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J Appl Physiol 95: 2171-2179, 2003. First published August 1, 2003; doi:10.1152/japplphysiol.00500.2003
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HIGHLIGHTED TOPICS
Physiology of Aging

Selected Contribution: Identification of differentially expressed genes between young and old rat soleus muscle during recovery from immobilization-induced atrophy

J. Scott Pattison,1 Lillian C. Folk,2 Richard W. Madsen,3 and Frank W. Booth1

1Departments of Biomedical Sciences and of Pharmacology and Physiology, and the Dalton Cardiovascular Institute University of Missouri at Columbia; 2Department of Veterinary Pathobiology and 3Department of Statistics, University of Missouri at Columbia, Columbia, Missouri 65211

Submitted 12 May 2003 ; accepted in final form 24 July 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
After cessation of hindlimb immobilization, which resulted in a 27-37% loss in soleus mass, the atrophied soleus muscle of young but not old rats regrows to its mass before treatment. We hypothesized that during remobilization the mRNA levels of growth potentiating factor(s) would be present in the soleus muscle of young (3- to 4-mo-old) but absent in old (30- to 31-mo-old) Fischer 344 x Brown Norway rats or that mRNAs for growth inhibitory factor(s) would be absent in young but present in old. Gene expression levels of >24,000 transcripts were determined by using Affymetrix RGU34A-C high-density oligonucleotide microarrays in soleus muscles at 3, 6, 10, and 30 days of remobilization after cessation of a 10-day period of hindlimb immobilization. Each muscle sample was applied to an independent set of arrays. Recovery-related differences were determined by using a three-factor ANOVA with a false discovery rate-adjustment of P = 0.01, which yielded 64 significantly different probe sets. Elfin, amphiregulin, and clusterin mRNAs were selected for further confirmation by real-time PCR. Elfin mRNA levels were less in old than in young rats at 6, 10, and 30 days of remobilization. Amphiregulin expression exhibited a unique spike on the 10th day of successful regrowth in young rats but remained unchanged old. Clusterin mRNA was unchanged in young muscles but was elevated on the 3rd, 6th, and 10th days of recovery in old soleus muscles. The mRNAs identified as differentially expressed between young and old recovery may modulate muscle growth that could highlight new candidate mechanisms to explain the failure of old soleus muscle to recover lost muscle mass.

aged; mRNA; rehabilitation; growth

THE INHERENT VALUE THAT SKELETAL muscle mass and strength play in overall health and homeostasis has been somewhat underappreciated. For example, the loss of skeletal muscle mass and strength is significant because it is associated with an increased incidence of death (30). Major modes by which skeletal muscle mass can be lost include physical inactivity, chronic health disorders, and aging (termed sarcopenia) (8). The average time course of sarcopenia is that by the age of 50 yr, ~10% of peak muscle mass is lost, and another 30% disappears by 80 yr of age (27). The health implications of muscle loss in the elderly are further exacerbated by the fact that aged populations seem to have a limited capacity to recover skeletal muscle mass after a bout of atrophy (29, 35). Skeletal muscles from old humans and animals also do not hypertrophy as well as their younger counterparts. In younger individuals, the exact time course for muscle fiber hypertrophy is not well documented but is thought to require at least 6-7 wk of regular resistive training at reasonably high intensity before increases in fiber cross-sectional area are deemed significant (33). Conversely, in humans averaging 87 yr of age, 10 wk of progressive resistance training increased thigh area by only 3% and knee strength by 200%, which Fiatarone et al. (16) attributed to an improved neural recruitment of existing, but underused, skeletal muscle, not actual hypertrophy. Animal studies further confirm the human observations in that skeletal muscle atrophied by hindlimb immobilization regrows on remobilization to its preatrophy size in younger (5, 9) but not older rats (8, 39). Some studies have found that mechanically overloaded skeletal muscle hypertrophies less in old than young rats (1, 4, 12). Thus the capacity of skeletal muscle to grow in response to increasing mechanical loads appears to be diminished in old human and animal subjects.

Inappropriate levels of factors permitting growth may underlie impaired muscle regrowth from atrophy at old age. For example, high-resistance exercise enhanced IGF-IEc mRNA (mechanogrowth factor) level in the vastus lateralis muscle of young, but not of old, human subjects (19). Delivery of exogenous IGF-I has been shown to rescue muscle from sarcopenia in old rodents (3, 8). Because many growth factors have been shown to alter muscle growth (17), the hypothesis was generated that a deficiency of multiple growth factors (an unknown "growth factor milieu") contributes to the incomplete regrowth of skeletal muscle in old animals. Another observation supporting a growth factor deficiency in old skeletal muscle is that a nearly complete or complete muscle regrowth is observed after myotoxin application (7). The effect of myotoxin treatment is to remove all muscle fiber protein and sarcolemma (sparing satellite cells, basal lamina, and nerves), which putatively releases growth factors for a massive regeneration process (7). Welle (38) contends that, although skeletal muscle has the capacity to regenerate itself, this process is not activated during the gradual age-related loss of skeletal muscle. In support of the aforementioned postulate, data from Fiatarone et al. (15) suggest that weight lifting did induce regeneration in the elderly, as evidenced by increased embryonic myosin heavy chain expression but was not sufficient to elicit muscle growth. Thus the hypothesis was generated that mRNAs for a subpopulation of growth factors would be differentially expressed between the regrowing soleus muscle of young rats and the nonregrowing soleus muscles of old rats. Affymetrix microarrays containing >24,000 genes and expressed sequence tags (ESTs) were employed to provide a global, unbiased determination of differential mRNA expression. To produce a muscle atrophied by inactivity for testing regrowth, young and old rats had their hindlimbs immobilized for 10 days, which resulted in a 27-37% atrophy of the soleus muscle. Changes in gene expression were then determined from soleus muscle samples at postimmobilization days 3, 6, 10, and 30. By doing a comprehensive mRNA assay, future hypotheses can be generated and subsequently tested, such as whether there is a group of genes involved in a common senescence program that prevent muscle growth that could potentially be altered, whether there are unidentified growth factors critical to muscle growth that are lacking in old muscle recovery, or whether there are potential molecular targets that can be manipulated to enhance growth of sarcopenic muscle or prevent sarcopenia altogether.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals. One hundred male Fischer 344 x Brown Norway Fl rats were obtained from the National Institute on Aging (Harlan Labs, Indianapolis, IN) and were killed at the ages of 3-4 mo (young, n = 50) and 30-31 mo (old, n = 50). Five rats were used per group (four groups: young controls, young immobilized/recovery, old controls, and old immobilized/recovery) at every time point (five time points: 0, 3, 6, 10, and 30 days) for a total of 20 groups. Half of the young and old rats were immobilized, whereas the other half of the animals were kept as controls with normal cage activity. The immobilization protocol has been previously published and is reviewed briefly next (5, 8). Before casting, animals were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (49 mg/ml), xylazine (6.2 mg/ml), and acepromazine (2.0 mg/ml) at a concentration of 0.10 ml/100 mg body wt. When rats became anesthetized, both hindlimbs were fixed from the waist down with wire mesh-reinforced plaster casts in the plantar-flexed position. Animals were maintained in the hindlimb-immobilized condition for 10 days. Rats were lightly anesthetized for removal of casts. All rats were housed 2-3 per cage and maintained on a 12: 12-h light-dark cycle, in which they received regular rat chow and water ad libitum. Before muscle removal, animals were anesthetized with an intraperitoneal injection of a cocktail containing ketamine (49 mg/ml), xylazine (6.2 mg/ml), and acepromazine (2.0 mg/ml) at a concentration of 0.123 ml/100 mg body wt. All animal experimental protocols were approved by the University of Missouri Animal Use Committee. Soleus muscles were excised, weighed, snap-frozen in liquid nitrogen, and subsequently powdered in a mortar and pestle cooled by liquid nitrogen. Both soleus muscles from a single rat formed a single observation, for which muscle RNA from a single animal was applied to an individual array. Although the recovery time points have not previously been published, the control (31) and the atrophy (J. S. Pattison, L. C. Folk, R. W. Madsen, T. E. Childs, E. E. Spangenburg, and F. W. Booth, unpublished observations) data have been published; these data sets found 682 and 739 differentially expressed probe sets, respectively, which were too large to be incorporated into a single paper.

Sample processing. Total RNA was extracted from an aliquot of muscle powder when put directly into TRIzol (Invitrogen; Carlsbad, CA) and homogenized on ice by using a Polytron homogenizer (Kinematica; Lucerne, Switzerland) on setting 7 for three pulses of 15 s each. RNeasy columns (Qiagen; Valencia, CA) were employed to further purify the extracted total RNA. Methods for sample preparation described in detail in the Affymetrix Expression Analysis Technical Manual (Santa Clara, CA) are briefly described next. cDNA synthesis was done on 10-µg aliquots of purified total RNA by using a T7-(dT)24 primer (100 pmol/µl). cDNA synthesis reactions were executed by using components of the Superscript Choice kit (Invitrogen; Carlsbad, CA), with all incubations done in a Mastercycler Gradient thermocycler (Eppendorf; Westbury, NY). The resulting double-stranded cDNA was quantified by use of a PicoGreen kit (Molecular Probes; Eugene, OR). One microgram of double-stranded cDNA was used in the in vitro transcription reaction employing biotinylated nucleotides and reagents provided in the BioArray high yield RNA transcript labeling kits (Enzo Diagnostics; Farmingdale, NY). The cRNA product was further purified by use of RNeasy columns (Qiagen). The purified biotinylated cRNA was then fragmented and subsequently hybridized to Affymetrix rat genome U34A, B, and C arrays. All arrays were analyzed by fluorescent intensity scanning according to Affymetrix protocols (Affymetrix Expression Analysis Technical Manual). The hybridization and scanning of the arrays were performed in the University of Missouri DNA Core Facility (Columbia, MO).

GeneChip analysis. The rat genome U34 array set contained 26,388 probe sets. The present experiment interrogated the relative abundance of ~24,000 genes and EST clusters (based on UniGene Build 34). Each probe set contained 16 perfectly matched (complementary) 25-mers, corresponding to different regions along the length of a transcript. Similarly, 16 mismatched pairs (encoding a single mutated base) that did not perfectly complement a mRNA's sequence were used as a measure of nonspecific background binding. Microarray Suite 5.0 software (Affymetrix) was used, which employs a one-sided Wilcoxon's signed-rank test to calculate a P value reflecting the significance of differences between the perfectly matched and mismatched probe pairs, on the basis of their fluorescence intensities. The resulting P values were used as a qualitative assessment of the ability to detect a given transcript, where P values < 0.04 were called "Present," P values between 0.04 and 0.06 were called "Marginal," and P values > 0.06 were called "Absent." Only probe sets that were Present or Marginal in >=60% of the samples for an experimental group were analyzed statistically. In total, 13,916 probe sets were sufficiently detected in at least one of the experimental groups, i.e., young or old soleus muscles. Microarray Suite 5.0 software (Affymetrix) utilizes statistically based algorithms to determine transcript abundance on the basis of fluorescence intensities (termed "signal"). The signal for each probe set was calculated as the one-step biweight estimate of the combined differences of all of the probe pairs in a probe set. All array data were normalized by scaling to target signal of 150. The calculated signal value was the variable utilized for all subsequent statistical analyses. Fold changes were calculated by dividing the mean signal intensities of the groups to be compared. In the past, microarray data analyses have been criticized as being "quite elusive about measurement reproducibility" (11). However, Bakay et al. (2) have reported that experimental error among Affymetrix microarrays is not a significant source of unwanted variability in expression profiling experiments (R2 = 0.979). Duplicate arrays were found to have small variability (R2 = 0.981) in the present experiment. Microarray data are available on the GEO database, in MIAME format (see Supplemental Data Tables 1 and 21).


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  		Table 1. Real-time PCR confirmed the microarray data that Elfin and amphiregulin mRNAs were significantly greater in the soleus muscles of young than the old rats whereas clusterin mRNA levels were significantly less in the young

 

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  		Table 2. Probe sets that had significant differences in expression as determined by a 3-factor ANOVA

 

Real-time PCR. Total RNA samples were purified over an RNeasy column (Qiagen). Total RNA was reverse transcribed into double-stranded cDNA during the previous cDNA synthesis for array sample processing with a T7-oligo(dT)24 primer. The resulting double-stranded cDNA was quantified by use of a PicoGreen kit (Molecular Probes). All 5' nuclease assays consisted of reactions containing 25 ng of cDNA, 250 nM MGB probe, 900 nM primers and Taqman7 Universal PCR Master Mix (ABI), in a 25 µl volume in duplicate with an ABI Prism 7000 sequence detection system. If a duplicate contained a range of >0.3 cycle time (CT), it was reassayed. All probe/primer combinations were designed using Primer-Express 2.0 (ABI) (Primer/probe sequences are in Supplementary Table 31). Real-time PCR data were analyzed for relative changes in expression by use of the {Delta}{Delta}CT method, according to User Bulletin no. 2 ABI PRISM 7700 sequence detection system. Relative efficiency plots were run to validate use of the {Delta}{Delta}CT method, where all slopes were <0.1. All targets were normalized to p38 mRNA expression. The expression of p38 was examined with an ANOVA on the factor combinations, examining the pairwise combinations of interest (young recovery vs. old recovery on days 3, 6, 10, and 30). Expression of p38 remained unchanged in the pairwise combinations of interest. Data are expressed as the calculated fold differences between different experimental groups (young control, young immobilized, old control, and old immobilized). Because of the limited amount of RNA isolated, some samples were exhausted before real-time PCR analysis, whereby the entire old 6-day control group was depleted; thus old controls from adjacent time points (3- and 10-day old controls) were used for statistical analyses of the gene expression changes at the 6th day of recovery. All other groups had n = 3-5 for real-time analyses.

Statistical methods. A three-factor ANOVA was employed to compare the signal values of young and old soleus groups (immobilized/controls) after 0, 3, 6, 10, and 30 days postimmobilization. Furthermore, a false discovery rate (FDR) adjusted P = 0.01 was applied to correct for the multiple ANOVAs performed on 13,916 probe sets that had been detected as present. As rats were killed in groups of five over a 40-day period, a one-way ANOVA was performed to determine whether any one control group differed from the other groups. Groups of young and old rats were analyzed separately. For real-time PCR analysis, the differences in {Delta}{Delta}CTs were analyzed with a 2 x 2 ANOVA at each time point with P < 0.05 set as significant for individual comparisons between different experimental groups (young control, young immobilized/recovery, old control, and old immobilized/recovery).

Database searching. The target sequences for the probe sets that differed significantly were analyzed with nucleotide BLAST analysis to identify known genes and to determine significant gene homologies with other species (http://www.ncbi.nlm.nih.gov/BLAST/). The target sequence is the region of a given gene or EST that was probed by the RG U34 arrays. Further information about a given sequence and its homologs and orthologs was procured from the Locuslink, Homologene, OMIM, mouse genome, rat genome, NetAffx, and Proteome databases (http://www.ncbi.nlm.nih.gov/LocusLink/, http://www.ncbi.nlm.nih.gov/HomoloGene/, http://www.ncbi.nlm.nih.gov/entrez/, http://www.informatics.jax.org/, http://rgd.mcw.edu/, https://www.affymetrix.com/analysis, https://www.incyte.com/proteome/databases). A gene's biological processes and molecular functions were classified by the defined gene ontologies given in the aforementioned databases.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
After 10 days of hindlimb immobilization, old and young rats lost comparable percentages of soleus mass: 27 and 37%, respectively (Pattison et al., unpublished observations). However, 30 days of recovery after the cessation of immobilization, young animals fully recovered their soleus mass to preatrophy size, whereas old animals recovered no significant muscle mass. Old soleus muscle mass did not differ between recovery days 0 and 30 (P = 0.948) (Fig. 1).



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  		Fig. 1. Failure of soleus muscles from old rats (B) to recover mass lost during 10 days of hindlimb immobilization (Imm), when the soleus muscles of young rats (A) show a full recovery to preatrophy mass from the same treatment. Wet weight of the soleus muscle (grams) is plotted as a function of recovery days after ending 10 days of hindlimb immobilization. Values are means ± SE; n = 5 rats/group. *P < 0.05.

 

A FDR adjustment for multiple testing was used, with the FDR taken to be 0.01. In this set of tests, the FDR level corresponded to an unadjusted P < 4.6 x 10-5. There were 64 probe sets that showed P < 4.6 x 10-5 in testing the overall model in the three-way ANOVA. With the use of an FDR adjustment for multiple testing at a level of 0.01, one would predict ~1 false positive out of the 64 significant differences. Three examples of differentially expressed mRNAs were selected for discussion, and their results are presented next. Elfin mRNA was less in the old rats at the 6th, 10th, and 30th recovery day (Figs. 2). Amphiregulin expression showed a unique spike in expression (~2-fold) in young soleus muscles after 10 days of reloading but no increase during recovery in the old soleus muscle (Fig. 3). Clusterin mRNA expression showed no change at any recovery time point in the soleus muscle of young rats while displaying increased expression in old soleus muscle at the 3rd, 6th, and 10th days of recovery (Fig. 4). Changes in mRNA expression for Elfin, amphiregulin, and clusterin were confirmed by real-time PCR (Table 1).



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  		Fig. 2. Elfin mRNA values in the soleus muscle are greater in young than old rats on the 6th, 10th, and 30th days of recovery from 10 days of hindlimb immobilization. Values are means ± SE; n = 5 rats/group. *Actual P < 4.6 x 10-5 from age-matched control.

 


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  		Fig. 3. Amphiregulin mRNA expression is elevated only in young soleus muscles at 10 days of recovery compared with old. Values are means ± SE; n = 5 rats/group. *Actual P < 4.6 x 10-5 from age-matched control.

 


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  		Fig. 4. Clusterin expression is elevated in old soleus muscles from days 3-10 of recovery, whereas no changes were observed in young. Values are means ± SE; n = 5 rats/group. *Actual P < 4.6 x 10-5 from age-matched control.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Numerous clinical conditions produce skeletal muscle wasting, each employing various combinations of physical inactivity, poor nutrition, increased cytokines, decreased anabolic factors, denervation, and reduced blood flow. Examples of these clinical conditions are catastrophic illness (cancer, chemotherapy, AIDS, etc.) (18), aging anorexia (18), hip fracture (29), and physical inactivity such as limb immobilization (8). Unfortunately, the recovery of muscle mass from these conditions seems to be limited in older humans. For example, the postoperative strength of 77-yr-old patients was impaired compared with 36-yr-old patients after surgery, in part because of diminished muscle mass (37). Significant ipsilateral quadriceps muscle atrophy (predominantly in type 2B and 2A fibers) occurred in 64-yr-old patients with chronic osteoarthritis of the hip. After total hip arthroplasty and rehabilitation, the wasting persisted 5 mo postoperatively judged by muscle ultrasound and strength measurements (35). Dependence on others to perform lower extremity physical activities of daily living tripled from prefracture level, after hip fracture, in 81-yr-old patients; this dependence remained doubled 2 yr postfracture (29). Depending on the population studied and function being assessed, an estimated 25-75% of those who are independently living before a fracture can neither walk independently nor achieve their previous level of independent living within 1 yr after their fracture (see Ref. 29 for references). The outcome of muscle wasting is low muscle strength, which Metter et al. (30) observed is associated with increased mortality, and they further stated that it was presumably a result of low muscle mass, which they did not measure. Here, we sought to examine differences in gene expression of young growing and old nonregrowing muscles independent of any clinical condition, thereby allowing us to examine only effects that are age induced on failed muscle regrowth from atrophy.

In the present study, an animal model of hindlimb immobilization was employed to produce atrophy of the soleus muscle for the purposes of testing the hypothesis that mRNAs for various growth factors would be differentially expressed between the regrowing soleus muscles of young rats and the nonregrowing soleus muscles of old rats after the immobilization was removed. The hypothesis was based on the failure of the soleus muscle of old rats to regrow after atrophy from hindlimb immobilization (8, 39), whereas young muscles did successfully regrow after immobilization (5, 9, Fig. 1 of present study). Growth factors were selected for the hypothesis because two groups have shown rescue of old skeletal muscle from atrophy by addition of exogenous IGF-I (3, 8), whereas the upregulation of endogenous growth factors does not appear to improve muscle growth (19). It was, therefore, deduced that an undefined growth factor milieu might be missing within the old, atrophied skeletal muscle and that microarrays could be employed as a global screen to identify potential genes for future study. Microarrays with ~24,000 genes and ESTs were employed for an unbiased assessment of the hypothesis. The strategy was that identification of differentially expressed mRNAs between regrowing young and nonregrowing old soleus muscles would provide unexpected new candidates for growth-stimulatory factors missing in the soleus muscle of old rats but present in young or inhibitory factors present in old that were absent in young.

Elfin mRNA was higher in the soleus muscle of young than old rats on recovery days 6, 10, and 30 (Fig. 2). Elfin (previously named CLIM1) is a member of the Enigma family of cytoplasmic proteins that contain a NH2-terminal PDZ domain and a series of COOH-terminal LIM domains (25). COOH-terminal LIM domain proteins are associated with the cytoskeleton and act as modular protein-binding interfaces mediating protein-protein interactions in the cytoplasm and nucleus by influencing the localization and activity of its specific protein partners (21, 23). In a blot of 16 human tissues, Elfin mRNA was expressed highest in the heart and next highest in skeletal muscle (26). Elfin colocalizes with {alpha}-actinin at Z-discs in the human myocardium (24), is expressed throughout the developing heart at embryonic day 8.5 (25), and colocalizes with actin stress fibers in C2C12 myoblasts (25). An emerging concept in muscle cell biology is that cytoskeletal-associated proteins may serve as molecular messengers that enable muscle cells to sense load or stretch and then signal a physiological response (10, 13). Elfin could be such a molecule. From the above context, we hypothesize that limited signaling from the Z-disc in the soleus muscle of old rats recovering from atrophy may contribute to its insufficient regrowth.

Amphiregulin mRNA was significantly increased in the soleus muscle at the 10th recovery day in only the young, but not in the old, rats (Fig. 3). Amphiregulin, also known as schwannoma-derived growth factor, is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells, and fibroblasts. Amphiregulin is also a mitogen for adult neural stem cells (15). Nerve terminal disruption, exposed junctional folds, and postsynaptic areas that contained little or no postjunctional folds were present at the neuromuscular junction of the soleus muscle on the 5th day of hindlimb immobilization, suggesting neural plasticity (14). Neuromuscular remodeling occurs in old skeletal muscle (6). Therefore, considering the above information, the lack of increase in amphiregulin mRNA in the old soleus muscle sets up the hypothesis for future studies that insufficient amphiregulin gene expression contributes to the failure of old skeletal muscle to recover from immobilization-induced atrophy because of insufficient neural neutrophic factors.

Whereas amphiregulin mRNA was significantly greater in the soleus muscle of young than in old rats at the 10th recovery day, the age effect was reversed for clusterin mRNA. Old rats had significantly more clusterin mRNA in their soleus muscles during recovery than young rats (Fig. 4). Peak expression of clusterin mRNA (~3-fold increase) was observed at 3 days of recovery and remained significantly elevated through 10 days of recovery in old soleus muscle recovery. Conversely, young soleus muscles showed no significant changes in clusterin expression from control levels in any of the time points measured. Clusterin (also known as complement lysis inhibitor, SP-40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, and apolipoprotein J) is a heterodimeric-conserved glycoprotein that is expressed in a wide variety of tissues and found in all human fluids (36). Its precise function is still uncertain, although it has been implicated in several diverse physiological processes such as developmental remodeling, apoptotic disease states as well as neurodegeneration in Alzheimer's disease, response to injury, and other stresses (22). Rat clusterin is expressed at high levels in dying cells, suggesting an involvement in the process of cell death, with a role in the terminal complement reaction acting as a control mechanism of the complement cascade; specifically, binding a C5b-C7 complex to the membrane of the target cell, and in this way inhibits complement-mediated cytolysis (34). However, clusterin can act to either promote or inhibit cell death, depending on the cellular context (22), e.g., mice devoid of the clusterin gene have 50% less brain injury after neonatal hypoxia injury (20), whereas clusterin limits progression of autoimmune myocarditis and protects the heart from postinflammatory tissue destruction (28). On the basis of the spike in clusterin mRNA only in the nonregrowing soleus muscle in old rats, the hypothesis is made that clusterin could play some inhibitory role, possibly signaling cell death, in the failure of old soleus muscles to regrow from atrophy after immobilization-induced atrophy ceases.

Other mRNAs identified as differentially expressed fell into a host of functional categories (listed in Table 2). Other known growth factor-related mRNAs identified with three-factor interactions included interleukin-15, IGF-binding protein-3, an EST similar to mouse pre-B-cell colony-enhancing factor, and an EST weakly similar to rat PDGF receptor-{alpha}. However, some of the aforementioned growth factor-related mRNAs seemed to show more of an atrophy-specific effect than a true recovery effect. The atrophy-specific effects have been reported in detail elsewhere (Pattison et al., unpublished observations). The hypothesis of the present study that a group of "growth factor milieu" mRNAs would be differentially regulated between young, regrowing and old soleus muscle failing to regrow was not conclusively supported (Table 2). However, the failure to find a disproportionate number of growth factor mRNAs should not be interpreted to mean that these are unimportant in soleus muscle regrowth, because changes in only a few critical growth factors could ameliorate the failed muscle regrowth of old soleus muscles after hindlimb immobilization.

In summary, the present analysis identified 64 new candidates, including 38 ESTs, whose inappropriate gene expression could play some role in the failure of old skeletal muscle to regrow to its preatrophy mass after ending immobilization. Among these, the growth factor amphiregulin supports our initial hypothesis that a deficiency of multiple growth factor mRNAs (an unknown growth factor milieu) contributes to the incomplete regrowth of skeletal muscle from inactivity-induced atrophy in old animals. Future work could show roles for Elfin between regrowing young muscle and nonregrowing old muscle gene expression in maintenance and growth of muscle mass. Finally, the upregulation in old skeletal muscle of clusterin mRNA, whose protein has been associated with increased cell death, could contribute to the poor regrowth of old atrophied muscle.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
ACKNOWLEDGMENTS
 REFERENCES
 
The research was supported by National Institute on Aging Grant AG-18881.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Dr. Gary Allen for providing access to the Bioinformatics Consortium at the University of Missouri and Dr. Mark McIntosh for leadership in establishment of the Affymetrix Core facility at the University of Missouri. We also thank Dr. Espen Spangenburg for thoughtful discussions and Aaron Wheeler for assistance in database searching.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. W. Booth, Univ. of Missouri, Dept. of Veterinary Biomedical Sciences, E102 Vet. Med. Bldg., 1600 E. Rollins, Columbia, MO 65211 (E-mail: boothf{at}missouri.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The supplementary materials for this article are available online at http://jap.physiology.org/cgi/content/full/00500.2003/DC1. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 

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