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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2005 as doi:10.1096/fj.04-3640fje. |
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* Clinical Sciences Division, Department of Medicine, University of Toronto, Toronto, Ontario, Canada;
Iobion Software Business Unit, Stratagene, Toronto, Ontario, Canada;
Department of Surgery, McMaster University, Hamilton, Ontario, Canada;
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Ontario, Canada; and Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario, Canada
1Correspondence: Room 7344, Medical Sciences Building, 1 Kings College Circle, University of Toronto, Toronto, Ontario M5S-1A8, Canada. E-mail: jane.batt{at}utoronto.ca
SPECIFIC AIMS
Skeletal muscle reinnervated after short periods of denervation recovers mass, form, and contractile function. In contrast, long-term denervation results in irreversible muscle impairment from permanent atrophy, fiber death, and fibrosis. We sought to investigate the molecular mechanisms underlying the irreversibility of the pathology of long-term denervated muscle. We performed transcriptional profiling of short- and long-term denervated muscle with cDNA microarrays and real-time RT-PCR. Analysis of the expression pattern of differentially regulated genes in specific gene ontology categories permitted delineation of signaling pathways and processes governing reversible and irreversible changes in short and long-term denervated muscle. We hypothesized that pathways unique to each of the two time points would be identified.
PRINCIPAL FINDINGS
Microarray analysis of RNA from rat gastrocnemius muscle denervated for 1 or 3 months was performed. Contralateral unoperated gastrocnemius muscle in each rat served as an internal control. 1132 clones of 15,264 National Institute of Aging clones present on the mouse cDNA microarrays were found to be differentially expressed in the 1 month denervated muscle by significance analysis of microarrays (SAM) one class analysis (693 up and 439 down, 
= 0.77, median number false significant 0.66). Of the up-regulated clones, 227 were unique known genes, and of the down-regulated clones 224 were unique.
In the 3 month denervated muscle, 721 clones were found to be differentially regulated (327 up and 394 down) by SAM one class analysis. Of these clones, 300 were unique known genes (79 up and 221 down, = 0.88, median number false significant 0.71). We used quantitative real-time RT-PCR and SDS-PAGE/Western blot analysis to validate the microarray results for some of the genes at both time points.
Differentially expressed genes identified by SAM analysis were grouped according to their designations in the Gene Ontology Database and analysis of the expression patterns within the groups highlighted biological networks activated or suppressed in the short- and long-term denervated muscle.
1. Cellular structural proteins and the extracellular matrix
Atrophy of the denervated muscle resulted in universal down-regulation of genes encoding structural proteins of the cytoskeleton, muscle filaments, and contractile proteins at 1 month. This included tropomyosin 2 and 3, 
actinin 2, syntrophin, actin-related homologue 1, with expansion of the list at the 3 month time point to include tubulin, tropomyosin 1, epsin, kinesin family member 23, and ß actin. Three ECM components, glypican-1, galectin-1, and decorin, were differentially regulated in either the 1 or 3 month denervated muscle. Glypican-1 and galectin-1 (both increased at 1 month, normal at 3 months) are positive regulators of muscle regeneration. Decorin (normal at 1 month, increased at 3 months) is a negative regulator of cellular proliferation and a putative negative regulator of muscle regeneration. The exclusive expression of positive regulators at 1 month correlates with the muscle’s initial myogenic response to denervation. Conversely, the simultaneous normalization of their levels and enhanced expression of a negative regulator at 3 months, correlates with, and may contribute to, the development of the irreversible pathology of long-term denervated muscle.
2. ATP production and glucose metabolism
Genes encoding mitochondrial enzymes that participate in ATP synthesis and/or electron transport were uniformly decreased in 1 and 3 month denervated muscles, including NADH dehydrogenase, ATP synthase mitochondrial F1/F0 complexes, creatine kinase mitochondrial 1, and cytochrome c oxidase. Global decreases in the expression of 13 glycolytic enzymes, 1 glycogenolytic enzyme, glucose transporters, and 7 enzymes of the tricarboxylic acid cycle were also seen. These data are in keeping with the known decrease in muscle ATP content and suppressed capacity to utilize glucose that develops in denervated muscle. These processes do not appear to contribute to the irreversible changes of long-term denervated muscle.
3. Protein degradation
In keeping with the published literature, analysis of the transcripts from the 1 month denervated muscle demonstrated activation of multiple proteolytic systems, including lysosomal proteases (i.e., cathepsin L), Ca-dependent proteases, aminopeptidases, and the critical ubiquitin-proteasome system. We noted up-regulation of the ubiquitin ligases atrogin-1 and MuRF1, which are recently recognized key players in multiple models of muscle atrophy, including short-term denervation. We also report the novel finding of the up-regulation of two other ubiquitin ligases, Nedd4 and Rnf11, and the ubiquitin-binding Valosin-containing protein (VCP). In 3 month denervated muscle, all proteases and ligases atrogin-1, MuRF1, and Rnf11 normalized their expression, while increased expression of Nedd4 and VCP persisted. This temporal pattern of expression of ubiquitin ligases suggests there may be a series of distinct ubiquitination pathways that work in parallel and/or sequentially during skeletal muscle atrophy postdenervation. Specifically, the ongoing atrophy of long-term denervated muscle appears to be mediated by degradation pathways distinct from acute denervation atrophy. The muscle targets of these enzymes presumably differ, thus offering an explanation as to why a series of ligases needs to be recruited during muscle catabolism.
4. Apoptosis
We found six proapoptotic genes to be differentially regulated in the 1 month denervated muscle. The gene for amyloid ß precursor protein (betaAPP) was up-regulated in 1 month denervated muscle, and apoptosis antagonizing transcription factor (Aatf) (a negative regulator of betaAPP synthesis), was down-regulated at 1 month. Regulation of betaAPP and Aatf has not been previously reported to be altered in other models of muscle atrophy. It is of interest since up-regulation of ß APP that occurs with denervation may serve as an immediate trigger of apoptosis specifically associated with denervation. None of the changes in proapoptotic genes differentially regulated in 1 month muscle persisted in 3 month denervated muscle, suggesting that transcriptional control of these proapoptotic genes is relevant to the earlier stages of atrophy, but does not contribute to the irreversible effects seen at 3 months.
5. Protein synthesis and translation factors
A marked and sustained increase was noted in eukaryotic translation elongation factor 1 1 (Eef1a1) in both 1 and 3 month denervated muscle. While this protein is an essential component of the eukaryotic translation apparatus, it has also been ascribed microtubule severing properties. Given its marked persistence in our 3 month denervated muscle and its severing properties, it is possible it may play a causative role in irreversibility of muscle atrophy secondary to long-term denervation.
Despite the muscle atrophy, transcript levels for some ribosomal proteins and 2 eukaryotic translation initiation factors (Eif2b5, Eif3s10) were marginally, but significantly up-regulated in the 1 and 3 month denervated muscle. Although protein synthesis is reduced in the early stages of denervation atrophy, as muscle weight stabilizes with denervation persistence, the muscle must maintain or increase expression of certain proteins associated with a stress and remodeling response. This may explain the finding of increased ribosomal expression.
6. Stress response
Oxidative stress response
The pattern of expression of all transcripts associated with an oxidative stress response was similar in the 1 and 3 month denervated muscle. In keeping with the reported literature, we observed significant increases in all of the mu subunits of glutathione-S-transferase present on the chip (Gstm1, m2 and m4). However, other stress response genes (i.e., glutathione peroxidase, selenoproteins) were either significantly down-regulated in our model or there was no difference in expression (thioredoxin). We do not see consistency of changes in the transcriptional profile of genes of the oxidative stress response that support a role for or against this process in the atrophy of 1 or 3 month denervated muscle.
Heat shock proteins/chaperones
We identified 10 heat shock proteins differentially regulated in the 1 month denervated muscle with increases in HSP 25, HSP 22, HSP 90, and HSP 4 and decreases in chaperonin, Cabc1, Fkbp3, calmegin, HSP74A, and Hrsp12.
In keeping with the reported literature, our work demonstrates that the heat shock stress response is mobilized by denervation, but the changes evident in 1 month denervated muscle are partially lost by 3 months, suggesting it does not play a critical role in the development of long-term denervation pathology. HSP 90 has neurite-promoting activity and its up-regulation in 1 month denervated muscle may stimulate nerve regeneration and muscle reinnervation.
7. Cell cycle regulators
We found multiple genes producing proteins that regulate cell cycle progression to be differentially expressed in the 1 and 3 month denervated muscle. These are of interest because the regenerative capacity of adult muscle, and initiation of myogenesis, rests with muscle satellite cells. In healthy adult muscle they are quiescent. In response to toxic and physiologic stimuli, satellite cells re-enter the cell cycle and undergo replication to form a large pool of new cells that fuse with newly formed myofibers or existing myofibers. Transcription factors that regulate the cell cycle may affect the denervated muscle’s capacity for regeneration and protection against atrophy. The transcripts of CDC25C, MKK7, and c-Jun (cell cycle promoters) were significantly up-regulated and ERK 3, retinoblastoma-like 1 (p107), Fin13, and E2f6 (cell cycle inhibitors) were significantly down-regulated in 1 month muscle, with return to baseline by 3 months.
This overall pattern of expression of cell cycle regulators in 1 month denervated muscle could stimulate muscle satellite cell proliferation to counteract the loss of mass. The resolution of these changes by 3 months may remove proliferating cells from the cell cycle and induce terminal differentiation of existing satellite cells and myoblasts. This loss of a proliferative response would prevent further increases in muscle mass and may be a deciding factor in limiting the myogenic response and regenerative capacity of 3 month denervated muscle.
CONCLUSIONS AND SIGNIFICANCE
Using cDNA microarray analysis, we have demonstrated differential patterns of gene expression unique to muscle following either short-term (1 month) or prolonged (3 month) denervation injury, supporting our primary hypothesis. We have conducted analyses of these expression patterns and delineated specific genes and biological association networks that may induce/permit development of irreversible denervation muscle pathology or alternatively protect the reinnervation and functional recovery potential of denervated muscle.
An altered profile of mediators of protein degradation in the 1 and 3 month denervated muscle suggests a temporality of recruitment of proteins involved in muscle degradation. The potential involvement of ubiquitin ligases Nedd4 and Rnf11 and the ubiquitin binding VCP are newly described. While atrogin-1 and MuRF1 partake in the induction of early denervation muscle atrophy, we have shown that their involvement is not sustained as denervation atrophy progresses. Nedd4 and VCP may be key regulators.
The short-lived up-regulation of positive regulators of the cell cycle and extracellular matrix components glypican-1 and galectin-1 that positively regulate muscle myogenesis and regeneration, in combination with the short-lived down-regulation of negative regulators of cell cycle progression, correlates with retention of the recovery potential of short-term denervated muscle. Resolution of these changes over time correlates with progressive loss of the regenerative potential of muscle if denervation persists. We have highlighted these and other significant candidate genes to assess a causative role in atrophy and/or loss of regenerative potential of long-term denervated muscle. We propose that a better understanding of both processes is essential to develop new treatments of denervation injuries and salvage denervated muscle.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3640fje;
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