Muscle fibroblasts and stem cells stimulate motor neurons in an age and exercise-dependent manner

Abstract

Exercise preserves neuromuscular function in aging through unknown mechanisms. Skeletal muscle fibroblasts (FIB) and stem cells (MuSC) are abundant in skeletal muscle and reside close to neuromuscular junctions, but their relative roles in motor neuron maintenance remain undescribed. Using direct cocultures of embryonic rat motor neurons with either human MuSC or FIB, RNA sequencing revealed profound differential regulation of the motor neuron transcriptome, with FIB generally favoring neuron growth and cell migration and MuSC favoring production of ribosomes and translational machinery. Conditioned medium from FIB was superior to MuSC in preserving motor neurons and increasing their maturity. Lastly, we established the importance of donor age and exercise status and found an age-related distortion of motor neuron and muscle cell interaction that was fully mitigated by lifelong physical activity. In conclusion, we show that human muscle FIB and MuSC synergistically stimulate the growth and viability of motor neurons, which is further amplified by regular exercise.

Keywords: aging; neural plasticity; neurodegeneration; sarcopenia; satellite stem cell; skeletal muscle; training.

FIGURE 1

Differential regulation of motor neuron gene expression by muscle stem cells and fibroblasts. (a) Representative image of MuSC (red = desmin) and FIB (blue = Te7) in vitro. Scale bar, 100 μm. (b) Experimental setup of direct coculture experiments: E14 rat motor neurons were plated on human primary MuSC or FIB for 1 day and analyzed by RNA sequencing. (c) Volcano plot showing differentially regulated human MuSC versus FIB genes. Blue and red are upregulated in FIB and MuSC, respectively. Cell type specific markers are highlighted. (d) Volcano plot showing differentially regulated rat genes upon exposure to human MuSC versus FIB. Blue and red are upregulated with FIB and MuSC, respectively. Top 10 lowest FDR, and Top 10 highest log2FoldDifference for each cell type is highlighted. (e) Pathway analysis showing top‐10 GO‐BP, GO‐CC and GO‐MF terms upregulated in motor neurons exposed to MuSC versus FIB. (f) Pathway analysis showing top‐10 GO‐BP, GO‐CC and GO‐MF terms upregulated in motor neurons exposed to FIB versus MuSC. Level of significance is indicated by color. Number of differentially regulated genes and total number of genes is indicated for each term. (c–f) Human n: Paired MuSC and FIB from 14 individuals. Rat n: Embryos from 7 dams. Statistics: Data were analyzed with DESeq2 and TopGO (elim), see bioinformatics section. FIB, muscle fibroblast; MuSC, muscle stem cell.

FIGURE 2

Motor neuron‐specific genes are upregulated by fibroblasts. Motor neuron‐specific genes identified from two single‐cell RNA sequencing data sets of murine spinal cord cells. (a) Volcano plot showing differentially regulated rat motor neuron‐specific genes from reference (Karlsen et al., 2023) upon exposure to human MuSC versus FIB. Blue and red are upregulated with FIB and MuSC, respectively. (b) Volcano plot showing differentially regulated rat motor neuron‐specific genes from reference (Delile et al., 2019) upon exposure to human MuSC versus FIB. Blue and red are upregulated with FIB and MuSC, respectively. Human n: Paired MuSC and FIB from 14 individuals. Rat n: Embryos from 7 dams. Statistics: Data were analyzed with DESeq2, see bioinformatics section. FIB, muscle fibroblast; MuSC, muscle stem cell.

FIGURE 3

Fibroblast conditioned medium preserves motor neurons and increases their maturity. (a) Experimental setup of conditioned medium experiments: E14 rat motor neurons were plated in Poly‐L‐Ornithine and laminin coated wells, in 40% conditioned medium from MuSC or FIB, and analysed after 1 and 2 days by immunocytochemistry and RNA sequencing. (b) One‐repetition maximum and muscle fiber cross‐sectional area. Data are means ± SEM. Statistics: Data were analyzed by unpaired two‐tailed t‐tests.* indicates p < 0.05, ****p < 0.0001. Representative image muscle cross‐section immunofluorescently stained with dystrophin. Scale bar, 100 μm. (c) Example of entire coverslip used for analysis. Insert shows zoomed‐in area. Scale bar, 250 μm. Magenta = ChAT, gray = Tau1. (d–g) Immunocytochemical analyses of Tau1+ and ChAT+ cells, either per area (d, f), or as a percentage of all cells (e, g), after 1 (d, e) and 2 (f, g) days. Data are means ± SEM. Human n: Old: 10, Young: 10. Rat n: 7. Statistics: Data were analyzed by two‐way repeated measures ANOVA (age group × cell type), with Fisher's LSD posthoc test. Main effects are written and post hoc tests indicated by *p < 0.05, **p < 0.01. (h) Differentially regulated rat genes upon exposure to human MuSC versus FIB after 1 day, displayed as paired values. Blue and red is FIB and MuSC, respectively. N: Old: 10, Young: 9. Rat n: 7. Data were analyzed with DESeq2, see bioinformatics section. FIB, muscle fibroblast; MuSC, muscle stem cell.

FIGURE 4

Sedentary aging is associated with reduced muscle performance and neuromuscular disturbance in vivo. (a) In vivo characterization of male participants. (b) Leg lean mass, measured by DEXA. (c) Isometric unilateral knee extension MVC, measured in a dynamometer. (d) Specific force, calculated as MVC per leg lean mass. (e) Muscle performance, measured as force exerted during repeated maximal knee extension concentric contractions, expressed relative to MVC. (f) CAF, measured in plasma by ELISA. Statistical analysis was conducted on log transformed values. (g) Muscle fiber cross sectional area measured using immunohistochemical analyses of muscle biopsy cross‐sections. All data are means ± SEM except CAF which is shown as geometric means with 95% CI. N: LLEX: 7, SED: 6, Young: 9. Statistics: Data were analyzed by unpaired two‐tailed t‐tests, with significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001. CAF, C‐terminal Agrin Fragment; DEXA, dual‐energy X‐ray absorptiometry; MVC, maximal voluntary contraction.

FIGURE 5

Lifelong exercise facilitates motor neuron survival in vitro. (a) Experimental setup of direct coculture experiments: Motor neurons were plated on MuSC and FIB isolated from muscles of Young, LLEX and SED, for 1 day and analyzed by immunocytochemistry and RNA sequencing. (b) Representative image cocultures stained with ChAT (red), Tau1 (green) and nuclei (gray). Scale bar, 100 μm. (c, d) Number of Tau1+ and ChAT+ cells, number of neurites and length of neurites in MuSC (c) and FIB (d) conditions. Data are shown as means ± SEM. Human n (MuSC): LLEX: 7, SED: 6, Young: 9. Human n (FIB): LLEX: 5, SED: 4, Young: 6. Rat n: 7. Statistics: Data were analyzed by unpaired two‐tailed t‐tests, with significance indicated by *p < 0.05. FIB, muscle fibroblast; MuSC, muscle stem cell.