Discoveries made with KCl-induced contractions have elucidated the more complex reactions involved in GPCRs signaling; once the mechanisms of smooth muscle Ca2+ sensitization and desensitization are fully understood, then the development of advanced treatments for vascular disorders such as hypertension, cerebral and coronary vasospasm, and vascular hyporeactivity following hemorrhagic shock may be possible. Studies have shown that KCl-induced contractions induce Ca2+-sensitization. Therefore, we tested the hypothesis that KCl induced Ca2+-sensitization is due to ROK activation by the increase in [Ca2+]i. To test this hypothesis, rabbit femoral arteries were permeabilized with 20µg/ml α-toxin and 1% Triton X-100 and subjected to different calcium concentrations in the presence or absence of various ROK inhibitors. For a comparison we also used various PKC and MLCK inhibitors and repeated these experiments in intact tissues. We found that either [Ca2+]i alone does not directly activate ROK or the permeabilization technique itself disrupts the normal ROK signaling system. Secondary findings revealed that α-toxin activates PKC pathways; in both chemically permeabilized preparations proteases also appear to be activated and MLCK is the primary kinase responsible for contraction.

Physical activity results in various types of stress in skeletal muscle including energetic, oxidative, and heat stress. Acute exposure to stress impairs skeletal muscle mitochondrial function. In contrast, chronic intermittent exposure to mild stress through exercise training results in increased mitochondrial content and respiratory capacity. While oxidative and energetic stress have received much attention regarding their long-term effect on skeletal muscle mitochondria, heat stress is not well understood. The purpose of this work was to investigate the effects of localized heat therapy on human skeletal muscle mitochondria, and to compare these effects to those of high-intensity interval exercise training. To accomplish this purpose, 35 subjects were assigned to receive 6 weeks of sham therapy, heat therapy, or exercise training; all localized to the quadriceps muscles of the right leg. Two-hour sessions of short-wave diathermy were used for the heat therapy, and identical sessions were used for sham therapy, but the diathermy units were not activated. Forty-minute sessions of single-leg extension, high-intensity interval training were used for the exercise intervention. All interventions took place three times per week. Muscle biopsies were performed at baseline, and after three and six weeks of intervention. Muscle fiber bundles were isolated and permeabilized for measurement of oxygen consumption via high-resolution respirometry. The primary finding of this work was that heat therapy improves mitochondrial respiratory capacity by 24.8 ± 6.2% compared to a 27.9 ± 8.7% improvement following exercise training. Both heat and exercise significantly increased mitochondrial respiration compared to baseline measures (p<0.05). Fatty acid oxidation and citrate synthase activity were also increased following exercise training by 29.5 ± 6.8% and 19.0 ± 7.4%, respectively (p<0.05). However, contrary to our hypothesis, heat therapy did not increase fatty acid oxidation or citrate synthase activity. Neither heat nor exercise training increased mitochondrial respiratory protein content. Overall these results suggest that heat therapy significantly improves mitochondrial function, but not to the same degree as exercise training.

Les jeunes oiseaux exposés au froid assurent leur homéothermie en stimulant les oxydations mitochondriales dans les muscles squelettiques. L’exposition prolongée au froid accroit les capacités de thermogenèse musculaire grâce à une plasticité bioénergétique mitochondriale dont le contrôle reste hypothétique. Chez les mammifères, des protéines de fusion (les mitofusines (Mfns) et OPA1(OPtic Atrophy 1)) participent au remaniement des réseaux dynamiques mitochondriaux dans de multiples types cellulaires. Le but de ce travail de thèse était de caractériser l’expression d’homologues aviaires des protéines de fusion mammaliennes et d’étudier leurs variations d’expression lors de la mise en place des processus bioénergétiques chez l’oiseau en croissance, lors d’une exposition aiguë ou prolongée au froid ou lors de challenges nutritionnels ou endocrines.Sur le plan méthodologique, une approche intégrative a été utilisée de l’animal entier (calorimétrie indirecte) à l’expression protéique (western blot) ou transcriptionnelle (RT-PCR) en passant par des mesures de la fonctionnalité bioénergétique sur des fibres musculaires perméabilisées et mitochondries isolées. Deux modèles animaux ont été utilisés, une espèce naturellement adaptée aux conditions extrêmes de l’Antarctique, le manchot Adélie (Pygoscelisadeliae), et un modèle de laboratoire, le canard de Barbarie (Cairina moschata). Nos résultats ont permis de caractériser chez l’oiseau l’expression de protéines de fusion (Mfn2, OPA1) immunoréactives homologues à celles des mammifères. Le séquençage d’une partie de la séquence codante des gènes codant les Mfns a montré une bonne similitude entre les espècesd’oiseaux et les mammifères. Chez le manchot, l’abondance relative de ces protéines dans lesmitochondries musculaires variait avec la croissance et l’exposition thermique en corrélation positiveavec les capacités bioénergétiques musculaires. Chez le canard, l’activité respiratoire et l’abondance relative de ces protéines étaient également corrélées suite à un jeûne de 60h ou, bien que dans une moindre mesure, après altération pharmacologique du statut thyroïdien.Ces résultats montrent pour la première fois chez l’oiseau l’expression de protéines homologues aux protéines de fusion des mammifères. L’association entre les variations d’expression de ces protéines et les modifications bioénergétiques du muscle squelettique indiquent qu’elles pourraient contribuer à la plasticité bioénergétique observée chez l’oiseau en croissance. Ces résultats suggèrent que des modifications potentielles de l’organisation des réseaux mitochondriaux musculaires pourraient contribuer aux réponses adaptatives des organismes face aux contraintes environnementales. / Cold-exposed young birds maintain their homeothermy by stimulating mitochondrial oxidations in skeletal muscle. Prolonged cold exposure enhances muscle thermogenic capacities through mitochondrial bioenergetics plasticity which control still remains hypothetical. In mammals, fusion proteins (mitofusins (Mfns) and OPA1 (Optic Atrophy 1)) contribute to the permanent and dynamic changes in mitochondrial networks in multiple cell types. The aim of our work was to characterize the expression of avian homologues of mammalian fusion proteins and to study the variations of their expression during the establishment of bioenergetics processes in growing birds, during an acute or a prolonged cold exposure and finally during nutritional or endocrine challenges. Methodologically, an integrative approach has been used from whole animal (indirect calorimetry) to protein (western-blot) or gene (RT-PCR) expression through measurements of the bioenergetics functionality of permeabilized muscle fibers and isolated mitochondria. Two animal models were used, a species naturally adapted to Antarctica harsh conditions, the Adélie penguin (Pygoscelis adeliae), and a laboratory model, the Muscovy duck (Cairina moschata).Our results allowed us to characterize, in birds, the expression of immunoreactive fusion proteins (Mfn2, OPA1) which were homologous to those of mammals. The sequencing of a part of the coding sequence of Mfns genes showed a great similitude between avian and mammalian species. In penguins, the relative abundance of these proteins in muscle mitochondria was modified by growth in the cold and was positively correlated with muscle bioenergetics capacities. In ducks, the respiratory activity and the relative abundance of these proteins were also correlated after a 60h fasting period or,though a lesser extent, after a pharmacological alteration of thyroid status. Our results show, for the first time in birds, the expression of proteins homologous to mammalian fusion proteins. The association between the changes in expression of these proteins and the bioenergetics modifications in skeletal muscle indicates that these proteins could contribute to thebioenergetics plasticity observed in growing chicks. These results suggest that potential modifications of the muscle mitochondrial network organization could play a role in the adaptive responses of organisms to the environmental constraints.