Heterogeneous Skeletal Muscle Cell and Nucleus Populations Identified by Single-Cell and Single-Nucleus Resolution Transcriptome Assays

Abstract

Single-cell RNA-seq (scRNA-seq) has revolutionized modern genomics, but the large size of myotubes and myofibers has restricted use of scRNA-seq in skeletal muscle. For the study of muscle, single-nucleus RNA-seq (snRNA-seq) has emerged not only as an alternative to scRNA-seq, but as a novel method providing valuable insights into multinucleated cells such as myofibers. Nuclei within myofibers specialize at junctions with other cell types such as motor neurons. Nuclear heterogeneity plays important roles in certain diseases such as muscular dystrophies. We survey current methods of high-throughput single cell and subcellular resolution transcriptomics, including single-cell and single-nucleus RNA-seq and spatial transcriptomics, applied to satellite cells, myoblasts, myotubes and myofibers. We summarize the major myonuclei subtypes identified in homeostatic and regenerating tissue including those specific to fiber type or at junctions with other cell types. Disease-specific nucleus populations were found in two muscular dystrophies, FSHD and Duchenne muscular dystrophy, demonstrating the importance of performing transcriptome studies at the single nucleus level in muscle.

Keywords: myonuclei heterogeneity; single-cell RNA-seq; single-nucleus RNA-seq; skeletal muscle; spatial transcriptomics.

FIGURE 1

Populations of mononuclear skeletal muscle cells and myotube nuclei with associated markers identified by single-cell and single-nucleus RNA-seq Populations and top markers of cells and nuclei from mononucleated cells or myotubes. In vitro populations include cell and nuclear populations identified from in vitro culture of biopsy derived cells or muscle cell lines. Development populations include those observed during development in mice. In vivo populations include those observed in human or mouse muscle. Created with BioRender.com.

FIGURE 2

Heterogenous myofiber nuclei with associated markers identified by high-throughput transcriptomics Populations and top markers of nuclei from myofibers identified through single-nucleus RNA-seq and low throughput spatial transcriptomics. Left is a generic myofiber with transcriptionally distinct nuclei labelled. All populations except fiber type specific, sarcomere assembly and lncRNA are influenced by their proximity to other cells, damage or the perimysium. Right are bag and chain fibers. Created with BioRender.com.