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

Abstract

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.

The delayed recovery of the remobilized rat tibialis anterior muscle reflects a defect in proliferative and terminal differentiation that impairs early regenerative processes

“…Figure (A) shows that mRNA levels for Sparc did not change in the immobilized TA at I8, but strongly increased in the remobilized TA at R1 and R10 (+115% and +158%, P < 0.05 vs. I8). In addition to its role in muscle regeneration, Sparc was suggested to bind to integrins (Itgs) in different cell types . We show here that mRNA levels for Itg‐α7 and ‐β1 increased in the immobilized TA at I8 (+218% and 93%, respectively, P < 0.05 vs. I0) and in the remobilized TA at R1 (+192% and 95%, respectively, P < 0.05 vs. I0), before being normalized at R6 ( Figure (B) and (C)).…”

“…The protein kinase Akt is involved in the control of protein turnover and, when inhibited, reduces protein synthesis and increases proteolysis . Akt phosphorylation and activity was either unchanged or reduced after cast immobilization in rodent or human soleus and GA muscles. A single study reported that Akt phosphorylation increased rapidly in the recovering soleus after cast removal .…”

Effect of bimagrumab on thigh muscle volume and composition in men with casting‐induced atrophy

“…In the study by Wall et al .,29 immobilization for 5 days induced muscle loss of ~3.5%, which increased to 8.4% when immobilization was extended to 14 days. While this magnitude of muscle loss can be recovered with little rehabilitation in healthy young men, it is difficult to overcome in elderly patients 14, 15, 16, 31…”

“…While this magnitude of muscle loss can be recovered with little rehabilitation in healthy young men, it is difficult to overcome in elderly patients. [14][15][16]31 Growing evidence suggests that the structural composition of a muscle is an important factor in its function. 10 With ageing, muscle strength is lost at a faster rate than lean mass, pointing to additional determinants of impaired contractility in older adults.…”

GAPDH and β-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle

“…Likewise, Lowe and colleagues (13) observed decreased GAPDH protein level in rat muscle with age that may be related to changes in fiber type composition. Additionally, GAPDH mRNA levels have been reported to be decreased after leg immobilization (16). In human adipose tissue it was demonstrated in obese and nonobese individuals that neither GAPDH nor ␤-tubulin are adequate as loading controls (19).…”