Patients who met the clinical diagnostic criteria for SIC and those who met the diagnostic criteria for sepsis on the day of hospital admission were included in the disease group and the disease control group, respectively. Healthy control group subjects were recruited from the general population upon completion of patient recruitment from Children's Hospital of Chongqing Medical University (Chongqing, China). The mean age of the three groups (healthy, Sepsis and SIC) was as follows: 6.08 (1-15), 2.67 (0-12) and 4.3 (0-16) years, respectively. The sex proportion (males/females) for each of the three groups was 60.0/40.0%. Patients and controls with malignant tumors, organ transplants, chronic viral infections (hepatitis, HIV), cirrhosis, chronic renal insufficiency or autoimmune diseases, and those who had used immunosuppressive drugs within the past 28 weeks were excluded from the study. A total of 33 children with SIC and 30 children with sepsis were included in the disease group and the disease control group, respectively, on the day of admission from Children's Hospital of Chongqing Medical University. Clinical data including vital signs, routine blood parameters, cardiac enzyme profiles, and liver and kidney function test data, were recorded. In addition, 23 healthy children were recruited as controls at the Physical Examination Center of the Children's Hospital of Chongqing Medical University. The complete date range for patient recruitment was from December 2021 to October 2022. Written informed consent was obtained from the parents of all of the patients in accordance with the ethical standards of The Declaration of Helsinki. The present study protocol was approved by the Clinical Research Ethics Committee of the Children's Hospital of Chongqing Medical University (approval no. 2021-353).

The SIC model was established via the intraperitoneal injection of lipopolysaccharide (LPS; cat. no. L8880; Beijing Solarbio Science & Technology Co., Ltd.) dissolved in sterile saline at a dose of 20 mg/kg. A total of 18 male C57BL/6J mice (age, 6-8 weeks; weight, 18-20 g) were purchased from the Animal Experiment Center of Chongqing Medical University (SCXK: 2022-0010). All of the mice were maintained under standard housing conditions: Temperature, 22±1°C; humidity, 50%; 12-h dark/light cycle; ad libitum access to food and water. After 3 days of acclimation, 18 mice were randomly divided into the following three groups: The sham group (n=6), the LPS experimental group (n=6) and the LPS + T3 experimental group (n=6). The sham group and LPS experimental group were injected intraperitoneally with saline or LPS solution, respectively, while the LPS + T3 group was pretreated with 80 mg/kg T3 (cat. no. T162132; Shanghai Aladdin Biochemical Technology Co., Ltd.) 24 h before injection with LPS. Since the half-life of T3 has been reported to be 24 h (31), and due to the survival status of the experimental animals after modeling, the duration of the experiment was 24 h. Animal healthy behavior and the humane endpoints, which were defined as a body temperature <33°C, inactivity of >3 h and/or a drop in weight of >20% of their original body weight, were monitored every 6 h during the study. Anesthesia and specific housing conditions were used during handling and intraperitoneal injection with LPS to ensure minimal pain, suffering and distress to animals. Each mouse was deeply anesthetized with an intraperitoneal injection of 10% chloral hydrate (350 mg/kg) prior to intraperitoneal injection with LPS, and no mouse exhibited signs of peritonitis following the administration of 10% chloral hydrate. Following intraperitoneal injection of LPS, the mice began to exhibit signs of infection, including ruffled fur, decreased activity, hunched posture and tachypnea. Subsequently, at 24 h post-injection, the 6 mice from the LPS group and the 6 mice from the LPS + T3 group were sacrificed. The mice in the sham group were euthanized 24 h after intraperitoneal injection of saline. The mice were euthanized by inhalation of 30% CO₂ for 5 min and death was verified by cervical dislocation. No moving, no breathing and pupil dilation of mice were assessed to confirm death. At the end of the experiment and after the mice were sacrificed, blood was collected from the retro-orbital vein and myocardial tissue samples were collected. The present study followed the animal experimentation guidelines (32) and was approved by the Animal Ethics Committee of the Children's Hospital of Chongqing Medical University (approval no. CHCMU-IACUC20210114036).

H9C2 cells, provided by The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences, were cultured in DMEM (4.5 g/l glucose; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a 5% CO₂ humidified incubator. The cells were divided into four groups: The control group, the LPS group, the LPS + T3 group and the LPS + SP600125 (SP; MedChemExpress) group. The concentration range was established according to the literature (33,34), and 80 ng/ml T3 and 10 µM SP (JNK inhibitor) were selected as the working concentrations The cells in the control group were cultured in complete medium containing 10% fetal bovine serum; the cells in the LPS group were incubated for 24 h after they reached 75% confluence, and the medium was then replaced with complete DMEM containing 10 µg/ml LPS at 37°C for 24 h to construct an in vitro model of SIC. In addition, the LPS + T3 and LPS + SP groups were pretreated with T3 for 24 h before LPS treatment or with SP for 30 min at 37°C, respectively. All of the cell experiments were independently repeated at least in triplicate.

Blood samples from patients, as well as mouse blood obtained from the orbital venous plexus, were centrifuged at 1,000 × g at room temperature for 15 min. According to the manufacturers' instructions, the levels of PLN, cardiac troponin I (cTnI), and T3 in the serum were determined via human-derived PLN (cat. no. JL15150; Shanghai Jianglai Biotechnology Co., Ltd.) and T3 (cat. no. JL13028; Shanghai Jianglai Biotechnology Co., Ltd.) ELISA kits, and mouse-derived cTnI (cat. no. E-EL-M1203c; Wuhan Elabscience Biotechnology Co., Ltd.), PLN (cat. no. JL24496; Shanghai Jianglai Biotechnology Co., Ltd.) and T3 (cat. no. E-EL-0079c;Wuhan Elabscience Biotechnology Co., Ltd.) ELISA kits. Albumin (ALB) and creatinine (CREA) were assessed using an automatic biochemical analyzer (Roche Cobas c701; Roche Diagnostics), procalcitonin (PCT) was assessed using an automatic chemiluminescence immunoassay analyzer (Cobas pro e801; Roche Diagnostics), and brain natriuretic peptide (BNP) was assessed using another automatic chemiluminescence immunoassay analyzer (Atellica IM 1600; Siemens).

After the mice were treated for 24 h to establish an in vivo model, they were sacrificed with 30% CO₂. The heart tissues were collected, rinsed with isotonic saline, and fixed in 4% paraformaldehyde for 12 h at room temperature. Subsequently, the tissues were dehydrated, embedded in paraffin, and were sectioned (8 µm) and stained with hematoxylin for 6 min and 1% eosin for 1 min, or Masson for 5 min at room temperature. The sections were then placed under a light microscope for observation and images were captured.

Cell viability and drug toxicity were measured using the Cell Counting Kit-8 (CCK-8) assay. The cells (4×10³/well) were placed in 96-well plates and were incubated according to the experimental design. Subsequently, 100 µl CCK-8 assay reagent (cat. no. M4839; AbMole BioScience) was diluted 10 times with serum-free culture medium and was incubated with the cells for 3 h. The optical density (OD) value was then detected at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.). All OD values were normalized by converting them to a percentage of the mean control value.

An apoptosis assay kit (cat. no. 556547; BD Biosciences) was used to detect apoptosis according to the manufacturer's instructions. All of the cells (including those in suspension) were collected as a precipitate, and 100 µl 1X binding buffer diluted in distilled water was added to resuspend the cell pellet. Subsequently, 5 µl FITC and 5 µl propidium iodide were added and vortexed sequentially, after which the samples were incubated at room temperature in the dark for 30 min. Finally, 400 µl 1X binding buffer was added to each tube, apoptosis was examined by flow cytometry (FACSCanto II; Becton, Dickinson and Company) and the data were analyzed using FlowJo (software version 10.8; FlowJo LLC).

The cells were placed in 24-well plates (5×10⁴/well) and were treated for 24 h. The cells were then fixed with 1 ml 4% paraformaldehyde for 10 min and were subjected to staining with crystal violet (cat. no. C0121; Beyotime Institute of Biotechnology) on a shaker at room temperature for 15 min. The cells were rinsed 3-5 times with PBS, air-dried, were placed under a light microscope for observation and images were captured. In addition, the OD value was detected at 570 nm using a microplate reader. All OD values were normalized by converting them to a percentage of the mean control value.

Intracellular calcium levels were detected using the calcium fluorescent probe Fluo-4AM (cat. no. S1060; Beyotime Institute of Biotechnology). The Fluo-4AM working solution (5 µM) was prepared according to the manufacturer's instructions, and the cells were incubated with it for 40 min at 37°C in the dark for fluorescent probe loading. The staining solution was then discarded, and PBS was added and the cells were incubated for a further 20 min. The cells were then observed and images were captured under a confocal microscope, and the mean fluorescence intensity was detected via flow cytometry (FACSCanto II) and the data were analyzed using FlowJo (software version 10.8). The values of the experimental groups were normalized to those of the control group.

A ROS assay was performed using DHE (cat. no. KGAF019; Nanjing KeyGen Biotech Co., Ltd.) and Hoechst 33258 (cat. no. C1011; Beyotime Institute of Biotechnology) fluorescent probe kits according to the manufacturers' instructions. Briefly, the cells were incubated with 5 µM DHE for 30 min at 37°C in the dark. To measure the levels of ROS, fluorescence images of the cells were captured under a confocal fluorescence microscope, or the cell suspension was collected in test tubes, and the mean fluorescence intensity of DHE was measured by flow cytometry (FACSCanto II) and the data were analyzed using FlowJo (software version 10.8). The values of the experimental groups were normalized to those of the control group.

The pcDNA3.1(+) plasmid overexpressing c-Jun contained the coding sequence region of the rat c-Jun gene from NCBI (https://www.ncbi.nlm.nih.gov/) and was constructed by Beijing Biomed Gene Technology Co., Ltd. An empty pcDNA3.1(+) plasmid was used as a negative control. The overexpression plasmids (900 ng/µl) were transfected into H9C2 cells (80% confluence) using the PolyJet™ transfection reagent (cat. no. SL100688; SignaGen Laboratories), according to the manufacturer's instructions, at 37°C for 6 h. The DNA and PolyJet were diluted separately at a ratio of 3:1 in serum-free DMEM, mixed, incubated for 15 min at room temperature and then added to the cell culture medium. After 12-18 h of transfection, the medium containing the PolyJet/DNA complex was removed and replaced with fresh serum-containing complete medium.

Total RNA was extracted from cell samples or myocardial tissue samples using an RNA extraction kit (cat. no. 9109; Takara Bio, Inc.), and single-stranded complementary DNA was synthesized using an RT kit (cat. no. RK20429; ABclonal Biotech Co., Ltd.). β-actin was selected as the reference gene according to previous studies (35,36). qPCR was performed using SYBR qPCR reagents (cat. no. RK21203; ABclonal Biotech Co., Ltd.), and the values of the average quantification cycle (Cq) were normalized to the expression of β-actin; finally, the relative mRNA expression levels were determined using the 2^−ΔΔCq method (37). The values of the experimental groups were normalized to those of the control group. The following qPCR cycling conditions were used: 3 min at 95°C, followed by 45 cycles at 95°C for 30 min, 95°C for 5 sec and 60°C for 30 sec. The primer details are shown in Table I.

Proteins were extracted from cells and myocardial tissues according to the instructions of the protein extraction kit (cat. no. KGP2100; Nanjing KeyGen Biotech Co., Ltd.), and their concentrations were determined with a BCA assay kit (cat. no. KGP902; Nanjing KeyGen Biotech Co., Ltd.). Proteins (60 µg) were separated by SDS-PAGE on 10 or 15% gels and were transferred to PVDF membranes, after which the protein samples were blocked with 5% skim milk powder for 2 h at room temperature. The membranes were subsequently incubated with the primary antibodies at 4°C overnight and with an HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the blots were rinsed three times with TBS-0.1% Tween (TBST; 5 min each wash) and 200 µl Pico ECL Ultrasensitive Substrate Chemiluminescent Detection Kit (cat. no. PA134-01; Beijing Biomed Gene Technology Co;) was added to scan the blot using the ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.), and the band intensities were measured using ImageJ software (v1.54; National Institutes of Health). The values of the experimental groups were normalized to those of the control group. Each experiment was performed in triplicate, and all samples were normalized to β-actin. The following antibodies diluted with TBST (1:500) were used: Rabbit anti-PLN (cat. no. ab219626; Abcam), rabbit anti-phosphorylated (p)-PLN (Ser16) (cat. no. AP0907; ABclonal Biotech Co., Ltd.), rabbit anti-p-PLN (Thr17) (cat. no. AF7278; Affinity Biosciences), rabbit anti-SERCA2 (cat. no. 381667; ZENBIO), rabbit anti-JNK (cat. no. ET1601-28; HUABIO), rabbit anti-p-JNK (cat. no. ET1609-42; HUABIO), rabbit anti-c-Jun (cat. no. ET1608-3; HUABIO), rabbit anti-p-c-Jun (cat. no. ET1608-4; HUABIO), mouse anti-β-actin (cat. no. R23613; ZENBIO), goat anti-rabbit-HRP (cat. no. SA00001-2 Proteintech Group, Inc.) and goat anti-mouse-HRP (cat. no. SA00001-1; Proteintech Group, Inc.).

The cardiac tissues from three mice in the sham and LPS groups were collected and used for proteomic analyses. The essential steps involved in tandem mass tagging (TMT) proteomics analysis are protein extraction, trypsin digestion, TMT labeling, HPLC fractionation, LC-MS/MS analysis and data search, which were performed as previously described (38). The proteomics analysis in the present study was facilitated by the Jingjie PTM Bioinformatics Team (Jingjie PTM BioLab Co. Ltd.).

Experiments were repeated at least three time, unless otherwise stated. Data are presented as the mean ± SD and were statistically analyzed using GraphPad Prism 8.0 (Dotmatics) and SPSS 27.0 (IBM Corp.) software. Comparisons between two groups were analyzed using unpaired Students' t-test or Welch's t-test on the basis of the results of the homogeneity test of variance (F test). Differences among three or more groups were analyzed using one-way analysis of variance followed by Tukey's test for pairwise comparisons or Dunnett's test for comparing each group to a control group. Receiver operating characteristic (ROC) curve analyses, and Spearman correlation analyses between PLN, and serum ALB, PCT and CREA, were performed in SPSS 27.0. P<0.05 was considered to indicate a statistically significant difference.

Hematoxylin and eosin and Masson staining of the cardiac tissue sections revealed inflammatory cell infiltration and myocardial fibrosis in the LPS group (Fig. 1A and B). The serological levels of cTnI were significantly greater in the LPS group than those in the control group (Fig. 1C). These results indicated that LPS successfully induced SIC in vivo. Furthermore, serum detection revealed a significant decrease in the concentration of T3 in response to LPS treatment (Fig. 1D). Compared with in the control group cells, the chosen concentration of 80 ng/ml T3 had the largest effect on cell viability; treatment with this resulted in a >80% increase in cell viability compared with the control (Fig. S1). Previous proteomics analyses of the hearts of normal mice and mice with sepsis (the LPS group) revealed that the expression of PLN, which is related to T3 regulation of cardiac calcium, was significantly increased in mice with sepsis (Fig. S2). Similarly, the results of WB and RT-qPCR further confirmed the significantly elevated levels of PLN in myocardial tissues and H9C2 cells in the LPS group (Fig. 1E-G). In vitro, the results of proliferation, apoptosis and inflammation assays revealed that LPS induced inflammatory damage to cardiomyocytes, as cells treated with LPS exhibited a decrease in cellular viability, an increased proportion of apoptotic cells and a reduction in cell growth density compared with in the control group (Fig. S3A-D). Moreover, serum PLN concentration was significantly elevated in the serum of mice with sepsis compared with that in the control group (Fig. 1I). Furthermore, analysis of serum T3 and PLN levels revealed a significant negative correlation between the two parameters in the mouse model of SIC (Fig. 1J). PLN is a critical regulator of calcium cycling and contractility in the heart, and impairs calcium recycling in the endoplasmic reticulum (30). In the present study, intracellular calcium overload increased, as indicated by Fluo-4AM green fluorescence staining, and was accompanied by abnormally elevated levels of ROS in the LPS group, as indicated by increased red fluorescence in the nucleus (Fig. S3E-H).

The results revealed that treatment with T3 significantly increased cell viability, reduced the proportion of apoptotic cells and promoted cell proliferation under LPS challenge (Fig. 2A-C). Additionally, T3 treatment significantly reduced the expression levels of the inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor α compared with in the LPS group (TNF-α) (Fig. 2D). Furthermore, the results of the present study revealed that, compared with in the LPS group, intracellular calcium accumulation was reduced and calcium overload was effectively alleviated after T3 intervention (Fig. 2E and G). In addition, compared with in the LPS group, T3 treatment reduced the intracellular ROS content and alleviated intracellular oxidative stress (Fig. 2F and H).

After T3 intervention in the mice, the serum concentrations of various factors were measured, revealing an increase in the concentration of T3, and a decrease in cTnI and PLN concentrations compared with in the LPS group (Fig. 3A). In addition, compared with in the LPS group, the mRNA expression levels of PLN in myocardial tissues were reduced, whereas SERCA2 expression was increased after T3 treatment (Fig. 3B). Tissue section staining showed that T3 treatment effectively reduced the infiltration of inflammatory cells in myocardial tissue in the LPS group (Fig. 3C), decreased the deposition of collagen fibers and reduced the number of fibroblasts in myocardial tissue (Fig. 3D).

The specific mechanisms by which T3 regulates PLN were subsequently investigated. According to the results of WB, treatment with T3 decreased the protein levels of PLN compared with in the LPS group in vivo and in vitro (Figs. 4A, B, S4A and B). In addition, it was revealed that since PLN expression decreased, the p-PLN/PLN ratio increased after T3 treatment (Fig. S4C and D). It has previously been reported that T3 can negatively regulate JNK phosphorylation, thereby inhibiting its proapoptotic effects (27). Therefore, the present study examined the levels of p-JNK and phosphorylation of its downstream target protein, c-Jun, following T3 intervention, and the results revealed that the phosphorylation of both JNK and c-Jun was decreased compared with in the LPS group (Figs. 4A, B, S4A and B).

To further explore the regulatory relationship between c-Jun and PLN, cells were transfected with a plasmid overexpressing c-Jun. Overexpression of c-Jun upregulated the mRNA and protein expression levels of PLN, and decreased the p-PLN/PLN ratio (Figs. 4C, D, and S4E).

According to the results of the CCK-8 assay, 10 µM was selected as the working concentration of the JNK inhibitor SP, as 10 µM SP had the least toxic effect on cells compared with the control (Fig. S1). Subsequently, it was used to confirm that T3 alleviates cardiomyocyte damage through the JNK/c-Jun pathway, as T3 and SP similarly reduced the phosphorylation levels of JNK and c-Jun (Fig. 5A). Further WB revealed that c-Jun phosphorylation levels were decreased after SP treatment and that total PLN protein levels were decreased, whereas the p-PLN/PLN ratio was increased compared with in the LPS group (Figs. 5A and S5). In comparison with the LPS group, cell apoptosis was decreased, and the intracellular synthesis of inflammatory factors was reduced following SP treatment (Fig. 5B and C). Furthermore, SP intervention decreased intracellular ROS levels and reduced intracellular calcium ion accumulation compared with in the LPS group (Fig. 5D and E).

To explore the clinical value of PLN, serum samples from patients were collected for analysis. The general data of the patients are listed in Table SI. The results revealed that in the SIC group, patients had significantly lower serum T3 levels and significantly higher PLN levels compared with those in the healthy group (Fig. 6A). ROC curves were constructed on the basis of the PLN, cTnI and BNP serum contents. PLN had a greater area under the curve, 0.9503 (95% CI: 0.9014-0.9993), compared with the other two markers, cTnI [0.9131 (95% CI: 0.8513-0.9748)] and BNP 0.8403 (95% CI: 0.7398-0.9408)], and the optimal threshold value was determined to be 436.8 pg/ml (Fig. 6B). In addition, PLN was negatively correlated with serum ALB, whereas it was positively correlated with serum PCT and serum CREA (Fig. 6C).