Walleye are an important recreational and commercial fish species that are distributed over an expansive geographic range across North America. However, its palatable white flesh and appeal to anglers have lead to declines in natural populations throughout Canada and the United States. These declines have prompted the idea that aquaculture may serve as a means of satisfying consumer demands and decreasing pressure on wild stocks. While culture programs exist for walleye, little is known about the growth physiology of walleye in a culture system. The goals of this thesis, therefore, were to develop a molecular marker that could be used to rapidly assess growth in juvenile walleye, and to make improvements to culture practices that will optimize growth. To begin, we examined the relationship between growth and the expression of the myosin heavy chain gene (MyHC) in the white muscle of juvenile walleye. The coding region of MyHC from the fast skeletal muscle of walleye was amplified using a full length cDNA. Growth was then characterized using traditional measurements of growth (length, weight and condition factor), as well as MyHC protein concentration and MyHC mRNA levels. Both MyHC mRNA and protein expression were highly correlated with faster growth in juvenile walleye. Over shorter time scales, the MyHC mRNA marker was sensitive enough to detect impacts of fasting that could not be detected using traditional measurements of growth. Next, MyHC mRNA quantification was applied to an aquaculture setting. Feed training is an important bottleneck during juvenile walleye culture that often leads to mortalities and cannibalism. These experiments showed that the brief fasting period during the diet switch from plankton to commercial pellet feed caused a significant decrease in MyHC mRNA levels. Furthermore, the success of feed training in terms of survivorship and growth potential increased significantly for larger fish. The final section of this thesis examined how acute and chronic temperature exposure impacted MyHC mRNA and protein expression. Results showed that the nature of the heat stress can significantly affect the MyHC response. These findings are important as the temperature stresses induced in these studies are common during the summer months in southern Ontario. / Thesis (Ph.D, Biology) -- Queen's University, 2009-02-19 12:30:51.406

Numerous references have suggested that alterations in exercise capacity during congestive heart failure (CHF) are not simply due to changes in myocardial function. In fact, recent evidence has indicated that reductions in skeletal muscle strength and endurance during CHF significantly impact exercise capacity of the CHF patient. Currently, it is believed that alterations in skeletal muscle phenotype, or more specifically a slow to fast transformation in phenotypic protein isoforms contribute to the reductions in muscle function. However, currently there are few data which directly document this slow to fast transformation of the skeletal muscle. Interestingly, it is well established that exercise training can cause changes in skeletal muscle phenotype, more specifically in the fast to slow direction. This is in direct contrast to what is known to occur during CHF. However, it is unclear if similar adaptations will result from training in a CHF patient. Also, it is not clear if the adaptations are due to alterations in the myocardium or changes directly imposed upon the muscle by the exercise training. Therefore, the purpose of this study was two-fold: 1) to clarify the changes in skeletal muscle myosin heavy chain (MHC) during CHF and 2) to determine if skeletal muscle can adapt to increased activity levels, utilizing functional overload (FO) without significantly improving cardiac function. In the first study the mixed plantaris muscles from rats afflicted with severe CHF demonstrated a significant (p<0.05) increase in fast MHC (e.g. IIb expression at the expense of IIx expression) compared to the control animal (SHAM). The mixed red gastrocnemius, regardless of the severity of CHF, exhibited significant (p<0.05) changes in all of the MHC isoforms. The slow soleus and fast white gastrocnemius did not display any significant changes in MHC expression. The changes in MHC isoform significantly correlated with indicators of disease severity, suggesting there may be an existing relationship between skeletal muscle MHC expression and alterations in myocardial function. In the second study, there were no differences exhibited between CHF and SHAM absolute or specific plantaris mass. There was a significant (p<0.05) 30% increase in both absolute and specific mass of the plantaris in the CHF-FO and SHAM-FO groups compared to the CHF and SHAM groups. There was a significant (p<0.05) 3.5% increase in slow MHC I expression and a significant (p<0.05) 6.5% decrease in fast MHC IIb expression in the CHF-FO group compared to the CHF group. In the SHAM-FO group, there was a significant (p<0.05) 4% increase in MHC I expression and a subsequent 8% decrease in fast MHC IIx+IIb in the SHAM-FO compared to the SHAM groups. There were no differences detected in the rates of Ca²⁺ uptake between the CHF-FO, SHAM, and SHAM-FO. However, Ca²⁺ uptake rates were significantly (p<0.05) elevated by 44% in the CHF group when compared to the other three groups. There were very few changes in plantaris SERCA 1 or 2 protein expression between the four groups. These data suggest that during CHF there are alterations in skeletal muscle isoform expression. However, at least some of the data suggest that changes in function are not always associated with changes in phenotype. Instead, it seems that the changes in Ca²⁺ handling may be due to an alteration in a regulatory mechanism. Also, the data indicate that skeletal muscle is adaptable to increases in activity levels without significantly altering myocardial morphology. / Ph. D.

Contractile and cytoskeletal protein concentrations have been shown to differ on the basis of fiber type in whole muscle homogenates. The purpose of this study was to compare the content of the intermediate filament protein, desmin, between type I and type IIa single muscle fibers from a mixed muscle in human subjects. Biopsies were taken from the vastus lateralis of six recreationally active males. Approximately 150 single muscle fibers were dissected from each sample and analyzed using SDS-PAGE to determine myosin heavy chain (MHC) composition. Following identification, muscle fibers were pooled into two groups (MHC I and MHC IIa). Desmin and actin content within the pooled samples was determined via immunoblotting. On average, muscle samples were composed of 51 ± 7 % type I, 2 ± 1% type I/IIa, 27 ± 5% type IIa, 19 ± 4% type IIa/IIx and 1 ± 1% type IIx MHC single fibers. Desmin and actin contents were 40% and 34% higher in type I fibers compared to type IIa fibers, respectively (P < 0.05). However the desmin to actin ratio was similar between pooled type I and IIa single muscle fibers within the vastus lateralis. These data suggest that desmin and actin content is a function of muscle fiber type. These differences in cytoskeletal protein content may have implications for differences in contractile function and eccentric damage characteristics between fiber types.

Cancer cachexia is a debilitating, paraneoplastic syndrome commonly associated with late stage malignancy. It is estimated that ~25% of cancer-related deaths are due directly to complications arising from cachexia (Barton, 2001). Cachexia manifests as severe body wasting, primarily due to the loss of skeletal muscle mass. This study tested the hypothesis that muscle atrophy associated with cancer cachexia could be attenuated by using a unilateral, functional overload (FO) model applied concurrently with tumor development. To accomplish this, Morris hepatoma MH-7777 cells were implanted in adult female, Buffalo rats (n = 12) and allowed to incubate for 6 weeks. FO surgeries (n = 12) were performed five days prior to MH-7777 cell implantation. Over the course of six weeks, healthy, age, sex and strain-matched, vehicle-injected rats (n = 12) gained ~5% of body weight compared to tumor-bearing rats that lost ~6% of body weight when adjusted for tumor mass. Tumor-bearing animals experienced significant atrophy to gastrocnemius, tibialis anterior, extensor digitorum longus, plantaris and diaphragm muscles. FO successfully reversed plantaris muscle atrophy in cachectic, tumor-bearing rats (n=5). FO plantaris masses were ~24% larger than contralateral controls. However, this hypertrophic response was not as great as FO plantaris muscles from healthy, sham-operated controls (~44% larger than contralateral controls, n=5). FO plantaris muscles from tumor-bearing rats had ~1.5 fold increase in myonuclei/fiber ratios compared those of sham-operated, tumor-bearing controls (n = 6). Therefore, cancer cachexia did not prevent myonuclear accretion necessary for skeletal muscle hypertrophy. Little data exists on adaptations to myosin heavy chain (MHC) isoforms in cachectic skeletal muscle. Plantaris muscles from tumor-bearing rats displayed decreased percentages of MHC type I compared to plantaris muscles from vehicle-injected controls (7% vs. 3%, respectively). However, FO plantaris muscles from tumor-bearing rats had an increased percentage of MHC type I and decreased percentage of MHC type IIb compared to sham-operated tumor-bearing rats, adaptations commonly seen in trained muscles. Therefore, cancer cachexia did not prevent the capability of skeletal muscle to respond normally to hypertrophic stimuli. This study also attempted to characterize a mechanism responsible for the hypertrophic response, increased myonuclei/fiber ratio and transition toward a slower MHC profile in FO plantaris muscles from tumor-bearing rats. Recently, the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, has been suggested as a critical factor regulating skeletal muscle growth and fiber-type dependent gene expression (Chin, 1998; Wu, 2000; Olson, 2000; Otis, 2001). The protein content of the catalytic subunit (CaNa) and the regulatory subunit (CaNb) of calcineurin were unchanged in plantaris muscles from tumor-bearing animals compared to healthy controls. Furthermore, total and specific (normalized to CaNa protein content) calcineurin phosphatase activity were not altered in any group. Therefore, calcineurin activity did not appear to be associated with the regulation of the morphological and physiological response of hypertrophying plantaris muscles in cachectic, tumor-bearing rats. Overall, this study indicated that atrophied plantaris muscles from tumor-bearing animals have a reduced capacity to hypertrophy potentially due to a decreased myonuclei/fiber ratio. Furthermore, it is unlikely that changes to mass and MHC isoform expression are associated with calcineurin phosphatase activity. / Ph. D.

Fully differentiated muscle fibers can undergo considerable phenotypic changes in order to adjust to changing conditions of the physiological environment. It is generally accepted that the electrical impulses a muscle receives play a role in modulating the quantities of metabolic proteins (glycolytic and oxidative enzymes) and types of contractile proteins (myosin heavy chain, MHC) that are expressed. Research has shown that decreased neuromuscular activation following spinal cord transection (ST) results in adaptations in the physiological characteristics of paralyzed muscles, including atrophy and an accompanying loss of force production, and transformations of contractile and metabolic proteins toward a more fatigable state. However, it remains unclear whether or not a strong interdependence of energy metabolism and MHC isoform composition persists. Therefore, the goal of this study was to identify and quantify relative myosin heavy chain (MHC) isoform expression and metabolic enzyme profile adaptations at multiple time points (1, 3 and 6 months) in soleus fibers of rats following spinal cord transection (ST). To accomplish this, female Sprague-Dawley rats (~150 g, n = 15) were subjected to complete transection of the spinal cord at a mid-thoracic level. Age and weight-matched, non-operated rats served as controls (n = 15). The soleus was processed for quantitative single fiber histochemical analyses for succinate dehydrogenase (SDH, oxidative marker) and a-glycerophosphate dehydrogenase (GPD, glycolytic marker) activities (~30 fibers/muscle) and immunohistochemical analysis for MHC isoform composition. The total number of soleus fibers analyzed was ~900. Oxidative capacity was increased in muscle fibers at all time points after ST. Specifically, SDH activity was significantly higher than controls by 142, 127 and 206% at 1, 3 and 6 months post-ST, respectively. ISDH, a measure of total oxidative power, also increased in muscle fibers at all time points after ST. For example, 6 months after ST ISDH activity was 93% higher than controls (91.8-3.8 vs. 47.6-0.9 OD x 10-3, respectively). Glycolytic capacity peaked one month after ST. Thereafter, glycolytic capacity of all fibers steadily declined. For example, by 6 months, GPD activity had declined by 76% compared to 1 month GPD activities (3.3-0.2 vs. 13.7-1.4 OD x 10-3, respectively). These data suggest that the increases in glycolytic capacity are transient as fibers transition toward a faster MHC phenotype and then return towards control levels as fibers of a given type become phenotypically stable. The GPD/SDH ratio, an index of metabolic substrate utilization, peaked at one month after ST (394-41) and significantly decreased at 3 months (224-10) and at 6 months (95-7) after ST. Therefore, a shift occurred such that a greater dependence on oxidative metabolism was apparent. These data suggest that the oxidative capacities of soleus muscle fibers are not compromised after ST. In fact, as the fibers transitioned toward faster MHC isoforms, the GPD/SDH ratio was maintained or decreased, suggesting a reliance on oxidative metabolism regardless of MHC isoform composition. This might imply a dissociation between the contractile and metabolic characteristics of paralyzed soleus muscle fibers. However, these data are consistent with previous data and suggest that the increased fatigability observed after chronic reductions in neuromuscular activity are not due to compromised capacities for ATP synthesis. / Master of Science

<p>The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study.</p><p>Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.</p>

A Disfunção temporomandibular (DTM) é um termo coletivo que abrange um largo espectro de problemas clínicos da articulação e dos músculos na área orofacial; estas disfunções são caracterizadas principalmente por dor, sons na articulação, e função irregular ou limitada da mandíbula. Os músculos da mastigação podem estar envolvidos na DTM de origem muscular, e por definição são os músculos que promovem o toque dental, portanto os elevadores da mandíbula: masseter, temporal, pterigóideos medial e lateral. A origem muscular da DTM é a mais prevalente, sendo portanto,o entendimento funcional e estrutural da composição dos músculos da mastigação essencial para a compreensão desta DTM. Este estudo tem como objetivo analisar a estrutura dos músculos da mastigação quanto a variação do calibre das fibras lentas e rápidas, densidade capilar e da expressão da miosina neonatal com a variação da idade. Foram estudadas 37 amostras dos músculos temporal e masseter (20 amostras do sexo masculino e 17 do sexo feminino) de autópsias do Serviço de Verificação de Óbitos de São Paulo com intervalo pós-mortem de até 18 horas, de ambos os gêneros e com idades divididas por décadas (1a a 9a décadas). Foram realizadas reações imunoistoquímicas com os anticorpos Ulex europaeus biotinilada aglutinina, anti-miosina neonatal, anti-miosina rápida, anti-miosina lenta para análise da expressão das proteínas. A avaliação foi feita por dois observadores, após calibração intra observador, (duas contagens no mesmo campo pelo mesmo observador em tempos diferentes) e inter observador (contagem do mesmo campo por dois observadores), até se atingir uma margem de erro menor que 10%. Em relação ao número de capilares/fibra, nos músculos masseter e temporal, da primeira a nona década, em média respectivamente foi de 1 e 0,7 respectivamente. O número de capilares por mm², nos músculos masseter e temporal, não variou ao longo das nove décadas estudadas, e o número de capilares por mm², foi significantemente maior no músculo masseter quando comparado ao temporal, (p=0,025). A miosina neonatal manteve-se presente embora com decréscimo em todas as décadas dos músculos masseter e temporal. Observou-se o diâmetro das fibras do tipo II menores que as fibras do tipo I / Temporomandibular disfunction (TMD) is a colletive term that refers to different clinical problems of the temporomandibular joint and the jaw muscles. These disfunctions are characterizied meanly by pain, joint sounds and irregular or limited mandibular function. The jaw muscles can be involved in the TMD of muscle etiology, and by definition they are the ones which provide the teeth touch, then the jaw elevators: masseter, temporalis, medial pterygoid and lateral pterygoid. As the muscular etiology is the most prevalent cause of TMD, the detailed understanding of structural and functional composition of the masticatory muscles is paramount to better comprehend TMD due to muscle disorder. This study has the aim to analyze the jaw muscle structure concerning capillarie density, neonatal myosin expression, and the cross sectional area of the fast and slow fibers in temporalis and masseter muscles in autopsy samples from 1st to 9th decades of age. Thirty seven temporalis and masseter muscles samples were studied (20 from male and 17 from female) from Serviço de Verificação de Óbitos of São Paulo. The specimens were divided by gender and ages. The samples were collected up to 18 hours post-mortem. Imunohistochemistry stainning were made with antibodies to analize the protein expression. The evaluations were made by two observers, after intra observer calibration (two evaluations on the same field by the same observer in different times) and inter observer (evaluation of the same field by two observers), unti getting less than 10% of error. The number of capillaries per fiber in the masseter and temporalis muscle was in average 1 and 0,7 respectively. The number of capillaries per mm² was significantly higher in the masseter when compared to temporalis muscle (p=0.025). The neonatal myosin was present in all decades in both muscles, and it was observed that the cross sectional area of the type II fibers was smaller than the type I fibers

Myocardial infarction results in tissue necrosis, leading to cell loss and ultimately to cardiac failure. Implantation of immature progenitor cells into the scar area may compensate for the cell loss and provides a new therapeutic avenue for infarct treatment. Premature myoblasts derived from skeletal muscle are one of the best candidates for this therapeutic purpose, because biopsies used for autologous cell therapy can be accessed easily, the isolated myoblasts can proliferate well in vitro, and the skeletal and cardiac muscles are structurally and functionally similar. In this study we investigated the survival and differentiation of the implanted skeletal myoblasts in the non-cryoinjured myocardium and the myocardial scar, using a syngeneic Lewis rat model. A therapeutic dose of 4x106 skeletal myoblasts/animal was implanted into the non-cryoinjured and scar tissue, and the fate of the implant was monitored at 12, 28 and 56 days after implantation by immunohistochemistry. We detected fast myosin heavy chain (fMHC) expression at each time point but significantly fewer positive cells in the scar than in the non-injured tissue. This was consistent with the staining patterns of slow myosin heavy chain (sMHC) and myogenin that overlapped with fMHC positive areas. Although the implanted myoblasts differentiated into skeletal muscle cells, they did not transdifferentiate into cardiac muscle, demonstrated by the absence of cardiac troponin I expression. During this analysis we developed a model, which could be useful to test new strategies for myoblast implantation (dosage, genetic modification, new injection technique etc.) designed to promote better engraftment of cultured myoblasts in the myocardial scar.

Muscles control body posture and movement by extrafusal and intrafusal (muscle spindle) fibres. The purpose of this thesis was to provide insight into the muscular basis for human jaw function at young age. Extrafusal and intrafusal fibres in the young masseter, and for comparison young biceps, were examined for composition of fibre types and myosin heavy chain (MyHC) isoforms by means of morphological, enzyme-histochemical, biochemical and immuno-histochemical techniques. For evaluation of plasticity during life span the data for young muscles were compared with previous reported data for adult and elderly muscles. The results showed significant differences in extrafusal fibre types and MyHC expression between young masseter and young biceps and between young masseter and masseter in adults and elderly. Compared with young biceps, young masseter was more intricate in composition of extrafusal MyHC expression. Muscle spindles were larger and more frequent in the masseter than in the biceps. Masseter and biceps muscle spindles showed fundamental similarities but also marked differences in MyHC expression. The results suggest that the young masseter is specialized in fibre types already at young age and shows a unique fibre type growth pattern. Whereas masseter extrafusal fibres display marked plasticity in fibre types and MyHC isoforms during life span muscle spindles/intrafusal fibres are morphologically mature already at young age and precede extrafusal fibres in growth and maturation. Results showed similarities in intrafusal MyHC expression between young masseter and biceps, but also differences implying muscle specific proprioceptive control. Differences in fibre types and MyHC expression between young masseter and young biceps extrafusal fibres are proposed to reflect diverse evolutionary and developmental origins and accord with the masseter and biceps being separate allotypes of muscle.

The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study. Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.