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. 2026 Aug 11;32:215. doi: 10.1186/s10020-026-01586-2

Sirtuin 4 knockout exacerbates cardiac injury in septic cardiomyopathy via activating the MAPK-JNK pathway

Na Li 1, Kangla Liao 1, Jiaxin Chen 1, Suxin Luo 1,✉, An He 1,✉, Shuting Chang 1,2,✉
PMCID: PMC13749821  PMID: 43004197

Abstract

Background

Septic cardiomyopathy (SCM) significantly contributes to mortality among septic patients, and its mechanism is closely related to abnormal inflammatory responses. Previous studies revealed the essential role of the Sirtuin4 gene in cardiomyocyte and myocardial function; however, the precise mechanisms underlying its involvement in SCM remain poorly understood.

Methods

Sepsis model was established by cecal ligation and puncture (CLP) in vivo. Survival rate and sepsis score were assessed. Myocardial function was evaluated by echocardiography, serum creatine kinase isoenzyme (CK-MB) concentration and TUNEL staining. In vitro, mouse cardiomyocytes HL-1 cell line was stimulated with lipopolysaccharide (LPS) to simulate an inflammatory environment. Transcriptomic sequencing identified differentially expressed genes (DEGs) and enriched signaling pathways in hearts from SIRT4-knockout CLP mice, and validated by Western blot and immunofluorescence.

Results

The expression of SIRT4 was significantly downregulated in both CLP-induced myocardium and LPS-treated HL-1 cells. SIRT4 knockout exacerbated CLP-induced mortality and myocardial dysfunction. Furthermore, transcriptomic and molecular biology experiments revealed that SIRT4 deficiency sharply activated the MAPK-JNK pathway. In vitro experiments further validated that SIRT4 knockdown aggravated LPS-induced inflammation in cardiomyocytes by activating the MAPK-JNK pathway. Conversely, inhibiting JNK effectively attenuated both the MAPK-JNK pathway activation and the cellular damage caused by SIRT4 deficiency. Additionally, SIRT4 deficiency activated the mediator AMPK, thereby upregulating the MAPK-JNK pathway in SCM.

Conclusion

SIRT4 knockout exacerbates cardiac injury in septic cardiomyopathy by activating the MAPK-JNK pathway, suggesting SIRT4 is a potential therapeutic target for sepsis-induced cardiomyopathy.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s10020-026-01586-2.

Keywords: Sirtuin4 (SIRT4), Septic cardiomyopathy, The MAPK‒JNK pathway, Inflammation

Introduction

Considerable progress has been achieved in research on septic cardiomyopathy (SCM) (Zhou et al. 2024). Its definition has expanded from simple functional impairment to a comprehensive diagnosis involving biomarkers and imaging (Singer et al. 2016). Globally, SCM is a major cause of death in patients with sepsis, with high incidence and significant association with poor prognosis (Hollenberg and Singer 2021). The primary challenge in clinical treatment lies in the complexity of its pathophysiological mechanisms, including mitochondrial dysfunction (Wu et al. 2023), calcium regulatory imbalance, autonomic nerve disorders, and persistent inflammatory responses (Xue et al. 2025). Currently, clinical management depends entirely on general sepsis treatment strategies (such as infection control, fluid resuscitation, and vasoactive drugs) due to the lack of targeted therapies. Effective means to directly repair myocardial function are still lacking.

Sirtuin4 (SIRT4) is a mitochondrial-localized NAD + -dependent deacetylase (Liu et al. 2023). In the past decade, research has expanded its recognized role from a simple metabolic regulator (Xue et al. 2023) (e.g., glutaminase) to a key regulator of cell fate and inflammation. SIRT4 has been identified as an important protective factor in heart diseases. Studies have demonstrated its critical role in models of heart failure (Byrne et al. 2025), myocardial hypertrophy (Luo et al. 2017), and diabetic nephropathy (Shi et al. 2017). The primary mechanisms through which SIRT4 regulates energy metabolism include inhibiting fatty acid oxidation, promoting glucose oxidation (Tao et al. 2018), and reducing mitochondrial oxidative stress (Luo et al. 2025). However, SIRT4’s functions extend far beyond metabolic regulation and may involve the fine modulation of immune-inflammatory responses through unclear mechanisms.

Recent evidence highlights the significance of SIRT4 in organ damage mediated by inflammatory responses, particularly through modulation of macrophage polarization. The absence of SIRT4 promotes macrophage differentiation toward an M1 pro-inflammatory state, amplifies NF-κB signaling, elevates inflammatory cytokine production (including TNF-α and IL-1β), and aggravates tissue inflammation and injury. Conversely, SIRT4 overexpression promotes the anti-inflammatory (M2) phenotype, alleviating inflammatory storms and multiple organ dysfunction in models such as sepsis and acute lung injury. Thus, SIRT4 represents a crucial nexus linking cellular metabolism and immune inflammation. Targeting SIRT4 provides a novel therapeutic strategy for treating heart diseases and inflammatory organ injuries. However, compared with family members such as SIRT1 (Deng et al. 2021) and SIRT3 (Deng et al. 2024), the role of SIRT4 in sepsis remains largely unexplored. Whether and how SIRT4 regulates classical inflammatory pathways to influence SCM progression remains an urgent scientific question.

Methods

Animals

Male 8-week-old C57BL/6 J mice (C57) and SIRT4 knockout mice (KO) were purchased from Shulaibao Biotechnology Co., Ltd. (Wuhan, China). All mice were housed under specific pathogen-free (SPF) conditions with a 12-h light/dark cycle and had free access to sterilized water and food. After one week of adaptive feeding, the mice were divided into four groups (n = 10 per group): C57 sham-operated group (C57 Sham), C57 cecal ligation and puncture group (C57 CLP), KO sham-operated group (KO Sham), and KO CLP group (KO CLP). To examine the effect of SIRT4 overexpression on cardiac injury, C57 mice were injected with adeno-associated virus serotype 9 (AAV9) encoding the Sirt4 gene (AAV9-Sirt4) or an empty vector (AAV9-NC). The mouse Sirt4 gene was cloned into the AAV9 vector by Genechem Co., Ltd. (Shanghai, China). Mice were divided into four groups: NC sham-operated group (C57 Sham), NC CLP-operated group (C57 CLP), AAV9-Sirt4 sham-operated group (AAV9 Sham), and AAV9-Sirt4 CLP-operated group (AAV9 CLP). AAV9 was administered via tail vein injection at a dose of 2 × 1011 viral genomes per mouse per month (Wang et al. 2023). All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines and were approved by the Animal Ethics Committee of Chongqing Medical University (Approval Number: IACUC-CQMU-2026–03076).

CLP model

As previously described, a sepsis model was established by CLP surgery (Ye et al. 2025). Briefly, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). A 1.5 cm incision was made along the lower quadrant of the abdomen, and the cecum was isolated. The distal three-quarters of the cecum was ligated with 4–0 silk sutures, and two punctures were made in the ligated cecum with a 22-gauge needle to induce sublethal sepsis. The cecum was then returned to the abdominal cavity, and the abdominal wall was closed in layers. The Sham group underwent the same surgical exposure without CLP. All mice were sacrificed 24 h post-operation, and tissues were collected for subsequent experiments.

Murine sepsis score

Murine sepsis severity was evaluated based on seven parameters, including general appearance, consciousness, activity, responsiveness to stimuli, ocular characteristics, respiratory rate, and breathing quality, as previously described (Shrum et al. 2014; Lu et al. 2023). Each parameter was scored on a scale of 0 to 4, with higher scores indicating greater severity. All scoring assessments were performed by two independent evaluators.

Echocardiography

Cardiac function was assessed by echocardiography using a Vevo 3100 imaging system. Under isoflurane anesthesia, two-dimensional M-mode echocardiographic images were acquired at the level of the papillary muscles. Core body temperature was continuously monitored and maintained with a heating pad throughout the procedure. Cardiac systolic and diastolic functions were evaluated by calculating the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and the E/A ratio.

Immunohistochemistry (IHC)

Immunohistochemical staining was performed on heart tissue sections that had been fixed in 4% paraformaldehyde, embedded in paraffin, and then cut. The tissue sections were incubated overnight at 4 °C with the following primary antibodies (all at 1:100 dilution): SIRT4 (ZENBIO, 862,208), TNF-α (ZENBIO, 346,654), phospho-MAPK (ZENBIO, 690,306), and phospho-JNK1 (ZENBIO, 690,280). The following day, the sections were incubated with the corresponding secondary antibodies for 1 h.

TUNEL staining

Myocardial apoptosis was evaluated by TUNEL staining according to the manufacturer's instructions (Abbkine Scientific, Wuhan, China). Briefly, fluorescein-conjugated dUTP was used to label DNA strand breaks, followed by nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). The slides were subsequently examined by laser scanning cytometry to determine the proportion of TUNEL-positive nuclei (Zhang et al. 2025).

Cell culture and transfection

The HL-1 cell line was cultured at 37 °C with 5% CO₂ in a specific culture medium (TCM-G783, HyCyte, China). To mimic sepsis-like conditions in vitro, cells were treated with LPS for 24 h. Compound C was acquired from the MedChemExpress (HY-13418A, USA). SIRT4 small interfering RNA (si-SIRT4) and negative control siRNA (si-NC) were transfected into cells using Lipomaster-2000 (Vazyme, Nanjing, China). The sequences of si-SIRT4 were: sense, 5′-CCAAGAAACUCCUCGUGAUTT-3′; antisense, 5′-AUCACGAGGAGUUUCUUGGTT-3′. The sequences of si-NC were: sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. After 48 h, knockdown efficiency was confirmed by western blotting.

Propidium iodide (PI)/Hoechst staining

Cells were seeded in 12-well plates at a density of 2.5 × 105 cells per well and subjected to the indicated experimental treatments. After treatment, the culture medium was removed, and cells were stained with PI/Hoechst solution (5 μg/mL, C1375S, Beyotime) for 30 min. Representative images were acquired using a fluorescence microscope.

Flow cytometry

Cell suspensions were washed twice with PBS, stained with the indicated antibodies for 30 min on ice, and then analyzed by flow cytometry. Apoptosis was assessed using an Annexin V-FITC/PI apoptosis detection kit (C1062M, Beyotime, China), with Annexin V labeled with FITC serving as the probe.

Immunofluorescence (IF)

Cell slides were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 5% bovine serum albumin (BSA) for 1 h. After washing three times with PBS, the slides were incubated with an anti-phospho-MAPK antibody overnight at 4 °C. The following day, the slides were incubated with Cy3-conjugated secondary antibody (1:500, Abcam, Cambridge, UK) for 2 h at room temperature in the dark. Subsequently, the samples were mounted with medium containing DAPI and imaged using a confocal microscope.

Western Blotting (WB)

Cardiac tissues and cardiomyocytes were lysed for protein analysis using RIPA buffer containing protease inhibitors (P10013, Beyotime). Proteins were separated by SDS-PAGE and transferred onto membranes. After blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-SIRT4 (1:1000, 69786S, Cell Signaling Technology), anti-IL-1β (1:1000, 16,806–1-AP, Proteintech), anti-phospho-MAPK (1:1000, R25238, ZENBIO), anti-MAPK (1:1000, R25239, ZENBIO), anti-JNK (1:1000, R22866, ZENBIO), anti-phospho-JNK1 (1:1000, R26311, ZENBIO), anti-IL-10 (1:1000, 60,269–1-Ig, Proteintech), anti-TNF-α (1:1000, 346,654, ZENBIO), anti-phospho-AMPK (1:1000, 310,044, ZENBIO), anti-AMPK (1:1000, R380431, ZENBIO), and anti-β-actin (1:20,000, 66,009–1-Ig, Proteintech). The membranes were then incubated with the corresponding secondary antibodies. Protein bands were visualized using a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA), and band intensities were quantified using ImageJ.

Statistical analysis

Data are presented as mean ± SEM. Statistical analysis was performed using Prism software (version 8.2.1). Comparisons between two groups were performed using Student's t-test, while differences among multiple groups were assessed by one-way ANOVA followed by Tukey's multiple comparisons test. Kaplan–Meier survival curves were generated, and the log-rank test was used to determine statistical significance. A P-value < 0.05 was considered statistically significant. "ns" indicates no significant difference.

Results

SIRT4 is downregulated in sepsis-induced mouse myocardium in vivo and in LPS-treated cardiomyocytes in vitro

To evaluate the expression of SIRT4 in the myocardium of mice with septic cardiomyopathy, we established a murine sepsis model using the CLP method. Immunohistochemical staining revealed that the expression level of SIRT4 was markedly decreased in the myocardium of SCM mice compared with the C57 Sham group (n = 3, P < 0.001) (Fig. 1A-B). This finding was further confirmed by Western blotting (Fig. 1C). The results showed that SIRT4 expression was significantly downregulated in the myocardium of the C57 CLP group relative to the C57 Sham group (n = 3, P < 0.05) (Fig. 1D). Furthermore, HL-1 cells were used to establish an in vitro SCM model by stimulation with different concentrations of LPS (Fig. 1E). The results demonstrated that SIRT4 expression in HL-1 cardiomyocytes was sharply decreased after stimulation with lipopolysaccharide (LPS, 1 μg/mL) (Fig. 1F). These results indicate a crucial role for SIRT4 in the myocardium and cardiomyocytes under SCM conditions.

Fig. 1.

Fig. 1

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SIRT4 expression is decreased in septic cardiomyopathy in vivo and in vitro. A, B Representative IHC images and quantification of myocardial SIRT4 expression in sham-operated and CLP-induced C57 mice (n = 3). C, D Representative immunoblotting and quantification of SIRT4 in myocardium of the C57 sham group and the C57 CLP group (n = 3). * P < 0.05, *** P < 0.0001 vs the C57 Sham group. E, F IB assessment and quantitative results of SIRT4 expression in HL-1 cardiomyocytes treated with various LPS concentrations to mimic SCM conditions in vitro (n = 3). * P < 0.05, ****P < 0.0001 vs the ctrl group

SIRT4 knockout aggravates cardiac dysfunction and mortality in septic cardiomyopathy

To investigate the role of SIRT4 in SCM, the wild-type (C57) mice and SIRT4-knockout (KO) mice subjected to CLP surgery, respectively (Fig. 2A). Successful SIRT4 knockout was confirmed by Western blotting (n = 5, P < 0.0001) (Fig. 2B). Survival curves revealed that the KO CLP group exhibited significantly lower survival rates than the C57 CLP group (n = 6, P < 0.0001) (Fig. 2C). Moreover, sepsis severity scores were significantly elevated in the C57 CLP group compared with the C57 Sham group (n = 5, P < 0.0001). Notably, the sepsis score was further increased in the KO CLP group relative to the C57 CLP group (Fig. 2D) (n = 5, P < 0.01). Moreover, the serum concentration of CK-MB was markedly elevated in the KO CLP group compared with the C57 CLP group (n = 6, P < 0.05) (Fig. 2E). Compared with the C57 Sham group, the C57 CLP group showed significantly decreased LVEF (n = 6, P < 0.05) and LVFS (n = 6, P < 0.01), as assessed by echocardiography (Fig. 2F). In addition, the E/A ratio was significantly reduced in the C57 CLP group (n = 6, P < 0.01). SIRT4 knockout further exacerbated the aforementioned myocardial dysfunction in the KO CLP group compared with the C57 CLP group (n = 6, P < 0.001). Morphological assessment revealed that left ventricular internal diameter at systole (LVID; s) (n = 6, P < 0.05) and diastole (LVID; d) (n = 6, P < 0.01) were increased in the C57 CLP group compared with the C57 Sham group (Fig. 2G). Importantly, SIRT4 knockout further aggravated the enlargement of LVID; s (n = 6, P < 0.01) and LVID; d (n = 6, P < 0.05) after CLP surgery compared with the C57 CLP group. Collectively, these results indicate that SIRT4 knockout aggravates cardiac dysfunction and mortality in septic cardiomyopathy.

Fig. 2.

Fig. 2

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SIRT4 knockout exacerbates mortality and cardiac dysfunction in septic cardiomyopathy. A Diagrammatic representation of the CLP and functional evaluation timeline in mice. B Immunoblotting confirmation of successful SIRT4 knockout in mouse myocardium from the C57 mice and the SIRT4-KO mice (n = 5). ****P < 0.0001 vs the C57 CLP group. C Survival analysis using Kaplan–Meier curves of mice within 24 h after CLP surgery (n = 6). ****P < 0.0001 vs the C57 CLP group. D Assessment of sepsis scores (n = 5). E Levels of serum creatine kinase-MB (CK-MB, U/L) across experimental groups (n = 6). F Representative echocardiographic M-mode images from each group. G Quantitative echocardiographic assessment of cardiac functional parameters (n = 6). *P < 0.05, ** P < 0.001, *** P < 0.0001, ****P < 0.0001 vs the C57 Sham group; #P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.0001 vs the C57 CLP group

SIRT4 knockout exacerbates inflammatory infiltration and apoptosis in septic cardiomyopathy

To investigate the role of SIRT4 knockout in myocardial histopathology, we performed corresponding experiments. As shown in Fig. 3 A, H&E staining revealed that infiltrating lymphocytes were significantly increased in the KO CLP group compared with the C57 CLP group. Statistical analysis demonstrated that the H score was significantly elevated in the C57 CLP group compared with the C57 Sham group (n = 6, P < 0.0001), and was further increased in the KO CLP group relative to the C57 CLP group (n = 6, P < 0.0001) (Fig. 3B). IHC staining showed that the TNF-α-positive area was markedly enlarged in the KO CLP group compared with the C57 CLP group (n = 3, P < 0.05) (Fig. 3C-D). These results indicate that inflammation was aggravated by SIRT4 knockout in SCM mouse myocardium. Furthermore, TUNEL staining demonstrated that myocardial apoptosis was exacerbated in the KO CLP group compared with the C57 CLP group (n = 6, P < 0.05) (Fig. 3E-F). Western blotting revealed that IL-1β expression was significantly upregulated in the C57 CLP group compared with the C57 Sham group (n = 3, P < 0.05), and was further elevated in the KO CLP group compared with the C57 CLP group (n = 3, P < 0.05) (Fig. 3G-H). These results collectively indicate that SIRT4 knockout exacerbates myocardial injury induced by septic cardiomyopathy.

Fig. 3.

Fig. 3

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SIRT4 knockout aggravates myocardial inflammation and apoptosis in septic cardiomyopathy. A, B Histopathological assessment by H&E staining and quantitative histological score(n = 10). Scale bar = 50 μm. C, D Immunohistochemical images and quantification of TNF-α expression in cardiac tissues (scale bar = 50 μm). E, F Immunofluorescent TUNEL (green) staining and quantification of apoptosis intensity in mouse cardiac sections (n = 6). G, H WB analysis and quantification of SIRT4 and IL-1β expression in mouse cardiac tissues from all groups (n = 3). *P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001 vs the C57 Sham group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs the C57 CLP group

SIRT4 overexpression alleviates cardiac dysfunction and mortality in septic cardiomyopathy

To explore the effect of SIRT4 overexpression in SCM, wild-type (C57) mice and SIRT4-overexpressing (AAV9) mice were subjected to CLP surgery (Fig. 4A). Successful SIRT4 overexpression was confirmed by Western blotting (n = 5, P < 0.0001) (Fig. 4B). Survival curves revealed that the AAV9 CLP group exhibited significantly higher survival rates than the C57 CLP group (n = 6, P < 0.01) (Fig. 4C). Moreover, the sepsis score was significantly lower in the AAV9 CLP group than in the C57 CLP group (n = 5, P < 0.0001) (Fig. 4D). The serum concentration of CK-MB was markedly reduced in the AAV9 CLP group compared with the C57 CLP group (n = 6, P < 0.0001) (Fig. 4E). Echocardiography indicated that SIRT4 overexpression effectively improved cardiac function (Fig. 4F), as evidenced by elevated LVEF (n = 6, P < 0.0001), LVFS (n = 6, P < 0.0001) and E/A ratio (n = 6, P < 0.01) compared with the C57 CLP group. Additionally, morphological parameters including LVID; d (n = 6, P < 0.0001) and LVID; s (n = 6, P < 0.0001) were significantly reduced by SIRT4 overexpression in SCM (Fig. 4G). These results suggest that SIRT4 overexpression improves cardiac function and reduces mortality in SCM.

Fig. 4.

Fig. 4

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SIRT4 overexpression attenuates mortality and cardiac dysfunction in septic cardiomyopathy. A Diagrammatic representation of the CLP and functional evaluation timeline in mice. B Immunoblotting confirmation of successful SIRT4 overexpression in mouse myocardium (n = 5). ****P < 0.0001 vs the C57 CLP group. C Survival analysis using Kaplan–Meier curves of mice within 24 h after CLP surgery (n = 6). **P < 0.01 vs the C57 CLP group. D Assessment of sepsis scores (n = 5). E Levels of serum creatine kinase-MB (CK-MB, U/L) across experimental groups (n = 6). F Representative echocardiographic M-mode images from each group. G Quantitative echocardiographic assessment of cardiac functional parameters (n = 6). *P < 0.05, ** P < 0.001, *** P < 0.0001, ****P < 0.0001 vs the C57 Sham group; #P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.0001 vs the C57 CLP group

SIRT4 overexpression attenuates inflammatory infiltration and apoptosis in septic cardiomyopathy

Next, we conducted histological experiments to investigate the role of SIRT4 overexpression in SCM. As shown in Fig. 5A-B, SIRT4 overexpression significantly reduced the H score elevated by CLP surgery (n = 6, P < 0.0001). IHC staining revealed that the TNF-α-positive area was markedly reduced in the AAV9 CLP group compared with the C57 CLP group (n = 6, P < 0.05) (Fig. 5C-D). These results indicate that myocardial inflammation was alleviated by SIRT4 overexpression in SCM. TUNEL staining demonstrated that myocardial apoptosis was significantly attenuated in the AAV9 CLP group compared with the C57 CLP group (n = 6, P < 0.05) (Fig. 5E-F). Western blotting showed that IL-1β expression was downregulated in the AAV9 CLP group compared with the C57 CLP group (n = 3, P < 0.0001) (Fig. 5G-H). Collectively, these results suggest that SIRT4 overexpression alleviates myocardial injury induced by CLP in SCM.

Fig. 5.

Fig. 5

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SIRT4 overexpression alleviates myocardial inflammation and apoptosis in septic cardiomyopathy. A, B Histopathological assessment by H&E staining and quantitative histological score (n = 10). Scale bar = 50 μm. C, D, Immunohistochemical images and quantification of TNF-α expression in cardiac tissues (scale bar = 50 μm) (n = 6). E, F Immunofluorescent TUNEL (green) staining and quantification of apoptosis intensity in mouse cardiac sections (n = 6). G, H WB analysis and quantification of SIRT4 and IL-1β expression in mouse cardiac tissues from all groups (n = 3). *P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001 vs the C57 Sham group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs the C57 CLP group

SIRT4 knockout activates the MAPK-JNK pathway in septic myocardium in vivo and in LPS-treated cardiomyocytes in vitro

To explore the mechanism underlying the role of SIRT4 in septic cardiomyopathy, we performed RNA-seq on cardiac tissues harvested from the KO CLP and C57 CLP groups. The heatmap displayed the differentially expressed genes (DEGs) between the two groups (Fig. 6A). The volcano plot identified 1254 upregulated genes and 1732 downregulated genes (Fig. 6B). KEGG enrichment analysis revealed 20 significantly altered signaling pathways (Fig. 6C). Of interests, the MAPK-JNK pathway was markedly altered in SCM mouse myocardium following SIRT4 knockout. Next, we performed biological experiments to validate the sequencing results. As shown in Fig. 6D-E, IHC staining demonstrated that the expression levels of phosphorylated MAPK (n = 3, P < 0.01) and phosphorylated JNK (n = 3, P < 0.01) were obviously elevated in SIRT4 KO mice compared with C57 mice after CLP surgery. Western blotting indicated that the MAPK-JNK pathway was significantly activated by SIRT4 knockout in septic myocardium (n = 3, P < 0.05) (Fig. 6F). Additionally, inflammatory cytokines were markedly elevated in septic myocardium of the SIRT4 knockout mice compared with the C57 mice (n = 3, P < 0.05) (Fig. 6G). Consistently, SIRT4 knockdown increased the activation of the MAPK-JNK pathway and the expression of inflammatory factors compared with the control group in LPS-treated HL-1 cells in vitro (n = 3, P < 0.05) (Fig. 6H-I). Collectively, these data reveal that SIRT4 deficiency activates MAPK-JNK signaling and enhances inflammatory responses in cardiomyocytes during septic cardiomyopathy.

Fig. 6.

Fig. 6

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MAPK-JNK signaling is activated by SIRT4 knockout in septic myocardium in vivo and in LPS-treated HL-1 in vitro. A Heatmap illustrating DEGs. Red denotes upregulation; blue denotes downregulation. B Volcano plot of DEGs (red dots: upregulated; blue dots: downregulated). C Top 20 enriched KEGG pathways of DEGs. D IHC staining and quantitative analysis for phosphorylated MAPK in mouse myocardium of the C57 CLP and the KO CLP groups (n = 3). Scale bar = 50 μm. E IHC staining and quantitative analysis for phosphorylated JNK in mouse myocardium of the C57 CLP and the KO CLP groups (n = 3). Scale bar = 50 μm. F, G Western blotting and quantitative analysis of the MAPK-JNK signaling pathway (F) and inflammatory factors (G) in myocardial of the C57 CLP group and the KO CLP groups (n = 3). *P < 0.05, ** P < 0.01 vs the C57 CLP group. H, I Western blotting and quantitative analysis of the MAPK-JNK signaling pathway (H) and inflammatory factors (I) in LPS-treated cardiomyocytes (n = 3). *P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001 vs the LPS + siNC group

JNK inhibition attenuates cardiac injury induced by SIRT4 deficiency in septic cardiomyopathy in vivo and in vitro

To investigate whether hyperactivation of the MAPK-JNK pathway is a key downstream mechanism mediating the effects of SIRT4 deficiency, we performed rescue experiments in cardiomyocytes using the JNK-specific inhibitor SP600125 (Chang et al. 2024). Western blotting analysis indicated that SP600125 markedly inhibited the activation of the MAPK-JNK pathway (n = 3, P < 0.0001) (Fig. 7A). Consistently, SP600125 reduced the expression of inflammatory factors induced by SIRT4 knockdown in HL-1 cells (n = 3, P < 0.0001) (Fig. 7A). Flow cytometry showed that SP600125 treatment significantly alleviated apoptosis in HL-1 cells co-stimulated with si-SIRT4 and LPS (n = 3, P < 0.05) (Fig. 7B). Hoechst/PI staining revealed that SP600125 markedly attenuated HL-1 cell injury caused by SIRT4 knockdown (n = 3, P < 0.0001) (Fig. 7C). In vivo, SP600125 treatment effectively reduced mortality in KO mice after CLP surgery (n = 6, P < 0.01) (Fig. 7D). Moreover, both the sepsis score (n = 5, P < 0.0001) (Fig. 7E) and serum CK-MB concentration (n = 6, P < 0.0001) (Fig. 7F) were significantly decreased by SP600125 treatment in KO mice subjected to CLP surgery. These findings indicate that specific inhibition of the MAPK-JNK pathway effectively rescues the excessive inflammatory response and cellular damage caused by SIRT4 deficiency.

Fig. 7.

Fig. 7

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JNK inhibition improves cardiac injury caused by SIRT4 depression in septic cardiomyopathy in vivo and in vitro. A Western blotting and statistical analysis of the MAPK-JNK pathway and inflammatory factors of HL-1 in different groups (n = 3). B Representative flow cytometry and cell apoptosis quantification (n = 3). C PI/Hoechst staining and quantitative analysis of LPS-treated HL-1 cells in different groups (n = 3). Scale bar = 200 μm.*P < 0.05, ** P < 0.01, ****P < 0.0001 vs the si-NC group; #P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.001 vs the si-SIRT4 group. D Survival analysis using Kaplan–Meier curves of mice within 24 h post-CLP (n = 6). **P < 0.01 vs the KO Ctrl group. E Assessment of sepsis scores in different groups post-CLP (n = 5). F, Levels of serum creatine kinase-MB (CK-MB, U/L) across experimental groups post-CLP (n = 6). ****P < 0.0001 vs the C57 Ctrl group; #### P < 0.0001 vs the KO Ctrl group

SIRT4 deficiency upregulates the MAPK-JNK pathway by activating AMPK in septic cardiomyopathy

To investigate the mechanism by which SIRT4 knockdown activates the MAPK-JNK pathway, we reviewed the literature and found that SIRT4 may participate in inflammatory stress through the activation of AMPK (Trujillo-Del Rio et al. 2026), and that AMPK plays an important role in JNK activation (Fan et al. 2023). Therefore, we examined whether SIRT4 knockdown could still activate the MAPK-JNK pathway following competitive inhibition of AMPK with Compound C (Vucicevic et al. 2011). Western blot analysis showed that in HL-1 cells under LPS stimulation, SIRT4 knockdown increased AMPK phosphorylation levels and total AMPK protein expression, and the MAPK-JNK pathway was activated (n = 3, P < 0.0001) (Fig. 8A–B). However, simultaneous inhibition of AMPK and knockdown of SIRT4 significantly suppressed the activation of the MAPK-JNK pathway. Furthermore, immunofluorescence staining revealed that inhibition of AMPK by Compound C combined with SIRT4 knockdown markedly reduced MAPK activation in HL-1 cells (Fig. 8C). Taken together, these results indicate that in cardiomyocyte inflammatory injury induced by septic cardiomyopathy, SIRT4 expression is decreased, and the reduction of SIRT4 induces AMPK activation, thereby upregulating the MAPK-JNK pathway and exacerbating inflammation.

Fig. 8.

Fig. 8

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SIRT4 knockout promotes the MAPK-JNK pathway via activating AMPK in septic cardiomyopathy. A, B Western blotting and statistical analysis of the MAPK-JNK pathway in LPS-treated HL-1 cells in different groups (n = 3). C Immunofluorescent imaging of phosphorylated-MAPK in HL-1 cells in different groups after LPS treatment. Scale bar = 20 μm. *P < 0.05, ** P < 0.01, ****P < 0.0001 vs the NC group; #P < 0.05, ## P < 0.01, ###P < 0.001, ####P < 0.001 vs the si-SIRT4 group

Discussion

Our study demonstrates that SIRT4, a mitochondrial NAD⁺-dependent deacetylase, plays a protective role against septic cardiomyopathy by suppressing AMPK-mediated MAPK-JNK signaling. Both in vivo and in vitro experiments revealed that SIRT4 expression was significantly downregulated in septic myocardium and LPS-treated cardiomyocytes. SIRT4 knockout exacerbated CLP-induced mortality, cardiac dysfunction, myocardial inflammatory infiltration, and apoptosis, whereas SIRT4 overexpression significantly alleviated these pathological changes. Mechanistically, SIRT4 deficiency activated AMPK, which led to excessive activation of the MAPK-JNK pathway. Conversely, pharmacological inhibition of JNK with SP600125 effectively attenuated myocardial injury and inflammation induced by SIRT4 deficiency both in vivo and in vitro. Furthermore, blockade of AMPK signaling with Compound C markedly suppressed MAPK-JNK pathway activation caused by SIRT4 knockdown. Taken together, these findings indicate that SIRT4 deficiency exacerbates cardiomyocyte apoptosis and inflammation in septic cardiomyopathy by activating the AMPK–MAPK-JNK signaling axis. Therefore, SIRT4 represents a promising therapeutic target for sepsis-induced myocardial injury.

SCM is characterized as an acute myocardial syndrome associated with sepsis, displaying high morbidity and mortality rates (Kakihana et al. 2016). Diverse mechanisms of cardiomyocyte death, such as apoptosis (Wang et al. 2015), pyroptosis (Li et al. 2019), necroptosis (Zhou et al. 2024), and ferroptosis (Li et al. 2020), contribute to SCM pathogenesis alongside elevated inflammatory mediators. Numerous studies have highlighted the MAPK-JNK cascade in the context of SCM. JNK, a key MAPK family member, plays a central role in mediating cardiac damage through pathways involving proapoptotic signal activation (Li et al. 2024), exacerbation of inflammatory cascades (Chen et al. 2018),and mitochondrial impairment (Jin et al. 2018). Furthermore, SIRT4 critically regulates mitochondrial function; its deficiency or suppression results in mitochondrial dysfunction, augmented reactive oxygen species (ROS) generation, and impaired metabolic regulation (Lin et al. 2024). These conditions strongly activate the JNK pathway. Consistent with these findings, the current study showed significantly reduced SIRT4 expression in myocardial tissue from CLP-induced SCM mice. Moreover, SIRT4 knockout further exacerbated cardiac dysfunction, apoptosis, and inflammatory infiltration induced by sepsis in vivo. However, whether and how SIRT4 regulates SCM remained unclear.

Subsequent experiments aimed to clarify the mechanism by which SIRT4 alleviates apoptosis during sepsis. The MAPK-JNK pathway was hypothesized to mediate myocardial injury associated with SIRT4 regulation, supported by transcriptomic data. The experiments confirmed the activation of MAPK-JNK signaling in myocardial tissue from CLP-treated SIRT4 KO mice. Furthermore, HL-1 cells stimulated with LPS to mimic sepsis in vitro (Wang et al. 2024) also showed activation of the MAPK-JNK pathway following SIRT4 depletion.

In line with previous research (Shen et al. 2017; Zhang et al. 2018), the JNK inhibitor SP600125 was selected for the current investigation. It has been previously established that SP600125 exerts protective effects in LPS-induced acute pulmonary injury (Zheng et al. 2014) and diabetic nephropathy (Zhang et al. 2018), primarily through its anti-inflammatory and antioxidative properties. Consistently, our study revealed that SP600125 effectively suppressed cardiomyocyte apoptosis and enhanced cellular viability via inhibition of MAPK-JNK signaling triggered by the depletion of SIRT4. Consequently, it appears that SIRT4 mitigates LPS-induced cardiomyocyte apoptosis in SCM by suppressing the activation of JNK. However, further research is warranted to precisely elucidate the interaction between SIRT4 and the JNK pathway. Our findings suggest that SIRT4’s protective effects against SCM largely depend on its ability to attenuate apoptotic processes mediated by MAPK-JNK signaling.

Earlier studies have reported that transgenic overexpression of SIRT1 (Kane and Sinclair 2018) or SIRT6 (Liu et al. 2021), administration of sirtuin-activating compounds (STACs) (Shen et al. 2024), or supplementation with NAD+ precursors (2022), can enhance organ function, physical resilience, resistance to diseases, and lifespan extension. Rational design of molecules capable of mitigating multiple diseases and potentially extending human lifespan is feasible (Bonkowski and Sinclair 2016). However, compared with other sirtuins such as SIRT1, the development of specific pharmacological agonists for SIRT4 remains limited. Nonetheless, multiple studies reported a vital role for SIRT4 in cardiac diseases. For instance, Zeng et al. (Zeng et al. 2018) previously demonstrated that SIRT4 protects mitochondria and reduces apoptosis in myocardial ischemia–reperfusion injury. Furthermore, SIRT4 has been demonstrated to counteract doxorubicin-induced cardiac toxicity through modulation of the AKT/mTOR/autophagy signaling cascade (He et al. 2022). The results obtained in our study further strengthen the translational relevance of targeting SIRT4 in therapeutic strategies for SCM. Modulating the SIRT4-JNK signaling axis could represent an innovative and effective therapeutic avenue for SCM management.

Nevertheless, this investigation also carries several limitations. Firstly, recent evidence highlights alternative regulated cell death mechanisms, including ferroptosis (Ye et al. 2025), as potential contributors to the initiation and progression of SCM. However, our study did not examine the roles of other cell death modalities such as pyroptosis, ferroptosis, or necroptosis in the cardioprotective effects conferred by SIRT4 during sepsis. Secondly, our findings are based on a single acute sepsis model in male mice, and sex‑specific or chronic models were not examined. Thirdly, the direct molecular interaction between SIRT4 and AMPK requires further elucidation. Finally, the lack of specific pharmacological SIRT4 agonists limits the translational applicability of our genetic overexpression approach. Future studies are required to comprehensively clarify the exact protective mechanisms associated with SIRT4 in SCM.

In summary, these findings indicate that SIRT4 knockout exacerbates cardiac injury in SCM by enhancing inflammatory responses and activating the AMPK-MAPK-JNK signaling pathway. Consequently, modulation of SIRT4 presents a potential therapeutic avenue for treating SCM.

Supplementary Information

Supplementary Material 1 . (125.7MB, zip)

Abbreviations

SCM

Septic cardiomyopathy

CLP

Cecal ligation and puncture

AAV9

Adeno-associated virus serotype 9

LPS

Lipopolysaccharide

DEGs

Differentially expressed genes

WB

Western blotting

SIRT4

Sirtuin4

LVFS

Left ventricular fractional shortening

LVEF

Left ventricular ejection fraction

CK-MB

Creatine kinase-MB

siRNA

Small interfering RNA

PI

Propidium iodide

IB

Immunoblotting

IF

Immunofluorescence

Authors’ contributions

Investigation, methodology and formal analysis, Na Li; software, Kangla Liao; validation, Shuting Chang and An He; resources, Suxin Luo; data curation, Jiaxin Chen; writing—original draft preparation, Na Li; writing—review and editing,Shuting Chang; visualization, Suxin Luo; supervision, An He; project administration, Na Li; funding acquisition, Na Li. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant number 82305006 and 82300477) and the Doctoral Research and Innovation Project of Chongqing Medical University (Grant number CYYY-BSYJSKYCXXM202414).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare that they have no conflicts of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Suxin Luo, Email: luosuxin@hospital.cqmu.edu.cn.

An He, Email: an.he@hospital.cqmu.edu.cn.

Shuting Chang, Email: 2023140093@stu.cqmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 . (125.7MB, zip)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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