
Protective role of triiodothyronine in sepsis‑induced cardiomyopathy through phospholamban downregulation
- Authors:
- Published online on: January 16, 2025 https://doi.org/10.3892/ijmm.2025.5488
- Article Number: 47
-
Copyright: © Xie et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Sepsis is a clinical syndrome of systemic dysfunctional metabolism triggered by infection (1). There are ~19 million cases of sepsis worldwide each year, with a mortality rate of 30%. Surveys have indicated that the incidence of sepsis and its morbidity rate are increasing annually (2,3). Sepsis can lead to functional impairment of multiple organs, with the heart being the most susceptible, accounting for 16-65% of cases and being the primary cause of death in patients with sepsis (4-6). The clinical term for sepsis accompanied by cardiac dysfunction is sepsis-induced cardiomyopathy (SIC) (7). SIC is a serious complication characterized by myocardial damage directly caused by the production of large amounts of inflammatory factors in the body after infection, by myocardial systolic depression, or by reduced cardiac output due to impaired myocardial energy metabolism (8).
Patients with sepsis usually have altered levels or functions of hormones, including thyroid hormones, insulin and adrenal glucocorticoids; low levels of triiodothyronine (T3) in particular are highly associated with mortality in patients with sepsis (9). Studies have shown that alterations in thyroid hormone levels or functions are often associated with liver and cardiovascular diseases (10,11). Thyroid hormones are synthesized by thyroid follicular epithelial cells and are stored in the follicular lumen (12); two biologically active thyroid hormones circulate in the body, namely, thyroxine and T3, with T3 being more active and having a greater affinity for receptors in the nuclei of target organs (13). The heart is one of the most important target organs for the action of thyroid hormones. Notably, thyroid hormones regulate calcium homeostasis in cardiomyocytes by modulating calcium channels and pumps, influencing physiological processes such as cardiomyocyte electrophysiology, mechanical contraction and energy metabolism (14,15).
The pathogenesis of SIC is complex and includes myocardial cell damage, increased release of proinflammatory factors, mitochondrial dysfunction and an imbalance in calcium homeostasis (16). Intracellular calcium cycling serves as a central regulator of myocardial contraction and diastole, ensuring proper myocardial cell function (17,18). The sarcoplasmic reticulum (SR) releases calcium ions into the cell cytoplasm to induce contraction and then recycles calcium ions back into the SR via the SR calcium ATPase (SERCA2) to achieve myocyte relaxation (19,20). This complex process is essential for excitation-contraction coupling to balance Ca2+ homeostasis, and abnormal Ca2+ handling caused by SR dysfunction in cardiomyocytes results in systolic dysfunction. Phospholamban (PLN), which is located in the SR, modulates SERCA2 activity and participates in intracellular calcium recycling, thereby influencing myocardial contractile and diastolic functions (21,22). When PLN binds to SERCA2, it decreases the affinity of the enzyme for calcium ions, preventing calcium re-entry into the SR and causing its accumulation in the cytoplasm. Phosphorylated-PLN fails to combine with SERCA2, facilitating calcium recycling from the cytoplasm to the SR. Thus, PLN maintains intracellular calcium homeostasis and thereby serves an important regulatory role in cardiac systolic-diastolic function (23-25). Numerous studies have indicated that, in addition to causing disturbances in myocardial contraction and relaxation, intracellular calcium overload can lead to various adverse effects, such as arrhythmia, cardiomyocyte apoptosis and mitochondrial dysfunction (26,27).
Current research on the role of PLN in SIC has focused primarily on its regulation of cardiac contractile function. Protein kinase A and the state of PLN itself, such as its protein pentameric form or monomers and the SERCA-PLN complex can influence the extent of PLN phosphorylation, thereby affecting cardiac function (28,29). Additionally, it has been reported that thyroid hormones are important regulatory factors at the transcriptional level of SERCA2, indirectly affecting the function of PLN by influencing the activity and expression of SERCA2 in cardiac muscle (30). However, in SIC, the specific molecular mechanism underlying the interaction between thyroid hormones and PLN has been largely overlooked. Research investigating the relationship between low T3 syndrome occurring in sepsis and calcium homeostasis is scarce (11).
Given the aforementioned findings, it may be hypothesized that T3 mitigates SIC by reducing PLN expression and calcium overload. The present study aimed to explore the relationship between low levels of T3 and PLN in SIC, and to further elucidate the specific mechanisms by which T3 regulates the expression of PLN. Additionally, the study endeavored to assess the clinical application of PLN in the context of SIC.
Materials and methods
Clinical patients and healthy controls
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).
Establishment of an animal model
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).
Establishment of cell models
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.
Detection of serological indicators
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).
Histological analysis
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 assay
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.
Apoptosis assay
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).
Crystal violet staining
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.
Calcium assay
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.
Reactive oxygen species (ROS) assay
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.
Plasmid construction and transfection
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.
Reverse transcription-quantitative PCR (RT-qPCR)
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.
Western blotting (WB)
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.).
Proteomics analyses
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.).
Statistical analysis
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.
Results
Serum T3 levels are significantly negatively correlated with myocardial PLN levels in sepsis
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).
T3 treatment alleviates cardiomyocyte damage in vitro and in vivo
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).
T3 reduces PLN expression through JNK/c-Jun signaling pathway inhibition
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).
Inhibition of the JNK/c-Jun signaling pathway reduces cardiomyocyte damage
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).
Clinical value of elevated PLN in patients with SIC
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).
Discussion
SIC is a critical condition involving the heart, and is associated with varying degrees of myocardial damage during the progression of sepsis, often leading to a poor prognosis and high mortality rate (7). The pathological mechanisms of SIC are complex and include inflammatory damage to cardiomyocytes, the release of nitric oxide and ROS, mitochondrial dysfunction and abnormal calcium regulation (16). Thyroid hormones are important indicators of disease severity and mortality, and patients with sepsis often experience thyroid dysfunction (39). In addition to their classical metabolic regulatory roles, thyroid hormones exert cardioprotective and reparative effects (40). Consistent with these findings, significantly low levels of T3 were observed in patients with SIC, as well as in in vivo and in vitro models of SIC, in the present study, and supplementation with T3 mitigated myocardial tissue lesions while reducing cardiomyocyte calcium overload and ROS levels.
PLN is a small transmembrane protein localized on the SR of cardiomyocytes that serves a key regulatory role in inhibiting Ca2+ transport, and thereby crucially influences cardiac contractile and diastolic functions (41). In cardiomyocytes, PLN reduces the affinity of SERCA2 for Ca2+, whereas the phosphorylation of PLN relieves this effect and promotes calcium recycling back into the SR (36). This process is vital for regulating myocardial contraction and diastole. Recently, Stege et al (42) revealed a prominent role for abnormal PLN protein distribution and SR/endoplasmic reticulum disorganization in the underlying mechanism of cardiomyopathy.
In the present study, abnormally elevated levels of PLN were observed in SIC. This elevation may have hindered the effective recycling of extracellular calcium into the SR, leading to calcium overload in cardiomyocytes and impairing cardiomyocyte diastolic function. This effect may be attributed to alterations in the phosphorylation levels of the Ser16 and Thr17 sites of PLN. Furthermore, the experimental results indicated that T3 decreased PLN expression and altered the phosphorylation ratio of PLN in cardiomyocytes in vivo and in vitro, which is consistent with the findings of previous studies (43,44); however, the specific mechanism is unknown.
It has previously been reported that T3 can protect against heart ischemia/reperfusion injury by modulating various intracellular kinase signaling pathways (45). The balance between the proapoptotic and prosurvival kinase signaling pathways is critical in myocardial injury and remodeling. It has previously been shown that T3 can significantly reduce the phosphorylation of the proapoptotic protein kinase JNK (46). Consistent with these findings, the current study revealed that activated JNK phosphorylation in SIC was significantly reduced by T3 treatment, indicating that T3 may inhibit the JNK pathway. JNK is a branch of the MAPK pathway that serves crucial roles in cell proliferation, migration, survival, senescence and stress responses. Despite its dual roles in apoptosis and survival regulation (47), the findings of the present study suggested that JNK inhibition may effectively prevent apoptosis and promote cell survival. c-Jun is the primary downstream target molecule of JNK and is a component of the activator protein-1 transcriptional complex, which is modulated by various protein kinases and has a regulatory role in apoptosis and stress responses (48). The JNK/c-Jun signaling pathway is directly associated with the development of various diseases, and its activation has been shown to have protective effects on ischemia/reperfusion injury in the liver, lung, brain and heart (49-51). The phosphorylation of c-Jun is a critical indicator of its activation (52). Only a few studies, such as Chin et al (53) and Song et al (54), have suggested a relationship between JNK/c-Jun and PLN in the context of myocardial ischemia/reperfusion or myocardial hypertrophy. In the present study, the results revealed increased c-Jun phosphorylation following JNK activation, indicating the activation of the JNK/c-Jun signaling pathway in SIC. Notably, concurrent changes in PLN activity were also observed upon inhibition of the JNK/c-Jun signaling pathway. Specifically, inhibition of this pathway led to decreased PLN expression and altered phosphorylation at its regulatory sites. To further investigate the relationship between the JNK/c-Jun pathway and PLN, plasmid transfection experiments were conducted to clarify whether c-Jun may act as an upstream transcription factor regulating PLN synthesis. The results showed that the mRNA and protein levels of PLN were found to be consistent with the expression changes of c-Jun.
The unknown cardiac dysfunction underlying SIC, combined with the complexities of the cardiovascular system, has resulted in the absence of standardized diagnostic criteria in clinical practice (55,56). PLN is specifically expressed in myocardial tissues and has a small molecular weight of 6 kDa in its monomeric form and 25 kDa in its pentameric form (57). This molecular weight is much lower than that of CK at 86 kDa or cTnI at 37 kDa, making PLN more readily released into the bloodstream during myocardial injury and demonstrating superior sensitivity as a biomarker in serum. The area under the ROC curve was 0.9503 for PLN, with a 95% confidence interval of 0.9014-0.9993, indicating that PLN has excellent diagnostic performance for SIC. Furthermore, serum PLN levels were revealed to be correlated with markers of liver and kidney injury, as well as inflammatory indicators. Therefore, it may be concluded that PLN holds clinical value in pediatric patients to a certain extent.
In summary, the present study identified abnormally decreased T3 and elevated PLN concentrations, with these concentrations showing a negative correlation in a mouse disease model. Moreover, elevated PLN levels were associated with impaired intracellular calcium recycling, leading to increased intracellular oxidative stress levels and subsequent cardiomyocyte injury. Exogenous supplementation with T3 reversed calcium overload and oxidative stress by inhibiting PLN and its downstream calcium regulatory factors, thereby reducing myocardial injury. The present study also revealed that T3 treatment suppressed the expression and activity of JNK and c-Jun, indicating that T3 may regulate PLN to mitigate myocardial injury through the JNK/c-Jun signaling pathway.
Notably, the current study has several limitations. First, the clinical sample size was small. Although the findings revealed increased PLN levels and decreased T3 levels across the three experimental groups, there was no negative correlation between PLN and T3 levels in clinical pediatric patients (data not shown). This discrepancy may be attributed to the incomplete development of the thyroid gland in children. There are significant differences in thyroid hormone levels between children and adults, while these variations tend to approach the adult reference ranges as age increases (58). Children have a significantly higher demand for thyroid hormones due to their growth requirements, which far exceeds that of adults. Additionally, because their thyroid glands are not yet fully mature, their hormonal regulatory capabilities are weaker. Consequently, the fluctuations in hormone levels under stress conditions are more pronounced in children compared with in adults. Furthermore, several factors such as obesity, smoking and lifestyle can influence thyroid hormone levels as age increases (59). To address this limitation, a larger sample size and a multicenter clinical trial are required to further validate the scientific hypothesis. Second, additional robust evidence, such as c-Jun knockdown, is needed to strengthen the credibility of this mechanism which involves the JNK/c-Jun signaling pathway in the regulatory mechanisms of T3. Thus, more direct experimental results including dual-luciferase reporter assays and other methodologies are needed to substantiate the interaction between c-Jun and PLN. Addressing these limitations will contribute to a more comprehensive understanding of the mechanisms underlying the observed effects of T3 and PLN on SIC. Finally, Figs. 2, 5 and S3 were derived from several distinct experiments, where experimental conditions and procedures may introduce a certain degree of error, hence the rate of apoptosis between H9C2 cells treated with LPS were not completely the same among these figures. Thus, in the analysis of each dataset, the data were normalized to the control group to minimize the impact of the difference to the greatest extent possible.
In conclusion, the present study demonstrated a negative correlation between low T3 levels and elevated PLN levels in a mouse model of SIC. The correlation may stem from the influence of PLN on intracellular calcium cycling and oxidative stress levels in cardiomyocytes, which are countered by T3 to improve cardiomyocyte function through the inhibition of PLN phosphorylation. Moreover, the findings of the current study suggested that the JNK/c-Jun signaling pathway may serve a crucial role in mediating the negative regulatory effects of T3 on PLN. Additionally, PLN was identified as a novel biomarker for SIC, which has significant potential for early diagnosis of SIC in pediatric patients.
Supplementary Data
Availability of data and materials
The data generated in the present study may be requested from the corresponding author. The data generated in the present study may be found in the iProX database under accession number PXD059227 or at the following URL: https://www.iprox.cn/page/project.html?id=IPX0010782000.
Authors' contributions
QX and QY analyzed the patient data regarding SIC and performed experiments on cell models. JZ contributed to study conception and design, BT analyzed and interpretated the data, and both were involved in drafting the manuscript or revising it. HXi, RW and HL performed the histological examination of the heart, and were major contributors in writing the manuscript. TC and HXu made substantial contributions to the conception and design of the research, analysis and interpretation of data, and the acquisition of funding. TC and HXu confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.
Ethics approval and consent to participate
The clinical study protocol was approved by the Clinical Research Ethics Committee of the Children's Hospital of Chongqing Medical University (approval no. 2021-353). Written informed consent was obtained from the parents of all patients. The animal experiments were approved by the Animal Ethics Committee of Children's Hospital of Chongqing Medical University (approval no. CHCMU-IACUC20210114036).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
The authors would like to thank Dr Qin Zhou and Mrs. Li Zhao (Department of Pediatric Research Institute, Children's Hospital of Chongqing Medical University) for their pathology help and assistance.
Funding
This work was supported by the China Postdoctoral Science Foundation (grant no. 2023M730447), the Chongqing Science & Technology Commission (grant no. CSTB2023NSCQ-MSX0603), the Chongqing Postdoctoral Research Program Special Grant (grant no. 2023CQBSHTB3048), the Chongqing Municipal Talent Program (grant no. cstc2024ycjh-bgzxm0024) and the Chongqing Medical Scientific Research Project (Joint project of Chongqing Health Commission and Science and Technology Bureau; grant no. 2025GDRC008).
References
Singh S, Mohan S and Singhal R: Definitions for sepsis and septic shock. JAMA. 316:4582016. View Article : Google Scholar : PubMed/NCBI | |
Gohil SK, Cao C, Phelan M, Tjoa T, Rhee C, Platt R and Huang SS: Impact of policies on the rise in sepsis incidence, 2000-2010. Clin Infect Dis. 62:695–703. 2016. View Article : Google Scholar : PubMed/NCBI | |
Mahla RS, Vincent JL and Sakr Y; ICON and SOAP Investigators: Sepsis is a global burden to human health: Incidences are underrepresented: Discussion on 'comparison of European ICU patients in 2012 (ICON) versus 2002 (SOAP)'. Intensive Care Med. 44:1197–1198. 2018. View Article : Google Scholar : PubMed/NCBI | |
Suzuki T, Suzuki Y, Okuda J, Kurazumi T, Suhara T, Ueda T, Nagata H and Morisaki H: Sepsis-induced cardiac dysfunction and β-adrenergic blockade therapy for sepsis. J Intensive Care. 5:222017. View Article : Google Scholar | |
Allison SJ: Sepsis: NET-induced coagulation induces organ damage in sepsis. Nat Rev Nephrol. 13:1332017. View Article : Google Scholar : PubMed/NCBI | |
Weiss SL, Peters MJ, Alhazzani W, Agus MSD, Flori HR, Inwald DP, Nadel S, Schlapbach LJ, Tasker RC, Argent AC, et al: Surviving sepsis campaign international guidelines for the management of septic shock and sepsis-associated organ dysfunction in children. Intensive Care Med. 46(Suppl 1): S10–S67. 2020. View Article : Google Scholar | |
Hollenberg SM and Singer M: Pathophysiology of sepsis-induced cardiomyopathy. Nat Rev Cardiol. 18:424–434. 2021. View Article : Google Scholar : PubMed/NCBI | |
Ehrman RR, Sullivan AN, Favot MJ, Sherwin RL, Reynolds CA, Abidov A and Levy PD: Pathophysiology, echocardiographic evaluation, biomarker findings, and prognostic implications of septic cardiomyopathy: A review of the literature. Crit Care. 22:1122018. View Article : Google Scholar : PubMed/NCBI | |
Melis MJ, Miller M, Peters VBM and Singer M: The role of hormones in sepsis: An integrated overview with a focus on mitochondrial and immune cell dysfunction. Clin Sci (Lond). 137:707–725. 2023. View Article : Google Scholar : PubMed/NCBI | |
Das BK, Agarwal P, Agarwal JK and Mishra OP: Serum cortisol and thyroid hormone levels in neonates with sepsis. Indian J Pediatr. 69:663–665. 2002. View Article : Google Scholar : PubMed/NCBI | |
Vidart J, Axelrud L, Braun AC, Marschner RA and Wajner SM: Relationship among low T3 levels, type 3 deiodinase, oxidative stress, and mortality in sepsis and septic shock: Defining patient outcomes. Int J Mol Sci. 24:39352023. View Article : Google Scholar : PubMed/NCBI | |
Jongejan RMS, Meima ME, Visser WE, Korevaar TIM, Van Den Berg SAA, Peeters RP and de Rijke YB: Binding characteristics of thyroid hormone distributor proteins to thyroid hormone metabolites. Thyroid. 32:990–999. 2022. View Article : Google Scholar : PubMed/NCBI | |
Salas-Lucia F and Bianco AC: T3 levels and thyroid hormone signaling. Front Endocrinol (Lausanne). 13:10446912022. View Article : Google Scholar : PubMed/NCBI | |
Cokkinos DV and Chryssanthopoulos S: Thyroid hormones and cardiac remodeling. Heart Fail Rev. 21:365–372. 2016. View Article : Google Scholar : PubMed/NCBI | |
Mastorci F, Sabatino L, Vassalle C and Pingitore A: Cardioprotection and thyroid hormones in the clinical setting of heart failure. Front Endocrinol (Lausanne). 10:9272020. View Article : Google Scholar : PubMed/NCBI | |
Wang R, Xu Y, Fang Y, Wang C, Xue Y, Wang F, Cheng J, Ren H, Wang J, Guo W, et al: Pathogenetic mechanisms of septic cardiomyopathy. J Cell Physiol. 237:49–58. 2022. View Article : Google Scholar | |
Shankar TS, Ramadurai DKA, Steinhorst K, Sommakia S, Badolia R, Thodou Krokidi A, Calder D, Navankasattusas S, Sander P, Kwon OS, et al: Cardiac-specific deletion of voltage dependent anion channel 2 leads to dilated cardiomyopathy by altering calcium homeostasis. Nat Commun. 12:45832021. View Article : Google Scholar : PubMed/NCBI | |
Capasso JM, Sonnenblick EH and Anversa P: Chronic calcium channel blockade prevents the progression of myocardial contractile and electrical dysfunction in the cardiomyopathic Syrian hamster. Circ Res. 67:1381–1393. 1990. View Article : Google Scholar : PubMed/NCBI | |
Selnø ATH, Sumbayev VV and Gibbs BF: IgE-dependent human basophil responses are inversely associated with the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Front Immunol. 13:10522902023. View Article : Google Scholar | |
Wang L, Myles RC, Lee IJ, Bers DM and Ripplinger CM: Role of reduced sarco-endoplasmic reticulum Ca2+-ATPase function on sarcoplasmic reticulum Ca2+ alternans in the intact rabbit heart. Front Physiol. 12:6565162021. View Article : Google Scholar | |
Carlson CR, Aronsen JM, Bergan-Dahl A, Moutty MC, Lunde M, Lunde PK, Jarstadmarken H, Wanichawan P, Pereira L, Kolstad TRS, et al: AKAP18δ anchors and regulates CaMKII activity at phospholamban-SERCA2 and RYR. Circ Res. 130:27–44. 2022. View Article : Google Scholar | |
Hamstra SI, Whitley KC, Baranowski RW, Kurgan N, Braun JL, Messner HN and Fajardo VA: The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function. Am J Physiol Cell Physiol. 319:C694–C699. 2020. View Article : Google Scholar : PubMed/NCBI | |
Kranias EG and Hajjar RJ: Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome. Circ Res. 110:1646–1660. 2012. View Article : Google Scholar : PubMed/NCBI | |
Sivakumaran V, Stanley BA, Tocchetti CG, Ballin JD, Caceres V, Zhou L, Keceli G, Rainer PP, Lee DI, Huke S, et al: HNO enhances SERCA2a activity and cardiomyocyte function by promoting redox-dependent phospholamban oligomerization. Antioxid Redox Signal. 19:1185–1197. 2013. View Article : Google Scholar : PubMed/NCBI | |
Lygren B, Carlson CR, Santamaria K, Lissandron V, McSorley T, Litzenberg J, Lorenz D, Wiesner B, Rosenthal W, Zaccolo M, et al: AKAP complex regulates Ca2+ re-uptake into heart sarcoplasmic reticulum. EMBO Rep. 8:1061–1067. 2007. View Article : Google Scholar : PubMed/NCBI | |
Steinberg C, Roston TM, Van der Werf C, Sanatani S, Chen SRW, Wilde AAM and Krahn AD: RYR2-ryanodinopathies: From calcium overload to calcium deficiency. Europace. 25:euad1562023. View Article : Google Scholar : PubMed/NCBI | |
Strubbe-Rivera JO, Schrad JR, Pavlov EV, Conway JF, Parent KN and Bazil JN: The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function. Sci Rep. 11:10372021. View Article : Google Scholar : PubMed/NCBI | |
Reddy LG, Autry JM, Jones LR and Thomas DD: Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure. J Biol Chem. 274:7649–7655. 1999. View Article : Google Scholar : PubMed/NCBI | |
Qin J, Zhang J, Lin L, Haji-Ghassemi O, Lin Z, Woycechowsky KJ, Van Petegem F, Zhang Y and Yuchi Z: Structures of PKA-phospholamban complexes reveal a mechanism of familial dilated cardiomyopathy. Elife. 11:e753462022. View Article : Google Scholar : PubMed/NCBI | |
Feyen DAM, Perea-Gil I, Maas RGC, Harakalova M, Gavidia AA, Arthur Ataam J, Wu TH, Vink A, Pei J, Vadgama N, et al: Unfolded protein response as a compensatory mechanism and potential therapeutic target in PLN R14del cardiomyopathy. Circulation. 144:382–392. 2021. View Article : Google Scholar : PubMed/NCBI | |
Sinha RA and Yen PM: Metabolic messengers: Thyroid hormones. Nat Metab. 6:639–650. 2024. View Article : Google Scholar : PubMed/NCBI | |
Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, Browne WJ, Clark A, Cuthill IC, Dirnagl U, et al: The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Boil. 18:e30004102020. View Article : Google Scholar | |
Feng X, Wang L, Zhou R, Zhou R, Chen L, Peng H, Huang Y, Guo Q, Luo X and Zhou H: Senescent immune cells accumulation promotes brown adipose tissue dysfunction during aging. Nat Commun. 14:32082023. View Article : Google Scholar : PubMed/NCBI | |
Jiang J, Zhou D, Zhang A, Yu W, Du L, Yuan H, Zhang C, Wang Z, Jia X, Zhang ZN and Luan B: Thermogenic adipocyte-derived zinc promotes sympathetic innervation in male mice. Nat Metab. 5:481–494. 2023. View Article : Google Scholar : PubMed/NCBI | |
Mahmood SR, Xie X, Hosny El Said N, Venit T, Gunsalus KC and Percipalle P: β-actin dependent chromatin remodeling mediates compartment level changes in 3D genome architecture. Nat Commun. 12:52402021. View Article : Google Scholar | |
Hunter T and Garrels JI: Characterization of the mRNAs for alpha-, beta- and gamma-actin. Cell. 12:767–781. 1977. View Article : Google Scholar : PubMed/NCBI | |
Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar | |
Zhang L and Elias JE: Relative protein quantification using tandem mass tag mass spectrometry. Methods Mol Biol. 1550:185–198. 2017. View Article : Google Scholar : PubMed/NCBI | |
Pantos C, Malliopoulou V, Paizis I, Moraitis P, Mourouzis I, Tzeis S, Karamanoli E, Cokkinos DD, Carageorgiou H, Varonos D and Cokkinos DV: Thyroid hormone and cardioprotection: study of p38 MAPK and JNKs during ischaemia and at reperfusion in isolated rat heart. Mol Cell Biochem. 242:173–180. 2003. View Article : Google Scholar : PubMed/NCBI | |
Lieder HR, Braczko F, Gedik N, Stroetges M, Heusch G and Kleinbongard P: Cardioprotection by post-conditioning with exogenous triiodothyronine in isolated perfused rat hearts and isolated adult rat cardiomyocytes. Basic Res Cardiol. 116:272021. View Article : Google Scholar : PubMed/NCBI | |
Vafiadaki E, Haghighi K, Arvanitis DA, Kranias EG and Sanoudou D: Aberrant PLN-R14del protein interactions intensify SERCA2a inhibition, driving impaired Ca2+ handling and arrhythmogenesis. Int J Mol Sci. 23:69472022. View Article : Google Scholar | |
Stege NM, de Boer RA, Makarewich CA, van der Meer P and Silljé HHW: Reassessing the mechanisms of PLN-R14del cardiomyopathy: From calcium dysregulation to S/ER malformation. JACC Basic Transl Sci. 9:1041–1052. 2024. View Article : Google Scholar : PubMed/NCBI | |
Dong J, Gao C, Liu J, Cao Y and Tian L: TSH inhibits SERCA2a and the PKA/PLN pathway in rat cardiomyocytes. Oncotarget. 7:39207–39215. 2016. View Article : Google Scholar : PubMed/NCBI | |
Gaique TG, Lopes BP, Souza LL, Paula GSM, Pazos-Moura CC and Oliveira KJ: Cinnamon intake reduces serum T3 level and modulates tissue-specific expression of thyroid hormone receptor and target genes in rats. J Sci Food Agric. 96:2889–2895. 2016. View Article : Google Scholar | |
Mattiazzi A, Tardiff JC and Kranias EG: Stress seats a new guest at the table of PLN/SERCA and their partners. Circ Res. 128:471–473. 2021. View Article : Google Scholar : PubMed/NCBI | |
Zeng B, Liao X, Liu L, Zhang C, Ruan H and Yang B: Thyroid hormone mediates cardioprotection against postinfarction remodeling and dysfunction through the IGF-1/PI3K/AKT signaling pathway. Life Sci. 267:1189772021. View Article : Google Scholar : PubMed/NCBI | |
de Castro AL, Fernandes RO, Ortiz VD, Campos C, Bonetto JH, Fernandes TR, Conzatti A, Siqueira R, Tavares AV, Schenkel PC, et al: Thyroid hormones improve cardiac function and decrease expression of pro-apoptotic proteins in the heart of rats 14 days after infarction. Apoptosis. 21:184–194. 2016. View Article : Google Scholar | |
Brennan A, Leech JT, Kad NM and Mason JM: Selective antagonism of cJun for cancer therapy. J Exp Clin Cancer Res. 39:1842020. View Article : Google Scholar : PubMed/NCBI | |
Vernia S, Morel C, Madara JC, Cavanagh-Kyros J, Barrett T, Chase K, Kennedy NJ, Jung DY, Kim JK, Aronin N, et al: Excitatory transmission onto AgRP neurons is regulated by cJun NH2-terminal kinase 3 in response to metabolic stress. Elife. 5:e100312016. View Article : Google Scholar : PubMed/NCBI | |
Manieri E, Folgueira C, Rodríguez ME, Leiva-Vega L, Esteban-Lafuente L, Chen C, Cubero FJ, Barrett T, Cavanagh-Kyros J, Seruggia D, et al: JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma. Proc Natl Acad Sci USA. 117:16492–16499. 2020. View Article : Google Scholar : PubMed/NCBI | |
Yang J, Do-Umehara HC, Zhang Q, Wang H, Hou C, Dong H, Perez EA, Sala MA, Anekalla KR, Walter JM, et al: miR-221-5p-mediated downregulation of JNK2 aggravates acute lung injury. Front Immunol. 12:7009332021. View Article : Google Scholar : PubMed/NCBI | |
Jaeschke A, Karasarides M, Ventura JJ, Ehrhardt A, Zhang C, Flavell RA, Shokat KM and Davis RJ: JNK2 is a positive regulator of the cJun transcription factor. Mol Cell. 23:899–911. 2006. View Article : Google Scholar : PubMed/NCBI | |
Chin KY, Silva LS, Darby IA, Ng DCH and Woodman OL: Protection against reperfusion injury by 3′,4′-dihydroxyflavonol in rat isolated hearts involves inhibition of phospholamban and JNK2. Int J Cardiol. 254:265–271. 2018. View Article : Google Scholar : PubMed/NCBI | |
Song Q, Schmidt AG, Hahn HS, Carr AN, Frank B, Pater L, Gerst M, Young K, Hoit BD, McConnell BK, et al: Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J Clin Invest. 111:859–867. 2003. View Article : Google Scholar : PubMed/NCBI | |
Sundqvist A, Voytyuk O, Hamdi M, Popeijus HE, van der Burgt CB, Janssen J, Martens JWM, Moustakas A, Heldin CH, Ten Dijke P and van Dam H: JNK-dependent cJun phosphorylation mitigates TGFβ- and EGF-induced pre-malignant breast cancer cell invasion by suppressing AP-1-mediated transcriptional responses. Cells. 8:14812019. View Article : Google Scholar | |
Martin L, Derwall M, Al Zoubi S, Zechendorf E, Reuter DA, Thiemermann C and Schuerholz T: The septic heart: Current understanding of molecular mechanisms and clinical implications. Chest. 155:427–437. 2019. View Article : Google Scholar | |
Funk F, Kronenbitter A, Hackert K, Oebbeke M, Klebe G, Barth M, Koch D and Schmitt JP: Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation. Cardiovasc Res. 119:1568–1582. 2023. View Article : Google Scholar : PubMed/NCBI | |
Babić Leko M, Gunjača I, Pleić N and Zemunik T: Environmental factors affecting thyroid-stimulating hormone and thyroid hormone levels. Int J Mol Sci. 22:65212021. View Article : Google Scholar | |
Silvestri E, Lombardi A, de Lange P, Schiavo L, Lanni A, Goglia F, Visser TJ and Moreno M: Age-related changes in renal and hepatic cellular mechanisms associated with variations in rat serum thyroid hormone levels. Am J Physiol Endocrinol Metab. 294:E1160–E1168. 2008. View Article : Google Scholar : PubMed/NCBI |