LMNA-Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-Derived iPSC Differentiation Support Cell Type and Lineage-Specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells

LMNA-Related DCM involves cardiomyocyte dysfunction resulting in heart failure or sudden death. It is caused by LMNA mutations encoding Lamin A/C defects involved in nuclear integrity and gene expression. To investigate possible mechanisms, we used iPSC derived from a LMNA c.357-2A>G Patient and her unaffected sister. We differentiated iPSC and conducted scRNA-seq for 4 Patient and 8 Control samples across seven time points from Day 0 to 30. Using a comprehensive bioinformatics workflow, we identified 110,521 high-quality cells and complex heterogeneity: ten main cell types, possible subtypes, and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes and Epicardium-Derived Cells (EPDC). By comparative analyses, we found differentially expressed genes (DEG) and enrichment supporting pathway dysregulation for ZNF genes and RNA polymerase II transcription in Pluripotent cells; BMP4 and TGF Beta/BMP signaling, sarcomere genes and cardiogenesis, CDH2 and EMT in Cardiomyocytes; LMNA and epigenetic regulation and DDIT4 and mTORC1 signaling in EPDC. In addition, top DEG included XIST, six imprinted genes, and gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our evidence supports disruption of epigenomic developmental programs in LMNA-Related DCM and also demonstrates current challenges of LMNA disease modeling using Patient-derived iPSC. scRNA-seq for 12 cell samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30 during iPSC differentiation to cardiomyocytes.

LMNA相关扩张型心肌病（LMNA-Related DCM）以心肌细胞功能障碍为核心病理表现，可导致心力衰竭或猝死。其致病机制为LMNA突变引发核纤层蛋白A/C（Lamin A/C）功能缺陷，而该蛋白参与维持细胞核完整性与基因表达调控。为探究该疾病的潜在发病机制，本研究使用了来自LMNA c.357-2A>G突变患者及其健康姐妹的诱导多能干细胞（induced pluripotent stem cell, iPSC）。我们对上述诱导多能干细胞进行定向分化，并对4例患者样本与8例对照样本开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），采样覆盖诱导分化第0天至第30天共7个时间节点。通过一套完整的生物信息学分析流程，我们共鉴定得到110521个高质量细胞，并揭示了复杂的细胞异质性：包含10种主要细胞类型、潜在细胞亚型，以及心脏祖细胞向心肌细胞、心外膜衍生细胞（Epicardium-Derived Cells, EPDC）分化的谱系分叉事件。通过比较分析，我们鉴定得到一系列差异表达基因（differentially expressed genes, DEG），并发现多条通路存在调控异常：在多能干细胞中，ZNF基因与RNA聚合酶II转录通路富集异常；在心肌细胞中，BMP4、TGF-β/BMP信号通路、肌节基因与心脏发生程序、CDH2与上皮间质转化（epithelial-mesenchymal transition, EMT）存在异常；在心外膜衍生细胞中，LMNA、表观遗传调控通路以及DDIT4与mTORC1信号通路存在异常。此外，排名靠前的差异表达基因包括XIST、6个印记基因，以及参与代谢、增殖与稳态维持的基因集。我们通过等位基因表达分析与蛋白质印迹（Western blot）实验验证了核纤层蛋白A/C单倍剂量不足现象。本研究结果证实LMNA相关扩张型心肌病中表观基因组发育程序遭到破坏，同时也揭示了利用患者来源诱导多能干细胞构建LMNA相关疾病模型所面临的现有挑战。本研究的单细胞RNA测序样本涵盖12份细胞样本（4份患者样本与8份对照样本），采集自诱导多能干细胞向心肌细胞分化过程中的7个时间节点：第0、2、4、9、16、19及30天。