Skeletal muscle can adapt its structure to cope with the mechanical and metabolic stresses placed on it by various amounts and patterns of human movement. The release of calcium into the cytoplasm of muscle fibres is thought to have an important role in these adaptations, yet the calcium-dependent signalling pathways involved haven’t been fully defined. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been presumed to drive mitochondrial biogenesis in skeletal muscle, but this has not been investigated in vivo. The experiments in this thesis aimed to address how CaMKII is activated in response to electrical stimulation of skeletal muscle and how CaMKII affects the muscle phenotype. A rat model was used for two main reasons: 1) it allowed for imposing well-defined stimulation patterns onto phenotypically homogenous muscle fibre populations under controlled conditions in situ, and investigating the molecular response to these stimulation patterns, and 2) it allowed for manipulation of CaMKII signalling in muscle fibres in vivo through the use of electro-assisted somatic gene transfer. It was hypothesised that CaMKII would be activated in a muscle and recruitment pattern specific manner. Furthermore, it was hypothesised that CaMKII overexpression would increase the expression of mitochondrial markers. In chapter 2, the effect of recruitment frequency on CaMKII phosphorylation in slow-twitch m. soleus and fast-twitch m. gastrocnemius medialis is investigated. Furthermore, the time course of CaMKII phosphorylation after muscle stimulation is studied. Chapter 3 presents a study into the effects of in vivo CaMKII overexpression in m. soleus and m. gastrocnemius on mitochondrial gene expression and muscle contractile function. The effects of CaMKII overexpression on skeletal alpha-actin transcription are presented in chapter 4. In chapter 5, a mathematical model of CaMKII activation in sarcomeres is described, and used to investigate the effects of CaMKII overexpression on calcium handling and on contractile properties of a muscle fibre. It was concluded that CaMKII is activated by very brief stimulation in a recruitment frequency-independent manner, and that increased CaMKII protein levels increase SERCA expression, but not mitochondrial gene expression.

The concept of skeletal muscle homeostasis - often viewed as the net balance between two separate processes, namely protein degradation and protein synthesis - are not occurring independently of each other, but are finely co-ordinated by a web of intricate signalling networks (Nader, 2005). Such signalling networks are in charge of executing environmental and cellular cues that ultimately determine whether muscle proteins are synthesised or degraded. Prolonged elevations of proinflammatory cytokines are closely associated with muscle wasting that occurs during the sarcopenia of ageing and in cachectic AIDS and cancer patients (Strle et a/. 2007). These clinical disorders occur along with a decline in IGF-I anabolic activity, which is consistent with in vitro findings in muscle progenitor cells (Strle et a/. 2007). Very low concentrations ofTNF-a (0.01-1 ng.ml") inhibit IGF-I-induced protein synthesis (Broussard et a/. 2003; Strle et al. 2004) and expression of the critical muscle differentiation factors, MyoD (Strle et a/., 2004) and myogenin (Broussard et al. 2003; Strle et a/. 2004). Potential treatments that might overcome TNF-a-induced hormone resistance in myoblasts are unknown. Increased activation of the IGF/insulin pathway is an attractive target for combating many of the cachectic conditions associated with muscle wasting. Using rodent skeletal muscle cell lines we have investigated TNF-a/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying the wasting process. We hypothesised that treatment of mouse myoblasts with TNF-a at specific doses ranging from high (20 ng.ml') to low (1.25 ng.ml") would result in dose-dependent block of differentiation and induction of apoptosis and that subsequent IGF-I co-incubations would stimulate myoblast survival and myotube formation. Objectives were to ascertain signalling pathways underpinning these outcomes. In contrast to our hypothesis, a novel role of IGF-I has been identified whereby eo-incubation of skeletal muscle C2 cells with IGF-I (1.5 ng.ml') and a non- apoptotic dose of TNF-a (1.25 ng.ml"; sufficient to block differentiation) unexpectedly were shown to facilitate a significant four-fold increase in myoblast death (P < 0.05). Specificity of the apoptotic potential of this growth factor was confirmed when neither bFGF-2 nor PDGF-BB (10 or 30 ng.ml' and 1.25 or 5 ng.rnl", respectively) were able to reveal the apoptotic potential of low dose TNF-a. By contrast, but in line with our II hypothesis, dosing with 10 ng.ml" TNF-a resulted in a block of differentiation and initiation of apoptosis, which was rescued by IGF-1. Preliminary signalling studies suggest that MAPK activation rather than the caspases are involved in the induction of death associated with low dose TNF-a (1.25 ng.mrl)/IGF-I incubation and therefore blocking the caspases would be without effect in this circumstance. The PI(3)K pathway is involved in the survival effects of high TNF-a (10 ng.mrl)/IGF co-incubations. Importantly, the rescue of death (regardless of the means required) did not facilitate differentiation and did not rescue the block of expression of IGF-ll or IGFBP-5 (produced by skeletal myoblasts as early events in their terminal differentiation and associated with preventing cell death) in our models. Using array technology we further established potential insulin survival and apoptotic genes that were upregulated in the above conditions and confirmed their expression with qRT-PCR. Of these genes three were selected to conduct gene silencing experiments. The gene silencing studies were effective in reducing expression of Adrald, Birc2 and Sirtl. Our findings suggest that inhibition of Adrald leads to an increase in myoblast death in conditions that are associated with myoblast survival and include basal conditions. This novel finding indicates Adrald expression to be essential for the general maintenance of myoblasts. This may be due to the multiple signalling pathways which the al-ARs regulate which include the PI(3)K-Akt pathway that is associated with growth and anti-apoptosis. Birc2 expression, which is upregulated in our cell model under conditions of myotoxic stress showed no significant effect on myoblast survival when suppressed. Associated with inhibition of apoptosis, it was hypothesised that inhibition of Birc2 would result in an increase in myoblast death however levels of damage were comparable to control myoblasts. Recent articles have stated that Birc, only when overexpressed above physiological levels, is associated with anti-apoptosis and consequently have proposed an alternative nomenclature that names the family after its distinctive structural feature, the BIR, rather than by inhibitor of apoptosis proteins lAPs (Silke & Vaux, 200 l; for review Srinivasula & Ashwell, 2008). Finally Sirtl, similar to Birc2 was highly expressed in conditions that induced the greatest incidence of myoblast death. Subsequent inhibition resulted in further increase in death which was not observed under basal conditions where myoblasts received DM alone. Unlike Adrald, this implicates Sirtl expression as a III survival mechanism which is specific for conditions associated with myotoxic stress. The mammalian Sirtl deacetylase was originally shown to modulate life-span in various species. However, the molecular mechanisms by which Sirtl increases longevity and with regard to the present study, survival, are largely unknown. In mammalian cells, Sirtl appears to control the cellular response to stress by regulating the FOXO family of Forkhead transcription factors. The FOXO family members are negatively regulated by the PI(3)K-Akt signalling pathway. Mammalian FOXOs control various biological functions, including cell cycle arrest, differentiation, repair of damaged DNA and apoptosis. Because the ability to regulate apoptosis and repair damage is correlated with increased organismal longevity and survival in many species these particular functions of FOXO transcription factors may be relevant to Sirtl ability to control longevity These experiments in myoblasts show that IGF-I (Lcng.ml') can facilitate apoptosis in the presence of non-a pop to tic doses ofTNF-a (1.25ng.mr\ which appears to depend not only on the upregulation of specific apoptosis genes (potentially downstream of MAPK) but also on the suppression of survival factors IGF-ll and IGFBP-5 which may also lie downstream of MAPK. These studies highlight the complex regulation of cell survival and cell death at the signalling level, as a consequence of interactions of one cytokine, TNF-a, and one growth factor, IGF-I. More information regarding the pathways involved in regulating their expression and activity will be necessary to fully understand the action of these molecules.

A major strategy to alleviate myopathic symptoms through enhancing muscle growth and regeneration is to inhibit the action of Myostatin, a TGF -β family member that inhibits muscle growth. Presently however, no study has expanded the morphological analysis of mouse skeletal muscle beyond a few muscles of the hind limb. Therefore I have initially undertaken an expansive analysis of the skeletal musculature of the mouse forelimb and highlighted the species-specific differences with the rat. Subsequently, I examine the musculature of the forelimb in both young and old wild type and Myostatin null mice and assess the potential beneficial effects of Myostatin deletion on muscle morphology and composition with ageing. I show that the mouse muscle displays a more glycolytic phenotype compared to the rat. I demonstrate that in the absence of Myostatin, the induced myofibre hyperplasia (increase in myofibre number), hypertrophy (increase in myofibre size) and glycolytic conversion (fibre type shift towards more glycolytic phenotype) all occur in a muscle-specific manner. Next, I examine the role of Myostatin deletion on the morphology of the nerve axon. I demonstrate that skeletal muscle hyperplasia in Myostatin null mouse is accompanied by an increase in nerve fibres in major nerves of both the fore and hind limb. In addition, I show that axons within these nerves undergo hypertrophy. Furthermore, I provide evidence that the age related neural atrophic process is delayed in the absence of Myostatin. I show that skeletal muscle hyperplasia in the Myostatin null is accompanied by an increase in the number of muscle proprioceptors. Crucially, I demonstrate that absence of Myostatin reduces the amount of the extracellular matrix connective tissue. Furthermore, Myostatin deletion perturbs age-related collagen formation. Finally, I show that lack of Myostatin increases the number and the proliferation potential of satellite cells (skeletal muscle stein cell). Collectively, these data conclude that Myostatin regulates skeletal muscle development.

The aim of this work was to investigate possible role of PKC in control of excitation-contraction coupling in phasic guinea pig and rat ureter smooth muscle. Immunohistochemistry and western blotting were used to identify the expression of four PKC isoforms α, β, δ and ε in ureter smooth muscle of both species. Photometric system combined with force measurement and electrical activity as well as confocal imaging of isolated ureteric myocites have been used in the functional studies. The effects of PKC activator PDBu and PKC inhibitor Ro320432 on force and Ca²⁺ induced by different modes of stimulation of ureter smooth muscle have been examined. d. It is established that expression and functional effects of PKC activation and inhibition in ureter smooth muscle were species dependent.

It is well documented that regular endurance exercise induces skeletal muscle mitochondrial biogenesis. However, the optimal training stimulus and nutritional intervention for which to maximize mitochondrial adaptations to endurance exercise is not well known. Developments in molecular techniques now permit the examination of the cell signalling responses to acute exercise therefore increasing our understanding of how manipulation of the training protocol and nutrient availability may enhance the training stimulus to a given bout of exercise. The primary aim of this thesis is to therefore characterise the skeletal muscle cell signalling responses thought to regulate mitochondrial biogenesis following an acute bout of high-intensity interval exercise and moderate- intensity continuous exercise. A secondary aim is to subsequently examine how manipulation of carbohydrate (CHa) availability may enhance the activation of key regulatory cell signalling pathways. The aim of the first study (Chapter 4) was to develop two exercise protocols of varied activity profile, which induced comparable total oxygen consumption and energy expenditure after being matched for average intensity, duration and distance ran. In a repeated measures and randomised design, eight active males performed an acute bout of high-intensity interval (HIT) running (6 x 3 min at 90 % V02max interspersed with 6 x 3 min at 50 % V02max also performed with a 7-min warm up and cool down at 70 % V02max) and an acute bout of moderate-intensity continuous (CaNT) running (50-m in continuous running at 70 % V02max). As a result of average intensity (70 % V02max) duration (50-min) and distance ran (9843 ± 176) being equal between protocols, total oxygen consumption (HIT; 162 ± 6, CaNT; 166 ± 10 L) and energy expenditure (HIT; 811 ± 30, CaNT; 832 ± 48 kcal) were matched between protocols (P > 0.05). Despite higher ratings of perceived exertion in HIT compared with CaNT (HIT; 14 ± 0.5, caNT; 13 ± 0.4 AU, P < 0.05), subjects reported greater ratings of perceived enjoyment in the HIT protocol (HIT; 87 ± 2, CaNT; 61 ± 4 AU, P < 0.05) according to the Physical Activity Enjoyment scale. By matching these two protocols for work done, these data therefore provided an appropriate framework for which to examine the molecular signalling responses of human skeletal muscle to acute HIT and CaNT. The aim of the second study (Chapter 5) was to characterise the skeletal muscle cell signalling responses associated with the regulation mitochondrial biogenesis following HIT and CaNT. In a repeated measures and randomised design, muscle biopsies (vastus lateralis) were obtained pre-, post- and 3 h post-exercise from ten active males who performed the HIT and CaNT protocols developed in Chapter 4. Despite the obvious difference in activity profiles between protocols, muscle glycogen (HIT; 116 ± 11, CaNT; 111 ± 17 mmol/kg dry wt) decreased similarly between protocols (P < 0.05), and phosphorylation (P-) of AMPK (HIT; 1.5 ± 0.3, CaNT; 1.5 ± 0.1) and p38MAPK (HIT; 1.9 ± 0.1, CaNT; 1.5 ± 0.2) increased immediately post-exercise before returning to baseline 3 h post exercise. P-p53 (HIT; 2.7 ± 0.8, CaNT; 2.1 ± 0.8) and expression of PGC-1a mRNA (HIT; 4.2 ± 1.7, CaNT; 4.5 ± 0.9) increased 3 h post-exercise in both HIT and CaNT though there were no difference between protocols (P > 0.05). Data therefore demonstrate comparable cell signalling responses between HIT and CaNT when matched for work done, average intensity, duration and distance ran. Furthermore, this is the first time exercise is shown to up-regulate p53 phosphorylation in human skeletal muscle therefore highlighting an additional pathway by which exercise may regulate mitochondrial biogenesis. Progressing from the role of the exercise stimulus in initiating mitochondrial biogenesis, the aim of the third study (Chapter 6) was to examine the effects of reduced CHO availability on modulating the exercise-induced activation of the cell signalling pathways as characterised in Chapter 5. Although HIT and CaNT protocols resulted in comparable signalling in Chapter 5, we chose HIT as our chosen exercise model given that it is perceived as more enjoyable than CaNT, has application for improving both human health and performance and also because of its relevance as a training modality for elite athletes in team and endurance sports. In a repeated measures and randomised design, muscle biopsies (vastus lateralis) were obtained from eight active males pre-, post and 3 h after performing an acute bout of high-intensity interval running with either high (HIGH) or low CHO availability (LOW). In LOW, subjects performed a bout of glycogen depleting exercise the night before and reported to the laboratory on the subsequent morning in a fasted state as well as restricting CHO before, during and after exercise. Subjects in HIGH CHO loaded for 24 h before reporting to the laboratory to perform HIT with CHO consumed before, during and after exercise. Resting muscle glycogen (HIGH, 467 ± 19; LOW, 103 ± 9 rnmol.kq" dw) and utilisation (HIGH, 142 ± 34; LOW, and 30 ± 12) was greater in HIGH compared with LOW (P < 0.05). Phosphorylation (P-) of ACCSer79 (HIGH, 1.4 ± 0.4; LOW, 2.9 ± 0.9), a marker for AMPK activity, and p53ser15 (HIGH, 0.9 ± 0.4; LOW, 2.6 ± 0.8) was higher in LOW immediately post- and 3 h post-exercise, respectively (P < 0.05). Before and 3 h post-exercise, mRNA content of PDK4, Tfam, COXIVand PGC-1a were greater in LOW compared with HIGH (P < 0.05) whereas CPT1 showed trend towards significance (P = 0.09). However, only PGC-1a expression was increased by exercise (P < 0.05) where 3-fold increases occurred independent of CHO availability. Data demonstrate that low CHO availability enhances p53 phosphorylation in a manner that may be related to upstream signalling through AMPK. Given the emergence of p53 as a potential molecular regulator of mitochondrial biogenesis, such nutritional modulation of contraction-induced p53 activation may have implications for both athletic and clinical populations. In summary, the work undertaken from the studies in this thesis provides novel information in relation to the regulation of exercise-induced cell signalling responses associated with mitochondrial biogenesis. Specifically, this is first report to examine cell-signalling responses to running exercise where comparable signalling between HIT and CaNT was observed when protocols are matched for average intensity and duration. Furthermore, these data provide the first report of an exercise-induced increase in p53 phosphorylation in which data demonstrate low CHO availability augments the exercise-induced increase in p53 signalling which may be related to upstream signalling through AMPK. Further studies would now benefit from addressing the nuclear and mitochondrial abundance of p53 in response to an acute exercise challenge as well as comprehensively examining how training status, exercise intensity and CHO availability affects p53 regulation and downstream target genes.

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.