*4.2. Ageing and Exercise Alter Acetylcholine Receptor Gene Expression*

One of our most marked findings is that the gene expression of the AChR γ subunit is robustly elevated in the skeletal muscle of elderly compared to young females. This coincides with this subunit being a functionally distinct foetal subunit [37–39], which is increasingly expressed following both denervation [39,40] and neurotransmitter blocking [41]. Interestingly, in our group of male participants the muscle homogenate gene expression of the γ subunit was acutely downregulated after the exercise bout but had already returned to baseline after one day. We were also able to detect this subunit, as well as α1, β1, and δ subunits, in both proliferating and differentiating cell cultures that were devoid of neural presence, meaning that satellite cell derived myonuclei can upregulate AChR gene expression without the presence of a nerve. As satellite cells have been shown to be crucial for maintaining the specialized post-synaptic region of the muscle fibre [23], it is interesting that we observed trends for an age effect in three out of the four subunits. It is worth noting that this is the case even with the conservative Bonferroni correction, but given that the age difference was not always in the same direction, it is possible that this is a real effect of age (and not an effect of general cell culture conditions), potentially reflecting an age-related satellite cell dysfunction that could negatively impact the maintenance of the NMJ. However, it should be noted that the cell cultures derived from satellite cells of the young subjects showed a significantly higher fusion index compared to the old subjects [25], indicative of a higher level of myotube maturity. Furthermore, we also observed a positive correlation between cell fusion index and gene expression levels of AChRγ (R = 0.74), MuSK (R = 0.75), and MHCe (R = 0.66) in the old group (Supplementary Figure S6). This would suggest that AChRγ gene expression in aneural cell cultures is increased concordant with myotube maturity and raises the possibility that the molecular differences we observed between the cell cultures from young and elderly muscle are determined by the extent of fusion. However we cannot rule out the opposite, i.e., that the lower gene expression levels contribute to the lower fusion index values.

Generally, our data show age and exercise effects on AChR γ subunit gene expression, in line with its suggested use in evaluating muscle fibre denervation in healthy individuals. In our previous study, we found a negative correlation between age and the AChR γ subunit in a large group of elderly men [20], which initially might seem to contradict our finding in the present study. However, it is important to acknowledge that a denervated muscle fibre is not in a "stable state", meaning that without the neural input the proteins will be degraded and the structure is gradually lost [34,35]. Ultimately, the muscle fibre completely disappears or is only present as a fraction of its former size and as such its contribution to the whole muscle gene expression profile will also decline.

Since our study includes one data set from males and the other from females, it is worth considering similarities in the pattern of the exercise response between the elderly male and female subjects, given that the day five timepoint of the females can be compared with the four- and seven-day timepoints of the male. In this way it seems that the α, δ, and γ subunits follow a similar pattern between the genders, with the first two subunits being upregulated in both male (α only a tendency) and female subjects after seven and five days, respectively, and the γ subunit being unaffected in both male and female subjects at these timepoints. The β subunit is consistently downregulated in the male subjects whereas this subunit is not affected in the elderly women five days after exercise. Whether this represents a true gender difference and what the functional significance might be however is unknown.

This study is to our knowledge the first to outline the gene expression time course for all AChR subunits following acute heavy resistance exercise and the first to analyse the expression of four out of five AChR subunits in both young and elderly individuals at rest and following acute exercise. The NMJ of humans is challenging to study molecularly since it is difficult to obtain actual human NMJs. Hence, we rely on extra-synaptic expression of various genes that are related to the NMJ. With this approach we observed that most subunits were found to be responsive to exercise, which would suggest that despite having reached an advanced age, there is a sustained tissue plasticity in terms of synthesizing new AChRs following an exercise stimulus. The subunit-specific responses also appear to be time-dependent, as some subunits were acutely reduced after exercise followed by a recovery phase, whereas others were downregulated for longer periods. The root of these widely diverging AChR subunit time courses is puzzling and, given evidence from animal studies that long-term exercise increases the size of the NMJ [11], it would be of interest to investigate the potential of lifelong exercise on NMJ adaptations in humans.

#### **5. Conclusions**

Taken together, these data support the concept that the loss of neural signal reverts certain muscle fibre proteins to an embryonic configuration (NCAM/MHCn/AChR γ) and that these markers are useful in evaluating the effectiveness of interventions to counteract the denervation-induced loss of muscle fibres in humans. Gene expression levels of the AChR γ subunit in particular repeatedly demonstrated sensitivity to age and exercise. The trends for age-related differences in the gene expression of AChR subunits in myotubes in cell culture were related to myogenic fusion index and potentially suggest a loss of satellite cell function in relation to the capacity to transcribe key molecules for NMJ stability. Finally, it can be speculated that the temporal manner of the AChR subunit gene expression response following exercise represents a beneficial stimulus for muscle mass preservation through strengthening of the NMJ.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/4/893/s1, Figure S1: Gene expression in muscle biopsies of elderly men receiving losartan (*n* = 13) or placebo (*n* = 12). mRNA data were normalized to RPLP0, log-transformed, and are shown as geometric means ± back-transformed SEM, relative to baseline (–10d). Data were analysed with a two-way repeated measures ANOVA (treatment/time). \* *p* < 0.05 vs. baseline. Tendencies are written; Figure S2: Panel showing four consecutive biopsy sections of an area with small MHCn-positive muscle fibres (a–d). The dotted squares (b,d) highlight the areas of the inserts (b1 <sup>+</sup> <sup>2</sup> and d1 <sup>+</sup> 2), in which a distinct dystrophin membrane is visible around the small fibre. Note that one of the positive muscle fibres is no longer visible in (c) and (d). MHCn-positive fibres are red, dystrophin, green, and nuclei, blue. MHCn, neonatal myosin heavy chain. Scale bars = 20 μm; Figure S3: Muscle fibre size of MHCn- and NCAM-positive fibres in biopsy cross-sections from the control leg in 12 young and 11 elderly women, pooled

and plotted on a logarithmic scale (a). The majority of the positive fibres were <100 μm2.Muscle fibres positive for MHCn and NCAM for young and elderly women in control and exercised legs (b). No differences between control and exercised leg was observed for any variable. MHCn, neonatal myosin heavy chain; NCAM, neural cell adhesion molecule; Figure S4: Cross section of a muscle biopsy from one subject stained with NCAM (green) and collagen XXII (red). Nuclei are blue. NCAM-positive fibres are found in close proximity of a tendon-like structure and collagen XXII positivity confirms this is a myotendinous junction. These fibres were excluded from the analysis. Scalebar is 500 μm; Figure S5: Differentiating cells relative to proliferating cells in control leg of young women. mRNA data were normalized to RPLP0 and are shown as geometric means ± SEM. \* *p* < 0.05 vs. proliferation; Figure S6: Myogenic fusion index correlates with cell culture mRNA levels of AChRγ, MuSK, and MHCe in rested leg of elderly (*n* = 10) but not young (*n* = 11) subjects. All mRNA data were log transformed and analysed with Pearson's correlation, with R and P values displayed.

**Author Contributions:** A.L.M., P.S., C.S., J.L.A., C.J.L.B., M.F.H., and M.K. contributed to the design of the project, while A.L.M., P.S., C.S., J.L.A., C.J.L.B., M.F.H., and M.K., S.M.J., E.B., A.K. acquired, analysed or interpreted the data of the project. C.S. and A.L.M. drafted the manuscript, and all authors gave intellectual feedback to the draft. All authors approve the final version of the manuscript to be published in *Cells* and are to be held accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding is gratefully acknowledged from The Nordea Foundation (Healthy Aging grant), The Danish Agency for Culture (FPK.2018-0036, FPK.2015-0020), The Lundbeck Foundation, The A.P. Møller Foundation for the Advancement of Medical Science, and Bispebjerg Hospital Research Funding.

**Acknowledgments:** The authors thank Anja Jokipii-Utzon and Camilla Brink Sørensen for excellent technical assistance with preparation of the muscle biopsies and the mRNA analysis. The monoclonal antibodies F1.652 (developmental MHC) and A4.951 (myosin heavy chain, human slow fibres), developed by Blau, H.M., were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH, and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

**Conflicts of Interest:** The authors declare no conflict of interest.
