Single nuclei profiling identifies cell specific markers of skeletal muscle aging, frailty, and senescence

Abstract

Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, which causes numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia or frailty include inflammation, muscle stem cell depletion, mitochondrial dysfunction, and loss of motor neurons, but whether there are key drivers of sarcopenia are not yet known. To gain deeper insights into age-related muscle loss, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, elderly, and frail human subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). This combined approach revealed changes in gene expression that occur with age and frailty in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in aged muscle. We validated several key genes changes in fixed human muscle tissue using digital spatial profiling. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with p21 cip1 -driven senescence in this subpopulation. Overall, our findings identify unique cellular subpopulations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related frailty.

Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

“…Our data indicates MYH6 (alpha isoform in cardiac muscle), MYH4 (primarily in rodent limb muscles) and MYH14 (a non-muscle myosin) were significantly different between Leiden groups, however the abundance of each of these isoforms was <1% of the total MYH content. This is in line with the current literature, which shows MYH6 and MYH4 to be completely absent or in very low abundance in human skeletal muscle fibers 1,7,30 . Nevertheless, the mean iBAQ-calculated quantity of MYH6 in our data was 5e-4 in Leiden group 1 and 8e-6 in Leiden group 0, which led to it being significantly different between groups despite the low amount.…”

“…We found that MYH protein quantities calculated using iBAQ from DIA-NN precursor data with subsequent actin normalization were more reliable in assigning MYH isoforms based on established knowledge of MYH subtype distributions within human skm compared to protein quantities from MaxLFQ. We characterized the differences in type 1 and 2A fiber types based on the quantities of the regulatory and structural proteins (troponins, tropomyosins, MYL proteins, Z-line linking proteins, and a sarcoplasmic reticulum protein) and found a majority of the proteins ( Figure 2 and Figure 5) to also be congruent with previous findings 3,30 . For example, MYL3, which we found to be significantly higher in Leiden 1 (mostly type 1 fibers), has been previously reported as 10 times more abundant in type 1 versus type 2A fibers 3 .…”

“…Expression of MYH4 ( Figure 3D ), which is thought to be found in rodent skm and only in a specialized muscle type in humans 29 , was 0.3% of total MYH content when protein quantities were computed using iBAQ, but 10% of total MYH when using MaxLFQ derived protein quantities. Other single cell human skm studies also observe negligible or no MYH4 present in fibers 30 . MYH6, which is found in cardiac muscle cells 31 , was low in both quantification strategies–0.3% of total MYH content when protein quantities were calculated using iBAQ and 0.8% of total MYH content when using MaxLFQ.…”

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

“…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).…”

“…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).…”