Stress-related heart damage may involve a protein disrupting calcium balance and mitochondrial health through specific cell pathways

Restraint stress (RS) is a classical model of psychological stress (Figure 1A). We first confirmed the successful establishment of the stress model by detecting serum corticosterone levels (Figure 1B). Then, we used echocardiography to evaluate the impact of stress on cardiac function. The results showed that cardiac output increased in acutely stressed mice, whereas left ventricular ejection fraction (LVEF) and cardiac output significantly decreased in chronically stressed mice. B-mode ultrasound revealed significant thickening of the left ventricular wall during both diastole and systole in chronically stressed mice. Meanwhile, M-mode echocardiography also showed increased cardiac output in acutely stressed mice and a significant reduction in LVEF and cardiac output in chronically stressed mice (Figure 1C–H). Biochemical assays revealed that stress elevated the levels of Cardiac Troponin I (cTnI) in both serum and left ventricular myocardium (LVM) tissue (Figure 1I,J). Moreover, chromotrope-2R brilliant green staining showed pathological changes such as ischemia and hypoxia in cardiomyocytes (Figure 1K). These results indicate that prolonged stress leads to cardiac dysfunction and myocardial injury.

Mitochondria are sensitive organelles in cardiomyocytes for detecting stress-induced injury, and their structural and functional alterations are a major cause of cardiomyocyte damage [19]. Using transmission electron microscopy, we observed that stress induced mitochondrial swelling, cristae dissolution, and fragmentation in cardiomyocytes (Figure 2A). Meanwhile, stress also caused abnormal expression of cytochrome C (Cyto-c) and Translocase of Outer Mitochondrial Membrane 20 homolog (TOM-20) (Figure 2B–D), indicating that stress induced mitochondrial damage and dysfunction in cardiomyocytes. Mitochondrial morphology is primarily regulated by fusion and fission processes and tightly controlled by quality control mechanisms that determine mitochondrial function [20,21,22]. To further investigate the cause of mitochondrial damage, we examined key proteins involved in mitochondrial quality control and found that chronic stress reduced the expression of mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and fusion proteins mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in cardiomyocytes (Figure 2B,E–G), while the expression of mitochondrial fission protein-1 (FIS1) and autophagy marker microtubule–associated proteins light chain 3 (LC3) was increased (Figure 2B,H,I), indicating that stress reduces mitochondrial biogenesis and fusion but promotes fission and autophagy, leading to marked disruption of mitochondrial quality control in mouse cardiomyocytes.

Abnormal cytosolic calcium levels are a key cause of impaired mitochondrial quality control [23,24,25]; we assessed calcium levels in cardiomyocytes and observed that both acute and chronic stress markedly elevated intracellular calcium levels (Figure 3A). Next, we analyzed critical calcium channel proteins in the sarcoplasmic reticulum (SR) and mitochondria (Figure 3B) and found no significant changes in SERCA2 or the mitochondrial permeability transition pore (mPTP)-regulating protein peptidyl-prolyl cis-trans isomerase D (PPID) after stress (Figure 3C,D), whereas expression of mitochondrial calcium uniporter (MCU) and FKBP12.6 proteins was markedly decreased (Figure 3E,F). FKBP12.6 acts as a regulatory protein for RyR2 channels in the SR, serving as a "molecular cap". Loss of FKBP12.6 leads to RyR2 dysfunction, leaving the channel persistently open or leaky, causing elevated cytosolic calcium. To investigate the mechanism of FKBP12.6 downregulation, we observed that its ubiquitination was markedly increased following stress (Figure 3G,H). We subsequently searched for proteins that might regulate FKBP12.6 ubiquitination and found that GLMN, a regulator of the E3 ubiquitin ligase complex, bound to FKBP12.6 (Figure 3I), and GLMN expression was reduced in response to stress (Figure 3J,K), suggesting that both acute and chronic stress diminish GLMN-mediated protection against FKBP12.6 ubiquitination, resulting in its degradation in cardiomyocytes.

Stress activates the HPA axis and markedly elevates circulating glucocorticoid levels (Figure 4A,B), prompting the investigation of whether this regulation occurs through glucocorticoid receptor-mediated transcriptional control of GLMN. Accordingly, we generated a cellular stress model by treating H9C2 cells with 500 μM glucocorticoid (GC) (Figure 4C,D) and used ChIP-qPCR to evaluate NR3C1 involvement in GLMN transcription. Glucocorticoid exposure significantly reduced GLMN mRNA expression in H9C2 cells (Figure 4E), suggesting that glucocorticoids repress GLMN transcription via NR3C1. We next established an H9C2 cell model with NR3C1 knockdown using siRNA (Figure 4F) and observed that reducing NR3C1 expression restored GLMN protein levels (Figure 4G,H) and significantly rescued stress-induced FKBP12.6 degradation mediated by ubiquitination (Figure 4I,J). In addition, we measured calcium levels in cardiomyocytes (Figure 4K) and assessed the expression of mitochondrial quality control-related proteins (Figure 4L), showing that silencing NR3C1 effectively blocked the stress-induced calcium overload, reversed the elevation of mitochondrial biogenesis protein PGC-1α (Figure 4M) and fission marker Fis1 (Figure 4N) caused by stress, and restored the stress-reduced expression of mitochondrial fusion proteins MFN1 and OPA1 (Figure 4O,P) and autophagy marker LC3 (Figure 4Q), thereby enhancing mitochondrial quantity (Figure 4R) and functional integrity (Figure 4S).

Male C57BL/6N mice (7–8 weeks, 22 ± 2 g) from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were acclimated for 7 days in SPF conditions (22 ± 2 °C, 12 h light/dark cycle). Animal protocols were approved by Hebei Medical University Ethics Committee (IACUC-Hebmu-2023011). Mice were randomly divided into four groups: control for 3 days (CON-3d), control for 14 days (CON-14d), restraint stress for 3 days (RS-3d), and restraint stress for 14 days (RS-14d), with n = 10 per group. Mice in the stress groups were restrained in cylindrical acrylic glass tubes (10 cm in length, 2.5 cm inner diameter) for RS. The tube body was provided with air holes and movable valves at both ends of the tube so that the mice could drill into the tube and could not move or turn freely [45]. RS groups were subjected to 6 h daily restraint (restraint tube model), where RS-3d represented acute stress with three consecutive days of restraint, and RS-14d represented chronic stress with fourteen consecutive days of restraint. Control groups experienced equivalent durations of food and water deprivation. At the endpoint, mice were anesthetized with 2% pentobarbital sodium (3.2 mL/kg, i.p.), blood was collected retro-orbitally, and they were euthanized. Hearts were excised and fixed in 4% paraformaldehyde, or rapidly frozen in liquid nitrogen and stored at −80 °C for future analysis.

H9C2 rat cardiomyocytes (STCC30008P) were purchased from Servicebio (Wuhan, China) and cultured in high-glucose DMEM (Viva Cell Biosciences, C3113-0500, Denzlingen, Germany) supplemented with 10% fetal bovine serum (ExCell Bio, FSP500, Shanghai, China) and 1% penicillin-streptomycin (Solarbio, P1400, Beijing, China). Cells were maintained in a humidified incubator at 37 °C with 5% CO₂. For stimulation, 500 μM glucocorticoids were added to the complete medium for 24 h; all other conditions remained consistent with standard culture procedures.

Small interfering RNAs (siRNAs) targeting NR3C1, a negative control siRNA, and a fluorescently labeled siRNA were obtained from RiboBio (Guangzhou, China). Transfections were performed using riboFECT™ CP Transfection Reagent (RiboBio, R10035.7) according to the manufacturer's protocol when the cell confluence reached 30–50%. After 48 h, transfection and knockdown efficiency were assessed by inverted fluorescence microscopy and RT-qPCR.

To ensure complete recovery of voluntary movement and minimize confounding variables, echocardiographic assessment was performed two hours post-RS modeling. Mice were lightly anesthetized with isoflurane and subjected to echocardiographic assessment of left ventricular systolic function using a Vevo 2100 system (VisualSonics, Toronto, ON, Canada) equipped with a 12–38 MHz linear transducer. Left ventricular systolic function was assessed via short-axis views obtained at the level of maximal left ventricular diameter to comprehensively evaluate RS-induced cardiac structural and functional alterations. B-mode imaging was used to evaluate ventricular wall thickness, while M-mode imaging assessed cardiac contractility. LVEF and cardiac output were quantitatively calculated by measuring left ventricle chamber dimensions during systole and diastole.

Mice were anesthetized via intraperitoneal injection of 2% pentobarbital sodium. Blood was collected retro-orbitally, and left ventricular myocardial tissues were harvested. Blood samples were centrifuged at 1000× g for 15 min at 4 °C. Serum and tissue samples were stored at −80 °C. According to the manufacturer's protocols, serum and cardiac tissue homogenates' cTnI levels, along with serum corticosterone levels, were quantified using a mice-specific cTnI ELISA kit (Cloud-Clone Corp, USEA478Mu, Wuhan, China) and a corticosterone ELISA kit (CAYMAN, 501320, Ann Arbor, MI, USA).

The isolated mouse heart was fixed in 10% formalin, and the tissue was subsequently dehydrated in steps of ethanol and embedded in paraffin. The heart wax block was sectioned into continuous sections (5 μm) for Chromotrope-2R brilliant green staining (LEAGENE, DK0007). Chromotrope-2R brilliant green staining is a sensitive method to assess cardiomyocyte injury. Slices were stained with Chromotrope-2R brilliant green staining for 10 min, fractionated three times in 2R fractionation solution for 5 min each, re-stained in bright green staining solution for 10 min, and finally observed under a light microscope (Olympus IX71; Mt. Olympus, Tokyo, Japan).

Ventricular tissue was rapidly dissected from isolated mice hearts on ice, fixed in 2.5% glutaraldehyde (pH 7.4), and transferred to the Electron Microscopy Laboratory at Hebei Medical University. Samples were dehydrated through graded acetone series and embedded in epoxy resin. For ultrastructural analysis, at least 400 mitochondria were randomly selected from five distinct regions per sample.

Mouse heart tissues were mechanically homogenized in a RIPA lysis buffer supplemented with protease and phosphatase inhibitors. After centrifugation at 14,000× g for 15 min at 4 °C, protein concentration was determined using a BCA assay kit. Proteins (8–12% SDS-PAGE) were separated and transferred onto PVDF membranes (Beyotime, FFP33/FFP24, Haimen, China) using a semi-dry transfer system (Trans-Blot Turbo). Membranes were blocked with 5% skim milk for 1 h, then incubated overnight at 4 °C with primary antibodies: OPA1 (1:1000, Proteintech, 27733-1-AP, Wuhan, China), PGC-1α (1:1000, Abclona, A12348), MFN1 (1:1000, Proteintech, 13798-1-AP), β-Tubulin (1:2000, Servicebio, GB11017-100), β-actin (1:1000, Servicebio, GB15003-100), GAPDH (1:5000, Servicebio, GB15004-100), LC3 (1:500, Proteintech, 14600-1-AP), Fis1 (1:1000, Proteintech, 10956-1-AP), TOM-20 (1:4000, Proteintech, 11802-1-AP), Cyt-C (1:1000, Abcam, ab133504), SERCA2 (1:1000, Proteintech, 27311-1-AP), MCU (1:1000, Proteintech, 26312-1-AP), PPID (1:500, Proteintech, 12716-1-AP), FKBP12.6 (1:2000, Proteintech, 15114-1-AP), GLMN (1:500, Proteintech, 32645-1-AP), and NR3C1 (1:1000, Proteintech, 24050-1-AP). Following incubation with IRDye 680RD anti-rabbit secondary antibody for 1 h at 37 °C, membranes were washed extensively with TBST. Protein bands were detected using the LI-COR Odyssey infrared imaging system, and relative expression levels were quantified using ImageJ software (v1.8.0, NIH, Bethesda, MD, USA).

Fluo-4, AM (Thermo Scientific, F14217, Waltham, MA, USA) was dissolved in 50 μL of DMSO (Thermo Scientific, D12345, Waltham, MA, USA) and diluted 1:1000 with HBSS (Report, RE0191, Shijiazhuang, China). Frozen sections or myocardial cell slides were incubated with the working solution at 37 °C in the dark for 30 min. Samples were then thoroughly washed with HBSS, counterstained with DAPI, and mounted using FluoroMount-G^®. Images were acquired using a confocal laser scanning microscope (Leica SP8, Wetzlar, Germany).

Left ventricular myocardial tissue was lysed in an IP lysis buffer (Servicebio, G2038) supplemented with protease inhibitor (Servicebio, G2008). Protein A/G magnetic beads (Servicebio, G3657) were pre-washed with a binding buffer. For immunoprecipitation, 1.5 mg of protein lysate was incubated with 10 μL of anti-FKBP12.6 antibody (Santa Cruz Biotechnology, sc-136962, Dallas, TX, USA) and 30 μL of protein A/G beads overnight at 4 °C. After magnetic separation, the beads were washed with a binding buffer, then mixed with a 4× SDS loading buffer and boiled at 95 °C for 10 min. The supernatant was collected by magnetic separation and subjected to immunoblotting analysis.

Cells were harvested and nuclei lysed to release chromatin, which was then fragmented by sonication. The sheared chromatin was incubated with a specific antibody against the target protein (Cell Signaling Technology, 3660S, Danvers, MA, USA), followed by addition of protein A/G magnetic beads (Cell Signaling Technology, 14209S, Danvers, MA, USA) to capture the antibody–protein–DNA complexes. After extensive washing to remove non-specific interactions, cross-links were reversed by heating at 65 °C in an SDS-containing buffer. Proteinase K digestion was performed to remove proteins, and the DNA was purified for subsequent qPCR analysis to identify target protein-bound genomic regions.

Total RNA was extracted from mouse heart tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), followed by chloroform extraction and isopropanol precipitation. As needed, complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit (RR047A; Takara Bio, Shiga, Japan). Real-time quantitative PCR (qRT-PCR) or quantitative PCR (q-PCR) was performed using the SYBR Green PCR kit (RR820A; Takara Bio) to assess the mRNA expression of NR3C1 and GLMN (primers from Sangon Biotech, Shanghai, China). Gene expression levels were normalized to Gapdh and calculated using the 2^−ΔΔCt method. The primer sequences were as follows:

GAPDH: Forward primer: 5′-GACATGCCGCCTGGAGAAAC-3′;

reverse primer: 5′-AGCCCAGGATGCCCTTTAGT-3′.

GLMN: Forward primer: 5′-CACGAAACCCTTTGTGGCAG-3′;

reverse primer: 5′-GCAAAACACCGGAAAGGGTC-3′.

NR3C1: Forward primer: 5′-TCCCCCTGGTAGAGACGAAG-3′;

reverse primer: 5′-ACTGTAGCTCCTCCCCTCAG-3′.

Cells were lysed using a RIPA lysis buffer (Report, RW0001, Shijiazhuang, China) supplemented with protease inhibitors (Report, RP-WA0120, Shijiazhuang, China). Ubiquitinated proteins were immunoprecipitated using an anti-ubiquitin antibody (1:100; Proteintech, 10201-2-AP) overnight at 4 °C, followed by incubation with protein A/G magnetic beads (Servicebio, G3657-1ML, Wuhan, China). After extensive washing, bound proteins were eluted by boiling in the sample buffer. The ubiquitination level of FKBP12.6 was assessed by Western blot using a specific anti-FKBP12.6 antibody (1:2000; Proteintech, 15114-1-AP).

All data were analyzed using GraphPad Prism (version 9). Results are presented as mean ± SEM. Statistical significance was assessed using normality/lognormality tests followed by one-way ANOVA. p < 0.05 was considered statistically significant. All experiments were independently repeated at least four times.