In 2018, a Tabula Muris of 20 mouse organs using scRNA-seq defined the first cellular atlas of skeletal muscle mononuclear cells [3]. This information is foundational because it (1) reinforced the extent of cellular diversity in skeletal muscle outside of the myofiber and (2) charted a path forward for using emerging single cell technology in skeletal muscle. In 2021, the first study leveraging scRNA-seq in the context of adult skeletal muscle adaptation to hypertrophic mechanical loading (i.e., resistance exercise mimetic) in mouse was published [4]. Satellite cells are the bona fide muscle stem cell population in skeletal muscle. This study provided initial evidence that satellite cell-derived myonuclei that fuse into myofibers during hypertrophy may differ from resident myonuclei. This work also expanded on how satellite cells communicate widely to other muscle-resident cells, such as FAPs, to control extracellular matrix (ECM) remodeling, inflammatory-related gene expression, and potential cell fate. The evidence suggests that intercellular communication from satellite cells can be mediated by extracellular vesicles (EVs) that deliver miRNAs such as miR-206 to target recipient cell mRNAs and affect cellular function [4]. Intercellular communication by satellite cells may help create a permissive environment for sustained myofiber growth during loading [4]. Using snRNA-seq (which contains myonuclei as well as nuclei from mononuclear cells in muscle) combined with progressive weighted wheel running (PoWeR) in mice, we further defined the role of satellite cells in the exercise training response [5]. Satellite cells positively influenced oxidative metabolism, ribosome biogenesis, and myofibril assembly related gene expression in myonuclei after 4 weeks of high-volume PoWeR, which was characterized by oxidative fiber type transitioning and hypertrophy [5]. Additional analysis suggested that (1) satellite cells activate but do not necessarily fuse during adult muscle adaptation to exercise, and (2) satellite cell depletion dysregulates myonuclear gene expression and causes “cryptic,” or transcriptionally ambiguous, myonuclei to emerge [6].

It is intuitive that the first studies using sc- and snRNA-seq focused on the influence of satellite cells on hypertrophic muscle adaptation given their myogenic potential. Additional scRNA-seq studies have since provided more holistic information on the function of various muscle mononuclear cells, as well as myofibers, during adaptation to exercise. Expanding on the findings of Joszi et al. > 20 years prior, work in 2021 using bulk RNA-seq in human muscle following resistance training and scRNA-seq in mouse in response to mechanical overload highlighted the role of macrophages in ECM remodeling [7]. This study collectively revealed that myofiber-derived leukemia inhibitor factor (LIF) specifically altered macrophage gene expression of the ECM remodeling gene matrix metalloproteinase 14 (MMP14) during hypertrophy to influence collagen turnover. Physical activity in mice has also been demonstrated to specifically target macrophages in muscle. scRNA-seq showed that chronic unweighted wheel running normalized dysregulated inflammatory gene expression in macrophages of aged mice, which restored muscle regenerative capacity [8]. Chronic unweighted wheel running in aged mice also reshaped aspects of gene expression to reflect that of younger mice; this included genes related to lipid metabolic processes and TNF-α signaling specifically in myofibers determined by single fiber RNA-seq [8]. Collectively, these initial studies highlight that the complex molecular circuitry following exercise training is cell type-specific and influenced by age.

In humans, scRNA-seq was recently used to explore muscle transcriptomic responses three hours following bouts of repeated high-intensity sprint exercise in two adult males and a female [9]. These analyses revealed a shift in mononuclear cell frequency—increased lymphocytes and monocytes and decreased proportion of endothelial cells—after intense exercise. Mesenchymal cells (likely FAPs) were most responsive to intense exercise, but most identified cell types that were profiled had an altered mRNA landscape. Activated satellite cells separated into distinct populations after exercise which may represent functional heterogeneity based on troponin gene expression (TNNI1: slow-twitch associated, or TNNI2: fast-twitch associated). Using scRNA-seq in an older male who previously mounted an effective hypertrophic response to a 14-week resistance exercise training program, Long et al. showed the heterogeneity of immune cells present in muscle 24 h following a single bout of high intensity resistance exercise [10]. These data broadly agree with the aforementioned murine exercise studies regarding mononuclear cells in muscle: macrophages, FAPs, and satellite cells are focal points in the muscle adaptive process. See Fig. 1 for an overview of the findings that we elaborated on in this manuscript.

Summary and interpretation of findings outlined in this article from published scRNA-seq and snRNA-seq from skeletal muscle exercise studies in mice and humans. EVs, extracellular vesicles; FAP, fibro-adipogenic progenitor cell; LIF, leukemia inhibitory factor; MMP14, matrix metalloproteinase 14; miRNA, microRNA. Created with BioRender.com