基于单细胞核RNA测序的Lmna N195K突变心肌病小鼠心脏特征分析

doi:10.3969/j.issn.1674-8115.2026.03.002

摘要/Abstract

摘要：

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。

关键词: Lmna N195K突变, 扩张型心肌病, 单细胞核测序, 差异表达分析, 受体－配体信号网络

Abstract:

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.

Key words: Lmna N195K mutation, dilated cardiomyopathy, single-nucleus RNA sequencing, differential expression analysis, receptor-ligand signaling network

中图分类号:

R542.2

王俊骁, 王雨泽, 张沙沙, 袁雨, 张冰, 李若谷. 基于单细胞核RNA测序的Lmna N195K突变心肌病小鼠心脏特征分析[J]. 上海交通大学学报（医学版）, 2026, 46(3): 275-290.

Wang Junxiao, Wang Yuze, Zhang Shasha, Yuan Yu, Zhang Bing, Li Ruogu. Analysis of cardiac features in Lmna N195K mutant mice using single-nucleus RNA sequencing[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(3): 275-290.

图/表 9

图1 LMNA p.N195K 基因敲入小鼠的构建及心脏功能表征Note： A. Schematic illustration of the CRISPR-Cas9 strategy utilized to generate Lmna p.N195K knock-in mice. The wild-type allele (ENSMUST0000003689.13) and the targeted allele containing the human p.N195K mutation in exon 3 are shown. B. Body weight curves of WT, N195K heterozygous (het), and homozygous (homo) mice at the age of 3‒4 months. C. Kaplan-Meier survival curves of WT, N195K het, and homo mice (log-rank test, P<0.001). D. Genotyping of Lmna N195K mutant mice (left: WT; right: Lmna N195K mutant); sequence validation of the corresponding alleles is shown below. E/F. Representative electrocardiograms and quantitative parameters (PR, QRS duration, QTc) in WT, N195K het, and homo mice at 2 (E) and 3 (F) months of age (x±s). G/H. Representative echocardiographic images and cardiac parameters, including EF, LVID; d, LVID; s, LVPW; d, and LVPW; s, in WT, N195K het, and homo mice at 2 (G) and 3 (H) months of age.

Fig 1 Generation of LMNA p.N195K knock-in mice and characterization of cardiac function

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR

Gene	Forward primer sequence (5′→3′)	Reverse primer sequence (5′→3′)
Anp	AGCTGGATCTTCGTAGGCTC	GCCGGTAGAAGATGAGGTCA
Bnp	GAGGTCACTCCTATCCTCTGG	GCCATTTCCTCCGACTTTTCTC
Myh6	CAAGCTCACTTGAAGGACACC	CACGATGGCGATGTTCTC
Myh7	AACCAGACGGCACTGAAGAG	TGCCCACTTTGACTCTAGGATG
Col1a1	GCTCCTCTTAGGGGCCACT	CCACGTCTCACCATTGGGG
Col1a2	GTAACTTCGTGCCTAGCAACA	CCTTTGTCAGAATACTGAGCAGC
Col2a1	GGGAATGTCCTCTGCGATGAC	GAAGGGGATCTCGGGGTTG
Col4a1	CTGGCACAAAAGGGACGAG	ACGTGGCCGAGAATTTCACC
Col4a2	GACCGAGTGCGGTTCAAAG	CGCAGGGCACATCCAACTT
Tgfb1	CTCCCGTGGCTTCTAGTGC	GCCTTAGTTTGGACAGGATCTG
Tgfb2	TCGACATGGATCAGTTTATGCG	CCCTGGTACTGTTGTAGATGGA
Tgfb3	CAGGCCAGGGTAGTCAGAG	ATTTCCAGCCTAGATCCTGCC

表2 基因型鉴定所用引物序列

Tab 2 Primer sequences used for genotyping

Primer name	Primer sequence
ms-lmna195-f(WT)	CTGAGAACAGGCTACAGACGC
h-lmna195-f(Lmna N195K)	GAGAACAGGCTGCAGACCA
lmna195-r(WT/ Lmna N195K)	GCTCTCAAACTCTCGCTGCT
LMNA195-tF1(Sanger)	GTGCCATGGGTAGAATGTGGGT
LMNA195-tR1(Sanger)	GAAAGGGTTAAGGGGAGACACT

图2 Lmna N195K 小鼠心脏组织病理学及分子改变Note： A. Gross morphology and HE staining of cardiac tissues (Upper panel: gross morphology; lower panel: HE staining. Scale bar: 5 mm for gross morphology, 1 mm for HE staining). B. WGA staining of cardiomyocyte membranes. Scale bar: 10 μm. C. Quantification of cardiomyocyte cross-sectional area from panel (B) (n≥600 cells). D. Sirius red staining for assessment of cardiac fibrosis. Scale bar: 100 μm. E. Quantification of fibrotic area from panel (D). F. TUNEL/DAPI/α-sarcomeric actin co-staining to identify apoptotic cardiomyocytes. Scale bar: 10 μm (20×), 5 μm (60×). G. Apoptotic cardiomyocytes (TUNEL+ CM) from panel (F). H. Immunoblot analysis of Lamin A/C protein in cardiac tissue; β-actin served as the loading control. I. Densitometric quantification of Lamin A/C protein relative to β-actin. J. Expression of fibrosis-related genes (Col1a1, Col1a2, Col2a1, Col4a1, Col4a2, Tgfb1, Tgfb2, Tgfb3) evaluated by quantitative PCR. K. Expression of heart failure markers (Anp, Bnp, Myh6, Myh7) assessed by quantitative PCR. Col1a1—Collagen, type I, alpha 1; Tgfb1-Transforming growth factor, beta 1.

Fig 2 Histopathological and molecular alterations in the hearts of Lmna N195K mice

图3 单细胞核RNA测序总体分析Note： A. UMAP plots of annotated cardiac cell nuclei from WT and Lmna N195K homozygous mice (cell clusters: 1-cardiomyocytes; 2-endothelial cells; 3-fibroblasts; 4-dendritic cells; 5-macrophages; 6-neurons activated; 7-neurons; 8-B cells; 9-T cells; WT: 10 036 nuclei; Lmna N195K homo: 14 279 nuclei). B. Volcano plot of differentially expressed genes (DEGs) between Lmna N195K homozygous and WT groups (red dots: upregulated DEGs; blue dots: downregulated DEGs). C. Bubble plot of DEGs across distinct cell types. D. Pie charts showing changes in the proportions of different cell types between WT and Lmna N195K homo groups (WT: 10 036 nuclei; Lmna N195K homo: 14 279 nuclei).

Fig 3 Global analysis of single-nucleus RNA sequencing (snRNA-seq)

图4 单核RNA测序提示 Lmna N195K 纯合突变导致的心肌细胞表达重塑与状态转变Note： A. UMAP plots of annotated cardiomyocyte nuclei (nuclei: 22 020) from WT and Lmna N195K homozygous mice. B. Spearman correlation analysis of all cardiomyocytes, showing expression correlations and transcriptional similarities among different subpopulations. C. Thirteen cardiomyocyte subclusters integrated (or categorized) into three major groups (CM1, CM2, and CM3), with proportions compared between WT and N195K homozygous groups, demonstrating LMNA mutation-induced shifts in cardiomyocyte composition. D. Bubble plot and GO enrichment analysis identifying significantly enriched biological processes and pathways in cardiomyocytes from LMNA mutant mice. E. Volcano plot of differentially expressed genes in cardiomyocytes between N195K homozygous and WT mice (red dots: upregulated genes; blue dots: downregulated genes). F/G. Pseudotime trajectory analysis of cardiomyocytes in WT and N195K homozygous groups, depicting transcriptional evolution under pathological conditions and potential cell fate transitions.

Fig 4 Single-nucleus RNA sequencing reveals transcriptional remodeling and state transitions of cardiomyocytes induced by the Lmna N195K homozygous mutation

图5 基于单核RNA测序的 Lmna N195K 突变导致心脏成纤维细胞状态改变及功能重塑分析Note： A. UMAP plots of annotated cardiac fibroblast nuclei from WT and Lmna N195K homozygous mice. B. Spearman correlation analysis of all cardiac fibroblasts showing expression correlations and transcriptional similarities among different subpopulations. C. Eleven cardiac fibroblast subclusters integrated (or categorized) into four major groups (FB1, FB2, FB3, and FB4), demonstrating LMNA mutation-induced shifts in fibroblast composition. D. GO enrichment analysis identifying significantly enriched biological processes and pathways in cardiac fibroblasts from LMNA mutant mice. E. Volcano plot of differentially expressed genes in cardiac fibroblasts between N195K homozygous and WT mice. F/G. Pseudotime trajectory analysis of cardiac fibroblasts in WT and N195K homozygous groups, depicting transcriptional evolution under pathological conditions and potential cell fate transitions.

Fig 5 Analysis of state alteration and functional remodeling of cardiac fibroblasts induced by Lmna N195K mutation based on snRNA-seq

图6 基于单核RNA测序的 Lmna N195K 突变致心脏内皮细胞状态改变及功能重塑分析Note： A. UMAP plots of annotated cardiac endothelial cell nuclei from WT and Lmna N195K homozygous mice. B. Spearman correlation analysis of all cardiac endothelial cells, showing expression correlations and transcriptional similarities among different subpopulations. C. Integration of 11 cardiac endothelial cell subclusters into five major groups (EC1, EC2, EC3, EC4, EC5) demonstrating LMNA mutation-induced alterations in endothelial cell composition. D. GO enrichment analysis identifying significantly enriched biological processes and pathways in cardiac endothelial cells from LMNA mutant mice. E. Volcano plot of differentially expressed genes in cardiac endothelial cells between N195K homozygous and WT mice. F/G. Pseudotime trajectory analysis of cardiac endothelial cells in WT and N195K homozygous groups, illustrating transcriptional evolution under pathological conditions and potential cell fate transitions.

Fig 6 Analysis of state alteration and functional remodeling of cardiac endothelial cells induced by Lmna N195K mutation based on snRNA-seq

图7 WT和 Lmna N195K 纯合突变型小鼠心脏信号通路的受体－配体联系网络Note： A. Bubble plot of receptor-ligand interactions in cardiac tissue in the WT group. B. Circos plot of PTPRM-related receptor-ligand interactions in cardiac tissue in the WT group. C. Bubble plot of receptor-ligand interactions in cardiac tissue in the Lmna N195K homozygous group. D—J. Circos plots of receptor-ligand interactions that were highly expressed in the Lmna N195K homozygous group.

Fig7 Receptor-ligand interaction networks in cardiac signaling pathways of WT and Lmna N195K homozygous mice

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