Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Sox9-coordinated cellular neighborhoods generate fibrosis.

Poorly regenerative organs deposit scar tissue to mend damage. Aggarwal et al. establish that transient Sox9 activity is necessary for early proximal tubule epithelial regeneration, while Trogisch et al. and Aggarwal et al. show that persistent Sox9 activity in epithelial and endothelial cells activates fibroblasts creating fibrotic microdomains in multiple organs.

Pirfenidone and nintedanib exert additive antifibrotic effects by the SPP1-AKT pathway in macrophages and fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted.

Targeting Interactions between Fibroblasts and Macrophages to Treat Cardiac Fibrosis.

Excessive extracellular matrix (ECM) deposition is a defining feature of cardiac fibrosis. Most notably, it is characterized by a significant change in the concentration and volume fraction of collagen I, a disproportionate deposition of collagen subtypes, and a disturbed ECM network arrangement, which directly affect the systolic and diastolic functions of the heart. Immune cells that reside within or infiltrate the myocardium, including macrophages, play important roles in fibroblast activation and consequent ECM remodeling. Through both direct and indirect connections to fibroblasts, monocyte-derived macrophages and resident cardiac macrophages play complex, bidirectional, regulatory roles in cardiac fibrosis. In this review, we discuss emerging interactions between fibroblasts and macrophages in physiology and pathologic conditions, providing insights for future research aimed at targeting macrophages to combat cardiac fibrosis.

A novel MT-ATP6 variant associated with complicated ataxia in two unrelated Italian patients: case report and functional studies.

BACKGROUND: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. METHODS: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. RESULTS: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. CONCLUSIONS: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.

Noncanonical WNT5A controls the activation of latent TGF-ß to drive fibroblast activation and tissue fibrosis.

Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

Lysosomal dysfunction and overload of nucleosides in thymidine phosphorylase deficiency of MNGIE.

Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Transcriptomic analysis identifies dysregulated pathways and therapeutic targets in PMM2-CDG.

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer.

Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Heterogeneity of primary and metastatic CAFs: From differential treatment outcomes to treatment opportunities (Review).

Compared with primary tumor sites, metastatic sites appear more resistant to treatments and respond differently to the treatment regimen. It may be due to the heterogeneity in the microenvironment between metastatic sites and primary tumors. Cancer­associated fibroblasts (CAFs) are widely present in the tumor stroma as key components of the tumor microenvironment. Primary tumor CAFs (pCAFs) and metastatic CAFs (mCAFs) are heterogeneous in terms of source, activation mode, markers and functional phenotypes. They can shape the tumor microenvironment according to organ, showing heterogeneity between primary tumors and metastases, which may affect the sensitivity of these sites to treatment. It was hypothesized that understanding the heterogeneity between pCAFs and mCAFs can provide a glimpse into the difference in treatment outcomes, providing new ideas for improving the rate of metastasis control in various cancers.

Downregulation of HMGCS2 mediated AECIIs lipid metabolic alteration promotes pulmonary fibrosis by activating fibroblasts.

BACKGROUND: Abnormal lipid metabolism has recently been reported as a crucial signature of idiopathic pulmonary fibrosis (IPF). However, the origin and biological function of the lipid and possible mechanisms of increased lipid content in the pathogenesis of IPF remains undetermined. METHODS: Oil-red staining and immunofluorescence analysis were used to detect lipid accumulation in mouse lung fibrosis frozen sections, Bleomycin-treated human type II alveolar epithelial cells (AECIIs) and lung fibroblast. Untargeted Lipid omics analysis was applied to investigate differential lipid species and identified LysoPC was utilized to treat human lung fibroblasts and mice. Microarray and single-cell RNA expression data sets identified lipid metabolism-related differentially expressed genes. Gain of function experiment was used to study the function of 3-hydroxy-3-methylglutaryl-Coa Synthase 2 (HMGCS2) in regulating AECIIs lipid metabolism. Mice with AECII-HMGCS2 high were established by intratracheally delivering HBAAV2/6-SFTPC- HMGCS2 adeno-associated virus. Western blot, Co-immunoprecipitation, immunofluorescence, site-directed mutation and flow cytometry were utilized to investigate the mechanisms of HMGCS2-mediated lipid metabolism in AECIIs. RESULTS: Injured AECIIs were the primary source of accumulated lipids in response to Bleomycin stimulation. LysoPCs released by injured AECIIs could activate lung fibroblasts, thus promoting the progression of pulmonary fibrosis. Mechanistically, HMGCS2 was decreased explicitly in AECIIs and ectopic expression of HMGCS2 in AECIIs using the AAV system significantly alleviated experimental mouse lung fibrosis progression via modulating lipid degradation in AECIIs through promoting CPT1A and CPT2 expression by interacting with PPARα. CONCLUSIONS: These data unveiled a novel etiological mechanism of HMGCS2-mediated AECII lipid metabolism in the genesis and development of pulmonary fibrosis and provided a novel target for clinical intervention.

Small-molecule targeting PKM2 provides a molecular basis of lactylation-dependent fibroblast-like synoviocytes proliferation inhibition against rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) play an important role in rheumatoid arthritis (RA)-related swelling and bone damage. Therefore, novel targets for RA therapy in FLS are urgently discovered for improving pathologic phenomenon, especially joint damage and dyskinesia. Here, we suggested that pyruvate kinase M2 (PKM2) in FLS represented a pharmacological target for RA treatment by antimalarial drug artemisinin (ART). We demonstrated that ART selectively inhibited human RA-FLS and rat collagen-induced arthritis (CIA)-FLS proliferation and migration without observed toxic effects. In particular, the identification of targets revealed that PKM2 played a crucial role as a primary regulator of the cell cycle, leading to the heightened proliferation of RA-FLS. ART exhibited a direct interaction with PKM2, resulting in an allosteric modulation that enhances the lactylation modification of PKM2. This interaction further promoted the binding of p300, ultimately preventing the nuclear translocation of PKM2 and inducing cell cycle arrest at the S phase. In vivo, ART obviously suppressed RA-mediated synovial hyperplasia, bone damage and inflammatory response to further improve motor behavior in CIA-rats. Taken together, these findings indicate that directing interventions towards PKM2 in FLS could offer a hopeful avenue for pharmaceutical treatments of RA through the regulation of cell cycle via PKM2 lactylation.

Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Single-cell insights: pioneering an integrated atlas of chromatin accessibility and transcriptomic landscapes in diabetic cardiomyopathy.

BACKGROUND: Diabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM's unique cell states and molecular landscape for targeted therapeutic interventions. METHODS: Single-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings. RESULTS: This study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation. CONCLUSION: Our integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.