Sub1 contributes to heart failure with preserved ejection fraction driven by aging in mice

Abstract

Heart failure with preserved ejection fraction (HFpEF) accounts for approximately half of all heart failure cases and predominantly affects older individuals, yet effective treatments remain limited. The molecular mechanisms linking cardiac aging to HFpEF are not fully understood. Here we show that the transcriptional regulator Sub1 is upregulated in aged hearts and in mouse models of HFpEF. Cardiac overexpression of Sub1 shortens lifespan, exacerbates diastolic dysfunction, and accelerates cardiac aging, whereas Sub1 knockdown delays cardiac aging and alleviates HFpEF features, even when initiated in aged mice. Mechanistically, Sub1 interacted with TAF9b and AROS to stabilize p53 by regulating its ubiquitination and acetylation, thereby promoting cardiomyocyte senescence. Furthermore, disrupting the Sub1–p53 interaction attenuates cardiomyocyte senescence in vitro. These findings identify Sub1 as a contributor to aging-associated HFpEF and provide insight into the molecular links between cardiac aging and disease progression.

Heart failure with preserved ejection fraction (HFpEF) predominantly affects older individuals. Here the authors report that the transcriptional regulator Sub1 promotes age-related HFpEF in mice, potentially via stabilizing p53 and subsequent cardiomyocyte senescence.

Introduction

Heart failure with preserved ejection fraction (HFpEF) accounts for nearly half of all heart failure cases and remains a major unmet clinical challenge due to the lack of effective therapies¹. Aging is the most important risk factor for HFpEF, and its prevalence is steadily increasing in parallel with the aging global population². HFpEF is therefore widely recognized as a geriatric syndrome and has become the most common form of heart failure among the elderly^3–5. It is frequently associated with comorbidities such as hypertension, diabetes mellitus, and obesity—all of which are known to accelerate cardiac aging⁶.

Aging induces profound structural and functional alterations in the heart, including cardiomyocyte hypertrophy, myocardial fibrosis, and diastolic dysfunction^7,8—hallmarks that closely resemble the pathological features of HFpEF⁹. Increasing evidence suggests that cellular senescence is a key driver of cardiac aging. Senescent cells progressively accumulate in the aging myocardium and contribute to tissue dysfunction through irreversible cell-cycle arrest and the secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP)^5,7. The buildup of senescent cardiomyocytes has been implicated in myocardial remodeling and impaired cardiac function during aging and HFpEF⁹.

Among the central regulators of cellular senescence, p53 plays a pivotal role in coordinating DNA damage responses, cell-cycle arrest, and senescence programs. However, excessive accumulation of senescent cells can drive tissue aging and age-related pathologies. Activation of p53 signaling is therefore closely linked to cardiac aging and age-associated cardiovascular diseases¹⁰. The upstream regulatory mechanisms controlling p53 activity in cardiomyocytes during cardiac aging remain incompletely understood.

Sub1 is a transcriptional coactivator involved in transcriptional regulation, oxidative stress responses, and maintenance of genomic stability^11–15. Previous studies have suggested that Sub1 may facilitate p53 recruitment to target loci and enhance its transcriptional activity in cancer cells¹⁶. Although Sub1 has been implicated in systemic aging phenotypes¹⁷, its role in cardiac aging and HFpEF remains largely unknown.

In this study, we identified a marked upregulation of Sub1 in aged hearts. Mechanistically, Sub1 promotes cardiac aging by disrupting the balance of p53 post-translational modifications (PTMs), leading to enhanced p53 stabilization and senescence signaling. Importantly, knockdown of Sub1 alleviated HFpEF phenotypes and attenuated cardiac aging, suggesting that Sub1 may represent a potential therapeutic target for age-related cardiac dysfunction.

Results

Age-dependent upregulation of Sub1 in myocardium drives HFpEF

To identify anti-aging therapeutic targets for HFpEF, we analyzed two myocardial RNA sequencing datasets (E-MTAB-7454 and PRJEB62450) comprising 15 non-HFpEF and 11 HFpEF samples. Cross-dataset analysis revealed 51 differentially expressed genes (DEGs) in HFpEF (Fig. 1a). Among these, Sub1 emerged as a prioritized candidate due to its significant upregulation in HFpEF myocardial tissues compared with controls (Fig. 1b). Sub1, previously identified as an aging-related gene¹⁷, prompted us hypothesize that it may serve as a potential anti-aging target for HFpEF. Single-cell RNA-seq analysis further revealed age-dependent Sub1 elevation in cardiomyocytes, correlating with age-related cardiomyocytes loss (Fig. 1c, Suppl. Fig. 1a, b). Those findings were reinforced by GTEx database analysis, where Sub1 mRNA expression in human hearts exhibited a progressive increase with advanced age (Fig. 1d), and showed strong positive correlations with aging-associated genes from the GenAge database (Fig. 1e). To validate these observations, we established in vitro senescence model in bleomycin-administrated HL-1 cardiomyocytes. Both qPCR and immunoblotting confirmed a time-dependent upregulation of Sub1 mRNA and protein, paralleled by elevated cardiac-specific senescence markers (Fig.1f, g, Suppl. Fig. 2a). Aligning with qPCR results, aged murine hearts (120-week-old) displayed significantly higher Sub1 protein levels compared to younger counterparts (8- and 60-week-old) (Fig. 1h). Collectively, these multi-layered analyses establish Sub1 as an aging-associated gene dynamically upregulated in HFpEF and senescence models.

Fig. 1. Sub1 in the heart exhibits an age-dependent increase and is associated with HFpEF.

a Identification of Sub1 as a novel anti-aging target in HFpEF. Differentially expressed genes (DEGs) in the E-MTAB-7454 dataset (n = 11 controls vs. 5 HFpEF) were overlapped with DEGs in the PRJEB62450 dataset (n = 4 controls vs. 6 HFpEF). b Sub1 expression levels in heart samples from non-HFpEF donors and HFpEF patients (n = 11 vs. 5 and n = 4 vs. 6, respectively). c Single-cell RNA sequencing analysis of Sub1 expression in cardiomyocytes from different age groups: middle-aged (20–59 years) and old (60–100 years) (n = 32567 and 36854 cells per group). The box represents the interquartile range (IQR), the center line indicates the median, and whiskers represent minimum and maximum values. d Sub1 expression in donor heart tissues from the GTEx database across the age range of 20–80 years (n = 14, 147, 154, 66, 26, and 22 per group). e Correlation analysis between Sub1 and aging-associated genes from the GenAge database using RNA-seq data of human heart samples from GTEx. The top 36 correlated genes are shown. f Sub1 mRNA levels in HL-1 cells treated with bleomycin for 6, 12, 24, 36, or 48 h ((n = 4 biological replicates). g Representative western blots of Sub1, the DNA damage marker γ-H2AX, and cell senescence markers (p53, p21, and p16) in HL-1 cells treated with bleomycin at the indicated time points ((n = 4 per group). h Representative western blots of Sub1 and cell senescence markers (p53, p21, p16) in mouse heart tissues at the indicated ages ((n = 4 biological replicates). For (b, c), P-values were determined by two-tailed unpaired Student’s t-test. For (d, f), p values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Sub1 overexpression accelerates cardiomyocyte senescence

The observed upregulation of Sub1 in aging cardiomyocytes prompted us to investigate its potential role in cardiac aging. To evaluate the effect of Sub1 on cardiomyocyte senescence in vitro, HL-1 cells were transduced with a Sub1-overexpressing lentivirus and treated with bleomycin. Compared to control cells, Sub1-overexpressing HL-1 cells exhibited elevated levels of senescence markers (p53, p21, p16) and DNA damage marker γ-H2Ax (Suppl. Fig. 3a). qRT-PCR further confirmed increased expression of cell cycle inhibitors (p16, p21) and senescence-associated secretory phenotype (SASP) factors (Suppl. Fig. 3b). Senescence-associated β-galactosidase (SA-β-gal) activity, a hallmark of cellular senescence, was assessed using both SA-β-gal and SPiDER-β-gal assays. Sub1-overexpressing cells displayed enhanced SA-β-gal activity following bleomycin treatment (Suppl. Fig. 3c, d). IF staining of senescence markers (p21) and genomic integrity markers (53BP1 and γ-H2Ax) yielded consistent results (Suppl. Fig. 3e). To further validate the role of Sub1, we knocked down its expression in HL-1 cells using Lenti-shSub1. Sub1 silencing mitigated bleomycin-induced senescence, as evidenced by reduced senescence marker expression and SA-β-gal activity (Suppl. Fig. 4). Collectively, these findings indicate that Sub1 overexpression promotes, while its knockdown attenuates, cardiomyocyte senescence.

Cardiomyocyte-specific overexpression of Sub1 accelerates cardiac aging and shortens lifespan

To investigate the role of Sub1 in cardiac aging in vivo, we generated cardiomyocyte-specific Sub1-overexpressing mice (Sub1 Tg) by crossing Sub1 Rosa flox/flox mice with Myh6-MerCreMer mice (Suppl. Fig. 5a). A natural aging model was established over time as outlined in Fig. 2a. At 8–12 weeks of age, tamoxifen was administered intraperitoneally to induce Sub1 overexpression specifically in cardiomyocyte. No significant differences in cardiac hypertrophy, aging markers, or cardiac function were observed between Sub1 Tg and Ctrl fl/fl mice at 25–35 weeks of age (Suppl. Fig. 6, Suppl. Table 3). However, at 70–80 weeks of age, Sub1 Tg mice began to exhibit abnormalities, primarily characterized by reduced activity (Suppl. Fig. 5b). While wild-type C57BL/6 J mice typically live for 110–120 weeks, Sub1 Tg mice showed a markedly shortened lifespan, averaging 85 weeks, significantly lower than that of Ctrl fl/fl mice. By 100 weeks, only 20% of Sub1 Tg mice survived, compared to 80% of controls (Fig. 2b). Histological analysis revealed pronounced cardiac hypertrophy and an increase in cardiomyocyte cross-sectional area in Sub1 Tg mice (Suppl. Fig. 5c, d). Markers of cardiac aging, including SA-β-gal, lipofuscin accumulation and p53/p21 expression, were significantly elevated in the hearts of Sub1 Tg mice relative to controls (Fig. 2c, d). TEM showed that Sub1 Tg mice had mitochondria with structural abnormalities, such as loss of cristae and reduced mitochondrial area (Suppl. Fig. 5g). Accordingly, the abnormal mitochondrial ultrastructure observed in the hearts of Sub1 Tg mice also leads to mitochondrial functional impairment (Suppl. Fig. 5h-j). Moreover, Sub1 Tg hearts displayed extensive lipofuscin deposition, further indicating premature cardiac aging (Fig. 2e). Functionally, Sub1 Tg mice showed mildly impaired systolic function, but the decline in diastolic function was more pronounced. Echocardiographic analysis revealed increased E/A and E/e’ values in Sub1 Tg mice, consistent with diastolic dysfunction (Fig. 2f, g, Suppl. Table 4). This reduced diastolic performance led to pulmonary congestion and reduced exercise capacity in Sub1 Tg mice (Suppl. Fig. 5e, f). At both the protein and mRNA levels, senescence markers were significantly upregulated in Sub1 Tg myocardial tissue compared to age-matched controls (Fig. 2h, i). RNA-seq analysis of heart tissues revealed differentially expressed genes (DEGs) enriched in aging-related pathways, such as cellular senescence and ATP metabolic process (Suppl. Fig. 7a–f). Other organs in Sub1Tg mice (plasma biomarker, liver, kidney, and skeletal muscle) did not exhibit premature aging (Suppl. Fig. 5k–m). Collectively, these findings demonstrate that cardiomyocyte-specific overexpression of Sub1 promotes premature cardiac aging and reduces overall lifespan.

Fig. 2. Cardiomycyte-specific Sub1 overexpression accelerates age-related cardiac remodeling and dysfunction.

a Schematic diagram of the experimental design showing natural aging in control floxed (Ctrl fl/fl) and Sub1 transgenic (Sub1 Tg) mice. b Kaplan–Meier survival analysis of Ctrl fl/fl ((n = 11 biological replicates) and Sub1 Tg mice ((n = 11 biological replicates) during natural aging. c Representative Sudan Black B and SPiDER β-gal staining in the hearts of aging Ctrl fl/fl and Sub1 Tg mice. Scale bars, 50 μm and 100 μm. Quantification of lipofuscin-positive and SPiDER β-gal-positive areas (%) ((n = 8 biological replicates). d Representative IF of p53 and p21 in the hearts of aging Ctrl fl/fl and Sub1 Tg mice. Scale bars, 50μm. Quantification of relative IF intensity ((n = 8 biological replicates). e Representative transmission electron microscopy (TEM) images of myocardial tissue from aging Ctrl fl/fl and Sub1 Tg mice (Lp, lipofuscin). Scale bars, 2μm, 1μm, and 0.5μm. Quantification of lipofuscin number and size using violin plots (n = 15 fields of view from 4 mice per group). f Representative echocardiographic images (M-mode, MV velocity, and tissue Doppler imaging [TDI]). g Quantification of left ventricular ejection fraction (LVEF), E/A ratio, and E/e′ ratio ((n = 8 biological replicates). h Representative western blots of Sub1, p53, p21, and p16 in heart tissues of aging Ctrl fl/fl and Sub1 Tg mice ((n = 6 biological replicates). i mRNA levels of Sub1, Nppb, Myh7, Il12a, Il6, Cdkn1a (p21), Cdkn2a (p16), Pot1a, and Tpp1 in heart of aging Ctrl fl/fl and Sub1 Tg mice (n = 6 per group). For (b), survival curves were analyzed using the Kaplan–Meier method and compared using a two-sided log-rank (Mantel–Cox) test. No adjustment for multiple comparisons was applied. For (c–e) and (g–i), p values were determined by two-tailed unpaired Student’s t-test. Data are presented as mean ± SD.

Global Sub1 deficiency delays cardiac aging phenotype

To further investigate the role of Sub1 in cardiac aging, we generated Sub1 knockdown mice (Sub1−/+) using the CRISPR/Cas9 system, as homozygous deletion of Sub1 (Sub1-/-) resulted in embryonic lethality (Suppl. Fig. 8a). In young mice, Sub1−/+ animals exhibited no significant differences in cardiac function or aging markers compared to Sub1 + /+ controls (Suppl. Fig. 9, Suppl. Table 5). However, by 120 weeks of age, 75% of Sub1−/+ mice remained alive, in contrast to only 40% of control mice, suggesting that partial Sub1 deficiency may extend lifespan (Fig. 3a, b). While Sub1-overexpressing mice displayed significant cardiac hypertrophy, Sub1−/+ mice did not show a corresponding improvement in hypertrophy, potentially due to age-related structural changes already being established (Suppl. Fig. 8b–d). Nonetheless, Sub1−/+ mice exhibited marked improvements in myocardial fibrosis (Suppl. Fig. 8d), exercise capacity (Suppl. Fig. 8e), pulmonary congestion (Suppl. Fig. 8f), and the expression of aging markers (Fig. 3c–e, h–i) and mitochondrial markers (Suppl. Fig. 8g–j), indicating that Sub1 knockdown delays cardiac aging. Although Sub1 knockdown did not significantly restore age-related declines in systolic function, it did improve diastolic function, as evidenced by echocardiographic measurements (Fig. 3f, g, Suppl. Table 6). Given the systemic nature of Sub1 knockdown, aging-related phenotypes in other organs were also moderately improved in Sub1−/+ mice (Suppl. Fig. 8k–m). Taken together, these findings suggest that partial Sub1 deficiency alleviates age-associated diastolic dysfunction and pathological cardiac remodeling, and may confer systemic anti-aging benefits.

Fig. 3. Global Sub1 konckdown delays age-associated cardiac remodeling and dysfunction.

a Schematic diagram depicting the procedure of natural aging Sub + /+ and Sub1 −/+ mice. b Kaplan–Meier analysis of the survival of Sub +/+ (n = 1(n = 12 biological replicates) and Sub1−/+ mice ((n = 13 biological replicates) in the natural aging process. c Representative Sudan Black B staining and SPiDER β-gal staining of the heart from aging Sub +/+ and Sub1 −/+ mice. Scale bars, 50 μm and 100 μm. Quantification of lipofuscin-positive (%) and SPiDER β-gal-positive (%). (n = 8 biological replicates). d Representative IF of p53 and p21 in the hearts of aging Sub +/+ and Sub1 −/+ mice. Scale bars, 50μm. Quantification of relative IF intensity (n = 8 biological replicates). e Representative TEM images of the myocardium of aging Sub +/+ and Sub1 −/+ mice. Lp, Lipofuscin. Scale bars, 2 μm, 1 μm and 0.5 μm. Quantification of lipofuscin number and size using violin plots (n = 15 fields of view from 4 mice per group). f Representative images of echocardiography (M-Mode, MV Velocity and TDI). g Statistical analysis of LVEF, E/A ratio and E/e’ ratio (n = 8 biological replicates). h Representative western blot of Sub1, p53, p21, and p16 in the heart from aging Sub +/+ and Sub1 −/+ mice (n = 6 biological replicates). i mRNA levels of Sub1, Nppb, Myh7, Il12a, Il6, Cdkn1a (p21), Cdkn2a (p16), Pot1a, and Tpp1 in heart of aging Sub +/+ and Sub1 −/+ mice. (n = 6 biological replicates). For (b), survival curves were analyzed using the Kaplan–Meier method and compared using a two-sided log-rank (Mantel–Cox) test. No adjustment for multiple comparisons was applied. For (c–e) and (g–i), p values were determined by two-tailed unpaired Student’s t-test. Data are presented as mean ± SD.

Knockdown of Sub1 protects HFpEF, an aging-driven CVD

HFpEF is the most common CVD in the elderly and is increasingly recognized as a true geriatric syndrome⁵. Given that Sub1 promotes cardiomyocyte senescence, we hypothesized that it might serve as a potential anti-aging therapeutic target for HFpEF. We established a “two-hit” HFpEF mouse model using 50–60-week-old mice to better capture age-related cardiac phenotypes (Fig. 4a). Considering the sex-related nature of HFpEF, both male and female mice were included in our experiments (Suppl. Fig. 11). In line with reduced blood pressure, Sub1−/+ mice exhibited decreased wet lung weight, indicating attenuated pulmonary congestion (Fig. 4b, c), along with improved exercise tolerance (Fig. 4d). Myocardial hypertrophy and fibrosis were also alleviated in Sub1−/+ mice (Fig. 4e, f, Suppl. Fig. 10a–c), with beneficial effects observed in both sexes (Suppl. Fig. 11a–h). Although HFpEF mice preserved systolic function, they displayed impaired diastolic function as evidenced by decreased E/A and E/e’ ratios, similar to age-related diastolic dysfunction. Notably, Sub1−/+ mice showed partial recovery of diastolic performance (Fig. 4j–l, Suppl. Fig. 11l–n, Suppl. Tables 7, 8). HFpEF mice also exhibited elevated expression of senescence markers, suggesting accelerated cardiac aging. Interestingly, we observed remarkable similarities in physiological, histological, and echocardiographic features between HFpEF and aged mice. Most importantly, Sub1 knockdown mitigated HFpEF phenotypes by slowing cardiac aging (Fig. 4g–i, m, n, Suppl. Fig. 11i–k, o, p). To further clarify the interplay between aging and HFpEF progression, we induced the HFpEF model in both young (20-week-old) and middle-age (60-week-old) mice. Aging mice developed HFpEF criteria more rapidly, indicating that aging accelerates HFpEF development (Suppl. Fig. 12). Low-dose doxorubicin, a common inducer of cardiac aging, abolished the protective effects of Sub1 knockdown in the HFpEF model (Fig. 5a–i, Suppl. Fig. 13, Suppl. Table 9). In summary, our findings support the concept that HFpEF is closely associated with cardiac senescence, and that targeting Sub1 may alleviate HFpEF symptoms by delaying heart aging.

Fig. 4. Knockdown of Sub1 protects HFpEF phenotypes by decelerating heart aging.

a Schematic diagram of the HFpEF modeling procedure. b–d Statistical analysis of SBP (systolic blood pressure), DBP (diastolic blood pressure) (b), wet/dry lung ratio (c) and running distance in exercise tolerance test (d) for Chow and HFpEF Sub1 + /+ and Sub1–/+ mice (n = 6 biological replicates). e Representative WGA staining of the heart from Chow and HFpEF mice. Scale bars, 50 μm. Quantification of cardiomyocyte cross-sectional area (n = 6 biological replicates). f Representative Masson’s trichrome staining of the heart from Chow and HFpEF mice. Scale bars, 50 μm. Quantification of fibrosis-positive (%) (n = 6 biological replicates). g Representative SPiDER β-gal staining of the heart from Chow and HFpEF mice. Scale bars, 50 μm. Quantification of SPiDER β-gal-positive (%) (n = 6 biological replicates). h Representative Sudan Black B staining of the heart from Chow and HFpEF mice. Scale bars, 100 μm. Quantification of lipofuscin-positive (%) (n = 6 biological replicates). i Representative TEM images of the myocardium from Chow and HFpEF mice. Lp, lipofuscin. Scale bars, 1 μm. Violin plot of lipofuscin number and size obtained using TEM analysis (n = 15 fields of view in 3 mice per group). j–l Representative echocardiographic images (M-mode, MV velocity, and TDI) and quantification of LVEF, E/A ratio, and E/e′ ratio (n = 6 biological replicates). m Representative western blot of Sub1, p53, p21, and p16 in the heart from Chow and HFpEF mice (n = 6 biological replicates). n mRNA levels of Cdkn1a (p21), Cdkn2a (p16), Nppb, and Nppa in hearts from Chow and HFpEF mice (n = 6 biological replicates). For (b–l, n), p-values were determined by two-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Fig. 5. Doxorubicin-induced aging abolishes Sub1-mediated protection in HFpEF mice.

a Schematic diagram of HFpEF modeling and doxorubicin (Dox) treatment. b, c Statistical analysis of SBP, DBP (b), and running distance in exercise tolerance test (c) of mice from groups G1 to G4 (n = 5 biological replicates). d Representative Masson’s trichrome and WGA staining of the heart of mice from G1 to G4. Scale bars, 50 μm. Quantification of fibrosis positive (%) and cardiomyocyte cross-sectional area in the heart (n = 5 biological replicates). e Representative Sudan Black B staining and SPiDER β-gal staining of the heart of mice from G1 to G4. Scale bars, 50 μm and 100 μm. Quantification of lipofuscin positive (%) and SPiDER β-gal positive (%) in the heart (n = 5 biological replicates). f Representative TEM images of the myocardium of mice from G1 to G4. Lp, Lipofuscin. Scale bars, 1 μm. Violin plot of Lipofuscin number and size obtained using TEM analysis (n = 15 fields of view in 5 mice per group). g Representative images of echocardiography (M-Mode, MV Velocity and TDI) and statistical analysis of E/A ratio and E/e’ ratio (n = 5 biological replicates). h Representative western blot of Sub1, p53, p21, and p16 in the heart of mice from G1 to G4 (n = 5 biological replicates). i mRNA levels of Cdkn1a (p21), Cdkn2a (p16), Il12a, and Il6 in hearts from chow-fed and HFpEF mice (n = 5 biological replicates). For (b–g, i), p-values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Identification of TAF9b and AROS as potential interacting proteins of Sub1 in cardiac senescence

As a transcriptional coactivator, Sub1 interacts with numerous nuclear proteins. To identify proteins that interact with Sub1 during cardiomyocyte senescence, we performed LC-MS/MS analysis on bleomycin-treated HL-1 cells transfected with Flag-Sub1 (Fig. 6a). Among the identified proteins, we focused on the top 15 significantly affected by bleomycin treatment, notably including p53, TAF9b, and AROS (Fig. 6b). Protein-protein interaction (PPI) analysis using the STRING database revealed p53 as the central node in the network (Suppl. Fig. 14a). Sub1 facilitates p53 binding to its target sites and enhances the transcription of p53-responsive genes¹⁵; p53 is well known for its role in inducing cell cycle arrest and cellular senescence¹⁸. Although direct interactions between Sub1 and TAF9b or AROS have not been previously reported, molecular docking analysis suggested promising binding potential between Sub1 and both TAF9b and AROS (Suppl. Fig. 14b-d). Functionally, TAF9b stabilizes p53 by inhibiting MDM2-mediated ubiquitination and degradation¹⁹, whereas AROS enhances SIRT1-mediated deacetylation of acetylated p53 at lysine 382 (Ac-p53(K382)), thereby repressing p53 activity²⁰. Our co-immunoprecipitation (Co-IP) assays in HL-1 and AMVMs confirmed the interactions between Sub1 and p53, TAF9b, and AROS (Fig. 6c, Fig. 7a, Suppl. Fig. 14e). Taken together, we hypothesize that Sub1 regulates cardiomyocyte senescence by modulating post-translational modifications (PTMs) of p53 through its interactions with TAF9b and AROS.

Fig. 6. Sub1 facilitates TAF9b–p53 interaction and suppresses ubiquitination-mediated p53 degradation.

a Schematic of quantitative proteomic screening to identify Sub1-binding proteins in a cellular senescence model. b Table of Sub1-interacting proteins significantly altered by bleomycin treatment, ranked by interaction ratio. c Co-immunoprecipitation (Co-IP) validating interactions between Sub1 and TAF9b or p53 in HEK293T cells (n = 3 independent experiments). d mRNA levels of p53 in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with bleomycin for 24 h (n = 4 per group). e, f Representative western blots and quantification of p53 protein turnover in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with cycloheximide (CHX, 10 μM) and bleomycin (n = 3 independent experiments). g Representative western blots of p53 in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with proteasome inhibitor MG132 (20 μM) and bleomycin (n = 3 independent experiments). h p53 immunoprecipitation showing ubiquitinated p53 levels in bleomycin-treated Sub1-overexpressing and Sub1-knockdown HL-1 cells (n = 3 independent experiments). i Schematic of the structural domains and interactions among Sub1, TAF9b, and p53. j, k Co-IP assays mapping the binding regions of TAF9b with Sub1 and Sub1 with TAF9b in HEK293T cells (n = 3 independent experiments). l Representative western blots of Sub1, TAF9b, p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b (n = 3 independent experiments). m Representative SPiDER β-gal staining of Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b. Scale bars, 20 μm (n = 6 per group). n p53 immunoprecipitation showing ubiquitinated p53 levels in Sub1-overexpressing HL-1 cells treated with bleomycin and si-TAF9b (n = 3 independent experiments). For (d), p-values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Fig. 7. Sub1 competes with SIRT1 for AROS binding and preserves acetylated p53.

a Co-IP assays validating the interaction between exogenous Sub1 and AROS in HEK293T cells (n = 3 independent experiments). b Representative western blots of Sub1 and acetylated p53 (K382) in Sub1-overexpressing and Sub1-knockdown HL-1 cells treated with bleomycin (n = 3 independent experiments). c Representative western blot of Sub1 and acetylated p53 (K382) in aged Ctrl fl/fl, Sub1 Tg and Sub1 + /+, Sub1 −/+ mice, (n = 3 independent experiments). d Representative western blots of acetylated p53 (K382) and SIRT1 in HL-1 cells transfected with increasing amounts of Flag-Sub1 plasmid (n = 3 independent experiments). e Schematic of the structural domains and interactions among Sub1, AROS, and SIRT1. f Co-IP mapping the binding regions of AROS with Sub1 and SIRT1 in HEK293T cells (n = 3 independent experiments). g Co-IP of SIRT1 in HEK293T cells co-transfected with Flag-Sub1 and HA-AROS plasmids (n = 3 independent experiments). h Representative SPiDER β-gal staining of Sub1-overexpressing HL-1 cells treated with bleomycin and HA-AROS. Scale bars, 20μm (n = 6 per group). i Representative western blots of Sub1, HA, acetylated p53 (K382), total p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin and HA-AROS (n = 3 independent experiments). j Representative western blots of Sub1, p53, p21, and p16 in Sub1-overexpressing HL-1 cells treated with bleomycin, Mutpep, and Acpep at the indicated concentrations (n = 3 independent experiments). k Quantification of p53 transcriptional activity on Cdkn1a (p21) promoter using luciferase assays in Sub1-overexpressing HL-1 cells treated with bleomycin, Mutpep, and Acpep, (n = 3 independent experiments). l Representative immunofluorescence (IF) images of cardiac organoids treated with bleomycin and indicated peptide, (n = 6 per group). For (k), p-values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. Data are presented as mean ± SD.

Sub1 mediates TAF9b binding to p53 and attenuates ubiquitin-mediated p53 degradation

Initial in vitro experiments showed that HL-1 cells overexpressing Sub1 displayed elevated p53 protein levels, whereas p53 mRNA remained unchanged following bleomycin stimulation (Fig. 6d), implying that Sub1 modulates p53 post-translationally.

LC-MS/MS analysis identified TAF9b as a Sub1-interacting protein, raising the possibility that Sub1 may facilitate the association between TAF9b and p53. To examine whether Sub1 affects p53 stability, cycloheximide (CHX) was used to block de novo protein synthesis and monitor p53 turnover. Sub1 overexpression markedly prolonged the half-life of p53 under bleomycin treatment, whereas Lenti-shSub1 accelerated p53 degradation (Fig. 6e–f). The proteasome inhibitor MG-132 restored p53 levels in shSub1 cells, indicating that Sub1 chiefly limits p53 degradation via the ubiquitin–proteasome pathway (Fig. 6g). Consistently, ubiquitin immunoprecipitation revealed reduced p53 ubiquitination in Sub1-overexpressing cells and enhanced ubiquitination in shSub1 cells (Fig. 6h, Suppl. Fig. 16b). Because ubiquitination can influence the subcellular localization of p53, we next examined its distribution. Both WB and immunofluorescence analyses showed increased nuclear accumulation of p53 in Sub1-overexpressing HL-1 cells, whereas Sub1 knockdown produced the opposite effect (Suppl. Fig. 15a–d), consistent with a role for Sub1 in limiting p53 ubiquitination and promoting its nuclear retention. To explore how Sub1 regulates this process, we investigated the interaction between Sub1, TAF9b, and p53. Domain mapping and co-immunoprecipitation assays suggested that the C-terminal domain (CTD) of Sub1 interacts with the N-terminal domain (NTD) of TAF9b, forming a Sub1–TAF9b–p53 complex (Fig. 6i)^19,21. Deletion of either the Sub1-CTD or the TAF9b-NTD disrupted this interaction, confirming the importance of these domains for complex formation (Fig. 6j–k). Functionally, in bleomycin-induced senescent HL-1 cells, siRNA-mediated TAF9b silencing attenuated the Sub1-mediated increase in p53 and senescence markers (Fig. 6l–m, Suppl. Fig. 16a), and restored p53 ubiquitination (Fig. 6n, Suppl. Fig. 16c, d). Collectively, these findings suggest that Sub1 facilitates the interaction between TAF9b and p53 and contributes to the stabilization of p53 by limiting its ubiquitination. This mechanism prolongs p53 stability and enhances p53-dependent pro-senescent signaling in cardiomyocytes.

Sub1 competitively binds AROS, inhibiting SIRT1 activation and preserving acetylated p53

AROS enhances the deacetylase activity of SIRT1, thereby repressing p53 transcriptional activity²⁰. Our LC-MS/MS and Co-IP analyses identified an interaction between Sub1 and AROS (Fig. 7a, Suppl. Fig. 14e), prompting us to investigate whether Sub1 influences p53 acetylation. As shown in Fig. 7b, c, Sub1 overexpression increased the level of Ac-p53 K382, a known SIRT1 substrate, in senescent cardiomyocytes and aged mice heart. Dose-dependent elevation of Ac-p53 was observed upon transfection of increasing amounts of Flag-Sub1 plasmid into HL-1 cells, without affecting total SIRT1 protein levels (Fig. 7d). Based on previous studies, we proposed a model in which Sub1 competitively binds to the N-terminal domain (NTD) of AROS (Fig. 7e), interfering with its ability to activate SIRT1. To test this hypothesis, we constructed an AROS-NT mutant and conducted Co-IP assays. Both Sub1 and SIRT1 were found to interact with AROS-NT (Fig. 7f). Notably, Sub1 overexpression disrupted the interaction between SIRT1 and AROS, supporting the notion of a competitive binding mechanism (Fig. 7g). To determine the functional relevance of this interaction in Sub1-induced cardiomyocyte senescence, we overexpressed AROS in Sub1-overexpressing HL-1 cells. AROS overexpression restored SIRT1 deacetylase activity, leading to reduced levels of Ac-p53 (K382) and attenuation of cellular senescence markers (Fig. 7h–I, Suppl. Fig. 16e). These findings suggest that Sub1 competitively binds to AROS, thereby preventing SIRT1 activation and preserving p53 acetylation, which contributes to its pro-senescence function.

Blocking the Sub1–p53 interaction attenuates cardiomyocyte senescence

Our previous data demonstrated that Sub1 reduces p53 ubiquitin-mediated degradation and prevents deacetylation of Ac-p53. In line with earlier reports that Sub1 can enhance p53 transcriptional activity²¹, we hypothesized that disrupting the interaction between Sub1 and p53 might mitigate cardiomyocyte senescence. A tri-acetylated peptide derived from the C-terminal region of p53 (residues 380–386; termed Acpep) has been reported to bind Sub1 with high affinity and competitively displace p53 from Sub1 in vitro^22,23. We synthesized Acpep and a control mutant peptide (Mutpep), in which the three acetylated lysine residues were replaced with leucine. HL-1 cells treated with bleomycin were supplemented with 1 mM or 2 mM Acpep or Mutpep. Acpep, but not Mutpep, effectively reversed the upregulation of senescence markers induced by Sub1 overexpression (Fig. 7j). To further explore whether Acpep modulates p53 transcriptional activity, we performed a dual-luciferase reporter assay targeting p21. Sub1 overexpression significantly enhanced p53-mediated transcriptional activation of the p21 promoter, whereas Acpep treatment markedly suppressed this effect (Fig. 7k). Then, we utilized cardiac organoids to validate the potential anti-aging therapeutic effects of Acpep (Fig. 7l, Suppl. Fig. 17a). Together, these results suggest that Sub1 promotes cardiomyocyte senescence by enhancing p53 transcriptional activation of p21, an effect that depends on disrupted p53 post-translational modifications. Blocking the Sub1–p53 interaction via acetylated peptides may represent a potential strategy to counteract cardiac aging.

Targeted Sub1 knockdown protects against age-related cardiac remodeling in aged mice

To determine whether Sub1 knockdown exerts therapeutic effects after the onset of aging, 90-week-old mice were administered AAV9 carrying cardiomyocyte-specific Sub1 shRNA via tail vein injection (Fig. 8a; Suppl. Fig. 18a, b). During the aging period from 70 to 90 weeks, prior to AAV delivery, no significant differences were observed in exercise capacity or cardiac function between groups. However, during the late aging phase (90 to 110 weeks), Sub1 knockdown resulted in marked improvements in exercise tolerance and diastolic function (Fig. 8b, f, g; Suppl. Fig. 18j). Notably, at 110 weeks of age, Sub1-deficient mice displayed enhanced physical activity compared to controls. Histological evaluation of hearts harvested at 110 weeks revealed that knockdown of Sub1 attenuated myocardial fibrosis and ameliorated age-associated structural remodeling (Increased SA-β-gal activity and lipofuscin accumulation, indicative of elevated aging markers) (Fig. 8c–e, h–i, Suppl. Fig. 18c–i), whereas no significant anti-aging effects were observed in non-cardiac tissues (Suppl. Fig. 18k–m). These findings indicate that cardiac-specific Sub1 silencing mitigates structural and functional deterioration associated with advanced cardiac aging.

Fig. 8. Targeted Sub1 knockdown protects against age-related cardiac decline in aged Mice.

a Schematic diagram of AAV9 administration in aged mice. b Running distance in exercise tolerance tests before and after AAV9 administration (n = 5 biological replicates). c Representative Sudan Black B staining and SPiDER β-gal staining of the heart from aging mice after AAV9 administration. Scale bars, 50 μm and 100 μm. Quantification of lipofuscin-positive (%) and SPiDER β-gal positive (%) (n = 5 biological replicates). d Representative IF of p53 and p21 in the hearts aging mice after AAV9 administration. Scale bars, 50 μm. Quantification of relative IF intensity (n = 5 biological replicates). e Representative TEM images of the myocardium of from aging mice after AAV9 administration. Lp Lipofuscin. Scale bars, 2 μm, 1 μm and 0.5 μm. Quantification of lipofuscin number and size using violin plots (n = 15 fields of view in 4 mice per group). f, g Representative images of MV Velocity and TDI of aging mice before and after AAV9 administration and statistical analysis of E/A and E/e’ ratio (n = 5 biological replicates). h Representative western blot of Sub1, p53, p21, and p16 in the heart from aging mice after AAV9 administration (n = 5 biological replicates). i mRNA levels of Nppb, Myh7, Il12, Il6, Cdkn1a (p21), Cdkn2a (p16), Pot1, and Tpp1 in the heart from aging mice after AAV9 administration (n = 4 biological replicates). For (b–g, i), p-values were determined by two-tailed unpaired Student’s t-test. Data are presented as mean ± SD.

Discussion

In this study, we propose a potential therapeutic strategy for HFpEF by targeting cardiac aging through Sub1 knockdown. Sub1 expression is elevated in the myocardial tissue of both aged individuals and HFpEF patients. Overexpression of Sub1 accelerates cardiac aging and shortens lifespan in mice, whereas Sub1 deficiency delays aging-associated structural and functional deterioration in the heart. By further investigating the relationship between HFpEF and aging, we demonstrate that aging is a key accelerating factor for HFpEF progression. Notably, HFpEF mice exhibit more severe cardiac aging phenotypes, which can be alleviated by Sub1 deletion. Mechanistically, Sub1 stabilizes p53 by preventing its ubiquitin-mediated degradation through interaction with TAF9b. Simultaneously, Sub1 competes with SIRT1 for binding to AROS, thereby preserving acetylated p53. This dual regulation disrupts the balance of p53 PTMs, ultimately promoting cardiac senescence. Importantly, pharmacologic disruption of the Sub1–p53 interaction using a synthetic peptide (Acpep) effectively attenuates cardiac aging in vitro. Collectively, our findings position Sub1 as a key node linking cardiac aging and HFpEF pathogenesis, and highlight Sub1 knockdown as a promising strategy to treat HFpEF by targeting cardiac aging (Fig. 9).

Fig. 9. Proposed mechanism by which Sub1 promotes cardiac aging and HFpEF progression.

Sub1 expression is elevated in aged and HFpEF hearts, where it accelerates cardiac senescence by stabilizing and maintaining acetylated p53. Mechanistically, Sub1 inhibits ubiquitin-mediated p53 degradation through interaction with TAF9b and competes with SIRT1 for AROS binding, thereby preserving p53 acetylation. Genetic or pharmacological inhibition of Sub1 alleviates cardiac aging and attenuates HFpEF progression.

Senescent cardiomyocytes exhibit elevated pacing frequency and augmented hypertrophy and fibrosis, culminating in characteristic manifestations of cardiac aging, such as lipofuscin deposition, myocardial wall thickening and stiffening, contributing to diastolic insufficiency in the elderly²³. Furthermore, it can secrete SASP factors, thereby inducing senescence in adjacent cell types, including fibroblasts²⁴. Cellular senescence has been increasingly implicated in the pathophysiology of age-related cardiovascular diseases, including HFpEF^5,25. HFpEF is the predominant form of heart failure in the elderly²⁶ and is now recognized as a systemic syndrome influenced by aging-related molecular and cellular dysfunctions across multiple organs²⁷. In this study, we establish a deeper mechanistic link between aging and HFpEF. First, aged mice and HFpEF model mice exhibit overlapping phenotypic features, particularly impaired diastolic function, suggesting shared underlying mechanisms. Second, induction of HFpEF in aged mice accelerates disease onset, indicating that aging serves as a key pathogenic amplifier of HFpEF. Third, we observed a marked increase in senescence markers in the myocardium of HFpEF mice, suggesting that HFpEF may, in turn, exacerbate cardiac aging. Together, these findings reinforce the concept of a bidirectional relationship between aging and HFpEF and support the notion that targeting myocardial senescence may offer a promising therapeutic avenue for HFpEF.

Given the bidirectional interplay between aging and HFpEF pathogenesis, targeting age-related molecular pathways holds significant therapeutic potential. Current pharmacological therapies proven effective for HFrEF, such as angiotensin-converting enzyme inhibitors (ACEIs) and β-blockers, have failed to show consistent benefit in patients with HFpEF²⁸. This underscores the urgent need for novel therapeutic strategies tailored to the distinct pathophysiology of HFpEF. Among emerging approaches, targeting biological aging has garnered increasing interest. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) represent the first class of drugs to demonstrate efficacy in HFpEF²⁹, with their cardioprotective effects attributed in part to anti-inflammatory and anti-senescent mechanisms^30,31. Similarly, nicotinamide supplementation, which promotes NAD⁺ homeostasis and combats cellular aging, has been shown to ameliorate diastolic dysfunction in preclinical models of aging, hypertension, and metabolic syndrome⁴. Metformin, a well-established metabolic modulator with anti-aging properties, has also demonstrated benefit in primate studies and has entered clinical trials for HFpEF³². In this context, our findings identify Sub1 as a potential modulator of cardiac senescence. Sub1 knockdown delays myocardial aging and alleviates HFpEF-related phenotypes in mice, highlighting its potential as a promising target for anti-aging–based therapeutic intervention in HFpEF.

One of the earliest recognized functions of p53 is the induction of cell cycle arrest and senescence. During physiological aging, p53 is stabilized and activated in response to accumulating DNA damage, leading to cell cycle arrest and cellular senescence. The activity of p53 is tightly regulated by various PTMs, including ubiquitination, phosphorylation, acetylation, methylation, and SUMOylation³³. Among these, ubiquitination, phosphorylation, and acetylation represent the most prominent modifications influencing p53 function by modulating its protein stability, subcellular localization, and interactions with cofactors¹⁸. The coordinated regulation of these PTMs is essential for the fine-tuning of p53 activity. MDM2, a principal negative regulator of p53, promotes its ubiquitination, thereby maintaining low basal p53 protein levels under non-stressed conditions or facilitating its nuclear export³³. Several upstream modulators, such as p14ARF, counteract MDM2-mediated suppression of p53³⁴. TAF9b has been reported to bind the N-terminal domain of p53, interfering with its interaction with MDM2. Overexpression of TAF9b reduces MDM2-mediated ubiquitination of p53 and consequently enhances p53 protein levels³⁵. In the present study, we identified Sub1 as a critical modulator bridging TAF9b and p53, facilitating the inhibitory effect of TAF9b on p53 ubiquitination. Furthermore, acetylation at K382 is essential for the transcriptional activity of p53 in mediating cell cycle arrest and senescence²⁰. Previous studies have shown that SIRT1-dependent deacetylation at this site suppresses p53 activity and cellular senescence^20,36. Here, we demonstrate that Sub1 impairs AROS-mediated activation of SIRT1, thereby preserving K382 acetylation and sustaining p53 function. Collectively, our findings reveal that Sub1 knockdown reestablishes the balance of p53 PTMs, thereby attenuating cardiac aging. Importantly, the beneficial effects of Sub1-targeted gene therapy are evident even when initiated in 90-week-old mice, suggesting that Sub1 inhibition holds therapeutic promise as an intervention strategy even at advanced stages of aging. Additionally, Sub1-overexpressing mice exhibit premature aging phenotypes and shortened lifespan, suggesting their utility as an accelerated aging model for aging-related research.

Although aging is an inevitable biological process, it can be decelerated through targeted interventions that attenuate age-related functional decline. Emerging anti-aging strategies include modulation of nutrient-sensing pathways (e.g., rapamycin), selective clearance of senescent cells, and manipulation of the gut microbiota³⁷. Among these, the targeted elimination of senescent cardiomyocytes using senolytics has garnered particular interest. Peptide-based senolytics have shown promise; for instance, a FOXO4-derived peptide disrupts the interaction between FOXO4 and p53, promoting p53 nuclear exclusion and thereby suppressing senescence³⁸. In the present study, we identified a peptide, Acpep, which inhibits the interaction between Sub1 and p53. By blocking this binding, Acpep impedes the transcriptional activation of senescence-associated genes by p53. These findings highlight Sub1 as a previously unrecognized regulatory node in cardiac aging and suggest that targeting the Sub1–p53 axis with peptide therapeutics may represent a viable strategy to ameliorate cardiac senescence.

This study has several limitations. First, the precise mechanism driving the upregulation of Sub1 during aging remains unclear. Using a transcription factor prediction tool, we identified several candidate transcription factors, including CREB1 and ATF2³⁹. However, under senescent conditions, overexpression of ATF2 or CREB1 did not affect Sub1 expression. The upstream factors responsible for the increased Sub1 expression during natural aging still require further investigation. Second, in response to DNA damage, p53 has been shown to bind the Sub1 promoter and enhance its transcription, while Sub1, in turn, facilitates p53 to promote transcription, suggesting a potential feed-forward loop⁴⁰. The pathological significance of this reciprocal regulatory interaction in cardiac aging requires further elucidation. Third, the anti-senescent effects of Acpep have been evaluated only in vitro. Future studies are needed to develop strategies for effective in vivo delivery and to assess its functional impact on cardiac aging.

In summary, our research identifies a novel pathway in which Sub1 regulates the PTMs balance of p53 through TAF9b and AROS, thereby influencing cardiac senescence. Aging and HFpEF mutually reinforce each other, and knockdown of Sub1 alleviates cardiac aging, thereby contributing to the treatment of HFpEF. Thus, Sub1 represents a potential anti-aging therapeutic target for HFpEF driven by aging.

Methods

Animals

The protocol for the animal study was in accordance with the institutional guidelines of the Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Approval Number: 2021-061 and 2023 − 154). C57BL/6 mice were purchased from SPF (Suzhou) Biotechnology Co., Ltd. All mice were housed in a temperature-controlled facility under a 12-hour light/dark cycle at 23 ± 3°C with 30–70% relative humidity. To induce HFpEF, 50- to 60-week-old mice were randomly assigned to either a standard chow diet or a high-fat diet (60% kcal from fat; Research Diets, D12492) combined with L-NAME (0.5 g/L in drinking water; Sigma, 51298-62-5) for 12 weeks⁴¹. Low-dose doxorubicin was used to induce cardiac aging by intraperitoneal administration at a dose of 2.5 mg/kg every other day for a total of six injections (cumulative dose: 15 mg/kg) over two weeks.

Generation of Cardiomyocyte-specific Sub1 overexpression mice

Cardiomyocyte-specific Sub1 overexpression (Sub1 Tg) mice were generated by targeting the Rosa26 locus with a CAG-LSL-Sub1-3×Flag construct using CRISPR/Cas9 technology. The targeting vector was constructed by In-Fusion cloning and contained a 3.3 kb 5′ homology arm, CAG promoter, loxP-STOP-loxP cassette, Sub1-3×Flag-WPRE-polyA sequence, and a 3.3 kb 3′ homology arm. Cas9 mRNA, gRNA, and the donor vector were microinjected into fertilized eggs to generate F0 mice. Six homologous recombinant F0 founders were identified by long-fragment PCR. Germline transmission was confirmed by breeding with wild-type mice, yielding 21 positive F1 offspring. Heterozygous conditional Sub1 overexpression mice showed no overt abnormalities. In these mice, the loxP-STOP-loxP cassette prevented transcription of the downstream Sub1 transgene. To achieve cardiomyocyte-specific induction, Rosa26-LSL-Sub1 mice were crossed with B6.FVB(129)-Tg(Myh6-cre/Esr1)1Jmk/J* mice (Myh6-MerCreMer). In the double-transgenic offspring, Cre recombinase is expressed specifically in cardiomyocytes but remains inactive until tamoxifen administration.

At 8 weeks of age, mice were administered tamoxifen (30 mg/kg/day; Sigma-Aldrich, #10540-29-1) dissolved in corn oil by intraperitoneal injection for 5 consecutive days to induce Cre-mediated excision of the loxP-STOP-loxP cassette. Control littermates received the same tamoxifen regimen. Experiments were initiated 2 weeks after the final tamoxifen injection to allow recovery from transient tamoxifen-related cardiac effects. Following induction, the STOP cassette was excised in cardiomyocytes, resulting in robust Sub1 overexpression driven by the CAG promoter. A schematic diagram of the strategy is shown in Suppl. Fig. 5a.

Generation of Sub1 −/+ mice

Systemic Sub1 knockout mice were generated via CRISPR/Cas9-mediated genome editing. A guide RNA targeting Sub1 was designed and transcribed in vitro, then co-injected with Cas9 mRNA into fertilized mouse embryos. The resulting F0 mice were genotyped by PCR and sequencing, and subsequently crossed with wild-type mice to produce F1 heterozygous offspring, which were also PCR-genotyped. Sub1−/+ mice were used for experiments because complete Sub1 knockout (Sub1 −/−) resulted in embryonic lethality. The generation strategy is illustrated in Suppl. Fig. 8a.

Echocardiography

Transthoracic echocardiography was performed to evaluate cardiac structure and function. Mice were anesthetized with isoflurane (3.0% for induction and 1.5% for maintenance). During image acquisition, animals were maintained on a heating platform. Ejection fraction (EF) and heart rate (HR) were measured using 2D-guided M-mode imaging in parasternal long axis view (Vevo F2, Fujifilm Visual Sonics, ON, Canada). Diastolic function was assessed in apical 4 chambers view by measuring peak Doppler velocities across the mitral valve during early (E wave) and late (A wave) diastole, early diastolic mitral annular velocity (e′), and deceleration time of early filling.

Blood pressure recordings

Systolic and diastolic blood pressure in conscious mice were assessed non-invasively using a tail-cuff system (CODA, Kent Scientific, CT, USA). To ensure consistency, animals were placed individually in restrainers on a pre-warmed platform, and measurements were carried out under stable physiological conditions. At least three independent readings were obtained for each mouse during each session. To reduce stress-related artifacts, mice were habituated to the procedure for three consecutive days prior to formal measurement⁴².

Exercise exhaustion test

After a 3-day acclimation period on the treadmill, mice performed an exhaustion test. The protocol started with running uphill at 5 m/min for 4 min, followed by 14 m/min for 2 min. Subsequently, the speed increased by 2 m/min every 2 min until the mice reached exhaustion, defined as the inability to resume running within 10 sec of contacting the electric grid. Running time and distance were recorded.

Histochemical analysis

Mice were deeply anesthetized with ketamine (100 mg/kg; Pfizer, NY, USA) and xylazine (10 mg/kg; Bayer, Leverkusen, Germany) via intraperitoneal injection, followed by cervical dislocation for euthanasia. Hearts were excised and immediately placed in 4% neutral-buffered formalin or OCT compound. Paraffin-embedded tissue sections (10 μm) were stained with Masson staining (ServiceBio, G1006), HE staining (ServiceBio, G1005), WGA staining (ServiceBio, G1730) and β-gal staining (ServiceBio, G1073) using a Leica RM2235 according to the manufacturer’s instructions. Frozen sections of heart tissue were obtained using a Leica CM1950. Sudan Black B staining (Haling, G2002) was then performed to determine the cardiac tissue lipofuscin content, and positive areas were calculated using ImageJ software⁴³. We scanned the tissue sections with the VS200 to obtain images.

Immunohistochemistry for 4-HNE

Paraffin-embedded mouse heart sections (4 μm) were deparaffinized, rehydrated, and subjected to antigen retrieval in citrate buffer (pH 6.0). After blocking endogenous peroxidase (3% H₂O₂) and nonspecific binding (5% BSA), sections were incubated with anti-4-HNE antibody (ServiceBio, GB150073-100, 1:200) overnight at 4 °C. HRP-conjugated secondary antibody and DAB were used for signal detection, followed by hematoxylin counterstaining. Images were acquired by VS200 and quantified using ImageJ.

Transmission electron microscopy

Small cubic tissue blocks ( ≤ 1 mm³) from the left ventricle were collected and initially fixed overnight at 4 °C in 2.5% glutaraldehyde prepared in 0.1 M sodium phosphate buffer (pH 7.4). The samples then underwent secondary fixation with 1% osmium tetroxide. Following this, tissues were dehydrated through a graded ethanol series and embedded in Epon-Araldite resin. Ultrathin sections ( ~ 50 nm) were sliced using an ultramicrotome (Leica, Wetzlar, Germany), stained sequentially with uranyl acetate and lead citrate, and subsequently visualized under a Hitachi H-7000 transmission electron microscope by BaiQianDu Bio (Wuhan, China).

SA-β-gal assay

Senescence-associated β-galactosidase (SA-β-gal) activity was assessed using two complementary methods: the Cellular Senescence Detection Kit SPiDER-β-gal (DOJINDO, SG02) and the Senescence β-Galactosidase Staining Kit (Beyotime, C0602).

For SPiDER-β-gal staining in cultured cells, cells were seeded on coverslips, washed twice with PBS, fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, and incubated with SPiDER-β-gal working solution (20 μM) for 30 min at 37 °C. Fluorescence images were acquired using a Leica DMi8 fluorescence microscope⁴⁴.

For colorimetric SA-β-gal staining, cells were seeded in 6-well plates and treated as indicated. After removal of the culture medium, cells were washed twice with PBS and stained according to the manufacturer’s instructions using the Senescence β-Galactosidase Staining Kit. Images were captured using a Leica bright-field microscope.

For tissue SA-β-gal staining, heart samples were embedded in OCT compound and snap-frozen in hexane cooled with dry ice. Cryosections (10 μm) were mounted on glass slides, fixed in 4% PFA for 20 min at room temperature, rinsed with PBS, and incubated with SPiDER-β-gal working solution (20 μM) at 37 °C for 1 h. Images were acquired using an Olympus VS200 slide scanner⁴⁵.

Bioinformatics analysis

The RNA sequencing datasets of heart biopsy samples from HFpEF patients (E-MTAB-7454 and PRJEB62450) were obtained from the ArrayExpress database and the European Nucleotide Archive, respectively. The “Limma” package was utilized to identify differentially expressed genes (DEGs) between the control and HFpEF groups (Padj < 0.05, |log2 FC | > 0.85). The Venn diagram was plotted using the SangerBox platform.

Cell culture

HL-1 cells (SCC065, Sigma-Aldrich), an immortalized mouse atrial cardiomyocyte cell line, and HEK-293T cells (SCSP-5209, National Collection of Authenticated Cell Cultures), were used in this study. Cell lines were maintained in a humidified incubator with 5% CO₂ at 37 °C, cultured high glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. All cell lines were routinely tested and confirmed to be free of mycoplasma contamination. We used 50 μg/ml bleomycin (MCE, HY-17565A) to induce the HL-1 for 24 h to construct a cardiomyocyte senescence model⁴⁶. MG-132 (2 μM, MCE, HY-13259) was applied for 24 h to inhibit proteasome activity. All plasmids and reagents generated in this study are available from the corresponding author upon request.

Plasmid construction and transfection

Full-length and truncated plasmids for Sub1, TAF9b, and Aros were constructed by GeneWiz Co., Ltd. (Suzhou, China). Transient transfection was performed using GeticoFect 3000 Transfection Reagent (Getico, 130101) according to the manufacturer’s protocol. Forty-eight hours post-transfection, cells were harvested for IP or WB.

Lentivirus production and transduction

Guide RNA (gRNA) sequences targeting Sub1 were designed using the CHOPCHOP database (https://chopchop.cbu.uib.no/) and cloned into the pLVX-IRES-Puro vector. Lentiviruses were generated by co-transfecting HEK293T cells with the packaging plasmids pMD2.G and psPAX2.

For transduction, HL-1 cells (1 × 10⁶ to 2 × 10⁶) were seeded into 6-well plates and infected with either Lenti-shSub1 or Lenti-Sub1 at a multiplicity of infection (MOI) of 20 in the presence of polybrene (Beyotime, C0351). After 24 h of incubation at 37 °C, cells were washed with PBS and cultured in complete medium for an additional 48 h before puromycin selection.

Small interfering RNA (siRNA) transfection

siRNAs were designed using the Invitrogen BLOCK-iT™ RNAi Designer. The siRNA targeting TAFb (5′-GTCGGATGCGTTTGACCAGAT-3′) was synthesized by Genescript Bioscience Co., Ltd. (Nanjing, China). Transfection was performed using GeticoFect 3000 Transfection Reagent (Getico, 130101) according to the manufacturer’s protocol. Twenty-four hours post-transfection, cells were treated with bleomycin for an additional 48 h.

Co-immunoprecipitation (Co-IP)

Protein-protein interactions were examined by Co-IP. Collect cell samples after adding 10 μM MG-132 for 4 h prior to harvest to inhibit proteasome activity and allow the accumulation of ubiquitinated p53. Then, cell samples were lysed in lysis buffer (Epizyme, PC105). Protein A/G magnetic beads (Selleck, B23201) were pre-incubated with either anti-p53 antibody (Abclonal, A25915) or rabbit IgG control antibody (Proteintech, 30000-0-AP). The antibody-conjugated beads were then incubated with cell lysates overnight at 4 °C to allow immunoprecipitation. After incubation, the beads were washed four times with washing buffer to remove nonspecific bindings. Immunocomplexes were eluted, resolved by SDS-PAGE, and subjected to immunoblotting with target-specific primary antibodies and secondary antibodies designed to minimize detection of IgG heavy and light chains (Vazyme, RA1008-01). For exogenous protein immunoprecipitation, Anti-Flag magnetic beads (Selleck, B26101) and Anti-HA magnetic beads (Selleck, B26102) were employed following the manufacturer’s protocols. All immunoprecipitation experiments were independently repeated three times.

Western blotting

Proteins were extracted from cultured cells and aging mouse hearts using RIPA lysis buffer (Epizyme, PC102) supplemented with 1% protease inhibitor cocktail (Epizyme, GRF101). Protein concentrations were determined using a BCA assay kit (Epizyme, ZJ101). Samples were denatured at 98 °C for 15 min, and 10 μg of total protein was separated on 8–15% SDS-PAGE gels, followed by transfer to PVDF membranes. Membranes were blocked with 5% BSA for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. Signals were detected and quantified using the GE ImageQuant LAS4000mini imaging system. A full list of antibodies is provided in Suppl. Table 1. All Western blot experiments were independently repeated three times.

qRT-PCR assay

RNA was extracted using the RNA isolation Total RNA Extraction Reagent (Vazyme, R401-01) according to the manufacturer’s instructions. Each sample’s 1000 ng total RNA was converted to cDNA with Hifair V Reverse Transcriptase (Yeasen, 11300ES92) and amplified using ChamQ SYBR qPCR Master Mix (Vazyme, Q341-02). Relative mRNA expression was calculated via the 2 − ΔΔCT method, using β-actin or GAPDH as controls. Primer pairs were synthesized by Tsingke Biotechnology, as detailed in Suppl. Table 2.

Half-life assays

To investigate the mechanism of p53 degradation, bleomycin-treated HL-1 cells were incubated with cycloheximide (a eukaryotic protein synthesis inhibitor; 20 μg/mL, MCE, HY-12320).

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 15 min, then treated with 0.4% Triton X-100 for membrane permeabilization. Afterward, nonspecific binding was blocked using normal goat serum for 1 h at room temperature. Cells were subsequently incubated with primary antibodies overnight at 4 °C. The next day, fluorescently labeled secondary antibodies were applied for 1 h in the absence of light. Nuclei were stained using DAPI, and fluorescence signals were visualized with fluorescence microscope. The antibodies used are listed in Suppl. Table 1.

Mass spectrometric analysis

HL-1 cells were transfected with Flag-Sub1 for 48 h and subsequently treated with bleomycin for 24 h. After lysis, immunoprecipitation was performed, and the bound proteins were excised and subjected to LC-MS/MS analysis by SpecAlly Biosciences Co., Ltd. (Wuhan, China).

Immunoprecipitated proteins were denatured, reduced, and alkylated in reaction buffer (1% sodium deoxycholate, 100 mM Tris-HCl pH 8.5, 10 mM TCEP, and 40 mM chloroacetamide) at 95 °C for 10 min. Samples were digested overnight at 37 °C with trypsin at a 1:50 (enzyme:protein, w/w) ratio. Digestion was terminated by acidification with trifluoroacetic acid. Peptides were purified using SDB desalting columns, dried under vacuum, and stored at −20 °C until analysis. Peptides were analyzed on a timsTOF Pro mass spectrometer (Bruker Daltonics) coupled to an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) with a CaptiveSpray ion source. Peptides were loaded onto a C18 trap column and separated on a reverse-phase C18 analytical column using a linear gradient of acetonitrile (0.1% formic acid) at a flow rate of 300 nL/min.

Mass spectrometry data were acquired in parallel accumulation–serial fragmentation (PASEF) mode over an m/z range of 100–1700. Ion mobility separation was performed with a scan range of 0.75–1.4 Vs/cm². Each acquisition cycle consisted of one full MS scan followed by 10 PASEF MS/MS scans. Singly charged ions were excluded, and dynamic exclusion was set to 0.4 min. Raw data were processed using MaxQuant with the Andromeda search engine against the UniProt human protein database. Trypsin/P was specified as the protease with up to two missed cleavages allowed. Carbamidomethylation (C) was set as a fixed modification, while oxidation (M), N-terminal acetylation, and deamidation (NQ) were set as variable modifications. The false discovery rate (FDR) was set to 1% at both peptide and protein levels. Label-free quantification (LFQ) was used for protein quantification. Proteins identified as contaminants or decoys were removed. Proteins showing a fold change > 4 between bait and control samples were considered candidate interactors. Functional enrichment analysis was performed using Gene Ontology (GO) and KEGG databases, and statistical significance was assessed using a two-sided hypergeometric test.

Molecular docking

Protein structures for molecular docking were predicted using AlphaFold 2, based on the UniProt IDs: AROS (Q86WX3), Sub1 (P53999), and TAF9B (Q9HBM6). Molecular docking was performed using the ClusPro server, with Sub1 set as the receptor and AROS or TAF9B as the ligands under default parameters. The top-ranked docking model was selected as the predicted binding conformation. Docked complexes were visualized and analyzed using PyMOL. Binding interactions were further evaluated using LigPlot+ v2.2.4, and interface analysis was conducted online via PDBePISA (Protein Interfaces, Surfaces and Assemblies).

RNA-sequence and data analysis

Total RNA was extracted from harvested Ctrl fl/fl and Sub1 Tg mice hearts. Samples were prepared in triplicate and processed at MajorBio (Shanghai, China). The DEGs were selected with a fold change > 1.5 or fold change < 0.667 and adjusted p-value < 0.05. The GO enrichment and GSEA analysis data were made in the MajorBio Cloud Platform (https://cloud.majorbio.com/page/tools.html).

Dual-luciferase reporter gene assay

The mouse p21 promoter (from −2001 to −1) was cloned into pGL3 vectors. Transient transfection and luciferase assays were performed. Renilla-Firefly Luciferase Dual Assay Kit (MCE, HY-K1013) was used to detect luciferase reporter gene expression.

Nuclear isolation

The preparation of nuclear and cytosolic proteins from HL-1 cells was performed using a commercial Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027)⁴⁷.

Peptide synthesis

Acpep (RRRRRRPKKKRKVH[Lys-Ac] [Lys-Ac] LMF [Lys-Ac]) and Mutpep (RRRRRRPKKKRKVHAAAMFK) were synthesized by Genescript (Nanjing, China). The sequences of the peptides refer to previous studies²².

AAV9 treatement

Cardiomyocyte-specific Adeno-Associated Virus 9 (AAV9)-shSub1 and AAV9-NC control viruses were created by OObio (Shanghai, China). The plasmid map is listed in Suppl. Fig. 17a. Each mouse received 5×10¹¹ viral genomes (VG) of AAV9 via tail vein injection, with injections repeated every four weeks to maintain effective viral transduction.

ELISA assay

Serum GDF-15 levels were measured using a commercially available mouse GDF-15 ELISA kit (WeiAoBio, EM30218). All reagents and samples were prepared at room temperature and the assay was performed according to the manufacturer’s instructions. Briefly, serum samples were diluted appropriately and added to antibody-precoated 96-well plates, followed by incubation for antigen binding. After washing to remove unbound components, horseradish peroxidase (HRP)–conjugated detection antibody was added and incubated. Plates were washed again and developed using TMB substrate. The reaction was stopped with stop solution, and absorbance was measured at 450 nm using a microplate reader. A standard curve was generated from serially diluted standards, and GDF-15 concentrations in samples were calculated based on the standard curve.

Determination of mitochondrial DNA content

Genomic DNA was extracted from mouse heart tissue using a commercial tissue DNA extraction kit (Tiangen, KG205), following the manufacturer’s protocol. Relative mitochondrial DNA (mtDNA) content was quantified by SYBR Green–based real-time qPCR using primers targeting the mitochondrial gene mt-Cytb and the nuclear gene β-globin as the reference. qPCR reactions were performed using 50 ng genomic DNA per well under standard cycling conditions. Relative mtDNA copy number was calculated by the ΔΔCt method. Primer pairs were listed in Suppl. Table 2.

Cardiac organoid generation

All procedures involving human induced pluripotent stem cells (hiPSCs) complied with relevant ethical regulations and were approved by Ethics committee of First affiliated hospital of Soochow University (No. 2017-264). The hiPSC lines used in this study were obtained from Ronovation Biotech, Hangzhou,China, with informed consent obtained where applicable. hiPSC-derived cardiac organoids were generated as the following method. Briefly, hiPSCs were maintained on Matrigel-coated plates in mTeSR1 medium (STEMCELL, #85850) and passaged using Accutase (Thermo, A1110501). For organoid formation, single-cell hiPSCs were seeded into ultra-low attachment 96-well round-bottom plates at a density of 5000–8000 cells per well in differentiation medium supplemented with 10 μM Y-27632 (Selleck, S1049) for the first 24 h to promote aggregation. On day 0, aggregates were treated with 6 μM CHIR99021 (Selleck, S2924) in RPMI 1640 supplemented with B27 minus insulin (Thermo, A1895601) for 24 h. On day 2, the medium was replaced with RPMI/B27 minus insulin containing 5 μM IWP-2 (Selleck, S7086) for 48 h. From day 4 onward, organoids were maintained in RPMI 1640 supplemented with B27 complete, with medium changes every 2–3 days.

Spontaneous contractile activity was typically observed between days 7 and 10, indicating successful cardiac differentiation. Cardiac organoids were cultured for up to 20 days and used for downstream analyses. Bleomycin (100 μg/ml), Acpep and Mutpep was added to the culture medium for 24 h before harvesting.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 10.0. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. One-way ANOVA followed by Bonferroni’s post hoc test was applied for multiple group comparisons, and two-way ANOVA with Bonferroni’s correction was used where appropriate. Survival curves were analyzed using the Kaplan–Meier method and compared using a two-sided log-rank (Mantel–Cox) test. A p-value < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

Z., Y.Z., J.W., and P.G. conceived and designed the study. D.Z., L.L., Y.Zhou, J.Wu, R.X., L.Li, X.Z., and H.L. performed the experiments. D.Z., L.L., Y. Zhou, J. Wang, K.D., and Z.D. analyzed the data. D.Z., Y.Z., and J.W. drafted the manuscript. Y.Z., J.W., and P.G. revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Peer review

Peer review information

Nature Communications thanks Mathias Mericskay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

This work was supported by the National Natural Science Foundation of China (82230009 and 82430016 to Y.Z.; 82470393 to J.W.; 82270265 and 82570295 to P.G.), the National Key Research and Development Program of China (2023YFA1800600 to Y.Z.), the Noncommunicable Chronic Diseases–National Science and Technology Major Project (2023ZD0503203 to Y.Z.).

Data availability

All data supporting the findings of this study are freely available without restriction. Source data are provided with this paper. The RNA-sequencing data generated in this study have been deposited in the NCBI BioProject database under accession number PRJNA1414796. The mass spectrometry proteomics data have been deposited in the iProX database under accession code IPX0013418000 and in the ProteomeXchange Consortium under identifier PXD080102. All other data supporting the findings of this study are available within the main text, Supplementary Information, and Source Data files. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains Supplementary material available at 10.1038/s41467-026-75237-9.