Abstract
BackgroundLimb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Whereas the knowledge of muscle mass signaling with immobilization has increased in recent years, the molecular regulation in the rehabilitation of immobilization-induced muscle atrophy is only sparsely studied. To investigate the phosphorylation and expression of candidate key molecular muscle mass regulators after immobilization and subsequent rehabilitation we performed two separate studies.MethodsWe immobilized the lower limb for 2 weeks followed by the in-house hospital standard physiotherapy rehabilitation (Study 1). Secondly, we conducted an intervention study using the same 2 weeks immobilization protocol during which protein/carbohydrate supplementation was given. This was followed by 6 weeks of rehabilitation in the form of resistance training and continued protein/carbohydrate supplementation (Study 2). We obtained muscle biopsies from the medial gastrocnemius prior to immobilization (PRE), post-immobilization (IMMO) and post-rehabilitation (REHAB) and measured protein expression and phosphorylation of Akt, mTOR, S6k, 4E-BP1, GSK3β, ubiquitin and MURF1 and mRNA expression of Atrogin-1, MURF1, FOXO1, 3 and 4 as well as appropriate housekeeping genes.ResultsIn both studies, no changes in protein expression or phosphorylation for any measured protein were observed. In Study 1, FOXO3 and FOXO4 mRNA expression decreased after IMMO and REHAB compared to PRE, whereas other mRNAs remained unchanged. Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies.ConclusionsIn neither study, the changes in muscle mass associated with immobilization and rehabilitation were accompanied by expected changes in expression of atrophy-related genes or phosphorylation along the Akt axis. Unexpectedly, we observed significant changes in several of the so-called housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies, thereby questioning the usefulness of these genes for normalization of RNA data purposes in muscle immobilization studies.
Highlights
Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function
For FOXO3 and FOXO4 this was manifested in the form of a downregulation at the IMMO time point (−40% for FOXO3 (p = 0.006) and −58% for FOXO4 (p = 0.026)) that persisted until the REHAB (2 W) time point (−19% for FOXO3 (p = 0.006) and −35% for FOXO4 (p = 0.009))
For Study 1, we hypothesized that the 2 weeks immobilization would decrease Akt and mammalian target of rapamycin (mTOR) signaling along with increased FOXO3, Atrogin-1 and Muscle Ring Finger protein 1 (MURF1) mRNA expression, reflecting the loss of muscle mass previously reported for this study [26]
Summary
Limb immobilization causes a rapid loss of muscle mass and strength that requires appropriate rehabilitation to ensure restoration of normal function. Two to three weeks of immobilization causes a loss of muscle mass of 5–10%, accompanied by a decrease in strength of 10–20% [6,7,8], and, individuals with injuries that require immobilization of a limb experience a rapid loss of muscle mass and strength. The recovery from such an injury can be complicated by the lag time for strength recovery and is especially true for vulnerable population groups including older and/or frail adults [9]. Discrepant results on the effect of amino acid supplementation on muscle loss with immobilization have been reported [12,13]
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