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Human skeletal muscle atrophy in amyotrophic lateral sclerosis reveals a reduction in Akt and an increase in atrogin-1

Bertrand Léger^*, Lodovica Vergani, Gianni Sorarù, Peter Hespel, Wim Derave, Charles Gobelet^*, Carla D'Ascenzio, Corrado Angelini and Aaron P. Russell^*,1

^* Clinique romande de réadaptation, SuvaCare, Sion, Switzerland;
Dipartimento di Scienze Neurologiche, Università di Padova, Istituto Veneto di Medicina Molecolare, Padova, Italy; and
Research Centre for Exercise and Health, Faculty of Kinesiology and Rehabilitation Sciences, K.U. Leuven, Leuven, Belgium

¹Correspondence: Clinique romande de réadaptation, SuvaCare, Av. Gd-Champsec 90, Sion 1951, Switzerland. E-mail: aaron.russell{at}crr-suva.ch

SPECIFIC AIMS

The molecular mechanisms influencing muscle atrophy in humans are poorly understood. Atrogin-1 and MuRF1, two ubiquitin E3-ligases, mediate muscle atrophy in rodents and cells and are suggested to be regulated by an Akt/Forkhead (FKHR) signaling pathway. However, the role of these "atrophy genes" and their suggested transcriptional regulators is not well understood in human skeletal muscle atrophy. The aim of the present study was to measure and compare the expression of atrogin-1 and MuRF1 and the activity of Akt and several of its catabolic (FKHR and FKHRL1) and anabolic (p70^s6k and GSK-3ß) targets in a human model of skeletal muscle atrophy; this model being amyotrophic lateral sclerosis (ALS).

PRINCIPAL FINDINGS

1. Confirmation of skeletal muscle atrophy
Skeletal muscle atrophy is a well known characteristic observed in ALS. In the present study skeletal muscle cross sectional area (CSA) of the type I and type II fibers was 35% (P=0.002) and 22% (P=0.03) smaller in the ALS than in control subjects, presenting a severe atrophy of the vastus lateralis muscle. Skeletal muscle atrophy was also confirmed in G93A ALS mice, when compared with control mice, as the mass of the tibialis anterior muscle was 50% lower (P<0.001).

2. Atrogin-1 and MuRF1
We observed that in skeletal muscle of the ALS patients, when compared with the healthy control subjects, a significant 170% and 340% increase, respectively, in atrogin-1 mRNA and protein content (Fig. 1 ). In the G93A mice, when compared with the wild-type mice, atrogin-1 mRNA and protein contents were significantly increased several fold. There was, however, no difference in MuRF mRNA levels for both the human and rodent groups.

View larger version (15K): [in this window] [in a new window]   Figure 1. Atrogin-1 mRNA (A) and protein (B) content in control subjects and ALS patients. **Significantly different from control subjects, (P<0.01). The Western blot image in B is of 2 representative control and ALS subjects.

3. Regulation of Akt and FKHR
As the transcription of atrogin-1 has been suggested to be regulated via an Akt/FKHR pathway we decided to measure Akt, FKHR (foxo1), and FKHRL1 (foxo3) mRNA and protein levels in the skeletal muscle of the ALS patients and controls. There was no difference in Akt, FKHR, and FKHRL1 mRNA levels in the ALS patients compared with the control subjects. However, at the protein level the ALS patients had a 68% lower content of the active phosphorylated Akt protein (P=0.02). Surprisingly, there was no difference in the nuclear protein contents of FKHR or FKHRL1 between the ALS and the control subjects (Fig. 2 ). As observed in the human samples, G93A mice had a reduction in the active Akt protein content, with no difference in the nuclear content of FKHRL1 when compared with wild-type mice.

View larger version (19K): [in this window] [in a new window]   Figure 2. Akt (A), FKHR (B) and FKHRL1 (C) in skeletal muscle from control subjects and ALS patients. *P < 0.05. The Western blot images are of 2 representative control and ALS subjects.

4. Regulation of p70^s6k and a GSK-3ß,
Akt has been shown to have an anabolic effect by activating several downstream kinases responsible for protein translation initiation and protein synthesis. The reduced Akt activity in the ALS patients would suggest that they have a lower quantity of phosphorylated p-70^s6k and a greater amount of GSK-3ß than control subjects. However, there was no difference in p-70^s6k or GSK-3ß mRNA and their phosphorylated protein contents in the ALS vs. control subjects.

CONCLUSIONS AND SIGNIFICANCE

Skeletal muscle atrophy is a devastating condition seen in many catabolic diseases such as cancer, diabetes, sepsis and AIDS, denervation, sarcopenia, and in neuromuscular disorders such as Duchenne muscular dystrophy and ALS. The molecular targets and signaling pathways influencing human muscle atrophy are not well understood. In the present study, we investigated for the first time in human skeletal muscle the regulation of several catabolic and anabolic targets believed to be crucial in the atrophy and/or hypertrophy processes. We used ALS as a human model of skeletal muscle atrophy and made comparisons with healthy controls subjects. Here we present several novel findings suggesting that 1) atrogin-1, but not MuRF1, are involved in human skeletal muscle atrophy as seen in ALS; 2) active Akt is reduced in human skeletal muscle atrophy and may be involved in the signaling cascade, which regulates atrogin-1; 3) although Akt activity is reduced in ALS downstream, catabolic factors such as FKHR and FKHRL1 or anabolic factors such as phosphorylated p70^S6K and phosphorylated GSK-3ß are unaffected.

The observation that atrogin-1, a muscle specific ubiquitin E3-ligase, was significantly increased in the atrophied skeletal muscle of ALS patients supports previous observations in cell and rodent models of muscle atrophy. Two previous studies have observed that atrogin-1 mRNA is increased in atrophied human skeletal muscle; however, no measurements of the atrogin-1 protein were made in these studies. It appears that the increases in atrogin-1 mRNA results in an increase in the translation of the atrogin-1 protein. Intuitively, this suggests that atrogin-1 would play a significant role in muscle protein degradation via ubiquitin-dependent proteolysis.

MuRF-1 is another muscle-specific ubiquitin E3-ligase. However, we did not observe any changes in its gene expression between the ALS and control subjects. It appears that skeletal muscle atrophy, observed in a neurological disorder such as ALS, is influenced more by atrogin-1 than by MuRF-1. This observation was also made after 2 wk of limb immobilization in humans, another model of skeletal muscle atrophy. However, several rodent and cell models of muscle atrophy, including immobilization, denervation, sepsis, incubation with dexamethasone, exposure to H₂O₂, and space flight, have all observed increases in MuRF1 mRNA. At present it is unknown whether MuRF-1 plays a role in human skeletal muscle atrophy.

We were interested in identifying which signaling pathways may be responsible for the increase in atrogin-1 mRNA and protein levels. Several studies investigating the transcriptional regulation of atrogin-1 have suggested that it is regulated via an Akt/FKHR pathway. It has been well documented that Akt can phosphorylate both FKHR and FKHRL1, resulting in their exclusion from the nucleus. However, in these pharmacological and genetic manipulation models of muscle atrophy, the phosphorylated active Akt protein was reduced while the FKHR and/or FKHRL1 translocation to the nucleus was increased. Once in the nucleus, FKHR and/or FKHRL1 bind to its forkhead element on the atrogin-1 promoter and increases atrogin-1 gene transcription. In support of these studies, we observed that Akt activity was significantly reduced in the ALS patients when compared with healthy controls. A reduced Akt activity in the ALS patients would suggest that they have a greater nuclear protein content of the active nonphosphorylated forms of the FKHR and FKHRL1 proteins. Surprisingly, we did not observe any difference in the nuclear protein content of both FKHR and FKHRL1 in the ALS when compared with the control subjects. In the present study, the same results for Akt and FKHRL1 and atrogin-1 protein were observed in the tibialis anterior muscle of SOD1 G93A transgenic mice. Our findings suggest that, in the ALS neuromuscular model of skeletal muscle atrophy, atrogin-1 regulation may not be via an Akt/FKHR pathway. It is possible that atrogin-1 may be regulated via another Akt-mediated transcription factor or via another signaling pathway altogether. Recent studies have suggested that atrogin-1 maybe regulated via a TNF/MAPK pathway in C2C12 cells stimulated with H₂O₂ and TNF, mice injected with recombinant TNF and in humans with acute quadriplegic myopathy however, the responsible transcription factor was not identified.

Because Akt was reduced in the ALS patients compared with control subjects, we investigated whether there was a concomitant regulation of downstream Akt target proteins responsible for protein translation and initiation, such as p70^s6k and GSK-3ß. Inhibition of Akt activity in myotubes blocks p70^s6k resulting in a decrease in muscle hypertrophy and growth. Surprisingly, we did not observe any difference in p70^s6k between the ALS and control subjects. It has previously been shown that p70^s6k can be activated directly by phosphoinositide-dependent protein kinase (PDK1), suggesting that Akt may be dispensable for signaling to p70^s6k. It is possible that p70^s6k activity is maintained by other mechanisms in an attempt to combat muscle atrophy. GSK-3ß can be phosphorylated by Akt, which leads to its inhibition and a consequential increase in protein synthesis. Therefore, we expected a decrease in phosphorylated GSK-3ß in the ALS patients when compared with the control subjects. However, GSK-3ß levels were not different between the two groups.

These results cannot be seen as being specific to the skeletal muscle atrophy observed in human or rodent ALS. However, the relevance of these results to ALS skeletal muscle atrophy should be mentioned. Although the initial skeletal muscle atrophy in ALS is a consequence of the degeneration in upper and lower motor neurons, local molecular signaling events may play an important role in the muscle atrophy observed in ALS. This is supported by the previous observation in ALS patients of an increase in the protein content of the 26S proteasome an integral component of the ATP-ubiquitin-dependent proteolytic complex. This finding, in combination with our observation of an increase in atrogin-1, suggests that the ATP-ubiquitin-dependent proteolytic system, may contribute to the skeletal muscle atrophy observed in ALS.

View larger version (39K): [in this window] [in a new window]   Figure 3. We have used ALS as a model of skeletal muscle atrophy. ALS presents a loss of upper and lower motor neurons, which leads to severe skeletal muscle atrophy. We have observed a decrease in the active Akt protein and an increase in atrogin-1 mRNA and protein in skeletal muscle of ALS patients and ALS mice. A reduction in Akt has been suggested as a major factor in the signaling required for an increase in atrogin-1 transcription. An increase in atrogin-1, a muscle-specific ubiquitin E3-ligase, suggests an increase in the activity of the ATP ubiquitin-dependent proteolytic complex, a principal factor involved in skeletal muscle atrophy.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5249fje;

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This Article Full Text (PDF) All Versions of this Article: 20/3/583 05-5249fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Léger, B. Articles by Russell, A. P. Search for Related Content PubMed PubMed Citation Articles by Léger, B. Articles by Russell, A. P.