目的·利用单细胞核RNA测序（single-nucleus RNA sequencing，snRNA-seq）技术，探究核纤层蛋白A/C基因第195位天冬酰胺替换为赖氨酸（Lamin A/C p.Asn195Lys，Lmna N195K）突变导致扩张型心肌病（dilated cardiomyopathy，DCM）小鼠模型心脏细胞的组成和分子特征变化，明确该突变对心脏多细胞生态系统的影响，并从细胞类型比例、基因表达谱、细胞间通信等方面系统描述其病理表型。方法·通过成簇规律间隔短回文重复序列及其相关蛋白9（clustered regularly interspaced short palindromic repeats-associated protein 9，CRISPR-Cas9）技术构建Lmna N195K纯合突变小鼠模型，并设立野生型对照。采用心脏超声和心电图评估心功能参数，通过苏木精-伊红（hematoxylin and eosin，HE）染色、天狼星红染色、小麦胚芽凝集素（wheat germ agglutinin，WGA）染色和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（terminal deoxynucleotidyl transferase dutp nick end labeling，TUNEL）凋亡检测分析心脏组织的病理变化。提取心脏组织细胞核进行snRNA-seq，使用Seurat（v4.4.0）进行细胞聚类、差异表达分析和细胞亚群鉴定，拟时序分析追踪细胞状态转变，CellChat（v1.6.1）推断细胞间通信网络。实时荧光定量逆转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction，RT-qPCR）及蛋白质印迹法（Western blotting，WB）分别检测纤维化标志物和LMNA蛋白表达水平。结果·Lmna N195K纯合突变小鼠表现出心腔扩张、收缩功能下降、严重心脏纤维化和高死亡率。组织学分析显示心肌细胞排列紊乱、纤维化面积显著增加、凋亡细胞增多，且突变组中LMNA蛋白表达降低。snRNA-seq共鉴定出9种心脏细胞类型，包括心肌细胞、内皮细胞、成纤维细胞等。突变组中代谢型心肌细胞（CM2）比例显著降低（P=0.005），收缩型心肌细胞（CM3）比例上升（P=0.004）；成纤维细胞几乎全部转化为促纤维化亚群FB4（P=0.001）；内皮细胞中高细胞外基质（extracellular matrix，ECM）分泌亚群EC3比例增加（P=0.007），提示内皮-间质转化（endothelial-to-mesenchymal transition，EndoMT）发生。差异表达分析发现多个与纤维化、能量代谢和心脏损伤相关的基因表达异常。细胞间通信分析显示，突变组中骨膜蛋白（periostin，POSTN）-整合素（integrin）、血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）-血管内皮生长因子受体1（vascular endothelial growth factor receptor 1，VEGFR1）以及层粘连蛋白亚基α2（laminin subunit alpha 2，LAMA2）-整合素（integrin）等受体－配体通路显著激活。结论·Lmna N195K突变引起心脏多细胞生态系统紊乱，主要表现为心肌细胞亚群比例失衡（代谢型减少、收缩型增加）、成纤维细胞向促纤维化表型转化、内皮细胞发生EndoMT并增强ECM分泌能力。这些变化通过异常的细胞间通信网络进一步协同促进心脏纤维化和病理性重塑，最终导致DCM的发生与发展。该研究从单细胞水平系统提示了Lmna N195K突变对心脏细胞组成和功能的影响，为理解该突变致DCM的细胞基础提供了重要数据支持。

Objective ·To investigate the cardiac cellular composition and molecular alterations in a dilated cardiomyopathy (DCM) mouse model carrying the Lamin A/C p.Asn195Lys (Lmna N195K) mutation, single-nucleus RNA sequencing (snRNA-seq) was employed to elucidate the mutation′s impact on the cardiac multicellular ecosystem. The pathological phenotypes were systematically characterized in terms of cell type proportions, gene expression profiles, and intercellular communication networks. Methods ·A homozygous Lmna N195K mutant mouse model was generated using clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) technology, with wild-type littermates serving as controls. Cardiac function was evaluated by echocardiography and electrocardiography, while histological analyses, including hematoxylin and eosin (HE) staining, Sirius Red staining, wheat germ agglutinin (WGA) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed to assess tissue pathology. Nuclei were isolated from cardiac tissue for snRNA-seq, and Seurat (v4.4.0) was applied for cell clustering, differential expression analysis, and subpopulation identification. Pseudotime analysis was used to trace cell state transitions, and CellChat (v1.6.1) was employed to infer intercellular communication networks. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting (WB) were conducted to assess the expression of fibrosis markers and LMNA proteins, respectively. Results ·Homozygous Lmna N195K mutant mice exhibited chamber dilation, impaired systolic function, severe cardiac fibrosis, and high mortality. Histological analysis revealed disorganized cardiomyocyte alignment, significantly increased fibrotic area, elevated apoptosis, and reduced LMNA protein expression in the mutant group. SnRNA-seq identified nine major cardiac cell types, including cardiomyocytes, endothelial cells, and fibroblasts. In the mutant group, the proportion of metabolically active cardiomyocytes (CM2) was markedly decreased (P=0.005), while contractile cardiomyocytes (CM3) were increased (P=0.004). Nearly all fibroblasts were converted into a profibrotic subpopulation FB4 (P=0.001). Among endothelial cells, the extracellular matrix (ECM)-secreting subpopulation EC3 was significantly expanded (P=0.007), suggesting the occurrence of endothelial-to-mesenchymal transition (EndoMT). Differential expression analysis revealed dysregulation of genes related to fibrosis, energy metabolism, and cardiac injury. Intercellular communication analysis indicated significant activation of receptor-ligand signaling pathways, including periostin(POSTN)-integrin, vascular endothelial growth factor A(VEGFA)-vascular endothelial growth factor receptor 1(VEGFR1), and Laminin Subunit Alpha 2(LAMA2)-Integrin signaling pathways. Conclusion ·The Lmna N195K mutation disrupts the cardiac multicellular ecosystem, characterized by cardiomyocyte subpopulation imbalance (decreased metabolic cells and increased contractile cells), fibroblast conversion to profibrotic phenotypes, and endothelial EndoMT with enhanced ECM secretion. These alterations, reinforced by aberrant intercellular communication networks, collectively drive fibrosis and pathological remodeling, ultimately leading to dilated cardiomyopathy. This study reveals the impact of Lmna N195K mutation on cardiac cell composition and function at the single cell level, offering critical insights into the cellular basis of LMNA related DCM.