背景:糖尿病心肌病(DCM)对健康构成了越来越大的威胁,糖尿病患者心力衰竭风险升高。了解DCM至关重要,成纤维细胞和内皮细胞在驱动心肌纤维化和导致心脏功能障碍中起关键作用。多模态单细胞谱分析的进展,例如scRNA-seq和scATAC-seq,为有针对性的治疗干预提供对DCM独特的细胞状态和分子景观的更深入的见解。
方法:使用CellRangerARCv2.0.1处理来自10xMultiome文库的单细胞RNA和ATAC数据。基因表达和ATAC数据进行Seurat和Signac过滤。鉴定了差异基因表达和可接近的染色质区域。转录因子活性用chromVAR估计,使用西塞罗计算顺式可访问性网络。将协同性连接与GeneHancer数据库进行比较。基因本体分析,生物过程评分,细胞间通讯分析,并进行基因-基序相关性以揭示复杂的分子变化。免疫荧光染色在石蜡包埋的组织上使用各种抗体来验证发现。
结果:本研究整合了从WT和DCM小鼠心脏获得的scRNA-seq和scATAC-seq数据,阐明整个糖尿病性心肌病进展过程中单细胞水平的分子变化。对整合数据进行稳健而准确的聚类分析,揭示了细胞比例的改变,显示内皮细胞和巨噬细胞减少,再加上DCM组的成纤维细胞和心肌细胞增加,表明纤维化和内皮损伤增强。染色质可达性分析揭示了细胞类型的独特模式,在心肌细胞中具有增强的转录活性。亚群分析强调了心肌细胞和成纤维细胞的明显变化,强调与脂肪酸代谢和心脏收缩有关的途径。以成纤维细胞为中心的沟通分析确定了与内皮细胞的相互作用,涉及VEGF受体。内皮细胞亚群表现出改变的基因表达,强调收缩和增长相关的途径。候选监管机构,包括Tcf21Arnt,Stat5a,Stat5b,被确认,表明它们在DCM发展中的关键作用。免疫荧光染色验证了细胞亚群的标记基因,证实PDK4、PPARγ和Tpm1是代谢模式改变的心肌细胞的标志物,激活的成纤维细胞和内皮细胞增殖受损。
结论:我们整合的scRNA-seq和scATAC-seq分析揭示了糖尿病心肌病中复杂的细胞状态和分子改变。确定的细胞类型特定的变化,转录因子,和标记基因提供了有价值的见解。该研究揭示了DCM的潜在治疗靶点。
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.
CONCLUSIONS: 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.