The Matricellular Protein CCN5 (WISP2) inhibits Cellular Senescence in Cardiac Myocytes and Fibroblasts

YongjoonJo1

MiyoungLee1

SungBinKim1

TaeHwanKwak2

DongtakJeong3

SeungPilJang4

JinWoo1✉EmailMoai27@gmail.com

Park1✉Emailwoojinpark@icloud.com

Department of Life SciencesGwangju Institute of Science and Technology (GIST)GwangjuRepublic of Korea

2BethphaGenGwangjuRepublic of Korea

3Department of Medicinal & Life Science, College of Science and Convergence TechnologyHanyang University-ERICAAnsanRepublic of Korea

4Center for Gene and Cell TherapyKorea Research Institute of Bioscience and BiotechnologyDaejeonRepublic of Korea

Yongjoon Jo¹, Miyoung Lee¹, Sung Bin Kim¹, Tae Hwan Kwak², Dongtak Jeong³, Seung Pil Jang⁴ and Woo Jin Park¹

¹ Department of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea

² BethphaGen, Gwangju, Republic of Korea

³ Department of Medicinal & Life Science, College of Science and Convergence Technology, Hanyang University-ERICA, Ansan, Republic of Korea

⁴ Center for Gene and Cell Therapy, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea

*Correspondence:

Seung Pil Jang, Moai27@gmail.com

Woo Jin Park, woojinpark@icloud.com

Keywords:

CCN5

WISP2

Cellular senescence

Cardiac myocytes

Cardiac fibroblasts

Abstract

Cardiovascular diseases remain the leading cause of global mortality. Cellular senescence has recently been implicated in the pathogenesis of various cardiovascular diseases. Our group has previously shown that the matricellular protein CCN5 is a potent anti-fibrotic molecule capable of inhibiting and reversing cardiac fibrosis. In this study, we investigated whether CCN5 can modulate cellular senescence in the heart utilizing three readouts: western blotting for p53 and p21, staining for senescence-associated β-galactosidase, and microscopic analysis of γH2AX-positive foci. CCN5 effectively inhibited doxorubicin-induced cellular senescence in both cardiac myocytes and fibroblasts. In addition, CCN5 suppressed cellular senescence in cardiac myocytes induced by the senescence-associated secretory phenotype factors secreted from cardiac fibroblast, and vice versa. CCN5 also restored the apoptotic response of senescent cells. Finally, CCN5 attenuated myocardial infarction-induced cellular senescence in mice. Collectively, our findings provide novel insights into the potential role of CCN5 in the development of anti-senescence therapies.

Introduction

Cardiovascular diseases, including cardiomyopathy, arrhythmia, and heart failure, remain a leading cause of global mortality, with their prevalence increasing alongside an aging population^1–3. Recent studies have shown that cellular senescence is closely linked to the pathogenesis and progression of diverse cardiovascular diseases by driving structural and functional alterations in the myocardium^4–6.

Stress-induced cellular senescence is a fundamental response to diverse insults such as DNA damage, mitochondrial dysfunction, and oncogenic activation. While it initially serves as a rapid protective mechanism, persistent senescence leads to detrimental consequences. Senescent cells are characterized by growth arrest, morphological changes, increased mitochondrial activity, and resistance to apoptotic stimuli. They also display a distinct phenotype known as the senescence-associated secretory phenotype (SASP), defined by the secretion of various cytokines, growth factors, and matrix metalloproteinases^7–9.

In the heart, senescent cells contribute to interstitial fibrosis, chronic inflammation, and extracellular matrix remodeling, thereby driving both diastolic and systolic dysfunction^4–6. The limited regenerative capacity of the myocardium further exacerbates these age-related changes, increasing vulnerability to stressors such as metabolic overload, hypertension, and ischemic injury. More specifically, senescent cardiac myocytes develop hypertrophy, mitochondrial dysfunction, impaired contractility, and abnormal conduction, ultimately leading to progressive myocardial dysfunction. Senescent cardiac fibroblasts undergo proliferative arrest yet remain metabolically active, characterized by enhanced secretion of SASP-associated factors^10,11.

Two primary strategies have been proposed for targeting senescent cardiac cells. The first employs senolytic drugs to selectively eliminate senescent cells, while the second utilizes senomorphic drugs to suppress deleterious features such as SASP^12,13. Although various nutritional and pharmacological interventions have been investigated^14–17, their effectiveness in preventing or reversing senescence-associated pathologies remains limited, underscoring the urgent need for novel therapeutic strategies.

Cell communication network (CCN) proteins (CCN1-6) are matricellular proteins that regulate diverse cellular processes, including fibrosis, angiogenesis, and wound healing. While most CCN proteins contain four distinct domains, IGFBP, vWC, TSP-1, and CT, CCN5 uniquely lacks the CT domain^18,19. This structural distinction has led to the hypothesis that CCN5 may function as an endogenous inhibitor of other CCN members. Supporting this idea, our group has shown that CCN5 inhibits cardiac hypertrophy and fibrosis, at least in part, by counteracting CCN2 activity^20–22. Previous studies further demonstrated that CCN1 and CCN2 promote fibroblast senescence^23–26. Therefore, we hypothesized that CCN5 may regulate cellular senescence by antagonizing CCN1 or CCN2.

In the present study, we investigated the role of CCN5 in cellular senescence using cultured rat cardiac myocytes and fibroblasts. Our results demonstrated that CCN5 suppressed cellular senescence induced by doxorubicin and by SASP factors in these cells. Moreover, CCN5 attenuated myocardial infarction (MI)-induced cellular senescence in the mouse heart. These findings suggest that CCN5 may serve as a potential therapeutic target for anti-senescence strategies in the heart.

Results

Doxorubicin (Dox) Induces Cellular Senescence in Cardiac Myocytes and Fibroblasts

Doxorubicin (Dox) is known to induce cellular senescence in diverse cell types primarily by eliciting genotoxic stress^27–29. To establish cellular senescence in cultured rat cardiac myocytes (H9c2) and cardiac fibroblasts, cells were treated with 100 nM Dox for 24 hours. Senescence was then assessed using three readouts. First, the expression of cell cycle regulators p53 and p21 was evaluated by western blotting. Second, senescent cells were quantified by microscopic detection of senescence-associated (SA) β-galactosidase activity. Third, accumulation of the DNA damage marker γH2AX was examined by immunostaining. All three assays consistently demonstrated that Dox effectively induced cellular senescence in both cardiac myocytes and fibroblasts (Supplementary Fig. 1). Accordingly, this protocol was employed in subsequent experiments to induce cellular senescence in vitro.

CCN5 inhibits Dox-induced Cellular Senescence in Cardiac Myocytes and Fibroblasts

We next examined whether CCN5 modulates Dox-induced cellular senescence in cardiac myocytes. After 24 hours of Dox treatment, the culture medium was replaced with fresh medium, and purified CCN5 protein (500 ng/mL) was added for an additional 24 hours (Fig. 1A). CCN5 treatment reduced the expression levels of p53 and p21 (Fig. 1B), decreased the number of SA-β-galactosidase-positive cells (Fig. 1C), and lowered the number of γH2AX foci compared with untreated cells (Fig. 1D). These results indicate that CCN5 suppresses cellular senescence in cardiac myocytes.

We then performed similar experiments in cardiac fibroblasts (Fig. 2A). As in cardiac myocytes, CCN5 treatment reduced the levels of senescent marker proteins (Fig. 2B), decreased the number of SA-β-galactosidase-positive cells (Fig. 2C), and diminished γH2AX foci (Fig. 2D). Together, these findings demonstrated that CCN5 inhibits Dox-induced cellular senescence in both cardiac myocytes and fibroblasts.

CCN5 Inhibits SASP-induced Cellular Senescence in Cardiac Myocytes and Cardiac Fibroblasts

Senescent cells secret diverse factors, including cytokines, growth factors, and matrix metalloproteinases, collectively termed SASP factors. These factors exert paracrine effects that drive neighboring healthy cells into cellular senescence. Previous studies have shown that SASP secreted from cardiac myocyte can induce cellular senescence in adjacent fibroblasts^30,31, and vice versa, thereby synergistically accelerating cellular senescence in the heart.

We first tested whether CCN5 prevents cellular senescence in cardiac myocytes upon the treatment with SASP secreted from cardiac fibroblasts (Fig. 3A). Cardiac fibroblasts were treated with Dox for 24 hours, followed by culture in fresh medium lacking Dox for an additional 24 hours. The resulting conditioned medium, enriched with SASP, was applied to cardiac myocytes in the presence or absence of purified CCN5 protein (500 ng/mL). Conditioned medium containing SASP induced cellular senescence in cardiac myocytes, as evidenced by elevated senescent markers (Fig. 3B), an increased number of SA-β-galactosidase-positive cells (Fig. 3C), and reduced γH2AX foci (Fig. 3D). All these effects were significantly suppressed by CCN5 treatment.

We next performed similar experiments to test whether CCN5 prevents cellular senescence in cardiac fibroblasts exposed to SASP secreted from cardiac myocytes (Fig. 4A). In this setting, SASP secreted from cardiac myocytes induced cellular senescence in cardiac fibroblasts, as shown by all readouts, which was markedly attenuated by CCN5 (Fig. 4B ~ D).

Collectively, these findings demonstrated that CCN5 inhibits senescence not only induced by Dox but also triggered by SASP secreted from neighboring cardiac cell types.

CCN5 Restores Apoptotic Responses in Senescent Cardiac Myocytes and Fibroblasts

Senescent cells are resistant to apoptotic stimuli, and their persistence is particularly detrimental in chronic disease states. We therefore tested whether CCN5 could restore apoptotic responsiveness in cardiac myocytes and fibroblasts. Cells were treated with Dox for 24 hours, followed by purified CCN5 protein for an additional 24 hours, as described above. Apoptosis was then induced by treatment with staurosporine (STS, 100 nM) for 9 hours (Fig. 5A, D)³².

Western blotting revealed that cleaved PARP (c-PARP) and cleaved caspase 3 (c-Caspase 3) were significantly elevated in non-senescent cells (Cont), but not in Dox-treated senescent cells (Dox). This implies that the senescent cardiac myocytes and fibroblasts are indeed resistant to STS. CCN5 treatment restored sensitivity to STS in both cardiac myocytes and fibroblasts, as indicated by increased c-PARP and c-Caspase 3 levels (Dox + CCN5) (Fig. 5B, E). Consistently, TUNEL assays showed a marked reduction in TUNEL-positive cells in senescent cells (Dox) compared with controls (Cont), which was significantly reversed by CCN5 treatment (Dox + CCN5) (Fig. 5C, F).

Together, these findings indicate that CCN5 normalizes the apoptotic response in senescent cardiac myocytes and fibroblasts.

CCN5 Inhibits Senescence in an in vivo Mouse Heart Model

We have previously shown that short-term expression of CCN5 via direct intramyocardial injection of modified mRNA encoding CCN5 (ModRNA-CCN5) significantly ameliorates structural and functional deterioration in mouse hearts subjected to myocardial infarction (MI)^33,34. Using the same model, we investigated whether CCN5 also modulates cellular senescence in the infarcted heart. Coronary artery ligation to induce MI and intramyocardial injection of ModRNA-CCN5 were performed sequentially on the same day. Hearts were harvested on day 7 for molecular and histological analyses (Fig. 6A).

ModRNA-mediated expression of CCN5 was confirmed by western blotting (Supplementary Fig. 2). MI markedly increased of p53 and p21 expression (Fig. 6B), the area and intensity of SA-β-galactosidase positive cells (Fig. 6C), and the number of γH2AX-foci (Fig. 6D) compared with sham-operated control hearts. CCN5 treatment significantly attenuated all of these cellular senescence-associated changes (Fig. 6B ~ D). These findings indicate that CCN5 effectively inhibits cellular senescence in vivo in mouse heart following MI.

Discussion

This study demonstrates that CCN5 inhibits cellular senescence in cardiac myocytes and fibroblasts induced by Dox, a widely used reagent for senescence induction. We further showed that CCN5 prevents cellular senescence in these cells driven by SASP factors secreted from neighboring cells in a reciprocal manner. SASP-mediated secondary senescence drives a positive feedback loop that accelerates the expansion of senescent cells in vivo⁵. The anti-senescent effect of CCN5 was further validated in an in vivo model of MI-induced cardiac senescence. Collectively, these findings suggest that CCN5 represent a promising therapeutic target for modulating cellular senescence in the heart.

Other CCN members have been reported to exert context-dependent roles in regulating cellular senescence. For example, CCN1 induces cellular senescence in multiple organs, including the skin, liver, and heart, and in diverse range of cell types, including fibroblasts, chondrocytes, and carcinoma cells^19,20. CCN2 has been shown to promote cellular senescence in skin fibroblast, which helps to restrict fibrosis during tissue repair^21,22. Furthermore, CCN1 and CCN3 have been directly implicated in the pathology of osteoarthritis by inducing chondrocyte senescence^35–37.

This study was motivated by our previous findings that CCN5 antagonizes CCN2 activity in both the heart^20,21 and eye^38–40. CCN2 expression is elevated in failing hearts under various pathological insults and is closely associated with cardiac fibrosis. We previously demonstrated that CCN5 inhibits cardiac fibrosis at least in part by downregulating CCN2. Similarly, CCN2 levels are elevated in the mouse retina under multiple pathogenic conditions and are linked to neovascularization and degeneration of retinal pigmented epithelium. In this contexts as well, CCN5 suppressed retinal pathologies while concomitantly reducing CCN2 expression. Based on these findings, we hypothesized that CCN5 may inhibit cellular senescence in the heart by antagonizing CCN2 and possibly other CCN family members.

Although our data clearly demonstrate that CCN5 inhibits cellular senescence in the heart, the underlying molecular mechanism remains unclear. For example, CCN2 expression levels were unaltered following treatment with Dox⁴¹ or SASP (data not shown). suggesting that suppression of the CCN2 is unlikely to account for the anti-senescence activity of CCN5. We previously showed that CCN5 reverses pre-formed cardiac fibrosis by actively inducing the reverse trans-differentiation of myofibroblasts¹⁹. By analogy, CCN5 may similarly trigger a reverse process of cellular senescence.

Given that CCN5 modulates cellular senescence induced by Dox and SASP, it may serve as a senomorphic strategy by preventing the onset of senescence. Moreover, through its ability to restore apoptotic sensitivity in senescent cells, CCN5 may also function as a senolytic strategy, promoting the removal of existing senescent cells.

Overall, this study elucidates an anti-senescent function CCN5 in the heart. Together with its previously identified anti-fibrotic activity, these findings position CCN5 as a promising therapeutic modality for a broad spectrum of heart diseases.

Methods

Cells and cell culture

Rat ventricular cardiac fibroblasts (Cell Applications) were cultured in FGM medium (Cell Applications). H9c2 cells were cultured in High-Glucose DMEM (HyClone, Cytiva) at 37°C in a 5% CO₂ incubator. HEK 293-F cells (Gibco, #R79007) were suspension-cultured in Freestyle 293 medium (Gibco, #12338018) at 37°C in an 8% CO₂ incubator with continuous shaking.

Purification of Recombinant Proteins

cDNAs encoding the full-length human CCN5 protein were subcloned into a pcDNA3.1-myc-his plasmid and subsequently transfected into HEK 293-F cells. One day prior to transfection, 1 x 10⁶cells were seeded into Freestyle 293 medium. On the day of transfection, plasmid DNA was diluted in Opti-MEM medium (Gibco, #51985034). FectoPRO transfection reagent (Polyplus, #116–001) was then added to the diluted plasmid DNA at a 1:1 ratio, and the mixture was incubated for 10 minutes at room temperature. The mixture was added to the cultured HEK 293-F cells. Three days’ post-transfection, the culture medium was collected and centrifuged at 2,000 × g for 10minutes. Proteins from the culture medium were purified using Capturem His-Tagged Purification Maxiprep Columns (Takara, #635715). The purified proteins were stored in a buffer containing 20 mM NaHPO₄, 150 mM NaCl, and 250 mM imidazole at -70°C⁴².

Induction of Senescence, CCN5 Treatment, and SASP-Containing Media Preparation

Senescence was induced by administering doxorubicin (100 nM) to cells in growth media for 24 hours. Purified CCN5 protein (500ng/ml) was added to cells for 24hours in media containing 0.1% FBS. Staurosporine (100nM) was applied to cells after CCN5 treatment for 9hours. To test SASP, following 24hours of doxorubicin treatment and a single wash with PBS, the medium was replaced with growth medium containing 0.1% FBS to remove doxorubicin. Cells were then incubated for 24 hours to generate SASP-containing conditioned media (CM). CM were treated with CCN5 protein for 24hour.

Western Blotting

Cell lysates were solubilized in RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, pH 8.0) supplemented with Protease Inhibitor Cocktail Set III (Merck Millipore, #535140). Cell lysates were quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, #23227), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, #IPVH00010). Blots were blocked with a 3% BSA solution and incubated with primary antibodies against p53(Abcam), p21(Abcam), p16 (Abcam, #ab32072), GAPDH (CST), beta-actin (Santa Cruz), PARP(CST), caspase3(CST) and cleaved-caspase3(CST) for 12–16 hours at 4°C. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBS-T), blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Scientific, #31460 for rabbit, #31430 for mouse), and washed again. Signals were developed using a EZ-Western Lumi Pico Kit (Dogenbio, Seoul, Korea) and Western Femto ECL Kit (FEMTO-100) and were detected by Amersham™ ImageQuant™ 800 (Cytiva).

Senescence-associated βgalactosidase (SA-βgal) assay

SA-βgal assay was assessed according to the manufacturer’s protocol (CST #9860). For in vitro SA-βgal assay, fibroblasts and myocytes were seeded on 6-well plates. Fluorescence microscopy and DIC images were captured from random fields in each well using an EVOS M7000. SA-βgal positive cells are normalized against Hoechst-positive nucleus. For in vivo SA-βgal assay, frozen heart tissues were outlined with ImmEdge® Hydrophobic Barrier PAP Pen (H-4000) and soaked in SA-βgal staining solution. Fluorescence microscopy and DIC images were captured using Olympus research slide scanner. All assays were performed in at least triplicate, with at least 300-500cells counted per sample from randomly selected fields. SA-βgal signals were analyzed using ImageJ software. The same settings (threshold, color threshold, brightness, analyze particles) were applied for each experimental set. Color Thresholding was applied to selectively retain the blue-colored SA-βgal stained regions. Thresholding process was employed to eliminate background noise, enabling the accurate determination and analysis of both nuclei and the SA-βgal stained areas.

Immunocytochemistry

Cells were seeded onto 16 mm coverslips. After CCN5 treatment, cells were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and blocked with a 5% BSA solution. Subsequently, cells were incubated with a primary antibody against γH2AX (JBW301, Merck), followed by incubation with Alexa Fluor 488 (Invitrogen, A11008) conjugated secondary antibodies. Hoechst 33342 dye was used for nuclear staining. Fluorescence microscopy images were captured using an Olympus fluorescent microscope. All assays were performed in at least triplicate, with at least 100 cells counted per sample from randomly selected fields. Immunofluorescence signals were analyzed using ImageJ software, applying the same settings (threshold, Gaussian distribution, analyze particles) for each experiment set. A Gaussian distribution-based image subtraction was applied to remove noise and extract foci. A thresholding process was also employed to eliminate background noise, enabling the accurate quantification and analysis of both nuclei and the γH2AX foci.

TUNEL Assay

TUNEL assay was performed according to the manufacturer’s instructions (DeadEnd Fluorometric TUNEL System (Promega, San Luis Obispo, CA, USA). The slides with seeded cells were treated with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT) and fluorescein UTP. The reaction was carried out in a humidified chamber at 37 ◦C for 1 hour. After the reaction, the slides were stained with Hoechst 33342 and examined under a fluorescent microscope (Olympus). Apoptotic signals and nuclear signals were counted using ImageJ software with a fixed threshold for each experiment, and the percentage of TUNEL-positive cells was calculated.

Animal Experiments and Myocardial Infarction Model

All animal experimental procedures were approved by the Institutional Animal Care and Use Committees of Gwangju Institute of Science and Technology (GIST).

All procedures were conducted in accordance with relevant guidelines and regulations for laboratory animals. This study is reported in accordance with ARRIVE guidelines. C57BL/6 mice (10–12-week-old males, 25–28 g body weight) were obtained from DBL Inc. (Eumseong, Korea). All efforts were made to minimize animal suffering. All mice were housed in an equipped animal facility with temperature of 18–23°C and humidity at 40–60%, under 12hous light/dark cycle, and had free access to food and water. To induce myocardial infarction model, mice were anesthetized by a mixture of 95 mg/kg ketamine (Yuhan, Korea) and 5 mg/kg xylazine (Bayer, Germany). The left side of the chest was shaved, and an intubation tube was inserted into the trachea. An incision was made in the fourth intercostal space, and the muscles were separated with scissors. The pericardium was gently removed. Myocardial infarction (MI) was induced by ligation of the left anterior descending (LAD) artery using a 7 − 0 black silk suture. The chest was sutured with 4 − 0 black silk. The same surgical procedure, without LAD ligation, was performed for the sham operation. Seven days post-surgery, isoflurane was used for anesthetization via inhalation before euthanasia. Mice were euthanized by cervical dislocation humanely performed by trained researchers in accordance with approved protocols. Harvested hearts were used for western blotting, senescence-associated β-galactosidase (SA-β-gal) assay, and immunohistochemistry.

Production of Modified mRNA-CCN5 and Injection

Polyadenylated and capped (CleanCap™ technology) modified RNAs (ModRNAs) were synthesized by TriLink Biotechnologies (San Diego, CA, USA) with full substitution of uridine with pseudo-uridine. The 5′ and 3′ untranslated regions (UTRs) were designed by the Zangi lab (New York, NY, USA). ModRNAs and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Waltham, MA, USA, #13778150) were separately diluted in Opti-MEM, mixed, and incubated for 15 minutes at room temperature. A total of 50 µg of the ModRNA mixture was directly injected into the endocardium adjacent to the infarcted area.

Histology and Immunohistochemistry

Harvested tissue samples were fixed in 4% paraformaldehyde for 72 hours at 4°C and incubated in 30% sucrose for 72 hours at 4°C as a cryoprotectant. The tissues were embedded in OCT compound (3801480, Leica, Germany). Frozen tissue blocks were sectioned at 8 µm thickness using a cryostat microtome (HM525NX, Thermo Scientific, USA). Sections were mounted on adhesive slides (J1800AMNZ, Epredia). For γH2AX, immunohistochemistry was performed, which included antigen retrieval by boiling in a pH 6 citrate buffer. Primary antibodies (JBW301, Merck, 1:250) were diluted in a 3% bovine serum albumin blocking solution and applied for incubation. Secondary Alexa Fluor 594 (Invitrogen, A11032) conjugated antibodies were then applied and visualized. Images were acquired using Olympus confocal microscopy (Olympus, Japan). Immunofluorescence signals were analyzed using ImageJ software applying the same settings (threshold, Gaussian distribution, analyze particles) for each experiment set. A Gaussian distribution-based image subtraction was applied to remove noise and extract foci. A thresholding process was employed to eliminate background noise, enabling the accurate quantification and analysis of both nuclei and the γH2AX foci.

Statistical Analysis

N numbers represent independent biological observations. All experiments were repeated independently at least three times. One-Way Analysis of Variance (ANOVA) was employed for statistical analyses to determine the significance of the data using GraphPad Prism 7 software (GraphPad Software, San Diego, CA). An asterisk (*, p ≤ 0.05) or a double asterisk (**, p ≤ 0.01) indicates statistical significance. Data are presented as the mean ± standard deviation.

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Supplementary Material 1

Author Contribution

Conceptualization, S.P.J. and W.J.P.; methodology, J.Y.J. and M.Y.L.; validation, S.B.K., J.Y.J. and M.Y.L.; formal analysis, J.Y.J.; investigation, J.Y.J.; data curation, S.B.K. and S.P.J.; writing—original draft preparation, J.Y.J.; writing—review and editing, J.Y.J., S.P.J. and W.J.P.; supervision, S.P.J. and W.J.P.; Resources, T.H.K. and D.T.J.; project administration, W.J.P.; funding acquisition, W.J.P.

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Acknowledgement

We thank Dr. YS. Oh (Central Research Facility of Gwangju Institute of Science and Technology) for technical assistance of the confocal microscopy. Figures 1 A, 2 A, 3 A, 4 A, 5A and 5D were created with BioRender.com. (https://www.biorender.com, https://BioRender.com/w1x0kdx). During the preparation of this manuscript, the authors used ChatGPT (version 5.0) to assist with proofreading and improving the readability of the manuscript. All content was reviewed and edited by the authors, who take full responsibility for the final version of the publication.

Author contributions statement

Funding

This study was supported by funding from the Korea–US Collaborative Research Fund (RS-2024-00466906) and the National Research Council of Science & Technology(NST) grant by the Korea government (MSIT) (No. GTL24021-000).

Competing interests

No potential conflicts of interest exist for other authors.

Data Availability

All relevant data are within this paper and its Supporting Information files. The datasets used or analyzed during this study are available from the corresponding authors upon reasonable request.

Figure Legends

Fig. 1

CCN5 inhibits Dox-induced Cellular Senescence in Cardiac Myocytes. (A) An experimental scheme is shown (created using Biorender). (B) H9c2 cardiac myocytes were treated with Dox, followed by CCN5 treatment. Cell lysates were immunoblotted for senescence marker proteins p53 and p21. Protein levels were quantified and normalized to GAPDH (n = 4). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) H9c2 cells were stained for SA-β-gal activity. Positive cells were counted in random fields and plotted as a percentages of total cell number (n = 3) (D) H9c2 cells were immunostained with antibodies against γH2AX (green) and counter-stained with Hoechst (blue). γH2AX foci were counted in random fields and plotted as a percentage of total nuclei (n = 3). Scale bar: 50 µm. Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01).

Fig. 2

CCN5 inhibits Dox-induced Cellular Senescence in Cardiac Fibroblasts. (A) An experimental scheme is shown (created using Biorender). (B) RCFs were treated with Dox, followed by CCN5 treatment. Cell lysates were immunoblotted for senescence marker proteins p53 and p21. Protein levels were quantified and normalized to GAPDH (n = 4). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) RCFs were stained for SA-β-gal activity. Positive cells were counted in random fields and plotted as a percentages of total cell number (n = 3) (D) RCFs were immunostained with antibodies against γH2AX (green) and counter-stained with Hoechst (blue). γH2AX foci were counted in random fields and plotted as a percentage of total nuclei (n = 3). Scale bar: 50 µm. Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01).

Fig. 3

CCN5 Inhibits Cellular Senescence of Cardiac Myocytes Induced by SASP from Fibroblasts. (A) An experimental scheme is shown (created using Biorender). (B) H9c2 cardiac myocytes were treated with Dox-media or CCN5. Cell lysates were immunoblotted for senescence marker proteins p53 and p21. Protein levels were quantified and normalized to GAPDH (n = 4). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) H9c2 cells were stained for SA-β-gal activity. Positive cells were counted in random fields and plotted as a percentages of total cell number (n = 3 ~ 4) (D) H9c2 cells were immunostained with antibodies against γH2AX (green) and counter-stained with Hoechst (blue). γH2AX foci were counted in random fields and plotted as a percentage of total nuclei (n = 3). Scale bar: 50 µm. Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01). Dox-media = Doxorubicin treated and SASP-containing conditioned medium

Fig. 4

CCN5 Inhibits Cellular Senescence of Cardiac Fibroblasts Induced by SASP from Cardiac Myocytes. (A) An experimental scheme is shown (created using Biorender). (B) RCFs were treated with Dox-media or CCN5. Cell lysates were immunoblotted for senescence marker proteins p53 and p21. Protein levels were quantified and normalized to GAPDH (n = 4). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) RCFs were stained for SA-β-gal activity. Positive cells were counted in random fields and plotted as a percentages of total cell number (n = 3) (D) RCFs were immunostained with antibodies against γH2AX (green) and counter-stained with Hoechst (blue). γH2AX foci were counted in random fields and plotted as a percentage of total nuclei (n = 3). Scale bar: 50 µm. Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01). Dox-media = Doxorubicin treated and SASP-containing conditioned medium

Fig. 5

CCN5 Restores Apoptotic Responses in Senescent Cardiac Myocytes and Fibroblasts. (A) An experimental schema is shown for B and C (created using Biorender). (B) H9c2 cardiac myocytes were treated with Dox, followed by CCN5 treatment and then STS. Cell lysates were immunoblotted for apoptosis marker proteins c-PARP and c-caspase3. Protein levels were quantified and normalized to GAPDH (n = 4 ~ 7). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) H9c2 were stained for TUNEL assay; TUNEL positive cells were counted in random fields and plotted as percentages of total cell number (N = 3). (D) An experimental schema is shown for E and F (created using Biorender). (E) RCFs were treated with Dox, followed by CCN5 treatment and then STS. Cell lysates were immunoblotted for apoptosis marker proteins c-PARP and c-caspase3. Protein levels were quantified and normalized to GAPDH (n = 4 ~ 7). Panel E is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (F) RCFs were stained for TUNEL assay; TUNEL positive cells were counted in random fields and plotted as percentages of total cell number (N = 3). Scale bar: 50 µm. Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01).

Fig. 6

CCN5 Inhibits Senescence in an in vivo Mouse Heart Model. (A) An experimental scheme is shown (created using Biorender). (B) Heart extract was immunoblotted for senescence marker proteins p53 and p21. Protein levels were quantified and normalized to GAPDH (n = 7). Panel B is a composite from separate blots for clarity. The uncropped blots are presented in Supplementary information. (C) Heart sections were stained for SA-β-gal activity. Positive cells were counted in random fields and plotted as a percentages of total cell number (n = 5) (D) Heart sections were immunostained with antibodies against γH2AX (purple) and counter-stained with Hoechst (blue). γH2AX foci were counted in random fields and plotted as a percentage of total nuclei (n = 4). Scale bar: 50 µm (500µm for 2x images). Data are shown as the mean ± SD. (one-way ANOVA; *p < 0.05, **p < 0.01).

Fig. 1

CCN5 inhibits Dox-induced Cellular Senescence in Cardiac Myocytes