DUSP1 Inhibits Mechanotransduction and MAPK Signalling to Alleviate Myocardial Cell Hypertrophy

ABSTRACT

Hypertrophic Cardiomyopathy (HCM) is one of the significant causes of heart failure. This study aimed to identify and validate potential therapeutic targets for HCM through bioinformatics analysis and in vitro and in vivo experiments. By analysing differentially expressed genes in HCM patients and combining multiple analytical approaches, key genes were screened. The functions of these genes were validated using AC16 cells under different mechanical force environments and Ang—II induction. HCM‐like pathological cardiac remodelling mouse model was established to evaluate myocardial hypertrophy, fibrosis and cardiac remodelling. The results demonstrated that DUSP1 expression was downregulated in the myocardium of HCM patients. Mechanical force transduction activated the MAPK pathway and overexpression of DUSP1 suppressed ERK/MAPK activation and attenuated mechanotransduction‐associated cardiomyocyte hypertrophy and hypertrophic gene expression. In vivo experiments showed that cardiac‐specific restoration of DUSP1 expression alleviated pathological cardiac remodelling and fibrosis. This study suggests that DUSP1 functions as an endogenous regulator of mechanically activated MAPK signalling, thereby limiting pathological myocardial hypertrophy. These findings identify a previously underappreciated role of DUSP1 in linking mechanical stress‐induced signalling with MAPK activation and pathological cardiac remodelling, suggesting DUSP1 restoration as a potential therapeutic strategy for HCM‐like cardiac remodelling.

Abbreviations

AAV

adeno‐associated virus

ACE2

angiotensin converting enzyme 2

Ang‐II

angiotensin II

BW

body weight

cTnT

cardiac troponin T

DEGs

differentially expressed genes

ECM

extracellular matrix

ERK

extracellular rignal‐regulated kinases

GEO

gene expression omnibus

GO

gene ontology

HCM

hypertrophic cardiomyopathy

HPA

human protein atlas

HW

heart weight

HW/BW

heart weight‐to‐body weight ratio

ISO

isoproterenol

LVW

left ventricular weight

LVW/BW

left ventricular weight‐to‐body weight ratio

NO

nitric oxide

PPI

protein–protein interaction

qRT‐PCR

quantitative real‐time polymerase chain reaction

RAS

renin‐angiotensin system

1. Introduction

Hypertrophic cardiomyopathy (HCM) is a primary autosomal dominant genetic disorder characterised by unexplained left ventricular hypertrophy, decreased left ventricular diastolic compliance and dynamic left ventricular outflow tract obstruction. It represents a significant cause of heart failure, arrhythmias and sudden death [1, 2]. HCM arises from over 2000 mutations in 11 or more genes encoding sarcomeric proteins, which code for proteins that form the thick and thin filament contractile components of the cardiac sarcomere or Z‐disk. The most commonly involved genes are those encoding β‐myosin heavy chain and myosin‐binding protein C [2]. As a global disease, HCM cases have been reported in 122 countries across all continents [2]. Early studies estimated the prevalence of HCM in the general population to be approximately 0.2%, while recent research suggests that the prevalence may reach 0.5%, with an incidence rate of 0.24–0.47 cases per 100 000 individuals per year in children [3, 4]. Clinically, HCM can manifest in various forms of symptomatic heart failure, including but not limited to dynamic left ventricular outflow tract obstruction, non‐obstructive HCM with restrictive cardiomyopathy features, and, in rare cases, ventricular arrhythmias and even sudden death [5]. The main treatments for HCM include β‐blockers, calcium channel blockers, cardiac myosin inhibitors, and, when necessary, invasive therapies such as myectomy, as well as anticoagulant therapy to prevent atrial fibrillation‐related complications [6]. However, these treatments do not effectively halt disease progression. Therefore, there is an urgent need to delve deeper into the genetic basis and pathophysiological mechanisms of HCM and explore new potential therapeutic targets.

In HCM, mechanotransduction, the mechanism by which cardiomyocytes sense and respond to extracellular mechanical forces, plays a crucial role in disease development [7, 8]. Mechanical stress is not only a consequence of abnormal myocardial contraction but also an important driver of pathological signalling activation and cardiac remodelling. HCM originates from mutations in genes encoding sarcomeric and Z‐disk proteins, including myosin, troponin, actin and myosin‐binding proteins [1, 2]. These proteins not only connect the contractile structures to the cytoskeleton and extracellular matrix (ECM) but also constitute the core structures for mechanosensing and signal transduction. Mutations in Z‐disk‐associated proteins are associated with various cardiomyopathies, including HCM [9, 10]. In the early progression of HCM, abnormal biophysical signals triggered by troponin cTnT‐R92Q mutations affect myocardial diastolic function through mechanotransduction, leading to coronary hemodynamic disturbances, angiogenic defects and fibrosis. These changes ultimately contribute to the cardiac remodelling process in HCM by modulating YAP protein expression and localization [11]. However, although multiple mechanosensitive pathways have been identified in HCM progression, the endogenous molecular mechanisms that prevent excessive activation of mechanical stress signalling remain poorly understood. Additionally, mutations in the cardiac myosin heavy chain (MYH7) gene result in enhanced myofilament contractility, which can affect the stability of muscle LIM protein (MLP) through mechanotransduction. This, in turn, abnormally activates the calcineurin‐NFAT signalling pathway, promoting cardiomyocyte hypertrophy [12]. Intriguingly, abnormal nitric oxide (NO) signalling not only increases cardiomyocyte sensitivity to mechanical forces, leading to intracellular Ca²⁺ dysregulation and promoting cardiomyocyte enlargement, but also activates calmodulin‐dependent protein kinase II (CaMKII), further disrupting cardiomyocyte sensitivity to Ca²⁺ and contractility, thereby exacerbating the pathological process in HCM [13].

The MAPK signalling pathway involves a series of intracellular cascades, including phosphorylation and dephosphorylation of multiple protein kinases, ultimately affecting gene expression, cell proliferation, differentiation, apoptosis and migration [14, 15]. The MAPK family includes ERK (extracellular signal‐regulated kinases), JNK (c‐Jun N‐terminal kinases) and p38 MAPK [16]. When cells are subjected to mechanical forces, such as pressure overload in cardiac myocytes, these forces can activate cell surface receptors and cytoskeletal proteins, such as integrins and cadherins [17, 18]. Activation of integrins prompts downstream Ras/Raf kinases to sequentially activate and catalyse the MEK/ERK cascade [19]. Notably, ERK has a protective effect during the early stages of cardiac hypertrophy, promoting beneficial cardiac remodelling and preventing cell death. However, in cardiac hypertrophy caused by chronic hypertension, ERK activation is associated with pathological hypertrophy [20]. A mouse model carrying a mutation that increases Raf1 activity demonstrates that the pathogenesis of HCM involves abnormal activation of the Erk1/2 signalling pathway and related symptoms can be treated with MEK inhibitors. This underscores the crucial role of Ras/Raf/MAPK signalling abnormalities in the development of HCM [21]. Although the activation of MAPK signalling in mechanically stressed cardiomyocytes has been well documented, whether endogenous MAPK inhibitory mechanisms participate in controlling mechanical stress‐induced cardiac remodelling remains unclear.

DUSP1 is a dual‐specificity phosphatase that has specificity for tyrosine and threonine, which can dephosphorylate ERK and thus inhibit the MAPK signalling pathway [22, 23]. Previous studies have demonstrated that DUSP1 functions as an important stress‐responsive regulator involved in inflammation, oxidative stress responses and cardiovascular remodelling through modulation of MAPK signalling. In this study, we conducted a comparative analysis of differentially expressed genes (DEGs) between patients with HCM and healthy myocardial tissues. We found that, in HCM myocardial tissues, mechanotransduction and the MAPK signalling pathway were significantly activated, while the expression of DUSP1 was significantly downregulated. However, whether DUSP1 expression is altered in human HCM myocardium and whether its downregulation contributes to mechanically induced pathological remodelling remain largely unknown. Based on these findings, we propose that reduced DUSP1 expression may contribute to the development of cardiomyocyte hypertrophy and restoring DUSP1 expression may alleviate pathological remodelling by restraining mechanotransduction‐associated MAPK activation.

2. Materials and Methods

2.1. Bioinformatics Analysis

To explore potential molecular mechanisms in HCM patients’ cardiac tissues, this study used a dataset from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) (GSE36961), which contains a case–control study comparing the mRNA transcriptome of HCM patients’ left ventricular muscle tissue to that of healthy control donors' hearts. The differences in gene expression levels between the two groups were evaluated using the limma package in R language. The adjusted p‐value (padj. value < 0.05) and absolute log₂ fold change (|log₂ Fold change| > 1) were used as screening criteria to identify DEGs between HCM patients and controls. These DEGs are considered potential key regulatory factors in the pathology of HCM. The DEGs were subjected to Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the Metascape online tool (https://metascape.org/) [24], to reveal potential biological functions and signalling pathways in HCM. The DEGs' protein–protein interaction (PPI) network was predicted based on physical and functional relationships using the STRING database (https://cn.string‐db.org/) [25], and visualised using Cytoscape software [26], or identification of key regulatory nodes. Furthermore, to gain further insight into the expression patterns of key interest genes, we retrieved their expression levels in different tissues and cell subpopulations from the Human Protein Atlas (HPA) database (https://www.proteinatlas.org/) [27].

2.2. Culture of AC16 Cardiomyocytes

The human cardiomyocyte line AC16 was obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in high‐glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 10 000 U/mL penicillin–streptomycin mixture (Gibco, USA) at 37°C in a 5% CO₂ incubator. The medium was replaced every 2 days and cells in the logarithmic growth phase with good growth were used for passaging. To ensure cell viability and stability, the thawed cells were passaged for no more than 30 days.

2.3. Analysis of Cardiomyocyte Surface Area

Cardiomyocyte membranes were fluorescently labelled using Dil cell membrane red fluorescent probe (Beyotime, C1036, China). Subsequently, the cardiomyocyte surface area was analysed using Image J, an open‐source image processing software based on Java. On the confocal images used, the software pixel length was calibrated to a μm scale. After background elimination, the boundaries of individual cells were marked using the region of interest tool. Multiple confocal images were assessed to ensure an adequate number of images for evaluation and the cell areas were extracted and compared.

2.4. RNA Extraction and Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR)

To accurately quantify the mRNA expression levels of genes, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, USA) and the integrity, quantity and purity of the RNA were checked using a spectrophotometer. cDNA synthesis was performed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). qRT‐PCR analysis was conducted using the SYBR Green Supermix Kit (Thermo Fisher Scientific, USA). The PCR reaction mixture contained DUSP1‐specific primers, cDNA template and SYBR Green Supermix. The mRNA expression levels were determined using the 2^−ΔΔCt method and normalised to the endogenous gene GAPDH. Each sample had 6 technical replicates to enhance data reliability. The primer sequences used in this study are provided in Table S1.

2.5. Western Blotting (WB)

Cells were lysed in RIPA lysis buffer containing protease inhibitors and further disrupted using ultrasound. Protein concentrations were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Proteins were separated by SDS‐PAGE and then transferred electrophoretically onto PVDF membranes. Subsequently, the membrane was blocked in a solution containing 5% skim milk powder for 1 h. Then, it was incubated overnight at 4°C with primary antibodies (DUSP1, Gene Tex, GTX54042; Raf1, abcam, ab181115; Raf1 phospho S259, abcam, ab173539; ERK1 + ERK2, abcam, ab184699; ERK1 phospho T202 + Y204 + ERK2 phospho T185 + Y187, abcam, ab278538; YAP1, abcam, ab52771; YAP1 phospho S127 antibody, abcam, ab76252; FAK, ab40794, Abcam; p‐FAK, ab81298, Abcam; p‐paxillin, ab277786, Abcam; GAPDH, ab9485, Abcam; diluted at a ratio of 1:1000). After being washed four times with TBST for 15 min each time, the membrane was incubated with diluted secondary antibody (Goat Anti‐Rabbit IgG H&L (HRP), Abcam, ab6721, diluted at a ratio of 1:2000) at room temperature for 1 h. Following another four washes with TBST, enhanced chemiluminescence was performed using an ECL kit (Thermo Fisher Scientific, USA). Each band was analysed using Image J software.

2.6. Immunofluorescence (IF)

To assess the expression and localization of YAP protein in cardiomyocytes, as well as changes in cytoskeletal structure, we conducted immunofluorescence staining experiments. Firstly, cultured AC16 human cardiomyocytes were fixed onto poly‐L‐lysine‐coated coverslips using 4% paraformaldehyde solution at room temperature for 15 min. Subsequently, cells were permeabilised with 0.1% Triton X‐100 for 10 min to enhance antibody penetration and blocked non‐specific binding sites with 3% bovine serum albumin for 30 min. Subsequently, the cells were incubated overnight at 4°C with the primary antibody against YAP (abcam, ab52771, diluted at a ratio of 1:1000) and p‐FAK (Abcam, ab81298 diluted at a ratio of 1:1000). The following day, after being washed three times with PBS, the cells were incubated with an Alexa Fluor 594‐labelled secondary antibody (ab150080, Abcam, diluted at a ratio of 1:2000) at room temperature in the dark for 1 h. To simultaneously visualise the cytoskeleton, we utilised Alexa Fluor 488‐labelled phalloidin (Phalloidin, C2201S, Beyotime, diluted at a ratio of 1:1000). After the incubation with the secondary antibody, the cells were stained with this reagent at room temperature in the dark for 30 min to label F‐actin. Finally, the cell nuclei were stained with DAPI (4′,6‐diamidino‐2‐phenylindole, C1002, Beyotime, diluted at a ratio of 1:1000) for 5 min. After completing all staining steps, the samples were thoroughly washed with PBS and the coverslips were then inverted and mounted onto slides containing antifade mounting medium. Fluorescent images were acquired using a fluorescence microscope and analysed quantitatively using ImageJ software.

2.7. Overexpression of DUSP1 in AC16 Cells

For the overexpression of DUSP1, the full‐length coding region of the DUSP1 gene was cloned into the pCMV6‐AC‐GFP vector (OriGene Technologies) to construct an overexpression plasmid. The constructed DUSP1 overexpression plasmid was transfected into AC16 cells using Lipofectamine 2000 transfection reagent (Invitrogen, USA) according to the manufacturer's instructions. Transfection efficiency was verified by qRT‐PCR and Western Blot analysis to confirm the overexpression. Additionally, cells transfected with an empty vector were used as a control (EV CTRL) to exclude any effects of the vector itself on the experimental results.

2.8. Establishment of the ISO‐Induced Cardiac Hypertrophy Mouse Model

To establish the ISO‐induced cardiac hypertrophy model, 6–8‐week‐old male C57BL/6 mice with similar body weights (BWs) were purchased from Vital River Laboratories. All animal experiments were conducted strictly in accordance with the ARRIVE (Animal Research: Reporting of in vivo Experiments) guidelines and approved by the Ethics Committee for Animal Experiments of the Experimental Animal Science Department of Fudan University (Approval Number: 2025‐HDYY‐074). After a 1‐week acclimatisation period under SPF‐grade conditions, the mice were randomly divided into four groups, each containing six mice to ensure statistical power: a normal control group (Normal), an HCM model group (HCM), a DUSP1 overexpression treatment group (DUSP1 OE) and a combined DUSP1 overexpression and ERK1/2 inhibitor treatment group (DUSP1 OE + ERK Inh). Mice in each group received subcutaneous injections of ISO at a dose of 1 mg/kg or an equal volume of saline as a control, twice daily with an interval of 8 h, for two consecutive weeks. To simulate early intervention, the treatment groups began receiving their respective treatments simultaneously with the ISO injections. Throughout the experiment, the health status and behavioural manifestations of the mice were closely monitored to ensure compliance with humanitarian principles.

At the end of the experiment, to ensure euthanasia of the mice in accordance with ethical standards and to minimise suffering, a two‐step method was used. Firstly, the mice were placed in a sealed container filled with CO₂ and the CO₂ concentration was gradually increased until the air was completely displaced, causing the mice to gradually lose consciousness. This process lasted for approximately 5 min until it was confirmed that the mice showed no signs of respiration or heartbeat. Subsequently, cervical dislocation was immediately performed to ensure rapid death of the mice, preventing any possibility of regaining consciousness. After euthanasia, the mice were weighed and their hearts were carefully removed, washed with pre‐cooled PBS to remove excess blood and weighed promptly. The upper part of the heart was used for histological analysis and fixed in 4% paraformaldehyde solution, while the lower part was processed on ice with TRIzol reagent for RNA extraction and subsequent PCR analysis.

2.9. Construction of a Mouse Model With Cardiac‐Specific Overexpression of DUSP1

To achieve cardiac‐specific overexpression of DUSP1, an Adeno‐Associated Virus (AAV) vector was selected as the gene delivery tool. In this study, the AAV9 serotype was used due to its highest transduction efficiency in cardiomyocytes [28]. A recombinant AAV9 vector containing the full‐length coding sequence of DUSP1 was constructed and placed under the control of the cardiac troponin T (cTnT) promoter to ensure specific expression of DUSP1 in cardiomyocytes. Mice received AAV‐DUSP1 vector via tail vein injection at doses ranging from 3.15 × 10¹⁰ to 1 × 10¹¹ virus particles per mouse to achieve efficient and durable gene expression in cardiac tissue while minimising potential side effects and toxic effects.

2.10. Heart Weight (HW) Index and Left Ventricular Index in Mice

At the end of the treatment, the BW of mice in each group was measured. After euthanasia, the HW was determined and the atria and right ventricles were trimmed and placed in 4°C PBS buffer for cleaning. The left ventricular weight (LVW) was then measured. The heart weight index (HW/BW) and left ventricular index (LVW/BW) were calculated.

2.11. Echocardiographic Assessment of Cardiac Function

Transthoracic echocardiography was performed to evaluate cardiac structure and function in mice from all experimental groups. Briefly, mice were anaesthetised with 1%–2% isoflurane and examined using a high‐frequency ultrasound imaging system equipped with a mouse‐specific transducer. Two‐dimensional and M‐mode echocardiographic images were acquired from parasternal long‐axis and short‐axis views. Left ventricular posterior wall thickness at diastole (LVPWd), interventricular septal thickness (IVSd), left ventricular internal diameter (LVID), ejection fraction (EF) and fractional shortening (FS) were measured to assess cardiac remodelling and systolic function. Doppler imaging was additionally performed to evaluate left ventricular outflow tract (LVOT) morphology and potential flow abnormalities. All measurements were obtained from at least three consecutive cardiac cycles by an investigator blinded to the experimental groups.

2.12. Haematoxylin and Eosin Staining

Following euthanasia of the experimental animals, heart tissue samples were carefully removed. The tissue samples were immediately immersed in 10% neutral buffered formalin for 48 h of fixation. Fixed tissue samples underwent gradual dehydration in a series of ethanol concentrations and were then cleared in xylene to remove moisture and residual fixatives. Next, the tissue blocks were infiltrated with molten paraffin multiple times to ensure thorough impregnation and subsequently sectioned into 4–6 μm thick slices using a microtome. Tissue sections were attached to preheated slides and stained with haematoxylin so that nuclei stained dark blue. After that, the cytoplasm and part of the extracellular matrix were counterstained with eosin to distinguish the different tissue components. During the staining process, additional steps such as rinsing and differentiation were performed to ensure uniform and clear staining. After staining, the sections were dehydrated with absolute ethanol, cleared with xylene and mounted with neutral gum for long‐term preservation.

2.13. Masson Staining

To assess collagen deposition and fibrosis in heart tissue, Masson trichrome staining was performed. Mouse heart tissue sections were dewaxed and hydrated, followed by sequential immersion in different staining solutions: first, the nuclei were briefly stained with Weigert iron haematoxylin solution to appear dark brown; then, muscle fibres were stained red with acid fuchsin solution; finally, the collagen fibres were specifically stained blue by phosphomolybdic acid differentiation and aniline blue staining. After staining, the sections were rapidly dehydrated, cleared and mounted with neutral balsam. The stained sections were observed under an optical microscope and quantitatively assessed using image analysis software.

2.14. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism 10 software. Initially, the data were tested for normal distribution and homogeneity of variances. Appropriate statistical methods were then selected based on the data characteristics: for comparisons among three or more groups, one‐way ANOVA combined with Tukey's multiple comparison test was used; for comparisons between two groups, Student's t‐test was employed and the nonparametric Mann–Whitney U test was chosen when the data did not conform to normal distribution. The significance levels were set as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, with all p‐values derived from two‐tailed tests. Each experiment was repeated at least three times to ensure the reproducibility and stability of the results.

3. Results

3.1. DEGs and Functional Enrichment Analysis in Myocardial Tissue of Patients With HCM

To explore the underlying molecular mechanisms of HCM, bioinformatics analysis was performed on the transcriptome information dataset GSE36961 from an HCM case–control study. padj. value < 0.05 and |log₂ Fold change| > 1 were used as criteria to identify DEGs between myocardial tissue from HCM patients and healthy control donors. The volcano plot (Figure 1a) revealed that a total of 28 genes were significantly upregulated and 63 genes were significantly downregulated in HCM patients, suggesting that these DEGs may be potential key regulators in the pathological process of HCM. Further functional enrichment analysis elucidated the roles of these DEGs in various biological processes. The tissue expression enrichment analysis of DEGs (Figure 1b) showed that they were mainly enriched in plasma cells, the cardiovascular system and the heart. The GO enrichment analysis revealed that DEGs were primarily involved in molecular functions (Figure 1c), including RAGE receptor binding, Wnt protein binding, calcium ion binding, etc.; biological processes (Figure 1d), such as growth regulation, regulation of heart contraction and negative regulation of phosphate metabolic process; and cellular components (Figure 1e), mainly focused on blood microparticles and endocytic vesicles. These molecular functions and biological processes may play crucial roles in HCM. The KEGG pathway enrichment analysis (Figure 1f) indicated that DEGs were mainly involved in biological processes such as complement and coagulation cascades and acute myeloid leukaemia.

FIGURE 1.

DEGs and functional enrichment analysis in myocardial tissue of patients with HCM. (a) Volcano plot of DEGs in myocardial tissue of patients with HCM. (b) Tissue expression enrichment analysis of DEGs. (c–e) GO enrichment analysis of DEGs. (c) Enrichment analysis of molecular functional of DEGs. (d) Enrichment analysis of biological processes. (e) Enrichment analysis of cellular component. (f) KEGG enrichment analysis of DEGs.

3.2. Age‐Dependent Downregulation of Dual Specificity Phosphatase DUSP1 in HCM and Its Negative Regulation of the MAPK Signalling Pathway

To further screen for potential key genes affecting HCM, we used the STRING database and Cytoscape software to screen and construct a PPI network for the top 20 genes with the highest number of interactions (Figure 2a). The cluster heatmap (Figure 2b) displayed the expression differences of the differential genes in myocardial tissue from adult HCM patients (HCMad), paediatric HCM patients (HCMch), healthy control adults (CTRLad) and healthy control children (CTRLch). Notably, angiotensin‐converting enzyme 2 (ACE2), which encodes a gene regulating the renin‐angiotensin system (RAS), was significantly upregulated in HCM patients and ACE2 gene polymorphisms were associated with the degree of left ventricular hypertrophy in HCM patients [29]. Additionally, the dual specificity phosphatase gene DUSP1, which encodes a phosphatase that dephosphorylates ERK and the integrin molecule‐encoding gene ITGB2 were significantly downregulated in HCM patients. Further single‐gene expression analysis (Figure 2c–e) showed that DUSP1 was not only significantly downregulated in HCM patients but also exhibited a more significant downregulation in adult HCM patients compared to paediatric HCM patients, demonstrating an age‐dependent decline. These results emphasise the potential important role of DUSP1 in HCM. Since mechanotransduction and the MAPK signalling pathway play crucial roles in normal myocardial tissue function, we further used VENN diagram intersection analysis (Figure 2f) to reveal the relationship between DEGs and the top 500 genes related to the MAPK signalling pathway and mechanotransduction from the GeneCards database (https://www.genecards.org/). The results showed that seven DEGs were related to the MAPK signalling pathway, including DUSP1; while ITGB2 and STAT3 were the two common intersection genes among the three. Based on these bioinformatics analysis results, we hypothesise that the downregulation of dual specificity phosphatase DUSP1 participates in the development of myocardial hypertrophy by relieving the original dephosphorylation inhibition of the MAPK pathway in myocardial tissue.

FIGURE 2.

Significant downregulation of DUSP1 in HCM and its negative regulation of the MAPK signalling pathway. (a) PPI network of the top 20 genes with the highest number of interactions encoding proteins. (b) Cluster heatmap of expression differences of differential genes in myocardial tissue from adult HCM patients (HCMad), paediatric HCM patients (HCMch), healthy control adults (CTRLad) and healthy control children (CTRLch). (c–e) Expression differences of ACE2, DUSP1 and ITGB2 in the aforementioned populations. (f) VENN intersection analysis of DEGs with genes related to the MAPK signalling pathway and mechanotransduction (MCTD). (g) Schematic diagram of the molecular mechanism involving integrins and DUSP1 in MAPK and mechanotransduction.

3.3. Role of Mechanotransduction in the Development of Human Cardiomyocyte Hypertrophy

To investigate the role of mechanotransduction in the process of cardiomyocyte hypertrophy, we selected the human cardiomyocyte line AC16 cells as the research subject. By coating cell culture plates with gradient concentrations of matrigel, we simulated ECM microenvironments with varying stiffness and crosslinking degrees, thereby providing a mechanical force microenvironment with progressively increasing intensity. After 48 h of culture, the cells were stained with Dil red fluorescent probe for cell membranes and it was observed that AC16 cells exhibited the most significant increase in surface area in the 12.5 mg/mL matrigel environment, mimicking mechanotransduction‐mediated myocardial hypertrophy (Figure 3a,b). To further simulate the in vivo pathological hypertrophic environment of hypertrophic cardiomyopathy (HCM), we treated the cells with a combination of 1 × 10⁻⁷ mol/L angiotensin II (Ang‐II) under the aforementioned conditions. The results demonstrated that the combined stimulation of Ang‐II and the mechanical force microenvironment significantly enhanced the hypertrophic response in AC16 cells (Figure 3c,d).

FIGURE 3.

Role of mechanotransduction in the development of human cardiomyocyte hypertrophy. (a, b) Effect of different concentrations of matrigel on AC16 cell volume, Scale bar = 100 μm, n = 3. (c, d) Combined treatment with matrigel and Ang‐II promotes AC16 cell hypertrophy, Scale bar = 100 μm, n = 3. (e) Effect of matrigel and Ang‐II treatment on mRNA expression levels of hypertrophic marker genes in cardiomyocytes, n = 6. (f) Immunofluorescence staining of mechanotransduction signalling factor YAP and F‐actin. Scale bar = 50 μm, (g, h) Bar graphs showing the fluorescence expression of YAP and F‐actin, n = 3.

To further investigate the role of mechanotransduction signalling in the process of cardiomyocyte hypertrophy, we employed qRT‐PCR technology to assess the mRNA expression levels of hypertrophy‐related marker genes, namely ANP, BNP and β‐MHC, in AC16 cells. The results revealed that, compared with the control group, the expression of these genes was significantly upregulated under mechanical stimulation induced by Matrigel. Notably, in the group treated with a combination of Matrigel and Ang‐II, the expression levels of ANP, BNP and β‐MHC exhibited the most pronounced increase (Figure 3e). To reveal the specific role of mechanotransduction signals in the process of cardiomyocyte hypertrophy, we performed immunofluorescence staining analysis (Figure 3f–h). The results demonstrated that the expression of YAP and cytoskeletal F‐actin, which are involved in mechanotransduction, increased significantly after treatment with Matrigel and Ang‐II. Quantitative analysis showed that the fluorescence intensities of YAP and F‐actin were significantly higher in the treated groups compared to the normal group, indicating that the mechanotransduction signalling pathway was activated during cardiomyocyte hypertrophy.

3.4. Inhibition of MAPK Signalling Pathway Reduced Cardiomyocyte Hypertrophy

To delve deeper into the molecular mechanisms underlying mechanotransduction in cardiomyocyte hypertrophy, this study analysed the activation status of the MAPK signalling pathway under mechanical stimulation using the Western Blot technique. The experimental results demonstrated that, compared with the untreated control group, mechanical force stimulation simulated by Matrigel significantly elevated the phosphorylation levels of RAF and ERK1/2 in AC16 cells, while concurrently reducing the phosphorylation level of YAP protein (Figure 4a,b, Figure S4). These findings suggest that mechanotransduction regulates the hypertrophic phenotype of cardiomyocytes by activating both the MAPK signalling pathway and the Hippo/YAP signalling pathway.

FIGURE 4.

Inhibition of the MAPK signalling pathway attenuates cardiomyocyte hypertrophy. (a, b) Western Blot bands and bar graphs showing the expression and phosphorylation levels of the MAPK signalling pathway and YAP under matrigel and Ang‐II stimulation, n = 3. (c, d) Inhibition of the MAPK pathway impedes the increase in cardiomyocyte area, Scale bar = 100 μm, n = 3. (e) Blocking the MAPK pathway inhibits the mRNA expression of hypertrophic marker genes in cardiomyocytes, n = 6. (f, g) Western Blot bands and bar graphs illustrating the effects of ERK1/2 inhibitor on the expression and phosphorylation of MAPK pathway components and YAP in cardiomyocytes, with n = 3.

To validate the role of the MAPK signalling pathway in mechanotransduction‐induced cardiomyocyte hypertrophy, the ERK1/2 inhibitor PD98059 was used to specifically inhibit the MAPK pathway. The results showed that inhibiting the MAPK signalling pathway significantly attenuated the degree of cardiomyocyte hypertrophy (Figure 4c,d). Furthermore, qRT‐PCR results revealed that combined stimulation with Matrigel and Ang‐II significantly upregulated the mRNA expression levels of hypertrophic marker genes such as ANP, BNP and β‐MHC in AC16 cells. However, upon the addition of an ERK1/2 inhibitor, the expression of these genes markedly decreased (Figure 4e). Western Blot results further demonstrated that the ERK1/2 inhibitor significantly reduced the phosphorylation levels of ERK1/2, thereby effectively suppressing the activation of the mechanotransduction/MAPK signalling axis downstream (Figure 4f,g, Figure S5). Collectively, these findings indicate that the MAPK signalling pathway plays a pivotal role in the process of cardiomyocyte hypertrophy mediated by mechanotransduction. Inhibition of this pathway not only downregulates the expression of hypertrophy‐related genes at the transcriptional level but also significantly alleviates the hypertrophic progression of cardiomyocytes, thereby validating the effectiveness of targeting the MAPK pathway as a potential therapeutic strategy for hypertrophic cardiomyopathy (HCM).

3.5. Inhibition of Cardiomyocyte Hypertrophy Progression by Overexpression of Dual Specificity Phosphatase DUSP1

To investigate the role of dual specificity phosphatase DUSP1 in the progression of cardiomyocyte hypertrophy, we examined the mRNA and protein expression levels of DUSP1 during cardiomyocyte hypertrophy (Figure 5a–c, Figure S6A). The results showed that under normal conditions, DUSP1 exhibited high mRNA and protein expression levels, whereas its expression was significantly downregulated during cardiomyocyte hypertrophy. This result aligns with the downregulation of DUSP1 expression observed in HCM patients through bioinformatics analysis, suggesting that DUSP1 may be involved in regulating the process of cardiomyocyte hypertrophy. Subsequently, we constructed AC16 cells overexpressing DUSP1 and validated the significant elevation of its mRNA and protein expression levels in these cells via PCR and Western Blot (Figure 5d–f, Figure S6B). To further investigate the function of DUSP1 in cardiomyocyte hypertrophy, we examined the effects of its overexpression on cell area and the expression of hypertrophic marker genes. The results demonstrated that, compared with the cardiomyocyte hypertrophy group, overexpression of DUSP1 significantly reduced the area of AC16 cells (Figure 5g,h), indicating that DUSP1 effectively inhibits the hypertrophic phenotype of cardiomyocytes in vitro. Furthermore, qRT‐PCR analysis revealed that overexpression of DUSP1 significantly decreased the mRNA expression levels of hypertrophy‐related genes such as ANP, BNP and β‐MHC (Figure 5i). Collectively, these findings confirm that DUSP1 exerts a significant inhibitory effect on cardiomyocyte hypertrophy, further supporting its critical role in regulating pathological myocardial hypertrophy.

FIGURE 5.

Downregulation of DUSP1 expression in hypertrophic cardiomyocytes and its involvement in the hypertrophic process. (a) Significant downregulation of DUSP1 mRNA expression during cardiomyocyte hypertrophy, n = 6. (b, c) Western blot bands and bar graph statistics of DUSP1 protein expression, n = 3. (d) PCR results validating the overexpression of DUSP1, n = 6. (e, f) Verification of DUSP1 overexpression by Western Blot, with n = 3. (g, h) Overexpression of DUSP1 attenuates the enlargement of cardiomyocyte area. Scale bar = 100 μm, n = 3. (i) Overexpression of DUSP1 reverses the expression of mRNA levels of hypertrophic marker genes in cardiomyocytes, n = 6.

3.6. Attenuation of Mechanotransduction‐Induced Cardiomyocyte Hypertrophy by Overexpression of DUSP1 Through Inhibition of the MAPK Pathway

To explore the effect of DUSP1 overexpression on mechanotransduction‐induced cardiomyocyte hypertrophy, we evaluated the protein expression and phosphorylation levels of RAF, ERK1/2 and YAP1 in cardiomyocytes under different treatment conditions using Western Blot (Figure 6a,b, Figure S7). The results showed that the levels of p‐RAF and p‐ERK1/2 were significantly elevated, while p‐YAP1 was significantly decreased in the Matrigel and Ang‐II treatment groups, indicating activation of mechanical stress‐associated signalling pathways. In contrast, DUSP1 overexpression markedly reduced the phosphorylation levels of ERK1/2 under mechanical stimulation, without completely reversing upstream mechanical signalling activation. These findings indicated that overexpression of DUSP1 alleviated mechanotransduction‐induced cardiomyocyte hypertrophy primarily by suppressing mechanically activated MAPK/ERK signalling rather than directly blocking the initiation of mechanical sensing signals. Furthermore, we observed the effects of DUSP1 overexpression on YAP and F‐actin through immunofluorescence staining (Figure 6c–e). The results revealed that the fluorescence intensities of YAP and F‐actin were significantly enhanced in the Matrigel and Ang‐II treatment groups, whereas the fluorescence intensity of F‐actin was markedly decreased in the DUSP1 overexpression group. These changes suggest that alterations in YAP‐associated signalling and cytoskeletal remodelling may occur downstream of reduced MAPK activation and attenuated hypertrophic responses. Collectively, these findings demonstrate that DUSP1 overexpression attenuates mechanical stress‐induced cardiomyocyte hypertrophy, which is associated with reduced MAPK/ERK activation and downstream hypertrophic remodelling.

FIGURE 6.

Attenuation of mechanotransduction‐induced cardiomyocyte hypertrophy by overexpression of DUSP1 through inhibition of the MAPK pathway. (a, b) Western Blot analysis reveals that overexpression of DUSP1 attenuates the expression of cardiomyocyte hypertrophy‐associated proteins induced by mechanotransduction through inhibiting the MAPK signalling pathway, with n = 3. (c–e) Immunofluorescence images and fluorescence intensity statistics showing the effects of overexpressing DUSP1 on YAP and F‐actin, Scale bar = 50 μm, n = 3.

3.7. DUSP1 Regulates Mechanotransduction‐Associated MAPK Activation Without Directly Altering Focal Adhesion Signalling

To further clarify whether DUSP1 directly regulates upstream mechanical sensing complexes or primarily affects downstream MAPK signalling, we examined the activation status of MAPK signalling and focal adhesion‐associated mechanotransduction components in mechanically stimulated AC16 cardiomyocytes.

Western blot analysis was performed to evaluate the phosphorylation levels of ERK1/2, FAK and paxillin under different treatment conditions. Compared with the Normal group, Matrigel combined with Ang‐II stimulation markedly increased the phosphorylation levels of ERK1/2, FAK and paxillin, indicating activation of mechanical stress‐associated signalling pathways. Overexpression of DUSP1 significantly reduced ERK1/2 phosphorylation induced by mechanical stimulation. However, DUSP1 overexpression did not substantially alter the phosphorylation levels of FAK and paxillin, suggesting that DUSP1 primarily regulates downstream ERK/MAPK activation rather than directly suppressing focal adhesion‐associated mechanosensing. Consistently, ERK inhibition further reduced ERK1/2 phosphorylation but showed limited effects on FAK and paxillin activation, whereas FAK inhibition markedly decreased FAK and paxillin phosphorylation, confirming that FAK functions as an upstream regulator of focal adhesion signalling.

To further investigate the spatial organisation of mechanical sensing structures, immunofluorescence staining was performed to assess p‐FAK, integrin and F‐actin remodelling. Mechanical stimulation induced prominent accumulation of integrin and p‐FAK signals at focal adhesion sites and promoted F‐actin reorganisation compared with the Normal group. DUSP1 overexpression partially reduced F‐actin remodelling but produced limited changes in integrin distribution and p‐FAK localization, further indicating that DUSP1 does not directly disrupt the formation or stability of focal adhesion complexes. In contrast, FAK inhibition markedly impaired integrin/FAK clustering and reduced F‐actin rearrangement, demonstrating the essential role of focal adhesion signalling in mechanical stress‐induced cytoskeletal remodelling.

Collectively, these findings define the molecular position of DUSP1 within the mechanotransduction signalling network. Mechanical stimulation activates the integrin/FAK/paxillin axis upstream of ERK/MAPK signalling, whereas DUSP1 acts mainly at the downstream MAPK amplification stage rather than directly regulating focal adhesion‐mediated mechanosensing. Therefore, YAP and F‐actin alterations following DUSP1 overexpression likely represent secondary effects of reduced MAPK activation and hypertrophic remodelling (Figure 7).

FIGURE 7.

DUSP1 regulates mechanically activated MAPK signalling without directly affecting focal adhesion mechanosensing. (a, b) Western blot analysis of ERK1/2, FAK and paxillin phosphorylation under mechanical stimulation and different inhibitor treatments. (c, d) Immunofluorescence staining of p‐FAK and F‐actin showing focal adhesion organisation and cytoskeletal remodelling. (e, f) Immunofluorescence staining of integrin and F‐actin showing focal adhesion organisation and cytoskeletal remodelling.

3.8. In Vivo Verification of Myocardial‐Specific Overexpression of DUSP1 in Alleviating HCM Progression

Given that DUSP1 is widely expressed in various tissues (Figure S1) and functions as an important regulator of cellular signalling, we established a cardiac‐specific DUSP1 overexpression model in mice with HCM‐like pathological cardiac remodelling to evaluate its effects on myocardial hypertrophy, fibrosis and cardiac function in vivo. Firstly, we recorded and observed the changes in mouse body weight over time (Figure 8a). Compared with the normal control group (Normal), the BW of mice in the HCM‐like remodelling group gradually decreased during the experimental period, while the BW of mice in the DUSP1 overexpression group (DUSP1 OE) and the DUSP1 overexpression + ERK inhibitor group (DUSP1 OE + ERK Inh) remained relatively stable. These findings suggest that DUSP1 overexpression partially prevented systemic deterioration associated with pathological cardiac remodelling. Secondly, we measured the HW/BW and LVW/BW in mice (Figure 8b,c). The HW/BW and LVW/BW ratios were significantly higher in the HCM‐like remodelling group than in the normal control group, while these ratios were significantly reduced in the DUSP1 overexpression and DUSP1 overexpression + ERK inhibitor groups, approaching normal levels. In addition, gross morphological examination of isolated hearts was performed to further validate cardiac remodelling (Figure S2). Compared with Normal mice, hearts from the HCM‐like remodelling group exhibited increased cardiac size and left ventricular enlargement, whereas DUSP1 overexpression markedly reduced these morphological abnormalities. Together, these results demonstrate that cardiac‐specific DUSP1 overexpression attenuates pathological cardiac hypertrophy in vivo. To further evaluate cardiac structure and function, transthoracic echocardiography was performed (Figure S3a). Compared with the Normal group, mice in the HCM‐like remodelling group exhibited increased left ventricular posterior wall thickness at diastole (LVPWd), accompanied by mild reductions in ejection fraction (EF) and fractional shortening (FS), indicating impaired cardiac remodelling and function (Figure S3b–d). In contrast, DUSP1 overexpression significantly reduced LVPWd and improved EF and FS values (Figure S3b–d). Doppler evaluation of the left ventricular outflow tract did not reveal obvious severe obstruction in this model, although structural remodelling was evident. Additionally, through HE staining and Masson staining of heart tissue (Figure 8d), we found that the cardiomyocytes in the HCM‐like remodelling group were arranged in a disordered manner with a high degree of fibrosis, while the cardiomyocytes in the DUSP1 overexpression and DUSP1 overexpression + ERK inhibitor groups were arranged more neatly and the degree of fibrosis was significantly reduced. Finally, we examined the mRNA expression levels of Dusp1, Anp, Bnp and β‐mhc in left ventricular tissues (Figure 8e). Our findings revealed a significant elevation in the expression of these genes in the HCM‐like remodelling group, whereas their expression levels were markedly reduced in both the overexpression DUSP1 group and the overexpression DUSP1 + ERK inhibitor group, approaching normal levels. Collectively, these findings demonstrate that myocardial‐specific restoration of DUSP1 expression suppresses pathological hypertrophic gene activation, reduces myocardial fibrosis and improves cardiac structure and function, thereby alleviating HCM‐like pathological cardiac remodelling.

FIGURE 8.

In vivo verification of myocardial‐specific overexpression of DUSP1 in alleviating HCM progression. (a) Line graph showing changes in BW over time in C57BL/6 mice, n = 6. (b, c) HW/BW and LVW/BW in mice, n = 6. (d, e) HE staining and Masson staining of mouse hearts. (e) qRT‐PCR was performed to detect the mRNA expression levels of Dusp1, Anp, Bnp and β‐mhc in the left ventricular tissue of the heart, with n = 6.

4. Discussion

HCM is a complex genetic myocardial disorder in which abnormal activation of mechanotransduction [7, 12] and the MAPK signalling pathway [20, 30] play pivotal roles. Although DUSP1 has been widely recognised as a negative regulator of MAPK signalling in various biological contexts [31], its specific role in regulating mechanically induced cardiac remodelling remains poorly understood. In this study, we extend the current understanding of DUSP1 by demonstrating its function in controlling mechanotransduction‐associated MAPK activation during pathological cardiac hypertrophy. The low expression of DUSP1 is closely associated with significantly activated mechanotransduction and MAPK signalling pathways in HCM myocardial tissue. Conversely, upregulation of DUSP1 effectively attenuates cardiomyocyte hypertrophy by suppressing ERK/MAPK activation and reducing downstream pathological remodelling responses.

Mechanotransduction serves as a bridge in the normal physiological functions of the heart, converting extracellular mechanical signals into intracellular molecular responses [32, 33]. By altering the stiffness and crosslinking degree of matrigel, our study simulated the effects of a gradient mechanical force environment on cardiomyocytes [34]. The results demonstrated that mechanotransduction signals significantly promoted the phenotypic changes associated with cardiomyocyte hypertrophy, which coincided with the upregulation of hypertrophic markers ANP, BNP and β‐MHC. Previous studies have demonstrated that mechanical stress activates multiple mechanosensitive pathways, including integrin‐associated signalling, YAP/TAZ signalling and MAPK cascades, which contribute to pathological cardiac remodelling [35]. However, the endogenous regulatory mechanisms that limit excessive mechanical stress‐induced signalling activation remain largely unknown. Further research revealed that mechanotransduction signals activate both the MAPK and Hippo/YAP signalling pathways, thereby regulating cardiomyocyte hypertrophy.

The MAPK signalling pathway plays a central role in cardiomyocyte hypertrophy and its abnormal activation is closely related to the pathological progression of HCM [36, 37]. We found that the phosphorylation level of ERK1/2 significantly increased in response to mechanotransduction signals. The use of ERK1/2 inhibitors effectively blocked the MAPK signalling pathway, significantly reducing the degree of cardiomyocyte hypertrophy and decreasing the expression levels of hypertrophic marker genes. These findings are consistent with previous studies demonstrating that excessive MAPK activation contributes to pathological myocardial remodelling [38]. Importantly, our study further suggests that MAPK activation represents a critical downstream effector of mechanically induced hypertrophic signalling.

As a key negative regulator of the MAPK signalling pathway [39, 40], DUSP1 has been implicated in cellular stress responses, inflammation and cardiovascular remodelling [41]. However, whether DUSP1 expression is altered in HCM myocardium and whether it participates in mechanical stress‐induced cardiac remodelling remain unclear. In this study, transcriptomic analysis of human HCM myocardial tissues revealed reduced DUSP1 expression accompanied by activation of mechanotransduction‐associated and MAPK signalling pathways. These findings suggest that loss of DUSP1‐mediated negative feedback may contribute to excessive activation of mechanically induced hypertrophic signalling. Overexpression of DUSP1 effectively inhibited cardiomyocyte hypertrophy and reversed the abnormal expression of hypertrophic marker genes. Mechanistically, our additional experiments demonstrated that DUSP1 primarily reduced ERK1/2 phosphorylation without markedly affecting upstream focal adhesion components, including FAK and paxillin. Therefore, DUSP1 does not appear to directly regulate the mechanical sensing machinery but rather functions as an endogenous brake that limits downstream ERK/MAPK amplification after mechanical stimulation. The alterations in F‐actin organisation observed following DUSP1 overexpression may therefore represent secondary consequences of reduced MAPK activation and attenuated hypertrophic remodelling. In vivo experiments further validated the protective role of DUSP1 in HCM. Mouse models overexpressing DUSP1 showed reduced HW index, left ventricular index and fibrosis, as well as decreased expression levels of markers indicating myocardial remodelling and impaired cardiac function, confirming the clinical potential of DUSP1 in alleviating the occurrence and development of HCM.

Despite the significant findings of this study on the role of DUSP1 in HCM, there are still some limitations. Firstly, the experiments were mainly based on cell lines and mouse models, which may not fully simulate the complex pathological processes of human HCM and further clinical validation is required. Secondly, the specific molecular mechanisms underlying the downregulation of DUSP1 expression with age remain unclear and need further investigation. Thirdly, although our findings establish DUSP1 as a regulator of mechanically activated MAPK signalling, whether DUSP1 interacts with specific mechanosensing complexes or other mechanotransduction‐related pathways requires further investigation. Additionally, although ERK1/2 inhibitor showed protective effects in vitro, further studies are needed to evaluate the translational potential and safety of targeting this pathway. Finally, this study focused on the effects of DUSP1 on mechanotransduction and the MAPK signalling pathway, with limited understanding of its interactions with other signalling pathways. Future research should comprehensively analyse the role of DUSP1 in the network of cardiac diseases.

In summary, this study identifies DUSP1 as an endogenous regulator of mechanically activated MAPK signalling and provides evidence that reduced DUSP1 expression contributes to pathological cardiac hypertrophic remodelling. Rather than directly blocking mechanical sensing structures, DUSP1 primarily limits downstream ERK/MAPK activation, thereby attenuating cardiomyocyte hypertrophy and fibrosis. These findings expand the current understanding of DUSP1 biology by linking its MAPK regulatory function with mechanical stress‐induced cardiac remodelling and suggest that restoration of DUSP1 activity may represent a potential strategy for controlling HCM‐like pathological remodelling.

Author Contributions

Xiao Zhong: formal analysis, roles/writing – original draft. Lingran Kong: data curation, roles/writing – original draft. Youjun Zhang: investigation. Jian Li: supervision. Shaofeng Guan: methodology, project administration, writing – review and editing. Xinkai Qu: conceptualisation, funding acquisition, project administration, writing – review and editing.

Funding

This research was supported by the Science and Technology Commission of Shanghai Municipality (23141901500) and the Shanghai Hospital Development Center (SHDC22023302).

Disclosure

The authors did not use generative AI or AI‐assisted technologies in the development of this manuscript.

Ethics Statement

This project has been approved by the Ethics Committee for Animal Experiments of the Experimental Animal Science Department of Fudan University (Approval Number: 2025‐HDYY‐074). All experiments were performed in accordance with the relevant guidelines and regulations.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Primer sequence.

Figure S1: Distribution of DUSP1 expression in various tissues and cell subsets throughout the body. (a) DUSP1 exhibits widespread expression in most tissues and cellular subpopulations throughout the body. (b) Pan‐cancer analysis of DUSP1 reveals that the dual specificity phosphatase DUSP1 is significantly downregulated in bladder urothelial carcinoma (BLCA), breast invasive ductal carcinoma (BRCA), cervical squamous cell carcinoma (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid carcinoma, papillary (THCA) and uterine corpus endometrial carcinoma (UCEC). Conversely, DUSP1 is significantly upregulated in kidney renal clear cell carcinoma (KIRC).

Figure S2: Gross pathology of the mouse heart.

Figure S3: Transthoracic echocardiography in mouse. (a) Echocardiographic views. (b) Statistical graph of LVPWd. (c) Ejection fraction. (d) Fractional shortening.

Figure S4: Figure 4a, b Full Membrane Image of Western Blot.

Figure S5: Figure 4f–g Full Membrane Image of Western Blot.

Figure S6: Figure 5b, e Full Membrane Image of Western Blot.

Figure S7: Figure 6a, b Full Membrane Image of Western Blot.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.