Degenerative and regenerative pathways underlying Duchenne muscular dystrophy revealed by single-nucleus RNA sequencing

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

Keywords: DMD mouse model; dystrophin; myofibers; myonuclei; skeletal muscle.

Fig. 1.

Creation and analysis of ΔEx51 Dmd mice. (A) CRISPR-Cas9 editing strategy used for generation of mice with dystrophin exon 51 deletion (ΔEx51). Mouse oocytes were injected with SpCas9 and 2 sgRNAs flanking exon 51. Upon deletion of exon 51, exon 52 (red) becomes out of frame with exon 50. (B) RT-PCR analysis of TA muscle to validate deletion of exon 51 (233-bp length). RT-PCR primers were in exons 48 and 53, and the amplicon size was 767 bp for WT mice and 534 bp for ΔEx51 mice. RT-PCR products are schematized on the right (n = 3). (C) Western blot analysis showing loss of dystrophin expression in the TA of ΔEx51 mice. Vinculin is the loading control (n = 3). (D) Dystrophin staining of the TA of WT and ΔEx51 mice. Dystrophin is shown in green. Nuclei are marked by DAPI stain in blue. (Scale bar, 100 μm.) (E) H&E staining of the TA of WT and ΔEx51 mice. Note extensive inflammatory infiltrate and centralized myonuclei in ΔEx51 muscle. (Scale bar, 100 μm.) (F) Heat map showing z-score–transformed expression of the differentially expressed genes between WT and ΔEx51 TA muscle (n = 3). (G) Selected top GO terms enriched in down- and up-regulated genes in ΔEx51 TA muscle.

Fig. 2.

snRNA-seq of TA muscle from WT and ΔEx51 mice. (A) Schematic of the experimental design for snRNA-seq on skeletal muscle nuclei. (B) UMAP visualization of all of the nuclei (11,222 nuclei, Left) from WT and ΔEx51 TA muscle colored by cluster identity. UMAPs depicting nuclei of WT TA muscle (7,013 nuclei, Center) and nuclei of ΔEx51 TA muscle (4,209 nuclei, Right). Percentages of nuclei in each cluster are indicated in the table. (C) Violin plots showing the expression of selected marker genes for each cluster of nuclei. (D) Heat map showing z-score–transformed average expression of the marker genes for each cluster of nuclei (Left). Selected top enriched GO terms of the marker genes for each cluster of nuclei (Right).

Fig. 3.

Analysis of gene expression changes in ΔEx51 myonuclei compared to WT myonuclei. (A) Number of up- and down-regulated genes in different clusters of ΔEx51 myonuclei. (B) Selected top GO terms enriched in up-regulated genes from the different clusters of ΔEx51 myonuclei. (C) Violin plots showing the differential expression of selected marker genes of protein ubiquitination between WT and ΔEx51 myonuclei. *Genes significantly differentially expressed. (D) Violin plots showing the differential expression of selected marker genes of apoptosis in WT and ΔEx51 myonuclei. *Genes significantly differentially expressed.

Fig. 4.

Analysis of gene expression dynamics in WT and ΔEx51 myonuclei. (A) Percentage of nuclei of clusters involved in the regeneration of skeletal muscle (Clusters MuSC, Myob, and RegMyon) in WT and ΔEx51 TA muscles. (B) UMAPs depicting the expression of Myh3, Myh8, and Mymk in the RegMyon cluster. (C) Pseudotime ordering of all of the nuclei of Clusters MuSC, Myob, and RegMyon. Each dot represents one nucleus (color-coded by its identity) and each branch represents one cell state. Activation of the MuSC cluster can lead to Myob fate A or to RegMyon fate B. (D) Heatmap showing z-score–transformed average expression of the differentially expressed gene dynamics toward Myob fate A and RegMyon fate B along pseudotime. The differentially expressed genes were clustered into four gene sets according to k-means. (E) Violin plots showing the expression of selected genes (involved in the regulation of cytoskeleton organization or encoding transcriptional factors) highly expressed in the nuclei of Cluster RegMyon. (F) Enrichment score of the top transcription factor binding motifs identified by iRegulon from the promoter regions of the marker genes of the Cluster RegMyon.