Establishment of a Novel in vitro Model of Sepsis-Induced Myocardial Injury Using Septic Serum: A Comprehensive Comparative Study

Introduction

Sepsis is a life-threatening syndrome characterized by a dysregulated host response to infection, leading to multisystem organ dysfunction. It is a major cause of mortality and morbidity in intensive care units (ICUs) worldwide and imposes a substantial medical burden.^1–3 Among the various complications, sepsis-induced myocardial injury is particularly common and lethal. Approximately 60% of septic patients experience myocardial injury, with associated mortality rates reaching 70–90%.^4,5 Despite recent advances, the lack of effective diagnostic biomarkers and targeted therapies remains a significant challenge, largely due to an incomplete understanding of the underlying molecular mechanisms.

To investigate the pathogenesis of sepsis-induced myocardial injury, the development of physiologically relevant and reliable in vitro models is essential. Currently, most studies utilize lipopolysaccharide (LPS) to induce inflammatory injury in cardiomyocytes and simulate the immune response associated with sepsis.^6–8 LPS activates Toll-like receptor 4 (TLR4), triggering downstream inflammatory cascades, excessive cytokine release, oxidative stress, and ultimately cardiomyocyte dysfunction.^9–12 While LPS-based models partially reproduce the inflammatory milieu of sepsis, they fail to reflect the systemic immune, metabolic, and neurohumoral alterations observed in clinical settings.^13–15 Similarly, tumor necrosis factor-α (TNF-α), a key proinflammatory cytokine in sepsis, promotes cardiomyocyte apoptosis, mitochondrial dysfunction, and impaired contractility through activation of NF-κB and other downstream pathways.^16,17 However, as with LPS, single-factor stimulation cannot replicate the complex biological environment of sepsis, which involves multiple cytokines, chemokines, and damage-associated molecular patterns acting in concert. Therefore, standardized and comprehensive in vitro models of sepsis-induced myocardial injury are still lacking. Finding a suitable in vitro model of sepsis-induced myocardial injury is crucial to explore the mechanism of sepsis-induced myocardial injury.

To address these limitations, we investigated the use of septic serum, which contains a dynamic and physiologically relevant mixture of inflammatory mediators, endotoxins, metabolic by-products, and immune components that better reflect the multifaceted pathophysiology of sepsis.^18–20 Septic serum may thus offer a more accurate and clinically relevant model of sepsis-induced myocardial injury.

In this study, we established in vitro models of sepsis-induced myocardial injury by treating cardiomyocytes with LPS, TNF-α, and septic serum. By assessing a range of cellular responses, including cell viability, cytotoxicity, inflammatory cytokine production, oxidative stress, apoptosis and myocardial injury markers, as well as performing transcriptomic profiling, we aimed to identify the most suitable model. Our findings provide a foundation for future mechanistic studies and the development of targeted therapeutic strategies for sepsis-induced myocardial injury.

Materials and Methods

Acquisition of Septic Serum

Healthy adult male C57BL/6J mice (weighing 25 ± 3 g) were randomly divided into two groups: a sham group (n=10) and a sepsis group (n=10). All procedures involving animals complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and were approved by the Fujian Medical University Institutional Animal Care and Use Committee (FJMU IACUC 2021–0336). LPS (Sigma, L2880, USA) was dissolved in normal saline at a concentration of 3 mg/mL, and 0.1 mL of this solution was administered intraperitoneally to each mouse in the sepsis group, a dose previously shown to induce systemic inflammation and cardiac injury.^21–23 The sham group received an intraperitoneal injection of an equivalent volume of normal saline. Cardiac structure and function were evaluated 24 hours after LPS injection (see Figure S1). Successful establishment of the sepsis-induced myocardial injury model was confirmed by the presence of typical systemic symptoms, such as decreased activity, hypothermia, and listlessness, along with evidence of cardiac structural and functional impairment consistent with established criteria. After establishing the sepsis-induced myocardial injury model, blood was collected immediately. Serum from ten mice per group was pooled to minimize individual variability, centrifuged to remove cellular debris, filtered, and stored at −80 °C for subsequent experiments. Control serum was similarly obtained from sham-operated mice and processed in parallel.

All invasive procedures were performed under anesthesia using isoflurane with oxygen. Buprenorphine (0.1 mg/kg, subcutaneous injection) was administered to relieve pain and minimize distress. To ensure animal welfare, mice were maintained on a heated pad during procedures and continuously monitored for signs of pain, distress, or abnormal behavior over the 24-hour observation period. Humane endpoints included severe hypothermia, profound lethargy, or unresponsiveness to stimuli. Animals meeting these criteria were euthanized immediately according to the AVMA Guidelines for the Euthanasia of Animals (2020), using an overdose of sodium pentobarbital. All personnel involved in animal care were specially trained in the use of anesthesia, analgesia, and animal monitoring techniques to ensure compliance with ethical guidelines and improve data reliability.²⁴ A total of 26 mice were included in this study. Of these, 20 mice were euthanized according to humane endpoints, while 6 mice died prior to meeting euthanasia criteria. Postmortem examination determined that their deaths were caused by multiple organ damage resulting from an excessive inflammatory response.

In vitro Model Construction

AC16 cardiomyocytes (Pricella, CL-0790) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells at passages 4–8 were used in all experiments. The control group received no treatment. To establish an in vitro model of sepsis-induced myocardial injury, cells were treated for 24 hours with either LPS (10 μg/mL; Sigma, L2880, USA), TNF-α (1 ng/mL; Pricella, PCK050, China), or septic serum (10 μL/mL) in the LPS, TNF-α, and serum groups, respectively. These concentrations were selected based on previous publications and preliminary experiments, which demonstrated that they effectively induce cardiomyocyte injury without causing excessive cytotoxicity.^25,26 A 24-hour treatment duration was chosen because it captures key cellular responses, including inflammatory activation and early markers of myocardial injury, while minimizing late-stage or nonspecific effects. The experimental scheme is illustrated in Figure 1. All experiments were conducted independently at least three times using biological replicates.

Figure 1 The flowchart of this study. Abbreviations: i.p, intraperitoneal; LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA kits were used to quantify the levels of Interleukin-1β (IL-1β) (Boster, EK0394, China), Interleukin-6 (IL-6) (Boster, EK0411, China) and Tumor necrosis factor-α (TNF-α) (Boster, EK0527, China) in mouse serum. In addition, culture supernatants were collected after 24 hours of treatment by centrifuging the media at 1000 × g for 5 minutes, followed by storage at −80 °C until cytokine analysis. ELISA kits were also employed to detect markers of myocardial injury in the cell supernatant, including brain natriuretic peptide (BNP) (Elabscience, E-EL-H6166, China), creatine kinase-MB (CK-MB) (CUSABIO, CSB-E05140h, China), and cardiac troponin I (cTnI) (CUSABIO, CSB-E05139h, China). All procedures were performed strictly in accordance with the manufacturers’ instructions. Cytokine and biomarker concentrations were normalized using standard reagents provided with each kit.

Hematoxylin-Eosin (HE) Staining

Ventricular myocardium was harvested from mice and immediately fixed in 4% paraformaldehyde at room temperature for 24 hours. The fixed tissue was subsequently dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 5 μm thickness were cut using a microtome and mounted onto glass slides. Sections were dewaxed, rehydrated, stained with hematoxylin for 5 minutes, followed by brief rinsing in distilled water and differentiation in acid alcohol. After counterstaining with eosin for 2 minutes, the slides were dehydrated, cleaned, and mounted with neutral resin. Histopathological alterations in myocardial tissue, including inflammatory cell infiltration, myocyte swelling, and disruption of structural integrity, were observed under a light microscope (Leica, RM2235, Germany).

Echocardiographic Examination

Mice were anesthetized using a mixture of isoflurane and oxygen, and anesthesia was confirmed prior to examination. Cardiac function was assessed using transthoracic echocardiography (Youkey, Guangdong, China). M-mode imaging was used to measure the left ventricular internal diameter at end-diastole (LVIDd) and at end-systole (LVIDs). Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were subsequently calculated based on these dimensions. To ensure consistency, all echocardiographic assessments were conducted by the same operator. Each parameter was measured in triplicate for each mouse to ensure accuracy and reproducibility.

Electrocardiograph (ECG) Monitoring

After anesthetizing the mice, electrodes were attached to each limb in accordance with the standard lead configuration. A stable lead II electrocardiogram (ECG) was continuously recorded for 5 minutes using the MD3000 biological signal acquisition and processing system (Zhenghua Bioinstrument, Huaibei, China). Care was taken to minimize movement and external disturbances to ensure accurate and artifact-free ECG data acquisition.

Cell Counting Kit-8 (CCK8)

Cell viability was assessed using the CCK-8 assay (Elabscience, E-CK-A362, China). Cardiomyocytes were seeded into 96-well plates at a density of 5×10⁴ cells per well and incubated overnight. Following 24 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 2 hours. The absorbance at 450 nm was then measured using a microplate reader (Tecan, Infinite M PLEX, Switzerland). Cell viability was expressed as a percentage relative to the untreated control group.

Lactatedehydrogenase (LDH) Release Assay

LDH release was measured using an LDH detection kit (Jiangcheng, Nanjing, A020-2-1, China) to evaluate cell membrane integrity. Following 24 hours of treatment, 100 μL of culture supernatant was collected from each well and transferred to a 96-well plate. Subsequently, 50 μL of LDH detection reagent was added to each well, and the plate was incubated at 37 °C in the dark for 30 minutes. Absorbance was measured at 490 nm using a microplate reader (Tecan, Infinite M PLEX, Switzerland). The percentage of LDH release was calculated to reflect the extent of cytotoxicity.

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from each group of cells (1 × 10⁵ cells/well) using an RNA extraction kit (Beyotime, R0017M, China). Complementary DNA (cDNA) was synthesized using a reverse transcription kit (Beyotime, D7170M, China) according to the manufacturer’s instructions. qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Kit (Vazyme, Q711-02, China). The thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 seconds, followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 30 seconds. A melting curve analysis was conducted at the end of the amplification cycles to verify the specificity of the products. GAPDH was used as the internal control gene. Relative mRNA expression levels were calculated using the 2^^−ΔΔCT method. Primer sequences are listed in Table S1.

Cell Bioenergetic Tests

AC16 cardiomyocytes were seeded into an XF96 cell culture microplate, and subjected to bioenergetic analysis after 24 hours of treatment. Prior to the assay, cells were washed twice with seahorse assay medium and incubated in a non-CO₂ incubator at 37 °C for 1 hour to allow for pH and temperature equilibration. The oxygen consumption rate (OCR) reagents were loaded into the appropriate ports of the sensor cartridge, which was then calibrated using the Seahorse XFe96 Analyzer (Agilent Technologies, USA). Upon successful calibration, the cell culture plate was loaded into the analyzer, and real-time measurements of OCR were recorded.

Reactive Oxygen Species (ROS) Assay Assessment

After 24 hours of treatment, cells in each group were washed with serum-free medium, followed by incubation with 10 μmol/L DCFH-DA fluorescent probe at 37 °C for 30 minutes in the dark. After incubation, cells were washed again to remove excess probe. ROS levels were then visualized and captured using a fluorescence microscope. Quantification of fluorescence intensity was performed using ImageJ software.

Flow Cytometry (FCM)

Apoptosis in AC16 cardiomyocytes was evaluated using flow cytometry. Briefly, 1×10⁶ cells from each group were seeded in 6-well plates and treated for 24 hours. Following treatment, cells were harvested, washed twice with PBS, and stained with Annexin V-FITC (Beyotime, C1062S, China) according to the manufacturer’s instructions. The cells were incubated in the dark at room temperature for 15 minutes, and apoptotic populations were subsequently analyzed using a flow cytometer.

Tunel Staining

Following treatment, cells in each group were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes. The TUNEL kit (Elabscience, E-CK-A321, China) was used to detect apoptosis rate, according to the manufacturer’s instructions. TUNEL-positive staining was observed, and the apoptosis rate was calculated.

Identification and Functional Enrichment Analysis of Differentially Expressed Genes (DEGs)

Transcriptomic analysis was performed to assess mRNA expression profiles across experimental groups, including three samples each from the control, LPS, TNF-α, and serum-treated groups (Table S2). The “Limma-voom” R package was used to analyze the DEGs. The DEGs with |log2 fold change (FC)| >0.583 and P value < 0.05 were considered statistically significant. Heatmaps and volcano plots of DEGs were generated using the “Pheatmap” and “ggplot2” R packages, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the “clusterProfiler” R package to evaluate biological processes (BP), molecular functions (MF), cellular components (CC), and signaling pathways. GO terms and KEGG pathways with P values < 0.05 were deemed significantly enriched.

Evaluation of the Diagnostic Value

Septic cardiomyopathy datasets (GSE79962 and GSE237861) were obtained from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Data were transformed using transcripts per million (TPM), averaged, normalized, and log-normalized [log(TPM+1)]. Gene set variation analysis (GSVA) was performed on the DEGs in the in vitro model of sepsis-induced myocardial injury treated with LPS, TNF-α, and septic serum in GSE79962 and GSE237861 datasets, respectively, and the GSVA score was calculated. Then, receiver operating characteristic curves (ROC) were plotted to evaluate the diagnostic value of DEGs in each group of sepsis-induced myocardial injury in vitro models for sepsis-induced myocardial injury.

Statistical Analysis

Statistical analyses were conducted using SPSS software (version 26.0) and R software (version 4.2.0). All data were presented as the mean ± standard deviation. One-way analysis of variance (ANOVA) was used to compare differences among multiple groups, followed by the least significant difference (LSD) method for pairwise comparisons. The Student’s t-test was applied for comparisons between two groups. The Grubbs test was used to detect outliers, and no data points were excluded from the final analysis. Data were assessed for normality using the Shapiro–Wilk test, with appropriate transformations applied if necessary. A p-value of less than 0.05 was considered statistically significant.

Results

Inflammatory Responses in Various in vitro Models of Sepsis-Induced Myocardial Injury

To evaluate the inflammatory response, we measured the expression levels of pro-inflammatory and anti-inflammatory genes in each group. Compared to the control group [IL-6 (1.01± 0.13); IL-1β (1.00 ± 0.07)], IL-6 and IL-1β expression was significantly upregulated in the LPS [IL-6 (1.34 ± 0.10); IL-1β (2.04 ± 0.15)], TNF-α [IL-6 (1.70 ± 0.08); IL-1β (2.69 ± 0.58)], and serum [IL-6 (1.93 ± 0.13); IL-1β (3.91 ± 0.88)] groups (P<0.05). IL-6 expression in the TNF-α group was significantly higher than in the LPS group (P<0.01), whereas no significant difference in IL-1β expression was observed between these two groups (P>0.05). Notably, both IL-6 and IL-1β expression levels were significantly higher in the serum group than in the LPS and TNF-α groups (P<0.05) (Figure 2A and B). Additionally, the expression of the anti-inflammatory gene TGF-β was significantly reduced in the LPS (0.55 ± 0.07), TNF-α (0.60 ± 0.09), and serum (0.57 ± 0.13) groups compared to the control group (1.02 ± 0.23) (P < 0.01), with no significant differences among the three treatment groups (P > 0.05) (Figure 2C). There were no significant differences in IL-10 expression among the control (1.02 ± 0.23), LPS (0.98 ± 0.22), and TNF-α (0.78 ± 0.01) groups (P>0.05). However, IL-10 expression was significantly lower in the serum group (0.44 ± 0.02) compared to the control, LPS, and TNF-α groups (P < 0.05) (Figure 2D). These findings suggest that the in vitro model of sepsis-induced myocardial injury established using septic serum effectively replicates the inflammatory milieu of sepsis in cardiomyocytes, including alterations in both pro-inflammatory and anti-inflammatory mediators.

Figure 2 Comparison of inflammatory responses in various in vitro models of sepsis-induced myocardial injury. (A–D) The expression levels of IL-6, IL-1β, TGF-β and IL-10 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α; IL-6, Interleukin-6; IL-1β, Interleukin-1β; TGF-β, Transforming growth factor-β; IL-10, Interleukin-10; ns, not significant.

Oxidative Stress in Various in vitro Models of Sepsis-Induced Myocardial Injury

Mitochondrial function and ROS levels were assessed to evaluate oxidative stress. As shown in Figure 3A, OCR measurements revealed that basal respiration was significantly reduced in the LPS (50.24 ± 5.34 pmol/min), TNF-α (47.31 ± 5.95 pmol/min), and serum (38.54 ± 6.25 pmol/min) groups compared to the control group (56.32 ± 2.91 pmol/min) (P<0.05). No significant difference was observed between the LPS and TNF-α groups (P>0.05), while basal respiration in the serum group was significantly lower than in both the LPS and TNF-α groups (P<0.01) (Figure 3B). ATP production was not significantly altered in the LPS (51.63 ± 3.74 pmol/min) or TNF-α (49.75 ± 4.64 pmol/min) groups compared to the control group (53.38 ± 5.85 pmol/min) (P > 0.05). However, the serum group showed a marked reduction in ATP production (34.91 ± 6.78 pmol/min) (P < 0.001) (Figure 3C). Maximal respiratory capacity was also significantly decreased in the LPS (74.16 ± 6.73 pmol/min), TNF-α (63.90 ± 5.06 pmol/min), and serum (46.17 ± 5.25 pmol/min) groups compared to the control group (107.57 ± 1.45 pmol/min) (P < 0.001), with the serum group exhibiting a significantly greater reduction than the other two groups (P < 0.001) (Figure 3D). Similarly, spare respiratory capacity was markedly decreased in the LPS (23.92 ± 5.58 pmol/min), TNF-α (16.59 ± 3.84 pmol/min), and serum (7.64 ± 2.85 pmol/min) groups relative to the control group (51.24 ± 2.96 pmol/min) (P < 0.001), and this reduction was most pronounced in the serum group (P < 0.001) (Figure 3E).

Figure 3 Comparison of oxidative stress responses in various in vitro models of sepsis-induced myocardial injury. (A–E) OCR for each group, as well as basal respiration, ATP production, maximal respiration, and spare respiratory capacity. (F) ROS levels in each group. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α; OCR, Oxygen consumption rate; ROS, Reactive oxygen species; ns, not significant.

ROS detection revealed no significant difference in ROS levels among the control (0.05 ± 0.03%), LPS (0.04 ± 0.02%), and TNF-α (0.05 ± 0.02%) groups (P > 0.05). In contrast, ROS levels in the serum group (0.11 ± 0.03%) were significantly higher than in the other three groups (P < 0.05) (Figure 3F).

These results indicate that septic serum treatment induces a more severe impairment of mitochondrial function and a greater degree of oxidative stress in cardiomyocytes compared to LPS or TNF-α stimulation alone.

Apoptotic Rates in Various in vitro Models of Sepsis-Induced Myocardial Injury

Apoptosis levels in cardiomyocytes were assessed to evaluate cellular damage induced by different modeling approaches. Flow cytometry analysis revealed that, compared to the control group (8.89 ± 0.23%), apoptosis rates were significantly increased in the LPS (19.09 ± 0.30%), TNF-α (22.50 ± 0.48%), and serum groups (23.48 ± 0.08%) (P<0.001). Notably, the serum group exhibited a significantly higher apoptosis rate than both the LPS group and TNF-α groups (P<0.001) (Figure 4A). Consistently, TUNEL staining revealed increased apoptotic areas in the LPS (0.05 ± 0.01%), TNF-α (0.07 ± 0.01%), and serum (0.25 ± 0.01%) groups compared to the control group (0.02 ± 0.01%) (P < 0.05). The apoptotic area was markedly higher in the serum group than in the LPS and TNF-α groups (P < 0.001), while no significant difference was observed between the LPS and TNF-α groups (P > 0.05) (Figure 4B). These findings suggest that septic serum induces a greater degree of cardiomyocyte apoptosis than LPS or TNF-α, highlighting the potential severity of systemic factors present in septic serum. However, the underlying molecular pathways responsible for this increased apoptosis warrant further investigation.

Figure 4 Comparison of apoptosis rates in various in vitro models of sepsis-induced myocardial injury. (A and B) The apoptosis level of each group. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α; ns, not significant.

Degree of Myocardial Injury in Various in vitro Models of Sepsis-Induced Myocardial Injury

The optimal concentrations of LPS, TNF-α, and septic serum for inducing cardiomyocyte injury were determined to be 10 μg/mL, 1 ng/mL, and 10 μL/mL, respectively (Figure 5A–C). At these optimal concentrations, cell viability was significantly reduced in the LPS (0.49 ± 0.02), TNF-α (0.44 ± 0.06), and serum (0.29 ± 0.02) groups compared to the control group. Notably, the decrease in cell viability was significantly greater in the serum group than in the LPS and TNF-α groups (P < 0.001) (Figure 5D). Correspondingly, LDH release in the supernatant, a marker of cell membrane damage, was elevated in the LPS (318.02 ± 60.89 U/L), TNF-α (353.09 ± 58.56 U/L), and serum (451.85 ± 52.58 U/L) groups, compared to the control group (181.23 ± 37.14 U/L) (P < 0.01). Although there was no significant difference in LDH levels between the LPS and TNF-α groups (P > 0.05), LDH release was significantly higher in the serum group than in either the LPS or TNF-α group (P < 0.05) (Figure 5E).

Figure 5 Comparison of the degree of injury in various in vitro models of sepsis-induced myocardial injury. (A–D) Cell viability of cardiomyocytes treated with LPS, TNF-α and septic serum. (E) LDH release level in each group. (F–H) The levels of BNP, cTnI and CK-MB in the supernatant of each group. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α; BNP, Brain natriuretic peptide; cTnI, cardiac troponin I; CK-MB, CreatineKinase-MB; ns, not significant.

To directly assess myocardial injury, we further examined key myocardial injury biomarkers in each group. There was no significant difference in BNP levels between the control (176.53 ± 8.30 pg/mL) and LPS (185.40 ± 12.78 pg/mL) groups (P > 0.05). However, BNP levels were significantly elevated in the TNF-α (238.14 ± 9.09 pg/mL) and serum (343.83 ± 37.27 pg/mL) groups compared to the control, with the serum group also showing significantly higher BNP levels than the TNF-α group (P < 0.05) (Figure 5F). Similarly, cTnI levels were markedly increased in the LPS (136.80 ± 4.37 pg/mL), TNF-α (168.80 ± 14.79 pg/mL), and serum (224.88 ± 3.45 pg/mL) groups compared to the control (90.16 ± 2.51 pg/mL), with the highest levels observed in the serum group (P < 0.001) (Figure 5G). CK-MB levels followed a similar trend, with significant increases observed in the LPS (6.80 ± 0.24 ng/mL), TNF-α (8.96 ± 0.19 ng/mL), and serum (10.98 ± 0.14 ng/mL) groups compared to the control (5.61 ± 0.14 ng/mL), and with the serum group showing significantly higher CK-MB levels than both the LPS and TNF-α groups (P < 0.001) (Figure 5H).

In summary, septic serum treatment resulted in more pronounced reductions in cardiomyocyte viability and greater elevations in injury biomarkers than LPS or TNF-α treatment, suggesting that this model more accurately recapitulates the pathophysiological features of sepsis-induced myocardial injury observed in clinical settings.

DEGs and Functional Enrichment Pathways in Various in vitro Sepsis-Induced Myocardial Injury Models

A total of 262 DEGs were identified in the LPS group, comprising 203 upregulated and 59 downregulated genes. In the TNF-α group, 237 DEGs were detected, including 195 upregulated and 42 downregulated genes. The serum group exhibited a markedly larger number of DEGs, totaling 706, with 664 upregulated and 42 downregulated genes (Figure 6A and B, Table S3). This broader transcriptional response in the serum group suggests that the septic serum model may more comprehensively reflect the gene expression alterations associated with sepsis, although the number of DEGs alone does not fully determine the fidelity of the model. Notably, analysis of the GSE79962 and GSE23781 datasets revealed that the serum group demonstrated greater diagnostic value for sepsis-induced cardiomyopathy compared to the LPS and TNF-α groups (Figure 6C).

Figure 6 Differentially expressed genes (DEGs) in various in vitro models of sepsis-induced myocardial injury. (A) Volcano plots of DEGs in each group. (B) Heatmaps of DEGs in each group. (C) Evaluation of the diagnostic value of DEGs in the septic cardiomyopathy datasets (GSE79962 and GSE23781). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α; AUC, area under the curve.

GO enrichment analysis revealed distinct biological processes among the three groups (Figure 7). In the LPS group, DEGs were primarily enriched in “ribonucleoprotein complex biogenesis”, “catalytic activity, acting on a nucleic acid”, “establishment of protein localization to the endoplasmic reticulum”, and “protein targeting to the ER” (Figure 7A). In the TNF-α group, enriched terms included “DNA repair”, “chromosome organization”, “antigen processing and presentation of endogenous peptide antigen via MHC class I”, and related processes (Figure 7B). DEGs in the serum group were mainly associated with “chromosomal region”, “cell division”, “negative regulation of mitochondrial membrane permeability”, and “negative regulation of mitochondrial outer membrane permeabilization involved in apoptotic signaling” (Figure 7C and Table S4).

Figure 7 Gene Ontology (GO) analysis of differentially expressed genes (DEGs) in various in vitro models of sepsis-induced myocardial injury. (A–C) GO analysis of DEGs treated with LPS, TNF-α, and septic serum. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α.

Similarly, KEGG pathway analysis identified distinct signaling pathways enriched in each group (Figure 8). In the LPS group, significant pathways included “mitophagy-animal”, “fatty acid elongation”, “antigen processing and presentation”, and the “insulin signaling pathway” (Figure 8A). DEGs in the TNF-α group were enriched in pathways such as “basal transcription factors”, “non-homologous end-joining”, “primary immunodeficiency” and “parathyroid hormone synthesis, secretion, and action” (Figure 8B). In contrast, the serum group showed enrichment in “cell cycle”, “homologous recombination”, “cGMP-PKG signaling pathway”, and “cellular senescence” pathways (Figure 8C and Table S5).

Figure 8 Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) in various in vitro models of sepsis-induced myocardial injury. (A–C) KEGG analysis of DEGs treated with LPS, TNF-α, and septic serum. Abbreviations: LPS, Lipopolysaccharide; TNF-α, Tumor necrosis factor-α.

Discussion

By comprehensively comparing inflammatory response, oxidative stress, myocardial injury, apoptosis, and gene expression across various in vitro models of sepsis-induced myocardial injury induced by LPS, TNF-α, and septic serum, we found that septic serum was the most effective inducer and could most comprehensively recapitulate the pathological features of septic myocardial injury. This model reflects a multifactorial injury mechanism, underscoring its potential as a more representative in vitro model.

The pathogenesis of sepsis-induced myocardial injury is complex and closely related to excessive inflammatory response, oxidative stress, apoptosis, and other mechanisms.^27–31 Inflammatory is a hallmark of organ dysfunction in sepsis, particularly within the cardiovascular system.³² Consistent with previous studies, both LPS and TNF-α induced increased production of pro-inflammatory cytokines without significantly affecting anti-inflammatory cytokines in cardiomyocytes, thus failing to replicate the multifaceted immune interactions observed clinically.³³ In contrast, treatment with septic serum not only enhanced pro-inflammatory cytokine expression but also suppressed anti-inflammatory cytokines, eliciting a more pronounced inflammatory response. This suggests that septic serum contains a broader array of cytokines, chemokines, and immune mediators, more accurately reflecting the dysregulated immune state characteristic of clinical sepsis. These findings highlight mechanistic differences from single-agent models. The imbalance between pro- and anti-inflammatory factors may exacerbate myocardial injury by sustaining inflammation, warranting further exploration into the downstream signaling pathways that mediate this dual effect.

Oxidative stress in sepsis leads to excessive production of ROS, which impair mitochondrial function and promote apoptosis, ultimately contributing to myocardial injury.^6,34,35 We observed that septic serum markedly increased ROS levels and mitochondrial dysfunction compared with LPS and TNF-α, indicating that the septic serum model is particularly suitable for investigating oxidative damage mechanisms in sepsis-induced myocardial injury.

The release of cardiac biomarkers such as BNP, CK-MB, and cTnI is commonly used to assess myocardial injury.^36–38 Our findings showed that these markers were significantly elevated in the septic serum group compared to the LPS and TNF-α groups, suggesting that the serum model more closely replicates the degree of cardiac injury observed in vivo, potentially due to the presence of abundant cardiotoxic factors in septic serum. However, it is important to note that increased biomarker levels reflect myocardial injury severity but do not necessarily mirror the full spectrum of sepsis-induced myocardial pathophysiology. The clinical relevance of these markers still requires further validation.³⁹

In terms of cytotoxicity, septic serum induced a greater reduction in cell viability and a higher degree of apoptosis than LPS or TNF-α, likely due to the combined effects of inflammatory, oxidative, and metabolic insults. This reinforces the utility of septic serum in modeling the multifactorial nature of sepsis-induced myocardial injury. However, the extent of injury does not necessarily equate to greater physiological relevance, and model selection must be carefully considered when simulating clinical scenarios.^40,41 Ultimately, the translational value of in vitro models must be evaluated in conjunction with functional, metabolic, and electrophysiological parameters.

Transcriptomic analysis revealed that the septic serum model yielded the highest number of DEGs, which were enriched in a broader range of biological processes, suggesting a more robust transcriptional response in cardiomyocytes. GO enrichment analysis indicated that DEGs in the septic serum group were associated with “negative regulation of mitochondrial membrane permeability” and “cell division” implying the involvement of mitochondrial dysfunction, apoptotic signaling, and dysregulated cell proliferation in septic myocardial injury.^42,43 KEGG pathway analysis further revealed enrichment in pathways such as “cell cycle”, “cGMP-PKG signaling”, and “cellular senescence”, suggesting roles for DNA damage responses, proliferative disorders, and premature cardiomyocyte senescence in the disease process.^44,45 Moreover, activation of the cGMP-PKG signaling pathway may represent a compensatory mechanism related to vasodilation and myocardial relaxation.^46,47 These data indicate that the septic serum model more accurately captures the phenotypic and molecular complexity of myocardial injury than LPS or TNF-α, which mainly triggered more limited responses related to immune activation and stress signaling.

Although LPS is widely used to induce sepsis models in vitro due to its availability and simplicity, our results suggest that LPS and TNF-α only partially mimic the multifaceted nature of clinical sepsis. In contrast, the septic serum model incorporates circulating toxins, inflammatory mediators, and metabolic dysfunctions that collectively drive myocardial injury. Nonetheless, due to its compositional complexity, future studies should focus on profiling the serum to identify key bioactive constituents responsible for cardiotoxicity. Establishing standardized serum preparation protocols will also be crucial for optimizing this model. Despite its advantages, limitations of the serum model must be acknowledged. First, the composition of septic serum may vary depending on the infection source, disease stage, and patient heterogeneity, potentially affecting reproducibility. Second, its multifactorial nature hinders attribution of specific effects to individual molecular components.

Several limitations should be considered. First, sepsis-induced myocardial injury is largely driven by cytokine release from macrophages, yet our study did not evaluate cardiomyocyte-macrophage interactions in co-culture systems. Second, functional assays assessing contractility or electrophysiological properties were not performed in the in vitro models. Third, although human cardiomyocytes were used, the transition from cellular models to clinical applicability remains limited. The in vivo complexity of sepsis cannot be fully replicated in vitro, and further validation using animal models and patient-derived cardiac tissues is needed to confirm the translational relevance of our findings.

Conclusion

The in vitro model of sepsis-induced myocardial injury established using septic serum demonstrates more pronounced pathological alterations across multiple parameters, indicating its superiority in faithfully replicating the complex pathophysiological features of sepsis-induced myocardial injury. By encompassing the multifactorial nature of sepsis, including inflammatory, oxidative, apoptotic, and metabolic components, this model provides a more physiologically relevant platform for mechanistic studies. It holds significant potential for advancing our understanding of sepsis-related cardiac dysfunction and for guiding the development of targeted therapeutic strategies.