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

Nedergaard A, Jespersen JG, Pingel J, Christensen B, Sroczynski N, Langberg H, Kjaer M, Schjerling P - BMC Res Notes (2012)

Bottom Line: In both studies, no changes in protein expression or phosphorylation for any measured protein were observed.Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Sports Medicine, Department of Orthopedic Surgery M, Bispebjerg Hospital, Copenhagen, Denmark. anders.fabricius.nedergaard@gmail.com

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.

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Data for Study 1. A: mRNA expression measured by RT-qPCR. mRNA data are normalized to geometric means of Cyclophillin A, β2-microglobulin and RPLP0 and are relative to individual PRE values. Target genes are presented in the left side panel, whereas putative housekeeping genes are presented in the right side panel. Data are presented as back-transformed mean ± SEM. Light gray columns represent IMMO and dark gray REHAB. B: Total protein and protein phosphorylation measured by Western blot, relative to individual PRE values. Data are presented as back-transformed mean ± SEM. Total Akt is represented twice as it was measured along with each phospho-Akt. Light gray columns represent IMMO and dark gray REHAB. * denotes a difference from PRE with 0.05 > p ≥ 0.01. ** denotes a difference from PRE with 0.01 > p ≥ 0.001. Underscored asterisks denote main (time) effects.
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Figure 2: Data for Study 1. A: mRNA expression measured by RT-qPCR. mRNA data are normalized to geometric means of Cyclophillin A, β2-microglobulin and RPLP0 and are relative to individual PRE values. Target genes are presented in the left side panel, whereas putative housekeeping genes are presented in the right side panel. Data are presented as back-transformed mean ± SEM. Light gray columns represent IMMO and dark gray REHAB. B: Total protein and protein phosphorylation measured by Western blot, relative to individual PRE values. Data are presented as back-transformed mean ± SEM. Total Akt is represented twice as it was measured along with each phospho-Akt. Light gray columns represent IMMO and dark gray REHAB. * denotes a difference from PRE with 0.05 > p ≥ 0.01. ** denotes a difference from PRE with 0.01 > p ≥ 0.001. Underscored asterisks denote main (time) effects.

Mentions: As for mRNA (Figure 2A), we observed significant time effects for FOXO3 (p = 0.004), FOXO4 (p = 0.013), GAPDH (p = 0.005), HADHA (p = 0.010) and S26 (p = 0.037) transcripts. 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)). GAPDH and HADHA were also downregulated at the IMMO time point (−53% for GAPDH (p = 0.004) and −40% for HADHA (p = 0.008)), but returned almost back to baseline expression at the REHAB time point. Despite manifesting a time effect, S26 was not significantly downregulated at either time point (Figure 2A).


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

Nedergaard A, Jespersen JG, Pingel J, Christensen B, Sroczynski N, Langberg H, Kjaer M, Schjerling P - BMC Res Notes (2012)

Data for Study 1. A: mRNA expression measured by RT-qPCR. mRNA data are normalized to geometric means of Cyclophillin A, β2-microglobulin and RPLP0 and are relative to individual PRE values. Target genes are presented in the left side panel, whereas putative housekeeping genes are presented in the right side panel. Data are presented as back-transformed mean ± SEM. Light gray columns represent IMMO and dark gray REHAB. B: Total protein and protein phosphorylation measured by Western blot, relative to individual PRE values. Data are presented as back-transformed mean ± SEM. Total Akt is represented twice as it was measured along with each phospho-Akt. Light gray columns represent IMMO and dark gray REHAB. * denotes a difference from PRE with 0.05 > p ≥ 0.01. ** denotes a difference from PRE with 0.01 > p ≥ 0.001. Underscored asterisks denote main (time) effects.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3405443&req=5

Figure 2: Data for Study 1. A: mRNA expression measured by RT-qPCR. mRNA data are normalized to geometric means of Cyclophillin A, β2-microglobulin and RPLP0 and are relative to individual PRE values. Target genes are presented in the left side panel, whereas putative housekeeping genes are presented in the right side panel. Data are presented as back-transformed mean ± SEM. Light gray columns represent IMMO and dark gray REHAB. B: Total protein and protein phosphorylation measured by Western blot, relative to individual PRE values. Data are presented as back-transformed mean ± SEM. Total Akt is represented twice as it was measured along with each phospho-Akt. Light gray columns represent IMMO and dark gray REHAB. * denotes a difference from PRE with 0.05 > p ≥ 0.01. ** denotes a difference from PRE with 0.01 > p ≥ 0.001. Underscored asterisks denote main (time) effects.
Mentions: As for mRNA (Figure 2A), we observed significant time effects for FOXO3 (p = 0.004), FOXO4 (p = 0.013), GAPDH (p = 0.005), HADHA (p = 0.010) and S26 (p = 0.037) transcripts. 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)). GAPDH and HADHA were also downregulated at the IMMO time point (−53% for GAPDH (p = 0.004) and −40% for HADHA (p = 0.008)), but returned almost back to baseline expression at the REHAB time point. Despite manifesting a time effect, S26 was not significantly downregulated at either time point (Figure 2A).

Bottom Line: In both studies, no changes in protein expression or phosphorylation for any measured protein were observed.Interestingly, we found significant changes in expression of the putative housekeeping genes GAPDH, HADHA and S26 with immobilization in both studies.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Sports Medicine, Department of Orthopedic Surgery M, Bispebjerg Hospital, Copenhagen, Denmark. anders.fabricius.nedergaard@gmail.com

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.

Show MeSH
Related in: MedlinePlus