Abstract

   Chronic psychological stress is closely related to myocardial diseases,
   but the pathological processes and related mechanisms are not yet
   clear. In this study, 5 batches of animal experiments are conducted to
   reveal the pathological process of chronic psychological stress on the
   heart, and found that cardiomyocyte hypertrophy is an early
   manifestation of cardiac damage. Furthermore, it is discovered that
   corticosterone and glucocorticoid receptor (GR) play an important role
   in mediating this phenomenon. Additionally, a new direct target gene of
   GR, Lama5, is identified and confirmed in vivo and in vitro that the
   corticosterone‐GR‐LAMA5 axis is a significant pathway mediating chronic
   psychological stress‐induced cardiomyocyte hypertrophy. Moreover, the
   potential of LAMA5 is validated as a biomarker for myocardial
   hypertrophy and impaired cardiac function in patients with depression.
   In summary, a novel hypertrophy‐related gene, Lama5 has been
   identified, that may play an important role in chronic psychological
   stress‐induced cardiomyocyte hypertrophy.

   Keywords: cardiomyocyte hypertrophy, chronic psychological stress,
   corticosterone, glucocorticoid receptors, laminin subunit alpha 5
     __________________________________________________________________

   In vivo experiments suggest that cardiomyocyte hypertrophy is an early
   manifestation of cardiac pathological changes induced by chronic
   psychological stress. In vivo and in vitro experiments suggest that the
   corticosterone‐glucocorticoid receptor‐LAMA5 axis mediates
   cardiomyocyte hypertrophy induced by chronic psychological stress. The
   clinical results suggest that LAMA5 is a potential specific marker of
   heart failure in depressed patients.

   graphic file with name ADVS-12-e14659-g007.jpg

1. Introduction

   Psychological stress exerts a significant impact on cardiovascular
   disease.^[ [44]^1 ^] For instance, it is well established that acute
   psychological stress can precipitate acute coronary syndrome^[ [45]^2 ,
   [46]^3 ^] or stress cardiomyopathy.^[ [47]^4 ^] Moreover, chronic
   psychological stress has been increasingly recognized as a contributing
   factor to hypertension^[ [48]^5 ^], atherosclerosis^[ [49]^6 ^], and
   other vascular‐related pathologies. However, the effects of chronic
   psychological stress on myocardial structure and function remain
   inadequately understood.

   In clinical practice, prevalent diseases characterized by chronic
   psychological stress as a primary etiological factor or clinical
   manifestation include anxiety and depression, which have been proven to
   be independent risk factors for various cardiovascular diseases,
   notably heart failure.^[ [50]^7 ^] The prevalence of anxiety and
   depression among heart failure patients significantly exceeds that
   observed in the general population, and individuals with these
   comorbidities frequently experience poorer prognoses.^[ [51]^8 , [52]^9
   ^] Nevertheless, the underlying pathological processes and mechanisms
   remain inadequately understood. Recent research has demonstrated that
   repeated exposure to social failure stress results in impaired cardiac
   muscle contraction performance and myocardial fibrosis in male rats.^[
   [53]^10 , [54]^11 ^] The underlying mechanism is hypothesized to
   involve the activation of the sympathetic nervous system and the
   hypothalamic‐pituitary‐adrenal axis, triggered by chronic psychological
   stress. This activation leads to an increase in stress hormone levels,
   including adrenaline and glucocorticoids (predominantly cortisol in
   humans and corticosterone in rodents). Nonetheless, additional evidence
   is required to substantiate these findings comprehensively.

   In this study, we conducted a comprehensive examination of the
   pathological processes in cardiac structure and function in rat models
   subjected to chronic psychological stress, utilizing five distinct
   batches. Our findings suggest that cardiomyocyte hypertrophy may serve
   as an early indicator of stress‐induced cardiac pathology.
   Subsequently, we conducted an analysis of transcriptomic alterations
   and serum hormone levels in heart tissues subjected to chronic
   psychological stress. This investigation led to the identification of a
   novel glucocorticoid receptor (GR) target gene, laminin subunit alpha 5
   (Lama5), and an exploration of its association with cardiomyocyte
   hypertrophy. Our findings suggest that the corticosterone
   (CORT)‐GR‐LAMA5 axis may represent a potential mechanism and
   therapeutic target for cardiomyocyte hypertrophy induced by chronic
   psychological stress.

2. Experimental Section

2.1. Rats

   For all animal studies, age‐matched male Wistar rats (purchased from
   Charles River) between 5 and 6 weeks of age were used. The rats were
   maintained in a specific pathogen‐free environment at a temperature of
   22 °C, with a 12‐h light/dark cycle. The experimental protocol received
   approval from the Experimental Animal Ethics Committee of Ningbo
   University (batch number: 12654), and all procedures adhered to the
   guidelines for the care and use of experimental animals as stipulated
   by the National Institutes of Health of the United States.

2.2. Chronic Psychological Stress Model

   Rats were randomly assigned to either a control group (n = 40) or a
   chronic mild stress (CMS) group (n = 40). CMS was a widely used
   depression animal model that simulates unpredictable, random adverse
   stimuli in daily human life. This study made slight modifications to
   the existing model methods.^[ [55]^12 ^] Following a 1‐week acclimation
   period, the rats in the CMS group were subjected to a 20‐week regimen
   of daily mild stressors, including: 18 h of food or water deprivation;
   24 h of wet bedding (200 mL of water per 300 g of bedding); 24 h
   without bedding material; 24 h with a 45° cage tilt; 5 min of tail
   clipping; 24 h of inverted photoperiod (reversed day and night cycle);
   36 h of continuous lighting; 30 min of cat meow stimulation (60dB) and
   bind for 1h. Each day, a random stressor was assigned, and the same
   stressor was applied to all rats in the experimental groups on that
   day. To enhance intra‐group comparability, the time difference in the
   onset of stress induction across all experimental groups was kept
   within 15 min. The daily stressor schedules for this study are
   documented in Table [56]S1 (Supporting Information). The rats in the
   control group were fed under standard conditions without exposure to
   stress. Throughout the duration of the experiment, the weight and
   overall health of the rats were systematically monitored. The rats from
   both groups were subdivided into five batches, with one batch being
   euthanized every 4 weeks. The whole heart tissues were weighed and then
   stored in a refrigerator at −80 °C or fixed in 4% paraformaldehyde. The
   fifth batch of fresh left ventricular tissues was taken for full
   transcriptome sequencing.

2.3. Sucrose Preference Test (SPT)

   Prior to the commencement of the experiment, a two‐day period of
   adaptive training was conducted to acclimate the rats to consuming a 1%
   sucrose solution. Sucrose preference tests were administered over a
   24‐h period on the first day of the experiment and subsequently every
   four weeks. During these tests, two drinking bottles—one containing a
   1% sucrose solution and the other containing plain water—were provided
   to measure the intake of both solutions over a 24‐h period. The sucrose
   preference index, defined as the ratio of sucrose solution intake to
   total water intake, was calculated to evaluate anhedonia in the rats.

2.4. Open Field Test (OFT)

   Open field tests were administered at four‐week intervals following the
   initiation of CMS to evaluate behavioral alterations in the rats. The
   OFT was a widely recognized method for assessing depression‐like
   behaviors as well as autonomous behaviors in novel environments. The
   experimental apparatus consisted of an open field box measuring 100 cm
   × 100 cm × 40 cm. Prior to each trial, the rats were positioned at the
   center of the box and permitted to explore freely for a duration of 5
   min. The movements of the rats were recorded via video surveillance,
   and the total distance traveled within the 5‐min period was quantified
   and analyzed using ANY‐Maze Software version 7.4 (Stoelting Co., USA).

2.5. Forced Swimming Test (FST)

   A cylindrical glass tank with dimensions of 30 cm in diameter and 60 cm
   in height was utilized, into which water was added to a depth of 45 cm.
   The water temperature was standardized to 26 °C prior to the
   commencement of each experimental trial. Rats were individually
   introduced into the tank for a duration of 6 min, during which their
   movements were recorded using a video surveillance system. Following
   the 6‐min period, the rats were removed from the tank, placed on dry
   bedding, and warmed using an infrared lamp. The durations of swimming,
   struggling, and immobility behaviors in the water were subsequently
   analyzed and quantified using ANY‐Maze Software version 7.4 (Stoelting
   Co., USA). The videos were analyzed manually, and the analyst was
   unaware of the grouping of the rats.

2.6. Echocardiography

   High‐frequency echocardiography (D6VET, Vinno) was employed at
   four‐week intervals to assess the cardiac function of the rats. The
   rats were anesthetized using 2% isoflurane and maintained on a heated
   mat to prevent hypothermia. Ultrasound gel was applied to the chest,
   and a high‐frequency probe (40 MHz) was utilized to obtain M‐mode and
   B‐mode images. Left ventricular ejection fraction (LVEF), fractional
   shortening (FS), diastolic interventricular septal (IVSd) thickness,
   and diastolic left ventricular posterior wall (LVPWd) thickness were
   recorded and analyzed.

2.7. Blood Pressure Measurement

   The systolic blood pressure of the same cohort of rats was assessed at
   4‐week intervals utilizing tail‐cuff plethysmography with the BP‐2010
   Blood Pressure Analysis System. Measurements were conducted prior to
   the commencement of behavioral experiments, between 8:00 and 10:00 AM.
   The procedure was acclimatized over a one‐week period preceding the
   formal data collection.

2.8. Hematoxylin and Eosin (HE) and Masson Staining

   The rat heart tissue was fixed in a 4% paraformaldehyde solution for 48
   h, followed by paraffin embedding. Transverse sections were then cut at
   5 µm intervals at the level of the left central ventricular papillary
   muscle. According to the experimental protocol, HE staining and Masson
   trichrome staining were performed using the HE staining kit (Solarbio)
   and the Masson trichrome staining kit (Solarbio), respectively. The
   morphology, thickness, and fibrosis of the myocardium were examined
   using a DMi1 Inverted Microscope (Leica). The proportion of the
   fibrotic area was quantified using ImageJ software.

2.9. Wheat Germ Agglutinin Staining

   Heart sections were stained with AF488‐labeled wheat germ agglutinin
   (WGA, AAT Bioquest) to evaluate the cross‐sectional area of
   cardiomyocytes. The myocardial cell area was visualized and documented
   using a fluorescence microscope (DMi8, Leica) and subsequently analyzed
   with ImageJ software. Each rat was photographed in the middle left
   ventricle of 4 fields, each field containing 50–100 cells, and the mean
   of all cells was taken as the myocardial cell area of the rat. The area
   of cardiomyocytes was compared by nested analysis.

2.10. Enzyme Linked Immunosorbent Assay (ELISA)

   A volume of 0.5 mL of blood was collected from the orbital sinus of the
   rats every 4 weeks. The samples were allowed to stand at room
   temperature for 1 h, followed by centrifugation at 2500 RPM for 15 min.
   The supernatant was then aliquoted and stored at −80 °C. According to
   the respective ELISA kit instructions, the serum from each batch of
   rats was analyzed for CORT (Elabscience), norepinephrine (Cusabio),
   serotonin (Cusabio), angiotensin II (Elabscience), aldosterone
   (Elabscience), and N‐terminal pro‐B‐type natriuretic peptide
   (NT‐proBNP, Elabscience).

2.11. Interventions of CORT‐GR‐LAMA5 In Vivo

   Wistar rats were randomly assigned to five experimental groups:
   control, CMS, CMS+ metyrapone, CMS+ relacorilant, and CMS+ LAMA5KD,
   with each group comprising eight mice. Prior to the commencement of the
   experiment, rats of the CMS+ LAMA5KD group were administered 200 µL of
   AAV carrying Lama5 shRNA via the tail vein at a viral titer of 2.93 ×
   10^13 vg mL^−1. The following LAMA5^− AAV was used, and detailed
   sequence information is available upon request: pcAAV‐cTnTo‐miR30shRNA
   (Lama5)‐WPRE (OBiO Technology). The serotype of the administered virus
   was MyoAAV1A. The virus utilized cardiac troponin T (cTnT) promoter to
   facilitate targeted aggregation of cardiomyocytes. Additionally, 200 µL
   of a non‐functional negative control virus was administered via tail
   vein injection to the remaining groups of rats. Following the
   initiation of the experiment, all rats, with the exception of the
   control group, were subjected to CMS stimulation for a duration of 12
   weeks, employing the same stimulation protocol as previously described.
   Among them, the CMS+ metyrapone group was given metyrapone
   (MedChemExpress) 25mg kg^−1 every 2 days, the CMS+ relacorilant group
   was given relacorilant (MedChemExpress) 30mg kg^−1 every 2 days, and
   the other groups were given the same volume of saline at the same time.
   During the final week of the experiment, the OFT, SPT, and
   echocardiography were conducted. Upon completion of the experimental
   procedures, the rats were anesthetized and euthanized. Subsequently,
   the entire heart tissue was then excised, weighed, and preserved either
   in 4% paraformaldehyde or rapidly frozen in liquid nitrogen at −80 °C
   for future analysis.

2.12. H9C2 Cell Line Culture

   H9C2 myocardial cell lines, procured from the Shanghai Cell Bank of the
   Chinese Academy of Sciences, were cultured in Dulbecco's Modified Eagle
   Medium (DMEM; Sigma–Aldrich) supplemented with 10% fetal bovine serum
   and 1% penicillin‐streptomycin. The cultures were maintained in a 5%
   CO[2] incubator at 37 °C, with medium changes performed every 2–3 days
   to ensure appropriate cell density.

2.13. Cellular Drug Intervention

   H9C2 cells were laid on 30% density plate, fresh medium was changed 24
   h later, and CORT (1µmol L^−1), relacorilant (1µmol L^−1), finerenone
   (10µmol L^−1), LY294002 (2µmol L^−1) and other drugs were given
   according to the group intervention, The medium and drugs were
   refreshed every 24 h, with a total drug exposure duration of 48 h. For
   transient siRNA transfection, lipo3000 (Thermo Fisher Scientific) was
   utilized to transfect GR/Lama5 siRNA according to the manufacturer's
   instructions. Subsequent drug treatments were administered as
   previously described. Scramble siRNA was used as the negative control;
   the specific sequences of the negative control siRNA and the siRNAs
   specific for GR and Lama5 are provided in Table [57]S2 (Supporting
   Information).

2.14. Establishment of Stable Cell Lines Overexpressing LAMA5

   In this experiment, BrainVTA Technology Biological Co., Ltd. was
   commissioned to synthesize a LAMA5‐overexpressing lentivirus
   (pLV‐U6‐sgRNA(Lama5)‐EFS‐EGFP‐T2A‐Puro‐CMV‐SV40NLS‐dCas9‐NLS‐VPR‐WPRE,
   BrainVTA). Following the determination of the lethal concentration of
   puromycin in H9C2 cells, both the LAMA5‐overexpressing lentivirus and
   the control virus were transfected according to the standard procedures
   specified by the company. 24 h later, fresh medium was replaced and
   1.5umol L^−1 purinomycin was given (every 24 h) for 7 consecutive days
   of screening, and a high‐purity Lama5 overexpression stable strain was
   obtained.

2.15. Dual Luciferase Reporter Gene Assay

   In this study, GenePharma was engaged to construct a [58]GPL4
   recombinant plasmid containing the Lama5 promoter sequence and a
   pcDNA3.1 recombinant plasmid containing the full‐length GR sequence.
   These plasmids were subsequently transfected into H9C2 cells utilizing
   lipo3000 (Thermo Fisher Scientific). After a 24‐h incubation period, a
   dual luciferase assay (GenePharma) was conducted in accordance with the
   manufacturer's protocol. Luciferase activity from both firefly and
   renilla luciferases was quantified using a BioTek microplate reader to
   assess the regulatory effect of GR on Lama5 promoter activity. The
   activity of firefly luciferase and renilla luciferase was quantified
   using a BioTek instrument, and the regulation of Lama5 promoter
   activity by GR was assessed. The relative activity of firefly
   luciferase was calculated based on the activity of renilla luciferase.

2.16. Immunofluorescence

   The cells were cultured in specialized cell culture dishes, fixed with
   4% paraformaldehyde for 10 min, and subsequently permeabilized with
   0.1% Triton X‐100 for 15 min. Tissue sections underwent standard
   dewaxing, rehydration, and antigen retrieval procedures, followed by
   blocking with phosphate‐buffered saline (PBS) containing 1% bovine
   serum albumin for 30 min. Primary antibodies were then added and
   incubated overnight at 4 °C. On the following day, the cells were
   incubated with fluorescently labeled secondary antibodies at a 1:400
   dilution for 1 h, and the nuclei were counterstained with DAPI for 10
   min. Fluorescence microscopy (Leica DMi8) was used to observe and
   photograph the localization and expression of target proteins in cells.

2.17. Western Blot (WB)

   Total cellular or tissue proteins were extracted using RIPA lysis
   buffer supplemented with a protease/phosphatase inhibitor cocktail
   (Epizyme Biotech). Protein concentrations were quantified utilizing a
   BCA assay kit (Epizyme Biotech). Following SDS‐PAGE, the protein
   samples were transferred onto a PVDF membrane (Sigma–Aldrich). The
   membrane was then blocked with 5% skim milk powder in PBS‐Tween 20
   buffer for 1 h and subsequently incubated overnight at 4 °C with
   specific primary antibodies. Primary antibodies used were as follows:
   anti‐MYH7 (1:1000, 22280‐1‐AP, Proteintech Group, Inc), anti‐CLO1A2,
   (1:5000, 66761‐1 ‐PBS, Proteintech Group, Inc), anti‐GR (1:5000,
   24050‐1‐AP, Proteintech Group, Inc), anti‐Histone H3 (1:3000,
   17168‐1‐AP, Proteintech Group, Inc), anti‐LAMA5 (1:1000, ab184330,
   Abcam), anti‐ Phospho‐PI3K (1:1000, #AF3242, Affinity Biosciences
   LTD.), anti‐PI3K (1:5000, 60225‐1‐Ig, Proteintech Group, Inc), anti‐
   Phospho‐Akt (1:1000, #4060, Cell Signaling Technology, Inc.), anti‐AKT
   (1:2000, 10176‐2‐AP, Proteintech Group, Inc), anti‐ITGB1 (1:2000,
   12594‐1‐AP, Proteintech Group, Inc), anti‐GAPDH (1:50000, 60004‐1‐Ig,
   Proteintech Group, Inc). The following day, an HRP‐labeled secondary
   antibody at a 1:500 dilution was incubated for 1 h. Color development
   was performed using an ECL kit from Epizyme Biotech. Protein bands were
   subsequently detected utilizing the ImageQuant LAS 500
   chemiluminescence imaging system (Bio‐Rad) and analyzed with ImageJ
   software.

2.18. Quantitative Polymerase Chain Reaction (qPCR)

   Total RNA was extracted from whole cells or tissues utilizing the
   TRIzol kit (Vazyme). Complementary DNA (cDNA) synthesis was performed
   using the reverse transcription kit (Vazyme), and amplification was
   conducted with the SYBR Green qPCR Master Mix (Vazyme). The target
   genes analyzed included GR, Lama5, and Myh7, among others. Relative
   gene expression levels were determined using the ΔΔCt method, with
   Gapdh serving as the reference gene. The primer sequences used in this
   study were as follows: rat Myh7 (forward: 5′‐ATGGACCTGGAGAACGACA‐3′;
   reverse: 5′‐ GCGTGCCTGAAGCTCTTT‐3′), rat Lama5 (forward:
   5′‐CACAGCAACCACACCACA‐3′; reverse: 5′‐ TCAGACATCCTCGGTAGGC‐3′), rat
   Gapdh (forward: 5′‐ TCTCTGCTCCTCCCTGTTC‐3′; reverse: 5′‐
   ACACCGACCTTCACCATCT‐3′).

2.19. Transcriptome Sequencing of Cardiac Tissue and H9C2 Cell

   The transcriptome sequencing experiment described in this study was
   performed by Shanghai OE Biotechnology Co., Ltd. Tissue sequencing
   included the left ventricular wall tissues of the control group (n = 5)
   and the CMS group (n = 10) in week 21, weighing ≈200mg. Cell sequencing
   was divided into control group, CORT group, and CORT+R group, with 5
   compound pores in each group, and the number of cells in each pore was
   greater than 1×10^6. The following is a summary of the procedural
   steps: Initially, total RNA was extracted from the samples.
   Subsequently, ribosomal RNA was removed utilizing a ribo‐zero kit. The
   RNA was then fragmented into short segments through the application of
   a fragmentation reagent. These RNA fragments served as templates for
   the synthesis of the first strand of complementary DNA (cDNA) using
   six‐nucleotide random primers. Subsequently, a two‐strand complementary
   DNA (cDNA) synthesis reaction system was established to synthesize the
   second strand of cDNA. The purified cDNA strand underwent a series of
   preparatory steps, including end repair, addition of adenine (A) tails,
   and ligation of sequencing adapters. Subsequently, the fragments were
   size‐selected, followed by PCR amplification. Following quality
   control, genome comparison, and transcription splicing of the initial
   sequencing data, a quantitative analysis was conducted to identify
   differentially expressed genes. Subsequently, gene function and pathway
   enrichment analyses, including Gene Ontology (GO), Kyoto Encyclopedia
   of Genes and Genomes (KEGG)^[ [59]^13 ^], and Gene Set Enrichment
   Analysis (GSEA), were performed to investigate the impact of chronic
   psychological stress on cardiac gene expression.

2.20. Clinical Information and Plasma Sample Collection

   Plasma samples were collected from healthy controls, depression (DP)
   patients with or without heart failure (HF), and HF patients without DP
   at Shaoxing Second Hospital. The clinical characteristics of the cohort
   are detailed in Table [60]1 . The protocol for the collection of human
   samples was approved by the Ethics Committee of Shaoxing Second
   Hospital (Ethics batch number: 2024052), and all participants provided
   written informed consent. Furthermore, ELISA kits (Elabsicence, China)
   were utilized to measure plasma cortisol and LAMA5 levels, following
   the manufacturer's protocols.

Table 1.

   Clinical information in our cohort.
   Clinical variables Control [n = 54] DP without HF [n = 51] DP with HF
   [n = 34] HF without DP [n = 62]
   Age (year) 60.60±8.25 63.12±8.12 65.97±8.69 65.19±6.35
   Gender (male) 24 (44.44%) 22 (43.14%) 14 (41.18%) 29 (46.77%)
   CHOL (mmol L^−1) 4.20±0.81 4.18±1.10 3.85±1.36 4.40±1.01
   LDL (mmol L^−1) 2.85±0.80 2.64±0.69 3.09±0.65 3.22±0.90
   Glu (mmol L^−1) 4.95±0.63 5.14±0.89 5.52±1.21 5.72±1.49
   BNP (pg mL^−1) 47.96±36.95 44.82±27.88 164.1±113.1 373.7±217.8
   cTnT (ng mL^−1) 0.003±0.003 0.004±0.003 0.006±0.009 0.006±0.007
   IVSd (mm) 9.36±1.17 9.26±1.10 10.57±1.42 10.52±1.49
   LVPWd (mm) 9.07±1.29 9.11±0.96 10.25±1.32 10.41±1.35
   LVEF (%) 67.51±6.95 68.20±8.63 53.13±11.32 49.20±9.89
   FS (%) 39.91±5.56 40.52±4.69 36.15±4.24 30.30±6.47
   [61]Open in a new tab

2.21. Screening of Diagnostic Biomarkers of DP Combined with HF

   Clinical parameters encompassing age, sex, history of underlying
   diseases, fasting blood glucose levels, B‐type natriuretic peptide
   (BNP), troponin, and ELISA results for cortisol and LAMA5 were
   collected. Least Absolute Shrinkage and Selection Operator (LASSO)
   regression analysis was employed to identify diagnostic markers for HF
   in patients with depression. This analysis ultimately identified two
   significant indicators: BNP and LAMA5.

2.22. Diagnostic Model for HF

   The construction of the nomogram was achieved based on the two
   biomarkers through the application of the “rms” package in R software.
   The assessment of diagnostic performance for HF entailed the generation
   of a receiver operation characteristic (ROC) curve to examine the
   nomogram. In addition, calibration curves and a decision curve analysis
   (DCA) enabled the evaluation of the predictive efficiency of the
   nomogram in HF.

2.23. Statistical Analysis

   Statistical analyses were conducted utilizing GraphPad Prism 9.5
   software. Measurement data were expressed as mean ± standard deviation,
   while categorical data were presented as counts (percentages). To
   assess the significance between groups, student's t‐test (two‐tailed)
   for comparisons between two groups, and one‐way analysis of variance
   (ANOVA) followed by Least Significant Difference (LSD) test (for equal
   variances) or Dunnett's test (for unequal variances) for comparisons
   involving three or more groups. Experiments with two categorical
   independent variables and one dependent variable were analyzed using
   two‐way ANOVA, followed by Bonferroni's multiple comparison tests. The
   numbers of sample sizes for each experiment were indicated in the
   figure legend. P‐values < 0.05 were considered significant, and
   significance for all figures was determined as ^* p < 0.05, ^** p <
   0.01, ^*** p < 0.001, and ^**** p < 0.0001.

2.24. Ethics Approval Statement

   All experiments involving humans were carried out in accordance with
   the Declaration of Helsinki. All procedures were performed in
   compliance with relevant laws and institutional guidelines. Ethics
   approval for the use of human plasma samples was provided by the Ethics
   Committee of Shaoxing Second Hospital (Ethics batch number: 2024052).
   Written informed consent was obtained from all patients before sample
   collection. And all animal experiments were approved by the
   Experimental Animal Ethics Committee of Ningbo University (batch
   number: 12654).

3. Results

3.1. CMS Induces Cardiomyocyte Hypertrophy in Rats

   We conducted five batches of CMS modeling lasting 21 weeks to observe
   the pathological processes in the heart due to chronic psychological
   stress. First, we assessed the behavioral changes in the CMS group of
   rats to confirm the level of stress on the mental aspect. Rats in the
   CMS group showed a significant decrease in body weight compared to the
   control group after the onset of stress (Figure [62]1A); the sucrose
   preference test showed a significant decrease in sucrose intake in the
   CMS group at week 9 and continued until the end of the experiment
   (Figure [63]1B), indicating anhedonia in the CMS rats; the open field
   test showed that the distance covered by the CMS group rats in 5 min
   was higher in the early stage than the control group, but lower in the
   later stage (Figure [64]1C), suggesting anxiety/depression in the CMS
   group; the forced swimming test showed a significantly longer
   immobility time in the CMS group rats within 6 min compared to the
   control group (Figure [65]1D), indicating a sense of hopelessness in
   the CMS rats.

Figure 1.

   Figure 1
   [66]Open in a new tab

   CMS induces cardiomyocyte hypertrophy in rats. A) Weight changes of
   control and CMS rats at 21 weeks (n=8‐40). B) Line graph showing
   sucrose intake ratio in control and CMS rats in sucrose preference test
   (n=5‐10). C) Line graph showing distance moved in the open field test
   for control and CMS rats in 5 min (n=6‐10). D) Line graph showing
   immobility time in the forced swimming test for control and CMS rats in
   4 min (n=5‐8). E) Gross images of the heart at week 9 and 21 in control
   and CMS rats. F) Line graph showing heart weight‐to‐body weight ratio
   in 5 batches of control and CMS rats (n=5‐10). G) Representative images
   of WGA staining in the heart at weeks 9 and 21 in control and CMS rats.
   H) Quantitative analysis of WGA‐labeled cardiomyocyte area in 5 batches
   of control and CMS rats (n=4‐5). I) Expression of MYH7 protein in
   control and CMS rats at weeks 13 and 21. J) Staining for Masson's
   trichrome in control and CMS rats at weeks 13 and 21. K) Proportion of
   collagen fibers in 5 batches of control and CMS rats (n=4‐6). L)
   Expression of COL1A2 protein in control and CMS rats at weeks 13 and
   21. M) Representative echocardiographic images of weeks 13 and 21 in
   control and CMS rats. N) Trend of LVEF changes in 5 batches of control
   and CMS rats (n=4‐10). O) Trend of FS changes in 5 batches of control
   and CMS rats (n=4‐10). CMS, chronic mild stress; WGA, wheat germ
   agglutinin; MYH7, myosin heavy chain 7; COL1A2, collagen type I alpha 2
   chain; LVEF, left ventricular ejection fraction; FS, fractional
   shortening.

   Next, we evaluated the pathological changes in the heart from various
   aspects. Figure [67]1E,F show that the heart weight‐to‐body weight
   ratio in the CMS group rats started to increase significantly from week
   9 compared to the control group. WGA staining revealed a significant
   increase in the surface area of cardiac muscle cells in the CMS group
   at 9 weeks and continued until the end of the experiment
   (Figure [68]1G,H). WB results showed a significant upregulation of the
   hypertrophic marker MYH7 in the heart tissue of the CMS group at week 9
   and continued until the end of the experiment (Figure [69]1I; Figure
   [70]S1A–J, Supporting Information). Masson's staining revealed a
   significant increase in fibrous tissue in the cardiac muscle tissue of
   the CMS group at week 13 and continued until the end of the experiment
   (Figure [71]1J,K). WB results showed a significant upregulation of the
   fibrotic marker COL1A2 in the heart tissue of the CMS group at week 13
   and continued until the end of the experiment (Figure [72]1L; Figure
   [73]S2A–J, Supporting Information). Echocardiography results showed a
   progressive decrease in LVEF and FS in the hearts of the CMS group
   rats, with a significant difference from week 13 (Figure [74]1M–O).
   However, there were no significant differences in diastolic
   interventricular septum thickness and left ventricular posterior wall
   thickness between the two groups throughout the study (Figure
   [75]S3A,B, Supporting Information). To further clarify the changes in
   ventricular thickness, we performed HE staining of heart sections
   (Figure [76]S3C–E, Supporting Information), which showed no
   statistically significant difference in ventricular wall thickness,
   although there was a trend of thickening in IVSd and LVPWd in the CMS
   group. We also observed changes in rat blood pressure and serum
   NTproBNP concentration. Blood pressure in the CMS group rats showed no
   significant difference compared to the control group in the first 4
   batches, only to show a statistically significant increase in week 21
   (Figure [77]S4A, Supporting Information), while serum NTproBNP showed
   no significant difference between the two groups (Figure [78]S4B,
   Supporting Information). Additionally, we measured the circulating
   concentrations of five stress‐related hormones, including CORT,
   adrenaline, serotonin, angiotensin II, and aldosterone. The results
   showed that CORT was consistently upregulated in CMS rats throughout
   the study (Figure [79]2A), adrenaline significantly increased in the
   early stress period (week 5) and gradually decreased to normal levels,
   indicating a strong adaptability of the sympathetic nervous system in
   chronic psychological stress (Figure [80]S4C, Supporting Information).
   The circulating levels of serotonin showed no significant differences
   in the five batches, while angiotensin II and aldosterone showed a
   trend of increase in the later stages of the study, but without
   statistical significance (Figure [81]S4D–F, Supporting Information).

Figure 2.

   Figure 2
   [82]Open in a new tab

   CORT may play an important role in CMS‐induced cardiomyocyte
   hypertrophy. A) Trend of serum CORT levels in 5 batches of control and
   CMS rats (n=4–8). B) PCA plot showing gene expression similarity
   between the control and CMS groups. C) Volcano plot displaying
   differential gene expression between control and CMS groups. D) Venn
   diagram showing the top 10 enriched GO terms from differential gene
   analysis. E) Bubble plot showing the top 20 KEGG‐enriched pathways from
   differential gene analysis. F) Relative mRNA expression of Myh7 (n=3)
   in H9C2 cells stimulated with different concentrations of CORT for 48
   h. G, H) Representative Western blot image (G) and quantification
   analysis (H) of MYH7 protein expression (n=3) in H9C2 cells stimulated
   with different concentrations of CORT for 48 h. I) Representative
   images of cell morphology (top) and WGA staining (bottom) in H9C2 cells
   stimulated with 1 µm CORT for 48 h. J) Quantitative analysis of cell
   surface area in WGA staining between control and CORT groups (n = 3).
   CMS, chronic mild stress; PCA, principal component analysis; GO, Gene
   Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MYH7, myosin
   heavy chain 7; CORT, corticosterone; WGA, wheat germ agglutinin.

   These results suggest that cardiomyocyte hypertrophy is an early and
   significant pathological phenotype in CMS rats. Therefore, we attempted
   to study the mechanisms and interventions for cardiomyocyte hypertrophy
   in CMS rats.

3.2. CORT Plays an Important Role in CMS Induced Cardiomyocyte Hypertrophy

   In order to explore the pathological mechanism of cardiomyocyte
   hypertrophy in CMS rats, we extracted heart tissue for whole
   transcriptome sequencing. In the mRNA sequencing results, quality
   control analysis showed a high consistency in mRNA transcript abundance
   among the samples overall (Figure [83]S5A,B, Supporting Information).
   2D (Figure [84]2B) and 3D (Figure [85]S5C, Supporting Information) PCA
   analysis revealed significant distribution differences between the CMS
   group and the control group. Differential gene analysis showed that in
   the hearts of CMS rats, 464 genes were significantly upregulated and
   635 genes were significantly downregulated (Figure [86]S5D, Supporting
   Information). The overall situation of differential gene expression and
   the TOP 30 are respectively shown in volcano plots (Figure [87]2C) and
   heat maps (Figure [88]S5E, Supporting Information). GO enrichment
   analysis of differential genes (Figure [89]2D) showed that among the
   top 10 entries, the most significant biological process was “response
   to glucocorticoid”. GSEA also found that the “response to
   glucocorticoid” pathway was significantly activated in the CMS group
   (Figure [90]S5F, Supporting Information). KEGG analysis (Figure [91]2E)
   revealed that differential genes were enriched in hypertrophy‐related
   signaling pathways such as “PI3K/AKT signaling pathway”, and a previous
   study has shown that the continuous activation of AKT is the main
   factor of volume‐loaded myocardial hypertrophy.^[ [92]^14 ^]

   In miRNA sequencing, the expression abundance of miRNAs showed high
   consistency (Figure [93]S6A,B, Supporting Information), and PCA plots
   indicated minimal differences between groups (Figure [94]S6C,
   Supporting Information). Differential expression analysis revealed
   significant differences in the expression levels of 15 miRNAs between
   groups (Figure [95]S6D, Supporting Information). Prediction of target
   genes for these 15 differential miRNAs and functional enrichment
   analysis of the related target genes showed in KEGG secondary
   classification (Figure [96]S7A, Supporting Information) that the top
   three organ systems enriched with target genes were “nervous system,”
   “endocrine system,” and “circulatory system”. KEGG pathway analysis
   further demonstrated (Figure [97]S7B, Supporting Information) that the
   target genes of differential miRNAs were involved in various
   cardiomyopathy, such as “dilated cardiomyopathy” and “hypertrophic
   cardiomyopathy”. Additionally, KEGG analysis again highlighted
   pathological manifestations related to high glucocorticoid expression,
   such as “Cushing's syndrome” and “synthesis and secretion of cortisol”.
   In previous studies, it was found that glucocorticoids such as
   dexamethasone induce cardiomyocyte hypertrophy^[ [98]^15 ^]; therefore,
   we believe that in CMS rats, sustained excessive secretion of CORT may
   be a promoting factor for cardiomyocyte hypertrophy.

   To validate whether CORT has a pro‐hypertrophic effect on
   cardiomyocytes, experiments were conducted in the rat cardiomyocyte
   cell line H9C2. Treatment with CORT at concentrations ranging from
   100nm to 100µm for 48 h, qPCR results showed (Figure [99]2F) that
   concentrations of 1–50µm CORT significantly increased the mRNA
   expression of the hypertrophic marker Myh7 in H9C2 cells. Western blot
   results indicated (Figure [100]2G) that stimulation with 1 and 10µm
   CORT significantly increased the expression of MYH7 protein in H9C2
   cells. Under the microscope (Figure [101]2I), it was evident that
   treatment with 1µm CORT induced significant morphological changes in
   H9C2 cells, as shown by WGA staining analysis (Figure [102]2J). 48 h of
   treatment with 1µm CORT resulted in a ≈40% increase in the cell surface
   area of H9C2 cells.

3.3. CORT Induces Hypertrophy Through Targeting GR in H9C2 Cells

   CORT is a primary glucocorticoid secreted by the adrenal glands of
   rodents. It can exert effects by binding to both GR and
   mineralocorticoid receptors (MR), as well as unclear non‐genomic
   effects.^[ [103]^16 ^] Previously used GR antagonists and MR
   antagonists mainly include mifepristone and spironolactone, where
   mifepristone acts as an antagonist to GR and progesterone receptors,
   and spironolactone acts as an antagonist to MR and androgen receptors,
   both of which have strong side effects. Therefore, in this study,
   intervention was done using the novel selective GR antagonist,
   relacorilant (abbreviated to “R” in group naming), and the selective MR
   antagonist, finerenone (abbreviated to “F” in group naming). In vitro
   experiments in which drug interventions were performed, the following
   groups were included: control group (drug solvent treatment), CORT
   group (CORT 1µm), CORT+R group (CORT 1µm and relacorilant 1µm), CORT+F
   group (CORT 1µm and finerenone 10µm), CORT+R+F group (CORT 1µm,
   relacorilant 1µm, and finerenone 10µm), and R group (relacorilant 1µm).
   qPCR and WB results (Figure [104]3A–C) showed that relacorilant
   significantly reduced the mRNA and protein expression of MYH7 induced
   by CORT, while finerenone could not effectively reverse the
   hypertrophic effect of CORT. Under the microscope (Figure [105]S8A,
   Supporting Information), it was observed that relacorilant could
   significantly inhibit the morphological changes induced by CORT in H9C2
   cells. The results of WGA staining (Figure [106]3D,E) suggested that
   relacorilant could antagonize the increase of H9C2 cell surface area
   induced by CORT, while finerenone had little effect. Since the
   activation of GR requires translocation into the nucleus to exert
   transcription factor effects, this process is termed “nuclear
   translocation.” Nuclear and cytoplasmic proteins were extracted for
   detection, and the results showed that after the addition of CORT, GR
   significantly increased in the cell nucleus (Figure [107]3F,G),
   significantly decreased in the cytoplasm (Figure [108]3H,I), and
   slightly decreased in total protein (Figure [109]S8B,C, Supporting
   Information). This suggests that CORT promotes nuclear translocation
   and depletion of GR, while relacorilant may block this process. Cell
   immunofluorescence results (Figure [110]3J) showed that after the
   addition of CORT, GR concentrated internally in the cell nucleus, and
   relacorilant could effectively reverse the nuclear translocation caused
   by CORT. Additionally, two siRNAs with strong knockdown effects on GR
   were screened by qPCR (Figure [111]S9A, Supporting Information).
   Quantification of cardiomyocyte cross‐sectional area by WGA staining
   (Figure [112]S9B,C, Supporting Information) indicates that GR knockdown
   significantly alleviates CORT‐induced hypertrophy. qPCR and WB results
   (Figure [113]3K–M) showed that knockdown of GR significantly alleviated
   the upregulation of MYH7 expression induced by CORT.

Figure 3.

   Figure 3
   [114]Open in a new tab

   CORT induces H9C2 cell hypertrophy through GR. A) Relative mRNA
   expression levels of Myh7 (n=3) in different groups of H9C2 cells. B,
   C) Representative Western blot image (B) and quantification analysis
   (C) of MYH7 protein expression (n=3) in different groups of H9C2 cells.
   D, E) Representative images of WGA staining (D) and quantification
   analysis (E) in different groups of H9C2 cells. F, G) Representative
   Western blot image (F) and quantification analysis (G) of nuclear GR
   protein expression (n=3) in different groups of H9C2 cells. H, I)
   Representative Western blot image (H) and quantification analysis (I)
   of cytoplasmic GR protein expression (n=3) in different groups of H9C2
   cells. J) Representative images of GR immunofluorescence staining and
   DAPI staining in different groups of H9C2 cells. K) Relative mRNA
   expression levels of Myh7 (n=3) in different groups of H9C2 cells. L,
   M) Representative Western blot image (L) and quantification analysis
   (M) of MYH7 protein expression (n=3) in different groups of H9C2 cells.
   GR, glucocorticoid receptor; MYH7, myosin heavy chain 7; CMS, chronic
   mild stress; PCA, principal component analysis; GO, Gene Ontology;
   KEGG, Kyoto Encyclopedia of Genes and Genomes; WGA, wheat germ
   agglutinin; CORT, corticosterone; R, relacorilant; F, finerenone.

   Transcriptional abundance (Figure [115]S10A, Supporting Information),
   PCA analysis (Figure [116]S10B, Supporting Information), and cluster
   analysis (Figure [117]S10C, Supporting Information) by H9C2 cells mRNA
   sequencing demonstrated intra‐group sample consistency and inter‐group
   variability, suggesting reproducibility of the intervention.
   Differential expression analysis (Figure [118]S10D, Supporting
   Information) showed that compared to the control group, there were 455
   upregulated genes and 187 downregulated genes in the CORT group; and
   compared to the CORT group, there were 182 upregulated genes and 431
   downregulated genes in the CORT+R group, with the overall distribution
   of genes shown in volcano plots (Figure [119]S10E,F, Supporting
   Information). GO analysis of the CORT versus control group (Figure
   [120]S11A, Supporting Information) indicated that the enriched main BP
   items for differential genes included “dexamethasone cell response”,
   “integrin‐mediated signaling pathway”, and “extracellular matrix
   remodeling”, while CC items mainly included “extracellular matrix” and
   “laminin5 complex”, suggesting a strong effect of CORT intervention on
   extracellular matrix remodeling in cardiomyocytes. KEGG analysis
   (Figure [121]S11B, Supporting Information) showed that differential
   genes were highly enriched in heart diseases such as “hypertrophic
   cardiomyopathy”, “dilated cardiomyopathy”, as well as pathways like
   “ECM‐receptor interaction” and “PI3K‐AKT signaling pathway”. Enrichment
   analysis of the CORT+R group compared to the CORT group showed results
   similar to the CORT versus control group findings (Figure [122]S11C,D,
   Supporting Information). GSEA further demonstrated that pathways
   related to cardiomyocyte hypertrophy, such as “hypertrophic
   cardiomyopathy”, “dilated cardiomyopathy”, and “PI3K‐AKT signaling
   pathway”, were activated in the CORT group (Figure [123]S11E,F,
   Supporting Information).

3.4. GR Promotes the Expression of Its Direct Target Gene (Lama5)

   As a transcription factor, GR participates in the transcriptional
   activation or inhibition of multiple downstream genes, and there is
   currently no clear association between known GR target genes and
   cardiomyocyte hypertrophy. Therefore, we attempted to identify new
   potential target genes involved in GR‐induced cardiomyocyte
   hypertrophy. We predicted GR target genes using three transcription
   factor databases, including database of human transcription factor
   targets (hTFtarget)^[ [124]^17 ^], gene transcription regulation
   database (GTRD)^[ [125]^18 ^] and cistrome data browser (CDB)^[
   [126]^19 ^], intersecting them with differentially expressed genes from
   rat heart tissue transcriptome, resulting in 93 differentially
   expressed potential GR target genes (Figure [127]4A). Analysis of
   protein‐protein interactions (PPI) was performed on the 93 potential
   target genes, and subsequently, utilizing the cytohuba algorithm to
   obtain the top 10 hub genes (Figure [128]4B). By intersecting the 10
   hub genes obtained from the animal sequencing results and the GR
   predicted target gene set analysis with the differential gene set of
   cell sequencing, we identified the top 2 genes showing the most
   significant fold change in expression at the cellular level (Lama5 and
   Txnip). LAMA5 is one of the non‐collagen components of the
   extracellular matrix^[ [129]^20 ^], and until recent years, there has
   been no related research in the field of heart disease. In 2023, a
   study on heart‐like organs discovered that LAMA5 is an important factor
   for the development and maturation of cardiac muscle cells, and its
   deficiency can lead to developmental abnormalities in fetal hearts.^[
   [130]^21 ^] Therefore, we are highly interested in investigating
   whether Lama5 is a direct target gene of GR and its role in
   cardiomyocytes. The results of qPCR and WB (Figure [131]4D–F) revealed
   that CORT intervention significantly increased the levels of Lama5 mRNA
   and protein expression, and this change could be reversed by using the
   GR antagonist, relacorilant. Furthermore, results from ChIP‐Seq
   indicated a high binding rate between GR and the gene sequence where
   Lama5 is located (Figure [132]4G). Results from a dual luciferase
   reporter gene assay (Figure [133]4H) demonstrated that the artificially
   constructed promoter sequence of Lama5 has the ability to initiate
   downstream luciferase expression, and increasing GR levels can enhance
   the transcriptional activity of the Lama5 promoter, significantly
   increasing luciferase expression.

Figure 4.

   Figure 4
   [134]Open in a new tab

   Lama5 is a direct target gene of GR. A) Venn diagram showing 93
   intersection genes between differentially expressed genes in rat heart
   tissue and GR target genes predicted by hTF, GTRD, and CDB databases.
   B) PPI analysis of the intersection genes, showing the common genes
   (marked in blue) and the top 10 hub genes (not marked in blue). C) Venn
   diagram showing the intersection of genes (Lama5 and Txnip) between hub
   genes and differentially expressed genes in H9C2 cells. D–F) Relative
   mRNA expression levels (D), representative Western blot image (E), and
   quantification analysis (F) of LAMA5 protein in H9C2 cells (n=3). G)
   ChIP‐Seq analysis in muscle cells treated with CORT from the CDB
   database, showing the binding regions of GR with Lama5 DNA. H)
   Dual‐luciferase reporter gene assay showing the relative luciferase
   intensity among different treatment groups (n=3). LAMA5, laminin
   subunit alpha 5; GR, glucocorticoid receptor; PPI, protein‐protein
   interaction; TXNIP, thioredoxin interacting protein; CORT,
   corticosterone; R, relacorilant; ChIP‐Seq, chromatin
   immunoprecipitation‐sequencing.

3.5. CORT‐GR Partially Induces Cardiomyocyte Hypertrophy Through LAMA5

   Four siRNAs targeting rat Lama5 were designed, with qPCR results
   (Figure [135]S12A, Supporting Information) indicating that siRNA5 and
   siRNA6 achieved higher knockdown efficiency. Quantification of
   cardiomyocyte cross‐sectional area by WGA staining (Figure [136]S12B,C,
   Supporting Information) indicates that Lama5 knockdown significantly
   alleviates CORT‐induced hypertrophy. qPCR results showed (Figure
   [137]5A) that siRNA intervention for Lama5 significantly reduced the
   mRNA expression levels of Lama5 and inhibited the upregulation of Myh7
   induced by CORT. At the protein level, the addition of the two siRNAs
   only slightly decreased the baseline level of LAMA5, but significantly
   reduced the further upregulation of LAMA5 induced by CORT and inhibited
   the upregulation of MYH7 protein expression induced by CORT
   (Figure [138]5B–D). Furthermore, we established a cell line with
   overexpression of LAMA5, where, under the microscope, some cells
   exhibited noticeable morphological signs of cellular hypertrophy
   (Figure [139]5E), and WGA staining revealed an average increase in cell
   surface area of 1.3 times compared to the control group
   (Figure [140]5F). qPCR results showed (Figure [141]5G) that in the
   stable Lama5 transfectants, the mRNA level of Lama5 was four times
   higher than that of the control group, and the expression level of Myh7
   increased by ≈80%. WB results indicated (Figure [142]5H,I) that in the
   stable Lama5 transfectants, the protein level of LAMA5 increased by
   80%, and the MYH7 protein expression level increased by around 20%.

Figure 5.

   Figure 5
   [143]Open in a new tab

   CORT/GR can induce cardiomyocyte hypertrophy through LAMA5. A) Relative
   mRNA expression levels of Lama5 and Myh7 in different treatment groups
   of H9C2 cells (n=3‐4). B) Representative Western blot images of LAMA5,
   MYH7, and GR in different treatment groups of H9C2 cells. C, D)
   Quantitative analysis of LAMA5 (C) and MYH7 (D) protein expression in
   different treatment groups of H9C2 cells (n=3). E) Representative cell
   morphology (top) and WGA staining images (bottom) of
   LAMA5‐overexpressing stable cells and NC group H9C2 cells. F)
   Quantitative analysis of WGA staining in Lama5‐overexpressing stable
   cells and NC group H9C2 cells (n=3). G–I) Relative mRNA expression
   levels of Lama5 and Myh7 (G), representative protein Western blot
   images (H), and quantitative analysis (I) in Lama5‐overexpressing
   stable cells and NC group H9C2 cells (n=3‐4). CORT, corticosterone; GR,
   glucocorticoid receptor; LAMA5, laminin subunit alpha 5; MYH7, myosin
   heavy chain 7; WGA, wheat germ agglutinin; NC, negative control.

3.6. Inhibition of LAMA5 Rescues Cardiomyocyte Hypertrophy In Vivo

   To further verify whether the CORT‐GR‐LAMA5 axis could mediate
   cardiomyocyte hypertrophy induced by CMS, we used metyrapone to inhibit
   CORT synthesis and relacorilant to antagonize the GR receptor in the
   CMS animal model. Additionally, we constructed an AAV carrying the cTnT
   gene to achieve targeted knockdown of Lama5 gene expression in rats'
   hearts. qPCR and WB results from rat hearts indicated (Figure
   [144]S13A–C, Supporting Information) that the total mRNA level of Lama5
   in the hearts infected with Lama5 knockdown (LAMA5KD) adeno‐associated
   virus was ≈50% of that in the control group, and the protein level was
   around 75% of that in the control group. The experimental groups
   included a control group, CMS group, CMS+metyrapone group,
   CMS+relacorilant group, and CMS+LAMA5KD group. The results showed
   (Figure [145]6A) that the body weights of rats in the four stress
   groups decreased to varying degrees, with the CMS group and CMS+LAMA5KD
   group showing the greatest decrease. The sucrose preference test
   results (Figure [146]6B) showed a decreased sucrose intake ratio in the
   four stress groups compared to the control group, with statistically
   significant differences. In the open field test (Figure [147]6C), the
   average distance moved by rats in the CMS/CMS+relacorilant/CMS+LAMA5KD
   groups over 5 min was significantly shorter than that of the control
   group, with statistically significant differences. The heart
   weight/body weight ratio was increased in the CMS group compared to the
   control group, and interventions with metyrapone/relacorilant and
   LAMA5KD mitigated this change (Figure [148]6E). Echocardiography
   results showed that the LVEF and FS of rats in the CMS group were
   significantly decreased compared to the control group, and
   metyrapone/relacorilant/LAMA5KD could alleviate the cardiac dysfunction
   caused by CMS (Figures [149]6D–G). WGA staining results showed that the
   surface area of cardiomyocytes in the hearts of rats in the CMS group
   was significantly increased compared to the control group, and
   metyrapone/relacorilant/LAMA5KD could reverse the cardiomyocyte
   enlargement induced by CMS (Figures [150]6H,I). WB results showed that
   the expression levels of LAMA5 and MYH7 were significantly increased in
   the hearts of rats in the CMS group, while metyrapone and relacorilant
   could inhibit the high expression of LAMA5 and MYH7 induced by CMS. In
   the CMS+LAMA5KD group, the expression of LAMA5 was decreased, and the
   protein level of MYH7 showed no significant difference compared to the
   control group (Figure [151]6J–L). Masson's trichrome staining revealed
   significant interstitial fibrosis in the hearts of rats in the CMS
   group compared to the control group, with similar fibrotic changes
   observed in the CMS+LAMA5KD group. Interestingly, the CMS+metyrapone
   and CMS+relacorilant groups could reverse the myocardial fibrosis
   induced by CMS (Figure [152]S13D,E, Supporting Information).

Figure 6.

   Figure 6
   [153]Open in a new tab

   Chronic psychological stress induces cardiomyocyte hypertrophy and
   cardiac dysfunction through the CORT‐GR‐LAMA5 axis. A) Trend of body
   weight changes in each group of rats (n=7‐8). B) Sucrose preference
   ratio at the end of the experiment in each group of rats (n=4). C)
   5‐min distance moved in the open field test at the end of the
   experiment in each group of rats (n=5). D) Representative images of
   echocardiography at the end of the experiment in each group of rats.
   E–G) HW/BW (E), LVEF (F), and FS (G) at the end of the experiment in
   each group of rats (n=4‐8). H, I) Representative images (H) and
   quantitative analysis (I) of WGA staining in cardiac myocytes of each
   group of rats (n=4). J) Representative Western blot images of LAMA5 and
   MYH7 in heart tissue of each group of rats. K, L) Quantitative analysis
   of LAMA5 (K) and MYH7 (L) protein expression in heart tissue of each
   group of rats (n=3). GR, glucocorticoid receptor; LAMA5, laminin
   subunit alpha 5; MYH7, myosin heavy chain 7; HW/BW, heart weight/body
   weight; LVEF, left ventricular ejection fraction; FS, fractional
   shortening; WGA, wheat germ agglutinin.

3.7. Correlation of Serum CORT and LAMA5 with Cardiac Function in Wistar Rats

   Given the hypertrophic effect of CORT and LAMA5 in CMS rats, we
   measured the serum concentrations of CORT and LAMA5 in the fourth and
   fifth batches of modeled rats to assess their biomarker value. As a
   comparison, we also measured the serum concentration of NTproBNP. The
   results showed that the serum levels of CORT and LAMA5 in the CMS group
   rats were significantly elevated compared to the control group, while
   the difference in NTproBNP levels was not statistically significant
   (Figure [154]7A–C). The serum levels of CORT and LAMA5 were
   significantly negatively correlated with LVEF, with correlation
   coefficients of −0.469 and −0.458, respectively, while NTproBNP showed
   no significant correlation with LVEF (Figure [155]7D–F). ROC analysis
   revealed that the serum levels of CORT and LAMA5 had AUC values of
   0.803 and 0.818, respectively, for diagnosing decreased LVEF, whereas
   NTproBNP had an area under the curve (AUC) of 0.606 for diagnosing
   decreased LVEF (Figure [156]7G–I).

Figure 7.

   Figure 7
   [157]Open in a new tab

   Correlation between serum CORT and LAMA5 levels with cardiac function
   in Wistar rats. A–C) Concentration analysis of serum CORT (A), LAMA5
   (B), and NTproBNP (C) in control and CMS rats (n=8‐15). D–F) Scatter
   plots showing the correlation between serum CORT (D), LAMA5 (E), and
   NTproBNP (F) levels with LVEF in rats (n=23). G–I) ROC analysis of
   serum CORT (G), LAMA5 (H), and NTproBNP (I) levels for the diagnosis of
   decreased LVEF in rats. CORT, corticosterone; LAMA5, laminin subunit
   alpha 5; NTproBNP, N‐terminal pro‐B‐type natriuretic peptide; CMS,
   chronic mild stress; LVEF, left ventricular ejection fraction; ROC,
   receiver operation characteristic.

3.8. LAMA5 is a Novel Diagnostic Marker for Myocardial Hypertrophy in
Patients with Depression

   In order to explore the biomarker value of glucocorticoids and LAMA5 in
   chronic psychological stress‐induced changes in cardiac structure and
   function, we recorded clinical data from DP without HF, DP with HF, HF
   without DP, and healthy control groups, and collected plasma samples
   for the determination of cortisol and LAMA5 concentrations. The
   baseline data of each group are shown in Table [158]1, with no
   significant differences in non‐disease‐specific indicators such as age
   and gender. As shown in Figure [159]8A, compared to the other groups,
   LAMA5 was significantly elevated in the DP with HF group, moderately
   elevated in the DP without HF group, while the levels of LAMA5 in the
   HF without DP group did not show significant differences compared to
   the control group. Cortisol concentration was higher in the DP with HF
   group and the DP without HF group compared to the control group (Figure
   [160]S14A, Supporting Information). BNP levels were slightly elevated
   in the DP with HF group and significantly elevated in the HF without DP
   group (Figure [161]8B). To further explore the diagnostic value of
   cortisol and LAMA5 for cardiac function in patients with depression, we
   conducted ROC and correlation analyses in patients of the control
   group, DP with HF group, and DP without HF group. The diagnostic ROC
   analysis for HF showed AUC values of 0.799 for LAMA5 and 0.646 for
   cortisol in diagnosing DP with HF (Figure [162]S14B, Supporting
   Information), while the diagnostic value for BNP was high at 0.824
   (Figure [163]8C). LAMA5 had a significant positive correlation with
   IVSd and LVPWd and a significant negative correlation with LVEF and FS
   (Figure [164]8D–G). Cortisol showed a significant negative correlation
   with LVEF, but no statistical significance in correlation with IVSd,
   LVPWd, and FS (Figure [165]S14C–F, Supporting Information). To explore
   the diagnostic model for DP with HF, we included multiple factors such
   as age, gender, underlying diseases, lipid levels, and blood sugar
   levels for LASSO regression to select the main variables (Figure
   [166]S14G, Supporting Information). The optimal diagnostic indicators
   were identified to be two, namely LAMA5 and BNP. The diagnostic
   nomogram is displayed in Figure [167]8H, calibration curves indicated
   relative reliability of the model (Figure [168]8I), DCA analysis showed
   positive clinical decision benefits for patients (Figure [169]8J), and
   the ROC analysis demonstrated a diagnostic AUC of 0.913 for the model
   (Figure [170]8K).

Figure 8.

   Figure 8
   [171]Open in a new tab

   Correlation and diagnostic value of plasma LAMA5 levels with cardiac
   function in patients with DP. A, B) Plasma levels of LAMA5 (A) and BNP
   (B) in patients in each group (n=34‐62). C) ROC analysis of BNP and
   LAMA5 for diagnosing DP with HF. D–G) Scatter plots showing the
   correlation between plasma LAMA5 levels in patients with IVSd (D),
   LVPWd (E), LVEF (F), and FS (G) (n=139). H) Forest plot of BNP and
   LAMA5 for diagnosing DP with HF. I–K) Calibration curve of diagnostic
   model (I), DCA analysis (J), and ROC analysis (K). LAMA5, laminin
   subunit alpha 5; DP, depression; BNP, B‐type natriuretic peptide; ROC,
   receiver operation characteristic; HF, heart failure; IVSd, diastolic
   interventricular septal; LVPWd, diastolic left ventricular posterior
   wall; LVEF, left ventricle ejection fraction; FS, fractional
   shortening; DCA, decision curve analysis.

3.9. The CORT‐GR‐LAMA5 Axis may Induce Cardiomyocyte Hypertrophy Regulating
the ITGB1‐PI3K‐AKT Pathway

   LAMA5 is an important component of the extracellular matrix and can
   bind to various integrin receptors to initiate downstream pathways and
   exert physiological effects. In cell sequencing results, GO analysis
   enriched multiple integrin‐related pathways such as “integrin‐mediated
   signaling” and “integrin binding,” while KEGG analysis enriched
   pathways including “ECM‐receptor interaction” and “PI3K/AKT.” Numerous
   studies have indicated the significant role of the integrin‐PI3K‐AKT
   pathway in cardiomyocyte hypertrophy. Therefore, we hypothesize that
   CORT‐GR‐LAMA5‐induced cardiomyocyte hypertrophy may be related to the
   ITGB1‐PI3K‐AKT pathway. We examined the activation status of PI3K/AKT
   in the animal model and found that in the CMS group, there was an
   upregulation of ITGB1/GAPDH, pPI3K/PI3K, and pAKT/AKT compared to the
   control group, while metyrapone, relacorilant, and knockdown of LAMA5
   partially inhibited the activation of the PI3K/AKT pathway (Figure
   [172]9A–D). Furthermore, we treated the H9C2 cell line with relacorilan
   and the PI3K inhibitor LY294002, and the results are shown in
   Figure [173]9E–H. After CORT intervention, the expression of ITGB1,
   LAMA5, MYH7, pPI3K, and pAKT was upregulated in the cells. Relacorilant
   intervention inhibited the CORT‐induced activation of PI3K/AKT, reduced
   the expression of LAMA5 and ITGB1, and attenuated the excessive
   upregulation of MYH7. However, under CORT combined with LY294002
   intervention, the expression levels of LAMA5 and ITGB1 remained
   elevated compared to the control group, while the activation of the
   PI3K/AKT pathway was inhibited, leading to a downregulation of MYH7,
   indicating that LY294002 could alleviate the CORT‐induced MYH7
   overexpression.

Figure 9.

   Figure 9
   [174]Open in a new tab

   The CORT‐GR‐LAMA5 axis may induce cardiomyocyte hypertrophy through the
   ITGB1‐PI3K‐AKT pathway. A) Representative Western blot images of pPI3K,
   PI3K, pAKT, AKT, and ITGB1 in heart tissue of each group of rats. B–D)
   Relative protein expression levels of pPI3K (B), pAKT (C), and ITGB1
   (D) in heart tissue of each group of rats (n=3). E) Representative
   Western blot images of PI3K/AKT/MYH7/LAMA5 in H9C2 cells of each group.
   F, G) Quantitative analysis of relative protein expression levels of
   pPI3K (F) and pAKT (G) in H9C2 cells of each group (n=3). H)
   Quantitative analysis of protein expression levels of ITGB1, LAMA5, and
   MYH7 in H9C2 cells of each group (n=3). CORT, corticosterone; GR,
   glucocorticoid receptor; LAMA5, laminin subunit alpha 5; ITGB1,
   integrin subunit beta 1; PI3K, phosphatidylinositol 3‐kinase; MYH7,
   myosin heavy chain 7.

4. Discussion

   In today's fast‐paced world, chronic stressors are abundant. According
   to the GBD 2021 report, depressive disorders and anxiety disorders
   ranked 2nd and 6th, respectively, in terms of causes of years lived
   with disability (YLD) across all age groups, with the combined YLD from
   both (98.8 million) exceeding the top‐ranked low back pain (70.2
   million).^[ [175]^22 ^] Therefore, further research is urgently needed
   in the field of anxiety/depression‐related physical and mental
   illnesses to address the increasing disease burden brought by a growing
   number of anxiety/depression patients. In the realm of cardiac
   structural diseases caused by chronic psychological stress, current
   research is relatively scarce, lacking comprehensive and continuous
   observations of disease progression. In this study, through five
   batches of chronic psychological stress lasting ≈5 months, we
   systematically observed the overall size/thickness, microscopic
   pathological manifestations, and functional changes of the hearts of
   CMS rats, and proposed for the first time that cardiomyocyte
   hypertrophy is the earliest cardiac pathological phenotype to appear
   throughout the entire CMS cycle, followed by mild interstitial fibrosis
   and contractile dysfunction. This is consistent with the common
   progression of various cardiomyopathies in clinical practice, where
   cardiomyocyte hypertrophy or myocardial fibrosis often appears earlier
   than the symptoms or signs of manifest cardiac dysfunction.^[ [176]^23
   ^] Additionally, it is noteworthy that when severe depression symptoms
   such as anhedonia were observed in the 9th week, cardiomyocyte
   hypertrophy was also detected, suggesting that cardiomyocyte
   hypertrophy and severe depression might occur simultaneously.
   Therefore, it is important to consider whether cardiomyocyte
   hypertrophy is already present in patients with severe depression, and
   monitoring the cardiomyocyte hypertrophy is particularly crucial for
   the early identification and intervention of the cardiac pathological
   progression in patients with anxiety/ion.

   However, the even more astonishing discovery is that in our rat model,
   cardiomyocyte hypertrophy appears earlier than the increase in blood
   pressure, indicating that the cardiomyocyte hypertrophy in CMS rats is
   not an adaptive change resulting from increased cardiac afterload. This
   phenomenon suggests the necessity to further explore the mechanisms of
   cardiomyocyte hypertrophy in CMS rats. Therefore, we conducted whole
   transcriptome sequencing of heart tissues from model and control rats
   to observe significant changes in molecular and biological processes.
   The enrichment analysis of differentially expressed mRNAs and miRNAs
   revealed the effects of glucocorticoids. Serum hormone testing in the
   rats from 5 batches revealed that NA increased in the early stages of
   stress but quickly recovered, indicating sympathetic adaptability in
   rats. Hormones such as 5HT/aldosterone/angiotensin II did not
   significantly increase in the early stages, while CORT increased
   significantly early in CMS and remained high until the end of the
   experiment. These results suggest that CORT may play an important role
   in cardiomyocyte hypertrophy induced by CMS. Studies have shown that
   the synthetic long‐acting glucocorticoid dexamethasone can induce
   hypertrophy in H9C2 cell lines and primary rat cardiomyocytes.^[
   [177]^15 ^] Unfortunately, previous related studies have used RU486 as
   a GR antagonist in experiments. RU486 not only binds to GR but also to
   progesterone receptors (PR) to exert contraceptive effects, and its
   affinity for PR is 13 times that of GR.^[ [178]^24 ^] Therefore, the
   role of CORT‐GR axis in inducing cardiomyocyte hypertrophy in CMS rats
   remains unclear, and the clinical utility of RU486 is limited due to
   its contraceptive side effects. Thus, in this study, we used
   relacorilant, a novel selective GR antagonist, in experiments.
   Additionally, we added a metyrapone group and found that inhibiting
   CORT synthesis can alleviate CMS‐induced cardiomyocyte hypertrophy,
   confirming the significant role of the CORT‐GR axis in cardiomyocyte
   hypertrophy in CMS rats.

   However, the specific mechanism by which CORT‐GR leads to cardiomyocyte
   hypertrophy is still unclear. Considering that GR is a transcription
   factor that is activated upon binding with CORT, playing a role in
   transcriptional activation or inhibition of downstream target genes by
   translocating into the nucleus.^[ [179]^25 ^] Therefore, we predicted
   GR target genes based on three databases and integrated tissue and cell
   sequencing data to ultimately identify 10 potential candidate target
   genes that play important roles in the heart. Among them, LAMA5 is a
   large (≈400 kD), evolutionarily conserved multi‐domain protein involved
   in forming the trimeric structure of laminin^[ [180]^26 ^], a major
   component of the extracellular matrix and basement membrane. Previous
   studies have shown that defects in LAMA5 lead to a variety of
   developmental abnormalities, including osteogenesis imperfecta,
   pulmonary hypoplasia, and nephrotic syndrome.^[ [181]^27 ^] Recent
   research has found that in cardiac‐like organs, LAMA5 is an important
   factor in promoting the maturation of cardiomyocytes, with a 20%
   decrease in myocardial contractile force following LAMA5 knockdown.
   Genetic knockout of LAMA5 is lethal during embryonic development, and
   mice carrying mutated LAMA5 genes (resulting in laminin polymerization
   defects) have a 20% reduction in the area of the left ventricular
   compact myocardium compared to wild‐type mice, suggesting the essential
   role of LAMA5 in the development and maturation of cardiomyocytes.^[
   [182]^21 ^] Therefore, we have a strong interest in understanding the
   role of LAMA5 in the heart. Through transcription factor
   dual‐luciferase reporter gene experiments and ChIP‐Seq data, we
   confirmed that Lama5 is a direct target gene of GR. Furthermore,
   through Lama5 knockdown and overexpression cell experiments, we
   discovered the important role of the CORT‐GR‐LAMA5 axis in
   cardiomyocyte hypertrophy, and animal experiments have confirmed this
   finding. Notably, inhibiting CORT reduced myocardial fibrosis in CMS
   rats, but LAMA5 knockdown did not. This suggests CORT may activate
   cardiac fibroblasts independently of LAMA5. A recent study found
   myocardial fibrosis in Beagle dogs given 4mg kg^−1 prednisolone (a
   synthetic glucocorticoid) for 84 days, which may explain reduced
   fibrosis in CMS rats treated with metyrapone or relacorilant, and
   further research is needed to understand the specific mechanism.^[
   [183]^28 ^] In addition, an interesting finding is that reported
   genetic defects in LAMA5 lead to infantile nephrotic syndrome, and most
   of these patients are resistant to corticosteroids.^[ [184]^29 ^] Since
   LAMA5 is a direct target gene of corticosteroid‐GR, this seems to
   explain the reason why children with LAMA5 defects exhibit resistance
   to corticosteroids.

   While we recognize the value of cardiomyocyte hypertrophy in
   identifying cardiac structural abnormalities caused by CMS, it is
   currently difficult to identify the occurrence of cardiomyocyte
   hypertrophy in clinical settings. Because myocardial cells are
   non‐regenerative cells, it is challenging to obtain cardiac tissue for
   histological observations in clinical practice, and non‐invasive tests
   such as electrocardiograms, echocardiograms, and cardiac magnetic
   resonance imaging mainly focus on overall structural changes in the
   heart, making it difficult to detect changes at the cellular level. As
   one of the main participants in CORT‐induced cardiomyocyte hypertrophy,
   the serological prognostic value of LAMA5 warrants further research.
   Therefore, we first analyzed the correlation and diagnostic value of
   LAMA5 with LVEF in rats at the animal level, compared to NTproBNP, and
   found that in rats with CMS‐induced cardiac dysfunction, LAMA5 had a
   higher diagnostic value for decreased LVEF than NTproBNP. Furthermore,
   we collected depressive patients with or without HF, as well as healthy
   individuals, and found that LAMA5 was significantly upregulated in
   depressed patients with HF, showing a significant negative correlation
   with LVEF. Using LASSO regression to screen LAMA5, BNP, cortisol, and
   various clinical indicators, we obtained a comprehensive model
   including LAMA5 and BNP. This model achieved a diagnostic ROC of 0.913
   for depressive patients with HF, demonstrating high diagnostic value.
   It is notable that, in contrast to BNP, LAMA5 does not exhibit a
   significant increase in HF patients without depression, indicating that
   LAMA5 is not a universal biomarker for myocardial hypertrophy or HF.
   The common HF is typically induced by underlying cardiac disorders,
   such as hypertension, during which the activation of the sympathetic
   nervous system and the renin‐angiotensin‐aldosterone system is
   predominantly involved, which is less associated with glucocorticoids.
   Thus, we contend that LAMA5, as a direct target of glucocorticoids,
   might not participate in the cardiomyocyte hypertrophy process
   resulting from other diseases but rather serves as a specific biomarker
   for psychogenic hypertrophy. This requires further validation through
   clinical studies with larger sample sizes in the future.

   In terms of treatment, we found that inhibiting CORT synthesis and GR
   antagonism can effectively inhibit cardiomyocyte hypertrophy and
   cardiac dysfunction caused by CMS. Among them, the new drug
   relacorilant is currently in multiple clinical trials for treating
   Cushing's syndrome, platinum‐resistant ovarian cancer, prostate cancer,
   and other diseases, showing promising therapeutic potential.^[ [185]^30
   , [186]^31 , [187]^32 ^] Compared to RU486, relacorilant not only lacks
   progesterone antagonist‐related complications, but also exhibits weaker
   feedback elevation of glucocorticoids after treatment than RU486, and
   no similar prolongation of the QT interval as seen with RU486 has been
   observed, demonstrating good safety.^[ [188]^33 ^] Furthermore,
   epidemiological surveys and clinical studies over the last two decades
   have shown a significant increase in the incidence and mortality of
   cardiovascular diseases in patients with Cushing's syndrome or
   long‐term use of exogenous glucocorticoids. Even in patients without
   clinical evidence of cardiovascular disease, cardiac magnetic resonance
   imaging has detected left ventricular hypertrophy, fibrosis, diastolic
   and systolic dysfunction in some patients with Cushing's syndrome, once
   again highlighting the adverse cardiovascular effects of
   glucocorticoids.^[ [189]^34 ^] A recent study found that out of 56
   patients with Cushing's syndrome, 28 exhibited signs of HF, and
   surgical treatment of the primary lesion led to the resolution of HF
   syndrome in 11 patients, suggesting that glucocorticoid‐induced
   myocardial changes may be reversible.^[ [190]^35 ^] However, there is
   limited research on drug interventions for Cushing's syndrome‐related
   heart diseases, and the therapeutic value of relacorilant for Cushing's
   syndrome and Cushing's syndrome‐related heart diseases is promising.
   Furthermore, LAMA5 is also one of the potential therapeutic targets for
   heart disease in patients with depression/Cushing's syndrome/exogenous
   glucocorticoid supplementation. Especially for patients receiving
   exogenous glucocorticoid supplementation, who require the therapeutic
   effects of GCs while also needing to prevent their adverse cardiac
   effects, LAMA5 may be a specific therapeutic target. However, there are
   currently no drugs targeting LAMA5, emphasizing the urgent need to
   develop small‐molecule drugs or miRNA formulations targeting LAMA5 for
   further research.

   Furthermore, our study initially identified the ITGB1‐PI3K‐AKT
   signaling pathway as a potential downstream mediator of LAMA5‐induced
   cardiomyocyte hypertrophy. However, it is important to note that LAMA5,
   as a component of the extracellular matrix, interacts with various
   proteins such as collagen and fibronectin, which may also significantly
   contribute to cardiomyocyte hypertrophy. Despite this, there has been
   limited research on the interactions between LAMA5 and other
   extracellular matrix components. In recent years, the differentiation
   of induced pluripotent stem cells (iPSCs) into cardiomyocytes has been
   extensively explored, with extracellular matrix proteins emerging as
   crucial mediators in both iPSC differentiation and the maturation of
   iPSC‐derived cardiomyocytes (iPSC‐CMs).^[ [191]^36 ^] Burridge et al.
   investigated the capacity of several extracellular matrix proteins and
   peptides to facilitate the formation of human iPSC‐CM monolayers and
   promote iPSC‐CM differentiation, including recombinant vitronectin, a
   synthetic vitronectin peptide, and recombinant human laminin‐521 (which
   is synthesized from LAMA5). Among the substrates evaluated, only the
   laminin‐based matrix demonstrated the ability to support long‐term
   adhesion (exceeding 15 days) of the iPSC‐CM monolayer.^[ [192]^37 ^]
   Subsequently, Yap et al. conducted a comparative analysis of the
   efficacy of laminin‐521 alone versus a combination of laminin‐521 and
   laminin‐221 in promoting the differentiation of human embryonic stem
   cells (hESCs) into cardiomyocytes. The study revealed that hESC‐derived
   cardiomyocytes produced using the combination of laminin‐521 and
   laminin‐221 exhibited a significantly higher purity (≈80%–90%
   cardiomyocytes) compared to those produced with laminin‐521 alone
   (≈30%–50% cardiomyocytes).^[ [193]^38 ^] This finding underscores the
   substantial impact that the combination of extracellular matrix
   proteins can have on the differentiation and maturation of
   cardiomyocytes. Moreover, this approach may serve as an effective
   strategy for investigating the interaction mechanism of LAMA5 and other
   extracellular matrix proteins.

   The limitations of this study mainly include the following aspects.
   First, despite conducting a 20‐week continuous CMS modeling, the
   decrease in LVEF in rats was not high enough to reach a level of overt
   HF, and NTproBNP did not significantly increase (although there was a
   gradual upward trend), therefore, the diagnostic value of LAMA5 for HF
   at the animal level is debatable. Second, it is difficult in clinical
   practice to find patients with clear heart dysfunction caused by
   depression. The patients we collected with depression comorbid with HF
   also had various underlying heart diseases, which introduced
   interference with the plasma levels of LAMA5. Thus, longer duration of
   CMS modeling to achieve significant heart dysfunction or new
   psychological stress models for stronger induction of cardiac changes,
   as well as high‐quality clinical studies with strict
   inclusion/exclusion criteria and long‐term follow‐up, are needed to
   explore the diagnostic value of LAMA5 in heart dysfunction caused by
   psychological stress. Moreover, considering the clinically intricate
   relationship between depression and HF, investigating the prognostic
   value of LAMA5 in HF patients comorbid with anxiety/depression may be a
   topic worth further exploration. In addition, due to the myocardial
   protective effect of estrogen, only male rats were used in the animal
   experiments in this study to avoid the interference of gender on the
   results, so the effect of gender on the cardiac pathological changes
   caused by CMS needs to be further studied and clarified. Lastly, the
   downstream mechanisms of LAMA5 in this study were only subject to
   relatively simple validation, and we plan to use iPSC‐derived
   cardiomyocytes and Lama5 knockout mice to further explore the
   hypertrophic mechanism of LAMA5 in future studies.

Conflict of Interest

   The authors declare no conflict of interest.

Author Contributions

   C.Z. and Y.S. contributed equally to this work. C.Z., J.L., and J.Z.
   did conceptualization. C.Z., Y.S., and X.J. did methodology. C.Z.,
   H.L., J.H., and Z.W. did investigation. J.L., J.Z., and C.H. did
   project administration. C.Z. prepared the original draft. J.L., H.Y.,
   and Q.X. reviewed and did editing. J.L. and Y.S. did funding
   acquisition. All authors have read and approved the final manuscript.

Supporting information

   Supporting Information
   [194]ADVS-12-e14659-s001.docx^ (28.6MB, docx)

Acknowledgements