ATF3 is required for the prevention of cardiomyopathy via the regulation of mitochondrial oxidative stress

ATF3 is required for the prevention of cardiomyopathy via the regulation of mitochondrial oxidative stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ATF3 is required for the prevention of cardiomyopathy via the regulation of mitochondrial oxidative stress Won-Ho Kim, Myong-Ho Jeong, Yideul Jeong, Su-Yeon Cho, Seung Hee Lee, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4485671/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Activating transcription factor 3 (ATF3) is a critical regulator for cellular stress responses and is implicated in stress-induced cardiac hypertrophy and fibrosis. However, the role of ATF3 in cardiomyopathy remains controversial. Here, we demonstrate that ATF3 plays a cardioprotective role by controlling mitochondrial oxidative stress in angiotensin II (Ang II)-triggered cardiomyopathy. The expression of ATF3 was significantly upregulated in hypertrophic hearts chronically infused with Ang II, which correlated with Ang II-treated cardiomyocytes. In neonatal mouse ventricular myocytes (NMVMs), Ang II-elicited hypertrophic responses were either aggravated or suppressed by ATF3 depletion or overexpression, respectively. Similar results were also obtained in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To analyze the direct role of ATF3 in cardiomyopathy, we generated mice with a cardiomyocyte-specific ATF3 deletion using a tamoxifen-inducible Cre-recombinase (αMHC-MerCreMer/loxP) system. In response to Ang II infusion, mice with cardiomyocyte-specific ablation of ATF3 (ATF3 cKO) exhibited aggravated cardiac hypertrophy and fibrosis concurrent with decreased fractional shortening and ejection fraction. In addition, the transcriptome analysis of control and cKO hearts revealed alterations in genes related to mitochondrial function and organization. In particular, the expression of Sirt3/Sod2 transcripts, well known as a mechanism for regulating mitochondrial oxidative stress, was increased in Ang II-infused mice, which was downregulated by the depletion of ATF3, suggesting the cardioprotective function of ATF3 through the improvement of mitochondrial function. These results suggest that ATF3 may be a potential therapeutic target for hypertrophic cardiomyopathy. Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy Biological sciences/Molecular biology/Transcriptomics Biological sciences/Cell biology/Cell signalling/Stress signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cardiovascular diseases (CVDs) have a major impact on global mortality rates. 1 Numerous studies on the structural and functional abnormalities of CVDs are being conducted to address these issues. Despite these efforts, the risk of CVDs continues to increase, further indicating the importance of research on the developmental process and basic mechanisms of CVDs. Emerging evidence indicates that mitochondrial dysfunction is a critical factor in the development of CVDs. Mitochondria are essential cellular organelles responsible for producing the majority of cellular ATP via oxidative phosphorylation. These organelles also provide cofactors such as heme, iron-sulfur clusters, amino acids, and nucleotides. 2 , 3 Moreover, mitochondria are the primary sites responsible for producing reactive oxygen species (ROS), 4 which can act as signaling molecules, in cells. However, excessive ROS can cause oxidative damage to proteins, lipids, and DNA. Oxidative damage can cause protein misfolding, which in turn induces the mitochondrial unfolded protein response (UPR mt ). Thus, maintaining proper mitochondrial function is crucial for cell survival and function. 5 The UPR mt is essential for safeguarding mitochondria through various pathways, including increasing the expression of mitochondrial chaperone proteins, promoting the degradation of damaged mitochondrial proteins and regulating mitochondrial biogenesis. 6 Several studies have suggested that the activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) axis plays a pivotal role in both the UPR mt and the endoplasmic reticulum UPR (UPR er ) to resolve the UPR, primarily by inducing ATF3. 7 , 8 , 9 , 10 ATF3 is a member of the ATF/cAMP response element-binding protein (CREB) family of transcription factors 11 and is rapidly induced in response to different types of stress, such as the UPR and oxidative stress. 8 , 12 ATF3 has been found to regulate various cellular processes as a transcriptional activator and repressor depending on the cellular context, cell type and presence of other cofactor protein complexes, 13 which is attributable to its dual and contradictory roles in a single organ. 14 , 15 Despite extensive research, the exact role of ATF3 in stress-induced cardiac hypertrophy and fibrosis remains unclear. Previous studies have indicated that ATF3 has a negative impact on CVDs, including stress-induced cardiac hypertrophy and fibrosis. 15 , 16 , 17 , 18 , 19 However, recent investigations have increasingly revealed that ATF3 also has protective effects against CVDs, 20 , 21 , 22 indicating its multifaceted role in cardiac dysfunctions and pathologies. 23 , 24 , 25 To elucidate the role of ATF3 in cardiac function, we utilized in vivo and in vitro experimental systems and analyzed Gene expression omnibus (GEO) datasets from single-nucleus RNA sequencing (snRNA-seq) of human hearts from healthy subjects and patients with myocardial infarction. Transient overexpression or depletion of Atf3 prevented or accelerated neonatal mouse cardiomyocyte (NMVM) hypertrophy, respectively. Analysis of snRNA-seq data from human hearts showed that ATF3 is predominantly expressed in ventricular cardiomyocytes especially in cells subjected to angiopoietin-like 4 (ANGPTL4), which can attenuate phenylephrine-induced myocardial hypertrophy. 26 Additionally, cardiomyocyte-specific Atf3-null (cKO) mice exhibited a dilated cardiomyopathy phenotype with reduced cardiac function and increased cardiac fibrosis upon angiotensin II (AngII) infusion. Moreover, transcriptome analysis revealed that, following AngII infusion, cKO mice exhibit significant dysregulation of genes involved in the regulation of cellular respiration, such as genes involved in organophosphate biosynthetic processes and mitochondrial oxidation. In particular, hearts from AngII-infused cKO mice exhibited reduced expression of mitochondrial chaperone proteins and Sirtuin 3 (Sirt3). A decrease in Sirt3 expression coincided with an increase in the protein levels of acetylated superoxide dismutase 2 (ac-Sod2), an inactivated form of Sod2, a major mitochondrial antioxidant protein. Additionally, overexpression of Atf3 in NMVMs increased the expression of Sirt3 at both the mRNA and protein levels and decreased cellular ROS accumulation. In conclusion, Atf3 deficiency in cardiomyocytes causes mitochondrial dysfunction due to a decrease in Sirt3/Sod2-mediated ROS scavenging, which likely contributes to cardiomyopathy. Methods Mice For generation of tamoxifen-inducible cardiac-specific Atf3 null mice, Atf3 tm2a/tm2a (from C57BL/6N- ATF3 tm2a(EUCOMM)Wtsi /H ) mice were crossed with FLP recombinase-expressing mice to promote recombination between FRT sites (Jax strain 003946), resulting in a floxed Atf3 allele ( Atf3 tm2c/tm2c , Atf3 f/f throughout the manuscript). Next, the Atf3 f/f mice were crossed with a MHC MerCreMer mice to generate aMHC MerCreMer ; Atf3 f/+ mice. The resulting aMHC MerCreMer ; Atf3 f/+ offspring were then backcrossed to Atf3 f/f mice to obtain aMHC MerCreMer ; Atf3 f/f ( Atf3 f/f−MCM throughout the manuscript) mice. For promotion of cardiac hypertrophy, 11 ~ 12-week-old Atf3 f/f and Atf3 f/f−MCM mice were infused with angiotensin II by subcutaneous implantation of an osmotic minipump (Alzet model 1002) for 14 days, followed by vehicle or tamoxifen injection (30 g/g body weight/day for 3 consecutive days, intraperitoneally) to induce Cre recombinase activity. In this procedure, the Atf3 +/+ ; aMHC MerCreMer mice were considered to control the heart phenotype for tamoxifen toxicity. This study was reviewed and carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) of the Korea Disease Control and Prevention Agency (KDCA) under the protocol approved by KDCA-IACUC-022-003. Echocardiography For echocardiographic analysis, the mice were anesthetized with 2% (vol/vol) isoflurane 1 day before sacrifice using a Vevo 2100 system (Fujifilm Visual Sonics, K-BIO Health). For analysis of cardiac parameters and functions, including the left ventricular internal diameter diastolic/systolic (LVID;d/s), interventricular septum diastolic/systolic (IVS;d/s), left ventricular posterior wall diastolic/systolic (LVPW;d/s), LV mass, fractional shortening (FS) and ejection fraction (EF), we analyzed cardiac M-mode images taken from the short axis view of the LV. Cell culture and transfection H9C2 (KCLB, 21446) cells were cultured in 10% FBS (fetal bovine serum) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin/streptomycin. NMVMs were isolated from postnatal day 1–2 Atf3 f/f mouse hearts on a C57BL/6J background and cultured as previously described. 27 hESCs were kindly provided by Professor JS Kang at Sungkyunkwan University (SKKU) and cultured as previously described. 28 For transient overexpression experiments, we used an ATF3 plasmid (Addgene, #26115) or an adenovirus expressing RFP-ATF3. Lipofectamine 2000 (Invitrogen, 11668) was used for the overexpression of plasmid DNA. For the adenoviral experiments, the cells were treated with AdATF3 at an MOI of 200 for 5 h, and adRFP was used as a control. For Atf3 knockdown in Atf3 f/f NMVMs, Cre recombinase Gesicle (TaKaRa, 631449) was used according to the manufacturer’s instructions. For induction of cellular hypertrophy in H9C2 and NMVMs, the cells were starved for 24 h and then treated with 1 µM AngII or 100 ng/ml of ET-1 for an additional 24 h. For induction of hypertrophy in hESC-CMs, the cells were treated with 100 ng/ml ET-1 for 24 h. Protein analysis Western blot analysis was performed as previously described. 29 Briefly, cultured cells or homogenized tissue samples were lysed in RIPA buffer (Pierce™, #89900) containing complete protease inhibitor cocktail (Sigma‒Aldrich, P8340). The samples were then subjected to SDS‒PAGE and immunoblotting using various primary antibodies. The primary and secondary antibodies used in this study are listed in Supplementary Table S2. Single-nuclear RNA sequencing (snRNA-seq) data analysis Previously published snRNA-seq data deposited in the Zenodo Data Archive ( https://zenodo.org/record/6578047 ) were used to assess the role of ATF3 in cardiomyocytes (CMs). All the snRNA-seq datasets were analyzed using the Seurat and CellChat pipelines. The datasets were integrated using the pipeline of the dimensionality reduction platform. The integrated cells underwent a quality control process to remove dead cells and doublets based on library size and cell dispersion. Subsequently, the integrated cells were normalized to counts per million (CPM). The filtered cells were then clustered using the Seurat pipeline with a resolution of 0.5. For data visualization, a dimensional reduction uniform manifold approximation and projection (UMAP) was generated through the Seurat function RunUMAP. Cluster identities were assigned based on the expression of the top marker genes and what is known from published literature. The differentially expressed genes in the snRNA-seq database were extracted based on the following conditions: 1 (p value 1.5. For PCA, selected genes in each group from RNA-seq samples were extracted based on a p value 2.0 and automatically clustered with 20 variances using the Seurat pipeline. RNA analysis Quantitative real-time PCR (qRT‒PCR) analysis was performed as previously described. 30 Total RNA was isolated from heart tissue samples and cultured cells using TRIzol reagent (Invitrogen, 5596026). cDNA was amplified using ReverTra Ace® qPCR RT Master Mix (TOYOBO, FSQ-201) following the manufacturer’s instructions. SYBR Premix Ex Taq (TaKaRa, RR420) was used to analyze gene expression on a QuantStudio™ 6 Flex System according to the manufacturer’s instructions. The primer sequences utilized in this study are presented in Supplementary Table S3. RNA sequencing was carried out on a NextSeq 550 platform (Illumina, Inc., USA). The analysis of RNA sequencing data was performed using ExDEGA v5.0.0.1 (e-Biogen) and the ClueGO/CluePedia, EnrichmentMap, and GeneMania plug-ins from Cytoscape (v3.10.0) software. Global gene expression was assessed by biological processes with gene set enrichment analysis using MSigDB v6.1 (> 1.3-fold, RC log2 > 2, P < 0.05) and displayed with the Flaski application ( http://flaski.app ). The Cytoscape (v3.1.0) ClueGo plugin was used to visualize enriched pathways associated with the biological pathway database. In brief, biological GO terms with medium specificity and a kappa score of 0.4 were explored. An enrichment/depletion method with a two-sided hypergeometric test was applied with Bonferroni step-down for each p value calculation. Enriched pathways with a p value < 0.05 were considered significant. Gene set enrichment analysis (GSEA) was performed to extract information on overrepresented gene ontology terms for various functional processes and signaling pathways between each sample. Visualization of significantly enriched GO terms of functional processes and signaling pathways between samples was performed with the Cytoscape plugin EnrichmentMap. The mapping of gene expression levels was performed using the GeneMania plugin. All GO terms of the network in our analysis were filtered with a p value less than 0.05 based on the pathway score. Coexpression networks were constructed using the WGCNA (v1.47) package in R (Langfelder and Horvath, 2008). After filtering genes, gene expression values were imported into WGCNA to construct coexpression modules using automatic network construction with default settings. Histology and immunostaining Histology and immunostaining of the heart sections were performed as previously described. 31 Briefly, animal hearts were harvested, embedded in optical cutting temperature (OCT), sliced to a thickness of 10 m, and stained with Masson’s trichrome following the manufacturer’s instructions (Abcam, ab150686). Immunostaining was performed as described previously. 32 Briefly, samples were fixed with 4% paraformaldehyde solution for 5 min and incubated with primary antibodies diluted 1:1000 in 2% BSA (wt/vol) in 0.2% PBST buffer overnight at 4°C following antigen retrieval with 20 µg/ml proteinase K (VIAGEN, #501-PK) treatment (10 mM Tris-HCl buffer, pH 8.0 for 15 min at 37°C). After incubation with the primary antibody, the samples were washed with PBS solution and incubated with secondary antibodies [diluted 1:1000 in 2% BSA (wt/vol) in 0.2% PBST buffer] for 1 h at room temperature, followed by Hoechst 33342 (Thermo Fisher Scientific, H3569) staining for nuclear analysis. For immunostaining of NMVMs or hESC-CMs, the cells were first fixed with 4% PFA for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. The cells were then blocked with 2% BSA solution in 0.2% PBST for 30 min and incubated with primary antibodies overnight at 4°C. Images were captured and processed with an EVOS M5000 system and an FV3000-ORS (Olympus Corp.) confocal microscope. The cell surface area and cross-sectional area were quantified with ImageJ software. Electron microscopy and toluidine staining For electron microscopy analysis, heart tissues were rapidly harvested and washed twice with cold 1x PBS. A segment of the posterior wall of the left ventricle approximately 1 mm 2 in size was excised and drop-fixed in modified Karnovsky's fixative solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate, pH 7.4) at 4°C overnight. The tissues were washed with 0.2 M sodium cacodylate buffer (pH 7.2) and postfixed with 2% aqueous osmium tetroxide for 1 h 30 min. The samples were then dehydrated through a series of increasing ethanol concentrations ranging from 50–100%. The tissues were subsequently washed with propylene oxide and embedded in Epon812 resin. For light microscopy, 1 m thick sections were prepared and counterstained with toluidine blue. For transmission electron microscopy (TEM), ultrathin sections (70 nm) were prepared and collected on 100 mesh copper grids. Images were captured with a LIBRA-120 microscope (Zeiss). The data were analyzed using ImageJ software. Mitochondrial analysis The following procedures were used to analyze mitochondrial respiration: NMVMs were seeded on Matrigel®-coated clear bottom 96 black-well plates at a density of 3 × 10 4 cells/ml and allowed to grow for 2 days. The cells were then treated with Cre recombinase Gesicles or transfected for ATF3 overexpression. After 24 h, the cells were serum-starved for 24 h and treated with 1 M AngII for 24 h. The cells were then stained with 2 µM JC-1 dye (Invitrogen, MP 03168) for 30 min, washed with Live Cell Imaging Solution (Invitrogen, A14291DJ), and imaged via an EVOS M5000 system. For JC-1 quantification, the SpectraMax® i3x system was used. For quantification of the mitochondrial DNA content, total DNA was extracted from the heart and NMVMs using the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, #9765) following the manufacturer’s instructions. The amount of mitochondrial DNA was measured by the ratio of mtCo1 to Ndufv1 using quantitative PCR. Reactive oxygen species (ROS) measurement For measurement of the level of ROS generated in cardiomyocytes following AngII treatment, the cells were treated with DCF-DA (Sigma, #D6883) for 40 min. After DCF-DA incubation, the cells were washed twice with 1X PBS before being observed. Live cell images were captured and analyzed using an EVOS M5000 system and ImageJ software, respectively. Statistical analysis Statistical differences between two or more groups were analyzed using either an unpaired two-tailed Student’s t test or one-way analysis of variance (ANOVA) with GraphPad Prism 9. The experiments were performed independently at least three times. The data are expressed as the means ± SDs or ± SEMs, as indicated in the figure legends. Results Atf3 is significantly increased in hypertrophic cardiomyocytes Due to conflicting findings regarding the expression and function of ATF3 in the progression of myocardial hypertrophy, we first investigated the expression of Atf3 in H9C2 cardiomyocytes after AngII treatment. Our findings revealed a significant increase in Atf3 protein and mRNA expression in the AngII-treated H9C2 cells compared to the control cells (Fig. 1 A-C). This increase was concurrent with increases in the expression of cardiac hypertrophy-related genes, atrial natriuretic peptide ( Anp) and brain natriuretic peptide ( Bnp) (Fig. 1 D and E). We also analyzed Atf3 expression in the heart following AngII infusion and found that the Atf3 protein level substantially increased after AngII injection and gradually decreased thereafter. In contrast, the expression of Anp and alpha-smooth muscle actin (a-Sma) began to increase on the 3rd and 5th days after AngII infusion, respectively (Fig. 1 F). This period may be the start of pathological cardiac remodeling in the heart. 33 The transcriptional level of Atf3 rapidly increased after AngII injection, peaking between days 3 and 7 (Fig. 1 G). Atf3 is essential for inhibiting cardiomyocyte hypertrophy To further clarify the function of ATF3 in cardiomyocytes, we used the Cre recombinase system to deplete Atf3 in cardiomyocytes by utilizing NMVMs isolated from mice with the Atf3 f/f genotype. The depletion of Atf3 by Cre protein treatment triggered an increase in the size of NMVMs, regardless of treatment with AngII (Fig. 2 A and B) or endothelin-1 (ET-1) (Figure S1 A and B), an inducer of cardiac hypertrophy. Similarly, AngII or ET-1 treatment increased the protein expression of Anp in NMVMs, and its expression was more significantly increased in Atf3-deficient NMVMs (Figs. 2 C and S1C). Further qPCR analysis confirmed a significant increase in the expression of Anp , Bnp , and beta-myosin heavy chain (b -Mhc ) in the Atf3-deficient NMVMs compared to the control cells (Fig. 2 D-G; Figure S1 D-G). Next, we investigated the protective effects of Atf3 overexpression on cardiomyocyte hypertrophy by infecting NMVMs with an adenovirus expressing RFP-tagged ATF3 (adATF3) or a control adenovirus (adRFP) after treatment with AngII or ET-1. In contrast to the hypertrophic response observed with Atf3 depletion, ATF3 overexpression reduced the size of cells treated with vehicle, AngII (Fig. 2 H and I) or ET-1 (Figure S1 H and I). Additionally, compared to the control-transfected cells, the ATF3-overexpressing cardiomyocytes exhibited reduced Anp induction triggered by AngII (Fig. 2 J) or ET-1 treatment (Figure S1 J). The mRNA expression of Anp , Bnp , and b -Mhc was also significantly reduced in the ATF3-overexpressing cells treated with vehicle, Ang-II (Fig. 2 K-M) or ET-1 (Figure S1 K-M). Although our in vitro results clearly demonstrated the role of Atf3 in cardiomyocyte hypertrophy, the fact that NMVMs isolated from mice are not composed solely of cardiomyocytes suggests that the observed effect of ATF3 expression on cardiomyocyte hypertrophy may be due to the role of cardiac fibroblasts, as previously reported. 21 To address this issue, we induced cardiomyocyte differentiation with h7-human embryonic stem cells (hESC-CMs) to obtain high-purity cardiomyocytes and observed the effect of ATF3 on cardiomyocyte hypertrophy. When the cellular hypertrophy of hESC-CMs was induced by treatment with 100 ng/ml of ET-1, ATF3 expression was significantly increased by approximately 1.5-fold compared to that in the control cells, which was consistent with the increased expression of the natriuretic peptide A ( NPPA ) and natriuretic peptide B ( NPPB ) transcripts, similar to the results obtained with H9C2 and NMVMs (Fig. 3 A-C). The protein expression of ATF3, NPPA, and ATF4 wasalso increased after ET-1 treatment (Fig. 3 D). Moreover, ATF3 overexpression via adATF3 transduction significantly reduced the size of cardiomyocytes, regardless of ET-1 treatment (Fig. 3 E and F). The adATF3-transduced cells exhibited decreased NPPA expression in response to ET-1 treatment (Fig. 3 G), and the mRNA expression of NPPA , NPPB , and b -MHC was also significantly reduced in the adATF3-transduced hESC-CMs (Fig. 3 H-J). Overall, these findings suggest that ATF3 expression in cardiomyocytes is important for its suppressive effect on cardiomyocyte hypertrophy. ATF3 is mainly expressed in the ventricular cardiomyocytes of human hearts A recent study revealed single-nucleus and spatial transcriptome profiles to elucidate the molecular alterations related to the progression of human heart disease. 34 To further elucidate the cell type responsible for ATF3 activity, we performed a dataset analysis of single-nucleus RNA-sequencing (snRNA-seq) results from human heart tissue from healthy controls and patients with myocardial infarction available in the accessible public domain ( https://zenodo.org/record/6578047 ). First, eleven major cardiac cell types were identified among clusters annotated with curated marker genes from the literature 34 , 35 , 36 , 37 and visualized with the uniform manifold approximation and projection (UMAP) algorithm (Fig. 4 A). The level and proportion of ATF3 expression differed depending on cardiac cell type based on the data analyzed by UMAP, violin plots, and bar plots (Fig. 4 B-D). Interestingly, contrary to previous reports, 21 the primary cells expressing the highest level of ATF3 in all human heart tissues were cardiomyocytes, not fibroblasts. Next, to investigate how cardiac stress affects ATF3 expression in different types of cardiac cells, we analyzed expression data from healthy human hearts and from patients with myocardial infarction. In addition, ATF3 expression in ventricular cardiomyocytes varied depending on the sampling region (control, border, fibrotic, ischemic, and remote zone) and the progression of myocardial infarction (myogenic, fibrotic, and ischemic events) (Fig. 4 E-H). To further examine the role of ATF3 in cardiomyocytes, we removed cardiomyocytes from healthy donor samples. We identified 7 cardiomyocyte clusters, each manually annotated by their ranked gene expression: PCDH7 + CM, GPC5 + CM, SLC44A5 + CM, A2M + CM, TSPAN9 + CM, BCL6 + CM, and ATF3 + CM which is mainly consisted of cells with high expression of ATF3 (Fig. 4 I-K). To investigate cellular crosstalk, we conducted receptor‒ligand interaction analysis using the CellChat pipeline (Fig. 4 L and M). ATF3 + CMs play dual roles as both signal senders in SEMA3 signaling, influencing all other cardiac clusters, and as signal acceptors for ANGPTL signaling from cardiomyocytes with highly expressed alpha-2-macroglobulin-positive cells (Fig. 4 N and O). ANGPTL4 can mitigate cardiac hypertrophy and fibrosis induced by phenylephrine and AngII, respectively. 26 , 38 Additionally, we found that this cluster has increased transcriptional activity of the SMAD1, BHLHE40 and NR4a1 (Nur77) transcription factors, which can also protect cardiomyocytes from ischemia‒reperfusion (IR) injury, 39 regulate mitochondrial ROS production 40 and maintain cardiomyocyte calcium homeostasis 41 , 42 respectively (Figure S2). Therefore, we hypothesize that ATF3 + CMs can inhibit maladaptive cardiac remodeling processes under stress conditions. Cardiac-specific Atf3 deletion causes dilated cardiomyopathy induced by AngII infusion To conduct a more comprehensive study, we observed how the heart function of mice is affected by the absence of Atf3 in myocardial cells. To induce specific Atf3 deficiency in cardiomyocytes, we generated Atf3 tm2c/tm2c;aMHC−MerCreMer mice ( Atf3 f/f−MCM ) in which the Atf3 gene was specifically removed from cardiomyocytes. At 5 days post-Ang II infusion, when Atf3 is highly expressed, Atf3 deficiency was induced by injecting tamoxifen (TMX) for 3 days to activate the Cre protein. As shown in Fig. 5 A, the TMX-injected Atf3 f/f−MCM (cKO) mice exhibited significantly reduced Atf3 expression with or without AngII infusion. In contrast to the 100% survival rate of the control mice, the survival rate of the cKO mice decreased to 90%, which further decreased to 70% in the presence of AngII infusion (Fig. 5 B). Atf3 deficiency exacerbated AngII-induced cardiac hypertrophy, resulting in a significant increase in heart weight (Fig. 5 C and D). In addition, Masson's trichrome staining revealed substantially increased myocardial fibrosis in the hearts of the cKO mice after AngII infusion (Fig. 5 E and F). Furthermore, a significant increase in cardiomyocyte size was observed in the hearts of the cKO mice after AngII infusion (Fig. 5 G and H). The mRNA levels of Anp , Bnp , and b -Mhc were strongly elevated in the hearts of the AngII-infused control and cKO mice (Fig. 5 I-K). Similarly, immunoblot analysis of the control and cKO mice followed by AngII infusion also revealed increased levels of Anp, a-Sma, and b-catenin in cKO mice (Fig. 5 L). To determine cardiac function, we performed echocardiographic analysis on the AngII-infused mice for 2 weeks. M-mode echocardiographic analysis revealed that 2 weeks of AngII infusion impaired cardiac function by significantly decreasing the ejection fraction (EF) and fractional shortening (FS) in the cKO mice compared to those in the control mice (Fig. 5 M-O). Furthermore, the systolic left ventricular internal dimension (LVID;s) was significantly increased in the hearts from the AngII-infused cKO mice compared to AngII-infused control mice (Table S1 ), indicating a dilated cardiac hypertrophy phenotype. These results indicate that Atf3 deficiency in cardiomyocytes exacerbates cardiac dysfunction caused by AngII infusion. Taken together, these data suggest that Atf3 might play a protective role against cardiomyopathy triggered by AngII. Cardiomyocyte-specific Atf3 depletion alters the transcriptome of genes related to mitochondrial dysfunction and the extracellular matrix composition To demonstrate the major pathway involved in abnormal cardiac remodeling in cKO mice, we performed 3' quantitative mRNA sequencing on hearts from both the control and cKO mice after AngII infusion. Despite the very low Atf3 protein expression under normal conditions, 278 upregulated and 286 downregulated genes were identified in the hearts from the Atf3 cKO mice (Figure S3A). A volcano plot showing the differential gene expression in the hearts from the cKO mice is presented in Figure S3B. Upregulated or downregulated genes are represented by yellow and blue dots, respectively. These genes include several critical genes related to cardiac hypertrophy, contractility, and heart failure, such as bone morphogenetic protein-4 ( Bmp4 ), 43 ATPase Na+/K + transporting subunit alpha 1 ( Atp1a1 ), 44 and angiotensin II receptor type 1a ( Agtr1a ). 45 Additionally, the expression of genes related to mitochondrial function, such as NADH:ubiquinone oxidoreductase complex assembly factor 5 ( Ndufaf5 ), 46 cytochrome c oxidase subunit 5A ( Cox5a ), 47 and nicotinamide nucleotide transhydrogenase ( Nnt ), 48 were significantly decreased. These data suggest that Atf3 depletion causes mitochondrial dysregulation. Additional differential gene expression analysis revealed altered pathways, including the establishment of protein localization to organelles and mitochondrial organization, which were ranked at the top of the Gene Ontology (GO):Biological Processes (GO:BP) list with statistical significance in a comparison with the Atf3 f/f control heart samples (Figure S3C). To determine the regulatory mechanisms underlying cardiac abnormalities in the hearts of the AngII-infused Atf3 cKO mice, we conducted a comparative analysis of 15,334 genes which were preprocessed the data by using the DESeq2 pipeline to remove entries with a read count of 15 or less and normalized and stabilized the variance. After these genes were filtered out, we calculated the distance (Fig. 6 A, upper) and correlation (Fig. 6 A, lower) between the samples using standard DESeq2 protocols. Pairwise correlation analysis (PCA) revealed a greater degree of similarity in gene expression between the cKO-AngII heart samples and the control and control-AngII heart samples. Next, we performed weighted gene coexpression network analysis (WGCNA), identified optimal cluster sets using dynamic tree cutting, characterized and constructed representative module eigengenes (Fig. 6 B), calculated the mean values for each module, represented them as single values (averages), and visualized them through heatmaps (Fig. 6 C). Further analysis focused on significant modules (Darkgreen, Grey60, Lightgreen, Turquoise), revealing the expression patterns of enriched genes through heatmap representation (Figures S4A and 6D). Using 2,330 genes enriched in the Turquoise module from the cKO-AngII mice, we performed gene set enrichment analysis (GSEA) and clustered them with similar GO terms using the EnrichmentMap algorithm. Through this process, we identified 287 nodes categorized into 9 GO groups (Fig. 6 E and F). These groups included cell substrate adhesion, cell matrix adhesion, cellular respiration and mitochondrial complex assembly as the top-ranked GO terms, suggesting that Atf3 plays a protective role against mitochondrial oxidative stress and cardiac fibrosis induced by AngII infusion (Fig. 6 G). To identify the genes associated with the top regulated GO terms, we examined the networks of nodes associated with 'cellular respiration' and 'cell matrix adhesion'. Using a radial layout algorithm, we verified that the 21 nodes associated with cellular respiration formed a network centered on the organophosphate biosynthetic process (Fig. 6 H). The expression levels of genes enriched in these ontologies are illustrated as heatmaps in Figs. 6 I and S5. These findings implicate the Nduf , Atg5 , and collagen gene families, suggesting that ATF3 is essential for preventing cardiac fibrosis and mitochondrial oxidative stress. Atf3 is required for maintaining mitochondrial function Notably, according to the RNA sequencing data, genes related to the oxidative phosphorylation system (OXPHOS) and mitochondrial quality control, such as heat shock protein 10 (Hsp10, also known as Hspe1) and Hsp60 (Hspd1), 49 , 50 , 51 NFE2-like BZIP transcription factor 1 (Nfe2l1, also known as Nrf-1), 52 translocase of the outer mitochondrial membrane complex subunit 20 (Tomm20), 53 estrogen-related receptor alpha (Esrra, also known as Err), 54 peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1), 55 , 56 overlapping proteolytic activity with m-AAA protease 1 (Oma1) 57 , 58 and caseinolytic peptidase P (ClpP), 59 were significantly altered (Figs. 6 I and S6). To confirm the dysregulation of mitochondrial oxidation, we performed transmission electron microscopy (TEM) analysis of the control and AngII-infused heart tissue (Fig. 7 A). Consequently, we found that the hearts from the AngII-infused cKO mice exhibited increased mitochondrial size and abnormal mitochondrial morphology (Fig. 7 B and C). Additionally, compared with the control heart tissue, the heart tissue from the AngII-infused cKO mice showed a significant decrease in mitochondrial DNA content (Fig. 7 D). Additionally, we performed a JC-1 mitochondrial membrane potential assay with control or ATF3-depleted NMVMs after AngII treatment. Compared with those of the controls, the red/green JC-1 ratio of the Atf3-depleted NMVMs decreased, indicating decreased mitochondrial function (Figure S7A and B). Consistent with these findings, compared with the control cells, the ATF3-depleted NMVMs also exhibited significantly decreased mitochondrial DNA content after AngII treatment (Figure S7C). To validate these findings, we examined the protein expression levels of OXPHOS complex subunits in vivo. As shown in Fig. 7 E and F, the expression of OXPHOS complex subunits in the cKO heart samples substantially decreased relative to those in the control heart samples. In summary, these findings suggest that the absence of Atf3 exacerbates cardiac dysfunction by impairing mitochondrial homeostasis. Atf3 can regulate ROS production through the Sirt3/Sod2 axis A previous study suggested that overexpression of Atf3 inhibits ROS production in primary hepatocytes and that hepatocyte-specific Atf3-null mice exhibit increased hepatic ROS levels. 60 Dysfunction of mitochondria in hearts of the AngII-infused cKO mice may be attributed to increased ROS accumulation resulting from the UPR mt , likely due to the reduced expression of mitochondrial chaperone proteins (Figure S6). Acetylation of the Sod2 protein regulates mitochondrial ROS production through a well-documented mechanism. There is an inverse correlation between Sod2 acetylation and Sod2 activity, and Sirt3, the major deacetylase in mitochondria, can activate Sod2 by deacetylating the lysine 68 residue of Sod2. 61 We therefore tested whether cKO hearts infused with AngII display reduced Sirt3 expression and increased Sod2 acetylation. As expected, the mRNA expression levels of Sirt3 and Sod2 were significantly increased in the AngII-infused control hearts, while these genes were stably expressed in the cKO hearts (Fig. 8 A and B). The Sirt3 protein expression level was significantly decreased in the AngII-infused cKO hearts (Fig. 8 C and D). Furthermore, ac-Sod2 protein levels were significantly increased in the cKO hearts following AngII infusion (Fig. 8 E). Since the acetylated Sod2 protein is inactive and cannot scavenge ROS, the increased acetylation of Sod2 might be attributable to increased intracellular ROS levels and the sequential activation of AKT and P70S6K. Additionally, a previous study suggested that Sirt3 overexpression can decrease the kinase activity of P70S6, 62 which is phosphorylated and activated by hypertrophy of cardiomyocytes. Interestingly, Atf3 deficiency exacerbated IR-induced liver inflammation by activating the mTOR/P70S6K/Hif-1alpha signaling pathway. 63 Therefore, we assessed the expression of pAKT and pP70S6K (Fig. 8 C, E, and F). The phosphorylation of P70S6K was significantly increased in the hearts of the AngII-infused cKO mice, whereas pAKT levels are not altered. (Fig. 8 C). Furthermore, Pgc-1a protein levels were significantly reduced in the hearts of the cKO-AngII mice (Fig. 8 C). Given that Sirt3 expression can be upregulated by Pgc-1a, 64 Atf3 depletion elicits dysregulation of the Pgc-1a/Sirt3/Sod2 axis, which is associated with cardiomyopathy. To determine whether the expression of Sirt3 and Sod2 is regulated by the presence or absence of Atf3 in primary cardiomyocytes, we depleted or overexpressed ATF3 in NMVMs followed by AngII treatment. Compared with those in the control cells, the Atf3-depleted NMVMs exhibited increased ac-Sod2 and pP70S6K protein levels, and Sirt3 and Sod2 transcript levels were significantly decreased after AngII treatment (Fig. 8 I-J). In contrast, in the ATF3-overexpressing NMVMs, Sirt3 expression was highly increased, and ac-Sod2 and pP70S6K protein levels were decreased compared to those in the control cells (Fig. 8 L). The mRNA expression of Sirt3 was also significantly increased by approximately 1.5-fold in the ATF3-overexpressing group compared to the control group (Fig. 8 M). Sod2 transcript levels in ATF3-overexpressing cells was similar to that in the RFP-overexpressing control cells (Fig. 8 N). Moreover, we examined whether the regulation of intracellular ROS levels depends on the presence or absence of ATF3 in H9C2 cardiomyocytes. The results showed that ROS levels were significantly greater in the ATF3-deficient H9C2 cells than in the control cells upon treatment with AngII (Fig. 8 O and P). Conversely, in the H9C2 cells transduced with adATF3, ROS levels were decreased after AngII treatment compared to those in the control cells (Fig. 8 Q and R). Furthermore, downregulated genes related to mitochondrial respiration, including those in the NDUF family and PGC-1a , in the hearts of the AngII-infused cKO mice were upregulated in the ATF3 + CM clusters from the human heart scRNA-seq data. In contrast, the expression of genes related to integrin and collagen superfamily genes related to ATF3 + CM clusters that were upregulated in the hearts of the AngII-infused cKO mice decreased (Figure S8). These results further supported that Atf3 is essential for the mitochondrial antioxidant mechanism governed by the Sirt3-Sod2 axis. In summary, our data collectively suggest that ATF3 in cardiomyocytes plays a crucial role in protecting cardiomyocytes from abnormal cardiac remodeling induced by AngII treatment by regulating the Pgc-1a/Sirt3/Sod2 axis and mitochondrial oxidative stress. Discussion Although many studies have revealed the role of ATF3 in cardiomyocytes through ATF3 overexpression or deletion, it is still unclear whether ATF3 protects against or facilitates the development of CVDs. 24 Several studies have suggested that ATF3 deficiency in mice leads to cardiac hypertrophy under pressure overload. Global ATF3 knockout exacerbated Ang II-induced cardiac hypertrophy, suggesting that ATF3 plays a protective role in the development of cardiac hypertrophy. The effect of this molecule is related to the ERK and JNK pathways and the ET-1 response. 22 , 65 , 66 In contrast, cardiac-specific ATF3 overexpression in transgenic mice led to various indicators of heart failure, including atrial enlargement, cardiac hypertrophy and fibrosis, reduced contractility and aberrant cardiac conduction. 16 Other studies reported that global ATF3-KO protected against the cardiac hypertrophy phenotype 18 and increased the survival rate after myocardial infarction. 17 These conflicting findings make it difficult to determine the function of ATF3 in cardiac hypertrophy. Moreover, ATF3 regulates multiple target genes and affects other cardiac resident cell types, as well as cardiomyocytes. Recently, overexpression of ATF3 specifically in cardiac fibroblasts was shown to strongly mitigate cardiac remodeling and heart failure by inhibiting the expression of Map2K3 and the subsequent p38-TGF-β signaling pathway. 21 Thus, it is very difficult to determine the exact function of ATF3 in cardiomyocytes under stress conditions associated with cardiac hypertrophy. Therefore, more studies are needed to elucidate the detailed regulatory mechanisms of cardiac hypertrophy mediated by ATF3 in cardiomyocytes. In this study, we demonstrated that ATF3 is required for the regulation of mitochondrial oxidative stress in cardiomyocytes. The ablation of ATF3 in cardiomyocytes led to a significantly increased hypertrophic response upon treatment with AngII and ET-1. In contrast, transient overexpression of ATF3 prevented the hypertrophic response in NMVMs and hESC-CMs. Similar to the in vitro results, hearts of the TMX-inducible cardiomyocyte-specific Atf3 knockout mice exhibited structural and functional defects, including cellular hypertrophy and fibrosis, as well as reduced cardiac function, as indicated by the decreased EF and FS. These data further supported the protective role of Atf3 in cardiomyocytes under stress conditions. Consistent with these findings, the RNA sequencing analysis also revealed changes in the expression of genes associated with the extracellular matrix, which are indicative of increased cardiac fibrosis in the hearts of the AngII-infused cKO mice. Interestingly, WGCNA and GSEA revealed that genes related to cellular respiration, such as Nduf and Atp5 superfamily genes and Pgc-1a , were significantly altered in the hearts of the AngII-infused cKO mice with abnormal mitochondrial structure and function. In a detailed analysis of mitochondrial dysfunction, we observed reduced expression of the Sirt3 protein and increased levels of acetylated Sod2 in the hearts of the AngII-infused cKO mice. This effect was reversed by ATF3 overexpression in NMVMs, suggesting that ATF3 can regulate mitochondrial oxidative stress, potentially through the UPR mt , by modulating Sirt3 and acetylated Sod2 protein levels. Recent studies have been conducted on transcriptional regulatory mechanisms at the single-cell level to elucidate the pathological development of heart diseases. In addition, spatial networking analysis between cells has enabled a better understanding of disease progression in multicellular tissues composed of diverse cell types. 34 , 67 , 68 These studies not only demonstrate gene expression patterns based on the presence or absence of a disease but also reveal the spatial and temporal expression and activity of specific gene clusters within each cell type. These results provide insights into the progression of diseases influenced by the spatial and temporal expression and activity of particular genes in specific contexts, going beyond a simple indication of the occurrence of a disease. By analyzing human heart snRNA-seq data, we identified cardiomyocytes as the primary cell type expressing ATF3 . The cluster of cardiomyocytes with high ATF3 expression (ATF3 + CM) expresses receptors that respond to the ANGPTL4 ligand, playing a crucial role in preventing abnormal cardiac remodeling induced by AngII infusion. 26 , 38 Additionally, we observed that this cluster has elevated transcriptional activity of the SMAD1, BHLHE40 and NR4a1 (Nur77) transcription factors, which can also prevent maladaptive cardiac remodeling, supporting our current findings. 39 , 40 , 41 , 42 In conclusion, our study addresses the importance of ATF3 in preventing maladaptive cardiac remodeling in cardiomyocytes in response to AngII and ET-1 treatments by regulating mitochondrial oxidative stress. This comprehensive approach ensures a more nuanced understanding of the intricate functions of proteins such as ATF3. Declarations Confilict of Interest None. Author contributions M.-H. Jeong, Y. Jeong, J.-S. Kang and W.-H. Kim designed the study. M.-H. Jeong, Y. Jeong and S.-Y. 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Single-cell and spatial transcriptomics: deciphering brain complexity in health and disease. Nat Rev Neurol 19, 346–362 (2023). Additional Declarations (Not answered) Supplementary Files ATF3SupplementalinformationforEMM.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4485671","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":315221946,"identity":"99f325e9-f614-41e9-85a4-5b6ad8bd80dd","order_by":0,"name":"Won-Ho Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYBACxhlsDMwMFQkQTgPxWs6QooVBAqiFsY0ULcyz29KkC+elyZm3Nz9gnLmHGIfNOXZMeua2HGOZM8cMGDc8I0bLjPS227zbKhJnSCQYMD44QLSWOUAt8s8/EKsl7dht3oYcoC08QIcRqSX9N8+xNGMJnpyCgzOI0WI4I83YmKcmWU6C/fjGhz1EaWlA4hCjgYFBnihVo2AUjIJRMLIBALDbOEj99v/AAAAAAElFTkSuQmCC","orcid":"","institution":"National Institute of Health","correspondingAuthor":true,"prefix":"","firstName":"Won-Ho","middleName":"","lastName":"Kim","suffix":""},{"id":315221947,"identity":"72857f95-a639-4914-90f2-093c42eded30","order_by":1,"name":"Myong-Ho Jeong","email":"","orcid":"https://orcid.org/0000-0001-8185-9869","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Myong-Ho","middleName":"","lastName":"Jeong","suffix":""},{"id":315221948,"identity":"2949b95e-d3ac-4f8a-b3c7-03a4ff50dffc","order_by":2,"name":"Yideul Jeong","email":"","orcid":"","institution":"Research Institute of Aging Related Disease, AniMusCure Inc.","correspondingAuthor":false,"prefix":"","firstName":"Yideul","middleName":"","lastName":"Jeong","suffix":""},{"id":315221949,"identity":"21a34b17-d802-4dfb-8e04-17c3f9508df7","order_by":3,"name":"Su-Yeon Cho","email":"","orcid":"","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Su-Yeon","middleName":"","lastName":"Cho","suffix":""},{"id":315221950,"identity":"f5dae34a-dc5c-4a9b-a067-23ae374d8614","order_by":4,"name":"Seung Hee Lee","email":"","orcid":"","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Seung","middleName":"Hee","lastName":"Lee","suffix":""},{"id":315221951,"identity":"4d1a92eb-bdad-45ae-b5a1-4f457e5b6570","order_by":5,"name":"Geun-Young Kim","email":"","orcid":"","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Geun-Young","middleName":"","lastName":"Kim","suffix":""},{"id":315221952,"identity":"87efad91-bed5-4952-94ba-fd25b965a5ac","order_by":6,"name":"Min-Ju Kim","email":"","orcid":"","institution":"National Institute of Health","correspondingAuthor":false,"prefix":"","firstName":"Min-Ju","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-05-27 14:20:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4485671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4485671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60341437,"identity":"231e7d9b-8073-4968-9038-628beec9a7c0","added_by":"auto","created_at":"2024-07-15 18:43:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1393658,"visible":true,"origin":"","legend":"\u003cp\u003eAtf3 is increased in H9C2 cardiomyocytes induced by AngII treatment.\u003c/p\u003e\n\u003cp\u003eA. Representative immunostaining images of PBS- or AngII-treated H9C2 cells. Scale bar: 25 mm. B. Immunoblot analysis of Atf3 and Anp expression in PBS- or AngII-treated H9C2 cells. a-Tubulin was used as a loading control. C-E. qRT‒PCR analysis of \u003cem\u003eAtf3\u003c/em\u003e and the cardiac hypertrophy markers \u003cem\u003eAnp\u003c/em\u003e and \u003cem\u003eBnp\u003c/em\u003e. N=6. F. Immunoblot analysis of Atf3, a-SMA and Anp in the control and AngII-infused WT mouse heart samples. Gapdh was used as a loading control. G. qRT‒PCR analysis of Atf3 in the mouse heart samples in panel F. *p\u0026lt;0.05, **p\u0026lt;0.001, ****p\u0026lt;0.0001. One-way ANOVA. The data represent ±SD.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/f823eb639bf899234266d98f.png"},{"id":60341439,"identity":"c28d87e4-d1f6-4e41-9abc-a4db80318ab5","added_by":"auto","created_at":"2024-07-15 18:43:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3293667,"visible":true,"origin":"","legend":"\u003cp\u003eAtf3 depletion causes mouse cardiomyocyte hypertrophy, while its overexpression attenuates AngII-induced cellular hypertrophy.\u003c/p\u003e\n\u003cp\u003eA. Representative fluorescence images of NMVMs treated with vehicle or Cre recombinase for 24 h following PBS or AngII treatment for 24 h. B. Measurement of the relative cell surface area as shown in panel (A). C. Immunoblotting of vehicle- or Cre-treated NMVMs treated with PBS or AngII for 24 h. a-Tubulin was used as a loading control. D. qRT‒PCR analysis of \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e and b\u003cem\u003e-Mhc\u003c/em\u003e levels in the control or Cre recombinase-treated NMVMs following PBS or AngII treatment. E. Representative immunostaining images of Ad-RFP- or Ad-RFP-ATF3-transfected NMVMs treated with PBS or AngII for 24 h. F. Quantification of the cell surface area, as shown in panel (E). G. Protein expression of Ad-RFP- or Ad-RFP-ATF3-transfected NMVMs treated with PBS or AngII for 24 h. -Tubulin was used as a loading control. H. Relative qRT‒PCR analysis of \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e and b\u003cem\u003e-Mhc\u003c/em\u003e levels in control or Ad-RFP-ATF3-transfected NMVMs treated with PBS or AngII for 24 h. n=3. For determination of statistical significance, one-way ANOVA was used. *p\u0026lt;0.05, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001. One-way ANOVA. The data represent ±SD. Scale bars: 20 mm (A and H) .\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/b47bdb3f3973457eb49289c2.png"},{"id":60341435,"identity":"cff929cf-7bcc-4360-8477-9b3975003bc2","added_by":"auto","created_at":"2024-07-15 18:43:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2092039,"visible":true,"origin":"","legend":"\u003cp\u003eATF3 overexpression can attenuate ET-1-induced cardiac hypertrophy in hESC-CMs.\u003c/p\u003e\n\u003cp\u003eA-C. Relative gene expression of \u003cem\u003eATF3\u003c/em\u003e, \u003cem\u003eNPPA\u003c/em\u003e and \u003cem\u003eNPPB\u003c/em\u003e in the control or ET-1-treated hES-CMs. D. Immunoblotting for ATF3, NPPA, ATF4 and a-SMA in the control or ET-1-treated hESC-CMs. Gapdh served as a loading control. E. Representative images of phalloidin (green)-stained hESC-CMs treated with the control or ET-1 for 24 h. Scale bar: 20 mm. F. Quantification of the relative cell surface area for panel E. G. Protein expression of ATF3, NPPA and TUBA1a in the adRFP- or adRFP-ATF3-overexpressing hESC-CMs treated with the control or ET-1 for 24 h. H‒J. qRT‒PCR analysis of \u003cem\u003eNPPA\u003c/em\u003e, \u003cem\u003eNPPB\u003c/em\u003e and b\u003cem\u003e-MHC\u003c/em\u003e in the control or ET-1-treated hESC-CMs overexpressing adRFP or adRFP-ATF3. For determination of statistical significance, one-way ANOVA was used. *p\u0026lt;0.05, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001. One-way ANOVA. The data represent ±SD.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/c320a7babb066bc5ebd2b903.png"},{"id":60341438,"identity":"43b4bd9a-4eac-452b-a647-443794e2a237","added_by":"auto","created_at":"2024-07-15 18:43:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3513230,"visible":true,"origin":"","legend":"\u003cp\u003eProfiles of human cardiomyocytes correlated with \u003cem\u003eATF3\u003c/em\u003e expression.\u003c/p\u003e\n\u003cp\u003eA. UMAP of snRNA-seq data from total cardiac cells (n=191,795). B. UMAP visualization of \u003cem\u003eATF3\u003c/em\u003e-expressing cardiac cells. C-D. Violin plots showing the \u003cem\u003eATF3\u003c/em\u003e expression level in total cardiac cells (C) and the proportion of each cell type (D). E. UMAP plot of \u003cem\u003eATF3 \u003c/em\u003eexpression in normal and myocardial infarcted heart tissue samples. F-H. Violin plots showing the \u003cem\u003eATF3 \u003c/em\u003eexpression levels in ventricular cardiomyocytes. I-J. Subclustering of ventricular cardiomyocytes isolated from normal control hearts annotated by their ranked gene expression. K. Expression of ATF3 in CM clusters. L. Circle plots of the significant ligand‒receptor (LR) interactions between CM clusters. M. The contribution of each LR pair shown in panel L. N-O. Circle plots for the SEMA3 (N) and ANGPTL (O) signaling pathways in each CM cluster and the communication score between interacting cell clusters (upper panel). The heatmap shows that rows and columns represent sources and targets, respectively. The bar plots on the right and at the top represent the total outgoing and incoming interaction scores, respectively (lower panel).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/0c49a13d901982dadcb2e45d.png"},{"id":60341440,"identity":"7009e374-57f4-4687-8a16-158308caa219","added_by":"auto","created_at":"2024-07-15 18:43:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6872271,"visible":true,"origin":"","legend":"\u003cp\u003eCardiac-specific ATF3 deficiency exacerbates cardiomyopathy induced by AngII infusion.\u003c/p\u003e\n\u003cp\u003eA. mRNA expression level of ATF3 in the indicated heart tissues. B. Survival rates of \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e (n=12, Cont), \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f-MCM\u003c/em\u003e\u003c/sup\u003e (n=12, cKO), Cont-AngII (n=12), and cKO-AngII (n=17) mice. Mice were injected with TMX (30 µg/g body weight/day for 3 consecutive days) to induce Cre activity. C. Representative images of hearts from the Cont and cKO mice infused with the control or AngII. D. Relative heart weights of the Cont and cKO mice infused with the control or AngII. E. Representative images of Masson’s trichrome staining of the Cont and cKO mice infused with the control or AngII. F. Quantification of the relative fibrotic area shown in panel (E). G. Representative confocal microscopy images of hearts from the Cont and cKO mice infused with the control or AngII. H. Quantification of the cross-sectional area (CSA) of the panel (G). I-K. Relative mRNA expression levels of \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e and b-\u003cem\u003eMhc\u003c/em\u003e in the indicated heart tissues. L. Immunoblotting analysis of Atf3, Anp, a-Sma and b-catenin protein expression in heart samples. Gapdh was used as a loading control. M. Representative echocardiographic images of the Cont and cKO mice infused with control PBS or AngII. N-O. The parameters of the echocardiogram of the ejection fraction; EF (N), fractional shortening; FS (O). For determination of statistical significance, one-way ANOVA was used. *p\u0026lt;0.05, **p\u0026lt;0.001, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001. One-way ANOVA. The data represent ±SD. Scale bars: 50 mm (E and G).\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/4451faff32e14d03113ebb5b.png"},{"id":60341441,"identity":"fd3634ca-2ee0-4e03-ba30-b6a6ddb2d9ef","added_by":"auto","created_at":"2024-07-15 18:43:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6638129,"visible":true,"origin":"","legend":"\u003cp\u003eCardiomyocyte-specific Atf3-depleted hearts show altered gene expression profiles related to cardiac fibrosis and mitochondrial oxidative stress induced by AngII infusion.\u003c/p\u003e\n\u003cp\u003eA. Visualization of the clustering pattern of gene expression in mouse heart samples via heatmaps (upper panel) and PCA plots (lower panel). B. WGCNA was performed on genes normalized in DESeq2. The heatmap shows the expression value of the module in each C. The average expression level of the modules in each sample is shown as a single value. D. Highly enriched genes in the cKO-AngII mice are shown as a heatmap (2330 genes). E. GO network for enriched genes in the turquoise module following GSEA. F. Statistics for the GO network in panel F. G. Up- and downregulated GO list based on NES. H. Graphical analysis of GO interactions for downregulated genes in the GO network in cKO-AngII heart samples. I. Heatmaps for enriched genes of GO terms in panel H\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/a3b7ffcea182814540904399.png"},{"id":60341436,"identity":"294130c9-b2f1-41a6-9ae8-8d2fe8998531","added_by":"auto","created_at":"2024-07-15 18:43:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2087387,"visible":true,"origin":"","legend":"\u003cp\u003eCardiomyocyte-specific Atf3 depletion causes impaired heart function via mitochondrial dysfunction.\u003c/p\u003e\n\u003cp\u003eA. Representative images for TEM analysis of the control or AngII-infused heart samples. Scale bar: 2 mm. B-C. Quantification of mitochondrial size and abnormalities in panel A. D. Relative mitochondrial DNA (\u003cem\u003emtCo1\u003c/em\u003e)-to-nuclear DNA (\u003cem\u003eNdufv1\u003c/em\u003e) ratio. The values from the control were set to 1. E. Representative immunoblotting images of OXPHOS complex subunits from the indicated groups. F. Quantification of protein expression in panel E. For determination of statistical significance, one-way ANOVA was used. *p\u0026lt;0.05, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001. One-way ANOVA. The data represent ±SD.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/e3101406fbb639c08b602a09.png"},{"id":60341434,"identity":"954cc9c8-2e55-4cd8-802b-33aa0c0f43e8","added_by":"auto","created_at":"2024-07-15 18:43:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3032132,"visible":true,"origin":"","legend":"\u003cp\u003eAtf3 is required for Sirt3-dependent Sod2 acetylation in vivo and in vitro\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA-B. qRT‒PCR analysis of \u003cem\u003eSirt3\u003c/em\u003e and \u003cem\u003eSod2\u003c/em\u003e in the indicated heart samples. C. Western blot analysis of the protein expression of Sirt3, ac-Sod2 and Sod2. Gapdh served as a control. D-H. Quantification of the protein expression levels of Sirt3 (D), acSod2 (E), pAKT (F), pP70S6K (G) and Pgc-1a (H). I. Protein analysis of the control or Cre Gesicle-treated NMVMs. The numbers under each protein show the relative intensity of the protein level. The values from the control were set to 1. J-K. qRT‒PCR analysis of \u003cem\u003eSirt3\u003c/em\u003e and \u003cem\u003eSod2\u003c/em\u003e in the indicated NMVMs. I. Protein analysis of the RFP- or RFP-ATF3-overexpressing NMVMs. The numbers under each protein show the relative intensity of the protein level. The values from the RFP control were set to 1. M-O. The RNA expression levels of \u003cem\u003eSirt3\u003c/em\u003eand \u003cem\u003eSod2\u003c/em\u003e in the indicated NMVMs. O and Q. Measurement of ROS in the ATF3-depleted or ATF3-overexpressing H9C2 cells treated with PBS or AngII. Scale bars: 20 mm. P and R. Quantification of the signal intensity in panels O and Q, respectively. For determination of statistical significance, one-way ANOVA was used. *p\u0026lt;0.05, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001. The data represent ±SD.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/c879527dfeb72d3dc68c83ea.png"},{"id":60343390,"identity":"53c4565c-2c82-40ee-ac1b-e79d4834e0e4","added_by":"auto","created_at":"2024-07-15 19:15:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40810889,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/e6a54954-0d8b-4e8e-9ddf-e506ff6c7f9d.pdf"},{"id":60341433,"identity":"e020537a-a188-47c6-81d5-da4f20fa3422","added_by":"auto","created_at":"2024-07-15 18:43:06","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3127462,"visible":true,"origin":"","legend":"","description":"","filename":"ATF3SupplementalinformationforEMM.docx","url":"https://assets-eu.researchsquare.com/files/rs-4485671/v1/38a52a6f8840e8cba695177b.docx"}],"financialInterests":"(Not answered)","formattedTitle":"ATF3 is required for the prevention of cardiomyopathy via the regulation of mitochondrial oxidative stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) have a major impact on global mortality rates.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Numerous studies on the structural and functional abnormalities of CVDs are being conducted to address these issues. Despite these efforts, the risk of CVDs continues to increase, further indicating the importance of research on the developmental process and basic mechanisms of CVDs.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that mitochondrial dysfunction is a critical factor in the development of CVDs. Mitochondria are essential cellular organelles responsible for producing the majority of cellular ATP via oxidative phosphorylation. These organelles also provide cofactors such as heme, iron-sulfur clusters, amino acids, and nucleotides.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Moreover, mitochondria are the primary sites responsible for producing reactive oxygen species (ROS),\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e which can act as signaling molecules, in cells. However, excessive ROS can cause oxidative damage to proteins, lipids, and DNA. Oxidative damage can cause protein misfolding, which in turn induces the mitochondrial unfolded protein response (UPR\u003csup\u003emt\u003c/sup\u003e). Thus, maintaining proper mitochondrial function is crucial for cell survival and function.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e The UPR\u003csup\u003emt\u003c/sup\u003e is essential for safeguarding mitochondria through various pathways, including increasing the expression of mitochondrial chaperone proteins, promoting the degradation of damaged mitochondrial proteins and regulating mitochondrial biogenesis.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Several studies have suggested that the activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) axis plays a pivotal role in both the UPR\u003csup\u003emt\u003c/sup\u003e and the endoplasmic reticulum UPR (UPR\u003csup\u003eer\u003c/sup\u003e) to resolve the UPR, primarily by inducing ATF3.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eATF3 is a member of the ATF/cAMP response element-binding protein (CREB) family of transcription factors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and is rapidly induced in response to different types of stress, such as the UPR and oxidative stress.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e ATF3 has been found to regulate various cellular processes as a transcriptional activator and repressor depending on the cellular context, cell type and presence of other cofactor protein complexes,\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e which is attributable to its dual and contradictory roles in a single organ.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Despite extensive research, the exact role of ATF3 in stress-induced cardiac hypertrophy and fibrosis remains unclear.\u003c/p\u003e \u003cp\u003ePrevious studies have indicated that ATF3 has a negative impact on CVDs, including stress-induced cardiac hypertrophy and fibrosis.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e However, recent investigations have increasingly revealed that ATF3 also has protective effects against CVDs,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e indicating its multifaceted role in cardiac dysfunctions and pathologies.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the role of ATF3 in cardiac function, we utilized in vivo and in vitro experimental systems and analyzed Gene expression omnibus (GEO) datasets from single-nucleus RNA sequencing (snRNA-seq) of human hearts from healthy subjects and patients with myocardial infarction. Transient overexpression or depletion of Atf3 prevented or accelerated neonatal mouse cardiomyocyte (NMVM) hypertrophy, respectively. Analysis of snRNA-seq data from human hearts showed that ATF3 is predominantly expressed in ventricular cardiomyocytes especially in cells subjected to angiopoietin-like 4 (ANGPTL4), which can attenuate phenylephrine-induced myocardial hypertrophy.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Additionally, cardiomyocyte-specific Atf3-null (cKO) mice exhibited a dilated cardiomyopathy phenotype with reduced cardiac function and increased cardiac fibrosis upon angiotensin II (AngII) infusion. Moreover, transcriptome analysis revealed that, following AngII infusion, cKO mice exhibit significant dysregulation of genes involved in the regulation of cellular respiration, such as genes involved in organophosphate biosynthetic processes and mitochondrial oxidation. In particular, hearts from AngII-infused cKO mice exhibited reduced expression of mitochondrial chaperone proteins and Sirtuin 3 (Sirt3). A decrease in Sirt3 expression coincided with an increase in the protein levels of acetylated superoxide dismutase 2 (ac-Sod2), an inactivated form of Sod2, a major mitochondrial antioxidant protein. Additionally, overexpression of Atf3 in NMVMs increased the expression of Sirt3 at both the mRNA and protein levels and decreased cellular ROS accumulation. In conclusion, Atf3 deficiency in cardiomyocytes causes mitochondrial dysfunction due to a decrease in Sirt3/Sod2-mediated ROS scavenging, which likely contributes to cardiomyopathy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eFor generation of tamoxifen-inducible cardiac-specific Atf3 null mice, \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2a/tm2a\u003c/em\u003e\u003c/sup\u003e (from C57BL/6N-\u003cem\u003eATF3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2a(EUCOMM)Wtsi\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/H\u003c/em\u003e) mice were crossed with FLP recombinase-expressing mice to promote recombination between FRT sites (Jax strain 003946), resulting in a floxed Atf3 allele (\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2c/tm2c\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e throughout the manuscript). Next, the \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003emice were crossed with a\u003cem\u003eMHC\u003c/em\u003e\u003csup\u003e\u003cem\u003eMerCreMer\u003c/em\u003e\u003c/sup\u003e mice to generate \u003cem\u003eaMHC\u003c/em\u003e\u003csup\u003e\u003cem\u003eMerCreMer\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/+\u003c/em\u003e\u003c/sup\u003e mice. The resulting \u003cem\u003eaMHC\u003c/em\u003e\u003csup\u003e\u003cem\u003eMerCreMer\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/+\u003c/em\u003e\u003c/sup\u003e offspring were then backcrossed to \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice to obtain \u003cem\u003eaMHC\u003c/em\u003e\u003csup\u003e\u003cem\u003eMerCreMer\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u0026minus;MCM\u003c/em\u003e\u003c/sup\u003e throughout the manuscript) mice. For promotion of cardiac hypertrophy, 11\u0026thinsp;~\u0026thinsp;12-week-old \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u0026minus;MCM\u003c/em\u003e\u003c/sup\u003e mice were infused with angiotensin II by subcutaneous implantation of an osmotic minipump (Alzet model 1002) for 14 days, followed by vehicle or tamoxifen injection (30 g/g body weight/day for 3 consecutive days, intraperitoneally) to induce Cre recombinase activity. In this procedure, the \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e;\u003cem\u003eaMHC\u003c/em\u003e\u003csup\u003e\u003cem\u003eMerCreMer\u003c/em\u003e\u003c/sup\u003e mice were considered to control the heart phenotype for tamoxifen toxicity. This study was reviewed and carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) of the Korea Disease Control and Prevention Agency (KDCA) under the protocol approved by KDCA-IACUC-022-003.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eFor echocardiographic analysis, the mice were anesthetized with 2% (vol/vol) isoflurane 1 day before sacrifice using a Vevo 2100 system (Fujifilm Visual Sonics, K-BIO Health). For analysis of cardiac parameters and functions, including the left ventricular internal diameter diastolic/systolic (LVID;d/s), interventricular septum diastolic/systolic (IVS;d/s), left ventricular posterior wall diastolic/systolic (LVPW;d/s), LV mass, fractional shortening (FS) and ejection fraction (EF), we analyzed cardiac M-mode images taken from the short axis view of the LV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and transfection\u003c/h2\u003e \u003cp\u003eH9C2 (KCLB, 21446) cells were cultured in 10% FBS (fetal bovine serum) in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with penicillin/streptomycin. NMVMs were isolated from postnatal day 1\u0026ndash;2 \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mouse hearts on a C57BL/6J background and cultured as previously described.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e hESCs were kindly provided by Professor JS Kang at Sungkyunkwan University (SKKU) and cultured as previously described.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e For transient overexpression experiments, we used an \u003cem\u003eATF3\u003c/em\u003e plasmid (Addgene, #26115) or an adenovirus expressing RFP-ATF3. Lipofectamine 2000 (Invitrogen, 11668) was used for the overexpression of plasmid DNA. For the adenoviral experiments, the cells were treated with AdATF3 at an MOI of 200 for 5 h, and adRFP was used as a control. For Atf3 knockdown in \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e NMVMs, Cre recombinase Gesicle (TaKaRa, 631449) was used according to the manufacturer\u0026rsquo;s instructions. For induction of cellular hypertrophy in H9C2 and NMVMs, the cells were starved for 24 h and then treated with 1 \u0026micro;M AngII or 100 ng/ml of ET-1 for an additional 24 h. For induction of hypertrophy in hESC-CMs, the cells were treated with 100 ng/ml ET-1 for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eProtein analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Briefly, cultured cells or homogenized tissue samples were lysed in RIPA buffer (Pierce\u0026trade;, #89900) containing complete protease inhibitor cocktail (Sigma‒Aldrich, P8340). The samples were then subjected to SDS‒PAGE and immunoblotting using various primary antibodies. The primary and secondary antibodies used in this study are listed in Supplementary Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSingle-nuclear RNA sequencing (snRNA-seq) data analysis\u003c/h2\u003e \u003cp\u003ePreviously published snRNA-seq data deposited in the Zenodo Data Archive (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zenodo.org/record/6578047\u003c/span\u003e\u003cspan address=\"https://zenodo.org/record/6578047\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e were used to assess the role of ATF3 in cardiomyocytes (CMs). All the snRNA-seq datasets were analyzed using the Seurat and CellChat pipelines. The datasets were integrated using the pipeline of the dimensionality reduction platform. The integrated cells underwent a quality control process to remove dead cells and doublets based on library size and cell dispersion. Subsequently, the integrated cells were normalized to counts per million (CPM). The filtered cells were then clustered using the Seurat pipeline with a resolution of 0.5. For data visualization, a dimensional reduction uniform manifold approximation and projection (UMAP) was generated through the Seurat function RunUMAP. Cluster identities were assigned based on the expression of the top marker genes and what is known from published literature. The differentially expressed genes in the snRNA-seq database were extracted based on the following conditions: 1 (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 2) fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5. For PCA, selected genes in each group from RNA-seq samples were extracted based on a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2 expression\u0026thinsp;\u0026gt;\u0026thinsp;2.0 and automatically clustered with 20 variances using the Seurat pipeline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA analysis\u003c/h2\u003e \u003cp\u003eQuantitative real-time PCR (qRT‒PCR) analysis was performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Total RNA was isolated from heart tissue samples and cultured cells using TRIzol reagent (Invitrogen, 5596026). cDNA was amplified using ReverTra Ace\u0026reg; qPCR RT Master Mix (TOYOBO, FSQ-201) following the manufacturer\u0026rsquo;s instructions. SYBR Premix Ex Taq (TaKaRa, RR420) was used to analyze gene expression on a QuantStudio\u0026trade; 6 Flex System according to the manufacturer\u0026rsquo;s instructions. The primer sequences utilized in this study are presented in Supplementary Table S3. RNA sequencing was carried out on a NextSeq 550 platform (Illumina, Inc., USA). The analysis of RNA sequencing data was performed using ExDEGA v5.0.0.1 (e-Biogen) and the ClueGO/CluePedia, EnrichmentMap, and GeneMania plug-ins from Cytoscape (v3.10.0) software. Global gene expression was assessed by biological processes with gene set enrichment analysis using MSigDB v6.1 (\u0026gt;\u0026thinsp;1.3-fold, RC log2\u0026thinsp;\u0026gt;\u0026thinsp;2, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and displayed with the Flaski application (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://flaski.app\u003c/span\u003e\u003cspan address=\"http://flaski.app\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The Cytoscape (v3.1.0) ClueGo plugin was used to visualize enriched pathways associated with the biological pathway database. In brief, biological GO terms with medium specificity and a kappa score of 0.4 were explored. An enrichment/depletion method with a two-sided hypergeometric test was applied with Bonferroni step-down for each p value calculation. Enriched pathways with a p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant. Gene set enrichment analysis (GSEA) was performed to extract information on overrepresented gene ontology terms for various functional processes and signaling pathways between each sample. Visualization of significantly enriched GO terms of functional processes and signaling pathways between samples was performed with the Cytoscape plugin EnrichmentMap. The mapping of gene expression levels was performed using the GeneMania plugin. All GO terms of the network in our analysis were filtered with a p value less than 0.05 based on the pathway score. Coexpression networks were constructed using the WGCNA (v1.47) package in R (Langfelder and Horvath, 2008). After filtering genes, gene expression values were imported into WGCNA to construct coexpression modules using automatic network construction with default settings.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunostaining\u003c/h2\u003e \u003cp\u003eHistology and immunostaining of the heart sections were performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Briefly, animal hearts were harvested, embedded in optical cutting temperature (OCT), sliced to a thickness of 10 m, and stained with Masson\u0026rsquo;s trichrome following the manufacturer\u0026rsquo;s instructions (Abcam, ab150686).\u003c/p\u003e \u003cp\u003eImmunostaining was performed as described previously.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Briefly, samples were fixed with 4% paraformaldehyde solution for 5 min and incubated with primary antibodies diluted 1:1000 in 2% BSA (wt/vol) in 0.2% PBST buffer overnight at 4\u0026deg;C following antigen retrieval with 20 \u0026micro;g/ml proteinase K (VIAGEN, #501-PK) treatment (10 mM Tris-HCl buffer, pH 8.0 for 15 min at 37\u0026deg;C). After incubation with the primary antibody, the samples were washed with PBS solution and incubated with secondary antibodies [diluted 1:1000 in 2% BSA (wt/vol) in 0.2% PBST buffer] for 1 h at room temperature, followed by Hoechst 33342 (Thermo Fisher Scientific, H3569) staining for nuclear analysis. For immunostaining of NMVMs or hESC-CMs, the cells were first fixed with 4% PFA for 15 min and then permeabilized with 0.2% Triton X-100 in PBS for 15 min. The cells were then blocked with 2% BSA solution in 0.2% PBST for 30 min and incubated with primary antibodies overnight at 4\u0026deg;C. Images were captured and processed with an EVOS M5000 system and an FV3000-ORS (Olympus Corp.) confocal microscope. The cell surface area and cross-sectional area were quantified with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eElectron microscopy and toluidine staining\u003c/h2\u003e \u003cp\u003eFor electron microscopy analysis, heart tissues were rapidly harvested and washed twice with cold 1x PBS. A segment of the posterior wall of the left ventricle approximately 1 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e in size was excised and drop-fixed in modified Karnovsky's fixative solution (2% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M sodium cacodylate, pH 7.4) at 4\u0026deg;C overnight. The tissues were washed with 0.2 M sodium cacodylate buffer (pH 7.2) and postfixed with 2% aqueous osmium tetroxide for 1 h 30 min. The samples were then dehydrated through a series of increasing ethanol concentrations ranging from 50\u0026ndash;100%. The tissues were subsequently washed with propylene oxide and embedded in Epon812 resin. For light microscopy, 1 m thick sections were prepared and counterstained with toluidine blue. For transmission electron microscopy (TEM), ultrathin sections (70 nm) were prepared and collected on 100 mesh copper grids. Images were captured with a LIBRA-120 microscope (Zeiss). The data were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial analysis\u003c/h2\u003e \u003cp\u003eThe following procedures were used to analyze mitochondrial respiration: NMVMs were seeded on Matrigel\u0026reg;-coated clear bottom 96 black-well plates at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/ml and allowed to grow for 2 days. The cells were then treated with Cre recombinase Gesicles or transfected for ATF3 overexpression. After 24 h, the cells were serum-starved for 24 h and treated with 1 M AngII for 24 h. The cells were then stained with 2 \u0026micro;M JC-1 dye (Invitrogen, MP 03168) for 30 min, washed with Live Cell Imaging Solution (Invitrogen, A14291DJ), and imaged via an EVOS M5000 system. For JC-1 quantification, the SpectraMax\u0026reg; i3x system was used. For quantification of the mitochondrial DNA content, total DNA was extracted from the heart and NMVMs using the MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa, #9765) following the manufacturer\u0026rsquo;s instructions. The amount of mitochondrial DNA was measured by the ratio of \u003cem\u003emtCo1\u003c/em\u003e to \u003cem\u003eNdufv1\u003c/em\u003e using quantitative PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReactive oxygen species (ROS) measurement\u003c/h2\u003e \u003cp\u003eFor measurement of the level of ROS generated in cardiomyocytes following AngII treatment, the cells were treated with DCF-DA (Sigma, #D6883) for 40 min. After DCF-DA incubation, the cells were washed twice with 1X PBS before being observed. Live cell images were captured and analyzed using an EVOS M5000 system and ImageJ software, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences between two or more groups were analyzed using either an unpaired two-tailed Student\u0026rsquo;s t test or one-way analysis of variance (ANOVA) with GraphPad Prism 9. The experiments were performed independently at least three times. The data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs or \u0026plusmn;\u0026thinsp;SEMs, as indicated in the figure legends.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAtf3 is significantly increased in hypertrophic cardiomyocytes\u003c/h2\u003e \u003cp\u003eDue to conflicting findings regarding the expression and function of ATF3 in the progression of myocardial hypertrophy, we first investigated the expression of Atf3 in H9C2 cardiomyocytes after AngII treatment. Our findings revealed a significant increase in Atf3 protein and mRNA expression in the AngII-treated H9C2 cells compared to the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). This increase was concurrent with increases in the expression of cardiac hypertrophy-related genes, atrial natriuretic peptide (\u003cem\u003eAnp)\u003c/em\u003e and brain natriuretic peptide (\u003cem\u003eBnp)\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). We also analyzed Atf3 expression in the heart following AngII infusion and found that the Atf3 protein level substantially increased after AngII injection and gradually decreased thereafter. In contrast, the expression of Anp and alpha-smooth muscle actin (a-Sma) began to increase on the 3rd and 5th days after AngII infusion, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). This period may be the start of pathological cardiac remodeling in the heart.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e The transcriptional level of \u003cem\u003eAtf3\u003c/em\u003e rapidly increased after AngII injection, peaking between days 3 and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAtf3 is essential for inhibiting cardiomyocyte hypertrophy\u003c/h2\u003e \u003cp\u003eTo further clarify the function of ATF3 in cardiomyocytes, we used the Cre recombinase system to deplete Atf3 in cardiomyocytes by utilizing NMVMs isolated from mice with the \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e genotype. The depletion of Atf3 by Cre protein treatment triggered an increase in the size of NMVMs, regardless of treatment with AngII (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B) or endothelin-1 (ET-1) (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and B), an inducer of cardiac hypertrophy. Similarly, AngII or ET-1 treatment increased the protein expression of Anp in NMVMs, and its expression was more significantly increased in Atf3-deficient NMVMs (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and S1C). Further qPCR analysis confirmed a significant increase in the expression of \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e, and beta-myosin heavy chain (b\u003cem\u003e-Mhc\u003c/em\u003e) in the Atf3-deficient NMVMs compared to the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-G; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-G). Next, we investigated the protective effects of Atf3 overexpression on cardiomyocyte hypertrophy by infecting NMVMs with an adenovirus expressing RFP-tagged ATF3 (adATF3) or a control adenovirus (adRFP) after treatment with AngII or ET-1. In contrast to the hypertrophic response observed with Atf3 depletion, ATF3 overexpression reduced the size of cells treated with vehicle, AngII (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and I) or ET-1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eH and I). Additionally, compared to the control-transfected cells, the ATF3-overexpressing cardiomyocytes exhibited reduced Anp induction triggered by AngII (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) or ET-1 treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eJ). The mRNA expression of \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e, and b\u003cem\u003e-Mhc\u003c/em\u003e was also significantly reduced in the ATF3-overexpressing cells treated with vehicle, Ang-II (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-M) or ET-1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eK-M).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough our in vitro results clearly demonstrated the role of Atf3 in cardiomyocyte hypertrophy, the fact that NMVMs isolated from mice are not composed solely of cardiomyocytes suggests that the observed effect of ATF3 expression on cardiomyocyte hypertrophy may be due to the role of cardiac fibroblasts, as previously reported.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e To address this issue, we induced cardiomyocyte differentiation with h7-human embryonic stem cells (hESC-CMs) to obtain high-purity cardiomyocytes and observed the effect of ATF3 on cardiomyocyte hypertrophy. When the cellular hypertrophy of hESC-CMs was induced by treatment with 100 ng/ml of ET-1, \u003cem\u003eATF3\u003c/em\u003e expression was significantly increased by approximately 1.5-fold compared to that in the control cells, which was consistent with the increased expression of the \u003cem\u003enatriuretic peptide A\u003c/em\u003e (\u003cem\u003eNPPA\u003c/em\u003e) and \u003cem\u003enatriuretic peptide B\u003c/em\u003e (\u003cem\u003eNPPB\u003c/em\u003e) transcripts, similar to the results obtained with H9C2 and NMVMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). The protein expression of ATF3, NPPA, and ATF4 wasalso increased after ET-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Moreover, ATF3 overexpression via adATF3 transduction significantly reduced the size of cardiomyocytes, regardless of ET-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). The adATF3-transduced cells exhibited decreased NPPA expression in response to ET-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), and the mRNA expression of \u003cem\u003eNPPA\u003c/em\u003e, \u003cem\u003eNPPB\u003c/em\u003e, and b\u003cem\u003e-MHC\u003c/em\u003e was also significantly reduced in the adATF3-transduced hESC-CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-J). Overall, these findings suggest that ATF3 expression in cardiomyocytes is important for its suppressive effect on cardiomyocyte hypertrophy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eATF3 is mainly expressed in the ventricular cardiomyocytes of human hearts\u003c/h2\u003e \u003cp\u003eA recent study revealed single-nucleus and spatial transcriptome profiles to elucidate the molecular alterations related to the progression of human heart disease.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e To further elucidate the cell type responsible for ATF3 activity, we performed a dataset analysis of single-nucleus RNA-sequencing (snRNA-seq) results from human heart tissue from healthy controls and patients with myocardial infarction available in the accessible public domain (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zenodo.org/record/6578047\u003c/span\u003e\u003cspan address=\"https://zenodo.org/record/6578047\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e First, eleven major cardiac cell types were identified among clusters annotated with curated marker genes from the literature\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and visualized with the uniform manifold approximation and projection (UMAP) algorithm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The level and proportion of ATF3 expression differed depending on cardiac cell type based on the data analyzed by UMAP, violin plots, and bar plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Interestingly, contrary to previous reports,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e the primary cells expressing the highest level of ATF3 in all human heart tissues were cardiomyocytes, not fibroblasts. Next, to investigate how cardiac stress affects ATF3 expression in different types of cardiac cells, we analyzed expression data from healthy human hearts and from patients with myocardial infarction. In addition, ATF3 expression in ventricular cardiomyocytes varied depending on the sampling region (control, border, fibrotic, ischemic, and remote zone) and the progression of myocardial infarction (myogenic, fibrotic, and ischemic events) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-H). To further examine the role of ATF3 in cardiomyocytes, we removed cardiomyocytes from healthy donor samples. We identified 7 cardiomyocyte clusters, each manually annotated by their ranked gene expression: PCDH7\u0026thinsp;+\u0026thinsp;CM, GPC5\u0026thinsp;+\u0026thinsp;CM, SLC44A5\u0026thinsp;+\u0026thinsp;CM, A2M\u0026thinsp;+\u0026thinsp;CM, TSPAN9\u0026thinsp;+\u0026thinsp;CM, BCL6\u0026thinsp;+\u0026thinsp;CM, and ATF3\u0026thinsp;+\u0026thinsp;CM which is mainly consisted of cells with high expression of \u003cem\u003eATF3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-K). To investigate cellular crosstalk, we conducted receptor‒ligand interaction analysis using the CellChat pipeline (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL and M). ATF3\u0026thinsp;+\u0026thinsp;CMs play dual roles as both signal senders in SEMA3 signaling, influencing all other cardiac clusters, and as signal acceptors for ANGPTL signaling from cardiomyocytes with highly expressed alpha-2-macroglobulin-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN and O). ANGPTL4 can mitigate cardiac hypertrophy and fibrosis induced by phenylephrine and AngII, respectively.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Additionally, we found that this cluster has increased transcriptional activity of the \u003cem\u003eSMAD1, BHLHE40\u003c/em\u003e and \u003cem\u003eNR4a1\u003c/em\u003e (Nur77) transcription factors, which can also protect cardiomyocytes from ischemia‒reperfusion (IR) injury,\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e regulate mitochondrial ROS production\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and maintain cardiomyocyte calcium homeostasis\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e respectively (Figure S2). Therefore, we hypothesize that ATF3\u0026thinsp;+\u0026thinsp;CMs can inhibit maladaptive cardiac remodeling processes under stress conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCardiac-specific Atf3 deletion causes dilated cardiomyopathy induced by AngII infusion\u003c/h2\u003e \u003cp\u003eTo conduct a more comprehensive study, we observed how the heart function of mice is affected by the absence of Atf3 in myocardial cells. To induce specific Atf3 deficiency in cardiomyocytes, we generated \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2c/tm2c;aMHC\u0026minus;MerCreMer\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u0026minus;MCM\u003c/em\u003e\u003c/sup\u003e) in which the \u003cem\u003eAtf3\u003c/em\u003e gene was specifically removed from cardiomyocytes. At 5 days post-Ang II infusion, when \u003cem\u003eAtf3\u003c/em\u003e is highly expressed, Atf3 deficiency was induced by injecting tamoxifen (TMX) for 3 days to activate the Cre protein. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the TMX-injected \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u0026minus;MCM\u003c/em\u003e\u003c/sup\u003e (cKO) mice exhibited significantly reduced \u003cem\u003eAtf3\u003c/em\u003e expression with or without AngII infusion. In contrast to the 100% survival rate of the control mice, the survival rate of the cKO mice decreased to 90%, which further decreased to 70% in the presence of AngII infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Atf3 deficiency exacerbated AngII-induced cardiac hypertrophy, resulting in a significant increase in heart weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). In addition, Masson's trichrome staining revealed substantially increased myocardial fibrosis in the hearts of the cKO mice after AngII infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and F). Furthermore, a significant increase in cardiomyocyte size was observed in the hearts of the cKO mice after AngII infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and H). The mRNA levels of \u003cem\u003eAnp\u003c/em\u003e, \u003cem\u003eBnp\u003c/em\u003e, and b\u003cem\u003e-Mhc\u003c/em\u003e were strongly elevated in the hearts of the AngII-infused control and cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI-K). Similarly, immunoblot analysis of the control and cKO mice followed by AngII infusion also revealed increased levels of Anp, a-Sma, and b-catenin in cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). To determine cardiac function, we performed echocardiographic analysis on the AngII-infused mice for 2 weeks. M-mode echocardiographic analysis revealed that 2 weeks of AngII infusion impaired cardiac function by significantly decreasing the ejection fraction (EF) and fractional shortening (FS) in the cKO mice compared to those in the control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM-O). Furthermore, the systolic left ventricular internal dimension (LVID;s) was significantly increased in the hearts from the AngII-infused cKO mice compared to AngII-infused control mice (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating a dilated cardiac hypertrophy phenotype. These results indicate that Atf3 deficiency in cardiomyocytes exacerbates cardiac dysfunction caused by AngII infusion. Taken together, these data suggest that Atf3 might play a protective role against cardiomyopathy triggered by AngII.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCardiomyocyte-specific Atf3 depletion alters the transcriptome of genes related to mitochondrial dysfunction and the extracellular matrix composition\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo demonstrate the major pathway involved in abnormal cardiac remodeling in cKO mice, we performed 3' quantitative mRNA sequencing on hearts from both the control and cKO mice after AngII infusion. Despite the very low Atf3 protein expression under normal conditions, 278 upregulated and 286 downregulated genes were identified in the hearts from the Atf3 cKO mice (Figure S3A). A volcano plot showing the differential gene expression in the hearts from the cKO mice is presented in Figure S3B. Upregulated or downregulated genes are represented by yellow and blue dots, respectively. These genes include several critical genes related to cardiac hypertrophy, contractility, and heart failure, such as bone morphogenetic protein-4 (\u003cem\u003eBmp4\u003c/em\u003e),\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e ATPase Na+/K\u0026thinsp;+\u0026thinsp;transporting subunit alpha 1 (\u003cem\u003eAtp1a1\u003c/em\u003e),\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and angiotensin II receptor type 1a (\u003cem\u003eAgtr1a\u003c/em\u003e).\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Additionally, the expression of genes related to mitochondrial function, such as NADH:ubiquinone oxidoreductase complex assembly factor 5 (\u003cem\u003eNdufaf5\u003c/em\u003e),\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e cytochrome c oxidase subunit 5A (\u003cem\u003eCox5a\u003c/em\u003e),\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e and nicotinamide nucleotide transhydrogenase (\u003cem\u003eNnt\u003c/em\u003e),\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e were significantly decreased. These data suggest that Atf3 depletion causes mitochondrial dysregulation. Additional differential gene expression analysis revealed altered pathways, including the establishment of protein localization to organelles and mitochondrial organization, which were ranked at the top of the Gene Ontology (GO):Biological Processes (GO:BP) list with statistical significance in a comparison with the \u003cem\u003eAtf3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e control heart samples (Figure S3C).\u003c/p\u003e \u003cp\u003eTo determine the regulatory mechanisms underlying cardiac abnormalities in the hearts of the AngII-infused Atf3 cKO mice, we conducted a comparative analysis of 15,334 genes which were preprocessed the data by using the DESeq2 pipeline to remove entries with a read count of 15 or less and normalized and stabilized the variance. After these genes were filtered out, we calculated the distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, upper) and correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, lower) between the samples using standard DESeq2 protocols. Pairwise correlation analysis (PCA) revealed a greater degree of similarity in gene expression between the cKO-AngII heart samples and the control and control-AngII heart samples. Next, we performed weighted gene coexpression network analysis (WGCNA), identified optimal cluster sets using dynamic tree cutting, characterized and constructed representative module eigengenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), calculated the mean values for each module, represented them as single values (averages), and visualized them through heatmaps (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Further analysis focused on significant modules (Darkgreen, Grey60, Lightgreen, Turquoise), revealing the expression patterns of enriched genes through heatmap representation (Figures S4A and 6D). Using 2,330 genes enriched in the Turquoise module from the cKO-AngII mice, we performed gene set enrichment analysis (GSEA) and clustered them with similar GO terms using the EnrichmentMap algorithm. Through this process, we identified 287 nodes categorized into 9 GO groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). These groups included cell substrate adhesion, cell matrix adhesion, cellular respiration and mitochondrial complex assembly as the top-ranked GO terms, suggesting that Atf3 plays a protective role against mitochondrial oxidative stress and cardiac fibrosis induced by AngII infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). To identify the genes associated with the top regulated GO terms, we examined the networks of nodes associated with 'cellular respiration' and 'cell matrix adhesion'. Using a radial layout algorithm, we verified that the 21 nodes associated with cellular respiration formed a network centered on the organophosphate biosynthetic process (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). The expression levels of genes enriched in these ontologies are illustrated as heatmaps in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and S5. These findings implicate the \u003cem\u003eNduf\u003c/em\u003e, \u003cem\u003eAtg5\u003c/em\u003e, and collagen gene families, suggesting that ATF3 is essential for preventing cardiac fibrosis and mitochondrial oxidative stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAtf3 is required for maintaining mitochondrial function\u003c/h2\u003e \u003cp\u003eNotably, according to the RNA sequencing data, genes related to the oxidative phosphorylation system (OXPHOS) and mitochondrial quality control, such as heat shock protein 10 (Hsp10, also known as Hspe1) and Hsp60 (Hspd1),\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e NFE2-like BZIP transcription factor 1 (Nfe2l1, also known as Nrf-1),\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e translocase of the outer mitochondrial membrane complex subunit 20 (Tomm20),\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e estrogen-related receptor alpha (Esrra, also known as Err),\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1),\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e overlapping proteolytic activity with m-AAA protease 1 (Oma1)\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and caseinolytic peptidase P (ClpP),\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e were significantly altered (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and S6). To confirm the dysregulation of mitochondrial oxidation, we performed transmission electron microscopy (TEM) analysis of the control and AngII-infused heart tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Consequently, we found that the hearts from the AngII-infused cKO mice exhibited increased mitochondrial size and abnormal mitochondrial morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and C). Additionally, compared with the control heart tissue, the heart tissue from the AngII-infused cKO mice showed a significant decrease in mitochondrial DNA content (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Additionally, we performed a JC-1 mitochondrial membrane potential assay with control or ATF3-depleted NMVMs after AngII treatment. Compared with those of the controls, the red/green JC-1 ratio of the Atf3-depleted NMVMs decreased, indicating decreased mitochondrial function (Figure S7A and B). Consistent with these findings, compared with the control cells, the ATF3-depleted NMVMs also exhibited significantly decreased mitochondrial DNA content after AngII treatment (Figure S7C). To validate these findings, we examined the protein expression levels of OXPHOS complex subunits in vivo. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F, the expression of OXPHOS complex subunits in the cKO heart samples substantially decreased relative to those in the control heart samples. In summary, these findings suggest that the absence of Atf3 exacerbates cardiac dysfunction by impairing mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAtf3 can regulate ROS production through the Sirt3/Sod2 axis\u003c/h2\u003e \u003cp\u003eA previous study suggested that overexpression of Atf3 inhibits ROS production in primary hepatocytes and that hepatocyte-specific Atf3-null mice exhibit increased hepatic ROS levels.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e Dysfunction of mitochondria in hearts of the AngII-infused cKO mice may be attributed to increased ROS accumulation resulting from the UPR\u003csup\u003emt\u003c/sup\u003e, likely due to the reduced expression of mitochondrial chaperone proteins (Figure S6). Acetylation of the Sod2 protein regulates mitochondrial ROS production through a well-documented mechanism. There is an inverse correlation between Sod2 acetylation and Sod2 activity, and Sirt3, the major deacetylase in mitochondria, can activate Sod2 by deacetylating the lysine 68 residue of Sod2.\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e We therefore tested whether cKO hearts infused with AngII display reduced Sirt3 expression and increased Sod2 acetylation. As expected, the mRNA expression levels of Sirt3 and Sod2 were significantly increased in the AngII-infused control hearts, while these genes were stably expressed in the cKO hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). The Sirt3 protein expression level was significantly decreased in the AngII-infused cKO hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and D). Furthermore, ac-Sod2 protein levels were significantly increased in the cKO hearts following AngII infusion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Since the acetylated Sod2 protein is inactive and cannot scavenge ROS, the increased acetylation of Sod2 might be attributable to increased intracellular ROS levels and the sequential activation of AKT and P70S6K. Additionally, a previous study suggested that Sirt3 overexpression can decrease the kinase activity of P70S6,\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e which is phosphorylated and activated by hypertrophy of cardiomyocytes. Interestingly, Atf3 deficiency exacerbated IR-induced liver inflammation by activating the mTOR/P70S6K/Hif-1alpha signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e Therefore, we assessed the expression of pAKT and pP70S6K (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, E, and F). The phosphorylation of P70S6K was significantly increased in the hearts of the AngII-infused cKO mice, whereas pAKT levels are not altered. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Furthermore, Pgc-1a protein levels were significantly reduced in the hearts of the cKO-AngII mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Given that Sirt3 expression can be upregulated by Pgc-1a,\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e Atf3 depletion elicits dysregulation of the Pgc-1a/Sirt3/Sod2 axis, which is associated with cardiomyopathy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the expression of Sirt3 and Sod2 is regulated by the presence or absence of Atf3 in primary cardiomyocytes, we depleted or overexpressed ATF3 in NMVMs followed by AngII treatment. Compared with those in the control cells, the Atf3-depleted NMVMs exhibited increased ac-Sod2 and pP70S6K protein levels, and \u003cem\u003eSirt3\u003c/em\u003e and \u003cem\u003eSod2\u003c/em\u003e transcript levels were significantly decreased after AngII treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI-J). In contrast, in the ATF3-overexpressing NMVMs, Sirt3 expression was highly increased, and ac-Sod2 and pP70S6K protein levels were decreased compared to those in the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). The mRNA expression of \u003cem\u003eSirt3\u003c/em\u003e was also significantly increased by approximately 1.5-fold in the ATF3-overexpressing group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM). \u003cem\u003eSod2\u003c/em\u003e transcript levels in ATF3-overexpressing cells was similar to that in the RFP-overexpressing control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eN). Moreover, we examined whether the regulation of intracellular ROS levels depends on the presence or absence of ATF3 in H9C2 cardiomyocytes. The results showed that ROS levels were significantly greater in the ATF3-deficient H9C2 cells than in the control cells upon treatment with AngII (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eO and P). Conversely, in the H9C2 cells transduced with adATF3, ROS levels were decreased after AngII treatment compared to those in the control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eQ and R). Furthermore, downregulated genes related to mitochondrial respiration, including those in the \u003cem\u003eNDUF\u003c/em\u003e family and \u003cem\u003ePGC-1a\u003c/em\u003e, in the hearts of the AngII-infused cKO mice were upregulated in the ATF3\u0026thinsp;+\u0026thinsp;CM clusters from the human heart scRNA-seq data. In contrast, the expression of genes related to integrin and collagen superfamily genes related to ATF3\u0026thinsp;+\u0026thinsp;CM clusters that were upregulated in the hearts of the AngII-infused cKO mice decreased (Figure S8). These results further supported that Atf3 is essential for the mitochondrial antioxidant mechanism governed by the Sirt3-Sod2 axis. In summary, our data collectively suggest that ATF3 in cardiomyocytes plays a crucial role in protecting cardiomyocytes from abnormal cardiac remodeling induced by AngII treatment by regulating the Pgc-1a/Sirt3/Sod2 axis and mitochondrial oxidative stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlthough many studies have revealed the role of ATF3 in cardiomyocytes through ATF3 overexpression or deletion, it is still unclear whether ATF3 protects against or facilitates the development of CVDs.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Several studies have suggested that ATF3 deficiency in mice leads to cardiac hypertrophy under pressure overload. Global ATF3 knockout exacerbated Ang II-induced cardiac hypertrophy, suggesting that ATF3 plays a protective role in the development of cardiac hypertrophy. The effect of this molecule is related to the ERK and JNK pathways and the ET-1 response.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e In contrast, cardiac-specific ATF3 overexpression in transgenic mice led to various indicators of heart failure, including atrial enlargement, cardiac hypertrophy and fibrosis, reduced contractility and aberrant cardiac conduction.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Other studies reported that global ATF3-KO protected against the cardiac hypertrophy phenotype\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and increased the survival rate after myocardial infarction.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e These conflicting findings make it difficult to determine the function of ATF3 in cardiac hypertrophy. Moreover, ATF3 regulates multiple target genes and affects other cardiac resident cell types, as well as cardiomyocytes. Recently, overexpression of ATF3 specifically in cardiac fibroblasts was shown to strongly mitigate cardiac remodeling and heart failure by inhibiting the expression of Map2K3 and the subsequent p38-TGF-β signaling pathway.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Thus, it is very difficult to determine the exact function of ATF3 in cardiomyocytes under stress conditions associated with cardiac hypertrophy. Therefore, more studies are needed to elucidate the detailed regulatory mechanisms of cardiac hypertrophy mediated by ATF3 in cardiomyocytes.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that ATF3 is required for the regulation of mitochondrial oxidative stress in cardiomyocytes. The ablation of ATF3 in cardiomyocytes led to a significantly increased hypertrophic response upon treatment with AngII and ET-1. In contrast, transient overexpression of ATF3 prevented the hypertrophic response in NMVMs and hESC-CMs. Similar to the in vitro results, hearts of the TMX-inducible cardiomyocyte-specific Atf3 knockout mice exhibited structural and functional defects, including cellular hypertrophy and fibrosis, as well as reduced cardiac function, as indicated by the decreased EF and FS. These data further supported the protective role of Atf3 in cardiomyocytes under stress conditions. Consistent with these findings, the RNA sequencing analysis also revealed changes in the expression of genes associated with the extracellular matrix, which are indicative of increased cardiac fibrosis in the hearts of the AngII-infused cKO mice. Interestingly, WGCNA and GSEA revealed that genes related to cellular respiration, such as \u003cem\u003eNduf\u003c/em\u003e and \u003cem\u003eAtp5\u003c/em\u003e superfamily genes and \u003cem\u003ePgc-1a\u003c/em\u003e, were significantly altered in the hearts of the AngII-infused cKO mice with abnormal mitochondrial structure and function. In a detailed analysis of mitochondrial dysfunction, we observed reduced expression of the Sirt3 protein and increased levels of acetylated Sod2 in the hearts of the AngII-infused cKO mice. This effect was reversed by ATF3 overexpression in NMVMs, suggesting that ATF3 can regulate mitochondrial oxidative stress, potentially through the UPR\u003csup\u003emt\u003c/sup\u003e, by modulating Sirt3 and acetylated Sod2 protein levels.\u003c/p\u003e \u003cp\u003eRecent studies have been conducted on transcriptional regulatory mechanisms at the single-cell level to elucidate the pathological development of heart diseases. In addition, spatial networking analysis between cells has enabled a better understanding of disease progression in multicellular tissues composed of diverse cell types.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e These studies not only demonstrate gene expression patterns based on the presence or absence of a disease but also reveal the spatial and temporal expression and activity of specific gene clusters within each cell type. These results provide insights into the progression of diseases influenced by the spatial and temporal expression and activity of particular genes in specific contexts, going beyond a simple indication of the occurrence of a disease. By analyzing human heart snRNA-seq data, we identified cardiomyocytes as the primary cell type expressing \u003cem\u003eATF3\u003c/em\u003e. The cluster of cardiomyocytes with high \u003cem\u003eATF3\u003c/em\u003e expression (ATF3\u0026thinsp;+\u0026thinsp;CM) expresses receptors that respond to the ANGPTL4 ligand, playing a crucial role in preventing abnormal cardiac remodeling induced by AngII infusion.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e Additionally, we observed that this cluster has elevated transcriptional activity of the \u003cem\u003eSMAD1, BHLHE40\u003c/em\u003e and \u003cem\u003eNR4a1\u003c/em\u003e (Nur77) transcription factors, which can also prevent maladaptive cardiac remodeling, supporting our current findings.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, our study addresses the importance of ATF3 in preventing maladaptive cardiac remodeling in cardiomyocytes in response to AngII and ET-1 treatments by regulating mitochondrial oxidative stress. This comprehensive approach ensures a more nuanced understanding of the intricate functions of proteins such as ATF3.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConfilict of Interest\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eM.-H. Jeong, Y. Jeong, J.-S. Kang and W.-H. Kim designed the study. M.-H. Jeong, Y. Jeong and S.-Y. Cho were involved in sample and data collection. M.-H. Jeong, Y. Jeong, S. H. Lee, G.-Y. Kim, M.-J. Kim, J.-S. Kang, and W.-H. Kim were involved in the data analysis and interpretation. M.-H. Jeong, Y. Jeong, J.-S. Kang and W.-H. Kim wrote the article, and all the authors critically revised the article.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis research was supported by the Korea National Institute of Health (2021-NI-022-02 and 2024-NI-012-00).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMetra M, Teerlink JR. Heart failure. Lancet 390, 1981\u0026ndash;1995 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLill R, \u003cem\u003eet al.\u003c/em\u003e The role of mitochondria in cellular iron-sulfur protein biogenesis and iron metabolism. Biochim Biophys Acta 1823, 1491\u0026ndash;1508 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWellen KE, Thompson CB. A two-way street: reciprocal regulation of metabolism and signalling. Nat Rev Mol Cell Biol 13, 270\u0026ndash;276 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurphy MP. 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Nat Rev Neurol 19, 346\u0026ndash;362 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4485671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4485671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivating transcription factor 3 (ATF3) is a critical regulator for cellular stress responses and is implicated in stress-induced cardiac hypertrophy and fibrosis. However, the role of ATF3 in cardiomyopathy remains controversial. Here, we demonstrate that ATF3 plays a cardioprotective role by controlling mitochondrial oxidative stress in angiotensin II (Ang II)-triggered cardiomyopathy. The expression of ATF3 was significantly upregulated in hypertrophic hearts chronically infused with Ang II, which correlated with Ang II-treated cardiomyocytes. In neonatal mouse ventricular myocytes (NMVMs), Ang II-elicited hypertrophic responses were either aggravated or suppressed by ATF3 depletion or overexpression, respectively. Similar results were also obtained in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To analyze the direct role of ATF3 in cardiomyopathy, we generated mice with a cardiomyocyte-specific ATF3 deletion using a tamoxifen-inducible Cre-recombinase (αMHC-MerCreMer/loxP) system. In response to Ang II infusion, mice with cardiomyocyte-specific ablation of ATF3 (ATF3 cKO) exhibited aggravated cardiac hypertrophy and fibrosis concurrent with decreased fractional shortening and ejection fraction. In addition, the transcriptome analysis of control and cKO hearts revealed alterations in genes related to mitochondrial function and organization. In particular, the expression of Sirt3/Sod2 transcripts, well known as a mechanism for regulating mitochondrial oxidative stress, was increased in Ang II-infused mice, which was downregulated by the depletion of ATF3, suggesting the cardioprotective function of ATF3 through the improvement of mitochondrial function. These results suggest that ATF3 may be a potential therapeutic target for hypertrophic cardiomyopathy.\u003c/p\u003e","manuscriptTitle":"ATF3 is required for the prevention of cardiomyopathy via the regulation of mitochondrial oxidative stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 18:42:48","doi":"10.21203/rs.3.rs-4485671/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2ab166f-ccbb-4a44-bc2b-d165375902d9","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33331201,"name":"Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy"},{"id":33331202,"name":"Biological sciences/Molecular biology/Transcriptomics"},{"id":33331203,"name":"Biological sciences/Cell biology/Cell signalling/Stress signalling"}],"tags":[],"updatedAt":"2024-07-15T18:43:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 18:42:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4485671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4485671","identity":"rs-4485671","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}