Calpain/calpastatin activities and substrate depletion patterns during hindlimb unweighting and reweighting in skeletal muscle
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Unloading of skeletal muscle by hindlimb unweighting (HU) is characterized by atrophy, protein loss, and an elevation in intracellular Ca2+ levels that may be sufficient to activate Ca2+-dependent proteases (calpains). In this study, we investigated the time course of calpain activation and the depletion pattern of a specific structural protein (desmin) with unloading and subsequent reweighting. Rats underwent 12 h, 24 h, 72 h or 9 days of HU, followed by reweighting for either 0, 12 or 24 h. Total calpain-like activity was elevated with HU in skeletal muscle (P < 0.05) and was further enhanced with reweighting (P < 0.05). The increases in calpain-like activity were associated with a proportional increase in activity of the particulate fraction (P < 0.05). Activity of the μ-calpain isoform was elevated with 12 and 24 h of HU (P < 0.05) and returned to control levels thereafter. With reweighting, activities of μ-calpain were elevated above control levels for all HU groups except 9 days (P < 0.05). In contrast, minimal changes in m-calpain and calpastatin activity were observed with HU and reweighting. Although desmin depletion levels did not reach statistical significance, a significant inverse relationship was found between the μ-calpain/calpastatin ratio and the amount of desmin in isolated myofibrils (R = −0.83, P < 0.001). The results suggest that calpain activation is an early event during unloading in skeletal muscle, and that the majority of the increase in calpain activity can be attributed to the μ-isoform.
KeywordsProtease Atrophy Hindlimb suspension Skeletal muscle
We thank Dr. Earl Noble for comments on the manuscript and Mr. Thomasz Dzialoszynski for technical assistance. This research was supported by a Natural Sciences and Engineering Research Council of Canada operating grant awarded to Dr. A. Belcastro.
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