RESUMO
Horses are particularly sensitive to excessive inflammatory reaction where myeloperoxidase, a marker of inflammation, may contribute to mitochondrial dysfunctions. This study investigated the interaction between myeloperoxidase and cultured primary equine skeletal myoblasts, particularly its effect on mitochondrial respiration combined or not with anoxia followed by reoxygenation (AR). We showed that active myeloperoxidase entered into the cells, interacted with mitochondria and decreased routine and maximal respirations. When combined with AR, myeloperoxidase caused a further decrease of these respiratory parameters while the leak increased. Our results indicate that myeloperoxidase amplifies the mitochondrial damages initiated by AR phenomenon and alters the mitochondrial function.
Assuntos
Respiração Celular , Hipóxia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mioblastos Esqueléticos/fisiologia , Oxigênio/metabolismo , Peroxidase/metabolismo , Animais , Células Cultivadas , CavalosRESUMO
Horses are outstanding athletes, performing in many different disciplines involving different kinds of efforts and metabolic responses. Depending on exercise intensity, their skeletal muscle oxygenation decreases, and the reperfusion at cessation of the exercise can cause excessive production of free radicals. This study on cultured primary equine myoblasts investigated the effect of different kinds of anoxia/reoxygenation (A/R) on routine respiration, mitochondrial complex I specific activity and free radicals production. Our data revealed that short cycles of A/R caused a decrease of all the parameters, opposite to what a single long period of anoxia did. A preconditioning-like effect could explain our first pattern of results whereas mild uncoupling could be more appropriate for the second one. Anyway, it seems that mitochondrial complex I could play a major role in the regulation of the balance between metabolic and antioxidant protection of the muscular function of athletic horses.
Assuntos
Radicais Livres/metabolismo , Mitocôndrias Musculares/metabolismo , Mioblastos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Cavalos , Hipóxia/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias Musculares/fisiologia , Proteínas Mitocondriais/metabolismo , Mioblastos/fisiologia , Oxigênio/metabolismo , Proteína Desacopladora 3RESUMO
Horses are particularly sensitive and exposed to excessive inflammatory responses evolving toward an important stimulation of polymorphonuclear neutrophils (PMNs). The aim of this work was to stimulate equine neutrophils in whole blood and to evaluate their response by measuring the release of total and active myeloperoxidase (MPO) and total elastase, considered as markers of neutrophil stimulation and degranulation. Because of the critical importance of the concomitant presence of LPS and TNF-α in equine pathological situations, we combined these two natural mediators to stimulate PMN and compared the response with those obtained after the PMN stimulation with each mediator used alone and well-known artificial stimulation systems such as 12-phorbol 13-myristate acetate (PMA) and the combination of cytochalasin B (CB) and N-formyl-methionyl-leucyl-phenylalanine (fMLP). All the activation systems, PMA, CB/fMLP, TNF-α, LPS and LPS/TNF-α, induced a significant release of total MPO in whole blood but only the combinations CB/fMLP and LPS/TNF-α significantly favored the release of active MPO. Regarding the total elastase, we did not observe a significant release in all the stimulated conditions except with PMA. It appears clearly that the choice of the neutrophil stimulation model is fundamental for the selection of potentially active pharmacological agents, especially on MPO activity.
Assuntos
Cavalos/sangue , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Peroxidase/metabolismo , Animais , Biomarcadores , Regulação Enzimológica da Expressão Gênica , Lipopolissacarídeos/metabolismo , Modelos Biológicos , Elastase Pancreática/genética , Peroxidase/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production. ANIMALS: 5 healthy horses (5 to 15 years old). PROCEDURES: Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3'-diaminobenzidine chromogen solution to detect peroxidase binding. RESULTS: Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenation-treated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses.