{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:43:42Z","timestamp":1785426222338,"version":"3.56.0"},"reference-count":43,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T00:00:00Z","timestamp":1781827200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004281","name":"National Science Centre","doi-asserted-by":"publisher","award":["2023\/51\/D\/NZ7\/00609"],"award-info":[{"award-number":["2023\/51\/D\/NZ7\/00609"]}],"id":[{"id":"10.13039\/501100004281","id-type":"DOI","asserted-by":"publisher"}]},{"award":["2023\/51\/D\/NZ7\/00609"],"award-info":[{"award-number":["2023\/51\/D\/NZ7\/00609"]}],"id":[{"id":"https:\/\/ror.org\/03ha2q922","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Autoimmune myocarditis frequently progresses to inflammatory cardiomyopathy through dysregulated immune\u2013stromal interactions. This study employs single-nuclei RNA-sequencing (snRNA-seq) to profile 46,233 cardiac nuclei from the experimental autoimmune myocarditis (EAM) mouse model at four timepoints: day 0 (healthy), day 14 (inflammation), day 21 (acute inflammation), and day 40 (late cardiac remodelling). Single-nuclei RNA profiling identified 18 transcriptionally distinct cell populations. Global cell\u2013cell communication analysis revealed a dramatic peak of intercellular signalling at day 14 (5907 interactions), with fibroblast subpopulations and macrophages as dominant hubs, followed by partial resolution at day 21 (2264 interactions) and renewed remodelling at day 40 (4862 interactions). Subclustering of the macrophage compartment identified five subpopulations: Mac-TLF, Mac-MHCII, Mac-rMHCII, Mac-ResL, and Classical Monocytes. Tissue-resident macrophages (Mac-TLF, CCR2-) dominated at healthy state (~55%) but were rapidly depleted at day 14, coinciding with a dramatic influx of recruited CCR2+ macrophages (Mac-rMHCII), which expanded to over 70% of the compartment and maintained dominance through day 40. At inflammation (day 14), the expanded Mac-rMHCII subpopulation displayed a strongly pro-inflammatory signature (Il1b, Stat2, Parp14, Apoe), and the overall macrophage compartment was enriched for cytokine response, Fc-gamma receptor, and Notch signalling pathways, while downregulating homeostatic and mitochondrial metabolic programmes, potentially contributing to impaired efferocytosis and cardiomyocyte dysfunction. Macrophage-centred communication networks expanded markedly at day 14 (1047 interactions), with resting fibroblasts (FB-R) as the primary signalling partner, driving pro-inflammatory stromal activation marked by upregulation of Ccl2, Ccl7, and Csf2. Intra-macrophage subcluster communication also intensified at this timepoint (447 interactions). These findings delineate the temporal and functional heterogeneity of cardiac macrophages during EAM progression and identify key immune\u2013stromal interactions driving pathological cardiac remodelling. The coexistence of pro-inflammatory and transitional reparative macrophage subsets highlights the limitations of broad immunosuppression and supports precision strategies targeting CCR2-mediated recruitment, the SPP1 signalling axis, and macrophage\u2013fibroblast crosstalk as therapeutic avenues in myocarditis and its progression.<\/jats:p>","DOI":"10.3390\/cells15121110","type":"journal-article","created":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T09:18:43Z","timestamp":1781860723000},"page":"1110","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cardiac Macrophages Exhibit Dynamic Heterogeneity and Functional Specialization During Experimental Autoimmune Myocarditis"],"prefix":"10.3390","volume":"15","author":[{"given":"Monika","family":"Stefanska","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, 60-637 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0602-0411","authenticated-orcid":false,"given":"Marta","family":"Kot","sequence":"additional","affiliation":[{"name":"Department of Transplantation, Faculty of Medicine, Institute of Paediatrics, Jagiellonian University Medical College, 30-663 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-1969-5242","authenticated-orcid":false,"given":"Damian","family":"Koterba","sequence":"additional","affiliation":[{"name":"Department of Computer Science, Faculty of Computer Science and Mathematics, Cracow University of Technology, 31-155 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1208-3291","authenticated-orcid":false,"given":"Joanna","family":"Zeyland","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, 60-637 Poznan, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Sinagra, G., Merlo, M., and Pinamonti, B. (2019). Historical Terminology, Classifications, and Present Definition of DCM. Dilated Cardiomyopathy: From Genetics to Clinical Management, Springer.","DOI":"10.1007\/978-3-030-13864-6"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Campodonico, J., Lauri, C., Pezzuto, B., Agostoni, P., and Vignati, C. (2025). Evolution in the Diagnosis and Treatment of Myocarditis in Recent Years: State of the Art. J. Clin. Med., 14.","DOI":"10.3390\/jcm14217661"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Uccello, G., Bonacchi, G., Rossi, V.A., Montrasio, G., and Beltrami, M. (2023). Myocarditis and Chronic Inflammatory Cardiomyopathy, from Acute Inflammation to Chronic Inflammatory Damage: An Update on Pathophysiology and Diagnosis. J. Clin. Med., 13.","DOI":"10.3390\/jcm13010150"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Lasica, R., Djukanovic, L., Savic, L., Krljanac, G., Zdravkovic, M., Ristic, M., Lasica, A., Asanin, M., and Ristic, A. (2023). Update on Myocarditis: From Etiology and Clinical Picture to Modern Diagnostics and Methods of Treatment. Diagnostics, 13.","DOI":"10.20944\/preprints202309.0327.v1"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e007405","DOI":"10.1161\/CIRCHEARTFAILURE.120.007405","article-title":"Management of Acute Myocarditis and Chronic Inflammatory Cardiomyopathy: An Expert Consensus Document","volume":"13","author":"Ammirati","year":"2020","journal-title":"Circ. Heart Fail."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Tkacz, K., Rolski, F., Stefa\u0144ska, M., W\u0119glarczyk, K., Szatanek, R., Siedlar, M., Kania, G., and B\u0142yszczuk, P. (2024). TGF-\u03b2 Signalling Regulates Cytokine Production in Inflammatory Cardiac Macrophages during Experimental Autoimmune Myocarditis. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25115579"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.immuni.2013.11.019","article-title":"Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation","volume":"40","author":"Epelman","year":"2014","journal-title":"Immunity"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1016\/j.celrep.2019.06.007","article-title":"The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis","volume":"28","author":"Hou","year":"2019","journal-title":"Cell Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1038\/s41590-018-0272-2","article-title":"Self-Renewing Resident Cardiac Macrophages Limit Adverse Remodeling Following Myocardial Infarction","volume":"20","author":"Dick","year":"2019","journal-title":"Nat. Immunol."},{"key":"ref_10","first-page":"S0828282X2600142X","article-title":"Cardiac Macrophages across Space and Time: Roles in Homeostasis, Disease, and Remodeling","volume":"2026","author":"Shrestha","year":"2026","journal-title":"Can. J. Cardiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1016\/j.cell.2017.03.050","article-title":"Macrophages Facilitate Electrical Conduction in the Heart","volume":"169","author":"Hulsmans","year":"2017","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.cell.2020.08.031","article-title":"A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart","volume":"183","author":"Santiago","year":"2020","journal-title":"Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1522","DOI":"10.1002\/eji.201847583","article-title":"Sca-1+ Cardiac Fibroblasts Promote Development of Heart Failure","volume":"48","author":"Chen","year":"2018","journal-title":"Eur. J. Immunol."},{"key":"ref_14","first-page":"102344","article-title":"Elucidating Cardiac Fibroblasts Heterogeneity and Activation during Experimental Autoimmune Myocarditis Using Spatial Transcriptomics","volume":"44","author":"Sarad","year":"2025","journal-title":"Biochem. Biophys. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/s13062-025-00621-2","article-title":"Macrophage-Derived SPP1 Exacerbate Myocardial Injury by Interacting with Fibroblasts in Viral Myocarditis","volume":"20","author":"Duan","year":"2025","journal-title":"Biol. Direct"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Chen, G., Jiang, H., Yao, Y., Tao, Z., Chen, W., Huang, F., and Chen, X. (2022). Macrophage, a Potential Targeted Therapeutic Immune Cell for Cardiomyopathy. Front. Cell Dev. Biol., 10.","DOI":"10.3389\/fcell.2022.908790"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e132","DOI":"10.1002\/cpz1.132","article-title":"Isolation of Nuclei from Mammalian Cells and Tissues for Single-Nucleus Molecular Profiling","volume":"1","author":"Nadelmann","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s13059-017-1382-0","article-title":"SCANPY: Large-Scale Single-Cell Gene Expression Data Analysis","volume":"19","author":"Wolf","year":"2018","journal-title":"Genome Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1038\/s41587-023-01733-8","article-title":"The Scverse Project Provides a Computational Ecosystem for Single-Cell Omics Data Analysis","volume":"41","author":"Virshup","year":"2023","journal-title":"Nat. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.cels.2019.03.003","article-title":"DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors","volume":"8","author":"McGinnis","year":"2019","journal-title":"Cell Syst."},{"key":"ref_21","unstructured":"Koterba, D. (2026, May 04). Doubletfinder-Py. Available online: https:\/\/github.com\/dam2452\/doubletfinder-py."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1016\/j.cell.2019.05.031","article-title":"Comprehensive Integration of Single-Cell Data","volume":"177","author":"Stuart","year":"2019","journal-title":"Cell"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2264","DOI":"10.1038\/s41592-025-02808-x","article-title":"Deep Generative Modeling of Sample-Level Heterogeneity in Single-Cell Genomics","volume":"22","author":"Boyeau","year":"2025","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5233","DOI":"10.1038\/s41598-019-41695-z","article-title":"From Louvain to Leiden: Guaranteeing Well-Connected Communities","volume":"9","author":"Traag","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5692","DOI":"10.1038\/s41467-021-25960-2","article-title":"Confronting False Discoveries in Single-Cell Differential Expression","volume":"12","author":"Squair","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"btad547","DOI":"10.1093\/bioinformatics\/btad547","article-title":"PyDESeq2: A Python Package for Bulk RNA-Seq Differential Expression Analysis","volume":"39","author":"Muzellec","year":"2023","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"btac757","DOI":"10.1093\/bioinformatics\/btac757","article-title":"GSEApy: A Comprehensive Package for Performing Gene Set Enrichment Analysis in Python","volume":"39","author":"Fang","year":"2023","journal-title":"Bioinformatics"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"The Gene Ontology Consortium, Aleksander, S.A., Balhoff, J., Carbon, S., Cherry, J.M., Drabkin, H.J., Ebert, D., Feuermann, M., Gaudet, P., and Harris, N.L. (2023). The Gene Ontology Knowledgebase in 2023. Genetics, 224, iyad031.","DOI":"10.1093\/genetics\/iyad031"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1613","DOI":"10.1038\/s41556-024-01469-w","article-title":"LIANA+ Provides an All-in-One Framework for Cell\u2013Cell Communication Inference","volume":"26","author":"Dimitrov","year":"2024","journal-title":"Nat. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Uhlig, M., Billig, S., Wienhold, J., and Schumacher, D. (2025). Pro-Fibrotic Macrophage Subtypes: SPP1+ Macrophages as a Key Player and Therapeutic Target in Cardiac Fibrosis?. Cells, 14.","DOI":"10.3390\/cells14050345"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2777","DOI":"10.1093\/cvr\/cvaf209","article-title":"Whole-Heart 3D Reconstruction of Mouse CVB3 Myocarditis Reveals Spatial and Transcriptomic Heterogeneity of Immune Foci","volume":"121","author":"Daoud","year":"2025","journal-title":"Cardiovasc. Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Barcena, M.L., Jeuthe, S., Niehues, M.H., Pozdniakova, S., Haritonow, N., K\u00fchl, A.A., Messroghli, D.R., and Regitz-Zagrosek, V. (2021). Sex-Specific Differences of the Inflammatory State in Experimental Autoimmune Myocarditis. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.686384"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.cpcardiol.2012.07.003","article-title":"Sex and Gender Differences in Myocarditis and Dilated Cardiomyopathy","volume":"38","author":"Fairweather","year":"2013","journal-title":"Curr. Probl. Cardiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.ymeth.2006.07.009","article-title":"Coxsackievirus-Induced Myocarditis in Mice: A Model of Autoimmune Disease for Studying Immunotoxicity","volume":"41","author":"Fairweather","year":"2007","journal-title":"Methods"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Enright, S., and Werstuck, G.H. (2024). Investigating the Effects of Sex Hormones on Macrophage Polarization. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25020951"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Trimarchi, G., Teresi, L., Licordari, R., Pingitore, A., Pizzino, F., Grimaldi, P., Calabr\u00f2, D., Liotta, P., Micari, A., and De Gregorio, C. (2024). Transient Left Ventricular Dysfunction from Cardiomyopathies to Myocardial Viability: When and Why Cardiac Function Recovers. Biomedicines, 12.","DOI":"10.3390\/biomedicines12051051"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"B\u0142yszczuk, P. (2019). Myocarditis in Humans and in Experimental Animal Models. Front. Cardiovasc. Med., 6.","DOI":"10.3389\/fcvm.2019.00064"},{"key":"ref_38","first-page":"CD004471","article-title":"Corticosteroids for Viral Myocarditis","volume":"2021","author":"Chen","year":"2013","journal-title":"Cochrane Database Syst. Rev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/s13052-023-01423-w","article-title":"Corticosteroid in Anti-Inflammatory Treatment of Pediatric Acute Myocarditis: A Systematic Review and Meta-Analysis","volume":"49","author":"Yao","year":"2023","journal-title":"Ital. J. Pediatr."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"549","DOI":"10.1016\/j.molimm.2011.10.002","article-title":"Dexamethasone Increases ROS Production and T Cell Suppressive Capacity by Anti-Inflammatory Macrophages","volume":"49","author":"Kraaij","year":"2011","journal-title":"Mol. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1995","DOI":"10.1093\/eurheartj\/ehp249","article-title":"Randomized Study on the Efficacy of Immunosuppressive Therapy in Patients with Virus-Negative Inflammatory Cardiomyopathy: The TIMIC Study","volume":"30","author":"Frustaci","year":"2009","journal-title":"Eur. Heart J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"102710","DOI":"10.1016\/j.autrev.2020.102710","article-title":"Efficacy of Immunosuppressive Therapy in Myocarditis: A 30-Year Systematic Review and Meta Analysis","volume":"20","author":"Cheng","year":"2021","journal-title":"Autoimmun. Rev."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1038\/s41569-020-00435-x","article-title":"Myocarditis and Inflammatory Cardiomyopathy: Current Evidence and Future Directions","volume":"18","author":"Ammirati","year":"2021","journal-title":"Nat. Rev. Cardiol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/12\/1110\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T06:45:56Z","timestamp":1782197156000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/12\/1110"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,19]]},"references-count":43,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["cells15121110"],"URL":"https:\/\/doi.org\/10.3390\/cells15121110","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,19]]}}}