AIM: One of the hallmarks of ageing muscle is the decreased ability to regenerate. This has been attributed to dysfunctional satellite cells that can result in reduced myogenic capacity. Although reduced myogenic capacity can impair muscle regeneration, other factors including restricted muscle repair programming are also at play (1). Muscle regeneration mirrors foetal development and thus, we hypothesise that the transcriptome (the full range of messenger RNA molecules expressed by an organism) involved in muscle development is affected by ageing, resulting in a diminished regeneration potential. The aim of this study was to develop a high-throughput in vitro model enabling cellular and molecular investigations of muscle regeneration across the life course.
Methods: Myotubes, differentiated from human myoblasts from an older donor (male, aged 68 years; from Promocell) and a younger donor (male, aged 20 years; from Lonza), were injured after exposure to 12% barium chloride. Myotube repair was assessed by morphological analysis of myotube fusion and width, cell cycle and the transcriptome. For morphological analysis, the myotubes were stained with phalloidin and DAPI (4´,6-diamidino-2-phenylindole) to label the cytoskeleton and nuclei of muscle cells. This enabled us to estimate the fusion index and myotube width. The cell cycle was investigated using the EdU (5-ethynyl-2 Ì-deoxyuridine) assay, which detects cells entering the S-phase (proliferative). In both morphological and proliferation assays, images were acquired with the Leica fluorescence microscope and analysed using ImageJ. Data were expressed as mean ± SEM. The transcriptome was assessed by RNA-seq. Timepoints for each analysis were pre-injury (control), post-injury, end of proliferation and end of differentiation (4 independent experiments). Statistical analyses of morphological and proliferation assays were performed using a two-sided unpaired t-test and RNA-seq using the DESeq2 R package (1.20.0).
Results: After repair, the fusion index (p=0.04) and myotube diameter (p=0.0008) were smaller in older myotubes compared to pre-injury (control). Younger myotubes exhibited a fusion index and width similar to their pre-injury state. With regards to the cell cycle, the number of EdU+ cells increased during the proliferation phase in myotubes derived from both young (p=0.00003) and aged (p=0.0008) myoblasts. Transcriptome analysis of older myotubes showed significant enrichment of the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, PI3K-Akt signalling, and of GO (Gene Ontology) biological processes associated with muscle development and the extracellular matrix. Many of the genes involved in muscle cell development were downregulated (Figure 1). In young myotubes, the most overrepresented KEGG pathways were cytokine receptor interaction and protein digestion and the most enriched GO biological process were extracellular matrix-related processes.
Conclusion: This model provides a high-throughput platform enabling cellular and molecular investigations of muscle regeneration across the life course. As expected, older myotubes showed impaired regeneration as evidenced by reduced myofusion index and width after repair. We postulate that this is due to the down-regulation of genes involved in muscle development and function.