Thesis (Ph. D.)--Washington State University, August 2007. / Includes bibliographical references.

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The mechanisms by which hormones and metabolites control muscle proteolysis remain unclear. The redox state of muscle has been investigated as a possible mediator of these effects. In diaphragms of traumatized and starved animals, faster proteolysis correlated with a more oxidized NAD(P) redox couple. In diaphragm, leucine lowered proteolysis and increased lactate/pyruvate ratios. The soleus and extensor digitorum longus (EDL) muscles from young growing rats had faster proteolysis and lower lactate/pyruvate ratios than of older rats. The lower proteolysis and high lactate/pyruvate ratios in larger muscles from older rats are not due to experimental oxygen diffusion limitations, but are probably inherent characteristics of the muscles themselves. Proteolysis, and medium and incubated tissue lactate/pyruvate ratios gave significant multiple correlations with the soleus (R = 0.939) and EDL (R = 0.973) weights. Up to four days of streptozotocin diabetes, the lactate/pyruvate ratios were lower and proteolysis was faster in soleus and EDL muscles compared to weight matched controls. Conversely, after six to 12 days, proteolysis was lower and lactate/pyruvate ratios were higher. In EDL of young fasted rats accelerated proteolysis was associated with lower lactate/pyruvate ratios. In older rats, proteolysis increased and lactate/pyruvate ratios decreased during short-term fasting. The reverse was true following long-term fasting, indicating a "protein-sparing" effect. In contrast, the soleus showed little change in redox state and proteolysis during starvation. Concentrations of reduced, oxidized and protein-bound glutathione were measured in normal, diabetic and fasting rats to determine whether the glutathione redox couple responded similarly to the NAD(P) redox couple. In muscles from fasted and diabetic rats decreases in reduced glutathione content lowered the reduced/oxidized glutathione ratio. The relationship between proteolysis and the glutathione redox state, however, was not strong. Likewise, incubations with insulin of leg muscles of fed, fasted, and diabetic muscles, decreased proteolysis but affected little the NAD and glutathione redox couples. Generally, in muscle accelerated proteolysis was associated with an oxidation of the NAD(P) redox couple and vice versa. These data suggest that, in muscle, the redox state may be important in the regulation of proteolysis.

No description available.

The effects of denervation, non-weight bearing (unloading) or immobilization on hindlimb muscle growth, protein and amino acid metabolism were studied. In the first 3 days after denervation or unloading, atrophy of the soleus was caused by a suppression of protein synthesis and an acceleration of protein degradation. Thereafter, further atrophy, up to 6 days was due to depressed protein synthesis only. The changes in both protein synthesis and degradation in the first three days accounted for 69% and 65%, respectively, of the total loss of protein and mass in 6 days of unloaded or denervated soleus. Over the 6-day period, denervated soleus lost more mass and protein than the unloaded muscle owing to the earlier onset and greater extent of proteolysis. In denervated soleus, both lysosomal and non-lysosomal proteolysis may be enhanced, whereas in the unloaded muscle possibly only non-lysosomal proteolysis was enhanced. In both cases non-lysosomal proteolysis may be mediated by Ca²⁺-activated neutral protease, partially as a result of Ca²⁺ release from sarcoplasmic reticulum. Possibly due to the lack of lysosomal proteolysis, the insulin receptor did not show apparent increased turnover with unloading, as suggested by increased insulin sensitivity of in vitro protein turnover in the unloaded soleus. In contrast, denervated soleus showed a normal response to insulin for in vitro protein turnover. These findings suggested a mechanistic difference of unloading and denervation atrophy of soleus. A decreased ratio of glutamine/glutamate in fresh muscle suggested that the synthesis of glutamine in soleus may be diminished by denervation just as by unloading. This diminution of glutamine synthesis was probably due to reduced availability of ammonia, as evidenced by the slow disappearance of ATP in incubated denervated soleus. Similiar to unloading, denervation led to a decrease in aspartate concentration. This decreased concentration apparently resulted in decreased rather than increased utilization of aspartate. Effects of stretch on unloaded soleus were particularly pronounced in the first two days. Thereafter, in the stretched, unloaded soleus protein degradation increased to nearly the same extent as did protein synthesis. Hence after two days, stretch seems to lose its effectiveness in mitigating the effects of unloading so that it may not be an adequate preventive measure of muscle wasting under non-weight bearing condition.

Young female rats treated with trenbolone demonstrated an increase in weight gain per day and overall weight increase during the treatment period. Trenbolone treated rats also experienced improved feed efficiency. Muscles removed from the lower hind limb of trenbolone treated rats had a greater DNA to protein ratio than muscles from control animals. However, there was no significant difference in wet muscle weight between trenbolone treated and control muscles. Satellite cells from untreated female rats were not responsive to trenbolone added in vitro. In studies utilizing serum free medium, trenbolone alone, and in the presence of growth factors, could not stimulate proliferation above controls. In similar serum free medium studies, satellite cells from trenbolone treated rats were more responsive to growth factors than cells from control rats.

The purpose of this study was to compare the growth response elicited by an acute bout of resistance exercise (RE) conducted on a traditional weight stack device (WS) and a flywheel device (FW). Eight recreationally trained males (25 ± 9 y, 77 ± 27 kg) performed 4 sets of 7 repetitions of bilateral knee extension on each exercise device separated by 7 days. Muscle biopsies were obtained from the vastus lateralis at rest and 4 hrs post-exercise to examine the expression of selected myogenic and proteolytic genes. RE increased (P < 0.05) mRNA expression of Myogenin (3.6 vs. 3.6 fold), and MyoD (2.2 vs. 2.0 fold) and decreased (P < 0.05) expression of Myostatin (1.4 vs. 1.5 fold) to a similar degree on both exercise devices. There was no change in the expression of Atrogin-1, MuRF-1 or MRF4 following RE on either device. The only device mediated difference in the expression of the selected genes was observed in Atrogin-1 which was lower following RE on the FW versus the WS device. The current data shows that in the initial hrs following RE, use of the FW is as effective as the traditional resistance training devices (WS) in promoting the induction of genes involved with muscle remodeling and growth. / School of Physical Education, Sport, and Exercise Science

Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.