Creatine kinase (CK) primarily serves as an energy buffer assisting in regulating ATP homeostasis through synthesis of ATP from ADP and phosphocreatine (PCr). This enzyme is bound in the sarcomere near sites of ATP consumption via acto-myosin ATPase (A‧M‧ATPase) and research in cardiac muscle has found that PCr can alter contractile performance (maximal isometric force and Ca²⁺ sensitivity). Based on this evidence, CK and A‧M‧ATPase may be coupled in skeletal muscle. Therefore the purpose of this investigation was to determine the influence of the CK system on contractile performance and energy utilization in skeletal muscle. When skinned fibers (membrane removed) were provided a limited supply of [ATP] (0.1 mM), this resulted in increased Ca²⁺ sensitivity. The addition of PCr to low ATP solutions restored Ca²⁺ sensitivity and allowed normal isometric force generation across a range of [Ca²⁺] via ATP synthesis by CK. This was also possible with only CK reaction substrates (ADP, PCr) in the absence of ATP. Based on these findings, endogenous CK activity in glycerol skinned skeletal muscle fibers is sufficient to permit normal function of the contractile apparatus. Energy utilization was studied by indirect assessment of ADP production. Decreased net ADP production as measured by NADH fluorescence revealed endogenous CK was able to convert ADP produced by A‧M‧ATPase to ATP in skeletal muscle across a range of both [Ca²⁺] and [ATP]. This was confirmed directly via high-performance liquid chromatography measurements of ATP and ADP by showing that skinned skeletal muscle bundles have sufficient endogenous CK activity to produce ATP from substrates (ADP, PCr) and the ability to maintain low [ADP] in the presence of PCr. This study adds to the evidence for specific compartmentation of CK near sites of ATP utilization and contributes to the body of knowledge on contractile performance in skinned skeletal muscle fibers. By showing how changing demands on skeletal muscle (through increased Ca²⁺) alters force production and Ca²⁺ sensitivity, these findings lend support for the importance of endogenous CK as a pathway of ATP regeneration in skeletal muscle. / Ph. D.

The influence of body size on metabolic rate, muscle enzyme activities, and the underlying patterns of mRNA for these enzymes were explored in an effort to explain the genetic basis of allometric variation in metabolic enzymes. Two pairs of sister species of centrarchid fishes were studied: black bass (largemouth bass, Micropterus salmoides and smallmouth bass, Micropterus dolomieui), and sunfish (pumpkinseed, Lepomis gibbosus and bluegill, Lepomis macrochirus). The goal was to assess the regulatory basis of both intraspecific and interspecific variation in relation to body size, as well as gain insights into the evolutionary constraints within lineages. Whole animal routine metabolic rate showed scaling coefficients not significantly different from 1, ranging from +0.87 to +0.96. However, there were significant effects of body size on the specific activities of oxidative and glycolytic enzymes. Mass-specific activity of the oxidative enzyme citrate synthase (CS) scaled negatively with body size in each species, with scaling coefficients ranging from -0.15 to -0.19 whereas the glycolytic enzyme pyruvate kinase (PK) showed positive scaling, with scaling coefficients ranging from +0.08 to +0.23. The ratio of mass-specific enzyme activity in PK to CS increased with body size, whereas the ratio of mRNA transcripts of PK to CS was unaffected, suggesting the enzyme relationships were not due simply to transcriptional regulation of both genes. The mass-dependent differences in PK activities were best explained by transcriptional regulation of the muscle PK gene; PK mRNA was a good predictor of PK specific enzyme activity within species and between species. Conversely, CS mRNA did not correlate with CS specific enzyme activities, suggesting post-transcriptional mechanisms may explain the observed inter-specific and intraspecific differences in oxidative enzymes. / Thesis (Master, Biology) -- Queen's University, 2007-10-31 11:55:28.757

Introduction: Recent evidence in isolated mitochondria and permeabilized muscle fibers in ex vivo using simultaneous measurements of O2 consumption and ATP production suggest that mitochondrial efficiency provides an additional mechanism to fine-tune oxidative phosphorylation rate to ATP demand in skeletal muscle. However, in the absence of a direct measurement of both VO2 and ATP synthesis from the same region of the contracting muscle, whether this mechanism plays a role in the skeletal muscle in vivo is still unknown. Purpose: Using a noninvasive approach combining phosphorus and proton magnetic resonance spectroscopy (31P/1H-MRS), the present study aimed to determine skeletal muscle ATP synthesis rate and muscle VO2 during a graded dynamic plantar flexion exercise to determine mitochondrial efficiency in contracting skeletal muscle contraction. Method: To measure mitochondrial efficiency under physiological conditions, we applied a recently developed methodological approach in ex vivo to human gastrocnemius muscle in vivo using 31P/1H-MRS noninvasive techniques. We conducted a series of constant workloads and ischemic protocols to assess oxidative ATP synthesis (ATPox) rate and Myoglobin-derived oxygen consumption (Mb-derived VO2). Specifically, during two separate visits, in 12 healthy, sedentary to recreationally active young male adults, we determined the ATPox rate by measuring the initial phosphocreatine (PCr) resynthesis rate during recovery and Mb-derived VO2 during 30 seconds of occlusion at the end of each given exercise workload. Results: The calculated mean power output during constant load performed by all subjects increased linearly at each incremental workload for both 1H and 31P visits. The mean percent coefficient of variation (CV%) at all exercise workloads (25% of WRmax= 6.0 ± 6.6, 50% of WRmax = 4.5 ± 2.7, 75% of WRmax = 4.9 ± 3.9, 100% = 7.0 ± 4.5) demonstrated high reliability and reproductivity in power output between each visit. The mean concentration of PCr consumption at the steady state remained constant between 25 and 50% of WRmax (28.6 ± 1.7 and 28.2 ± 1.4, respectively) but increased linearly beyond 50% of WRmax (75% = 22.9 ± 1.4 mM and 100%= 18.0 ± 1.4 mM, respectively). Mean intracellular pH was not significantly different between 25 and 50% of submaximal workloads (6.98 ± 0.02 and 6.97 ± 0.02, respectively). Intracellular pH dropped to 6.94 ± 0.02 during the last min of exercise at 75% of WRmax and further decreased at 100% of WRmax, (6.87 ± 0.03). The percentage of Mb oxygenation level and partial pressure of oxygen(PO2) at the steady state exhibited a consistent linear decline with increasing workload. Accordingly, a distinct and strong linear relationship was found between the MbO2 and workload (r2 = 0.71). Similarly, as the exercise workload increased, the ATPox synthesis rate also increased linearly throughout all exercise workloads (r2 = 0.45). In contrast, there was no significant change in Mb-derived VO2 with increased exercise workload averaged over a 30 s (r2 = 0.36) and 10 s time frame (r2 = 0.17). As a result, there was no significant correlation between ATPox synthesis rate and Mb-derived VO2 across exercise intensities. However, at rest, the P/O ratio in the gastrocnemius muscle was 1.95 ± 0.68, consistent with theoretical values and previous studies in mice. Conclusion: Despite the decrease in MbO2 (%), Mb-derived VO2 from both 30 and 10 s averages remained relatively constant during the ischemic protocol, likely due to O2 availability limitation induced by the prolonged occlusion and the slow time-resolution for measuring the dMb signal, which precluded the quantification of mitochondrial efficiency during exercise. However, mitochondrial efficiency calculated at rest was in agreement with previously documented values using other methodologies and thus can provide an additional parameter to more comprehensively evaluate mitochondrial function in vivo.

Au cours de la croissance et de la maturation, le muscle squelettique subit de nombreux changements. Principalement on constate une augmentation considérable de la masse musculaire concomitante à l’augmentation de la capacité de force et plusieurs résultats suggèrent que la maturation affecte la fonction musculaire à la fois au niveau de la commande motrice et du métabolisme énergétique. Pour des raisons éthiques évidentes, la fonction musculaire n’a été que peu étudiée chez l’enfant. Malgré leur caractère strictement non-invasif les techniques de résonance magnétique n’ont été que peu utilisées pour caractériser cette fonction chez l’enfant et les résultats controversés ne permettent pas de dégager un consensus. Dans ce travail la fonction musculaire de l’enfant sain a été étudiée in vivo par Imagerie et Spectroscopie de Résonance Magnétique. Par IRM, nous nous avons mis en évidence que la capacité de force volontaire maximale d’un muscle reste proportionnelle à sa taille de l’enfance à l’âge adulte. Par ailleurs nos résultats obtenus par SRM du P31 ont clairement montré que la capacité oxydative et la production d’ATP mitochondriale était augmentée avant la puberté, illustrant que les enfants sollicitaient plus leur métabolisme aérobie que les adultes pour répondre à la demande énergétique pour une intensité donnée. De plus, nos résultats ont montré que la filière énergétique de la glycolyse anaérobie était pleinement mature dès l’enfance. Enfin, face à la difficulté pour mettre en place des études longitudinales chez l’homme nous avons développé un protocole expérimental permettant le suivi longitudinal de la fonction musculaire au cours du développement chez le rat. / Growth and maturation are accompanied by important changes in skeletal muscle function (e.g. muscle mass and strength dramatically increase). Moreover, some evidences strongly suggest that maturation significantly affects skeletal muscle function both at the neural drive and energetics levels. For ethical reasons, few studies have been performed in children. Despite their non traumatic aspect the MR techniques, it has been barely used in this context. In this work, the skeletal muscle function of healthy children has been characterized in vivo using MRI and 31P-MRS. Our results refuted the hypothesis of a motor drive immaturity in children. We did not report any change in the relationship between muscle volume measured by MRI and maximum isometric strength or in specific strength from childhood to adulthood. The ability of a given muscle volume to produce force seems not to change during growth. Then, we investigated whether development affects muscle energetics using 31P-MRS comparing prepubescent boys and men. Our results showed that, for a similar total energy cost, the aerobic contribution to ATP production was significantly higher in boys and compensated for by a reduced PCr breakdown while glycolysis was similar whatever the age. In addition, the recovery rate of PCr after the standardized exercise was faster in boys illustrating a higher maximal oxidative capacity before puberty. Finally, our understanding of skeletal muscle function in children is still limited by the difficulty to perform longitudinal studies. In that respect, we have initiated an original protocol allowing the longitudinal investigation of the gastrocnemius muscle throughout development in rat.