Heat stress (HS) stimulates mitochondrial reactive oxygen species (ROS) production and protein degradation in skeletal muscle. The present study investigated the stimulatory effects of HS-induced mitochondrial ROS production on the ubiquitin-proteasome protein degradation system in primary cultured avian muscle cells. Cells were isolated from the breast muscle of neonatal chicks, and then grown for 48 h. Thereafter, the cells were subjected to 37ï¿½C or 41ï¿½C (HS). Exposure to 6 h of HS treatment significantly decreased the cellular protein content compared to that of normal cells, an effect was completely suppressed by the addition of a proteasome-specific inhibitor. Whereas the mRNA levels of the 20S proteasome C2 subunit, which is one of the subunits of the 26S proteasome, did not change at any time during HS treatment (1, 3, 6 h), the mRNA levels of atrogin-1 and muscle ring-finger protein 1, both of which are muscle-specific ubiquitin ligases, increased after 1 h of HS but then decreased to near-normal values with time. Intracellular ROS production (the sum of H2O2, hydroxyl radicals, peroxyl radicals, peroxynitrite) did not change in the 1 h HS-exposed cells, but was significantly increased after 3 h and 6 h of HS. Mitochondrial superoxide production was significantly increased after 1 h of HS, which might increase the mRNA expression of ubiquitin ligase in muscle cells. In cells pretreated with 4-hydroxy TEMPO, which is able to decrease mitochondrial superoxide production, the increases in mitochondrial superoxide production and ubiquitin ligase mRNA levels observed after 1 h of HS were suppressed. The protein content of these cells was not decreased, which was observed after the longest period of HS (6 h). These findings suggest that mitochondrial superoxide production may play an important role in activating the ubiquitin-proteasome system, probably via the induction of ubiquitin ligases, in HS-exposed muscle cells.
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