INTRODUCTION

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INNERVATION OF SKELETAL MUSCLE fibers during development induces the redistribution of preexisting fetal acetylcholine receptors (AChRs; composed of ₂ subunits) from the entire sarcolemmal membrane to the subsynaptic membrane (4). Nerve-induced clustering of fetal AChRs is also associated with the disappearance of AChRs harboring the -subunit and the selective expression of AChR -subunit gene by myonuclei underlying the newly formed neuromuscular junction (4). Thus innervated (adult) muscle fibers either do not express (37) or express very low levels (30, 34) of the AChR -subunit mRNA. Presence of the -subunit in the AChR characterizes the adult subtype (composed of ₂ subunits) (29, 37) and functionally serves to decrease the length of channel-opening bursts and the duration of miniature end-plate potentials while increasing the conductance and Ca²⁺ permeability of end-plate channels, compared with AChRs containing the -subunit (20). In short, the innervation of skeletal muscle fibers during development results in alterations of AChR subunit composition, function, and localization.

Expression of the AChR -subunit is driven in part by the myogenic transcription factor myogenin (15). Myogenin dimerizes with E proteins to bind with two E boxes in the -subunit promoter (8). Innervated adult skeletal muscle expresses little or no myogenin; however, its expression is increased on denervation (5). Expression of AChR -subunit (40) mRNA and protein (13) is also increased with denervation, and the dogma is that myogenin is driving transcription of the AChR -subunit gene (15). We have previously (26) reported an upregulation of myogenin mRNA in the atrophying tibialis anterior muscle of old rats and hypothesized that AChR -subunit mRNA would be upregulated in old atrophying skeletal muscle. Supporting this hypothesis is the observation of Pestronk et al. (32) in 1980. They reported a 73% increase in the density of extrajunctional AChRs concomitant with denervation-like changes in neuromuscular junction morphology of soleus muscles from 28-mo-old relative to 18-mo-old rats. Because the expression of the AChR -subunit is restricted to the subsynaptic membrane, and because denervation of adult skeletal muscle results in renewed expression of -subunit containing AChRs throughout the entire muscle fiber membrane (23), the observations of Pestronk et al. (32) suggested to us that the AChR -subunit mRNA could be reexpressed or expressed at detectable levels in skeletal muscles of old rats. However, pretranslational events underlying the upregulation of extrajunctional AChRs in muscles of old rats have not been described.

Whereas both old humans and rats lose whole motor units (25), -motoneurons (19, 35), and muscle fibers (18, 22, 24, 33), it has also been reported that muscle atrophy (11) and -motoneuron loss (17) begin earlier and become more pronounced with aging in the lower than in the upper half of the body. Thus we compared a skeletal muscle from both the rat hindlimb and forelimb to determine whether an age-related atrophy would be present in the hindlimb, but not the forelimb, muscle. We hypothesized that increases in AChR -subunit and myogenin mRNAs would be associated with atrophying muscle in the hindlimb. Furthermore, we hypothesized that these increases would be of lesser magnitude in a nonatrophying muscle of the forelimb.

	MATERIALS AND METHODS

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Animals. Specific pathogen-free Fischer 344/Brown Norway F1 generation cross male rats were obtained from the National Institutes of Health-Aging Program (Harlan, Indianapolis, IN); they were aged 2 mo (young), 18 mo (adult), and 31 mo (old) (n = 10 rats/age group), which are age designations employed in gerontological studies (7). Animals were housed two per microisolator cage and maintained in a barrier-controlled, positive-pressure room at 21°C with a 12:12-h light-dark cycle. All food, water, and bedding were sterilized by autoclave. All animal protocols were approved by the Institutional Animal Welfare Committee, University of Texas Health Science Center. Generation of positive control mRNA for myogenin and AChR -subunit expression was accomplished by partially denervating the right hindlimb of an adult rat. Briefly, a 3-mm segment of the sciatic nerve at L₄ was excised and removed. At day 7 postaxotomy, both left (unoperated control) and right (partially denervated) gastrocnemius muscles were harvested, trimmed of excess connective tissue, rapidly frozen in liquid nitrogen, weighed, and stored at 80°C until further analysis.

Muscle sampling. Rats were anesthetized with an intramuscular injection (1.4 ml/kg) of a mixture containing ketamine (54 mg/ml), xylazine (2.2 mg/ml), and acepromazine (3.5 mg/ml). The entire biceps brachii and gastrocnemius muscles of both forelimbs and hindlimbs were removed, trimmed of excess connective tissue, and rapidly frozen in liquid nitrogen. Muscles were weighed and subsequently stored at 80°C until further analysis. Rats were euthanized by cervical dislocation while under anesthesia.

Selection of the biceps brachii for comparison with the gastrocnemius muscle was made for the following reasons: 1) the biceps brachii and gastrocnemius muscles have a similar composition of fiber types; 2) both the biceps brachii and gastrocnemius muscles show signs of atrophy in old age; and 3) in a previous report (26), a 41% atrophy of the tibialis anterior muscle was noted in some of the same rats employed in this study, which suggested that neither flexor nor extensor function affected atrophy of these leg muscles.

Total RNA isolation and transfer. For isolation of total RNA, muscles were powdered under mortar and pestle and cooled in liquid nitrogen. Approximately 250 mg of muscle were homogenized in Trizol (BRL), and total RNA was isolated by using the guanidine thiocyanate method of Chomczynski and Sacchi (6). Twenty micrograms of RNA isolated from each muscle were loaded onto a denaturing 1% agarose gel [1× 3-(N-morpholino)propanesulfonic acid, 6.7% formaldehyde] and electrophoresed at 5 V/cm for 2.5 h. The integrity and concentration of the RNA were confirmed by visual inspection of ethidium bromide-stained 18S and 28S rRNAs. The RNA was then transferred to a nylon membrane by capillary action.

Probe generation. Separate vectors (pSP72) containing 1.8 (rAChR10) and 2.3 (rAChR3) kb of rat - and -subunit cDNA, respectively, were generously supplied by Dr. Vite Witzemann (40). The entire AChR -subunit cDNA was used as a probe for Northern blot analysis. The AChR -subunit fragment (366 bp) used as a DNA probe was cut from pSP72 by using EcoR I and Pst I restriction enzymes. The 4.1-kb vector (pBS-MGN#1) containing 1.4 kb of rat myogenin cDNA was generously supplied by Dr. Victor K. Lin (41). The 1.4-kb fragment of myogenin cDNA was cut from the vector by using EcoR I restriction enzyme. To correct for mRNA-loading differences and transfer efficiency, we employed a 1.2-kb fragment of the mouse 18S RNA gene (Ambion). After restriction enzyme digestion, all cDNA fragments were separated in a 1.2% agarose gel and purified from the agarose by using GeneClean (Bio101).

Probes for Northern blot analysis were generated by random priming. Briefly, 20-50 ng of each cDNA fragment was combined with 1 µl of deoxynucleotide triphosphate (dNTP) random hexamer d(N₆)TP (Pharmacia), and a final volume of 15.5 µl was achieved with double-distilled H₂O. This cocktail was heated to 95°C for 5 min then cooled on ice for 5 min. After a brief spin, 2.5 µl of DNA polymerase buffer (Promega), 1 µl of dNTPs (25 mM, minus CTP), 5 µl of [-³²P]dCTP (10 mCi/ml; Amersham), and 1 µl of Klenow fragment (1 unit; Promega) were added, mixed, and incubated at 37°C for 30 min. Free nucleotides were separated from probe fragments via spin column (Sepharose G50, Sigma Chemical). Specific activities of the probes were determined by scintillation counting and ranged from 1-3 × 10⁹ counts · min¹ · µl¹.

Hybridization. Probe hybridization was carried out for 3 h at 66-68°C in Quikhyb (Stratagene). The specific activity of hybridization ranged between 1-2 × 10⁶ counts · min¹ · ml¹ of Quikhyb. After hybridization, blots were washed two times for 15 min in 2× saline-sodium citrate (SSC) + 0.1% SDS at room temperature, then once for 15 min in 0.5× SSC + 0.1% SDS at 50°C, and finally for 15 min in 0.1× SSC + 0.1% SDS at 50°C. Blots were subsequently exposed on X-ray film at 80°C.

Data collection and analysis. Autoradiographs were quantitated by densitometric scanning (BioImage, Millipore) as integrated optical density (IOD). The IOD values of 18S mRNA were used to correct for loading differences for myogenin, -, and -subunit IOD mRNAs. Data are expressed as mean IODs relative to a percentage of 18S RNA IOD.

Statistics. Statistical analysis was performed by using a one-way analysis of variance. Significant differences among the means were detected by using the Tukey-Kramer post hoc test with the level of significance set at P  0.05. Values are expressed as means ± SE.

	RESULTS

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Body and muscle mass. The body masses of adult (511.4 ± 28.7 g) and old (559.6 ± 22.3 g) Fischer 344/Brown Norway F1 generation cross rats were significantly greater than those of young animals (274.4 ± 15.4 g; n = 10 rats/group). However, body masses were not statistically different between adult and old rats. Wet weights of biceps brachii muscles from adult and old rats were not different from each other but were significantly greater than biceps brachii muscles from young rats (n = 5 rats/group; Fig. 1A). In contrast, gastrocnemius muscle masses from old rats were significantly less than in muscles from young and adult rats (n = 10 rats/group; Fig. 1B). Gastrocnemius muscle masses between young and adult rats were not statistically different.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Muscle masses of young (2-mo-old), adult (18-mo-old), and old (31-mo-old) Fischer 344/Brown Norway F1 generation cross rats. Masses of biceps brachii muscles (A) of adult and old rats were not significantly different from each other but were significantly more than in the young group (n = 5 rats/group). Wet weights of gastrocnemius muscles (B) were significantly less in the old group than in either the young or adult groups (n = 10 rats/group). * P  0.05.

Myogenin and AChR mRNAs. Levels of myogenin mRNA detected in biceps brachii muscles were not different between young, adult, and old rats (Figs. 2 and 3A). In contrast, levels of myogenin mRNA detected in gastrocnemius muscles of old rats were significantly elevated 11- and 2.75-fold above levels in young and adult rats, respectively. The expression of myogenin mRNA in gastrocnemius muscles of adult rats suggests a trend toward but was not statistically different from levels expressed in young rats (P = 0.08). As a positive control for myogenin mRNA detection, partially denervated gastrocnemius muscle yields a level of myogenin mRNA 1.8-fold higher than mRNA from gastrocnemius muscles of old rats (Fig. 2).

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Representative Northern blot analysis of myogenin, -subunit of acetylcholine receptor (AChR), -subunit of AChR mRNAs, and 18S rRNA in biceps brachii and gastrocnemius muscles of young (Y = 2-mo-old), adult (A = 18-mo-old), and old (O = 31-mo-old) Fischer 344/Brown Norway F1 generation cross rats. Same membrane was used to determine myogenin, AChR -subunit, AChR -subunit, and 18S rRNA (in that order) after stripping previous probe and autoradiogram exposure to verify absence of radioactivity. Approximate sizes (in kb) of detected mRNAs were determined from relative position to 18S rRNA (1.9 kb). On right, partially denervated (PD) and its contralateral unoperated (CPD) gastrocnemius muscle.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Myogenin (A), AChR -subunit (B), and AChR -subunit (C) mRNA levels in biceps brachii (solid bars, n = 5) and gastrocnemius (open bars; n = 10) muscles of young (2-mo-old), adult (18-mo-old), and old (31-mo-old) Fischer 344/Brown Norway F1 generation cross rats. Values for each mRNA have been normalized to 18S rRNA and are expressed as intensity of optical density units (IOD) per IOD of 18S rRNA (%18S IOD). * P  0.05. Myogenin and -subunit of AChR mRNAs are significantly higher in gastrocnemius muscles of old than of young or adult rats.

In a similar fashion, the expression of AChR -subunit mRNA in young, adult, and old rats paralleled myogenin mRNA expression (Figs. 2 and 3B). AChR -subunit mRNA expressions in biceps brachii muscles from young, adult, and old rats were not different. However, in gastrocnemius muscles, the AChR -subunit mRNA expression from old rats was elevated 9.8- and 4.7-fold higher than in young and adult rats, respectively. Expression levels for the AChR -subunit mRNA in gastrocnemius muscle from young and adult rats were not statistically different. In partially denervated gastrocnemius muscle, AChR -subunit mRNA expression was elevated 13-fold higher than levels detected in mRNA from gastrocnemius muscles of old rats (Fig. 2). The IODs for AChR -subunit mRNA in young and adult rats were much lower relative to myogenin or AChR -subunit mRNAs. In addition, no signal for AChR -subunit mRNA was detectable in some animals in the young and adult gastrocnemius groups and in all age groups for biceps brachii muscle, (Figs. 2 and 3), which may contribute to the variability observed.

In contrast to expression of myogenin and AChR -subunit mRNAs in gastrocnemius muscles, Northern blot analysis of AChR -subunit mRNA in biceps brachii muscles from young, adult, and old rats did not reveal statistical differences. (Fig. 2 and Fig. 3C). Similarly, no differences among age groups existed for AChR -subunit mRNA in the gastrocnemius muscle. Unlike expression of AChR -subunit mRNA, AChR -subunit mRNA expression and accumulation at the end plate is believed to be largely independent of the nerve or electrical muscle activity (38). Thus our inability to measure a significant change in AChR -subunit mRNA levels in gastrocnemius muscles from old rats was not surprising.

	DISCUSSION

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Skeletal muscle denervation has been used as a model system to study the molecular mechanisms by which innervation and muscle activity regulate synaptic gene expression (1). In surgically denervated skeletal muscle of young animals, all of the following increase: 1) myogenic transcription factor mRNAs, including those of MyoD, myogenin, and MRF4 (5); 2) sensitivity of the muscle fiber to acetylcholine (2); 3) expression of fetal AChRs along the entire length of the sarcolemmal membrane within 48 h of the denervation (9); and 4) the levels of the -, -, -, and -subunit mRNAs (9). In fact, surgical denervation of young or adult skeletal muscle is classically associated with increased expression of AChR -subunit mRNA (1, 12-14, 36-39). Furthermore, previous investigators have shown that an elevation in the transcription factor myogenin transactivates the AChR -subunit promoter by forming heterodimers on its E-box regulatory sites (21, 31). We report here for the first time in gastrocnemius muscle from old animals a renewal of AChR -subunit mRNA expression.

How might AChR -subunit mRNA increase in atrophied muscles of old rats? Witzemann et al. (39) compared the effects of surgical denervation and of presynaptic and postsynaptic pharmacological blockade for neuromuscular transmission on changes in AChR -subunit mRNA expression in skeletal muscle. They concluded that two different neural signals are responsible for the downregulation of AChR -subunit mRNA in the innervated muscle fiber: 1) an inhibitory neural-derived signal that presumably acts locally at the end plate and 2) a muscle-derived signal that is linked to nerve-induced activity that acts along the whole fiber length. In normally innervated fibers, -subunit mRNA is decreased to undetectable levels and is only slightly elevated in muscles kept electrically inactive by mere disuse of the neuromuscular synapse (postsynaptic pharmacological blockade). However, in muscle fibers inactivated by surgical denervation or by presynaptic blockade of neuromuscular transmission, the -subunit is abundant along the entire fiber length. We speculate that nerve terminal retraction of some muscle fibers from old rats does not result in reinnervation. Under these conditions, the denervated fiber would atrophy and die. We hypothesize that muscle atrophy in our old rats is a secondary event to alterations at the neuromuscular junction.

What evidence is there for denervation or denervation/reinnervation events to have occurred in muscle fibers of old rats? The report of Pestronk et al. (32) provides multiple indexes of skeletal muscle denervation in the soleus muscles of 28-mo-old Wistar rats, such as increased extrajunctional AChRs, the multiple innervation of end plates, single preterminal axons innervating 6-10 muscle fibers, grouped muscle fiber atrophy, and an increased incidence of muscle fiber-type grouping. In addition, Fagg et al. (10) found that a high degree of terminal sprouting and >50% of preterminal axons exhibit nodal sprouts in soleus muscles of 27-mo-old Sprague-Dawley rats. Marsh et al. (27) observed a decreased isometric tension, an increased incidence and degree of tetanic fade (a rapid waning of tension despite continued stimulation) in the tibialis anterior muscle in old, but not young, Fischer 344/Brown Norway F1 cross rats. These observations suggest that there are age-associated alterations at the neuromuscular junction. Our data for myogenin and AChR -subunit mRNA from old rats are consistent with and extend these morphological and functional observations of denervation by providing support at the molecular level.

Skeletal muscle atrophy (11) and -motoneuron loss (17) begin earlier and become more pronounced with age in the hindlimbs than in the forelimbs of rats. Both voluntary and involuntary force in old humans decreased more in the triceps surae group than in the elbow flexor muscles (28). Consistent with observations in the rat, we report a 17.5% decline in gastrocnemius (hindlimb) muscle mass of the 31-mo-old Fischer 344/Brown Norway F1 generation cross rats, whereas the biceps brachii (forelimb) muscle mass from the same rats did not change. We extend on our previous observation of increased myogenin mRNA in atrophying old muscle by our finding of less drastic changes in the mRNAs of myogenin and the -subunit of the AChR in a nonatrophying old muscle (biceps brachii). Moreover, our previous finding of increased myogenin mRNA expression in the atrophied tibialis anterior muscles of old rats (26) is now further supported by our present observation of an increase in both myogenin and AChR -subunit mRNA levels in the atrophied gastrocnemius muscle. The association between muscle atrophy and increases in the mRNAs for myogenin and the -subunit of the AChR provides additional evidence that these two events (atrophy in old muscle and compensatory changes of mRNAs in the direction induced by surgical denervation) are related. Not as drastic of a change in myogenin and AChR -subunit mRNAs in the nonatrophying biceps brachii muscle, relative to changes in the gastrocnemius muscle of the same rat, implies that increases in these mRNAs are related more to loss of muscle mass in old rats than to the chronological age of the animal. Because Hashizume and Kanda (18) reported that the number of medial gastrocnemius, but not ulnar, -motoneurons decreased in 27-mo-old Fischer 344 rats, we speculate that denervation may be more prevalent in the gastrocnemius than in biceps brachii muscle of 31-mo-old rats in the present study.

Considered in concert, our findings indicate that muscle atrophy with aging is associated with an upregulation of myogenin and AChR -subunit mRNAs, whereas AChR -subunit mRNA levels are unchanged. Our observation of elevated AChR -subunit mRNA in atrophied muscles of old rats is a novel molecular observation. In disused skeletal muscle, the intact nerve-muscle interface suffices to drastically inhibit AChR -subunit mRNA levels even in electrically silent muscle (35), which is markedly different from the robust increase observed in surgically denervated muscle. This has led to the idea that in innervated skeletal muscle AChR -subunit mRNA is downregulated by an activity-independent neuronal inhibitory signal (39). Therefore, we speculate that the upregulation of AChR -subunit mRNA in gastrocnemius muscle from old rats is related to a retraction of the nerve terminal. Upon muscle fiber denervation with age, the neuronal inhibitory signal may be lost and -subunit mRNA expression is subsequently increased. Because denervation/reinnervation events are a continuous process throughout the lifespan of an animal, one could speculate that the efficiency of reinnervation is greatly impaired with age in the gastrocnemius, relative to the biceps brachii muscle. Hall and Sanes (16) have speculated on the physiological significance of -subunit AChRs in muscles of embryonic rats, stating that the -subunit may be more effective at depolarizing smaller muscle fibers than are -subunit AChRs. A similar speculation could be made for small denervated fibers in old muscles. What advantage this offers the muscle is open for further speculation. Whereas it is not known whether the mechanisms that underlie the age-associated synapse loss (3) are presynaptic, postsynaptic, or both, our present observations indicate that some of the potential postsynaptic mechanisms are changing in a direction consistent with the denervation of individual fibers. We hypothesize that increases in AChR -subunit and myogenin mRNA happen in elderly humans, in whom losses of total motor units, -motoneurons, muscle fibers, mass, and strength are occurring.