Effects of 2 weeks lower limb immobilization and two separate rehabilitation regimens on gastrocnemius muscle protein turnover signaling and normalization genes

BACKGROUND: Limb 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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: In 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.