

2.7. Serpine1 Affects Fibroblast Activation Through TGF-β and FAK Signaling Pathways

Functional enrichment analysis revealed that downregulated genes were significantly enriched in regulation of cell–substrate adhesion, extracellular matrix organization, and response to transforming growth factor beta signaling pathway (Figure 8A). Moreover, Serpine1 plays a critical role in the process of myocardial fibrosis and cell adhesion. To further investigate whether Serpine1 interacts with the TGF-β pathway and cell adhesion during cardiac fibrosis, we performed siRNA-mediated knockdown of Serpine1 and lentivirus-mediated overexpression of Serpine1 in CFs, followed by either CHIR99021/FGF2 or AngII treatment. The expressions of key proteins involved in this pathway, including collagens, Smad2, phosphorylated Smad2 (p-Smad2), Smad3, phosphorylated Smad3 (p-Smad3), FAK and phosphorylated FAK (p-FAK) in CFs were assessed. Western blot analysis demonstrated that knockdown of Serpine1 significantly reduced the protein levels of ColI, ColIII, Serpine1 and α-SMA, while the total Smad2/3 levels were slightly decreased and the levels of phosphorylated Smad2/3 (key effectors of canonical TGF-β signaling) were markedly reduced by CHIR99021 and FGF2 treatment or Serpine1 knockdown. In addition, both CHIR99021 combined with FGF2 and knockdown of Serpine1 can reduce the total protein and phosphorylation levels of FAK in fibroblasts. Conversely, although no significant change was observed for αSMA and ColI, the protein levels of ColIII, pSmad2/3, FAK and p-FAK were significantly increased by Serpine1 overexpression, particularly with AngII treatment (Figure 8B). These findings suggest that Serpine1 can significantly affect activation of cardiac fibroblasts by suppressing TGF-β and FAK.

2.8. CHIR99021 and FGF2 Regulate Serpine1 via the TGF-β and FAK Signaling Pathway

To explore whether CHIR99021 combined with FGF2 inhibits cardiac fibroblast activation through the regulation of TGF-β and FAK on Serpine1, we performed an overexpression Serpine1 in normal CFs and subsequently treated the cells with CHIR99021 and FGF2 in combination. Western blotting analysis showed that the overexpression of Serpine1 markedly enhanced the protein expression levels of ColIII and α-SMA, as well as those of Smad2/3 and FAK in fibroblasts. Notably, the combined treatment with CHIE99021 and FGF2 inhibited the protein level of Serpine1 in CFs. In addition, it also effectively attenuated the expression of Smad2/3 and FAK proteins and the upregulation of ColIII and α-SMA induced by Serpine1 (Figure 9A,B). However, CHIR99021 and FGF2 markedly suppressed the expression of endogenous Serpine1 while exerting minimal influence on the exogenously introduced Serpine1. Together, our results suggest that the activation of cardiac fibroblasts was significantly promoted by Serpine1 overexpression and CHIR99021/FGF2 can attenuate Serpine1-induced activation of cardiac fibroblasts by inhibiting TGF-β and FAK signaling pathway.

3. Discussion

Cardiac fibrosis is closely associated with heart failure; it not only plays a pivotal role in the onset and progression of cardiovascular diseases but also represents a final common pathway in many primary cardiac disorders [2]. Multiple pathological factors, including myocardial injury and pressure overload, contribute to the development of cardiac fibrosis [19]. Additionally, cardiac fibrosis is intricately linked to various cytokines, primarily including TGF-β, angiotensin II, and reactive oxygen species [20]. These mediators promote cardiac fibrosis through both shared and distinct signaling pathways [21,22].

The development of myocardial fibrosis is mainly attributed to the excessive proliferation of cardiac fibroblasts and their transformation into myofibroblasts, leading to extracellular matrix accumulation in the myocardium and resulting in cardiac remodeling [23]. However, the key molecular mechanisms underlying the overproliferation of cardiac fibroblasts and abnormal collagen synthesis remain unclear, which significantly hampers both basic research and the clinical prevention and treatment of myocardial fibrosis [23,24].

GSK-3 is a serine/threonine protein kinase. Numerous studies have shown that GSK-3 functions as a central regulator involved in various biological processes, including tumor metastasis, cell proliferation, inflammatory responses, cellular stress, and apoptosis [25,26,27]. The use of small-molecule GSK-3 inhibitors has shown great therapeutic potential in treating a variety of diseases [13]. CHIR99021, an aminopyrimidine derivative, effectively inhibits the activity of GSK-3. Studies have demonstrated that CHIR99021 plays an important role in promoting the differentiation of CFs into cardiac precursor cells [12]. Notably, CHIR99021 shows strong potential for reprogramming CFs into cardiomyocytes to replace lost myocardial cells, offering promising prospects for cardiac regeneration [28].

FGF2, a member of the mammalian fibroblast growth factor family, exhibits cytokine-like properties and has demonstrated potent antifibrotic effects in both in vivo and in vitro models [29,30,31]. Previous studies have shown that FGF2 can inhibit BMP4-mediated smooth muscle cell differentiation through the BMP4/Smad signaling pathway and maintain the progenitor cell characteristics of PMPCs [32]. Similarly, our previous experiments found that FGF2 markedly suppressed α-SMA expression in CFs and reduced the secretion of type I and type III collagen. Moreover, CHIR99021 could effectively inhibit the differentiation of fibroblasts into myofibroblasts, which might be an attractive therapeutic option for inhibiting myocardial fibrosis. However, the synergetic therapeutic efficacy of the combined use of these two factors has not yet been explored.

Immunofluorescence and Western blotting experiments confirmed that CHIR99021 combined with FGF2 significantly inhibited the secretion levels of α-SMA, ColI and ColIII in CFs compared to either treatment alone, indicating that the combination of FGF2 and CHIR99021 resulted in a stronger inhibitory effect on CF activation. Furthermore, Calcein AM cell viability assays showed that both CHIR99021 and FGF2 enhanced the viability of CFs. Notably, their combination produced an even greater effect on promoting CF viability. In addition, animal experiments of myocardial infarction have shown that the combination of CHIR99021 and FGF2 has the effect of inhibiting fibroblast collagen production and protecting myocardial cells in vivo, which is manifested as a significant reduction in infarct area and an improvement in the degree of fibrosis.

To investigate the underlying mechanism of CHIR99021 and FGF2 inhibiting fibroblast activation, we treated CFs in vitro with both compounds and performed differential gene expression analysis, revealing that most fibrosis-related genes were significantly downregulated in the CHIR99021 treatment group. qRT-PCR confirmed significant reductions in the expression of Acta2, Tgfβ1, Col1a1, Col3a1, and other fibrotic genes. Following treatment with CHIR99021 and FGF2, we observed significant changes in genes involved in the MAPK and TGF-β signaling pathways—pathways known to regulate cell proliferation, cell–cell interactions, extracellular matrix synthesis, tissue repair, and focal adhesion. Functional annotation of the DEGs showed reductions in cell adhesion, migration, proliferation, and processes related to reactive oxygen species metabolism and hypoxia. The expression of genes involved in ECM organization was also inhibited. RNA-seq analysis revealed a significant reduction in Serpine1 expression in the CHIR99021/FGF2 group, along with decreased expression of other fibrosis-related genes such as Eln, Acta2, Ddr2, Pdgfrb, Ltbp4, Fbln5, Tgfβ3, Mmp2, Tgfβ1, Itgb1, and Col1a1. These findings indicate that the combined treatment suppresses ECM remodeling and fibroblast activation at the transcriptional level.

Serpine1, a member of the serpin gene family, serves as a major physiological inhibitor of plasminogen activators and has been shown to be associated with fibrosis in multiple organ systems [33,34,35]. In this study, we observed elevated Serpine1 expression in fibrotic cardiac tissue from mice following myocardial infarction. Considering the function of Serpine1 in regulating the clearance of ECM through matrix metalloproteinases in fibroblasts, it is clear that it is directly related to cardiac interstitial fibrosis. Besides this, it remains unclear whether Serpine1 also participates in fibrosis by activating cardiac fibroblasts to secrete collagen. In addition, combination treatment with CHIR99021 and FGF2 protected against cardiac remodeling and inhibited collagen deposition and serpine1 expression in fibrotic areas. Based on this, we speculate that CHIR99021 in combination with FGF2 inhibits the activation of fibroblasts and their ability to secrete collagen by regulating Serpine1.

Further analysis revealed substantial upregulation of Serpine1 accompanied with the upregulation of fibroblast activation-related markers such as α-SMA and ColIII in AngII-treated CFs, indicating that increased expression primarily arises from stimulation by profibrotic factors. Notably, the combination treatment of CHIR99021 and FGF2 markedly suppressed the AngII-induced upregulation of Serpine1 in CFs. Subsequent knockdown of Serpine1 expression resulted in significant inhibition of CFs activation and collagen expression, whereas overexpression of Serpine1 led to increased collagen production. However, CHIR99021 combined with FGF2 can inhibit the expression of Smad2/3 and FAK and can significantly suppress the expression of collagen and α-SMA caused by the upregulation of Serpine1. Together, our research indicates that CHIR99021 and FGF2 cooperatively regulate Serpine1 through the modulation of the TGF-β and FAK signaling pathways, thereby inhibiting the activation of cardiac fibroblasts.

TGF-β is closely related to the regulation of cellular growth fibrosis and differentiation [36]. More importantly, Serpine1 is one of the most important target genes of the TGF-β/Smad signaling pathway [37,38]. However, whether TGF-β takes part in the impact of CHIR99021 and FGF2 on fibroblast activation by inhibiting Serpine1 remains in doubt. Of note, the CHIR99021/FGF2 group demonstrated upregulated expression levels of TGF-β downstream targets (Col1a1, Col3a1, Acta2), as well as high Serpine1 expression, suggesting a crosstalk between TGF-β downstream targets and Serpine1. Further mechanistic study also showed that both phosphorylation and total level of Smad2 and Smad3 were regulated by Serpine1; the decrease in Smad2 and Smad3 phosphorylation is due to the decrease in t-Smad2 and t-Smad3, suggesting that CHIR99021 and FGF2 regulate Serpine1 expression via the TGF-β signaling pathway. In addition, CHIR99021 and FGF2 can also affect the activation of fibroblasts by regulating FAK activation, indicating that Serpine1 may be involved in FAK-related ECM synthesis. FAK can integrate various signals, including fibronectin, growth factors and extracellular matrix, etc., thereby promoting or inhibiting cell proliferation and migration. Therefore, we believe that multiple complex mechanisms are involved in this process, involving multiple regulatory factors. Taken together, these findings suggest that CHIR99021 combined with FGF2 inhibits cardiac fibroblast activation by reducing Serpine1-mediated extracellular matrix synthesis and attenuating TGF-β and FAK dependent profibrotic signaling.

4. Materials and Methods

4.1. Animals

Sprague-Dawley rats were obtained from the Experimental Animal Centre of Xinxiang Medical University, and male C57BL/6 mice (8 weeks old) were purchased from Henan Skbex Biology Company (Anyang, China). All animal experiments were conducted in accordance with the policies and regulations of the National Institutes of Health Office of Laboratory Animal Welfare. The experiments were approved by the Xinxiang Medical University Animal Supervision Committee (Approval No. XYLL-20240152) and performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

4.2. Cell Culture and Identification

CFs were isolated from neonatal Sprague-Dawley (SD) rats following a modified protocol [39]. Briefly, left ventricles were excised and digested with 0.05% collagenase II (Gibco, Geel, Belgium) and 0.07% trypsin (Beyotime, Shanghai, China) at 37 °C for 30 min. The cell suspension was centrifuged at 1000 rpm for 5 min, and the supernatant was plated in Petri dishes. After 45 min, unattached cells were removed, and adherent cells were washed twice. Cells were cultured in complete DMEM (10% FBS, 1% Pen-Strep), and third-passage cells were used for analysis. CF-specific markers Vimentin (ab92547, Abcam, 1:250, Shanghai, China) and DDR2 (A01698-1, Boster, 1:200, Wuhan, China) were detected by immunofluorescence staining.

4.3. Cell Viability Assay

To determine the optimal CHIR99021 (M1692, Abmole, China) concentration, CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (1, 5, 10, 20, and 40 μM). Viability was assessed at 24, 48, and 72 h using a Cell Counting Kit-8 (CCK8, MCE, Shanghai, China), and OD450 values were measured with a Multiskan microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed in triplicate and repeated three times. CFs were also treated with CHIR99021 (10 μM) or FGF2 (20 ng/mL) for 48 h, followed by incubation with 2 μM Calcein AM at 37 °C for 45 min. After washing with DMEM, cells were observed under a fluorescence microscope (Leica DMI3000 B, Leica, Wetzlar, Germany), and fluorescence intensity was analyzed using ImageJ (version 1.54).

4.4. Immunofluorescence Staining

CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 (HA500124, HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China). Cell nucleus staining was performed using the DAPI staining solution (ID22502, Solarbio, 0.5 μg/mL, Beijing, China). Positive cells were quantified in five random fields per group at 200× magnification, and positive rates were calculated as follows: Positive rate (%) = (number of marker-positive cells/total DAPI-stained cells) × 100. Quantification was performed using ImageJ.

4.5. Hydroxyproline Content Determination

To assess collagen metabolism, hydroxyproline content in the medium was quantified. CFs were seeded equally, treated, and incubated with serum-free DMEM for 24 h. Supernatants were collected by centrifugation and analyzed using a hydroxyproline assay kit (Jiancheng Bioengineering, Nanjing, China), following the manufacturer’s instructions.

4.6. Transcriptome Sequencing and Bioinformatics Analysis

Total RNA was extracted from control and CHIR99021 + FGF2 groups using Trizol (Invitrogen, Waltham, MA, USA), with three biological replicates per group. RNA-seq and bioinformatics analysis were conducted by GENEWIZ (Suzhou, China) as previously described [31]. Differentially expressed genes (Q-value < 0.01, |log2 fold change| ≥ 2) were analyzed. Hierarchical clustering was performed using MeV software (version 4.9.0), and GO and GSEA analyses were conducted using the clusterProfiler package in R (v3.6) [40].

4.7. qRT-PCR Analysis

Total RNA was extracted with Trizol (Invitrogen, Waltham, MA, USA), and cDNA synthesis was performed using a Thermo Fisher RNA PCR kit. qRT-PCR was conducted on a LightCycler 480 II system (Roche, Mannheim, Germany) using SYBR Green Master Mix (Takara, Tokyo, Japan). PCR conditions: 95 °C for 2 min; 45 cycles at 95 °C for 5 s, 60 °C for 60 s, and 72 °C for 30 s. Relative gene expression was calculated using the 2^−ΔΔCt^ method and normalized to Gapdh. Primer sequences are listed in Supplementary Table S1.

4.8. Animal Models

Forty male C57BL/6 mice (6–8 weeks old, 20–25 g) were housed in a pathogen-free facility (20–23 °C, 12 h light/dark cycle). Mice were randomly divided into sham-operated (n = 20/group) and myocardial infarction (MI) groups (n = 40/group). MI was induced via permanent ligation of the left anterior descending artery (LAD) under isoflurane anesthesia (Rwd Life Science, Shenzhen, China). Successful infarction was confirmed by a pale ischemic region. Sham-operated mice underwent thoracotomy without LAD ligation. On the fifth day after the operation, mice in MI group were randomly divided into two groups and received intraperitoneal injections of 0.2 mL CHIR99021 (10 μM) + FGF2 (20 ng/mL) and 0.2 mL DMSO, respectively. The drug was administered every three days until the 20th day after MI.

4.9. Masson Staining

Mouse hearts were harvested and sectioned at 8 μm thickness. Masson’s staining was performed using a commercial kit (G1340, Solarbio, China). Collagen deposition was visualized and analyzed using a digital panoramic scanning microscope (Pannoramic MIDI, Budapest, Hungary).

4.10. Lentivirus Infection

Lentiviruses (Ov-Serpine1 and Si-Serpine1; GenePharma Inc., Suzhou, China) carrying eGFP reporters were used to infect CFs at an MOI of 20 in the presence of 5 μg/mL polybrene. After 72 h, infection efficiency was confirmed by fluorescence microscopy. Cells were selected with puromycin (2 μg/mL) for 3 days before further analysis.

4.11. Western Blotting Analysis

Cells were lysed using RIPA buffer and diluted with sodium dodecyl sulfate (SDS) loading buffer (Beyotime, China). Total proteins were separated by 10% SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Biosharp, Beijing, China). The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline and incubated overnight at 4 °C with the following primary antibodies: anti-Serpine1 (HA500124, HuaBio, 1:1000, China), anti-α-SMA (ET1607-53, HuaBio, 1:1000, China), anti-ColI (HA722517, HuaBio, 1:1000, China), anti-ColIII (HA720050, HuaBio, 1:1000, China), anti-Smad2 (ET1604-22, HuaBio, 1:5000, China), anti-p-Smad2 (ET1612-32, HuaBio, 1:5000, China), anti-Smad3 (#9523, CST, 1:1000, Danvers, MA, USA), anti-p-Smad3 (#9520, CST, 1:1000, Danvers, MA, USA), anti-FAK (ET1602-25, HuaBio, 1:5000, China), anti-p-FAK (ET1610-34, HuaBio, 1:1000, China) and anti-GAPDH (ab8245, Abcam, 1:20,000, Waltham, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Beyotime, 1:10,000, China) for 1 h at room temperature. Protein bands were visualized using an ECL detection system (Amersham Biosciences, Princeton, NJ, USA), and band intensity was quantified using ImageJ software.

4.12. Statistical Analysis

GraphPad Prism software (version 10.6) was used for statistical analysis. All data are presented as mean ± standard deviation (SD). Comparisons between two groups were performed using Student’s t-test, while comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05 (* p < 0.05, ** p < 0.01, and *** p < 0.001).

5. Conclusions

In summary, we demonstrate for the first time that the combination of CHIR99021 and FGF2 effectively inhibit collagen deposition in myocardial tissue and reduce myocardial cells apoptosis, which may contribute to improvements in cardiac remodeling. This synergistic effect inhibits the activation of cardiac fibroblasts by reducing Serpine1-mediated extracellular matrix synthesis and weakening TGF-β and FAK dependent pro-fibrotic signaling. Our data also suggests that targeting Serpine1 for the combined chemical treatment may be a potential strategy for the treatment of myocardial fibrosis in the future. Future research directions could include testing the efficacy of these growth factors in clinical combination use and optimizing their doses, which may further improve the treatment effect relevant to clinical practice.