**4. Discussions**

The most notable findings of the present study was that the skeletal muscle of elderly individuals, with morphological signs of ageing as demonstrated by a reduced type II muscle fibre CSA and a heightened number of denervated muscle fibres, has a significantly elevated gene expression level of the denervation-responsive AChR γ subunit and MHCn as compared to young healthy individuals. Our data also suggest an age effect on the capacity of satellite cell-derived myotubes to transcribe AChR genes, which is fundamental for NMJ maintenance. Furthermore, we provide novel insight into the transient changes in gene expression of all five muscle AChR subunits following heavy resistance exercise in healthy elderly human skeletal muscle. Together these data support the role of exercise in stimulating the stability of the NMJ, but also indicate age-related changes, even in healthy elderly individuals.

#### *4.1. Muscle Fibre Denervation in Elderly Humans*

Healthy elderly women with clear signs of ageing (lower type II muscle fibre CSA and lower muscle strength), also show a significantly heightened number of denervated muscle fibres compared to young healthy women, as evidenced by a greater proportion of fibres positive for NCAM and MHCn. When a muscle fibre loses its neural input, the plasticity of the peripheral nervous systems allows for adjacent nerve sprouts to attempt to re-innervate denervated muscle fibres through nerve sprouting [31]. It is believed that the increased synthesis of NCAM in denervated muscle fibres facilitates this innervation process [32,33]. Denervated muscle fibres will also revert into an immature myosin heavy chain configuration, as we found more MHCn-positive fibres in old subjects compared to the young. Furthermore, we also observed a substantial 10-fold higher gene expression level of MHCn in the muscle tissue of the elderly compared to the young females, reflecting a persisting synthesis of this distinct myosin isoform. Importantly, it should be noted that there is not a complete overlap between MHCn- and NCAM-positive stained fibres, which suggests that the rate at which these proteins aggregate in the muscle fibres following denervation might differ. In terms of denervated muscle fibre morphology, we observed a persisting MHCn and NCAM protein presence in even the smallest of muscle fibres (<75 μm2). These miniature fibres are easily missed during regular biopsy assessments and could represent long-term denervated fibres that had atrophied over time [34] and undergone deterioration of muscle proteins [35], but maintained an increased and long-lasting cytoplasmic expression of MHCn [36] and NCAM [32]. The length of these miniature muscle fibres is a matter of uncertainty. We have previously been able to follow such fibres through 400 μm of consecutive biopsy sections in a selected subject [20]. In a sub analysis in the present study, we searched for MHCn-positive fibres through four consecutive sections and found that 13, 32, and 39% of the fibres had disappeared after 1, 2, and 3 sections, respectively. This implies a substantial number of miniature fibres ends, which could indicate that long-term denervated fibres are gradually degraded both transversally and longitudinally.
