In Vitro Regeneration Capacity In Response To Chemical Injury Is Compromised In Aged Vs Young Muscle Cells

Healthy skeletal muscle can regenerate after ischemic, mechanical or toxin-induced injury. However, age-related changes in skeletal muscle cells and their niche may impair the regeneration potential. While strategies to regenerate muscle are being developed, in vitro models to investigate the regenerative potential after injury in aged versus young muscle cells are lacking. PURPOSE: This study aims to develop in vitro muscle models of injury and regeneration using cells from older and young donors. METHODS: Differentiated C2C12 murine and human myoblasts from old and young donors were injured after exposure to 12% barium chloride for 6 h. Regeneration was assessed by morphological analysis including myotube width, number of nuclei and fusion index. For that cells were stained with phalloidin and DAPI to label the cytoskeleton and nuclei of muscle cells, respectively. Images were acquired with the Leica fluorescence microscope and analysed using ImageJ. Timepoints were pre-injury (control), post-injury, end of proliferation and end of differentiation. RESULTS: The number of myonuclei post-injury remained unaltered compared to pre-injury among all cell lines (p > 0.05). Three days post-injury, nuclei number reached significance only in murine (p = 0.0006) and young (p = 0.03) muscle cells, whereas aged myotubes showed a significant increase in the total number of myonuclei at the end of the regeneration period (p = 0.01). Myotube diameter at the end of the regeneration recovered to pre-injury state in C2C12 and young but was significantly smaller in aged cells (p = 0.04). CONCLUSION: Delayed peak in proliferation and significantly smaller myotubes in aged compared to young and murine muscle cells at the end of the experiment could be attributed to declined proliferation rate of resident satellite cells that causes suspended myogenesis and regenerative response in older muscle cells. Barium chloride injury model is an easily reproducible in vitro approach that could be applied to study age and/or disease-related regenerative changes in different cell lines. KEYWORDS: regeneration, injury, skeletal muscle, ageing