TEAD1 trapping by the Q353R-Lamin A/C causes dilated cardiomyopathy

Abstract

Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R-LMNA-related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA-related DCM.

Fig. 1.. Immature intracellular structure of Lmna^Q353R/WT mice.

(A) Family tree of the DCM cohort with the LMNA mutation (p.Q353R). Shapes filled with black color indicate the patients with DCM. The squares indicate the men and the circles the women. Diagonal lines indicate the family members that have died. (B) Sanger sequencing of genomic DNA of a patient (III-1) and a healthy sibling in the cohort (III-2), including the mutation site. (C) Hematoxylin and eosin staining analysis of Lmna^WT/WT and Lmna^Q353R/WT knock-in murine hearts on embryonic day 17.5 (E17.5). (D) Immunostaining of troponin T (TnT) in Lmna^WT/WT and Lmna^Q353R/WT knock-in mice on E17.5. DAPI, 4′,6-diamidino-2-phenylindole. (E) Electron microscopic images showing structure of left ventricular wall in an Lmna^WT/WT mouse on E17.5. Fb, fibroblast; Ec, endothelial cell; Nc, nucleus; Sm, sarcomere; Mt, mitochondria; Z, z-disc. (F) Electron microscopy images showing structure of left ventricular wall in an Lmna^Q353R/WT knock-in mouse on E17.5. Ds, desmosome; G, Golgi body; WT, wild type.

Fig. 2.. Immature transcriptional dysregulation.

(A) The uniform manifold approximation and projection (UMAP) plot of scRNA-seq data of all cardiac cells derived from WT and Lmna^Q353R/WT knock-in mice’s hearts at E13.5 and E17.5. All cardiac cells were classified into 13 cell clusters (clusters 0 to 12). WBC, white blood cell; RBC, red blood cell. (B) The UMAP plot of scRNA-seq data of cells annotated as CMs. CMs were classified into eight clusters (CM clusters 0 to 7). (C) Bar plot showing the distribution of CM clusters in each sample. WT, Lmna^WT/WT; Q353R, Lmna^Q353R/WT. (D) Violin plot showing gene expression levels of Tnnt2, representative marker genes of CM cluster 0, marker genes for ventricular, marker genes for atrial CM, genes associated with cell cycle, and genes associated with collagen grouped by CM clusters. (E) Heatmap showing the results of GO enrichment analysis for marker genes of each cluster.

Fig. 3.. TEAD1 regulates CM maturation.

(A) The UMAP plot of scATAC-seq data of all cardiac cells from WT and Lmna^Q353R/WT knock-in mice at E17.5. All cardiac nuclei were classified into 10 clusters (clusters 0 to 9). (B) The UMAP plot pf scATAC-seq data colored by transferred CM subcluster labels of scRNA-seq data at E17.5 in Fig. 2B. (C) Bar plots showing the distribution of CM subclusters in WT and Lmna^Q353R/WT knock-in mice at E17.5. (D) Motif activities of TEAD1 as visualized on the UMAP plot. (E) Violin plots showing the expression levels of TEAD family genes in all CMs. (F) Violin plots showing the expression levels of TEAD family genes in each CM cluster.

Fig. 4.. Immature intracellular structure of Lmna^Q353R/WT iPSCMs.

(A) Electron microscopic images showing morphological properties of isogenic control (Con) and LMNA^Q353R/WT (Q353R) of iPS cell–derived CMs (iPSCMs). (B) Sarcomere density of iPSCMs counted per 625 μm². n = 100 (WT, Q353R; data were obtained from five differentiation batches). *P < 0.05. (C) Representative image of the long and short axis of an iPSCM nucleus (left). Aspect ratio of iPSCM nuclei is also shown (right). n = 72 (WT) and 82 (Q353R); data were obtained from five differentiation batches. *P < 0.05.

Fig. 5.. Transcriptional dysregulation of TEAD1.

(A) GO enrichment analysis of genes with higher H3K4me3 peaks in Con than in Q353R. (B) Motif enrichment analysis of regions with higher H3K4me3 peaks in Con than in Q353R. TF, transcription factor. (C) Venn diagram depicting the number of TEAD1 target genes and GO enrichment analysis of Con-specific TEAD1 target genes. (D) Representative genome browser view of TEAD1 bound regions in CUT&RUN analysis of Con (green) and Q353R (red). (E) Venn diagram depicting the number of overlapping genes between down-regulated genes in Q353R iPSCMs in RNA-seq and TEAD1 target genes in CUT&RUN analysis. DEG, differentially expressed gene. (F) Heatmap showing the expression levels of 39 genes involved in CM maturation among the overlapping genes identified in (E). The names of GO biological process to which each gene belongs are also listed.

Fig. 6.. TEAD1 trapping at the nuclear membrane.

(A) Scheme of the binding protein screening experiment. (B) Result of binding protein screening. Proteins are ordered according to the strength of interaction with mutant Q353R Lamin A/C. (C) Western blot of DDDDK-tag and TEAD1 using co-immunoprecipitated sample. Protein samples extracted from iPSCMs cells were pulled down using an anti-DDDDK tag antibody. Con, isogenic control iPSCMs; Q353R, LMNA^Q353R/WT iPSCMs; IP, immunoprecipitation; IB, immunoblot. (D) Immunostaining of Lamin A/C and TEAD1 in Con and LMNA p.Q353R iPSCMs. Con, isogenic control iPSCMs; Q353R, LMNA^Q353R/WT iPSCMs. Scale bars, 5 μm. (E) Quantification of TEAD1 intensity at the nuclear periphery of iPSCM. n = 14. *P < 0.05. M is the average value of the fluorescence intensity profile, and P is the fluorescence intensity profile on the nuclear membrane. By calculating and comparing the P/M value, significant differences between WT and Q353R were measured. WT: 2.13 ± 0.52; Q353R: 2.68 ± 0.48. (F) The principal components analysis (PCA) plot of bulk RNA-seq data of samples obtained from Con (n = 4), Q353R (n = 4), dimethyl sulfoxide (DMSO)–treated Q353R (n = 3), and TT-10–treated Q353R iPSCMs (n = 3). (G) GO term enrichment analysis of the top 200 genes sorted by principal component 2 (PC2) score, indicating rescued genes by TT-10 treatment. (H) Contractile properties of iPSCM microtissues (n = 6 per group). Q353R + TT-10, LMNAQ353R/WT iPSCMs treated with TT-10. *P <0.05; **P < 0.01. (I) (Top) Representative calcium transient images of iPSCMs, recorded for 10 s. (Bottom) Calcium transient analysis of the iPSCMs (n = 7 per group). *P <0.05; **P < 0.01. (J) Violin plot showing the expression level (log₁₀FPKM) of representative TEAD1 target genes in single CMs from patients with DCM and myocarditis and control subjects. MYL2 is shown as a representative non-TEAD1 target gene.