Although several lines of evidence link muscle-derived oxidants and inflammation to skeletal muscle wasting via regulation of apoptosis and proteolysis, little information is currently available on ...muscle repair. The present work was designed to study oxidative stress response, inflammatory cytokines, apoptotic, or proteolytic pathways during the early (1 and 5 days) and later (14 days) stages of the regrowth process subsequent to 14 days of hindlimb unloading. During the early stages of reloading, muscle mass recovery (day 5) was facilitated by transcriptional downregulation (day 1) of pathways involved in muscle proteolysis mu-calpain, atrogin-1/muscle atrophy F-box (MAFbx), and muscle RING finger-1/(MuRF1) mRNA and upregulation of an autophagy-related protein Beclin-1 (day 5). At the same time, oxidative stress (glutathione vs. glutathione disulfide ratio, superoxide dismutase, catalase activities) remained still enhanced, whereas the increased uncoupling protein 3 gene expression recovered. Increased caspase-9 (mitochondrial-driven apoptosis) and decreased caspase-12 (sarcoplasmic reticulum-mediated apoptosis) activation was also normalized at early stages (day 5). Conversely, the receptor-mediated apoptotic pathway initiated by ligand-induced (tumor necrosis factor-alpha, TNF-alpha) binding and promoting the activation of caspase-8 remained elevated until 14 days. Our data suggest that at early stages, muscle repair is mediated via the modulation of mitochondrial-driven apoptosis and muscle proteolysis. Despite full muscle mass recovery, oxidative stress and TNF-alpha-mediated apoptotic pathway are still activated till later stages of muscle remodeling.
Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca
2+
homeostasis is affected in that situation remains obscure. We ...show here that there is a 20% atrophy of the fast-type
flexor digitorum brevis
(FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca
2+
current was found similar in fibres from control and HU animals. In fibres loaded with the Ca
2+
dye indo-1, the value for the rate of Ca
2+
decay after the end of 5–100-ms-long voltage-clamp depolarisations from −80 to +10 mV was found to be 30–50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca
2+
removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca
2+
-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of Ca
2+
transients accompanied by an increase in resting Ca
2+
and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca
2+
removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.
Calcium-dependent signalling pathways are believed to play an important role in skeletal muscle atrophy, but whether intracellular Ca(2+) homeostasis is affected in that situation remains obscure. We ...show here that there is a 20% atrophy of the fast-type flexor digitorum brevis (FDB) muscle in rats hind limb unloaded (HU) for 2 weeks, with no change in fibre type distribution. In voltage-clamp experiments, the amplitude of the slow Ca(2+) current was found similar in fibres from control and HU animals. In fibres loaded with the Ca(2+) dye indo-1, the value for the rate of Ca(2+) decay after the end of 5-100-ms-long voltage-clamp depolarisations from -80 to +10 mV was found to be 30-50% lower in fibres from HU animals. This effect was consistent with a reduced contribution of both saturable and non-saturable components of myoplasmic Ca(2+) removal. However, there was no change in the relative amount of parvalbumin, and type 1 sarco-endoplasmic reticulum Ca(2+)-ATPase was increased by a factor of three in the atrophied muscles. Confocal imaging of mitochondrial membrane potential showed that atrophied FDB fibres had significantly depolarized mitochondria as compared to control fibres. Depolarization of mitochondria in control fibres with carbonyl cyanide-p-trifluoromethoxyphenylhydrazone induced a slowing of the decay of Ca(2+) transients accompanied by an increase in resting Ca(2+) and a reduction of the peak amplitude of the transients. Overall results provide the first functional evidence for severely altered intracellular Ca(2+) removal capabilities in atrophied fast-type muscle fibres and highlight the possible contribution of reduced mitochondrial polarisation.
L’exposition à une situation d’hypokinésie induit une atrophie fonctionnelle et phénotypique du musclesquelettique. Différents mécanismes sont suggérés contribuer à ce phénomène de plasticité ...musculaire,incluant en particulier des modifications de l’homéostasie calcique et de la production d’espèces réactivesde l’oxygène qui, en relation avec certains processus inflammatoires, activeraient des voies d’apoptose etde protéolyse musculaire. Le présent travail a porté un intérêt spécifique à ces phénomènes dans le cadrede l’atrophie musculaire induite par une hypokinésie ainsi que dans la récupération après cessation de ceprotocole. À l’aide d’approches cellulaires nous montrons que l’extrusion du calcium cytoplasmique estconsidérablement ralentie dans les fibres musculaires atrophiées. Cet effet, lié au moins en partie à unecontribution altérée des mitochondries, pourrait jouer un rôle dans l’activation de voies protéolytiquescalcium-dépendantes. Dans un deuxième temps, nous avons étudié l’évolution du niveau de stressoxydant et de l’expression de différentes cytokines ainsi que de marqueurs de la voie apoptotiquecaspase-dépendante et de la protéolyse musculaire au cours du phénomène de récupération après la fin del’hypokinésie. Les résultats montrent que le retour à la normale de la masse musculaire est facilité lors dela phase précoce (1-5 jours) de récupération via la modulation de l’apoptose mitochondriale et de laprotéolyse musculaire. Par contre le stress oxydant et la voie apoptotique impliquant TNF-a persistentjusqu’à 14 jours de récupération, alors que la masse musculaire est déjà reconstituée.
Exposure to hypokinesia induces a functional and phenotypic atrophy of skeletal muscle. Several types ofmechanisms have been suggested to contribute to this plasticity phenomenon, including changes inintracellular calcium handling and in production of reactive oxygen species which, together withinflammatory processes, would activate muscle apoptosis and proteolysis pathways. The present workspecifically focussed on these mechanisms within the framework of a model of hypokinesia-inducedatrophy and of recovery from this atrophy. Measurements on isolated muscle cells revealed that the rateof myoplasmic calcium extrusion was considerably reduced in atrophied muscle fibres. This effect whichappears to be, at least in part, related to an altered mitochondrial contribution, may play a pivotal role inthe activation of calcium-dependent proteolysis pathways. We then studied the time course of changes inoxidative stress as well as in the expression level of several cytokines and proteins specifically involvedin proteolysis and caspase-dependent apoptotic pathways, along the course of recovery from atrophy. Ourresults demonstrate that, at early stages (1-5 days) of recovery, muscle re-growth is mediated via themodulation of mitochondrial-driven apoptosis and muscle proteolysis. In contrast, oxidative stress and theTNF-a related apoptotic pathway remain activated until late stages (14 days) of recovery, at a time whenmuscle mass has already recovered.
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