(2024) 22:361
Zhang et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03593-8

BMC Medicine

RESEARCH

Open Access

Hyperactivation of ATF4/TGF‑β1 signaling
contributes to the progressive cardiac fibrosis
in Arrhythmogenic cardiomyopathy caused
by DSG2 Variant
Baowei Zhang1†, Yizhang Wu1†, Chunjiang Zhou1, Jiaxi Xie2, Youming Zhang1, Xingbo Yang3, Jing Xiao3,
Dao Wu Wang4, Congjia Shan5, Xiujuan Zhou2, Yaozu Xiang3* and Bing Yang1*

Abstract
Background Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized with progressive
cardiac fibrosis and heart failure. However, the exact mechanism driving the progression of cardiac fibrosis and heart
failure in ACM remains elusive. This study aims to investigate the underlying mechanisms of progressive cardiac fibrosis in ACM caused by newly identified Desmoglein-2 (DSG2) variation.
Methods We identified homozygous DSG2F531C variant in a family with 8 ACM patients using whole-exome sequencing and generated Dsg2F536C knock-in mice. Neonatal and adult mouse ventricular myocytes isolated from Dsg2F536C
knock-in mice were used. We performed functional, transcriptomic and mass spectrometry analyses to evaluate
the mechanisms of ACM caused by DSG2F531C variant.
Results All eight patients with ACM were homozygous for DSG2F531C variant. Dsg2F536C/F536C mice displayed cardiac
enlargement, dysfunction, and progressive cardiac fibrosis in both ventricles. Mechanistic investigations revealed
that the variant DSG2-F536C protein underwent misfolding, leading to its recognition by BiP within the endoplasmic
reticulum, which triggered endoplasmic reticulum stress, activated the PERK-ATF4 signaling pathway and increased ATF4
levels in cardiomyocytes. Increased ATF4 facilitated the expression of TGF-β1 in cardiomyocytes, thereby activating cardiac
fibroblasts through paracrine signaling and ultimately promoting cardiac fibrosis in Dsg2F536C/F536C mice. Notably, inhibitionof the PERK-ATF4 signaling attenuated progressive cardiac fibrosis and cardiac systolic dysfunction in Dsg2F536C/F536C mice.
Conclusions Hyperactivation of the ATF4/TGF-β1 signaling in cardiomyocytes emerges as a novel mechanism
underlying progressive cardiac fibrosis in ACM. Targeting the ATF4/TGF-β1 signaling may be a novel therapeutic target
for managing ACM.
Keywords Arrhythmogenic cardiomyopathy, Desmoglein 2, Cardiac fibrosis, Transforming growth factor β1,
Activating transcription factor 4†

Baowei Zhang and Yizhang Wu contributed equally as first authors.

*Correspondence:
Yaozu Xiang
yaozu.xiang@tongji.edu.cn
Bing Yangbingyang@tongji.edu.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as longas you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article orparts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicatedotherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
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Zhang et al. BMC Medicine(2024) 22:361

BackgroundArrhythmogenic cardiomyopathy (ACM) is an inherited
cardiomyopathy characterized by ventricular arrhythmias, high risks of sudden death and progressive heart
failure [1]. Implantable cardioverter defibrillators andradiofrequency ablation have significantly reduced therisk of ventricular arrhythmias and sudden death, however, patients with ACM are still threatened by progressive heart failure. The pathological features of ACM aremarked by cardiomyocyte loss and cardiac fibrosis [2],which may contribute to the progressive heart failure.
Genetic variations in genes encoding desmosomal proteins account for approximately two-thirds of ACM cases[3]. Desmoglein 2 (DSG2), a transmembrane protein
encoded by DSG2, is an important component of desmosome complex [4]. Since its discovery as a causative gene
for ACM in 2006 [5], DSG2 has consistently ranked as thesecond most prevalent pathogenic gene associated with
ACM [6]. Additionally, ACM patients with pathogenic
DSG2 variations have high likelihood of left ventricularfibrosis and cardiac systolic dysfunction [7]. While previous studies have highlighted the involvement of disrupted
desmosomal adhesion, Wnt signaling, and inflammationin ACM [8–13], the mechanism underlying the progressive cardiac fibrosis in ACM caused by pathogenic DSG2
variations remains unclear. Furthermore, there is currently a lack of effective treatments to mitigate or reverse
the progressive cardiac dysfunction in ACM.
The endoplasmic reticulum (ER), the primary site forthe folding and post-translational modification of membrane proteins, plays a crucial role in maintaining protein homeostasis in eukaryotic cells [14]. However, underconditions of inflammation, hypoxia, or gene variation,
accumulated misfolded or unfolded proteins in the ER
could induce ER stress and activate the unfolded protein
response (UPR), promoting apoptosis, inflammation,
fibrosis, and other detrimental effects in various diseases[15]. However, the role of UPR in ACM caused by variant
DSG2 is unclear.
Previous studies, including our own, have identified
the DSG2 missense variant c.1592 T > G (rs200484060)
in patients with ACM [16–19]. This variant leads to thesubstitution of phenylalanine with cysteine at codonsite 531 (p. F531C), which is located in the extracellularanchoring domain of DSG2. In silico analysis indicateda high likelihood of this missense variant having a detrimental impact on the structure and function of the protein (Polyphen: probably damaging; SIFT: deleterious).
Nevertheless, the pathogenicity and underlying mechanisms of this variant in ACM are still not fully understood. Given that DSG2 is a transmembrane protein, we
postulated that the UPR may be activated and involved

Page 2 of 23

in the progressive cardiac fibrosis in ACM caused by the
DSG2F531C variant.
In the present study, we conducted genetic sequencing of a five-generation family affected by ACM. All eightpatients diagnosed with ACM were homozygous for
DSG2F531C variant. Subsequently, we developed a knockin mouse model carrying the mouse-equivalent form
(Dsg2F536C) of this variant. Homozygous knock-in mice
exhibited overt cardiac phenotypes highly similar to theclinical manifestations of ACM. Through mechanistic
investigations, we demonstrated that the variant DSG2(vDSG2) in cardiomyocytes was misfolded and triggered
protein kinase R like ER kinase (PERK)—activating transcription factor 4 (ATF4) signaling, leading to an increasein the expression of transforming growth factor β1 (TGFβ1). This activated cardiac fibroblasts via paracrine signaling, ultimately promoting cardiac fibrosis in Dsg2F536C/
F536C
mice. Inhibition of the PERK-ATF4 signaling attenuated the elevated TGF-β1 levels, ameliorated the progressive cardiac fibrosis and dysfunction in Dsg2F536C/
F536C
mice. These findings highlight the hyperactivationof the ATF4/TGF-β1 signaling in cardiomyocytes as anovel mechanism driving progressive cardiac fibrosis in
ACM, suggesting that targeting the ATF4/TGF-β1 signaling could serve as a potential therapeutic strategy for
ACM.

Methods
Human study

This study involved 48 individuals from a large family in
China. The proband was diagnosed with ACM duringhospitalization for ventricular tachycardia at the First
Affiliated Hospital of Nanjing Medical University. All
family members were clinically evaluated according to
the guideline for management of ACM, which included
clinical history, 12-lead electrocardiography (ECG) andtransthoracic echocardiography [20, 21]. In addition,
24-h ambulatory ECG and cardiac magnetic resonanceimaging (MRI) were performed on some participants.
This study was approved by the Ethics Committee of theFirst Affiliated Hospital of Nanjing Medical University(NO.2011-SR-014) and informed consent was obtained
from all participants prior to evaluation and testing.
Genomic DNA from all family members was extracted
from peripheral blood samples using QIAamp DNA
Blood Mini Kits (Qiagen). Sanger sequencing was performed to analyze the five desmosome-encoding genes(DSG2, DSC2, JUP, PKP2, DSP) as well as the TMEM43
and PLN genes, as previously reported [16]. To further
explore whether additional genes beyond those mentioned above were associated with ACM, whole-exome
sequencing was performed in the proband using theZhang et al. BMC Medicine(2024) 22:361

Agilent SureSelect Human All Exon V6 Kit on the Hiseq
X Ten. The ACMG criteria were applied to evaluate thepathogenicity of identified variants. Novel nonsynonymous variants were assessed in a population of 950 ethnically matched healthy controls.
Animal experiments

Animal experiments were approved by the Institutional
Animal Care and Use Committee of Nanjing Medical
University (approval number: IACUC-14030160). All animal procedures were performed in accordance with the
National Institutes of Health Guide for the Care and Useof Laboratory Animals. General anesthesia was induced
using a 1%-3% inhaled isoflurane/oxygen mixture. The
mice were housed under specific pathogen-free conditions with 12-h dark–light cycles and had free access tofood and water. Animals of both sexes were used in theexperiments without bias. All mice were euthanized bycervical dislocation after deep anesthesia using a 5% isoflurane/oxygen mixture.
Dsg2F536C mice were generated using homologous
recombination technology as previously described [22,23]. Briefly, the targeting construct, which included the
F536C point variant and a neomycin cassette flanked by
Loxp sites, was designed and electroporated into embryonic stem cells (ESCs) (Additional file 1: Fig. S3a). After
genotyping by Southern blotting, the targeted ESCs were
injected into C57BL/6 J blastocysts to generate mousechimeras (Additional file 1: Fig. S3b). By crossing thechimeras with CMV-Cre mice, the neomycin cassette
was removed, and the heterozygous Dsg2F536C/WT progeny were generated after backcrossing with C57BL/6 J
mice. This backcrossing process was repeated for at least
4 generations to eliminate background diversity. Subsequently, heterozygous intercrosses of Dsg2F536C/WT mice
were used to obtain cohorts of homozygous Dsg2F536C/
F536C, heterozygous Dsg2F536C/WT and wild-type Dsg2WT/
WT
mice for this variant. Further sequencing analysis forthe polymerase chain reaction (PCR) product confirmed
the positive mutation leading to the substitution of phenylalanine with cysteine (Additional file 1: Fig. S3c). Subsequently, PCR strategy was carried out to identify thewild-type allele (368 bp) and the mutant allele (466 bp)(Additional file 1: Fig. S3d).
Echocardiography was performed under inhaled anesthesia with 1% to 3% isoflurane/oxygen mixture. The
mice were maintained at an appropriate state, with heart
rate at about 450 bpm and temperature at about 37 °C.
Images were obtained using a mouse transthoracic
echocardiography system equipped with a 30-MHz linear transducer (Vevo2100; VisualSonics Inc) at a sweep
speed of 200 mm/s. The left ventricular geometry andsystolic function were assessed in the short-axis view

Page 3 of 23

at the level of the papillary muscles. All the procedures
were performed by the same researcher, who was blinded
to the genotype.
Electrocardiographic measurements were performed
under the same anesthesia conditions as the echocardiography. Limb-lead surface ECGs were recorded using
subcutaneous 23-gauge needle electrodes attached toeach limb. The ECGs were continuously recorded by
the Powerlab acquisition system (AD Instruments) andanalyzed with IOX Software (EMKA) and Chart5 Pro
analysis software (AD Instruments). To accurately measure the duration and amplitude of the P wave and QRS
complex, we selected ECG segments with a stable baseline and excluded any instances of arrhythmias. The
P wave was marked from the beginning of the P wave
to the point where it returned to the iso-electrical line.
The QRS complex was measured from the onset of thefirst deflection of the QRS complex to the point where
the last returned to the baseline [24]. The measurement
and analysis of ECG parameters were performed by thesame professional cardiac electrophysiologist, who was
blinded to the genotype. After ECG recording, the mice
were euthanized and their hearts were excised for further
study, either by freezing at -80 °C or by fixing in 4% paraformaldehyde (PFA)/OCT Compound (Sakura).
Histological analysis

Frozen Sects. (5 μm) were used for immunofluorescencestaining. The sections were fixed with 4% PFA for 1 h andthen washed with PBS. Antigen retrieval was performedby boiling in sodium citrate for 2 min, followed by permeabilization with 1% Triton X-100 (Sigma-Aldrich)for 15 min. After washing with PBS, the sections were
blocked with 1% bovine serum albumin (BSA) for 45 min
and incubated with primary antibodies at 4℃ overnight.
The sections were then washed in PBS and incubatedwith the secondary antibodies for 1 h at room temperature. Following additional PBS washes, the sections were
stained with DAPI (Invitrogen) and mounted with Prolong Gold Antifade Mountant (Invitrogen). The slides
were scanned with laser scanning confocal microscopy(Nikon) and analyzed with Image Pro Plus 6.0.
To assess the extent of fibrosis throughout the ventricles, the paraffin-embedded hearts were serially sectioned from the apex to the base into 10 µm slices. At
least five mice of the same age and genetic backgroundwere included in each group to assess cardiac fibrosis
and ventricular wall thickness (n ≥ 5 in each group). For
each heart, eight levels of paraffin sections from the apex
to the base were selected for Masson’s trichrome staining (Nanjing Jiancheng Bioengineering Institute), andobserved under a light microscope. The collagen volume fraction (CVF) and ventricular wall thickness wereZhang et al. BMC Medicine(2024) 22:361

measured using Image Pro Plus 6.0. The CVF was calculated as the ratio of the collagen area to the total tissue
area in each slice. The CVF of the entire heart was determined by averaging the CVF values from the 8 selected
tissue sections. The measurement method for ventricularwall thickness is the same as in our previous publication[25]. Briefly, the thickness of the left ventricular free wall,
the interventricular septum, and the right ventricular
free wall were measured at the level of the left ventricular papillary muscles. The minimal value among thesemeasurements was selected as the final ventricular wall
thickness.
Paraffin Sects. (5 μm) were prepared and stained for
immunohistochemistry according to standard procedures. After baking at 60 °C for 30 min, the sections weredeparaffinized, underwent antigen retrieval, and were
subjected to endogenous peroxidase and protein blocking. Primary and secondary antibody incubations were
performed sequentially, followed by DAB staining andhematoxylin counterstaining. Isotype control antibody
staining was performed simultaneously. The slides werethen scanned with light microscopy and analyzed using
Image Pro Plus 6.0.
Plasmid construction and transfection

HEK293T cells were purchased from ATCC and cultured
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The pcDNA3.1 vector containing
cDNA of mouse Dsg2 and a C-terminal 3xFlag epitope
was purchased from Youbio Bio lnc. PCR with high-fidelity DNA polymerase (Q5 High-Fidelity 2X Master Mix,
NEB) was performed to construct the plasmid with a
Dsg2 site mutation. After confirming the Dsg2 variant by
DNA sequencing, pcDNA3.1 vectors with wild-type Dsg2
and variant Dsg2 were used to transfect HEK293T cells(Lipo 8000 transfection reagent, Beyotime Bio.) to overexpress the wild-type DSG2 (wtDSG2) and vDSG2.
HL‑1 cells culture and lentiviral transfection

HL-1 cells were cultured in dishes coated with gelatin/fibronectin and maintained in Claycomb culture
medium supplemented with 10% FBS, 0.1 mM norepinephrine, 2 mM l-glutamine and 1% penicillin/streptomycin. Wild-type and variant mouse Dsg2 cDNA
with AscI and PacI restriction sites were cloned from
pcDNA3.1-mDsg2-3 × Flag (C). Both Dsg2 cDNA werecloned back into pCDH plasmid containing 3xFlag tag
and ampicillin resistance selection marker. These vectors
were transformed into DH5α competent cells, and single
clones were selected from LB Agar plates to grow in the
LB broth. After verification by PCR and DNA sequencing, plasmids were purified using the Plasmid Midi Kit

Page 4 of 23(QIAGEN). Plasmids containing inserted Dsg2 cDNA
were mixed with psPAX-2 and VSVG plasmids at a ratioof 10:3:2, and used to transfect HEK293T cells with EZ
Trans reagent. Six hours post-transfection, the culture
medium was replaced with fresh DMEM complete culture medium. Virus particles were harvested from theculture medium of HEK 293 T cells after 48 h incubation. The viral medium was mixed with HL-1 cell culture
medium at a ratio of 1:1 to transfect the HL-1 cells. After48 h of lentiviral transfection, fresh HL-1 cell culture
medium containing puromycin (2ug ­mL−1) was used to
select HL-1 cells transfected with lentivirus. Stable clones
were amplified after 10 days of puromycin selection andwere identified by qPCR and immunoblotting.
Knockdown of Atf4 in HL‑1 cells via CRISPR‑Cas9

Knockdown of Atf4 was performed using the CRISPRCas9 system. The sgRNA sequence was designed using
the online CRISPR guide design software (Benchling,https://​www.​bench​ling.​com) and synthesized by Songon
Biotech (Shanghai). After phosphorylation and annealing, the sgRNA containing the BsmBI restriction site wascloned into the lenti-CRISPRv2-puro vector, followed bytransformation into DH5α-competent cells and plasmids
purification. The supernatant containing lentivirus particles from HEK293T cells was collected 48 h after cotransfection with lenti-CRISPRv2, psPAX-2 and VSVG
plasmids. HL-1 cells were then transfected with the viralsupernatant for 48 h and subsequently selected withpuromycin (2ug ­mL−1) for 10 days. The knockdown ofAtf4 was verified by Western blotting.
Primary ventricular myocyte isolation and treatment

Adult mice ventricular myocytes (AMVM) were isolatedusing Langendorf-free method [26]. Briefly, mice wereanesthetized by inhaling 1% to 2% isoflurane/oxygen
mixture, and the chest was opened to expose the heart.
The descending aorta was cut, followed by injection of
7 ml EDTA buffer into the right ventricular. After isolation of the heart from the chest, 10 ml EDTA buffer, 3 ml
perfusion buffer, and 30 to 40 ml collagenase buffer weresequentially injected into the left ventricular at the samesite. Both ventricles were gently pulled into 1 ­mm3 tiny
pieces using forceps, and then the cardiomyocytes weredissociated by gentle trituration. The enzyme activity ofcollagenase was inhibited by stop buffer, and the cell suspension was passed through a 100-μm filter. The cardiomyocytes were isolated from other non-cardiomyocytesby 4 times gravity settling, which included 3 intermediate calcium reinduction buffers to restore the physiological condition of the cardiomyocytes. The cells in every
supernatant were collected as non-cardiomyocytes.

Zhang et al. BMC Medicine(2024) 22:361

For obtaining neonatal mice ventricular myocytes(NMVM) and fibroblasts, hearts were excised from the
0–3 days old Dsg2WT/WT or Dsg2F536C/F536C mice pups,
and rinsed with cold PBS. After removing atria tissue,
both ventricular were sheared and digested in 0.25%
Trypsin (EDTA free) for 7—9 h at 4 ℃, followed by digestion in DMEM medium supplemented with 0.1% collagenase type-II and 1% BSA for 10 min at 37℃. The buffer
solution was then transferred to a tube with DMEM and
10% FBS to stop the digestion. This process was repeated
3–4 times until the cardiac tissue was fully digested. After
passing the 100-μm filter, the solution was centrifuged
at 800 rpm for 5 min, and the pellet was resuspended in
DMEM supplemented with 10% FBS and 1% penicillin/
streptomycin. The cells were plated in a 6-cm dish andincubated at 37 °C for 90 min to allow the fibroblasts toadhere. Then the supernatant containing cardiomyocytes
was collected, plated in 12-well culture plate at 1*106
cells per well, and maintained in medium containing
DMEM, M199, 10% FBS, 1% penicillin/streptomycin, and100 μM 5-Bromo-2’-deoxyuridine for further study. For
the p-PERK inhibition assay, NMVMs from Dsg2F536C/
F536C
mice were treated with GSK2606414 for 24 h, subsequently maintained in normal culture medium for 24 h,and subjected to protein assay. For adenovirus transfection, NMVMs were transfected adenovirus for 12 h, subsequently maintained in normal culture medium for 24 hand subjected to protein assay.
Immunoblotting, immunoprecipitation and mass
spectrometry

Heart tissue samples and cells sample were lysed withRIPA buffer (Cell Signaling Technology) supplemented
with protease and phosphatase inhibitor cocktail (Roche).
The protein fractions were separated by centrifugationand the concentrations were determined by the Pierce
BCA kit (Pierce). Protein denaturation was achieved bymixing with Laemmli sample buffer supplemented with
10% β-mercaptoethanol (Sigma-Aldrich) and boiled.
Equal amounts of protein extracts were loaded and electrophoresed on SDS–polyacrylamide gels, followed byblotting onto pre-equilibrated Immobilon-P polyvinylidene fluoride membrane (Millipore). The protein
fractions were probed with primary antibodies overnightat 4 °C, followed by incubation with HRP-conjugated secondary antibody. The bands were detected using SuperSignal West Femto substrate (Pierce) on a ChemiDoc
Imaging system (Bio-Rad). Band intensity quantification
was performed using ImageJ. To identify ubiquitinationof vDSG2, immunoprecipitation with anti-Flag antibody was conducted in HL-1 cells with stable expression of vDSG2 and wtDSG2. HL-1 cells were lysed with
IP lysis buffer (Pierce) supplemented with protease andPage 5 of 23

phosphatase inhibitor cocktail (Roche). Lysates wereseparated by centrifugation, and the supernatants wereincubated with anti-Flag antibody and tumbled overnight at 4 °C. Protein A agarose beads (Cell Signaling
Technology) were then added into the lysates, and tumbled for 2 h at 4 °C. After being rinsed with IP lysis buffer
five times, the bead-bound proteins were solubilizedwith Laemmli sample buffer supplemented with 10%β-mercaptoethanol and boiled to denature, followed byimmunoblotting to probe the target protein.
To screen for potential vDSG2-interacting proteins,cell lysates purified by immunoprecipitation with antiFlag antibody in HL-vDSG2 cells were loaded onto SDSPAGE gels and stained with Coomassie Brilliant Blue
(Beyotime). Bands were exercised from the gel, digested,and subjected to mass spectrometry analysis via online
nano-reversed phase liquid chromatography (Oebiotech).
The MS/MS data were analyzed using ProteomeDiscoverer 2.5.
Quantitative real‑time polymerase chain reaction (qPCR)

Total RNA samples were extracted from mouse heart tissue and HL-1 cells using Trizol reagent (Invitrogen) and
the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. A total of 1000 ng mRNA was used tosynthesize cDNA using iScript reagent (Bio-Rad). Quantitative PCR (qPCR) was performed using SYBR Greenmix (Bio-Rad). The 2­ −ΔΔCT method was employed to estimate the mRNA levels of target genes, calculating foldchanges relative to the expression of GAPDH or β-actin,
which served as endogenous controls.
RNA‑sequencing and data analysis

Bulk RNA-sequencing (RNA-Seq) was conducted to
explore the potential mechanisms underlying the pathogenesis of ACM caused by Dsg2F536C. Total RNA wasextracted from the hearts of Dsg2F536C/F536C, Dsg2F536C/
WT, and Dsg2WT/WT mice at 10 weeks of age. Sequencing was performed on the Hiseq-PE150 instrument
using paired-end sample preparation chemistry. The raw
RNA-Seq reads were aligned to the mm10 genome using
Hisat2 (version 2.1.0) with parameters ‘–no-unal –nomixed –no-discordant’. Gene count values were quantified using Featurecounts (version 2.0.0). Gene transcript
per million (TPM) values were generated by custom R
scripts. DESeq2 (version 1.28.1) was utilized for differential analyses with cutoffs FoldChange >  = 2 and adjusted
P value <  = 0.05. Gene ontology (GO) analysis for differentially expressed genes was performed using DAVID(version 6.8). Gene Set Enrichment Analysis (GSEA)
employed the ‘GSEA’ function of the clusterProfiler R
package (version 4.6.0).

Zhang et al. BMC Medicine(2024) 22:361

Page 6 of 23

Fig. 1 Clinical characteristics and the family pedigree of the patients with the c.1592 T > G (p.Phe531Cys) variant in DSG2. a Conventional
electrocardiography showed T-wave inversion and epsilon waves (black arrows) in the precordial leads. b Transthoracic echocardiography showedthinning of the apical myocardium of the right ventricle, the formation of aneurysm (arrow in the upper figure), and the widening of rightventricular outflow tract (arrow in the lower figure). c Fibrosis in both the left ventricle (arrow in the upper figure) and right ventricle (arrowin the lower figure) identified by cardiac LGE + MRI. d Family pedigree of the ACM patients with the c.1592 T > G (p.Phe531Cys) variant in DSG2. The
filled symbols represented clinically diagnosed ACM patients, and open symbols were heathy individuals without clinical characteristics of ACM.
The proband was marked with an arrow ((III-9)). e Sanger sequencing was performed to validate the variant identified by targeted sequencing
and whole-exome sequencing. f The variant (F531C) was located in an evolutionary highly conservative area across multiple species

snRNA‑seq analysis

An available processed single-nucleus RNA sequencing (snRNA-seq) dataset containing human ACM andnormal heart tissues was downloaded from CellxGene(https://​cellx​gene.​czisc​ience.​com/​colle​ctions/​e7534​2a8-​
0f3b-​4ec5-​8ee1-​245a2​3e0f7​cb) [27]. Cardiac cell types
were already well-clustered and annotated in the study,
and we used the annotations directly from the dataset’s metadata. Data from normal ventricular tissues
was extracted and visualized with Dimplot using Seurat(4.2.0). The expression levels of DSG2 in different cellclusters were showed with FeaturePlot and VlnPlot.
Statistical analysis

Continuous variables are presented as mean ± standard
deviation. The Shapiro–Wilk normality test was utilized
to assess data distribution. For normally distributed data,
unpaired t-tests (for two datasets) or one-way ANOVA
with Tukey’s multiple comparison test (for three datasets) was employed. Alternatively, the Mann–Whitney U
test (for two datasets) or Kruskal–Wallis test with Dunn’s
multiple comparison test (for three datasets) was wereused for non-normally distributed data. All data were
processed and analyzed using GraphPad Prism 8.0.2,
with significance set at a two-tailed p-value < 0.05.

Results

Identifying DSG2F531C variant in an ACM family

Clinical evaluations were performed systematically for
all family members, eight of whom were diagnosed with
ACM according to the updated diagnostic criteria for
ACM [20, 21]. All patients with ACM presented with palpitations and dyspnea, and the eldest sister of the index
patient died suddenly at the age of 50 (III-1) (Additional
file 2: Table S1). The mean age of onset of symptomatic
ACM was 50.5 ± 6.38 years. Typical characteristics ofsurface ECG in patients with ACM include complete
right bundle branch block, prolonged terminal activation duration of QRS (≥ 55 ms) in lead V1-V3, typicalepsilon wave, abnormal inverted T-waves in precordial leads (Fig. 1a, Additional file 2: Table S1), and frequent ventricular premature complex and/or ventriculartachycardia with left bundle branch block morphology(Additional file 1: Fig. S1, Additional file 2: Table S1).

Zhang et al. BMC Medicine(2024) 22:361

Echocardiographic findings indicated diffuse right ventricular dilation and regional akinesia or aneurysm in theright ventricular free wall in all patients with ACM, threeof whom had abnormalities in the left ventricle (II-1, III3, and III-9) (Fig. 1b, Additional file 2: Table S1). Cardiac
MRI revealed a paradoxical movement of the apical myocardium of the right ventricle, systolic myocardial ectasia,
aneurysm formation in the right ventricle, and fibrosis inboth ventricles (Fig. 1c, Additional file 2: Table S1).
The grandparents (I-1 and I-2) and parents (II-I & II-2) of
the proband were consanguineous (Fig. 1d). We identified
a homozygous DSG2 missense variant, c.1592 T > G, in theproband (III-9, Fig. 1d, e). This missense variant leads to anamino acid change p.F531C, which is considered detrimental according to PolyPhen-2 and SIFT analyses. This variant
was observed in 0.093% of alleles (heterozygous state) fromindividuals of East Asian descent in the Exome Aggregation Consortium. In addition, we identified only one heterozygous DSG2F531C variant among 950 healthy controls.
Bioinformatic analysis confirmed that the amino acid at thevariant site was highly conserved among DSG2 homologs inlower organisms and vertebrates, and even in other members of the cadherin family (Fig. 1f, Additional file 1: Fig.
S2a). Furthermore, whole-exome sequencing in the proband
revealed no additional pathogenic or likely pathogenic
variants associated with ACM. In addition, we performed
cascade screening of the remaining family members. Interestingly, all the family members diagnosed with ACM were
homozygous carriers of this variant. All heterozygous carriers (12 members) were absent from the ACM clinical characteristics (Fig. 1d, Additional file 2: Table S1).
Homozygous Dsg2F536C mice presented with cardiac
enlargement, systolic dysfunction, and progressive cardiac
fibrosis

Phenylalanine at the 536th position of mouse DSG2 washomologous to phenylalanine at the 531st position ofhuman DSG2 (Additional file 1: Fig. S2b). Therefore, we

Page 7 of 23

generated Dsg2F536C knock-in mice using homologous
recombination technology to validate the pathogenicity of Dsg2F536C variant and investigate its mechanism in
ACM pathogenesis (Additional file 1: Fig. S3). Echocardiography was performed to assess the cardiac geometry
and function in homozygous Dsg2F536C/F536C, heterozygous Dsg2F536C/WT, and Dsg2WT/WT mice at 10 weeks ofage, which were considered adult mice. Left ventricularejection fraction and left ventricular fractional shortening were markedly lower in Dsg2F536C/F536C mice than in
Dsg2WT/WT, accompanied by an increased left ventricularinternal diameter (Fig. 2a, b). In addition, the left ventricular wall thickness was lower in Dsg2F536C/F536C mice
(Additional file 1: Fig. S4). These findings indicated that
Dsg2F536C/F536C mice had abnormalities in cardiac geometry and function. Electrocardiography showed increased
P-wave duration and amplitude in Dsg2F536C/F536C mice at
10 weeks of age as well as prolonged QRS duration anddecreased QRS amplitude (Fig. 2c, d), implying depolarization and conduction abnormalities in Dsg2F536C/
F536C
mice. No differences in echocardiography or electrocardiography were observed between Dsg2WT/WT and
Dsg2F536C/WT mice (Fig. 2a-d, Additional file 1: Fig. S4).
The loss of cardiomyocytes and replacement of fibrous
tissue are the most important hallmarks of ACM pathology. Masson’s trichrome staining demonstrated obvious
fibrotic infiltration in both ventricles of Dsg2F536C/F536C
mice (Fig. 2e). The CVF of hearts from Dsg2F536C/F536C
mice was significantly higher than that of hearts from
Dsg2F536C/WT and Dsg2WT/WT mice (Fig. 2f ). Fibrosis was
mainly located at the free wall of both ventricles, butless so in the interventricular septum (Fig. 2f ). The freewalls of both ventricles in Dsg2F536C/F536C mice were significantly thinner than those in Dsg2F536C/WT and Dsg2WT/
WT
mice (Fig. 2g). Furthermore, we examined the fibrosis
patterns in the hearts of Dsg2F536C/F536C mice. Replacement fibrosis secondary to cardiomyocyte loss, interstitial fibrosis, and perivascular fibrosis were all evident(See figure on next page.)
Fig. 2 Homozygous Dsg2F536C mice had cardiac enlargement, systolic dysfunction, and progressive cardiac fibrosis. a Representative imagesof M-mode echocardiography in 10-week-old Dsg2WT/WT (WT), Dsg2F536C/WT (Het) and Dsg2F536C/F536C (Hom) mice. b Quantification analysis of LV
geometry (LVDD and LVDS) and systolic function (LVEF and LVFS) among 10-week-old WT, Het and Hom mice. N = 13 in WT group, N = 16 in Het
group, and N = 19 in Hom group. c Representative images of ECG recorded in 10-week-old WT, Het and Hom mice. d Quantification of the duration
and amplitude of P wave and QRS complex among 10-week-old WT, Het and Hom mice. N = 6 in each group. e Representative images of Masson’s
staining in 10-week-old WT, Het and Hom mice. N = 6 in each group. f Quantification of cardiac collagen volume fraction in 10-week-old WT,
Het and Hom mice. g Quantification of thickness of both free ventricular walls in 10-week-old WT, Het and Hom mice. h Representative imagesof cardiac replacement fibrosis, interstitial fibrosis, and perivascular fibrosis in 10-week-old Hom mice. i Representative images of cardiac
progressive fibrosis in Hom mice. j Quantification of cardiac collagen volume fraction in Hom mice from 2-week-old to 10-week-old (N = 5
in each group). ***: p < 0.001 compared with the hearts from 2-week-old mice, ###: p < 0.001 compared with the hearts from 4-week-old mice. k
Quantification of ventricular wall thickness in Hom mice from 2-week-old to 10-week-old (N = 5 in each group). * p < 0.05 compared with the hearts
from 2-week-old mice, **p < 0.01 compared with the hearts from 2-week-old mice, ***: p < 0.001 compared with the hearts from 2-week-old mice,###: p < 0.001 compared with the hearts from 4-week-old mice. Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used
to analyze the data

Zhang et al. BMC Medicine(2024) 22:361

Fig. 2 (See legend on previous page.)

Page 8 of 23

Zhang et al. BMC Medicine(2024) 22:361

in the hearts of Dsg2F536C/F536C mice (Fig. 2h). To clarify
the disease course and progression of ACM, hearts from
Dsg2F536C/F536C, Dsg2F536C/WT and Dsg2WT/WT mice atearly stages (2 and 4 weeks) were excised for Masson’s trichrome staining. Obvious fibrosis was observed in both
ventricles of 4-week-old Dsg2F536C/F536C mice (Fig. 2i,
Additional file 1: Fig. S5a). Unexpectedly, fibrosis wasobserved in some Dsg2F536C/F536C mice at 2 weeks of age
and was locally scattered in the epicardium of both ventricles (Fig. 2i). Therefore, cardiac fibrosis in Dsg2F536C/
F536C
mice had several characteristics: presence at a very
early stage (2 weeks), early involvement of both ventricles, rapid progression from the epicardium to the endocardium (Fig. 2i, j). In addition, the thicknesses of both
ventricles in Dsg2F536C/F536C mice decreased progressively(Fig. 2k, Additional file 1: Fig. S5c). Consistent with theechocardiography and electrocardiography results, nopathological characteristics of ACM were observed in thehearts of Dsg2F536C/WT mice, indicating that heterozygous
Dsg2F536C carriers might be exempt from ACM.
Both the pedigree investigation and animal study
showed that ACM was absent in heterozygous DSG2F531C
carriers, which differed from most desmosome generelated ACM cases [28]. To confirm this finding, apharmacological stress study with isoproterenol (ISO)intraperitoneal injection (20 mg ­Kg−1 body weight for
2 weeks), which was used to increase mechanical stressand accelerate the progression of ACM [29], was performed in Dsg2F536C/WT and Dsg2WT/WT mice (Additional file 1: Fig. S6a). Although echocardiography results
showed impaired cardiac systolic function after a twoweek ISO injection, the changes were similar between
Dsg2F536C/WT and Dsg2WT/WT mice (Additional file 1: Fig.
S6b, c). Interstitial fibrosis induced by ISO was observedin the hearts of both Dsg2F536C/WT and Dsg2WT/WT mice,but there was no significant difference in the cardiac
fibrosis between the two groups (Additional file 1: Fig.
S6d, e). In addition, the distribution of cardiac fibrosis induced by ISO in Dsg2F536C/WT mice was mainly inthe endocardium and septum, which was different fromPage 9 of 23

that in Dsg2F536C/F536C mice, but was similar to the distribution of cardiac fibrosis in WT mice treated with ISO(Additional file 1: Fig. S6d, e). In addition, there wereno significant differences in the thickness of either ventricle between Dsg2F536C/WT and Dsg2WT/WT mice (Additional file 1: Fig. S6d, e). Therefore, fibrosis in the heartsof Dsg2F536C/WT mice was due to the direct pro-fibrotic
effects of ISO [30], which was also present in WT mice.
These results indicate that ACM was absent from heterozygous DSG2F531C variant carriers, even under severe
mechanical stress and sympathetic hyperactivity.
DSG2‑F536C was degraded through the ubiquitin–
proteasome system

Western blotting and immunohistochemistry were performed to investigate the effect of the variant on the level
and location of DSG2. Unexpectedly, there was nearly
undetectable DSG2 in immunoblotting in hearts from
Dsg2F536C/F536C mice (Fig. 3a). Immunohistochemistrydemonstrated that DSG2 mainly located at desmosomesin the hearts from Dsg2WT/WT and Dsg2F536C/WT mice, but
was absent in Dsg2F536C/F536C mice (red arrow in Fig. 3b).
The levels of Dsg2 mRNA from the hearts of Dsg2F536C/
F536C
mice were similar to those in Dsg2F536C/WT and
WT/WT
Dsg2
mice (Additional file 1: Fig. S7a), excluding
the possibility of abnormal transcription or degradation of the variant Dsg2 mRNA. The overexpression ofvDSG2, tagged with a C-terminal 3xFlag, in HEK293T
cells effectively ruled out the possibility that the antibody
was unable to recognize vDSG2 (Additional file 1: Fig.
S7b). We speculated that vDSG2 might be modified anddegraded after translation. 3D models of wtDSG2 andvDSG2 protein were constructed using AlphaFold2 [31].
Root Mean Square Deviation (RMSD) was used to quantify the differences between the structure of wtDSG2
and vDSG2. Although there was no significant change
in protein structure at the variant site (RMSD = 0.876),
the global structure of vDSG2 was significantly altered(RMSD 16.679, Fig. 3c, Additional file 1: Fig. S7c). Further comparison of the three-dimensional structures of(See figure on next page.)
Fig. 3 DSG2-F536C was degraded via ubiquitin–proteasome system. a Representative western blot and quantification of DSG2 protein levelsin the hearts from 10-week-old Dsg2WT/WT (WT), Dsg2F536C/WT (Het) and Dsg2.F536C/F536C (Hom) mice. N = 6 in each group. b Representative imagesof immunohistochemical staining and quantification of DSP and DSG2 in the hearts of 10-week-old WT, Het, and Hom mice. c The predicted 3Dmodel of wild-type DSG2 protein (left) and the variant DSG2-F536C protein (right) via AlphaFold2. d Representative western blot and quantificationof DSG2-Flag protein levels in HL-1 cells (Ctrl), HL-1 cells with stable expression of vDSG2 (vDSG2), and HL-1 cells with stable expression of wtDSG2(wtDSG2). N = 3 in each group. e Mass spectrometry analysis revealed several candidates interacting with vDSG2. f Representative western blot
and quantification of the ubiquitination of DSG2-Flag followed immunoprecipitation with anti-Flag antibody. N = 3 in each group. g Representative
western blot and quantification of DSG2-Flag protein levels in HL-1 cells with stable expression of vDSG2 treated with DMSO, MG132 (10 μM for 8 h),
or CQ (25 μM for 8 h). N = 3 in each group. h Representative western blot and quantification of DSG2-Flag in HL-1 cells with stable expressionof vDSG2 or vDSG2 with lysine site mutation. N = 3 in each group. Ordinary one-way ANOVA with Turkey’s multiple comparisons test or Unpaired
t-test were used to analyze the data, * p < 0.05, **: p < 0.01, ***: p < 0.001

Zhang et al. BMC Medicine(2024) 22:361

Fig. 3 (See legend on previous page.)

Page 10 of 23

Zhang et al. BMC Medicine(2024) 22:361

vDSG2 and wtDSG2 revealed abnormal folding in theintracellular segment (amino acids 630–781) of vDSG2(red segment in vDSG2 and yellow segment in wtDSG2in Additional file 1: Fig. S7d). To validate the degradation of vDSG2 in cardiomyocytes, HL-1 cells with stable
expression vDSG2-Flag (HL-vDSG2) and wtDSG2-Flag(HL-wtDSG2) were generated by lentiviral transfection
and puromycin screening. The mRNA levels of Dsg2 weresimilar between HL-vDSG2 and HL-wtDSG2 cells (Additional file 1: Fig. S7e), but the protein levels of DSG2-Flag
in HL-vDSG2 cells were significantly decreased (Fig. 3d),which was in accordance with the nearly undetectable
DSG2 in the hearts from Dsg2F536C/F536C mice.
Mass spectrometry analysis of proteins purified byimmunoprecipitation with an anti-Flag antibody in HLvDSG2 cells showed that ubiquitination-related proteins(UBC, UBAP2L, TRIM21, USP10) were potential candidates interacting with vDSG2 (Fig. 3e, Additional file 3:
Table S2), suggesting that vDSG2 might be modified
by ubiquitin in cardiomyocytes. The ubiquitin–proteasome system (UPS) and autophagy-lysosome system are
the major pathways for clearing misfolded and aggregation-prone proteins in cardiomyocytes [32]. We purified
DSG2-Flag via immunoprecipitation with an anti-Flag
antibody in HL-1, HL-vDSG2, and HL-wtDSG2 cells tocompare the ubiquitination levels between vDSG2 and
wtDSG2. There was a robust increase in the ubiquitination of vDSG2 compared to that of wtDSG2 (Fig. 3f ). HLvDSG2 cells were then treated with proteasome inhibitor(MG-132) and lysosomal inhibitor (chloroquine, CQ).
The levels of DSG2-Flag increased significantly with
MG-132 treatment but remained unchanged after CQ
treatment (Fig. 3g). In contrast, the DSG2-Flag levels in
HL-wtDSG2 cells did not increase after MG-132 treatment (Additional file 1: Fig. S7f ). These results validated
that UPS contributed to the degradation of the DSG2F536C variant protein in cardiomyocytes.
Four lysines in mouse DSG2 (K698, K712, K783, andK987) were predicted to be modified by ubiquitin using
PhosphoSitePlus. Each predicted lysine was mutated toan arginine to identify the ubiquitination sites involvedin the degradation of vDSG2. The K698R mutation
attenuated the degradation of vDSG2 in HL-vDSG2 cells(Fig. 3h), indicating that K698 is the ubiquitination site
contributing to the degradation of vDSG2.
Primary activation of TGF‑β1/SMADs signaling
in the hearts from Dsg2F536C/F536C mice

Degradation of the DSG2-F536C variant protein via the
UPS may impair the desmosome structure and function, a common mechanism underlying ACM caused
by desmosomal genetic variations, which is insufficient to match the high risk of progressive heart failure

Page 11 of 23

in ACM caused by DSG2 variants. RNA sequencing ofbulk hearts was performed to investigate the potential
unique mechanisms underlying the pathogenesis of
ACM caused by Dsg2F536C. Generally, the transcriptome of Dsg2F536C/F536C mice showed significant differences compared with Dsg2F536C/WT mice and Dsg2WT/WT
mice (Additional file 1: Fig. S8a). Of the 878 differentiallyexpressed genes (DEGs) between Dsg2F536C/F536C andDsg2F536C/WT mice, 753 genes were upregulated and 125
genes were downregulated in Dsg2F536C/F536C mice. Of
the 1230 DEGs between Dsg2F536C/F536C and Dsg2WT/WT
mice, 1074 genes were upregulated and 156 genes were
downregulated in Dsg2F536C/F536C mice. However, there
were only 3 DEGs between Dsg2F536C/WT and Dsg2WT/WT
mice (Additional file 1: Fig. S8b-c), providing molecularevidence for the absence of ACM in Dsg2F536C/WT mice.
Furthermore, 630 genes were upregulated in Dsg2F536C/
F536C
mice compared with both Dsg2F536C/WT and Dsg2WT/
WT
mice (Additional file 1: Fig. S8c). GO analyses showedthat genes with increased expression were enriched forfunctional terms related with inflammatory response, celladhesion, extracellular remodeling and cellular responseto transforming growth factor beta stimulus (Additional
file 1: Fig. S8d). Given that cardiac fibrosis is the primary pathological feature of ACM mice and considering
the central role of the TGF-β signaling pathway in cardiac fibrosis, we further confirmed the activation of the
TGF-β1 signaling pathway in the hearts of Dsg2F536C/F536C
mice through GSEA (Fig. 4a-c, Additional file 4). Western blotting showed markedly increased levels of TGF-β1
in Dsg2F536C/F536C hearts compared to those in Dsg2WT/
WT
and Dsg2F536C/WT hearts (Fig. 4d) as well as elevatedphosphorylation levels of SMAD2/3 (Fig. 4e, f ). These
results indicated that the canonical TGF-β1 pathway
was activated and might be an important mechanism in
ACM.
As the master regulator of fibrosis, TGF-β1 plays apivotal role in cardiac fibrosis and is upregulated understress, inflammation, fibrosis, and myocardial injury[33]. The elevated TGF-β1 may be the primary cause
or secondary effect of cardiac fibrosis in 10-week-old
Dsg2F536C/F536C mice. We compared TGF-β1 levels in thehearts of Dsg2F536C/F536C, Dsg2F536C/WT and Dsg2WT/WT
mice at 1 week of age, when cardiac fibrosis was absent
in Dsg2F536C/F536C mice. The results showed a trend ofelevated TGF-β1 expression in the hearts of Dsg2F536C/
F536C
mice before the onset of fibrosis (Additional file 1:
Fig. S8e). Furthermore, TGF-β1 levels in NMVM from
Dsg2F536C/F536C mice were significantly increased, butwere similar between neonatal mouse cardiac fibroblasts
from Dsg2F536C/F536C and Dsg2WT/WT mice (Fig. 4g). A
previous dataset found that DSG2 was mainly expressedin human cardiomyocytes, but not in fibroblasts

Zhang et al. BMC Medicine(2024) 22:361

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Fig. 4 Primary activation of TGF-β1/SMADs signaling in hearts from Dsg2F536C/F536C mice. a-b GSEA indicated that response to TGF-beta stimulus
pathway was activated in the hearts in 10-week-old Dsg2F536C/F536C (Hom) mice compared with Dsg2WT/WT (WT) or Dsg2.F536C/WT (Het) mice. c
Heatmap of DEGs in cellular response to TGF-beta stimulus pathway. d Representative western blot and quantification of TGF-β1 protein levelsin the hearts from 10-week-old WT, Het and Hom mice. N = 4 in each group. e–f Representative western blot and quantification of the ratioof p-SMAD2/SMAD2 (e) and p-SMAD3/SMAD3 (f) in the hearts from 10-week-old WT, Het and Hom mice. N = 4 in each group. g Representative
western blot and quantification of TGF-β1 protein levels in NMVM and NMFB from WT and Hom mice. N = 3 in each group. h Representative western
blot and quantification of TGF-β1 protein levels in HL-1 cells (Ctrl), HL-1 cells with stable overexpression of variant DSG2 (vDSG2) and HL-1 cellswith stable overexpression of wild-type DSG2 (wtDSG2). N = 3 in each group. Mann–Whitney U test was used to investigate the difference of TGF-β1
in NMFB between WT and Hom mice in panel g. Otherwise, ordinary one-way ANOVA with Turkey’s multiple comparisons test or Unpaired t-test
were used to analyze the data, * p < 0.05, **: p < 0.01, ***: p < 0.001(Additional file 1: Fig. S9a-c) [27]. Immunoblotting also
showed undetectable DSG2 levels in cardiac fibroblasts
isolated from neonatal rats (Additional file 1: Fig. S9d).

Together, these results suggested that elevated TGFβ1 levels in the hearts from Dsg2F536C/F536C mice might
be directly due to the DSG2-F536C variant protein inZhang et al. BMC Medicine(2024) 22:361

cardiomyocytes, which was further corroborated in HLvDSG2 cells (Fig. 4h).
Cardiac fibroblast activation and differentiation intomyofibroblasts are hallmarks of cardiac fibrosis [34].
Due to the low expression of DSG2 in cardiac fibroblasts, we hypothesized that cardiac fibroblasts were passively activated by TGF-β1 released by cardiomyocytes
in Dsg2F536C/F536C mice. To investigate this, we treatedneonatal mouse cardiac fibroblasts with conditioned
medium from HL-1, HL-vDSG2 and HL-wtDSG2 cells,
respectively. We observed enhanced migration ability (Additional file 1: Fig. S9e), increased expression of
α-SMA (Additional file 1: Fig. S9f ), and activation of thecanonical TGF-β1/SMADs signaling (Additional file 1:
Fig. S9g, h) in cardiac fibroblasts treated with conditioned medium from HL-vDSG2 cells. These results indicated that increased levels of TGF-β1 in cardiomyocytes
could activate cardiac fibroblasts and promote cardiac
fibrosis through paracrine signaling.
DSG2‑F536C promoted TGF‑β1 expression by inducing
endoplasmic reticulum stress in cardiomyocytes

The mechanism underlying the increased expression of
TGF-β1 induced by the DSG2-F536C variant in cardiomyocytes remains unclear. Mass spectrometry analysis
showed that HSPA5 (also known as BiP) might interact with vDSG2 (Fig. 3e, Additional file 3: Table S2),
which was validated by co-immunoprecipitation (Co-IP)(Fig. 5a). As an important chaperone, BiP is responsible
to bind with misfolded protein in ER. The interaction
between vDSG2 and BiP indicated that vDSG2 enteredinto the ER, and was recognized as misfolded protein.
The interaction between BiP and misfolded protein could
induce ER stress and activate the UPR. Increased levelsof BiP in NMVM from Dsg2F536C/F536C mice, as well as
in HL-vDSG2 cells, implied that the DSG2-F536C variant protein could induce ER stress in cardiomyocytes(Fig. 5b, Additional file 1: Fig. S10a). Elevated levels of
BiP and C/EBP homologous protein (CHOP) were also
observed in the cardiac tissue of 1-week and 10-week-old
Dsg2F536C/F536C mice (Fig. 5c, d), providing evidence of
ER stress in the hearts of DSG2F536C/F536C mice. Cytoskeleton associated protein 4 (CKAP4), a marker protein of
sheet-like cisternal ER [35], was shown to be increasingly expressed in the hearts of 10-week-old Dsg2F536C/
F536C
mice, presenting with an increased intensity andarea of CKAP4 in cardiomyocytes on immunofluorescence staining (Fig. 5e). Immunoblotting also revealed
increased CKAP4 levels in the hearts of 10-week-old
Dsg2F536C/F536C mice (Fig. 5f ). These results indicatedER expansion in cardiomyocytes from Dsg2F536C/F536C
mice, which is an important characteristic of ER stress[36]. Furthermore, immunoblotting revealed that BiP

Page 13 of 23

levels were significantly higher in adult mouse ventricular myocytes than in non-cardiomyocytes (Fig. 5g), supporting that ER stress induced by vDSG2 was mainly in
cardiomyocytes.
To investigate the relationship between ER stress andelevated TGF-β1 in cardiomyocytes, HL-1 cells weretreated with brefeldin A (BFA) to induce ER stress. The
findings revealed elevated TGF-β1 levels in BFA-treatedcardiomyocytes compared to those in control cells(Additional file 1: Fig. S10b). Furthermore, by overexpressing BiP to inhibit ER stress in HL-vDSG2 cells [37],we observed a reduction in the expression of TGF-β1(Fig. 5h). These results demonstrated that ER stress could
promote the expression of TGF-β1 in cardiomyocytes.
DSG2‑F536C promoted the expression of TGF‑β1
via the PERK‑ATF4 signaling in cardiomyocytes

There are three pathways of UPR in mammals: PERKATF4, IRE1-XBP1s, and ATF6 signaling (Fig. 6a) [38].
Considering that ATF4, XBP1s, and cleaved ATF6 are
the main transcription factors downstream of the UPR
pathway, we assessed the protein levels of these transcription factors in HL-vDSG2 cells. Immunoblotting
showed increased levels of ATF4 (Fig. 6b), unchanged
levels of XBP1s, and decreased levels of cleaved ATF6 in
HL-vDSG2 cells (Additional file 1: Fig. S11a). Immunoblotting of cardiac tissue also showed that only ATF4 levels were significantly increased in 10-week-old Dsg2F536C/
F536C
mice (Fig. 6d, Additional file 1: Fig. S11b). In addition, the ratios of phosphorylated PERK (p-PERK)/PERK
and p-eIF2α/eIF2α in both HL-vDSG2 cells and cardiac tissue from 10-week-old Dsg2F536C/F536C mice wereincreased parallelly (Fig. 6c, d). These results demonstrate that only the PERK-ATF4 pathway was activated byvDSG2 and might contribute to the increased expressionof TGF-β1 in cardiomyocytes.
In order to investigate the cause-and-effect relationship between ATF4 and TGF-β1, we overexpressed ATF4
in cardiomyocytes and found that ATF4 overexpression increased TGF-β1 levels in HL-1 cells and NMVM
(Fig. 6e, f ). ATF4 knockdown via CRISPR-Cas9 did not
significantly change TGF-β1 in HL-1 (Additional file 1:
Fig. S11c, d). However, ATF4 knockdown attenuatedBFA-induced expression of TGF-β1 in HL-1 cells (Additional file 1: Fig. S11e). In parallel, the levels of TGF-β1
were decreased significantly after ATF4 knockdown
in HL-vDSG2 cells (Fig. 6g). Knockdown of ATF4 wasalso performed in NMVM from Dsg2F536C/F536C mice via
adenovirus, and the results validated that ATF4 knockdown ameliorated the enhanced expression of TGF-β1
in cardiomyocytes (Fig. 6h). Furthermore, GSK2606414,
an inhibitor of p-PERK [39], attenuated the increased
expression of ATF 4 and TGF-β1 in HL-vDSG2 cells

Zhang et al. BMC Medicine(2024) 22:361

Page 14 of 23

Fig. 5 DSG2-F536C promoted the expression of TGF-β1 via inducing endoplasmic reticulum stress in cardiomyocytes. a Co-IP demonstrated
that BiP could interact with vDSG2 in HL-1 cells. b Representative western blot and quantification of BiP protein levels in NMVM from Dsg2F536C/
F536C(Hom) and WT mice. N = 6 in WT group, N = 5 in Hom group. c Representative western blot and quantification of BiP and CHOP levels
in hearts from 10-week-old Dsg2WT/WT(WT), Dsg2F536C/WT (Het) and Dsg2F536C/F536C (Hom) mice. N = 3 in each group. d Representative western
blot and quantification of BiP and CHOP protein levels in the hearts from1-week-old WT, Het and Hom mice. N = 3 or 4 in each group. e
Representative immunofluorescence and quantification of CKAP4 intensity and area in hearts from 10-week-old WT, Het and Hom mice. N = 3
in each group. f Representative western blot and quantification of CKAP4 protein levels in hearts from 10-week-old WT, Het and Hom mice. g
Representative western blot and quantification of BiP and cTnT protein levels in adult mouse cardiomyocytes (CM) and non-cardiomyocytes (NCM)from 10-week-old Dsg2.F536C/F536C mice. h Representative western blot and quantification of TGF-β1 protein levels in HL- vDSG2 cellswith overexpression of BiP. N = 3 in each group. Kruskal–Wallis test with Dunn’s multiple comparison test was used to analyze the relative ratioof BiP/β-Tubulin in panel d. Otherwise, ordinary one-way ANOVA with Turkey’s multiple comparisons test or Unpaired t-test was used to analyze
the data, * p < 0.05, **: p < 0.01, ***: p < 0.001

Zhang et al. BMC Medicine(2024) 22:361

in a dose-dependent manner (Fig. 6i, Additional file 1:
Fig. S11f ), which was further validated in AMVM and
NMVM from Dsg2F536C/F536C mice (Fig. 6j). Together,
these results demonstrated that DSG2-F536C promoted
the expression of TGF-β1 via PERK-ATF4 signaling in
cardiomyocytes.
The p‑PERK inhibitor ameliorated cardiac fibrosis
and cardiac systolic dysfunction in Dsg2F536C/F536C mice

The p-PERK inhibitor GSK2606414 showed profoundeffects on the alleviation of increased TGF-β1 expression
induced by DSG2-F536C in cardiomyocytes, prompting
us to investigate its effects on cardiac fibrosis and cardiac systolic dysfunction in Dsg2F536C/F536C mice. Four-to
five-week-old Dsg2F536C/F536C mice were randomly treatedwith DMSO (control group) or GSK2606414 (GSK
group, Fig. 7a). After 4 weeks of treatment, GSK2606414
retarded progressive cardiac enlargement, thinning of
the ventricular wall, and cardiac systolic dysfunction in
Dsg2F536C/F536C mice compared with DMSO (Fig. 7b, c).
Masson’s staining demonstrated that the administrationof GSK2606414 significantly reduced cardiac fibrosis andimproved the thickness of the left ventricular free wall(Fig. 7d, e). Immunoblotting verified that GSK2606414
treatment decreased ATF4, TGF-β1 levels andp-SMAD2/SMAD2 ratio in the hearts of Dsg2F536C/F536C
mice (Fig. 7f, g). All these results indicated ATF4/TGFβ1 signaling played an important role in ACM caused by
Dsg2F536C variant, and inhibition of ATF4/TGF-β signaling could attenuate the progressive cardiac fibrosis andsystolic dysfunction in Dsg2F536C/F536C mice.

Discussion
In this study, we identified the DSG2F531C variant inpatients with ACM from a large family that were all
homozygous carriers of the DSG2F531C variant. To elucidate the pathogenic role of this variant, we generated
Dsg2F536C knock-in mice and found homozygous mice

Page 15 of 23

presenting typical characteristics of ACM, including cardiac enlargement, cardiac systolic dysfunction, and cardiac fibrosis in both ventricles. However, these features
were absent in mice with the heterozygous Dsg2F536C
variant, even under chronic pharmacological stresswith ISO. Mechanistic investigations demonstrated that
DSG2-F536C was misfolded, recognized by the chaperone BiP in ER, and was degraded via the UPS. Meanwhile, the variant protein induced ER stress, activated
PERK-ATF4 signaling, and subsequently enhanced theexpression of TGF-β1 in cardiomyocytes, which activatedcardiac fibroblasts in a paracrine manner and promoted
cardiac fibrosis in ACM. Inhibition of the PERK-ATF4
pathway attenuated the increased TGF-β1 expression,
progressive cardiac fibrosis, and systolic dysfunction in
Dsg2F536C/F536C ACM mice (Fig. 8).
Although previous studies have identified homozygous
DSG2F531C variant in ACM patients, its pathogenicity
and genetic mode of inheritance remain uncertain [16–19]. This study identified DSG2F531C variant in a large
family with 8 ACM patients. The pedigree investigation
revealed that the DSG2F531C variant might contribute to
ACM through an autosomal recessive inheritance pattern, distinguishing it from the majority of pathogenic
variations in desmosomal genes [40]. Accordingly, in
Dsg2F536C knock-in mice, we observed that only miceharboring homozygous variants presented a severe andfully penetrant ACM phenotype, whereas Dsg2F536C/
WT
mice presented no abnormalities in cardiac structure, systolic function, histology, or molecular signature. Competitive and high-intensity endurance exercise
could increase the likelihood of developing ACM in heterozygous desmosomal variant carriers, mainly becauseof the high activity of the sympathetic nervous system
during exercise [41, 42], which could be simulated bypharmacological stress with chronic infusion of ISO[29]. In this study, there were no differences in cardiac
geometry, function, or histological findings between(See figure on next page.)
Fig. 6 DSG2-F536C induced the expression of TGF-β1 via PERK-ATF4 signaling pathway. a Schematic showing the three UPR pathways
in mammals. Created with Biorender.com under academic license. b Representative western blot and quantification of ATF4 protein levels in Hl-1
cells (Ctrl), HL-1 cells with stable expression of vDSG2 (vDSG2), and HL-1 cells with stable expression of wtDSG2 (wtDSG2). N = 3 in each group. c
Representative western blot and quantification of p-PERK/PERK ratio, and p-eIF2α/eIF2α ratio in Hl-1 cells (Ctrl), HL-vDSG2 (vDSG2), and HL-wtDSG2(wtDSG2). N = 3 in each group. d Representative western blot and quantification of the levels of ATF4, p-PERK/PERK ratio, and p-eIF2α/eIF2α ratio
in hearts from 10-week-old Dsg2WT/WT (WT), Dsg2F536C/WT (Het) and Dsg2F536C/F536C (Hom) mice. N = 4 in each group. e Representative western blot
and quantification of the levels of TGF-β1 protein levels in HL-1 cells with overexpression of ATF4 via lentivirus. N = 3 in each group. f Representative
western blot and quantification of the levels of TGF-β1 protein levels in NMVM with overexpression of ATF4 via adenovirus. N = 3 in each group.
g Representative western blot and quantification of the levels of TGF-β1 protein levels in HL-vDSG2 cells with ATF4 KD. N = 3 in each group. h
Representative western blot and quantification of the levels of TGF-β1 protein levels in NMVM with ATF4 KD. NMVM were isolated from Dsg2F536C/
F536C
mice. N = 3 in each group. i Representative western blot and quantification of the levels of TGF-β1 protein levels in HL-vDSG2 treatedwith GSK2606414 (0.4 μM for 24 h). N = 3 in each group. j Representative western blot and quantification of the levels of TGF-β1 protein levels
in AMVM and NMVM treated with GSK2606414 (0.2 μM for 24 h). AMVM and NMVM were isolated from Dsg2F536C/F536C mice. N = 3 in each group.
Ordinary one-way ANOVA with Turkey’s multiple comparisons test was used to analyze the data, * p < 0.05, **: p < 0.01, ***: p < 0.001

Zhang et al. BMC Medicine(2024) 22:361

Fig. 6 (See legend on previous page.)

Page 16 of 23

Zhang et al. BMC Medicine(2024) 22:361

Dsg2F536C/WT and Dsg2WT/WT mice after 2-week ISO
administration. Accordingly, Dsg2F536C variant may be
associated with autosomal recessive inherited ACM.
This finding is of great significance for the managementof family members who are heterozygous DSG2F531C variant carriers. Family members with pathogenic gene variants but without phenotypes are recommended to avoid
competitive or high-intensity endurance exercise [20]. In
this study, Dsg2F536C/F536C mice presented no features of
ACM after chronic and high-intensity pharmacologicalstress, indicating that exercise recommendations might
be provided on an individual basis to some pathogenic
gene carriers.
Activation of quiescent fibroblasts by TGF-β1 andsubsequent remodeling of extracellular matrix proteins
are key pathophysiological processes of cardiac fibrosis[33]. A previous study demonstrated Dsg2W2A pathogenic variant could activate and release TGF-β1 throughintegrin-αV/β6 pathway, leading to cardiac fibrosis andACM [12]. The present study not only identified the activation of TGF-β1 signaling but also revealed that theDsg2F536C variant could directly promoted the expression of TGF-β1 in cardiomyocytes. The increased TGFβ1 activated the canonical TGF-β1 pathway in fibroblasts
by increasing the phosphorylation of SMAD2/3, which
was transferred into the nucleus, promoted the expression of extracellular matrix-related genes, and led to cardiac fibrosis [43]. Moreover, we identified that elevated
TGF-β1 from cardiomyocytes harboring homozygous
Dsg2F536C variant was one of the primary causes of cardiac fibrosis in ACM. Interestingly, cardiac fibroblasts
from Dsg2F536C/F536C mice exhibited similar TGF-β1 levels to those from WT mice. This suggested that cardiac
fibroblasts were passively activated by TGF-β1 secreted
from adjacent cardiomyocytes in Dsg2F536C/F536C mice.
Previous studies demonstrated that the loss of plakophilin-2 and the mutation of transmembrane protein 43
enhanced the expression of TGF-β1 in cardiomyocytes in
ACM [44, 45]. However, the mechanism underlying theincreased expression of TGF-β1 caused by desmosomal
gene variants is unclear.

Page 17 of 23

A previous study showed the presence of ER stress inthe hearts of ACM mice, yet the mechanisms underlying ERS and its significance in ACM remain unclear [46].
In this study, it was found that DSG2-F536C was misfolded and interacted with BiP in the ER. The interactionof DSG2-F536C and BiP could release the three stresssensors, PERK, ATF6, and IRE1 at the ER membrane,
which would activate the 3 corresponding downstream
signaling and induce ER stress. The elevated levels of
BiP, CHOP, and ER expansion in cardiomyocytes verified ER stress in the hearts of Dsg2F536C/F536C mice. Further investigation revealed that ER stress upregulated theexpression of TGF-β1 in cardiomyocytes, consequently
promoting cardiac fibrosis in Dsg2F536C/F536C mice. The
correlation between ER stress and increased TGF-β1
levels was shown in Crohn’s disease [47]. However, itremains unclear how ER stress leads to the elevated levelsof TGF-β1. Increased ATF4 levels with unchanged levels of ATF6 and XBP1s in cardiac tissues from Dsg2F536C/
F536C
mice and HL-vDSG2 cells indicated that only the
PERK-ATF4 pathway was selectively activated in cardiomyocytes harboring the homozygous Dsg2F536C variant.
This may be related to chronic ER stress induced by thecontinuous expression of the vDSG2. A previous study
showed that activities of IRE1α—XBP1s and the ATF6
pathway were attenuated while the PERK-ATF4 pathway remained active during chronic ER stress [48], whichmight be related to the inhibition of global protein translation [49].
After disassociation from BiP in the ER lumen, PERK
normally form homodimers and undergo autophosphorylation. This is followed by the phosphorylationof eIF2α. Phosphorylated eIF2α functions to inhibit
the translation of cap-dependent mRNA, while promoting the translation of a subset of mRNA with small
upstream open reading frames, including ATF4 [50,51]. As a transcription factor, ATF4 binds to the target
gene promoters, thereby facilitating the transcription ofgenes involved in apoptosis, redox regulation, and aminoacid metabolism [52, 53]. Studies have reported conflicting results on the role of ATF4 in the heart. Some

(See figure on next page.)
Fig. 7 Inhibition of ATF4/TGF-β1 signaling ameliorated cardiac dysfunction and cardiac fibrosis in Dsg2F536C/F536C mice. a Schematic diagram
showing the treatment strategy with GSK2606414 in Dsg2F536C/F536C mice. Created with Biorender.com under academic license. b Representative
images of M-mode echocardiography of LV geometry and systolic function in Dsg2F536C/F536C mice at baseline and after 4-week treatment
with GSK2606414 or DMSO. c Quantification of LV geometry (LVDD and LVDS) and systolic function (LVEF and LVFS) in Dsg2F536C/F536C miceat baseline and after 4-week treatment with GSK2606414 or DMSO. N = 4 in control group and N = 9 in GSK2606414 group. d Representative imagesof Masson’s staining in Dsg2F536C/F536C mice after 4-week treatment with GSK2606414 or DMSO. Scale bar 1 mm. e Quantification of global fibrosis
and wall thickness of both ventricles in Dsg2F536C/F536C mice after 4-week treatment with GSK2606414 or DMSO. N = 5 in GSK group, and N = 3
in control group. f-g Representative western blot (f) and quantification (g) of TGF-β1, ATF4 protein levels and p-SMAD2/SMAD2 ratio in hearts
from Dsg2F536C/F536C mice after 4-week treatment with GSK2606414 or DMSO. N = 4 in GSK group, and N = 3 in control group. Unpaired t-test wereused to analyze the data, * p < 0.05, **: p < 0.01, ***: p < 0.001, ns, not significant

Zhang et al. BMC Medicine(2024) 22:361

Fig. 7 (See legend on previous page.)

Page 18 of 23

Zhang et al. BMC Medicine(2024) 22:361

Page 19 of 23

Fig. 8 Schematic conclusion of the main findings of the study. Dsg2F536C in the cardiomyocyte leads to the misfolding of the variant protein, whichis recognized by the chaperon BiP in the ER, followed by translocating to cytosol to be degraded via ubiquitin–proteasome pathway. Meanwhile,
the variant protein activated the PERK-ATF4 signaling pathway, and subsequently increase the expression of TGF-β1 in cardiomyocytes, which
promotes cardiac fibrosis and heart failure in ACM via activating cardiac fibroblasts. Notably, inhibition of the PERK/ATF4 signaling could attenuate
the increased TGF-β1 levels and cardiac fibrosis in ACM mice. Created with Biorender.com under academic license

researchers have found that ATF4 had cardioprotective
effects under hemodynamic and mitochondrial stress via
its antioxidant effects [54, 55]. However, ATF4 overexpression in cardiomyocytes induced lethal cardiac atrophy by enhancing autophagy [56]. Another study foundthat increased ATF4 levels mediated the impaired cardiomyocyte maturation induced by the RYR2 variant [57].
Our study showed that ATF4 accelerated cardiac fibrosis
by increasing TGF-β1 levels in cardiomyocytes. In addition, the expression of TGF-β1 in cardiomyocytes could
be reversed by a p-PERK inhibitor or ATF4 knockdown,
indicating that the activation of PERK/ATF4 signaling
was a novel mechanism underlying the elevated TGF-β1
levels in ACM. In vivo experiments validated the beneficial effects of a p-PERK inhibitor in attenuating progressive cardiac fibrosis and cardiac systolic dysfunction in
Dsg2F536C/F536C mice.

In addition to UPR, The ER associated degradation is
the main protein degradation systems to deal with misfolded proteins in ER to attenuate the ER stress. Most of
the misfolded proteins in ER are translocated to cytosol,
where they are targeted with ubiquitin label by ubiquitin cascade reaction, and followed by the degradationin the proteasome [58]. Increased levels of vDSG2-Flag
following MG132 treatment, along with elevated ubiquitination levels of vDSG2 in HL-V cells, indicate thatvDSG2 may be degraded through the ER-associateddegradation pathway. Targeting the ubiquitination anddegradation of proteins has emerged as a new therapeutic strategy [59]. Mass spectrometry analysis identified
TRIM21 and USP10 as potential candidates interactingwith vDSG2. TRIM21 is an intracellular ubiquitin ligase[60], whereas USP10 functions as a deubiquitinating
enzyme [61]. These findings suggest that TRIM21 andZhang et al. BMC Medicine(2024) 22:361

USP10 may play roles in the ubiquitination and degradation of DSG2, highlighting their potential as therapeutic targets for DSG2-related ACM in the future.

Conclusions
Hyperactivation of the ATF4/TGF-β1 signaling in cardiomyocytes is a novel driver of progressive cardiac fibrosis
in ACM. The identification of these mechanisms provides
potential therapeutic targets for ACM management.
Abbreviations
ACM	Arrhythmogenic cardiomyopathy
AMVM	Adult mouse ventriclar myocytes
ATF4	Activating transcription factor 4
ATF6	Activating transcription factor 6
BFA	Brefeldin A
BiP	Immunoglobulin heavy chain binding protein
BSA	Bovine serum albumin
CHOP	C/EBP-homologous protein
CKAP4	Cytoskeleton associated protein 4
CVF	Collagen volume fraction
CQ	Chloroquine
DEGs	Differentially expressed genes
DMEM	Dulbecco’s modified Eagle’s medium
DSG2	Desmoglein-2
DSP	Desmoplakin
ECG	ElectrocardiographyeIF2α	Eukaryotic translation initiation factor 2α
ESC	Embryonic stem cell
FBS	Fetal bovine serumGO	Gene ontology
GSEA	Gene set enrichment analysis
HSPA5	Heat shock protein family A member 5
IRE1	Inositol requiring enzyme 1ISO	Isoproterenol
NMVM	Neonatal mouse ventricular myocytes
PERK	Protein kinase R like ER kinase
PFA	Paraformaldehyde
RMSD	Root Mean Square Deviation
TGF-β1	Transforming growth factor β1
UPR	Unfolded protein response
UPS	Ubiquitin-proteasome system
vDSG2	Variant DSG2WT	Wild type
wtDSG2	Wild-type DSG2XBP1s	Sliced X-box binding protein 1

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03593-8.
Additional file 1: Figure S1. Representative ECG of persistent ventriculartachycardia in an ACM patient with DSG2F531C variant. Figure S2.
Comparation of amino acids sequence of DSG2 across the cadherin family
number and between human and mouse. (a) The variant (F531C) was
located in an evolutionary highly conservative area across the cadherinfamily proteins. (b) The Phe 536 of mouse DSG2 (black arrow) was
homologue to the Phe 531 of human DSG2 (red arrow). Figure S3.
Generation of Dsg2F536C variant mice. (a) Generation of the variant allele.
(b) Southern blot analysis on four initially screened neomycin-resistantembryonic stem (ES) clones. The left panel displayed HpaI digest (19.9 K
and 10 K) and the right panel displayed NheI digest for Neo (14.2 K). Clone
B6 served as the wild-type ES clone, while clones E11 and E1 wereidentified as correctly targeted clones. Clone B10, however, wasdetermined to be an incorrectly targeted clone. (c) Sequencing result toPage 20 of 23

showed correct change of TTC (encoding phenylalanine) to TGC(encoding cysteine). (d) Genotyping. The PCR products were 466 bp for
Homozygous (Dsg2F536C/F536C) and 368 bp for wild-type (Dsg2WT/WT).
Figure S4. Comparison of left ventricular free wall thickness among
10-week-old Dsg2WT/WT (WT), Dsg2F536C/WT(Het), and Dsg2F536C/F536C(Hom) mice. Ordinary one-way ANOVA and Turkey’s multiple
comparisons test was used to analyze the data, ***: p < 0.001; ns, notsignificant. N = 13 in WT group, N = 16 in Het group, and N = 19 in Homgroup. Figure S5. Homozygous Dsg2F536C/F536C mice showed significant
biventricular fibrosis. (a) Representative images of Masson’s staining in
4-week-old Dsg2WT/WT (WT), Dsg2F536C/WT (Het) and Dsg2F536C/F536C (Hom)mice. (b) Quantification of biventricular CVF, thickness of both freeventricular walls and interventricular septum in 4-week-old WT, Het andHom mice. N = 5 in each group. ***: p < 0.001, **: p < 0.01, *: p < 0.05. (c)Quantification of interventricular septum thickness in Dsg2F536C/F536C
mice from 2- to 10-week-old. N = 5 in each group. #: p < 0.05 comparedwith the heart of 4-week-old mice; ns, not significant. One-way ANOVA
and Turkey’s multiple comparisons test was used to analyze the data.
Figure S6. Failed to induce the phenotypes of ACM under the chronicalpharmacological stress with ISO in heterozygous Dsg2F536C/WT mice. (a)Schematic diagram showing the strategy of the chronically pharmacological stress with ISO. Created with Biorender.com under academic
license. (b) Representative images of M-mode echocardiography of LV
geometry and systolic function in WT and Dsg2F536C/WT (Het) mice after
2-week treatment with ISO. ns, not significant. (c) Quantification of LV
geometry (LVDD, LVDS, thickness of free wall and interventricular
septum) and systolic function (LVEF and LVFS) in WT and Het mice after
2-week treatment with ISO. N = 6 in WT group, and N = 8 in Het group.
ns, not significant. (d) Representative images of Masson’s staining in WT
and Het mice after 2-week treatment with ISO. Scale bar 1 mm. (e)Quantification of global fibrosis and wall thickness of both ventricles in
WT and Het mice after 2-week treatment with ISO. N = 6 in WT group,
and N = 8 in Het group. ns, not significant. Unpaired t-test was used toanalyze the data. Figure S7. DSG2-F536C was degraded via theubiquitin–proteasome signaling. (a) Relative mRNA levels of Dsg2 in thehearts of 10-week-old WT, Het and Hom mice. N = 3 in each group. (b)Representative western blot and quantification of DSG2-Flag in
HER293T cells transfected with plasmids with mutant or WT mice Dsg2
cDNA and a C-terminal 3xflag epitope. The DSG2-Flag was detected byanti-DSG2 and anti-Flag antibody respectively. N = 3 in each group. (c)The predicted 3D model of extracellular and transmembrane domains(55th—619th amino acid) of wild-type DSG2 protein (blue) and thevariant DSG2-F536C protein (green) via AlphaFold2. The black dashed
box indicated a similar secondary structure at the variant site, and the
purple indicated the mutated amino acid. (d) Comparison of thethree-dimensional structures between vDSG2 (green) and wtDSG2(blue). The red segments indicate the regions of abnormal folding invDSG2, whereas the yellow segments represent the corresponding
normal structure in wtDSG2. (e) Relative mRNA levels of Dsg2 in HL-1
cells, HL-1 cells with stable expression of variant DSG2 (vDSG2) and wild
type DSG2 (wtDSG2). N = 3 in each group. (f ) Representative western
blot and quantification of DSG2-Flag and β-Catenin protein levels in
HL-wtDSG2 treated with MG132. N = 3 in both groups. Kruskal–Wallis
test with Dunn’s multiple comparison test was used to analyze
DSG2-Flag levels in panel f. Otherwise, One-way ANOVA and Turkey’s
multiple comparisons test or Unpaired t test was used to analyze thedata. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ns, not significant. Figure S8.
Activation of TGF-β1 pathway in the hearts of 10-week-old Dsg2F536C/
F536C
mice. (a) Heatmap of differentially expressed genes (DEGs) in thehearts of 10-week-old Dsg2WT/WT (WT), Dsg2F536C/WT (Het) and Dsg2F536C/
F536C(Hom) mice. N = 4 in each group. (b) Volcano plot and quantification of DEGs between different groups. N = 4 in each group. (c) Venn
diagram between Hom vs Het and Hom vs WT mice. N = 4 in eachgroup. (d) Gene Ontology (GO) analyses for upregulated genes in thehearts of 10-week-old Hom mice compared with both WT and Het
mice. (e) Representative western blot and quantification of TGF-β1
protein levels in the hearts from 1-week-old WT, Het and Hom mice.
N = 3 in each group. Kruskal–Wallis test with Dunn’s multiple
comparison test was used to analyze the data. Figure S9. Overexpression of DSG2-F536C in HL-1 cells significantly promoted fibroblastZhang et al. BMC Medicine(2024) 22:361

activation via SMADs signaling. (a) UMAP map of snRNA-seq displaying 9
distinct cellular clusters. (b) Feature plot showing expression of DSG2
among different cellular clusters. (c) Violin plot showing DSG2 expression
in human hearts. (d) Representative western blot and quantification of
DSG2 levels in neonatal rat ventricular myocytes (NRVM) and neonatal rat
fibroblasts (NRFB). N = 3 in both groups. ***: p < 0.001. (e) The conditioned
medium from HL-vDSG2 cells promoted NMFB migration by woundhealing assay compared with conditioned medium from HL-1 andHL-wtDSG2 cells. N = 3 in each group. *: p < 0.05 compared with HL-1, **:p < 0.01 compared with HL-1 cells, #: p < 0.05 compared with HL-wtDSG2cells. (f ) Representative western blot and quantification of α-SMA protein
levels in NMFB treated with conditioned medium of HL-1, HL-vDSG2 andHL-wtDSG2 cells. N = 4 in each group. *: p < 0.05. (g-h) Representative
western blot and quantification of p-SMAD2/SMAD2 ratio (g) andp-SMAD3/SMAD3 ratio (h) in NMFB treated with conditioned medium
from HL-1, HL-vDSG2 and HL-wtDSG2 cells (red arrow indicates p-SMAD3).
N = 4 in each group. ***: p < 0.001. Kruskal–Wallis test with Dunn’s multiple
comparison test was used to analyze p-SMAD3 in the panel h. Otherwise,
One-way ANOVA and Turkey’s multiple comparisons test or Unpaired t
test was used to analyze the data. Figure S10. DSG2-F536C promoted theexpression of TGF-β1 via inducing endoplasmic reticulum stress in
cardiomyocytes. (a) Representative western blot and quantification of BiP
protein levels in HL-1 cells, HL-1 cells with stable expression of variant
DSG2 (vDSG2), and HL-1 cells with stable expression of wild type DSG2
(wtDSG2). N = 3 in each group. (b) Representative western blot andquantification of TGF-β1 protein levels in HL-1 cells treated with placeboor brefeldin A (BFA, 2.5ug/L for 12 h). N = 3 in each group. One-way
ANOVA and Turkey’s multiple comparisons test or unpaired t-test wasused to analyze the data, **: p < 0.01, ***: p < 0.001. Figure S11. DSG2F536C promoted the expression of TGF-β1 in cardiomyocytes via
PERK-ATF4 pathway. (a) Representative western blot and quantification of
XBP1s and ATF6-50KD in HL-1 (Ctrl), HL-vDSG2 and HL-wtDSG2 cells. N = 3
in each group. (b) Representative western blot and quantification of
ATF6-50KD, XBP1u and XBP1s in the hearts of 10-week-old Dsg2WT/WT(WT), Dsg2F536C/WT (Het) and Dsg2F536C/F536C (Hom) mice. N = 3 in eachgroup. (c) Representative western blot and quantification of ATF4 in HL-1
cells treated with CRISPR-Cas9 with sgRNA targeting ATF4. N = 3 in eachgroup. (d) Representative western blot and quantification of TGF-β1 in
HL-1 cells with ATF4-KD. N = 3 in each group. (e) Representative western
blot and quantification of ATF4 and TGF-β1 protein levels in HL-1 cells with
ATF4-KD under ER stress induced by BFA. N = 3 in each group. (f )Representative western blot and quantification of ATF4 and TGF-β1 in
HL-vDSG2 cells treated with different doses of GSK2606414. N = 3 in eachgroup. One-way ANOVA and Turkey’s multiple comparisons test was used
to analyze the data. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Table S3. Lists ofprimary antibodies used in this study. Table S4. Primary sequence forgenotyping. Table S5. Primary sequence for real time quantitative PCR.
Table S6. Target for RNAi.
Additional file 2: Table S1. Clinical characterization of DSG2-F531C
mutation-positive family members.
Additional file 3: Table S2. Results of mass spectrometry analysis of purified protein by immunoprecipitation with anti-Flag antibody in cultured
cardiomyocytes with stable expression vDSG2.
Additional file 4. GSEA of DEGs between Dsg2F536C/F536C and Dsg2F536C/WT or
Dsg2WT/WT mice.
Additional file 5. Uncut blot
Additional file 6. ARRIVE guidelines checklist.
Acknowledgements
Not applicable.
Authors’ contributions
Conceptualization: BZ, YX and BY. Methodology: BZ, YW, CS and CZ. Investigation: BZ, YW, CZ and JX1; Software: YZ, JX2, and XY. Writing – Original Draft:
BZ and YW. Writing – Review & Editing: CS, BZ, DWW, YX and BY. Resources:
BY, DWW, and XZ. Funding acquisition: XZ, and BY. Supervision: YX and BY. All
authors read and approved the final manuscript.

Page 21 of 23

Funding
This work was supported by grants from the Top-level Clinical Discipline
Project of Shanghai Pudong District Grant/Award (PWYgf2021-01), The healthscience and technology project of Shanghai Pudong (PW2023A-17), the Science and Technology Plan Project of Health Commission of Jiangxi Province(202312389), National Natural Science Foundation of China (No. 81070158 to
BY, No. 82370323 to BY, No. 81170161 to XZ), Health Commission of Shanghai
Pudong District, China (PW2019D-1), and Shanghai Committee of Science andTechnology, China (19DZ1930402).
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
This study was approved by the Ethics Committee of the First Affiliated Hospitalof Nanjing Medical University (NO.2011-SR-014) and the informed consent was
obtained from all the participants before undergoing evaluation and testing.
Animal experiments were approved by the Institutional Animal Care and Use
Committee of Nanjing Medical University (approval number: IACUC-14030160).
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Cardiology, Shanghai East Hospital, Tongji University Schoolof Medicine, 150 Jimo Road, Pudong, Shanghai 200120, P.R. China. 2 Department of Cardiology, the First Affiliated Hospital of Nanjing Medical University,
300 Guangzhou Road, Nanjing 210029, P.R. China. 3 Shanghai East Hospital,
School of Life Sciences and Technology, Tongji University, 150 Jimo Road,
Pudong, Shanghai 200120, P.R. China. 4 State Key Laboratory of Reproductive Medicine, the Centre for Clinical Reproductive Medicine, Departmentof Cardiology, the First Affiliated Hospital of Nanjing Medical University, 300
Guangzhou Road, Nanjing 210029, P.R. China. 5 Model Animal Research Center,
Nanjing University, Nanjing, China.
Received: 18 March 2024 Accepted: 27 August 2024

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