Thesis (M.Ed.)--Bowling Green State University, 2005. / Document formatted into pages; contains vii, 54 p. : ill. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Outre son rôle dans le métabolisme du cycle de l’urée, la citrulline possède de nombreuses propriétés notamment celle de stimuler la synthèse protéique musculaire. Or, la synthèse protéique est un poste de dépense énergétique important de la cellule. Nos travaux ont ainsi exploré, à la fois in vivo et in vitro, les effets de la citrulline sur le métabolisme énergétique afin de comprendre comment l’activation de la synthèse protéique musculaire par cet acide aminé est coordonnée avec le métabolisme énergétique. Nos résultats ont permis de mettre en évidence que la citrulline module bien le métabolisme énergétique, notamment via une réorientation des flux énergétiques au profit de la synthèse protéique. De plus, nos travaux ont précisé les effets de la citrulline sur le métabolisme protéique avec notamment un effet synergique de la citrulline et de l’exercice sur la synthèse protéique et sur la performance. Enfin, ces travaux ont permis d’explorer pour la première fois in vitro les effets de la citrulline (et de la leucine) sur le sécrétome musculaire. Nous avons ainsi démontré que la citrulline module le sécrétome musculaire et mis en évidence la complexité de régulation des protéines sécrétées par les acides aminés. En conclusion, nos travaux sont une contribution à la meilleure compréhension de la régulation musculaire du métabolisme protéino-énergétique par la citrulline. / Besides his role in the metabolism of the urea cycle, citrulline has many properties including the ability to stimulate muscle protein synthesis. However, protein synthesis is an important item of cell energy expenditure. Our work has explored both the in vivo and in vitro effects of citrulline on energy metabolism in order to understand how the activation of muscle protein synthesis by this amino acid is coordinated with energy metabolism. Our results have shown that citrulline modulates energy metabolism via a reorientation of energy flux in favor of protein synthesis. Moreover, our work has clarified citrulline effects on protein metabolism with a synergistic effect of citrulline and exercise on protein synthesis and performance. Finally, this work allowed to explore for the first time citrulline (and leucine) effects on muscle secretome. We have thus demonstrated that citrulline modulates muscle secretome and highlighted how complex is the regulation of secreted proteins by amino acids. In conclusion, our work contributes to a better understanding on muscle regulation of protein-energy metabolism by citrulline.

No description available.

There are two opposing schools of thought concerning the source of calcium ions for the initiation and maintenance of contractions of smooth muscle. Bohr (1964) and Woodward et al. (1970) believe that the calcium for the initiation of contraction is released from bound intracellular stores, whereas Somlyo et al. (1969) believe that most of the activator calcium comes from the extracellular fluid. It was felt that the determination of the source(s) and sink(s) of calcium ions in arteriolar smooth muscle would be required for an understanding of the control of peripheral blood pressure, and so experiments to obtain this information were carried out using the rabbit anterior mesenteric portal vein as a model of arteriolar smooth muscle. Spontaneous contractions of the vein stop within one minute after the addition of EGTA to the bath and can be returned by simply raising the extracellular calcium concentration. The addition of 1 mM MnC1₂, or raising the MgC1₂ concentration above 5 mM, or raising the CaC1₂ concentration above 10 mM all inhibit spontaneous activity; this inhibition can be rapidly reversed by the addition of appropriate amounts of EGTA. The removal of free extracellular calcium either by adding EGTA or placing the vein in calcium-free solution inhibits the responses to all agonists within five minutes. If the calcium concentration is reduced from 2.5 mM to 0.1 mM, the responses to agonists are greatly decreased. If one sets the response in normal Kreb's solution to each concentration of agonist equal to 100 per cent, then the relative reduction of responses in low calcium solution is inversely proportional to both the potency and concentration of the agonist used. If, however, one produces contractions by adding calcium to tissues bathed in calcium-free solution containing noradrenaline, then the curves of the relative response versus calcium concentrations are independent of the concentration of noradrenaline. The addition of either EGTA or manganese to a tissue already contracted in response to any agonist produces a rapid relaxation to a decreased, but sustained tension. The degree of relaxation is proportional to the concentration of manganese or EGTA added. The addition of manganese is also able to inhibit the initiation of responses to noradrenaline, KC1, serotonin, histamine and procaine. The inhibition by manganese of the responses to noradrenaline, KC1, and serotonin but not histamine and procaine can be reversed by increasing the extracellular calcium concentration. The addition of MnC1₂ or LaCl₃ does not selectively inhibit a slow phase of the contraction to noradrenaline in the mesenteric portal vein as it does in aorta (Van Breeman, 1969). The effect of adding MnCl₂ is similar to the effect of decreasing extracellular calcium, in that the relative inhibition of response is inversely proportional to the potency and concentration of agonist used. The effect of altering pH is the same on contractions produced by each agonist tested; lowering the pH below 7.4 inhibits the responses, raising it above pH 7.4 potentiates the responses. If the vein is placed in calcium-free solution containing EGTA for 10 minutes, the addition of CaC1₂ produces a contraction. The response to calcium is transient unless the final concentration of the added calcium is 5 mM or greater; at these higher calcium concentrations the response is biphasic; an initial transient response is followed by a slow tonic response. The addition of stock solutions to produce final concentrations of 5 mM Mg⁺⁺ or 0.5 mM Mn⁺⁺ or 0.1 mM Ca⁺⁺ in the bath after the EGTA treatment abolishes the transient responses to calcium but has little effect on the tonic portion of a contraction produced by 10 mM Ca⁺⁺. When these EGTA experiments are carried out in low sodium solution (17% of normal or less) the response to 2.5 mM Ca⁺⁺ which is normally transient, becomes instead a sustained contraction. It is concluded then that: 1. Calcium ions themselves are able to control the permeability of the membrane to calcium. 2. The initiation of responses to all agonists probably involves the release of membrane-bound calcium and the influx of extracellular calcium. Differences in efficacy are probably due to differences in ability to release the membrane-bound calcium. 3. A continued influx of calcium is required to maintain a contraction produced by any agonist. 4. Manganese competes with calcium at a membrane site to inhibit the initiation of contraction, and to relax a tissue which is already contracted. 5. Responses to all agonists are potentiated at a pH greater than 7.4, and decreased below pH 7.4. 6. The relaxation process in the mesenteric-portal vein seems to depend in some manner upon extracellular sodium ions. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Mitochondrial dysfunction is a common feature in a wide range of disorders and diseases from obesity, diabetes, cancer to cardiovascular diseases. The overall goal of my doctoral research has been to investigate mitochondrial metabolic dysfunction in skeletal and cardiac muscles in the context of chronic disease development. Perinatal nutrition is well known to affect risk for insulin resistance, obesity, and cardiovascular disease during adulthood. The underlying mechanisms however, are poorly understood. Previous research from our lab showed that the in utero maternal undernutrition mouse model is one in which skeletal and cardiac muscle physiology and metabolism is impaired. Here we used this model to study the impact of in utero undernutrition on offspring skeletal primary muscle cells and to determine if there is a cell autonomous phenotype. Metabolic analyses using extracellular flux technologies revealed a shift from oxidative to glycolytic metabolism in primary myotubes. Gene expression profiling identified significant changes in mRNA expression, including an upregulation of cell stress and OXPHOS genes and a downregulation of cell division genes. However, there were no changes in levels of marker proteins for mitochondrial oxidative phosphorylation (OXPHOS). Findings are consistent with the conclusion that susceptibility to metabolic disease in adulthood can be caused at least in part by muscle defects that are programmed in utero and mediated by impaired mitochondrial function. In my second project, the effects of the absence of glutaredoxin-2 (Grx2) on redox homeostasis and on mitochondrial dynamics and energetics in cardiac muscle from mice were investigated. Previous work in our lab established that Grx2-deficient mice exhibit fibrotic cardiac hypertrophy, and hypertension, and that complex I of OXPHOS is defective in isolated mitochondria. Here we studied the role of Grx2 in the control of mitochondrial structure and function in intact cells and tissue, as well as the role of GRX2 in human heart disease. We demonstrated that the absence of Grx2 impacts mitochondrial fusion, ultrastructure and energetics in mouse primary cardiomyocytes and cardiac tissue and that provision of the glutathione precursor, N-acetylcysteine (NAC) did not restore glutathione redox or prevent impairments. Furthermore we used data from the human Genotype-Tissue Expression consortium to show that low GRX2 expression is associated with increased fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, our results indicate that GRX2 plays a major role in cardiac mitochondrial structure and function, and protects against left ventricle pathologies in humans. In my third project, we collaborated with cardiac surgeon, Dr. Calum Redpath, of the Ottawa Heart Institute to study atrial mitochondrial metabolism in atrial fibrillation patients with and without type 2 diabetes (T2DM). T2DM is a major risk factor for atrial fibrillation, but the causes are poorly understood. Atrial appendages from coronary artery bypass graft surgery were collected and analyzed. We showed an impaired complex I respiration in diabetic patients with atrial fibrillation compared to diabetic patients without atrial fibrillation. In addition, and for the first time in atrial fibrillation patients, mitochondrial supercomplexes were studied; results showed no differences in the assembly of the “traditional” complexes but a decrease in the formation of “high oligomeric” complexes. A strong trend for increased protein oxidation was also observed. There were no changes in markers for OXPHOS protein levels. Overall findings reveal novel aspects of mitochondrial dysfunction in atrial fibrillation and diabetes in humans. Overall, our results reveal that in utero undernutrition affects the programming of skeletal muscle primary cells, thereby increasing susceptibility to metabolic diseases. In addition, we show that GRX2 impacts cardiac mitochondrial dynamics and energetics in both mice and humans. Finally, we show impaired mitochondrial function and supercomplex assembly in humans with atrial fibrillation and T2DM. Ultimately, understanding the mechanisms causing mitochondrial dysfunction in muscle tissues during chronic disease development will increase our capacity to identify effective prevention and treatment strategies.

<p> Local renin-angiotensin systems (RASs) have been described in many mammalian tissues. However, the role of angiotensin II (Ang II) in skeletal muscle is poorly understood with initial reports suggesting it may function to regulate overload-induced hypertrophy. Therefore, the purpose of this thesis was to 1) investigate the potential that adult skeletal muscle and muscle stem cells possess a local RAS. 2) Describe its role in regulating skeletal muscle regeneration and growth following injury and 3) demonstrate its capacity to regulate muscle stem cell activity and myogenesis. We report that cultured primary and C2C12 myoblasts and myotubes possess a local Ang II signalling system evidenced by the differential expression of angiotensinogen, angiotensin converting enzyme (ACE), and both angiotensin type 1 and 2 (AT1, AT2) receptors. Interestingly, myoblasts demonstrated the capacity to produce Ang II in spite of lacking renin expression. Furthermore, angiotensin receptors demonstrated differential localization with AT1 associated with actin filaments in proliferating myoblasts, and localized to the nucleus in differentiated myotubes. We also report that a local angiotensin system is present in vivo and responsive to myotrauma as cardiotoxin injection (to induce muscle injury) resulted in the increased staining intensity of angiotensinogen and AT1 during myogenesis with a progressive downregulation throughout the regenerative timecourse. </p> <p> To investigate the effects of Ang II signalling blockade on muscle growth and regeneration we induced muscle injury in mice supplemented with captopril (ACE inhibitor) or mice devoid of the AT1 a receptor. Histological analysis revealed that ACE inhibition resulted in a decreased muscle fibre growth, increased proportion of small myofibres, an inability to accrete myonuclei and a robust hyperplasia of muscle fibres. Similarly, AT1 a receptor ablation resulted in decreased muscle fibre growth following injury suggesting that these effects are receptor specific. </p> <p> To investigate the mechanisms underlying these effects we assessed the role of Ang II in regulating muscle satellite cell function. In vitro experiments revealed that Ang II had the ability to regulate the early response of satellite cells to muscle injury by acting as a potent transcriptional activator of quiescent myoblasts and directing their subsequent migration. Furthermore, these migratory effects were mediated through an Ang 11-induced increase in matrix metalloproteinase 2 (MMP2) content and reorganization of the actin cytoskeleton. Interestingly, Ang II may also participate in the fusion of myoblasts as captopril treatment suppressed the expression of markers of differentiation (myogenin) and maintained the expression of markers of proliferation (Pax7, Myf5). In agreement with this, IHC analysis revealed that ACE inhibition also induced a strong trend for a decrease in the proportion of myogenin positive cells following injury. Collectively, these results implicate the activation of local Ang II signalling system as a pleiotropic regulator of skeletal muscle growth. </p> / Thesis / Doctor of Philosophy (PhD)

Molluscan adductor muscles display thick filament regulation. A calcium binding site (regulatory domain) near the neck of the myosin molecule is responsible for controlling the ATPase activity, hence the rate of contraction. In the absence of calcium, the ATPase activity is highly suppressed. When calcium binds to the regulatory domain the inhibition is relieved allowing contraction to occur. Steady-state measurements are insufficient to characterise the ATPase activity in detail because of the dominant contribution from unregulated myosin molecules. Therefore spectoscopic techniques, allied to transient kinetic analysis were used to determine the effect of calcium on the various steps of the HMM ATPase mechanism. ATP, ADP and calcium binding to HMM caused small, but measurable, enhancements (upto 8%) in the proteins tryptophan fluorescence. Stopped-flow fluorescence spectroscopy allowed the kinetics of binding and dissociation to be elucidated. Calcium bound to, and dissociated from, HMM rapidly (108 M-1 S-1 and 25 S-1 respectively). When calcium was bound to the regulatory domain the affinity of the active site for nucleotide was reduced, an effect seen as an increase in the rate constant for nucleotide dissociation. Fluorescent ATP analogues, based on formycin were synthesised. These nucleotides displayed large fluorescence enhancements on binding to HMM (upto 500%). Turnover of FTP by HMM was suppressed 100-fold by the removal of calcium, as determined by transient kinetic measurements. The large fluorescence enhancements seen on binding of various formycin nucleotides allowd the effect of calcium on the association and dissociation processes to be examined in great detail. Binding was found to be a complex, multistep process in which the presence of calcium increased the rate of interconversion of the various HMM/nucleotide complexes by several orders of magnitude.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Science