Single nuclei transcriptomics of muscle reveals intra-muscular cell dynamics linked to dystrophin loss and rescue

Abstract

In Duchenne muscular dystrophy, dystrophin loss leads to chronic muscle damage, dysregulation of repair, fibro-fatty replacement, and weakness. We develop methodology to efficiently isolate individual nuclei from minute quantities of frozen skeletal muscle, allowing single nuclei sequencing of irreplaceable archival samples and from very small samples. We apply this method to identify cell and gene expression dynamics within human DMD and mdx mouse muscle, characterizing effects of dystrophin rescue by exon skipping therapy at single nuclei resolution. DMD exon 23 skipping events are directly observed and increased in myonuclei from treated mice. We describe partial rescue of type IIa and IIx myofibers, expansion of an MDSC-like myeloid population, recovery of repair/remodeling M2-macrophage, and repression of inflammatory POSTN1 + fibroblasts in response to exon skipping and partial dystrophin restoration. Use of this method enables exploration of cellular and transcriptomic mechanisms of dystrophin loss and repair within an intact muscle environment. Our initial findings will scaffold our future work to more directly examine muscular dystrophies and putative recovery pathways.

Distinct myofibre domains of the human myotendinous junction revealed by single nucleus RNA-seq

“…This gave rise to a total of 30 subclusters including 4 MTJ-myonuclei subclusters; 26 were new subclusters (indicated by 'Sub' in the cluster names) and 4 original (Cytoplasm, Myo_9, Myo_10, SatCell) (Figure 4A, Supplementary Figure S4, Supplementary Data S3). To compare cell-cluster markers we include in Supplementary Figure S5 a comparison between our 30 nuclei-subclusters and cell-types or nuclei-types reported in the literature (Harvey et al 2019;Dos Santos et al 2020;Kendal et al 2020;Rubenstein et al 2020;De Micheli et al 2020a, b;Barruet et al 2020;Chemello et al 2020;He et al 2020;Kim et al 2020;Petrany et al 2020;Perez et al 2021;Wen et al 2021;Eraslan et al 2022;Scripture-Adams et al 2022;Yan et al 2022). This showed a good alignment between our clusters compared to snRNA-seq (Perez et al 2021;Eraslan et al 2022) and scRNA-seq (Rubenstein et al 2020;He et al 2020) in human skeletal muscle, and in snRNA-seq (Dos Santos et al 2020;Chemello et al 2020;Wen et al 2021) and scRNA-seq (De Micheli et al 2020b) in mice.…”

A protocol for single nucleus RNA-seq from frozen skeletal muscle

“…Skeletal muscle is a complex tissue composed of around 40% of an adult human's mass (Frontera & Ochala, 2015). It contains many cell types, including satellite cells, myotubes, myogenic precursors, fibroadipogenic progenitors, fibroblasts, ECs, pericytes, adipocytes, immune cells, and smooth muscle (De Micheli et al, 2020;Rubenstein et al, 2020;Orchard et al, 2021;Scripture-Adams et al, 2022). Single-cell sequencing technologies are becoming an important tool to interrogate the individual transcriptomes of cell types in otherwise complex heterogenous tissues.…”

“…Increasingly, this technique is proving to be well suited for simultaneously examining many cell types, uncover rare disease states, and identify subpopulations with distinct functions (Birnbaum, 2018). Few studies have examined human skeletal muscle at the single-cell level (Barruet et al, 2020;De Micheli et al, 2020;Rubenstein et al, 2020;Orchard et al, 2021;Scripture-Adams et al, 2022), however, its utility is clear for basic and medical science studies of muscle in health and disease.…”

“…Few articles have applied snRNAseq to skeletal muscle (Zeng et al, 2016;Dos Santos et al, 2020;Jiang et al, 2020;Kim et al, 2020;Petrany et al, 2020;Orchard et al, 2021;Eraslan et al, 2022). Of these, three groups isolated nuclei directly from mouse muscle (Dos Santos et al, 2020;Kim et al, 2020;Petrany et al, 2020), two used cell cultures (Zeng et al, 2016;Jiang et al, 2020), and four used human skeletal muscle (Orchard et al, 2021;Eraslan et al, 2022;Perez et al, 2022;Scripture-Adams et al, 2022), although Orchard and colleagues focussed on analyzing accessible chromatin regions by means of ATAC-seq. To isolate nuclei directly from muscle, these reports used fiber dissection and dounce homogenization (Dos Santos et al, 2020;Scripture-Adams et al, 2022), or a mix of various homogenization methods and detergents (Petrany et al, 2020;Orchard et al, 2021;Eraslan et al, 2022).…”

“…Of these, three groups isolated nuclei directly from mouse muscle (Dos Santos et al, 2020;Kim et al, 2020;Petrany et al, 2020), two used cell cultures (Zeng et al, 2016;Jiang et al, 2020), and four used human skeletal muscle (Orchard et al, 2021;Eraslan et al, 2022;Perez et al, 2022;Scripture-Adams et al, 2022), although Orchard and colleagues focussed on analyzing accessible chromatin regions by means of ATAC-seq. To isolate nuclei directly from muscle, these reports used fiber dissection and dounce homogenization (Dos Santos et al, 2020;Scripture-Adams et al, 2022), or a mix of various homogenization methods and detergents (Petrany et al, 2020;Orchard et al, 2021;Eraslan et al, 2022). One recently published work used fresh muscle samples with a proprietary instrument specifically designed for sc/snRNAseq (S2 Genomics) (Perez et al, 2022).…”