It has been shown that the IGF-I gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. To study the roles of environmental mechanical cues on gene programming and splicing in developing muscle, an in vitro 3D cell culture system was employed. C2C12 skeletal myoblasts were grown in 3D collagen cultures. The presence of this extracellular matrix component, the application of uniaxial strain (produced endogenously by the myoblasts) and a high concentration of cells was shown to enhance differentiation in these cultures. These differentiated myotube cultures were then subjected to different regimens of exogenous mechanical strain. IGF-IEa, which initiates the fusion of myoblasts to form myotubes, was found to be constitutively expressed in myoblasts and myotubes and its expression up-regulated by a single ramp stretch of one hour duration but reduced by repeated cyclical stretch. In contrast, MGF which is involved in the proliferation of mononucleated myoblasts, that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was up-regulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of understanding the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.

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The loss of muscle mass with age (Sarcopenia) has received growing attention over the past decade. Despite efforts to provide a universal definition with clinically meaningful cut-off points for diagnosis, there is no clear consensus on how to best quantify and assess the impact of loss of muscle mass and function on functional limitations. Whilst most previous studies have used dual energy x-ray absorptiometry (DXA) to quantify this loss, chapter 2 of this thesis shows that DXA underestimates the loss of muscle mass with age in comparison to the gold standard MRI. Muscle mass per se is not enough to determine whether a person has an exceptionally low muscle mass, as it can be readily seen that a healthy tall person will have a larger muscle mass than a small person. Clinicians and researchers thus need an index of muscle mass that takes differences in stature into account and also gives an objective cut off point to define low muscle mass. In Chapter3, we show that femur volume does not significantly differ between young and old. We used this observation to introduce a new index: thigh muscle mass normalised to femur volume, or the muscle to bone ratio. This index allows the examination of the true extent of muscle atrophy within an individual. In previous studies the appendicular lean mass (determined with DXA) divided by height squared appeared to be a relatively poor predictor of functional performance. In Chapter 4, the index introduced in Chapter 3, the muscle to bone ratio, proved to be a somewhat better predictor of functional performance in the overall cohort. This was, however, not true when examining the intra-group relationships. A similar situation applied to the maximal muscle strength. In older adults, the parameter which predicted functional performance best was muscle power per body mass, measured during a counter-movement jump. Chapter 5 shows that part of the larger loss power and force than muscle mass is attributable to a left-ward shift of the torque-frequency relationship, indicative of a slowing of the muscle, and reduction in maximal voluntary activation, as assessed using the interpolated twitch technique in older adults. Chapter 5 also shows that the fatigue resistance during a series of intermittent contractions was similar in young and older adults. However, older adults could sustain a 50% maximal voluntary contraction force longer than young people. Part of this discrepancy maybe due to an age-related slowing of the muscle.

Increasing the temperature of the exercising muscle, passively or actively, leads to alterations in the contractile properties of the muscle, importantly an increase in power output. There is limited information, however, regarding the metabolic changes, if any, occurring within the muscle at higher temperatures and how these are related to the contractile changes occurring within the muscle and how such changes may, or may not, affect the efficiency of the working muscles. The greater power output produced during maximal sprint cycling, after a passive increase in Tm, was associated with an increase in the rate of anaerobic ATP turnover and muscle fibre conduction velocity. Further investigation revealed that this greater anaerobic ATP turnover within the muscle was the result of a greater activity of type HA fibres in the cadence range of 160-180 revs. min⁻¹. When the external power output of the muscle remains constant during more prolonged cycling exercise, performed at 60 revs. min⁻¹, there was also a greater rate of anaerobic ATP turnover in the first 2 min of exercise, with no differences in the remainder of exercise after passive elevation of Tm. There were no differences in the aerobic energy contribution or the kinetics of the V0₂ response between T. conditions. These changes led to a decrease in mechanical efficiency in the first 2 min of exercise, which was associated with a tendency for a greater PCr degradation in type I fibres. When T. was elevated via prior intense exercise there was decrease in mechanical efficiency, during 6 min of heavy exercise, at both 60 and 120 revs. min⁻¹. There was also a greater "absolute" primary amplitude and decrease in the slow component after prior exercise, with the response being greater at 120 revs. min⁻¹. The present research has demonstrated that whilst an increase in T. leads to a greater power output, during maximal exercise, mechanical efficiency is reduced as exercise progresses beyond a few seconds. Furthermore, at faster pedal rates T. affects type IIA fibres whilst at slower pedal rates (60 revs. min⁻¹) there appears to be a preferential effect on type I fibres, highlighting the velocity specific effect of Tm.

The mechanism whereby anabolic androgens affect the myogenic development of skeletal muscle is incompletely understood. Three different forms of assessing elements of this process were utlilised. Firstly, experiments were designed to test whether androgens had any effect on the proliferation and differentiation of C2C 12 cells; then assessment of changes in gene expression after a specified time in differentiation medium was made. Secondly, the effect on androgens on gene expression in differentiating human primary cultured cells was tested has not been reported previously. The third type of study involved a system that used microarray technology to consider whether a candidate gene could be indentified that may be associated with a clinical condition of unexplained spontaneous muscle hypertrophy. Objectives: 1) To assess effects of androgen +1- IGF-l on expression of IGF-l mRNA in muscle-derived precursor cells. 2) To identify potential candidate genes involved in excessive muscle hypertrophy in an adult male subject. Methods:C2C12 or human muscle-derived cells were differentiated in the presence of androgen (testosterone, T, 0-500nM or dihydrotestosterone, DHT, 0-300nM) and/or IGF -1 (10-50ng/ml). Immunostaining and RT -PCR were used to assess the effect of androgens and IGF -l on muscle-specific proteins and the dynamics of relevant gene expression respectively. A microarray methodology was employed to assess the skeletal muscle gene expression of clinical subjects with spontaneous muscle hypertrophy. Results: Significantly increased myogenicity occurred in human muscle-derived cells exposed to IGF-l (p<O.02) after 3 days' (early) differentiation. After 7 days' differentiation, supraphysiological doses of T (+) and IGF-l (+++) were both found to increase fusion index without an observable synergistic effect (p<O.05). Both T and DHT increased IGF-l mRNA generation (p<O.OOOI), an effect attenuated by flutamide (p<O.05), whereas exogenous IGF-l (p<O.OOOI) reduced IGF-l mRNA concentrations relative to control. In addition, T and IGF-l reduced SOCS -2 mRNA expression (p<O.05). In the microarray study, in a single subject with idiopathic muscle hypertrophy, evidence for downregulation of SOCS-2, a negative regulator of growth hormone (GH)/IGF -1 signalling cascade was detected. Conclusions: The mechanism whereby androgens induce muscle hypertrophy may be linked to local IGF -1 generation and/or dynamics of SOCS-2 activity. We describe novel methodologies that may assist in attempting to elucidate the mechanisms of hypertrophy associated with anabolic steroids in skeletal muscle.