CDCP1 Deletion Protects Against Pressure Overload-Induced Cardiac Dysfunction and Fibrosis in Mice

CDCP1 Deletion Protects Against Pressure Overload-Induced Cardiac Dysfunction and Fibrosis in Mice | 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 CDCP1 Deletion Protects Against Pressure Overload-Induced Cardiac Dysfunction and Fibrosis in Mice Naveen Pereira, Rachad Ghazal, Akshatha N. Srinivas, Min Wang, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9236741/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Human genomic studies link reduced CUB domain-containing protein 1 (CDCP1) expression with myocardial recovery in heart failure. While CDCP1 regulates cardiac fibroblast proliferation in vitro, it’s in vivo role in cardiac fibrosis remains unclear. Using a Cdcp1 -knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload–expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in Cdcp1 -KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that CDCP1 knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target. Biological sciences/Genetics/Genomics/Medical genomics Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy Biological sciences/Molecular biology/Transcriptomics cardiac fibrosis CDCP1 heart failure mouse model spatial transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Cardiac fibrosis is a hallmark pathological feature in various cardiovascular diseases and a major driver of heart failure progression. 1 – 3 This process is characterized by excessive extracellular matrix (ECM) accumulation that is mediated by activated cardiac fibroblasts, 4 and contributes significantly to myocardial stiffness and dysfunction. Despite its clinical significance, effective therapies targeting cardiac fibrosis remain limited. 5 , 6 Chronic pressure overload induces left ventricular (LV) hypertrophy and progressive interstitial ECM accumulation. 7 , 8 This excessive collagen deposition elevates filling pressures, compromises chamber compliance, and predisposes to arrhythmias and heart failure progression. 9 Mechanistically, cardiac fibrosis involves resident cardiac fibroblast proliferation, migration and transdifferentiation into α-smooth muscle actin (αSMA)-expressing myofibroblasts, which secrete collagens resulting in adverse cardiac remodeling. 10 Current antifibrotic therapies with demonstrated clinical efficacy remain elusive, 11,12 stressing the need to identify novel molecular targets. We previously performed a genome-wide association study (GWAS) for myocardial recovery in patients with recent onset heart failure. 11 , 12 That GWAS identified a genetic locus (index SNP rs6773435) that is an expression quantitative trait locus (eQTL) for the CUB domain-containing protein 1 ( CDCP1 ) gene in cultured fibroblasts. 13 Reduced CDCP1 expression was associated with improved left ventricular ejection fraction (LVEF) in heart failure patients. We further demonstrated that CDCP1 expression is upregulated in human cardiac fibroblasts in response to profibrotic stimuli and that its knockdown attenuates fibroblast proliferation. 11 However, its role in cardiac fibrosis and remodeling in vivo remains undefined. In this study, we investigated the role of CDCP1 in cardiac fibrosis using a pressure overload-induced murine model with Cdcp1 gene knockout (KO). This model recapitulates the chronic neurohormonal stress milieu characteristic of heart failure, providing a biologically relevant context to functionally interrogate CDCP1 in cardiac fibrosis. Cdcp1 -KO mice demonstrated attenuated pathological remodeling in response to chronic angiotensin II/phenylephrine (AngII/PE) infusion, preserved cardiac function, and reduced fibrotic burden compared to wild-type (WT) controls. Transcriptomic analysis revealed that Cdcp1 deletion suppresses profibrotic and proinflammatory gene networks. Spatial transcriptomics revealed that Cdcp1 deletion induces region-specific alterations in fibrogenic gene signatures within the myocardium, including reduced inflammation and extracellular matrix organization associated cardiomyocyte subpopulations and attenuation of growth factor and TGF-β signaling associated fibroblast subtypes. Our findings establish Cdcp1 as a critical mediator of cardiac fibrosis, providing the direct functional evidence of its role in cardiac pathophysiology and supporting its potential as a therapeutic target for cardiac fibrosis. METHODS Animal Model and Ethical Approval All procedures conformed to the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services) and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Mayo Clinic. Mice on an FVB/NJ background (The Jackson Laboratory, Bar Harbor, ME) were housed under specific pathogen-free (SPF) conditions with controlled temperature (22 ± 1°C) and humidity (55 ± 5%), on a 12-h light/dark cycle. Standard rodent chow and water were provided ad libitum . Throughout the study, all efforts were made to minimize discomfort during procedures such as ear tagging, tissue sampling for genotyping, and anesthesia for osmotic pump implantation. Generation of Cdcp1 Knockout Mice Cdcp1 -null mice were generated on an FVB/NJ genetic background by CRISPR-Cas9 editing to remove exon 1 of Cdcp1 gene. Four guide RNAs were designed to flank exon 1: 5g-1 (5’-GTAGATGGTCTGGGACCTCG-3’), 5g-2 (5’-GGGGGGGTCATCACAACATG-3’), 3g-1 (5’-GGGATACTCGATTGGGACGT-3’), and 3g-2 (5’-GAGAACGTCCTCCTAAGGCT-3’). Genotyping was performed using primers DY164 (forward: 5’-GCATGGGCTTCTGTTTCTGT-3’) and DY165 (reverse: 5’-GCACGGACAGCTAAAATGGT-3’). Founder mice harboring exon deletion confirmed by PCR genotyping of ear DNA were bred to homozygosity to obtain Cdcp1 −/− (KO) offspring and littermate-matched WT controls. Both male and female mice, aged 8–12 weeks, were used in all experiments. Mice were randomly assigned to saline or AngII/PE treatment groups, with males and females distributed as evenly as possible across genotypes and treatment conditions. Mice were monitored daily for overall health, body weight, and signs of distress. Angiotensin II/Phenylephrine Infusion Model To induce pressure overload and cardiac fibrosis, osmotic minipumps (ALZET → model 1002, DURECT Corporation, Cupertino, CA) were implanted subcutaneously at 10 weeks of age under 1.5% isoflurane anesthesia. Minipumps were pre-loaded to deliver either saline (0.9% NaCl) as a control, or AngII (1.2 µg/g/day; Sigma-Aldrich, Cat. No. A9525, St. Louis, MO, USA) plus phenylephrine (PE) HCl (35 µg/g/day; Sigma-Aldrich, Cat. No. P6126-5G) for 28 days. Incisions were closed with a single 5 − 0 nylon suture, and mice were placed in warmed recovery chambers postoperatively. Four experimental cohorts were established: Saline_WT (WT mice receiving saline infusion), Saline_KO ( Cdcp1 −/− mice receiving saline), AngII/PE_WT (WT mice receiving AngII/PE), and AngII/PE_KO ( Cdcp1 −/− mice receiving AngII/PE). All animals were monitored for any adverse effects, including changes in mobility or grooming. No mortality or overt toxicity was observed in any group during the 4-week infusion period. A separate cohort of mice was monitored for 14 weeks to examine the effect of Cdcp1 deletion on long-term survival during pressure overload. For all other endpoints (echocardiography, histology and transcriptomics), mice were euthanized at 4 weeks post-implantation. Echocardiographic Assessment of Cardiac Function Transthoracic echocardiography (TTE) was performed at baseline and at 4 weeks post-minipump implantation using the Vevo → F2 Imaging System (FUJIFILM VisualSonics, Toronto, Canada) equipped with a 46 − 20 MHz linear-array transducer. Mice were lightly anesthetized (1-2.5% isoflurane), positioned supine on a warming platform, and the heart rate was maintained between 450–600 bpm to minimize anesthesia-induced cardio depression through adjustment of inhaled isoflurane concentration. Two-dimensional (2D) and M-mode images were acquired in the parasternal short-axis view at the mid-papillary level. Left ventricular (LV) mass index (LVMI), LV ejection fraction (LVEF), and LV fractional shortening (LVFS) were calculated from M-mode measurements as previously described. 13 All acquisitions and their analysis were conducted in a blinded fashion to the animal’s genotype and treatment group. Histopathology and Fibrosis Quantification After the 28-day infusion period, mice were euthanized by CO 2 inhalation, in accordance with approved IACUC protocol. Hearts were rapidly excised, rinsed in 10X Phosphate-Buffered Saline (PBS), and fixed in 10% neutral-buffered formalin at room temperature over 24 hours. Fixed tissues were embedded in paraffin and sectioned at 6 µm thickness. Collagen deposition was assessed histologically by Picrosirius Red staining (Polysciences Inc., Warrington, PA) following the manufacturer’s protocol. Light microscopy images were captured at 20X and 40X magnification (Nikon, Tokyo, Japan). Quantification of cardiac fibrosis was determined by analyzing Picrosirius Red-positive area in ≥ 5 randomly selected mid-myocardial fields per sample, excluding large epicardial vessels. Bulk-tissue RNA Sequencing Snapfrozen LV tissues were pulverized under liquid nitrogen. Total RNA was extracted using Quick-RNA™ Miniprep Kit (Zymo Research, Cat. No. R2052). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and library preparation was carried out using the NEBNext → Ultra ™ II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), following the manufacturer’s guidelines. Multiplexed libraries were loaded onto an Illumina NovaSeq™ 6000 flow cell (Illumina, San Diego, CA, USA) and sequenced in a 2⋅150 bp paired-end format. Base calling and raw data processing were managed by the NovaSeq Control Software (NCS). The raw reads were quality-checked, trimmed and aligned to Mus musculus reference genome (GRCm39: GCF_000001635.27) using STAR (v2.5.2b). Read counts were assigned to annotated genes with featureCounts (Subread v1.5.2) counting only the uniquely mapped reads in exonic regions. Differential expression analysis was performed using DESeq2 (R/Bioconductor) with the Wald test applied to obtain log 2 fold changes and P -values. Genes with an adjusted P -value 1 were designated as differentially expressed. Functional gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package on a ranked list of gene symbols ordered by log₂ fold‑change from DESeq2 differential expression. Spatial Transcriptomic Assays Formalin-fixed, paraffin-embedded (FFPE) heart blocks were sectioned at 5 µm thickness. Tissues with RNA integrity number (RIN) > 6 were selected for spatial transcriptomics using the Visium Spatial Gene Expression platform (10x Genomics, Pleasanton, CA). Tissue sections with picrosirius red were placed on capture areas (≈5000 barcoded spots per 6.5⋅6.5 mm area, spot diameter: 55 µm, center-to-center distance: 100 µm), and imaged with a Leica Aperio VERSA (Leica Microsystems, Wetzlar, Germany) at 20X resolution. Permeabilization conditions were optimized according to the Tissue Optimization protocol (10x Genomics CG000238). Spot-based RNA capture, reverse transcription, cDNA amplification, and library construction were performed according to the Visium Spatial Gene Expression Slide & Reagent Kit (10x Genomics). Resultant libraries were sequenced on an Illumina NovaSeq™ 6000 system. Spatial Transcriptomic Data Analysis Raw FASTQ files were processed to quantify spot-level gene expression. Spots on tissue were manually adjusted with Loupe browser (10x Genomics). The feature barcode expression matrices were analyzed and visualized in Seurat v5.1.0 (R/CRAN). Samples were normalized using sctransform method with additional log normalization and scaled based on gene counts. Harmony integration method was used for inter-sample comparison. Uniform Manifold Approximation and Projection (UMAP) was used for 2D visualization of clusters. Gene signatures distinguishing each cluster or subpopulation were identified using Seurat’s “FindMarkers” with a false discovery rate (FDR) 0.25. To characterize the cell-type proportions of spatial spots, Seurat’s integrated anchor-based deconvolution method was employed using previously published and annotated single-cell RNAseq data (GEO accession number: GSE120064) 14 . We used the broad cell type labels (cardiomyocytes, fibroblasts, etc.) and the subtype annotations (CM4, CM6, etc.) to deconvolve the spots. Differential expression analysis was conducted using Seurat “FindMarkers” function across conditions and cell types. Genes with absolute average log 2 fold change > 1 and adjusted P -value (Benjamini-Hochberg method) < 0.05 were considered significantly differentially expressed. GSEA in GO terms was performed with clusterProfiler and org.Mm.eg.db R packages. Cell Culture Cryopreserved adult human ventricular fibroblasts (HVFs) (Cell Applications, Inc) were cultured in Cardiac Fibroblast growth medium. Cells were subcultured using 0.25% trypsin when they reached 70% confluence and maintained at 37°C in a 5% CO 2 humidified incubator. HVFs between the passages 2 to 6 were used for all in vitro experiments. CDCP1 Transient Knockdown Cells were transfected with 25 nM siGENOME SMARTpool siRNAs (Dharmacon) targeting CDCP1 using Lipofectamine RNAiMAX Reagent (ThermoFisher). Profibrotic Stimulation HVFs were serum-starved overnight and treated with Recombinant human PDGF-BB (20 ng/ml), TGF-β (10 ng/ml), and Angiotensin II (100 ng/ml) in serum free DMEM. Reconstitution buffer was used as vehicle control. Depending on the experimental design, cells were harvested at 48 h, 72 h or 5 days post-stimulation for RNA extraction or immunofluorescence staining. Real-time Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from HVFs using Quick-RNA MicroPrep Kit (Zymo Reseacrh). qRT-PCR was performed using a one-step Power SYBR Green RNA-to-CT kit (Applied Biosystems) on StepOne PCR system (Thermo Fisher). Ct values were normalized to the reference gene GAPDH, and the relative quantification was calculated using the ΔΔCt method. Immunofluorescence HVFs were seeded in 96-well plates and cultured to 50% confluence prior to treatment with profibrotic stimuli. Following treatment, intact cells were either processed directly for immunofluorescence or subjected to decellularization to isolate cell-derived extracellular matrix (ECM). For intact-cell staining, nuclei were first labeled with Hoechst 33342 (1:1000) for 30 min at room temperature. For ECM preparation, cells were decellularized using a solution containing 20 mM ammonium hydroxide and 0.5% Triton X-100 for 5 min at room temperature. Both intact cells and decellularized ECM were gently washed three times with 1× PBS and fixed with 10% formalin for 10 min at room temperature. After fixation, samples were washed and blocked in 1× PBST (1% BSA, 0.1% Triton X-100) supplemented with 3% normal goat serum for at least 3 h or overnight at 4°C. Samples were then incubated overnight at 4°C with a rabbit anti–collagen I primary antibody (NB600-408G; 1:500). Following PBS washes, samples were incubated with Alexa Fluor 488–conjugated goat anti-rabbit secondary antibody (1:1000) together with DAPI (1:1000) for 1 h at room temperature. After final washes, collagen I immunofluorescence in both intact fibroblasts and decellularized ECM was imaged and quantified using a Cytation 5 imaging system. Statistical Considerations Sample sizes for each experimental group were determined based on power calculations and anticipated effect sizes. All data are presented as mean ± standard deviation (SD). Two-way ANOVA (genotype ⋅ treatment) was used to assess main effects and interactions for echocardiographic and morphometric parameters across the four experimental groups. Pre-specified pairwise comparisons were performed using unpaired two-tailed Student’s t -tests. Two-way ANOVA with Sidak’s post hoc test was used for longitudinal body weight analysis. Survival analysis was performed using the log-rank (Mantel-Cox) test. For histological fibrosis quantification, an unpaired two-tailed Student’s t -test was applied. A P -value < 0.05 was considered statistically significant. GraphPad Prism (GraphPad Software, San Diego, CA) and R (v4.2.2) were used for statistical calculations and data plotting. RESULTS Generation and baseline characterization of Cdcp1 KO mice To investigate the role of Cdcp1 in cardiac remodeling and fibrosis, we generated Cdcp1 -KO mice using CRISPR-Cas9 editing to remove exon 1 of the Cdcp1 gene (Fig. 1 A). PCR genotyping confirmed the successful deletion of the targeted region by showing amplification products of 1,771 bp for the WT allele and a truncated product approximately 501 bp for the KO allele (Fig. 1 B). Homozygous Cdcp1 -KO mice were obtained at expected Mendelian ratios and were viable without overt congenital anomalies. To assess whether Cdcp1 deletion affected baseline growth and development, we monitored body weight from weaning to adulthood. Longitudinal body weight measurements from 3 to 11 weeks of age showed comparable growth between WT (n = 14, 9 males, 5 females) and Cdcp1 -KO mice (n = 10, 7 males, 3 females) ( Supplementary Fig. 1A ). Two-way ANOVA showed no significant main effect of genotype on body weight ( P = 0.478) and no significant Age⋅Genotype interaction ( P = 0.986). Sidak’s post hoc comparisons confirmed that there were no significant differences between genotypes at any time point ( P > 0.05). To examine the effect of Cdcp1 deletion on long-term survival during pressure overload, a separate cohort of mice was monitored for 14 weeks after osmotic minipump implantation ( Supplementary Fig. 1B ). All mice in the Saline WT (n = 9), Saline KO (n = 10), and AngII/PE KO (n = 9) groups had 100% survival throughout the observation period. In contrast, three deaths occurred in the AngII/PE WT group (n = 12), one at 13 weeks and two at 14 weeks post-implantation of AngII/PE infusion pump (log-rank test, P = 0.056). For chronic pressure overload, we implemented a 4-week experimental protocol (Fig. 1 C). This timepoint captures the period of active fibrotic remodeling characteristic of the AngII/PE model, during which interstitial collagen accumulation precedes the onset of overt systolic dysfunction. Mice were monitored weekly for body weight and general health assessment. At the experimental endpoint, cardiac function was reassessed by echocardiography, followed by euthanasia for heart tissue collection and morphometric analysis. Collectively, these results demonstrated successful generation of viable Cdcp1 -KO mice with normal baseline growth and development. Cdcp1 deletion attenuates cardiac hypertrophy and fibrosis after pressure overload To investigate the impact of Cdcp1 deletion on cardiac structure and function during pressure overload, we performed serial TTE at baseline and after 4 weeks of chronic AngII/PE infusion. At baseline, the left ventricular (LV) mass index (LVMI) was comparable across all groups (Fig. 1 D). AngII/PE infusion induced significant cardiac hypertrophy in WT mice, with increased LVMI compared to saline-treated WT control (3.65 ± 0.45 mg/g vs. 3.05 ± 0.50 mg/g, P = 0.016) (Fig. 1 E). This hypertrophic response was significantly attenuated in AngII/PE-treated Cdcp1 -KO mice compared to AngII/PE-treated WT (3.17 ± 0.30 mg/g vs. 3.65 ± 0.45 mg/g, P = 0.012) (Fig. 1 E), and Cdcp1 deletion prevented the increase in LVMI from baseline observed in WT mice after pressure overload (-0.17 ± 0.53 mg/g vs. 0.35 ± 0.39 mg/g, P = 0.027) (Fig. 1 F). Consistent with the 4-week timepoint capturing active fibrotic remodeling prior to systolic dysfunction, LV ejection fraction (LVEF) and LV fractional shortening (LVFS) remained within normal ranges across all groups, though AngII/PE-treated Cdcp1 -KO mice showed trends toward higher values compared with AngII/PE-treated WT mice ( Supplementary Fig. 1C-D ). Histological analysis of Picrosirius red-stained heart sections revealed that AngII/PE treatment significantly increased collagen deposition in WT mice compared to saline controls, as expected (15.8 ± 0.5% vs. 7.9 ± 0.4%, P < 0.0001) (Fig. 1 G). By contrast, this fibrotic response was markedly attenuated in Cdcp1 -KO mice (9.4 ± 0.4% vs. 15.8 ± 0.5%, P < 0.0001), representing a 41% reduction in fibrotic burden (Fig. 1 H). Saline-treated Cdcp1 -KO and WT groups showed comparably low collagen-positive areas (7.6 ± 1.8% vs. 7.8 ± 2.2%), indicating no fibrotic changes due to Cdcp1 deletion alone. Collectively, these findings demonstrate that Cdcp1 deletion attenuates pressure overload-induced cardiac hypertrophy and fibrosis, the structural precursors to heart failure progression. RNA-seq identifies Cdcp1 -dependent fibrotic and inflammatory gene programs in the left ventricle To understand the molecular mechanisms underlying the effects of Cdcp1 deletion, we performed RNA-seq using LV tissues from all experimental groups. Differential gene expression (DEG) analysis comparing AngII/PE-treated WT to saline-treated WT mice revealed 1,857 DEGs including 1,342 upregulated and 515 downregulated genes (Fig. 2 A). There was significant upregulation of cardiac stress markers ( Nppb , Tnnt3 ), pro-fibrotic and ECM remodeling factors ( Lox , Postn , Col1a1 , Adamts8 ), and inflammatory mediators ( Ccl8 , Gals3 ), which is consistent with the pathological remodeling typically observed in pressure overload. No transcriptional differences were found when comparing saline-treated Cdcp1 -KO and WT mice, suggesting that Cdcp1 deletion alone has no effect on baseline cardiac gene programs ( Supplementary Fig. 2 ). However, when we compared AngII/PE-treated Cdcp1 -KO with AngII/PE-treated WT mice, we found 783 DEGs including 424 downregulated and 359 upregulated genes (Fig. 2 B). Cdcp1 deletion in the context of pressure overload was associated with significant downregulation of pro-fibrotic factors ( Ctgf , Lox , Col1a1 ) and inflammatory mediators ( Ccl7 , Ccl12 , Il21r , Il6 ). Gene set enrichment analysis (GSEA) of GO Biological Process terms using a ranked gene list ordered by log₂ fold-change identified extracellular structure organization, regulation of inflammatory response, and leukocyte migration among the most significantly positively enriched pathways in AngII/PE-treated versus saline-treated WT mice, consistent with the observed cardiac remodeling (Fig. 2 C). When the same analysis was applied to the AngII/PE-treated Cdcp1 -KO versus WT mice, extracellular matrix organization, external encapsulating structure organization, and immune-related terms were negatively enriched, while mitochondrial gene expression and respiration pathways were positively enriched (Fig. 2 D), suggesting that CDCP1 may also be involved in regulating immune responses in pressure-overloaded hearts. Furthermore, AngII/PE treatment markedly increased expression of ECM genes ( Col1a1 , Col1a2 , Col3a1 ), fibroblast activation markers ( Vim , Ctgf ), matrix remodeling enzymes ( Mmp2 , Mmp14 ), inflammatory chemokines ( Ccl7 , Ccl12 ), the cardiac-stress marker Tnnt3 , and Cdcp1 itself in WT heart LVs, whereas the increase in expression of each of these genes was significantly attenuated in Cdcp1 -KO heart LVs (Fig. 2 E). Spatial Transcriptomics Reveals Region- and “Cell Type”-Specific Remodeling Suppressed by Cdcp1 KO in Pressure Overload To investigate the spatial distribution of transcriptional changes associated with Cdcp1 deletion during pressure overload, we performed spatial transcriptomic profiling of cardiac sections from all four experimental groups ( Supplementary Fig. 3 ). Across all samples, we captured a total of 7,889 spatial barcoded transcriptomic spots (Saline_WT: 1,857; Saline_KO: 1,915; AngII/PE_WT: 2,193; AngII/PE_KO: 1,924), with median gene counts per spot ranging from 2,049 to 3,710. UMAP embedding calculated on the first 30 principal components showed distinct separation of spots according to sample of origin before integration (Fig. 3 A), reflecting systematic transcriptomic differences across the samples. Using a reference-guided deconvolution approach, we inferred the relative contributions of major cardiac cell types, across the myocardial sections, including cardiomyocytes, fibroblasts, endothelial cells, and immune cells (Fig. 3 B). Cardiomyocytes were the predominant cell type across all groups (67.2–88.1%), followed by fibroblasts (5.2–17.9%), endothelial cells (4.3–16.2%), and T cells (0.5–2.1%). Quantification of cell-type proportions suggested a consistent reduction in fibroblast abundance in Cdcp1 -KO hearts compared with WT controls, observed both under saline conditions and following AngII/PE treatment. While AngII/PE-treated WT hearts exhibited a marked expansion of predicted fibroblasts (18%), this increase was entirely blunted in Cdcp1 -KO hearts (6%) ( Fig. 3 C ). Together, these findings indicate that Cdcp1 deletion is associated with reduced fibroblast representation in the myocardium particularly in the context of pressure overload. Consistent with these compositional changes, spatial expression mapping of extracellular matrix–associated genes revealed reduced fibrogenic signaling in Cdcp1 -deficient hearts. Expression of Col1a2, Fn1, Mmp2 , and Loxl2 were prominently enriched in AngII/PE-treated WT hearts but substantially diminished in AngII/PE-treated Cdcp1 -KO hearts (Fig. 3 D), indicating suppression of ECM production and matrix remodeling transcriptional programs in the absence of Cdcp1 . To further resolve cellular heterogeneity, we next examined subpopulation structure within major cardiac cell types. Deconvolution analysis identified multiple transcriptionally distinct subclusters of cardiomyocytes, fibroblasts, and endothelial cells, whose relative proportions differed across experimental groups (Fig. 3 E). Marker gene analysis confirmed robust and cell-type–specific expression patterns defining each subcluster (Fig. 3 F). Five distinct cardiomyocyte subtypes (CM2, CM4, CM6, CM7, and CM8) were identified across all experimental groups ( Supplementary Fig. 4A ). Quantitative analysis revealed that AngII/PE-treated WT hearts exhibited a relative enrichment of the CM4 cardiomyocyte subtype, which was enriched for inflammatory response, extracellular matrix organization, collagen fibril organization, and wound healing pathways ( Supplementary Fig. 6A ). In contrast, this cell type population was reduced in AngII/PE-treated Cdcp1 -KO hearts, whereas the CM6 subtype, which was enriched for muscle cell differentiation, myofibril assembly, cell-substrate adhesion, and integrin-mediated signaling pathways ( Supplementary Fig. 6B ), was increased in Cdcp1 -KO hearts compared with AngII/PE-treated WT ( Supplementary Fig. 4B ). Similarly, deconvolution of fibroblast and endothelial compartments identified multiple transcriptionally distinct subtypes whose proportions differed across experimental conditions ( Supplementary Fig. 4A ). Notably, all the three profibrotic fibroblast subtypes (FB5, FB8, and FB9) were expanded in AngII/PE-treated WT hearts but attenuated in Cdcp1 -KO hearts, while endothelial subtypes showed more modest but consistent shifts in abundance ( Supplementary Fig. 4B ). Among fibroblast subpopulations, FB5 showed the most prominent Cdcp1 -dependent differences. Gene set enrichment analysis revealed that FB5 was enriched for biological processes related to response to growth factors, response to TGF-β, regulation of cell migration, and vasculature development (Fig. 3 G). Notably, FB5 exhibited high expression of collagen and ECM-associated genes, consistent with a profibrotic fibroblast state (Supplementary Fig. 4C) . This subpopulation was prominently expanded in AngII/PE-treated WT compared to Cdcp1 -KO hearts, linking the presence of Cdcp1 expression to the emergence of collagen-producing fibroblast niches during pressure overload In summary , these analyses demonstrate Cdcp1 deletion attenuates pathological cardiac remodeling, reduces histological fibrosis, and is associated with diminished fibroblast expansion and ECM gene expression. These results are consistent with our prior demonstration that Cdcp1 contributes to cytokine driven expansion of cardiac fibroblasts. 11 To determine whether CDCP1 directly influences human fibroblast fibrogenic programs, we next examined CDCP1-dependent extracellular matrix programs in human ventricular fibroblasts. CDCP1 regulates growth factor-induced extracellular matrix related gene expression and collagen deposition in human ventricular fibroblasts To directly test fibroblast fibrogenic responses, we examined the effects of CDCP1 knockdown in human ventricular fibroblasts (HVFs) exposed to profibrotic stimuli. HVFs were treated with platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-β (TGF-β), and angiotensin II, all of which are key signaling pathways enriched in FB5 fibroblasts in vivo . Stimulation with individual fibrotic cues led to a significant induction of CDCP1 expression (Fig. 4 A), suggesting CDCP1 as a fibroblast-intrinsic, stress-responsive gene involved in profibrotic signaling pathways. Silencing of CDCP1 markedly attenuated the induction of extracellular matrix–associated genes across all fibrotic stimuli, including COL1A1 , CTGF , and LOX (Fig. 4 B). These genes represent core components of collagen production, matrix cross-linking, and fibroblast activation, consistent with the transcriptional programs enriched in FB5 fibroblasts identified in spatial transcriptomic analyses. At the protein level, immunofluorescent staining of intact HVFs demonstrated a robust reduction in collagen I production following CDCP1 knockdown under profibrotic stimulation (Fig. 4 C). Furthermore, analysis of decellularized extracellular matrix confirmed a corresponding decrease in collagen I deposition (Fig. 4 D), indicating that CDCP1 regulates not only fibroblast gene expression but also synthesis and deposition of collagen I, the predominant component of fibrotic scar in the heart, by activated fibroblasts. Together, these findings establish CDCP1 as a fibroblast-intrinsic regulator of growth factor driven extracellular matrix gene expression and collagen deposition, providing a mechanistic link to the attenuated fibrotic remodeling seen in Cdcp1 -deficient hearts. DISCUSSION In this study, we demonstrate that CDCP1 is a regulator of extracellular matrix production in cardiac fibroblasts. Global Cdcp1 deletion attenuates pressure overload-induced cardiac hypertrophy, collagen deposition, suppresses ECM gene expression, and reduces pro-fibrotic fibroblast subpopulations, a finding that is supported by CDCP1 knockdown in human cardiac fibroblasts. Consistent with prior GWAS findings linking lower CDCP1 expressions to improved cardiac function in patients with heart failure 11 , 12 , we found that Cdcp1 KO attenuated pressure overload-induced increases in LV mass, whereas LVEF and LVFS showed a trend toward improved systolic function. This is consistent with early fibrotic remodeling preceding systolic dysfunction 15 in this model. 16 , 17 Cdcp1 KO mice exhibited significantly reduced collagen deposition compared to wild-type controls in a pressure overload model, with no evidence of baseline fibrosis in saline-treated hearts, indicating that CDCP1 is dispensable under physiological conditions but contributes to maladaptive remodeling during cardiac stress. Bulk RNA sequencing revealed that Cdcp1 deletion suppressed the expression of a broad array of fibrosis-associated genes, including Col1a1 , Postn , Lox , and Ctgf , as well as inflammatory chemokines such as Ccl7 , Ccl12 , and Il6 . These results suggest that Cdcp1 may act upstream of canonical fibrotic and inflammatory gene programs, potentially through modulation of fibroblast activation and ECM remodeling pathways. Mechanistically, our prior in vitro studies demonstrated that CDCP1 is upregulated in human cardiac fibroblasts in response to PDGF-BB stimulation and that CDCP1 knockdown suppresses fibroblast proliferation via reduced AKT phosphorylation. 11 The present in vivo findings, specifically the reduced fibroblast abundance observed in Cdcp1 -KO hearts by spatial transcriptomics, are consistent with this proliferation-dependent mechanism. PDGFRα is uniquely expressed by cardiac fibroblasts and is essential for their survival through PI3K signaling. PDGFRα loss causes approximately 50% reduction in resident cardiac fibroblasts within days. 18 Importantly, controlled reduction of PDGFRα + fibroblasts (60–80%) has been shown to preserve cardiac function following AngII/PE-induced cardiac fibrosis, 19 supporting the therapeutic potential of targeting this pathway. Our prior data suggests that CDCP1 functions as a modulator of PDGF-AKT signaling in cardiac fibroblasts, and its deletion may attenuate sustained AKT activation required for fibroblast expansion and survival. To further define the spatial and cell-type–specific impact of Cdcp1 deletion, we leveraged spatial transcriptomics. Deconvolution analysis showed that Cdcp1 -KO hearts had reduced fibroblast abundance and altered spatial distribution under pressure overload. Importantly, Cdcp1 deletion led to a marked reduction of the FB5 and FB8 fibroblast subtypes, which were associated with TGF-β signaling, growth factor responses, vascular remodeling, and migration. The transcriptional profile of FB5 – enriched in ECM organization, growth factor signaling, and localized to interstitial and perivascular regions – corresponds to the THBS4+/CILP + pro-fibrotic fibroblast populations recently characterized in pressure overload models. 20 , 21 These populations, which express Thbs4 , Cilp , Postn , and Cthrc1 , represent a pressure overload-specific fibroblast state distinct from classical α-SMA + myofibroblasts observed after myocardial infarction. 22 Our prior findings suggest that CDCP1 plays a role in the emergence or maintenance of these ECM-producing fibroblast populations through AKT-mediated survival signaling. 11 Cardiomyocyte subpopulation analysis revealed more layers of Cdcp1 -dependent remodeling. In WT hearts under pressure overload, the emergence of CM4 – a pro-fibrotic cardiomyocyte subtype enriched in Wnt signaling, ECM remodeling, and inflammatory gene signatures – was pronounced. However, this population was significantly reduced in Cdcp1 -KO hearts. Instead, Cdcp1 -deficient hearts displayed enrichment of CM6, a cardiomyocyte subtype characterized by signatures of muscle cell development, cell-substrate adhesion, and myofibril assembly, suggesting a potentially reparative or adaptive remodeling phenotype. The spatial co-localization of CM4 with FB5 clusters in WT hearts, ( Supplementary Figs. 4 and 5 ) and the coordinated reduction of both populations in Cdcp1 -KO hearts, suggests that CDCP1 may regulate pathological fibroblast-cardiomyocyte crosstalk during pressure overload. Our findings place CDCP1 within a growing class of transmembrane proteins validated as cardiac fibrosis targets. For instance, fibroblast activation protein (FAP) emerged as a promising therapeutic target. FAP-specific CAR T cells have been shown to reduce fibrosis and improve function in the AngII/PE model, 23 and more recently, in vivo generation of transient FAP-targeted CAR T cells via CD5-targeted lipid nanoparticle (LNP)-mediated delivery of modified mRNA enabled transient, controllable intervention. 24 Depletion of αv integrin in PDGFRβ + cells confers protection against cardiac fibrosis, and small molecule inhibitors similarly attenuate established fibrosis via the shared SRC-PI3K/AKT pathway. 25 IL-11 receptor (IL-11RA) KO mice are protected from cardiac fibrosis, and neutralizing antibodies demonstrate preclinical efficacy against it. 26 Like these targets, CDCP1 is a cell-surface transmembrane protein with limited cardiomyocyte expression, rendering it suitable for antibody-based therapeutic strategies currently under development in oncology. 27 , 28 Importantly, CDCP1 may have context-dependent roles across different fibrotic pathways, cells and organs. In lung fibroblasts, CDCP1 knockdown enhanced TGF-β1-induced myofibroblast differentiation, 29 in contrast to our findings in cardiac fibroblasts, where CDCP1 promotes PDGF-driven proliferation and is necessary for TGF-β mediated ECM synthesis. This organ specific function warrants further mechanistic investigation. Several limitations of this study should be acknowledged. First, the use of a global Cdcp1 knockout mouse model precludes definitive attribution of the observed phenotypes to specific cell types; although convergent in vivo and in vitro data implicate fibroblasts, cell type–specific deletion (e.g., fibroblast- or cardiomyocyte-restricted models) will be necessary to delineate causal mechanisms and intercellular contributions more precisely. Notably, however, the use of a global knockout may also enhance translational relevance, as a pharmacologic strategy targeting CDCP1 would likely exert systemic effects rather than cell type–restricted modulation. Second, the AngII/phenylephrine infusion model recapitulates aspects of pressure overload–induced remodeling but does not fully capture the heterogeneity of human heart failure etiologies, limiting direct translational generalizability. Third, the primary analyses were conducted at an early time point (4 weeks), when fibrotic remodeling precedes overt systolic dysfunction, and therefore do not establish long-term functional consequences or effects on advanced heart failure phenotypes. Fourth, spatial transcriptomic resolution is limited by spot size and reliance on computational deconvolution, which may obscure finer cellular heterogeneity and introduce inference bias in cell-type assignments. Fifth, while human ventricular fibroblast experiments support a conserved, fibroblast-intrinsic role for CDCP1, these reductionist systems do not fully recapitulate the multicellular myocardial environment or systemic influences present in vivo. Finally, although CDCP1 emerges as a potential therapeutic target, the safety, tissue specificity, and efficacy of CDCP1-directed interventions—particularly given its context-dependent roles across organs—remain to be established in translational and large-animal studies. From a translational perspective, CDCP1 is a promising therapeutic target for cardiac fibrosis. As a transmembrane protein with an accessible extracellular domain, it is amenable to antibody-based therapies already in development for oncologic indications, 27,28 which could potentially be repurposed for this indication. Moreover, Cdcp1 deletion is well-tolerated at baseline and elicits pathological phenotypes only under stress, suggesting a favorable therapeutic window. Human genetic data from the UK Biobank PheWAS link CDCP1 variants to heart failure mortality, 11 and conserved mechanisms in human cardiac fibroblasts reinforce its translational potential. In conclusion, our study provides direct in vivo evidence that CDCP1 attenuates pressure overload-induced cardiac fibrosis. By integrating genetic, histological, and spatial transcriptomic approaches, we demonstrate that Cdcp1 deletion suppresses pro-fibrotic THBS4 + /CILP + -like fibroblast populations, attenuates pathological cardiomyocyte states, and preserves cardiac structure. Mechanistically, CDCP1 functions as an important mediator of multiple pro-fibrotic stimuli that drive fibroblast collagen synthesis. These insights provide a mechanistic basis for targeting CDCP1 to attenuate cardiac fibrosis and thus heart failure progression. Declarations Data and Code Availability The bulk RNA sequencing data generated during this study are available in the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE296565. The spatial transcriptomics data is available under accession number GSE303631. All analysis scripts and computational pipelines used in this study are publicly available at https://github.com/irenemaring/CDCP1_PressureOverload. No custom algorithms were developed. All code for standardized analyses (Seurat, DESeq2,) is publicly accessible through GitHub or CRAN/Bioconductor repositories. Conflict of Interest: The authors declare no conflicts of interest related to this study. Sources of Funding: Rachad Ghazal is supported by the National Heart, Lung, and Blood Institute T32HL007111 grant. Min Wang is supported by the National Institute of General Medical Sciences T32GM008685 grant. Acknowledgements: We acknowledge the Mayo Clinic Department of Comparative Medicine. We also thank the Mayo Clinic Spatial Multiomics Core for the probe-based 10x Genomics Visium CytAssist assays. Contributions: Conceptualization: MW, DL, DJT, NLP; Methodology: RG, ANS, MW, IM, RH, TO, CET, DYL, AJ, HV, LJL, JM; Investigation: ANS, MW, LW, WS; Manuscript writing: RG, ANS, JJLC; Manuscript editing: CMR, TO, CW, DJB, GS, RW, DL, DJT, NLP References Humeres C, Frangogiannis NG. Fibroblasts in the Infarcted, Remodeling, and Failing Heart. JACC Basic Transl Sci . 2019;4:449–467. doi: 10.1016/j.jacbts.2019.02.006 Travers JG, Kamal FA, Robbins J, Yutzey KE, Blaxall BC. Cardiac Fibrosis: The Fibroblast Awakens. Circ Res . 2016;118:1021–1040. doi: 10.1161/CIRCRESAHA.115.306565 Pfeffer MA, Shah AM, Borlaug BA. Heart Failure With Preserved Ejection Fraction In Perspective. Circ Res . 2019;124:1598–1617. doi: 10.1161/CIRCRESAHA.119.313572 Tallquist MD, Molkentin JD. Redefining the identity of cardiac fibroblasts. Nat Rev Cardiol . 2017;14:484–491. doi: 10.1038/nrcardio.2017.57 Gonzalez A, Schelbert EB, Diez J, Butler J. 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Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation. Am J Physiol Lung Cell Mol Physiol. 2018;314(5):L695-L707. doi: 10.1152/ajplung.00205.2017 Additional Declarations There is NO Competing Interest. Supplementary Files SUPPLEMENTARYFIGURESlegends.docx SupplementaryFigure4.png Supplementary Dataset 4 SupplementaryFigure2.tif Supplementary Dataset 2 SupplementaryFigure5.png Supplementary Dataset 5 SupplementaryFigure6.tif Supplementary Dataset 6 SupplementaryFigure1.tif Supplementary Dataset 1 SupplementaryFigure3.tif Supplementary Dataset 3 FullUncroppedGel.pdf Supplementary Information: Full Uncropped Gel Images Cite Share Download PDF Status: Under Review 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. 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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-9236741","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631960970,"identity":"e202222e-8fcb-410f-9990-9b1156659a1e","order_by":0,"name":"Naveen Pereira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYLCCBATTBkqzEa8ljUgtSOAwYS3m7L0PHzzcYwdknDF88HHH+Wj+GckHGD6UHcapxbLnuLFBwrNkICPH2HDmmdu5M26kJTDOOIdbi8GNNDaJhAPMDAYH0tKkedtu5zbcyDFg5m3Do+X+M/YfCQfqGQzOP0v//bftXO58kJa/+LTcYGNjSDhwGMhIPsbM2HYgdwNICyMeLZY9acxAhx3nMbjx+LBkb1ty7sYzzxIO9pxLx6nFnP0Y48cfB6rlDM4nNn742WaXO+948sEHP8qscTsMSvMghAQSGA7gVI+kBQnw49UwCkbBKBgFIxAAADm1XJMRdY1RAAAAAElFTkSuQmCC","orcid":"","institution":"Mayo Clinic","correspondingAuthor":true,"prefix":"","firstName":"Naveen","middleName":"","lastName":"Pereira","suffix":""},{"id":631960971,"identity":"8094e3a5-0983-4660-96c6-7d3c1eb27042","order_by":1,"name":"Rachad Ghazal","email":"","orcid":"","institution":"Mayo Clinic","correspondingAuthor":false,"prefix":"","firstName":"Rachad","middleName":"","lastName":"Ghazal","suffix":""},{"id":631960972,"identity":"8fe437bf-6912-4684-8424-7468fbe2aeee","order_by":2,"name":"Akshatha N. 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Four guide RNAs (5g-1, 5g-2, 3g-1, 3g-2) were designed to flank and excise exon 1. Genotyping primers (DY164, DY165) are indicated. \u003cstrong\u003eB,\u003c/strong\u003e PCR genotyping results confirming successful deletion of \u003cem\u003eCdcp1\u003c/em\u003e. Amplification yielded a 1,771 bp product for the wild-type (WT) allele and a truncated \u0026lt;501 bp product for the KO allele. \u003cstrong\u003eC\u003c/strong\u003e, Experimental timeline and procedures. At baseline (week -1), mice underwent transthoracic echocardiography and initial body weight measurement. At week 0, osmotic minipumps were subcutaneously implanted for chronic infusion of either saline or AngII/PE (1.2 µg/g/day AngII, 35 µg/g/day PE). Four experimental groups were studied. Animals were monitored weekly for body weight and general health. At the endpoint (week 4), cardiac function was reassessed by echocardiography, followed by euthanasia for collection of heart tissues and measurement of heart and body weights. \u003cstrong\u003eD\u003c/strong\u003e, Left ventricular mass index (LVMI, mg/g, calculated as LV mass divided by body weight) at baseline prior to AngII/PE infusion, \u003cstrong\u003eE\u003c/strong\u003e, LVMI at 4 weeks post AngII/PE infusion. \u003cstrong\u003eF\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eChange in LVMI (Δ mg/g from baseline to 4 weeks). For D-F: Saline WT, n=9; Saline KO, n=10; AngII/PE WT, n=9; AngII/PE KO, n=10. Data presented as mean±SD. Statistical analysis was performed using two-way ANOVA (genotype ´ treatment) followed by pre-specified pairwise comparisons using unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e-tests. \u003cstrong\u003eG\u003c/strong\u003e, Micrographs showing Picrosirius red-stained ventricular cross-sections from WT and \u003cem\u003eCdcp1\u003c/em\u003e-KO mice after 4 weeks of saline or AngII/PE infusion. Scale bars: 1 mm. \u003cstrong\u003eH\u003c/strong\u003e, Quantification of collagen-positive area (%) in each group (Saline WT, n=48; Saline KO, n=42; AngII/PE WT, n=48; AngII/PE KO, n=30). Data points represent individual fields with mean±SD overlay. Statistical significance was determined using unpaired Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/0ec7eb0a4c544be7dc9aa9c3.png"},{"id":108977069,"identity":"dc9575bf-aba1-402d-8043-69ccf7bd0638","added_by":"auto","created_at":"2026-05-11 11:30:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6754987,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic Profiling Identifies \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCdcp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Dependent Pathways Driving Myocardial Fibrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;A,\u003c/strong\u003e Volcano plot of differentially expressed genes (DEGs) in WT mice treated with saline versus AngII/PE. \u003cstrong\u003eB,\u003c/strong\u003e Volcano plot of DEGs in \u003cem\u003eCdcp1\u003c/em\u003e-KO versus WT mice after AngII/PE treatment. \u003cstrong\u003eC,\u003c/strong\u003e GSEA of Gene Ontology (GO) Biological Process terms using ranked gene list ordered by log\u003csub\u003e2\u003c/sub\u003e fold-change from the AngII/PE WT versus saline WT comparison. \u003cstrong\u003eD,\u003c/strong\u003e GSEA of GO Biological Process terms using a ranked gene list from the AngII/PE \u003cem\u003eCdcp1-\u003c/em\u003eKO versus AngII/PE WT comparison. For C and D, the top 5 positively and top 5 negatively enriched terms by adjusted P-value are shown. Dot size indicates gene set size; dot color indicates adjusted P-value. \u003cstrong\u003eE,\u003c/strong\u003e Heatmap showing expression profiles of extracellular matrix-related genes, inflammatory mediators, and selected markers (\u003cem\u003eCdcp1, Tnnt3\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/74ebc60e01507b62bfb8a0f4.png"},{"id":109081068,"identity":"5637c1f8-80b0-4187-bc29-dc1fa5de0b7f","added_by":"auto","created_at":"2026-05-12 11:56:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12221191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial transcriptomic analysis reveals \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCdcp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-dependent alterations in cardiac cell-type composition under pressure overload.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eUMAP embedding of spatial transcriptomic spots calculated on the first 30 principal components prior to integration, colored by sample of origin.\u003cstrong\u003eB,\u003c/strong\u003e Global spatial deconvolution mapping the distribution of identified cell types across heart tissue sections (Cardiomyocytes: CM; fibroblasts: FB; endothelial cells: EC; T-cells). Color scale indicates inferred proportion of spot corresponding to the cell type. \u003cstrong\u003eC, \u003c/strong\u003eTable showing mean ± SD of deconvoluted cell type proportions per spot across all four groups. \u003cstrong\u003eD, \u003c/strong\u003eSpatial dot plots showing expression of extracellular matrix-associated genes (\u003cem\u003eCol1a2, Fn1, Mmp2, Loxl1\u003c/em\u003e) \u003cstrong\u003eE, \u003c/strong\u003eRelative proportions of major cell subtype populations across saline WT, saline KO, AngII/PE WT, and AngII/PE KO samples. \u003cstrong\u003eF,\u003c/strong\u003e Dot plot of marker gene expression used to define cardiomyocyte, fibroblast, and endothelial subpopulations. \u003cstrong\u003eG, \u003c/strong\u003eSpatial distribution and\u003cstrong\u003e \u003c/strong\u003eGSEA for GO biological process of FB5 fibroblast subpopulation in AngII/PE WT versus KO spots.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/03781dfa045a0bec8f2db84c.png"},{"id":108836068,"identity":"9b80355f-4aab-41c2-823e-ca98d2bad13e","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10769395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCdcp1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency attenuates profibrotic gene expression and collagen deposition in human ventricular fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHVFs were treated with profibrotic stimuli PDGF-BB (20 ng/mL), TGF-β (10 ng/mL), or angiotensin II (100 ng/mL), individually for 48 h. \u003cstrong\u003eA, \u003c/strong\u003e\u003cem\u003eCDCP1\u003c/em\u003e expression was assessed by qRT-PCR. \u003cstrong\u003eB, \u003c/strong\u003emRNA levels of extracellular matrix-associated genes (\u003cem\u003eCOL1A1, CTGF, LOX) \u003c/em\u003eafter CDCP1 knockdown. \u003cem\u003eGAPDH \u003c/em\u003ewas used as internal control. \u003cstrong\u003eC,\u003c/strong\u003e Representative immunofluorescence images of collagen I in intact HVFs following profibrotic stimulation upon CDCP1 knockdown. \u003cstrong\u003eD,\u003c/strong\u003e Immunofluorescence staining of collagen I in decellularized fibroblast-derived extracellular matrix.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/d3dee9b80540dcad4d02eef3.png"},{"id":109204520,"identity":"979a1eac-2560-400e-95a7-bf9e4526dd34","added_by":"auto","created_at":"2026-05-13 15:00:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33755377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/d6a6490b-56da-4b68-b936-a36b58f85d5f.pdf"},{"id":108836065,"identity":"ffe5bdf5-b760-483c-8263-ec1a40c2e83a","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16218,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYFIGURESlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/4debc816bdfa66d107039b7a.docx"},{"id":108836066,"identity":"bcb80167-085a-453f-a0de-dbfbd136a046","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13815432,"visible":true,"origin":"","legend":"Supplementary Dataset 4","description":"","filename":"SupplementaryFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/c571b527b0415b7471194c87.png"},{"id":108836069,"identity":"949f1796-127b-4e95-a5fa-bb598316e1d4","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":678576,"visible":true,"origin":"","legend":"Supplementary Dataset 2","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/51b3e6d055987d4111f57b83.tif"},{"id":109067891,"identity":"64ef8096-edaf-4973-a165-2167cc451186","added_by":"auto","created_at":"2026-05-12 10:02:27","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13244304,"visible":true,"origin":"","legend":"Supplementary Dataset 5","description":"","filename":"SupplementaryFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/ee3daeaaa3272b61f01bbe06.png"},{"id":108977493,"identity":"4a983cb9-a90f-4225-b4c5-637d8befa51c","added_by":"auto","created_at":"2026-05-11 11:31:54","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2246516,"visible":true,"origin":"","legend":"Supplementary Dataset 6","description":"","filename":"SupplementaryFigure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/49b4d888515e9bb575cddcaf.tif"},{"id":108977299,"identity":"2c0ba2af-1a06-4c9b-84ab-37ad3d7891f4","added_by":"auto","created_at":"2026-05-11 11:31:16","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":29114670,"visible":true,"origin":"","legend":"Supplementary Dataset 1","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/e09059754675a335afe222c8.tif"},{"id":108836072,"identity":"1a9ecc48-7956-41ad-98cf-ed10b836e75e","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":726640,"visible":true,"origin":"","legend":"Supplementary Dataset 3","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/f65ca055e45a804273bcc9c6.tif"},{"id":108836073,"identity":"6c4798b3-7c9a-4571-b893-25b8fc8ea007","added_by":"auto","created_at":"2026-05-08 22:57:27","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":34464,"visible":true,"origin":"","legend":"Supplementary Information: Full Uncropped Gel Images","description":"","filename":"FullUncroppedGel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9236741/v1/3819d4ad9db2b60d25b5f528.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CDCP1 Deletion Protects Against Pressure Overload-Induced Cardiac Dysfunction and Fibrosis in Mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCardiac fibrosis is a hallmark pathological feature in various cardiovascular diseases and a major driver of heart failure progression.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This process is characterized by excessive extracellular matrix (ECM) accumulation that is mediated by activated cardiac fibroblasts,\u003csup\u003e4\u003c/sup\u003e and contributes significantly to myocardial stiffness and dysfunction. Despite its clinical significance, effective therapies targeting cardiac fibrosis remain limited.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Chronic pressure overload induces left ventricular (LV) hypertrophy and progressive interstitial ECM accumulation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e This excessive collagen deposition elevates filling pressures, compromises chamber compliance, and predisposes to arrhythmias and heart failure progression.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Mechanistically, cardiac fibrosis involves resident cardiac fibroblast proliferation, migration and transdifferentiation into α-smooth muscle actin (αSMA)-expressing myofibroblasts, which secrete collagens resulting in adverse cardiac remodeling.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Current antifibrotic therapies with demonstrated clinical efficacy remain elusive,\u003csup\u003e11,12\u003c/sup\u003e stressing the need to identify novel molecular targets.\u003c/p\u003e \u003cp\u003eWe previously performed a genome-wide association study (GWAS) for myocardial recovery in patients with recent onset heart failure.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e That GWAS identified a genetic locus (index SNP rs6773435) that is an expression quantitative trait locus (eQTL) for the \u003cem\u003eCUB domain-containing protein 1\u003c/em\u003e (\u003cem\u003eCDCP1\u003c/em\u003e) gene in cultured fibroblasts.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Reduced \u003cem\u003eCDCP1\u003c/em\u003e expression was associated with improved left ventricular ejection fraction (LVEF) in heart failure patients. We further demonstrated that \u003cem\u003eCDCP1\u003c/em\u003e expression is upregulated in human cardiac fibroblasts in response to profibrotic stimuli and that its knockdown attenuates fibroblast proliferation.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e However, its role in cardiac fibrosis and remodeling \u003cem\u003ein vivo\u003c/em\u003e remains undefined.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the role of CDCP1 in cardiac fibrosis using a pressure overload-induced murine model with \u003cem\u003eCdcp1\u003c/em\u003e gene knockout (KO). This model recapitulates the chronic neurohormonal stress milieu characteristic of heart failure, providing a biologically relevant context to functionally interrogate CDCP1 in cardiac fibrosis. \u003cem\u003eCdcp1\u003c/em\u003e-KO mice demonstrated attenuated pathological remodeling in response to chronic angiotensin II/phenylephrine (AngII/PE) infusion, preserved cardiac function, and reduced fibrotic burden compared to wild-type (WT) controls. Transcriptomic analysis revealed that \u003cem\u003eCdcp1\u003c/em\u003e deletion suppresses profibrotic and proinflammatory gene networks. Spatial transcriptomics revealed that \u003cem\u003eCdcp1\u003c/em\u003e deletion induces region-specific alterations in fibrogenic gene signatures within the myocardium, including reduced inflammation and extracellular matrix organization associated cardiomyocyte subpopulations and attenuation of growth factor and TGF-β signaling associated fibroblast subtypes. Our findings establish \u003cem\u003eCdcp1\u003c/em\u003e as a critical mediator of cardiac fibrosis, providing the direct functional evidence of its role in cardiac pathophysiology and supporting its potential as a therapeutic target for cardiac fibrosis.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Model and Ethical Approval\u003c/h2\u003e \u003cp\u003eAll procedures conformed to the Guide for the Care and Use of Laboratory Animals (U.S. Department of Health and Human Services) and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Mayo Clinic. Mice on an FVB/NJ background (The Jackson Laboratory, Bar Harbor, ME) were housed under specific pathogen-free (SPF) conditions with controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity (55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%), on a 12-h light/dark cycle. Standard rodent chow and water were provided \u003cem\u003ead libitum\u003c/em\u003e. Throughout the study, all efforts were made to minimize discomfort during procedures such as ear tagging, tissue sampling for genotyping, and anesthesia for osmotic pump implantation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of\u003c/b\u003e \u003cb\u003eCdcp1\u003c/b\u003e \u003cb\u003eKnockout Mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eCdcp1\u003c/em\u003e-null mice were generated on an FVB/NJ genetic background by CRISPR-Cas9 editing to remove exon 1 of \u003cem\u003eCdcp1\u003c/em\u003e gene. Four guide RNAs were designed to flank exon 1: 5g-1 (5\u0026rsquo;-GTAGATGGTCTGGGACCTCG-3\u0026rsquo;), 5g-2 (5\u0026rsquo;-GGGGGGGTCATCACAACATG-3\u0026rsquo;), 3g-1 (5\u0026rsquo;-GGGATACTCGATTGGGACGT-3\u0026rsquo;), and 3g-2 (5\u0026rsquo;-GAGAACGTCCTCCTAAGGCT-3\u0026rsquo;). Genotyping was performed using primers DY164 (forward: 5\u0026rsquo;-GCATGGGCTTCTGTTTCTGT-3\u0026rsquo;) and DY165 (reverse: 5\u0026rsquo;-GCACGGACAGCTAAAATGGT-3\u0026rsquo;). Founder mice harboring exon deletion confirmed by PCR genotyping of ear DNA were bred to homozygosity to obtain \u003cem\u003eCdcp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e (KO) offspring and littermate-matched WT controls. Both male and female mice, aged 8\u0026ndash;12 weeks, were used in all experiments. Mice were randomly assigned to saline or AngII/PE treatment groups, with males and females distributed as evenly as possible across genotypes and treatment conditions. Mice were monitored daily for overall health, body weight, and signs of distress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAngiotensin II/Phenylephrine Infusion Model\u003c/h3\u003e\n\u003cp\u003eTo induce pressure overload and cardiac fibrosis, osmotic minipumps (ALZET\u003csup\u003e\u0026rarr;\u003c/sup\u003e model 1002, DURECT Corporation, Cupertino, CA) were implanted subcutaneously at 10 weeks of age under 1.5% isoflurane anesthesia. Minipumps were pre-loaded to deliver either saline (0.9% NaCl) as a control, or AngII (1.2 \u0026micro;g/g/day; Sigma-Aldrich, Cat. No. A9525, St. Louis, MO, USA) plus phenylephrine (PE) HCl (35 \u0026micro;g/g/day; Sigma-Aldrich, Cat. No. P6126-5G) for 28 days. Incisions were closed with a single 5\u0026thinsp;\u0026minus;\u0026thinsp;0 nylon suture, and mice were placed in warmed recovery chambers postoperatively. Four experimental cohorts were established: Saline_WT (WT mice receiving saline infusion), Saline_KO (\u003cem\u003eCdcp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice receiving saline), AngII/PE_WT (WT mice receiving AngII/PE), and AngII/PE_KO (\u003cem\u003eCdcp1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice receiving AngII/PE). All animals were monitored for any adverse effects, including changes in mobility or grooming. No mortality or overt toxicity was observed in any group during the 4-week infusion period. A separate cohort of mice was monitored for 14 weeks to examine the effect of \u003cem\u003eCdcp1\u003c/em\u003e deletion on long-term survival during pressure overload. For all other endpoints (echocardiography, histology and transcriptomics), mice were euthanized at 4 weeks post-implantation.\u003c/p\u003e\n\u003ch3\u003eEchocardiographic Assessment of Cardiac Function\u003c/h3\u003e\n\u003cp\u003eTransthoracic echocardiography (TTE) was performed at baseline and at 4 weeks post-minipump implantation using the Vevo\u003csup\u003e\u0026rarr;\u003c/sup\u003e F2 Imaging System (FUJIFILM VisualSonics, Toronto, Canada) equipped with a 46\u0026thinsp;\u0026minus;\u0026thinsp;20 MHz linear-array transducer. Mice were lightly anesthetized (1-2.5% isoflurane), positioned supine on a warming platform, and the heart rate was maintained between 450\u0026ndash;600 bpm to minimize anesthesia-induced cardio depression through adjustment of inhaled isoflurane concentration. Two-dimensional (2D) and M-mode images were acquired in the parasternal short-axis view at the mid-papillary level. Left ventricular (LV) mass index (LVMI), LV ejection fraction (LVEF), and LV fractional shortening (LVFS) were calculated from M-mode measurements as previously described.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e All acquisitions and their analysis were conducted in a blinded fashion to the animal\u0026rsquo;s genotype and treatment group.\u003c/p\u003e\n\u003ch3\u003eHistopathology and Fibrosis Quantification\u003c/h3\u003e\n\u003cp\u003eAfter the 28-day infusion period, mice were euthanized by CO\u003csub\u003e2\u003c/sub\u003e inhalation, in accordance with approved IACUC protocol. Hearts were rapidly excised, rinsed in 10X Phosphate-Buffered Saline (PBS), and fixed in 10% neutral-buffered formalin at room temperature over 24 hours. Fixed tissues were embedded in paraffin and sectioned at 6 \u0026micro;m thickness. Collagen deposition was assessed histologically by Picrosirius Red staining (Polysciences Inc., Warrington, PA) following the manufacturer\u0026rsquo;s protocol. Light microscopy images were captured at 20X and 40X magnification (Nikon, Tokyo, Japan). Quantification of cardiac fibrosis was determined by analyzing Picrosirius Red-positive area in \u0026ge;\u0026thinsp;5 randomly selected mid-myocardial fields per sample, excluding large epicardial vessels.\u003c/p\u003e\n\u003ch3\u003eBulk-tissue RNA Sequencing\u003c/h3\u003e\n\u003cp\u003eSnapfrozen LV tissues were pulverized under liquid nitrogen. Total RNA was extracted using Quick-RNA\u0026trade; Miniprep Kit (Zymo Research, Cat. No. R2052). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and library preparation was carried out using the NEBNext\u003csup\u003e\u0026rarr;\u003c/sup\u003e Ultra\u003csup\u003e\u0026trade;\u003c/sup\u003e II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA), following the manufacturer\u0026rsquo;s guidelines. Multiplexed libraries were loaded onto an Illumina NovaSeq\u0026trade; 6000 flow cell (Illumina, San Diego, CA, USA) and sequenced in a 2\u0026sdot;150 bp paired-end format. Base calling and raw data processing were managed by the NovaSeq Control Software (NCS). The raw reads were quality-checked, trimmed and aligned to Mus musculus reference genome (GRCm39: GCF_000001635.27) using STAR (v2.5.2b). Read counts were assigned to annotated genes with featureCounts (Subread v1.5.2) counting only the uniquely mapped reads in exonic regions. Differential expression analysis was performed using DESeq2 (R/Bioconductor) with the Wald test applied to obtain log\u003csub\u003e2\u003c/sub\u003e fold changes and \u003cem\u003eP\u003c/em\u003e-values. Genes with an adjusted \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and an absolute log\u003csub\u003e2\u003c/sub\u003e fold change\u0026thinsp;\u0026gt;\u0026thinsp;1 were designated as differentially expressed. Functional gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package on a ranked list of gene symbols ordered by log₂ fold‑change from DESeq2 differential expression.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSpatial Transcriptomic Assays\u003c/h2\u003e \u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) heart blocks were sectioned at 5 \u0026micro;m thickness. Tissues with RNA integrity number (RIN)\u0026thinsp;\u0026gt;\u0026thinsp;6 were selected for spatial transcriptomics using the Visium Spatial Gene Expression platform (10x Genomics, Pleasanton, CA). Tissue sections with picrosirius red were placed on capture areas (\u0026asymp;5000 barcoded spots per 6.5\u0026sdot;6.5 mm area, spot diameter: 55 \u0026micro;m, center-to-center distance: 100 \u0026micro;m), and imaged with a Leica Aperio VERSA (Leica Microsystems, Wetzlar, Germany) at 20X resolution. Permeabilization conditions were optimized according to the Tissue Optimization protocol (10x Genomics CG000238). Spot-based RNA capture, reverse transcription, cDNA amplification, and library construction were performed according to the Visium Spatial Gene Expression Slide \u0026amp; Reagent Kit (10x Genomics). Resultant libraries were sequenced on an Illumina NovaSeq\u0026trade; 6000 system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSpatial Transcriptomic Data Analysis\u003c/h3\u003e\n\u003cp\u003eRaw FASTQ files were processed to quantify spot-level gene expression. Spots on tissue were manually adjusted with Loupe browser (10x Genomics). The feature barcode expression matrices were analyzed and visualized in Seurat v5.1.0 (R/CRAN). Samples were normalized using sctransform method with additional log normalization and scaled based on gene counts. Harmony integration method was used for inter-sample comparison. Uniform Manifold Approximation and Projection (UMAP) was used for 2D visualization of clusters. Gene signatures distinguishing each cluster or subpopulation were identified using Seurat\u0026rsquo;s \u0026ldquo;FindMarkers\u0026rdquo; with a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a log\u003csub\u003e2\u003c/sub\u003e fold change threshold\u0026thinsp;\u0026gt;\u0026thinsp;0.25. To characterize the cell-type proportions of spatial spots, Seurat\u0026rsquo;s integrated anchor-based deconvolution method was employed using previously published and annotated single-cell RNAseq data (GEO accession number: GSE120064)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We used the broad cell type labels (cardiomyocytes, fibroblasts, etc.) and the subtype annotations (CM4, CM6, etc.) to deconvolve the spots. Differential expression analysis was conducted using Seurat \u0026ldquo;FindMarkers\u0026rdquo; function across conditions and cell types. Genes with absolute average log\u003csub\u003e2\u003c/sub\u003e fold change\u0026thinsp;\u0026gt;\u0026thinsp;1 and adjusted \u003cem\u003eP\u003c/em\u003e-value (Benjamini-Hochberg method)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significantly differentially expressed. GSEA in GO terms was performed with clusterProfiler and org.Mm.eg.db R packages.\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eCryopreserved adult human ventricular fibroblasts (HVFs) (Cell Applications, Inc) were cultured in Cardiac Fibroblast growth medium. Cells were subcultured using 0.25% trypsin when they reached 70% confluence and maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. HVFs between the passages 2 to 6 were used for all \u003cem\u003ein vitro\u003c/em\u003e experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCDCP1\u003c/b\u003e \u003cb\u003eTransient Knockdown\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCells were transfected with 25 nM siGENOME SMARTpool siRNAs (Dharmacon) targeting \u003cem\u003eCDCP1\u003c/em\u003e using Lipofectamine RNAiMAX Reagent (ThermoFisher).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProfibrotic Stimulation\u003c/h2\u003e \u003cp\u003eHVFs were serum-starved overnight and treated with Recombinant human PDGF-BB (20 ng/ml), TGF-β (10 ng/ml), and Angiotensin II (100 ng/ml) in serum free DMEM. Reconstitution buffer was used as vehicle control. Depending on the experimental design, cells were harvested at 48 h, 72 h or 5 days post-stimulation for RNA extraction or immunofluorescence staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eReal-time Polymerase Chain Reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from HVFs using Quick-RNA MicroPrep Kit (Zymo Reseacrh). qRT-PCR was performed using a one-step Power SYBR Green RNA-to-CT kit (Applied Biosystems) on StepOne PCR system (Thermo Fisher). Ct values were normalized to the reference gene GAPDH, and the relative quantification was calculated using the ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eHVFs were seeded in 96-well plates and cultured to 50% confluence prior to treatment with profibrotic stimuli. Following treatment, intact cells were either processed directly for immunofluorescence or subjected to decellularization to isolate cell-derived extracellular matrix (ECM). For intact-cell staining, nuclei were first labeled with Hoechst 33342 (1:1000) for 30 min at room temperature. For ECM preparation, cells were decellularized using a solution containing 20 mM ammonium hydroxide and 0.5% Triton X-100 for 5 min at room temperature. Both intact cells and decellularized ECM were gently washed three times with 1\u0026times; PBS and fixed with 10% formalin for 10 min at room temperature. After fixation, samples were washed and blocked in 1\u0026times; PBST (1% BSA, 0.1% Triton X-100) supplemented with 3% normal goat serum for at least 3 h or overnight at 4\u0026deg;C. Samples were then incubated overnight at 4\u0026deg;C with a rabbit anti\u0026ndash;collagen I primary antibody (NB600-408G; 1:500). Following PBS washes, samples were incubated with Alexa Fluor 488\u0026ndash;conjugated goat anti-rabbit secondary antibody (1:1000) together with DAPI (1:1000) for 1 h at room temperature. After final washes, collagen I immunofluorescence in both intact fibroblasts and decellularized ECM was imaged and quantified using a Cytation 5 imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Considerations\u003c/h2\u003e \u003cp\u003eSample sizes for each experimental group were determined based on power calculations and anticipated effect sizes. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Two-way ANOVA (genotype \u0026sdot; treatment) was used to assess main effects and interactions for echocardiographic and morphometric parameters across the four experimental groups. Pre-specified pairwise comparisons were performed using unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests. Two-way ANOVA with Sidak\u0026rsquo;s post hoc test was used for longitudinal body weight analysis. Survival analysis was performed using the log-rank (Mantel-Cox) test. For histological fibrosis quantification, an unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied. A \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. GraphPad Prism (GraphPad Software, San Diego, CA) and R (v4.2.2) were used for statistical calculations and data plotting.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eGeneration and baseline characterization of\u003c/b\u003e \u003cb\u003eCdcp1\u003c/b\u003e \u003cb\u003eKO mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of \u003cem\u003eCdcp1\u003c/em\u003e in cardiac remodeling and fibrosis, we generated \u003cem\u003eCdcp1\u003c/em\u003e-KO mice using CRISPR-Cas9 editing to remove exon 1 of the \u003cem\u003eCdcp1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). PCR genotyping confirmed the successful deletion of the targeted region by showing amplification products of 1,771 bp for the WT allele and a truncated product approximately 501 bp for the KO allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Homozygous \u003cem\u003eCdcp1\u003c/em\u003e-KO mice were obtained at expected Mendelian ratios and were viable without overt congenital anomalies. To assess whether \u003cem\u003eCdcp1\u003c/em\u003e deletion affected baseline growth and development, we monitored body weight from weaning to adulthood. Longitudinal body weight measurements from 3 to 11 weeks of age showed comparable growth between WT (n\u0026thinsp;=\u0026thinsp;14, 9 males, 5 females) and \u003cem\u003eCdcp1\u003c/em\u003e-KO mice (n\u0026thinsp;=\u0026thinsp;10, 7 males, 3 females) (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). Two-way ANOVA showed no significant main effect of genotype on body weight (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.478) and no significant Age\u0026sdot;Genotype interaction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.986). Sidak\u0026rsquo;s post hoc comparisons confirmed that there were no significant differences between genotypes at any time point (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). To examine the effect of \u003cem\u003eCdcp1\u003c/em\u003e deletion on long-term survival during pressure overload, a separate cohort of mice was monitored for 14 weeks after osmotic minipump implantation (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). All mice in the Saline WT (n\u0026thinsp;=\u0026thinsp;9), Saline KO (n\u0026thinsp;=\u0026thinsp;10), and AngII/PE KO (n\u0026thinsp;=\u0026thinsp;9) groups had 100% survival throughout the observation period. In contrast, three deaths occurred in the AngII/PE WT group (n\u0026thinsp;=\u0026thinsp;12), one at 13 weeks and two at 14 weeks post-implantation of AngII/PE infusion pump (log-rank test, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056). For chronic pressure overload, we implemented a 4-week experimental protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This timepoint captures the period of active fibrotic remodeling characteristic of the AngII/PE model, during which interstitial collagen accumulation precedes the onset of overt systolic dysfunction. Mice were monitored weekly for body weight and general health assessment. At the experimental endpoint, cardiac function was reassessed by echocardiography, followed by euthanasia for heart tissue collection and morphometric analysis. Collectively, these results demonstrated successful generation of viable \u003cem\u003eCdcp1\u003c/em\u003e-KO mice with normal baseline growth and development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCdcp1\u003c/b\u003e \u003cb\u003edeletion attenuates cardiac hypertrophy and fibrosis after pressure overload\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of \u003cem\u003eCdcp1\u003c/em\u003e deletion on cardiac structure and function during pressure overload, we performed serial TTE at baseline and after 4 weeks of chronic AngII/PE infusion. At baseline, the left ventricular (LV) mass index (LVMI) was comparable across all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). AngII/PE infusion induced significant cardiac hypertrophy in WT mice, with increased LVMI compared to saline-treated WT control (3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 mg/g vs. 3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 mg/g, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This hypertrophic response was significantly attenuated in AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO mice compared to AngII/PE-treated WT (3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 mg/g vs. 3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 mg/g, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and \u003cem\u003eCdcp1\u003c/em\u003e deletion prevented the increase in LVMI from baseline observed in WT mice after pressure overload (-0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53 mg/g vs. 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 mg/g, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.027) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistent with the 4-week timepoint capturing active fibrotic remodeling prior to systolic dysfunction, LV ejection fraction (LVEF) and LV fractional shortening (LVFS) remained within normal ranges across all groups, though AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO mice showed trends toward higher values compared with AngII/PE-treated WT mice (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C-D\u003c/b\u003e). Histological analysis of Picrosirius red-stained heart sections revealed that AngII/PE treatment significantly increased collagen deposition in WT mice compared to saline controls, as expected (15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5% vs. 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). By contrast, this fibrotic response was markedly attenuated in \u003cem\u003eCdcp1\u003c/em\u003e-KO mice (9.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% vs. 15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), representing a 41% reduction in fibrotic burden (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Saline-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO and WT groups showed comparably low collagen-positive areas (7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% vs. 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%), indicating no fibrotic changes due to \u003cem\u003eCdcp1\u003c/em\u003e deletion alone. Collectively, these findings demonstrate that \u003cem\u003eCdcp1\u003c/em\u003e deletion attenuates pressure overload-induced cardiac hypertrophy and fibrosis, the structural precursors to heart failure progression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq identifies\u003c/b\u003e \u003cb\u003eCdcp1\u003c/b\u003e\u003cb\u003e-dependent fibrotic and inflammatory gene programs in the left ventricle\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo understand the molecular mechanisms underlying the effects of \u003cem\u003eCdcp1\u003c/em\u003e deletion, we performed RNA-seq using LV tissues from all experimental groups. Differential gene expression (DEG) analysis comparing AngII/PE-treated WT to saline-treated WT mice revealed 1,857 DEGs including 1,342 upregulated and 515 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). There was significant upregulation of cardiac stress markers (\u003cem\u003eNppb\u003c/em\u003e, \u003cem\u003eTnnt3\u003c/em\u003e), pro-fibrotic and ECM remodeling factors (\u003cem\u003eLox\u003c/em\u003e, \u003cem\u003ePostn\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eAdamts8\u003c/em\u003e), and inflammatory mediators (\u003cem\u003eCcl8\u003c/em\u003e, \u003cem\u003eGals3\u003c/em\u003e), which is consistent with the pathological remodeling typically observed in pressure overload. No transcriptional differences were found when comparing saline-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO and WT mice, suggesting that \u003cem\u003eCdcp1\u003c/em\u003e deletion alone has no effect on baseline cardiac gene programs (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). However, when we compared AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO with AngII/PE-treated WT mice, we found 783 DEGs including 424 downregulated and 359 upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). \u003cem\u003eCdcp1\u003c/em\u003e deletion in the context of pressure overload was associated with significant downregulation of pro-fibrotic factors (\u003cem\u003eCtgf\u003c/em\u003e, \u003cem\u003eLox\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e) and inflammatory mediators (\u003cem\u003eCcl7\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e, \u003cem\u003eIl21r\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e). Gene set enrichment analysis (GSEA) of GO Biological Process terms using a ranked gene list ordered by log₂ fold-change identified extracellular structure organization, regulation of inflammatory response, and leukocyte migration among the most significantly positively enriched pathways in AngII/PE-treated versus saline-treated WT mice, consistent with the observed cardiac remodeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). When the same analysis was applied to the AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO versus WT mice, extracellular matrix organization, external encapsulating structure organization, and immune-related terms were negatively enriched, while mitochondrial gene expression and respiration pathways were positively enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that CDCP1 may also be involved in regulating immune responses in pressure-overloaded hearts. Furthermore, AngII/PE treatment markedly increased expression of ECM genes (\u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eCol1a2\u003c/em\u003e, \u003cem\u003eCol3a1\u003c/em\u003e), fibroblast activation markers (\u003cem\u003eVim\u003c/em\u003e, \u003cem\u003eCtgf\u003c/em\u003e), matrix remodeling enzymes (\u003cem\u003eMmp2\u003c/em\u003e, \u003cem\u003eMmp14\u003c/em\u003e), inflammatory chemokines (\u003cem\u003eCcl7\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e), the cardiac-stress marker \u003cem\u003eTnnt3\u003c/em\u003e, and \u003cem\u003eCdcp1\u003c/em\u003e itself in WT heart LVs, whereas the increase in expression of each of these genes was significantly attenuated in \u003cem\u003eCdcp1\u003c/em\u003e-KO heart LVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSpatial Transcriptomics Reveals Region- and \u0026ldquo;Cell Type\u0026rdquo;-Specific Remodeling Suppressed by\u003c/b\u003e \u003cb\u003eCdcp1\u003c/b\u003e \u003cb\u003eKO in Pressure Overload\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the spatial distribution of transcriptional changes associated with \u003cem\u003eCdcp1\u003c/em\u003e deletion during pressure overload, we performed spatial transcriptomic profiling of cardiac sections from all four experimental groups (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Across all samples, we captured a total of 7,889 spatial barcoded transcriptomic spots (Saline_WT: 1,857; Saline_KO: 1,915; AngII/PE_WT: 2,193; AngII/PE_KO: 1,924), with median gene counts per spot ranging from 2,049 to 3,710. UMAP embedding calculated on the first 30 principal components showed distinct separation of spots according to sample of origin before integration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), reflecting systematic transcriptomic differences across the samples. Using a reference-guided deconvolution approach, we inferred the relative contributions of major cardiac cell types, across the myocardial sections, including cardiomyocytes, fibroblasts, endothelial cells, and immune cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Cardiomyocytes were the predominant cell type across all groups (67.2\u0026ndash;88.1%), followed by fibroblasts (5.2\u0026ndash;17.9%), endothelial cells (4.3\u0026ndash;16.2%), and T cells (0.5\u0026ndash;2.1%). Quantification of cell-type proportions suggested a consistent reduction in fibroblast abundance in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts compared with WT controls, observed both under saline conditions and following AngII/PE treatment. While AngII/PE-treated WT hearts exhibited a marked expansion of predicted fibroblasts (18%), this increase was entirely blunted in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts (6%) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e).\u003c/b\u003e Together, these findings indicate that \u003cem\u003eCdcp1\u003c/em\u003e deletion is associated with reduced fibroblast representation in the myocardium particularly in the context of pressure overload. Consistent with these compositional changes, spatial expression mapping of extracellular matrix\u0026ndash;associated genes revealed reduced fibrogenic signaling in \u003cem\u003eCdcp1\u003c/em\u003e-deficient hearts. Expression of \u003cem\u003eCol1a2, Fn1, Mmp2\u003c/em\u003e, and \u003cem\u003eLoxl2\u003c/em\u003e were prominently enriched in AngII/PE-treated WT hearts but substantially diminished in AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating suppression of ECM production and matrix remodeling transcriptional programs in the absence of \u003cem\u003eCdcp1\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further resolve cellular heterogeneity, we next examined subpopulation structure within major cardiac cell types. Deconvolution analysis identified multiple transcriptionally distinct subclusters of cardiomyocytes, fibroblasts, and endothelial cells, whose relative proportions differed across experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Marker gene analysis confirmed robust and cell-type\u0026ndash;specific expression patterns defining each subcluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Five distinct cardiomyocyte subtypes (CM2, CM4, CM6, CM7, and CM8) were identified across all experimental groups (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). Quantitative analysis revealed that AngII/PE-treated WT hearts exhibited a relative enrichment of the CM4 cardiomyocyte subtype, which was enriched for inflammatory response, extracellular matrix organization, collagen fibril organization, and wound healing pathways (\u003cb\u003eSupplementary Fig.\u0026nbsp;6A\u003c/b\u003e). In contrast, this cell type population was reduced in AngII/PE-treated \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts, whereas the CM6 subtype, which was enriched for muscle cell differentiation, myofibril assembly, cell-substrate adhesion, and integrin-mediated signaling pathways (\u003cb\u003eSupplementary Fig.\u0026nbsp;6B\u003c/b\u003e), was increased in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts compared with AngII/PE-treated WT (\u003cb\u003eSupplementary Fig.\u0026nbsp;4B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, deconvolution of fibroblast and endothelial compartments identified multiple transcriptionally distinct subtypes whose proportions differed across experimental conditions (\u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). Notably, all the three profibrotic fibroblast subtypes (FB5, FB8, and FB9) were expanded in AngII/PE-treated WT hearts but attenuated in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts, while endothelial subtypes showed more modest but consistent shifts in abundance (\u003cb\u003eSupplementary Fig.\u0026nbsp;4B\u003c/b\u003e). Among fibroblast subpopulations, FB5 showed the most prominent \u003cem\u003eCdcp1\u003c/em\u003e-dependent differences. Gene set enrichment analysis revealed that FB5 was enriched for biological processes related to response to growth factors, response to TGF-β, regulation of cell migration, and vasculature development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Notably, FB5 exhibited high expression of collagen and ECM-associated genes, consistent with a profibrotic fibroblast state \u003cb\u003e(Supplementary Fig.\u0026nbsp;4C)\u003c/b\u003e. This subpopulation was prominently expanded in AngII/PE-treated WT compared to \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts, linking the presence of \u003cem\u003eCdcp1\u003c/em\u003e expression to the emergence of collagen-producing fibroblast niches during pressure overload\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn summary\u003c/b\u003e, these analyses demonstrate \u003cem\u003eCdcp1\u003c/em\u003e deletion attenuates pathological cardiac remodeling, reduces histological fibrosis, and is associated with diminished fibroblast expansion and ECM gene expression. These results are consistent with our prior demonstration that \u003cem\u003eCdcp1\u003c/em\u003e contributes to cytokine driven expansion of cardiac fibroblasts.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e To determine whether CDCP1 directly influences human fibroblast fibrogenic programs, we next examined CDCP1-dependent extracellular matrix programs in human ventricular fibroblasts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCDCP1\u003c/b\u003e \u003cb\u003eregulates growth factor-induced extracellular matrix related gene expression and collagen deposition in human ventricular fibroblasts\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo directly test fibroblast fibrogenic responses, we examined the effects of \u003cem\u003eCDCP1\u003c/em\u003e knockdown in human ventricular fibroblasts (HVFs) exposed to profibrotic stimuli. HVFs were treated with platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-β (TGF-β), and angiotensin II, all of which are key signaling pathways enriched in FB5 fibroblasts \u003cem\u003ein vivo\u003c/em\u003e. Stimulation with individual fibrotic cues led to a significant induction of \u003cem\u003eCDCP1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting \u003cem\u003eCDCP1\u003c/em\u003e as a fibroblast-intrinsic, stress-responsive gene involved in profibrotic signaling pathways. Silencing of \u003cem\u003eCDCP1\u003c/em\u003e markedly attenuated the induction of extracellular matrix\u0026ndash;associated genes across all fibrotic stimuli, including \u003cem\u003eCOL1A1\u003c/em\u003e, \u003cem\u003eCTGF\u003c/em\u003e, and \u003cem\u003eLOX\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These genes represent core components of collagen production, matrix cross-linking, and fibroblast activation, consistent with the transcriptional programs enriched in FB5 fibroblasts identified in spatial transcriptomic analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the protein level, immunofluorescent staining of intact HVFs demonstrated a robust reduction in collagen I production following \u003cem\u003eCDCP1\u003c/em\u003e knockdown under profibrotic stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, analysis of decellularized extracellular matrix confirmed a corresponding decrease in collagen I deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating that \u003cem\u003eCDCP1\u003c/em\u003e regulates not only fibroblast gene expression but also synthesis and deposition of collagen I, the predominant component of fibrotic scar in the heart, by activated fibroblasts. Together, these findings establish CDCP1 as a fibroblast-intrinsic regulator of growth factor driven extracellular matrix gene expression and collagen deposition, providing a mechanistic link to the attenuated fibrotic remodeling seen in \u003cem\u003eCdcp1\u003c/em\u003e-deficient hearts.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we demonstrate that CDCP1 is a regulator of extracellular matrix production in cardiac fibroblasts. Global \u003cem\u003eCdcp1\u003c/em\u003e deletion attenuates pressure overload-induced cardiac hypertrophy, collagen deposition, suppresses ECM gene expression, and reduces pro-fibrotic fibroblast subpopulations, a finding that is supported by \u003cem\u003eCDCP1\u003c/em\u003e knockdown in human cardiac fibroblasts.\u003c/p\u003e \u003cp\u003eConsistent with prior GWAS findings linking lower \u003cem\u003eCDCP1\u003c/em\u003e expressions to improved cardiac function in patients with heart failure\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, we found that \u003cem\u003eCdcp1\u003c/em\u003e KO attenuated pressure overload-induced increases in LV mass, whereas LVEF and LVFS showed a trend toward improved systolic function. This is consistent with early fibrotic remodeling preceding systolic dysfunction\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e in this model.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eCdcp1\u003c/em\u003e KO mice exhibited significantly reduced collagen deposition compared to wild-type controls in a pressure overload model, with no evidence of baseline fibrosis in saline-treated hearts, indicating that \u003cem\u003eCDCP1\u003c/em\u003e is dispensable under physiological conditions but contributes to maladaptive remodeling during cardiac stress. Bulk RNA sequencing revealed that \u003cem\u003eCdcp1\u003c/em\u003e deletion suppressed the expression of a broad array of fibrosis-associated genes, including \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003ePostn\u003c/em\u003e, \u003cem\u003eLox\u003c/em\u003e, and \u003cem\u003eCtgf\u003c/em\u003e, as well as inflammatory chemokines such as \u003cem\u003eCcl7\u003c/em\u003e, \u003cem\u003eCcl12\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e. These results suggest that \u003cem\u003eCdcp1\u003c/em\u003e may act upstream of canonical fibrotic and inflammatory gene programs, potentially through modulation of fibroblast activation and ECM remodeling pathways.\u003c/p\u003e \u003cp\u003eMechanistically, our prior \u003cem\u003ein vitro\u003c/em\u003e studies demonstrated that \u003cem\u003eCDCP1\u003c/em\u003e is upregulated in human cardiac fibroblasts in response to PDGF-BB stimulation and that \u003cem\u003eCDCP1\u003c/em\u003e knockdown suppresses fibroblast proliferation via reduced AKT phosphorylation.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e The present \u003cem\u003ein vivo\u003c/em\u003e findings, specifically the reduced fibroblast abundance observed in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts by spatial transcriptomics, are consistent with this proliferation-dependent mechanism. PDGFRα is uniquely expressed by cardiac fibroblasts and is essential for their survival through PI3K signaling. PDGFRα loss causes approximately 50% reduction in resident cardiac fibroblasts within days.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Importantly, controlled reduction of PDGFRα\u003csup\u003e+\u003c/sup\u003e fibroblasts (60\u0026ndash;80%) has been shown to preserve cardiac function following AngII/PE-induced cardiac fibrosis,\u003csup\u003e19\u003c/sup\u003e supporting the therapeutic potential of targeting this pathway. Our prior data suggests that \u003cem\u003eCDCP1\u003c/em\u003e functions as a modulator of PDGF-AKT signaling in cardiac fibroblasts, and its deletion may attenuate sustained AKT activation required for fibroblast expansion and survival.\u003c/p\u003e \u003cp\u003eTo further define the spatial and cell-type\u0026ndash;specific impact of \u003cem\u003eCdcp1\u003c/em\u003e deletion, we leveraged spatial transcriptomics. Deconvolution analysis showed that \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts had reduced fibroblast abundance and altered spatial distribution under pressure overload. Importantly, \u003cem\u003eCdcp1\u003c/em\u003e deletion led to a marked reduction of the FB5 and FB8 fibroblast subtypes, which were associated with TGF-β signaling, growth factor responses, vascular remodeling, and migration. The transcriptional profile of FB5 \u0026ndash; enriched in ECM organization, growth factor signaling, and localized to interstitial and perivascular regions \u0026ndash; corresponds to the THBS4+/CILP\u0026thinsp;+\u0026thinsp;pro-fibrotic fibroblast populations recently characterized in pressure overload models.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e These populations, which express \u003cem\u003eThbs4\u003c/em\u003e, \u003cem\u003eCilp\u003c/em\u003e, \u003cem\u003ePostn\u003c/em\u003e, and \u003cem\u003eCthrc1\u003c/em\u003e, represent a pressure overload-specific fibroblast state distinct from classical α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts observed after myocardial infarction.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Our prior findings suggest that \u003cem\u003eCDCP1\u003c/em\u003e plays a role in the emergence or maintenance of these ECM-producing fibroblast populations through AKT-mediated survival signaling.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCardiomyocyte subpopulation analysis revealed more layers of \u003cem\u003eCdcp1\u003c/em\u003e-dependent remodeling. In WT hearts under pressure overload, the emergence of CM4 \u0026ndash; a pro-fibrotic cardiomyocyte subtype enriched in Wnt signaling, ECM remodeling, and inflammatory gene signatures \u0026ndash; was pronounced. However, this population was significantly reduced in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts. Instead, \u003cem\u003eCdcp1\u003c/em\u003e-deficient hearts displayed enrichment of CM6, a cardiomyocyte subtype characterized by signatures of muscle cell development, cell-substrate adhesion, and myofibril assembly, suggesting a potentially reparative or adaptive remodeling phenotype. The spatial co-localization of CM4 with FB5 clusters in WT hearts, (\u003cb\u003eSupplementary Figs.\u0026nbsp;4 and 5\u003c/b\u003e) and the coordinated reduction of both populations in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts, suggests that \u003cem\u003eCDCP1\u003c/em\u003e may regulate pathological fibroblast-cardiomyocyte crosstalk during pressure overload.\u003c/p\u003e \u003cp\u003eOur findings place \u003cem\u003eCDCP1\u003c/em\u003e within a growing class of transmembrane proteins validated as cardiac fibrosis targets. For instance, fibroblast activation protein (FAP) emerged as a promising therapeutic target. FAP-specific CAR T cells have been shown to reduce fibrosis and improve function in the AngII/PE model,\u003csup\u003e23\u003c/sup\u003e and more recently, \u003cem\u003ein vivo\u003c/em\u003e generation of transient FAP-targeted CAR T cells via CD5-targeted lipid nanoparticle (LNP)-mediated delivery of modified mRNA enabled transient, controllable intervention.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Depletion of αv integrin in PDGFRβ\u003csup\u003e+\u003c/sup\u003e cells confers protection against cardiac fibrosis, and small molecule inhibitors similarly attenuate established fibrosis via the shared SRC-PI3K/AKT pathway.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e IL-11 receptor (IL-11RA) KO mice are protected from cardiac fibrosis, and neutralizing antibodies demonstrate preclinical efficacy against it.\u003csup\u003e26\u003c/sup\u003e Like these targets, \u003cem\u003eCDCP1\u003c/em\u003e is a cell-surface transmembrane protein with limited cardiomyocyte expression, rendering it suitable for antibody-based therapeutic strategies currently under development in oncology.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eImportantly, \u003cem\u003eCDCP1\u003c/em\u003e may have context-dependent roles across different fibrotic pathways, cells and organs. In lung fibroblasts, \u003cem\u003eCDCP1\u003c/em\u003e knockdown enhanced TGF-β1-induced myofibroblast differentiation,\u003csup\u003e29\u003c/sup\u003e in contrast to our findings in cardiac fibroblasts, where \u003cem\u003eCDCP1\u003c/em\u003e promotes PDGF-driven proliferation and is necessary for TGF-β mediated ECM synthesis. This organ specific function warrants further mechanistic investigation.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. First, the use of a global Cdcp1 knockout mouse model precludes definitive attribution of the observed phenotypes to specific cell types; although convergent in vivo and in vitro data implicate fibroblasts, cell type\u0026ndash;specific deletion (e.g., fibroblast- or cardiomyocyte-restricted models) will be necessary to delineate causal mechanisms and intercellular contributions more precisely. Notably, however, the use of a global knockout may also enhance translational relevance, as a pharmacologic strategy targeting CDCP1 would likely exert systemic effects rather than cell type\u0026ndash;restricted modulation. Second, the AngII/phenylephrine infusion model recapitulates aspects of pressure overload\u0026ndash;induced remodeling but does not fully capture the heterogeneity of human heart failure etiologies, limiting direct translational generalizability. Third, the primary analyses were conducted at an early time point (4 weeks), when fibrotic remodeling precedes overt systolic dysfunction, and therefore do not establish long-term functional consequences or effects on advanced heart failure phenotypes. Fourth, spatial transcriptomic resolution is limited by spot size and reliance on computational deconvolution, which may obscure finer cellular heterogeneity and introduce inference bias in cell-type assignments. Fifth, while human ventricular fibroblast experiments support a conserved, fibroblast-intrinsic role for CDCP1, these reductionist systems do not fully recapitulate the multicellular myocardial environment or systemic influences present in vivo. Finally, although CDCP1 emerges as a potential therapeutic target, the safety, tissue specificity, and efficacy of CDCP1-directed interventions\u0026mdash;particularly given its context-dependent roles across organs\u0026mdash;remain to be established in translational and large-animal studies.\u003c/p\u003e \u003cp\u003eFrom a translational perspective, \u003cem\u003eCDCP1\u003c/em\u003e is a promising therapeutic target for cardiac fibrosis. As a transmembrane protein with an accessible extracellular domain, it is amenable to antibody-based therapies already in development for oncologic indications,\u003csup\u003e27,28\u003c/sup\u003e which could potentially be repurposed for this indication. Moreover, \u003cem\u003eCdcp1\u003c/em\u003e deletion is well-tolerated at baseline and elicits pathological phenotypes only under stress, suggesting a favorable therapeutic window. Human genetic data from the UK Biobank PheWAS link \u003cem\u003eCDCP1\u003c/em\u003e variants to heart failure mortality,\u003csup\u003e11\u003c/sup\u003e and conserved mechanisms in human cardiac fibroblasts reinforce its translational potential.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides direct \u003cem\u003ein vivo\u003c/em\u003e evidence that \u003cem\u003eCDCP1\u003c/em\u003e attenuates pressure overload-induced cardiac fibrosis. By integrating genetic, histological, and spatial transcriptomic approaches, we demonstrate that \u003cem\u003eCdcp1\u003c/em\u003e deletion suppresses pro-fibrotic THBS4\u003csup\u003e+\u003c/sup\u003e/CILP\u003csup\u003e+\u003c/sup\u003e-like fibroblast populations, attenuates pathological cardiomyocyte states, and preserves cardiac structure. Mechanistically, \u003cem\u003eCDCP1\u003c/em\u003e functions as an important mediator of multiple pro-fibrotic stimuli that drive fibroblast collagen synthesis. These insights provide a mechanistic basis for targeting \u003cem\u003eCDCP1\u003c/em\u003e to attenuate cardiac fibrosis and thus heart failure progression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData and Code Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bulk RNA sequencing data generated during this study are available in the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE296565. The spatial transcriptomics data is available under accession number GSE303631. All analysis scripts and computational pipelines used in this study are publicly available at https://github.com/irenemaring/CDCP1_PressureOverload. No custom algorithms were developed. All code for standardized analyses (Seurat, DESeq2,) is publicly accessible through GitHub or CRAN/Bioconductor repositories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest: \u003c/strong\u003eThe authors declare no conflicts of interest related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of Funding:\u003c/strong\u003e Rachad Ghazal is supported by the National Heart, Lung, and Blood Institute T32HL007111 grant. Min Wang is supported by the National Institute of General Medical Sciences T32GM008685 grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements: \u003c/strong\u003eWe acknowledge the Mayo Clinic Department of Comparative Medicine. We also thank the Mayo Clinic Spatial Multiomics Core for the probe-based 10x Genomics Visium CytAssist assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MW, DL, DJT, NLP; Methodology: RG, ANS, MW, IM, RH, TO, CET, DYL, AJ, HV, LJL, JM; Investigation: ANS, MW, LW, WS; Manuscript writing: RG, ANS, JJLC; Manuscript editing: CMR, TO, CW, DJB, GS, RW, DL, DJT, NLP\u003cbr clear=\"all\"\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHumeres C, Frangogiannis NG. 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While CDCP1 regulates cardiac fibroblast proliferation in vitro, it\u0026rsquo;s in vivo role in cardiac fibrosis remains unclear. Using a \u003cem\u003eCdcp1\u003c/em\u003e-knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload\u0026ndash;expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in \u003cem\u003eCdcp1\u003c/em\u003e-KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that \u003cem\u003eCDCP1\u003c/em\u003e knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target.\u003c/p\u003e","manuscriptTitle":"CDCP1 Deletion Protects Against Pressure Overload-Induced Cardiac Dysfunction and Fibrosis in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 22:57:21","doi":"10.21203/rs.3.rs-9236741/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"387d92e4-1810-48f9-a52c-c8974043ebc5","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-11T16:32:19+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-06T14:48:41+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-01T02:01:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"15","date":"2026-04-30T18:51:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-29T18:16:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-29T14:44:17+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67266194,"name":"Biological sciences/Genetics/Genomics/Medical genomics"},{"id":67266195,"name":"Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy"},{"id":67266196,"name":"Biological sciences/Molecular biology/Transcriptomics"}],"tags":[],"updatedAt":"2026-05-08T22:57:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 22:57:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9236741","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9236741","identity":"rs-9236741","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}