Skeletal muscle atrophy caused by immobilization is a major challenge for patient rehabilitation. The development of therapies designed to prevent or attenuate skeletal muscle atrophy will fill an important medical need. Many therapeutic trials focusing on the TNFα-NFκB pathway have been undertaken with the purpose of reducing the impacts of muscle diseases. For example, anti-TNFα antibodies (Remicade™) have been show to interfere with TNFα activity and consequently reduce the breakdown of dystrophic muscles [33
]. A systemic treatment with curcumin, an NFκB inhibitor, has been shown to stimulate muscle regeneration after traumatic injury [34
]. The use of a synthetic double-stranded oligodeoxynucleotide as a cis-element to block the binding of NFκB to promoter regions has been shown to inhibit cachexia in a mouse tumor model [35
]. Another strategy to interfere with the NFκB signaling pathway involves the use of proteasome inhibitors. Proteasome inhibitors have been shown to inhibit IκBα degradation and prevent NFκB activation [27
]. Since the activity of the NFκB canonical pathway relies on the proteasome machinery, we sought to determine whether the use of the proteasome inhibitor MG132 during immobilization-induced stimulus would enhance physical performance.
We recently showed that the inflammatory molecule TNFα is involved in skeletal muscle atrophy using an immobilized hindlimb mouse model [14
]. TNFα is known to increase the activity of the canonical NFκB pathway in conditions associated with muscle weakness, including sepsis, cancer, and ageing [36
] and to cause protein degradation in cultured myotubes [5
]. In fact, the ubiquitin-proteasome pathway has been shown to be of major importance in the catabolic signaling of TNFα leading to the breakdown of muscle protein [40
]. Ubiquitinated proteins are selectively targeted for degradation through the 26S proteasome pathway by tissue-specific E3 ligases [12
] that catalyze the transfer of activated ubiquitin. The muscle-specific E3 ligases MuRF-1 and Atrogin/MAFbx are part of atrophy [13
]. A review of the literature revealed slight differences in the results regarding the genes involved in the proteasome degradation pathway when it is perturbed by an increase in NFκB activity. MuRF1 mRNA, but not Atrogin/MAFbx mRNA, is upregulated in the gastrocnemius muscle of transgenic mice with constitutive NFκB activity [8
]. Judge et al. electrotransferred a dominant negative IκBα into the soleus muscle of mice that were subsequently tested in an unloading atrophy model. They showed that Atrogin/MAFbx but not MuRF-1 is regulated by the NFκB pathway in the soleus muscle [11
]. On the other hand, Bodine et al. (2001) used disuse, denervation, and hindlimb suspension models of atrophy to show that both MuRF1 and Atrogin/MAFbx are upregulated in the gastrocnemius muscle [13
]. These slight differences may be due to the different types of muscle studied, the backgrounds of the animals used, and the different models used to induce muscle atrophy.
In the present work, we showed that the proteasome inhibitor MG132 interfered with the NFκB canonical pathway of TNFα-treated C2C12 cells by protecting IκBα from degradation and thus preventing the activation of NFκB transcription. Lastly, we showed that MG132 prevents the TNFα-induced up-regulation of MuRF1 and Atrogin-1/MAFbx in vitro. Our results demonstrated that MG132 protects IκBα from degradation, which confirmed the central role of the proteasome machinery in the activation of the NFκB canonical pathway [41
We used an immobilization model that induced skeletal muscle atrophy to show that a treatment with the proteasome inhibitor MG132 prevented the immobilization-induced atrophy of the TA muscle. These results correlated with the histological analysis showing that MG132 preserved myofiber size during immobilization. The proteasome inhibitor Velcade™ (also known as PS-341 or Bortezomib™) has been shown to partially but significantly reduce the denervation-induced atrophy of the soleus muscle [22
]. The authors concluded that the partial inhibition was due to an inadequate dose of Velcade™ or other proteolytic mechanisms that are not inhibited by the drug, such as the Ca2+
-dependent protease or lysosomal pathways, which are also known to be involved in muscle wasting [43
]. In addition to inhibiting proteasome activity, MG132 has been reported to repress certain lysosomal cysteine proteases and calpains [44
], which might also explain the total rescue of muscle mass in our study.
The immobilization-induced model is known to cause an early inflammatory process characterized by the up-regulation of TNFα, IL-1, and IL-6 mRNA, which are targets of the canonical NFκB pathway [45
]. Interestingly, treating mice with MG132 prevented the up-regulation of these pro-inflammatory genes during immobilization. Likewise, the levels of MuRF-1 and Atrogin/MAFbx mRNA, which are targets of NFκB, increased after immobilization. This effect was prevented by MG132, which completely inhibited the expression of MuRF-1 mRNA. Surprisingly, unlike the in vitro experiment, MG132 did not completely inhibit Atrogin/MAFbx mRNA expression in immobilized mice, but did significantly attenuate it. We cannot rule out the involvement of another mechanism in the increase in Atrogin/MAFbx mRNA levels in vivo. There is some evidence suggesting that disuse muscle atrophy induced by unloading is associated with the activation of an alternative NFκB pathway distinct from the pathway seen with cachexia where TNFα is responsible for muscle atrophy [48
]. Despite the fact that we observed an up-regulation of the canonical NFκB pathway target genes for TNFα, IL-1, and IL-6 [45
], it is possible that there is a cross-over between with the non-canonical NFκB pathway, which is independent of IκBα. Other investigators have shown that TNFα mediates the upregulation of atrogin/MAFbx expression in the skeletal muscle via p38MAPK [10
]. These conflicting findings suggest that multiple signaling pathways mediate muscle wasting, and highlight the complexity of the molecular pathways involved in the regulation of skeletal muscle atrophy.
To correlate the morphological, histological, and molecular results with the functional properties of TA muscles from MG132-treated mice, we evaluated the time to exhaustion performance of mice. Our results showed that the endurance of MG132-treated mice was significantly higher than that of vehicle-treated control mice after 4 days of remobilization. However, no significant differences were observed after 11 days of remobilization. Indeed, after 11 days, the time to exhaustion was similar to that of mice that had never had their hindlimbs immobilized.