**1. Introduction**

The rate of loss of muscle mass increases with advancing age [1], and ultimately leads to impaired physical function in elderly individuals [2–4]. This age-dependent decline in muscle mass is partly due to a loss of individual muscle fibres [5] as a result of muscle fibre denervation [6–8]. While physical exercise is recognized as a strong countermeasure against the loss of muscle mass and has consistently been shown to maintain physical function and health in the last ten years of life [9,10], it is currently unclear whether denervation can be ameliorated or reversed by exercise.

It has been shown in animals that exercise causes positive adaptations to the neuromuscular junction (NMJ) that to some extent can attenuate the age-related degeneration of the NMJ [11]. Changes in expression of acetylcholine receptors (AChRs) with acute exercise have been suggested to indicate NMJ remodelling in animals [12,13] and represent a potential target for studying this in humans [14]. AChR are present in abundance at the NMJ [15] and are almost non-existent in the extra-synaptic region of the muscle fibre [16]. Upon experimental denervation, however, the α1, β1, γ, and δ subunits increase extra synaptically [16–19], raising the possibility that these AChR subunits can be used as indicators of denervation associated with ageing. We recently observed a correlation between age and gene expression levels of the foetal γ AChR subunit in a large group (*n* = 70) of healthy elderly men ranging in age from 65 to 94 years, in conjunction with tissue markers of muscle fibre denervation, neural cell adhesion molecule (NCAM) and neonatal myosin (MHCn), at the protein level [20]. Direct comparisons with a younger cohort as well as the potential for exercise to influence AChR expression patterns are however lacking.

One of the challenges for ageing skeletal muscle is related to the decline in satellite cell function with age. Not only is satellite cell function important for tissue repair and maintenance, but it also has potential implications for maintenance of the NMJ, where myonuclei at this site must be capable of carrying out the specialization necessary to complete the formation of the NMJ. This includes producing a high concentration of AChRs at the membrane and a clustering of myonuclei, which become transcriptionally specialized and distinct from adjacent extra-synaptic myonuclei [21,22]. Whether this capacity declines with age is currently unknown. Satellite cells have been shown to play a vital role in maintaining the post-synaptic region in mice, both in terms of myonuclear clusters of AChRs and re-innervation of the regenerating NMJ [23,24]. In this context it is interesting that we have recently observed a poorer fusion capacity of satellite cells derived from old women compared to young women, accompanied by a distinctly different molecular profile throughout the myogenic program [25]. It remains unknown, however, to what extent this dysfunction in human satellite cells has implications for NMJ maintenance with increasing age.

Based on the above, the main purpose of this study was to investigate the influence of age and exercise on molecular markers of NMJ stability and muscle fibre denervation in healthy elderly individuals. An additional focus was to determine how ageing would alter the capacity of myonuclei in cell culture to produce key transcriptional elements for NMJ formation.

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

This study is based on muscle biopsies collected from two studies, on 12 young and 12 elderly women [25], and on 25 elderly men [26], respectively. Both studies were approved by The Committees on Health Research Ethics for The Capital Region of Denmark (Ref: H-15017223, H-3-2012-081). All procedures conformed to the Declaration of Helsinki of 1975, revised in 2013, and the subjects gave written informed consent before participation. All participants were healthy, non-smokers, non-obese, and did not perform strenuous physical exercise on a regular basis. The men were part of a randomized controlled trial investigating the effect of the blood pressure-lowering medication losartan on the muscle response to exercise, where half of the participants received losartan and the other half placebo. Given the general lack of drug effect, the two groups were merged in the present study (separate group data are also provided for reference in online Supplementary Figure S1).

All participants performed a maximal strength test in a Leg Extension machine (M52, TechnoGym, Cesena, Italy) to determine the one-repetition maximum (1 RM), which was used to determine the load lifted during the subsequent bout of heavy resistance exercise. The Leg Extension exercise protocols consisted of both concentric and eccentric contractions. First, 4–5 sets of 12 concentric contractions at 70% of 1 RM were performed, followed by four sets of 4–6 eccentric contractions at 110% of 1 RM, as

previously described [25,26]. The exercise was performed with one leg only, leaving the contralateral leg as a control. No other exercise was allowed during the study period.

#### *2.2. Muscle Biopsies*

For all participants, muscle biopsies were obtained from the vastus lateralis muscle, under local anaesthetic (1% lidocaine), using the percutaneous needle biopsy technique of Bergström [27], with five 6-mm needles and manual suction. Pieces of muscle tissue were aligned, embedded in Tissue-Tek, and then frozen in isopentane, pre-cooled in liquid nitrogen, and stored at −80 ◦C. The men had six muscle biopsies taken over 17 days, at the following time points: −10 and −3 days before exercise from the control, non-exercised leg, and from the exercised leg at +4.5 h and on days +1, +4, and +7 post exercise. The day −3 sample was excluded from the current study since its purpose was to investigate a potential effect of losartan in the rested state and is therefore superfluous in the current context. The young and elderly women had muscle biopsies collected from each leg five days after exercise, from which a part was embedded as described above and a part was used for cell culture, where myoblasts were plated in 12-well plates for three days of proliferation (12,000 cells per well), or three days of proliferation followed by four days of differentiation (20,000 cells per well), as previously described in detail [25].

#### *2.3. RNA Extraction*

100 cryo sections, 10 μm thick, from the embedded muscle tissue were homogenized in 1 mL of TriReagent (Molecular Research Center, Cincinnati, OH, USA) containing five stainless steel balls of 2.3 mm in diameter (BioSpec Products, Bartlesville, OK, USA), and one silicon-carbide sharp particle of 1 mm (BioSpec Products), by shaking in a FastPrep®-24 instrument (MP Biomedicals, Illkirch, France) at speed level four for 15 s. Cell culture cells were dissolved directly in the Trireagent. Bromo-chloropropane was added in order to separate the samples into an aqueous and an organic phase. Following isolation of the aqueous phase, RNA was precipitated using isopropanol. The RNA pellet was then washed in ethanol and subsequently dissolved in 20 μL RNAse-free water. Total RNA concentrations and purity were determined by spectroscopy at 260, 280, and 240 nm. Good RNA integrity was ensured by gel electrophoresis.

#### *2.4. Real-Time RT-PCR*

mRNA targets related to innervation were analysed for the current study. The specific primers are given in Table 1. Total RNA (500 ng for muscle and 150 ng for cell culture) was converted into cDNA in 20 μL using OmniScript reverse transcriptase (Qiagen, Redwood City, CA, USA) and 1 μM poly-dT (Invitrogen, Naerum, Denmark) according to the manufacturer's protocol (Qiagen). The same pool of cDNA used previously for the cells in culture [25] and the male muscle tissue [26] was used here. For each target mRNA, 0.25 μL cDNA were amplified in a 25-μL SYBR Green polymerase chain reaction (PCR) containing 1 × Quantitect SYBR Green Master Mix (Qiagen) and 100 nM of each primer (Table 1). The amplification was monitored real time using the MX3005P Real-time PCR machine (Stratagene, San Diego, CA, USA). The Ct values were related to a standard curve made with known concentrations of cloned PCR products or DNA oligonucleotides (UltramerTM oligos, Integrated DNA Technologies, Inc., Leuven, Belgium) with a DNA sequence corresponding to the sequence of the expected PCR product. The specificity of the PCR products was confirmed by melting curve analysis after amplification. Ribosomal Protein Lateral Stalk Subunit P0 (RPLP0) mRNA was chosen as internal control. To validate this use, another unrelated "constitutive" mRNA, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), was measured and normalized with RPLP0. In the cell culture experiment GAPDH mRNA normalized to RPLP0 mRNA was constant, indicating that RPLP0 (and GAPDH) was indeed constant and suitable for normalization. However, in tissue the GAPDH/RPLP0 ratio was lower in the elderly female subjects and one and four days after exercise in the males, showing either a GAPDH decrease or a RPLP0 increase. However, the decrease in GAPDH was not reflected

in the general pattern of the other mRNA when normalized to RPLP0, arguing against a general normalization error. We therefore chose to use retain RPLP0 for normalization. The GAPDH mRNA data from cell culture of the females and tissue of the males have been used as internal control in already published papers [25,26].

**Table 1.** Primers used for PCR. RPLP0: Ribosomal Protein Lateral Stalk Subunit P0; GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase; AChR: acetylcholine receptor; MuSK: muscle-specific-kinase; MHCn: neonatal myosin; MHCe: embryonic myosin heavy chain.

