Hypertrophy-stimulated myogenic regulatory factor mRNA increases are attenuated in fast muscle of aged quails

Dawn A. Lowe1, Troy Lund2, and Stephen E. Alway1,3

Departments of 1 Anatomy and 2 Medical Microbiology and Immunology, College of Medicine, and 3 Institute on Aging, University of South Florida, Tampa, Florida 33612

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Myogenic regulatory factors (MRFs) are a family of skeletal muscle-specific transcription factors that regulate the expression of several muscle genes. This study was designed to determine whether MRF transcripts were increased in hypertrophy-stimulated muscle of adult quails and whether equivalent increases occurred in muscles of older quails. Slow-tonic anterior latissimus dorsi and fast-twitch patagialis muscles of adult, middle-aged, aged, and senescent quails were stretch overloaded for 6, 24, or 72 h, with contralateral muscles serving as controls. RNase protection assays showed that MRF4 and MyoD transcript levels were increased and myogenin and Myf5 transcripts were induced in stretch-overloaded muscles. However, MRF4 and MyoD increases were significantly attenuated in patagialis muscles of older quails. RT-PCR analyses of three MRF-regulated genes showed that increases in the transcription of these genes occurred with stretch overload, but the increases were less in muscles of older quails. In summary, attenuated MRF responses in muscles from aged animals may partially explain why muscles from older animals do not hypertrophy to the same extent as muscles from younger animals.

aging; stretch overload; skeletal muscle hypertrophy; MyoD; MRF4

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

HYPERTROPHY OF SKELETAL MUSCLE involves an increased rate of synthesis and accumulation of proteins, and therefore increased transcription of muscle genes is necessary. The mechanisms by which nuclei increase transcription of specific skeletal muscle mRNA in response to a hypertrophy stimulus are not known. We suspect that a family of skeletal muscle-specific transcription factors called myogenic regulatory factors (MRFs) is involved. This family is composed of four members that were identified on the basis of their ability to convert nonmuscle cells to myoblasts: MRF4/Myf6/herculin (33), MyoD (7), myogenin (39), and Myf5 (4). These factors belong to a larger basic helix-loop-helix (bHLH) class of transcription factors. The basic region binds DNA, whereas the HLH region is involved in homo- or heterodimerization with other HLH proteins. Heterodimerization with a ubiquitous E protein is most common, and this dimerization increases the efficiency of binding to target E boxes that are present in the promoter region of several skeletal muscle genes (30). E box-containing genes, which MRFs have been shown to bind and regulate, include alpha -actin (28), desmin (20), nicotinic ACh receptor (AChR) (31), muscle creatine kinase (17), troponin I (21), and myosin light chain (36). Because increases in structural and contractile proteins (e.g., desmin and troponin I), as well as muscle-specific enzymes (e.g., muscle creatine kinase), are necessary for muscle to hypertrophy and remain functional, we hypothesize that MRFs are upregulated in response to a hypertrophy stimulus. Additionally, hyperplasia often accompanies hypertrophy (1, 2, 5, 19), and this would require increases in AChR proteins to enable neural connections to the new fibers.

MRFs play pivotal roles in establishing the myogenic lineage and in controlling terminal differentiation (23), and thus most studies of the MRFs have been conducted from a developmental perspective. In general, Myf5 and MyoD are required for the determination of myoblasts, myogenin is critical for differentiation, and the function of MRF4 appears to be in fully differentiated fibers. The roles of MRFs in regenerating muscle and in maintaining muscle phenotype have also been investigated. Muscle regeneration involves activation of satellite cells, the only myogenic cells in adult skeletal muscle. Proliferation and differentiation of these cells are similar to events that occur during embryogenesis, including increased expression of MRFs (34). MyoD and myogenin have also been implicated in regulating muscle fiber type, as myogenin accumulates in slow-twitch fibers and MyoD accumulates in fast-twitch fibers (13, 35). Few studies have investigated MRFs in fully differentiated, hypertrophic muscle (14, 22). The first objective of this study was to determine whether skeletal muscle stimulated to hypertrophy by stretch overload had an increased expression of MRFs and whether increases in the transcription of genes regulated by MRFs occurred.

Protein synthesis and accumulation are attenuated in skeletal muscle of older individuals (24). Consequently, muscles of aged animals have a reduced ability to hypertrophy. For example, 30 days of overload resulted in a 44% increase of muscle mass in adult quails but only a 26% increase in aged quails (19). It is not known which step(s) in the chain of processes that lead to an increase in muscle protein content is responsible for the attenuation. It is possible that specific genes are not activated in hypertrophy-stimulated muscle in aged animals. This would occur if transcription factors, such as the MRFs, are lower or have reduced activity in these muscles. Two studies have shown MRF expression in muscle from aged animals (27, 29). In those studies it was reported that basal levels of MRF transcripts were higher in hindlimb muscles from aged mice (29) and rats (27) than in muscles from younger mice and rats, respectively. However, it remains to be determined whether MRF expression in muscle from older animals can be elevated further with a hypertrophy stimulus. Thus our second objective of this study was to determine whether hypertrophy-stimulated muscle in aged and senescent animals showed increases in MRF expression and transcription of genes regulated by MRFs similar to that in younger animals. The final objective was to define whether MRFs were regulated differently in fast- and slow-twitch muscles during hypertrophy.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals and experimental protocol. Japanese quails (Couturnix couturnix Japonica) of four different ages were studied. Quails were obtained from a breeder (Willow Games Farms) at 16-18 mo of age and used as the oldest animals in this study when they reached 28 mo of age (senescent). Eggs produced by the quails were incubated and hatched in an incubator (Humidaire Incubator, New Madison, WI). Chicks were housed in a chicken brooder for 2 wk and then housed five to seven quails per cage, along with the older quails, at 20-23°C with a 12:12-h light-dark cycle. Chicks were studied when they reached 4 (adult), 8 (middle aged), and 18 mo (aged). The life span of Japanese quails is ~2.5 yr, with a mortality rate of ~60% by 28 mo (38). Quails in our colony have lived as long as 32 mo. Japanese quails are physically and sexually mature at 6 wk and show no maturational changes in carcass composition or body weight beyond 2 mo after hatching (25, 26, 38). Body weight did not differ among adult, middle-aged, aged, and senescent quails in this study: 173 ± 28, 176 ± 22, 170 ± 23, and 174 ± 20 (SD) g, respectively (P = 0.92, n = 9 for middle-aged group and 21 for adult, aged, and senescent groups). It was important that the quails were at a stable body weight during these experiments so that hypertrophy-stimulated growth was not confounded by normal growth of the animals. Male and female quails were used in these studies, because hypertrophic responses are not different between the sexes in these animals (S. E. Alway, unpublished observations).

Muscle hypertrophy was induced by stretch overload of the right wing, as described previously (2). The left wing served as a contralateral control. After 6, 24, or 72 h of stretch overload (n = 7 per age group per time, except for the middle- aged group, where n = 3 per time), quails were anesthetized with pentobarbital sodium (35 mg/kg ip). The anterior latissimus dorsi (ALD) and patagialis (Pat) muscles from both wings were excised and immediately frozen in liquid nitrogen to preserve RNA quality and then stored at -80°C.

Riboprobes. Quail cDNA clones for MyoD (qmf1-cc509) (6), myogenin (qmf2-cc527) (32), and Myf5 (qmf3-cc528) (32), provided by Dr. Charles Emerson (Fox Chase Cancer Center, Philadelphia, PA), were subcloned to make riboprobes that could be used simultaneously in RNase protection assays (RPAs). MyoD riboprobes were made from a Dde I-EcoR I 267-bp fragment of qmf1, myogenin probes from a Hind III-BamH I 203-bp fragment of qmf2, and Myf5 probes from a Sac II-EcoR I 408-bp fragment of qmf3. Restriction fragments were cloned into pBluescript KS vectors (Stratagene, La Jolla, CA). For the quail MRF4 riboprobe, total RNA from quail muscle was reverse transcribed (Superscript II RNase H- RT, Life Technologies) using oligo(dT) primers (Promega, Madison, WI) and then amplified by PCR. Oligonucleotides used for PCR were derived from the chicken MRF4 mRNA sequence (9) and produced a 330-bp PCR product (5'-GGCTGGATCAGCAGGACAAA and 3'-AGGGCCGTTCGCCGGGGGGA; annealing temperature 62°C). The resulting cDNA was cloned using the pCR-Script Amp SK(+) Cloning Kit (Stratagene), and the insert was verified by DNA sequencing; the quail 330-bp cDNA was 98% homologous to chicken MRF4 mRNA. A 150-bp segment of quail glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified by RT-PCR. The lower primer was synthesized, including a T3 phage promoter element (Table 1). The resulting PCR product was then transcribed directly. Riboprobes were transcribed using biotin-14-CTP and T7 or T3 RNA polymerase (BrightStar BIOTINscript Kit, Ambion) and then gel purified. In the MyoD, myogenin, and Myf5 riboprobes, 100% of the CTP was biotinylated; only 50 and 10% of the CTP was biotinylated in the MRF4 and GAPDH riboprobes, respectively. Each probe was verified for specificity by Northern blot analyses using 25 µg of total RNA from a control Pat muscle; each probe detected a single band at ~1.2 kb.

                              
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Table 1.   Primers for PCR given in 5'-3' direction

RNA analyses. Total RNA was isolated from ALD and Pat muscles with use of TriReagent (Molecular Research Center, Cincinnati, OH), which is based on the guanidine thiocyanate method. Frozen muscles were mechanically homogenized in 1 ml of TriReagent. Extracted RNA was solubilized in RNase-free H2O and quantitated in duplicate by absorbance at 260 nm. Twenty-five micrograms of RNA from an ALD muscle or 40 µg of RNA from a Pat muscle were used for each RPA. Any remaining RNA was stored at -80°C. RPAs were done according to the directions from the manufacturer (HybSpeed RPA, Ambion). Positive and negative control samples contained all five riboprobes and either yeast mRNA or mouse liver mRNA. Protected fragments were not observed when control samples were digested with RNase (negative controls), and full-length riboprobes (>= 10% larger than protected fragments) were observed when RNase digestion was omitted (positive controls). Century Marker Template (Ambion) was transcribed using biotin-14-CTP and used as size standards in the RPAs. Protected fragments, controls, and standards were electrophoresed on 5% acrylamide-8 M urea gels, electroblotted onto positive-charged nylon membranes, and immobilized by ultraviolet cross-linking. Chemiluminescence detection was used (BrightStar BioDetect Nonisotopic Detection Kit, Ambion), and exposure to film was 2-4 h. Signals were quantitated on a densitometer (model 620, Bio-Rad, Hercules, CA), and each MRF signal was normalized by the GAPDH signal in that lane. This normalization accounted for variability in initial amounts of RNA used in the assays, losses during the assays, or blotting inefficiencies. Preliminary experiments were done to ensure that 1) riboprobes were used in excess of the message they were to detect and 2) the resulting signals were within the linear range of the detection system and the film (i.e., each probe was biotinylated such that all 5 resulting signals could be analyzed simultaneously).

RT-PCR was performed using 1 µg of total RNA, 250 ng of oligo(dT) primer, and 100 units of RT (Superscript II RNase H- RT) in a total volume of 10 µl. Control RT reactions that contained no RT were done. Thirty cycles of PCR were carried out using 1 µl of cDNA generated from the RT, 100 ng of each primer (specific primers are listed in Table 1), and 2.5 units of Taq DNA polymerase (Sigma Chemical, St. Louis, MO) in a total volume of 50 µl. GAPDH was amplified in the same PCRs and used as an internal control to verify that equal amounts of cDNA were used in PCRs and that equal amounts of PCR products were loaded onto agarose gels. Annealing temperatures given in Table 1 for desmin, muscle creatine kinase, and AChR were used in the PCRs. The resulting signals were visualized after separation on 1.5% agarose gels with use of SYBR Green I nucleic acid gel stain (Molecular Probes, Eugene, OR), scanned using an optical scanner (model 840, Molecular Dynamics, Sunnyvale, CA), and then quantitated using ImageQuant software. Preliminary experiments were conducted to ensure that PCR was performed within the linear range. Control RT reactions were PCR amplified to confirm that DNA did not contaminate the RNA; no bands were detected when these reactions were visualized on agarose gels with SYBR Green I staining. In addition, no extraneous bands were detected in any of the PCRs.

DNA analyses. DNA was extracted from the same Pat and ALD muscle homogenates from which the RNA was isolated. The extraction was done according to the directions provided with the TriReagent. After precipitation, DNA was solubilized in 40 mM NaOH and quantitated in duplicate by absorbance at 260 nm.

Statistical analyses. MRF data are expressed as percent differences of MRF-to-GAPDH ratios between contralateral control and stretch-overloaded muscles. Values are means ± SE. One-way ANOVA (age) and Tukey post hoc tests were used to determine whether differences between basal MRF and GAPDH mRNA levels occurred. Two-way ANOVA (age × time) and Tukey post hoc tests were used to determine whether differences between the means for percent changes in nucleic acid contents and MRF mRNA levels occurred. An alpha -level of 0.05 was used for all tests.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Nucleic acid content of ALD muscles. RNA content of ALD muscles from adult quails was increased after 6, 24, and 72 h of stretch overload compared with contralateral muscles (P <=  0.013; Fig. 1) and was increased in muscles from senescent quails after 24 and 72 h of stretch overload (P <=  0.04). RNA content increases in 72-h stretch-overloaded ALD muscles were affected by age (P = 0.001; Fig. 1). RNA content of control ALD muscles was not different between adult and senescent quails: 32 ± 2 and 34 ± 2 µg/muscle, respectively (P = 0.58).


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Fig. 1.   Changes in RNA (top) and DNA (bottom) content of anterior latissimus dorsi (ALD) muscles from adult and senescent quails after 6, 24, and 72 h of stretch overload relative to contralateral control values. Values are means ± SE. RNA and DNA content changes were affected by age (P <=  0.002): * significantly different from adult at same respective time point.

DNA content of ALD muscles was not affected by 6 or 24 h of stretch overload (P <=  0.17; Fig. 1). DNA content was greater in 72-h stretch-overloaded ALD muscles than in contralateral control muscles (P <=  0.003), and the increase was affected by age (P = 0.002; Fig. 1). DNA content of control ALD muscles was not different between adult and senescent quails: 21 ± 2 and 23 ± 2 µg/muscle, respectively (P = 0.52).

Nucleic acid content of Pat muscles. After 6 h of stretch overload, RNA content was increased in Pat muscles from adult animals (P = 0.01; Fig. 2), and after 24 h of stretch overload, RNA content was increased in muscles from adult, middle-aged, and aged animals (P <=  0.04). RNA content of Pat muscles from quails of all ages was increased after 72 h of stretch overload compared with contralateral muscles (P <=  0.01). Increases in RNA content after 24 and 72 h of stretch overload were less in Pat muscles from senescent quails than in those from younger quails (P = 0.04; Fig. 2). RNA content of control Pat muscles was not different among adult, middle-aged, aged, and senescent quails: 122 ± 9, 127 ± 5, 132 ± 6, and 118 ± 4 µg/muscle, respectively (P = 0.35).


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Fig. 2.   Changes in RNA (top) and DNA (bottom) content of patagialis (Pat) muscles from adult, middle-aged, aged, and senescent quails after 6, 24, and 72 h of stretch overload relative to contralateral control values. Values are means ± SE. RNA and DNA content changes were affected by age (P <=  0.04): * significantly different from adult at same respective time point; ** significantly different from middle-aged at same respective time point.

DNA content of Pat muscles was not affected by 6 or 24 h of stretch overload regardless of age (P >=  0.19; Fig. 2). DNA contents were increased after 72 h of stretch overload in muscles from quails of all ages (P <=  0.04), and the increases were affected by age (P = 0.01; Fig. 2). DNA content of control Pat muscles was not different among adult, middle-aged, aged, and senescent quails: 79 ± 8, 84 ± 11, 90 ± 13, and 83 ± 11 µg/muscle, respectively (P = 0.65).

RPAs. All five riboprobes were used and detected simultaneously in each RPA (Fig. 3). Protected fragments of MRF4, MyoD, and GAPDH were detected in all muscle samples. Myf5 was detected in ~60% of the ALD muscles, and myogenin was detected only in 72-h stretch-overloaded ALD muscles. Fewer than 20% of the Pat muscles expressed detectable levels of myogenin or Myf5 mRNA, but when they were detected it was always in stretch-overloaded muscle (Fig. 3).


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Fig. 3.   Representative results of RNase protection assays. Simultaneous detection of Myf5, MRF4, MyoD, myogenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs are shown in 72-h stretch-overloaded (Stretch) and contralateral control (Control) Pat muscles of adult quails. In each assay, 40 µg of total RNA were used.

GAPDH mRNA was a valid internal control, because levels were not different between stretch-overloaded and contralateral control muscles within each age group (P >=  0.16 for ALD muscles and P >=  0.25 for Pat muscles). No difference in GAPDH levels was detected between ALD muscles from adult and senescent quails (P = 0.53). However, levels of GAPDH mRNA were ~25% greater in Pat muscles from adult and middle-aged animals than in Pat muscles from aged and senescent animals (P = 0.02). Because GAPDH was used as an internal control in the RPAs and the MRF data (relative to GAPDH) were expressed as within-animal differences, the change in GAPDH mRNA with age was not a confounding factor.

Basal MRF levels. Control muscle MRF mRNA levels (basal levels) were not different among age groups in ALD muscles (P >=  0.48), but a difference was detected in Pat muscles (P <=  0.02). MRF4 and MyoD mRNA levels in Pat muscles from senescent quails were 48 and 50% greater, respectively, than levels from adult quails. In Pat muscles from middle-aged and aged quails, MRF4 and MyoD mRNA basal levels were intermediate to those from adult and senescent quails but not significantly different from any group.

Stretch overload-induced MRF changes in ALD muscles. Initially, only ALD muscles from adult and senescent quails were investigated. The time of stretch overload did not affect increases in MRF4, MyoD, or Myf5 mRNA levels (P >=  0.13). When collapsed across time, MRF4, MyoD, and Myf5 mRNA levels were significantly elevated in response to stretch overload in ALD muscles from adult and senescent animals (P <=  0.03; Fig. 4). However, no differences in MRF4, MyoD, or Myf5 mRNA increases between ALD muscles from adult and senescent quails were found (P >=  0.38); therefore, ALD muscles from middle-aged and aged quails were not studied.


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Fig. 4.   Densitometric quantitation of MRF4, MyoD, and Myf5 mRNA increases in ALD muscles from adult and senescent animals. All myogenic regulatory factor values were normalized by GAPDH values in same lane and are expressed as percent increases above contralateral control values. Values are means ± SE. Myogenic regulatory factor values were not different between muscles stretch overloaded for 6, 24, or 72 h; therefore, these data within adult and senescent groups were pooled (n = 21 per age group for MRF4 and MyoD and n = 11-15 per age group for Myf5). MRF4, MyoD, and Myf5 mRNAs were increased (i.e., significantly different from zero) in stretch-overloaded muscles in both age groups. MRF4, MyoD, and Myf5 mRNA increases were not affected by age (P >=  0.38).

Stretch overload-induced MRF changes in Pat muscles. MRF4 and MyoD mRNA increases were not different between Pat muscles stretch overloaded for 6, 24, and 72 h within any age group (P >=  0.34), and no interaction between time of stretch overload and age was detected (P >=  0.25). Therefore, data from the three stretch-overload times were pooled for further analyses. MRF4 mRNA levels in stretch-overloaded Pat muscles (collapsed across time) were significantly elevated above contralateral control levels at each age (P <=  0.005). However, MRF4 increases in muscles from older animals were less than those in muscles from younger animals (P = 0.02; Fig. 5). MyoD mRNA was also increased at each age after stretch overload (P <=  0.04), and the increase was significantly affected by age (P = 0.02; Fig. 5).


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Fig. 5.   Densitometric quantitation of MRF4 and MyoD mRNA increases in Pat muscles from adult, middle-aged, aged, and senescent quails. MRF4 and MyoD values were normalized by GAPDH values in same lane and are expressed as percent increases above contralateral control values. Values are means ± SE. MRF4 and MyoD values were not different between muscles overloaded for 6, 24, or 72 h; therefore, these data within each age group were pooled (n = 21 per age group, except for middle-aged group, where n = 9). MRF4 and MyoD mRNAs were increased (i.e., significantly different from zero) in stretch-overloaded muscles in all age groups. MRF4 and MyoD mRNA increases were affected by age (P <=  0.02): * significantly different from adult; ** significantly different from adult and middle-aged.

RT-PCR of transcripts whose genes are regulated by MRFs. Analyses of desmin, muscle creatine kinase, and AChR mRNA were done to determine whether changes in transcription of some genes regulated by MRFs occurred with stretch overload. Because quantities of RNA were limited, the sensitive method of RT-PCR was performed using the primers listed in Table 1 and RNA isolated from 72-h stretch-overloaded and control Pat and ALD muscles from adult and senescent animals. Stretch-overloaded muscles from adult and senescent quails had greater desmin signals than the contralateral control muscles (Fig. 6). The desmin signal increase in Pat muscle from the younger quails was greater than that from the older quails. No differences in stretch-induced desmin signal increases were observed between adult and senescent ALD muscles (Fig. 6). These same patterns of increases were observed with muscle creatine kinase (Fig. 7) and AChR signals (Fig. 8). A second set of 72-h stretch-overloaded and control Pat and ALD muscles from adult and senescent quails was analyzed to confirm the RT-PCR results shown in Figs. 6-8. The amplification results were analogous to those shown.


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Fig. 6.   Desmin RT-PCR results of 72-h stretch-overloaded (S) and contralateral control (C) Pat and ALD muscles from adult and senescent quails. Desmin and GAPDH were amplified in the same PCR, and 2 µl of PCR product were loaded per lane. Gels were stained with SYBR Green I nucleic acid gel stain, and the image was captured using a PhosphoImager.


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Fig. 7.   Muscle creatine kinase RT-PCR results of 72-h stretch-overloaded (S) and contralateral control (C) Pat and ALD muscles from adult and senescent quails. Muscle creatine kinase and GAPDH were amplified in the same PCR, and 1.5 µl of PCR product were loaded per lane. Gels were stained with SYBR Green I nucleic acid gel stain, and the image was captured using a PhosphoImager.


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Fig. 8.   AChR RT-PCR results of 72-h stretch-overloaded (S) and contralateral control (C) Pat and ALD muscles from adult and senescent quails. AChR and GAPDH were amplified in the same PCR, and 5 µl of PCR product were loaded per lane. Gels were stained with SYBR Green I nucleic acid gel stain, and the image was captured using a PhosphoImager.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

General. We have emphasized our findings in the Pat muscle over those in the ALD muscle because of their different fiber type compositions. The ALD muscle is composed of tonic fibers (2, 5), and thus its relevance to mammalian muscle is unclear. The Pat muscle, however, is a twitch muscle that is composed primarily of fibers similar to type II mammalian fibers (19), and so it is probable that findings in this muscle can be extrapolated more readily to mammalian muscle.

It is not surprising that MyoD transcripts were detected in all Pat muscles, because MyoD is prevalent in fast-twitch fibers (13, 35). Likewise, very few of these muscles showed transcripts of myogenin, the MRF that is prevalent in slow-twitch fibers (13, 35). We did not consistently detect myogenin mRNA in the slow-tonic ALD muscle, except after 72 h of stretch overload. This makes sense in light of the facts that 1) ALD muscle is a slow-tonic muscle and myogenin is more prevalent in slow-twitch muscle and 2) myogenin is primarily involved in differentiation and thus is normally expressed at time points later than Myf5 and MyoD (23). MRF4 is predominantly expressed in fully differentiated muscle fibers (12). Correspondingly, all muscles in our study had relatively high levels of MRF4 transcripts.

Muscle hypertrophy. RNA content in adult ALD and Pat muscles was increased as early as 6 h after stretch overload, and DNA content was increased after 72 h. The increases indicate that the stretch overload did apply a hypertrophy stimulus to these muscles, and the data are comparable to other reports of nucleic acid content changes in chicken and rat muscles after stretch (10, 18). Earlier studies conducted in our laboratory have characterized muscle mass and fiber number increases that occur in ALD (1, 2, 5) and Pat (19) muscles from adult and aged quails after stretch overload.

The roles of MRFs in mature skeletal muscle have not been delineated, despite the finding that each has a unique role during embryonic skeletal muscle development (23). In our study, MyoD and MRF4 mRNA levels were elevated and myogenin and Myf5 mRNAs were induced after stretch overload, indicating that they all take part in the response to a hypertrophy stimulus. MyoD and MRF4 mRNAs were elevated by 100-500% after 6-72 h of stretch overload in adult ALD and Pat muscles. Although we observed no increase of any MRF mRNA in quail Pat muscle after 0.5 h of stretch in situ (unpublished observations), the MRF mRNA responses after 6-72 h of stretch overload are similar in magnitude to those observed in stretched ALD muscle of adult chickens (8) and adult rat hindlimb muscles after the muscles were casted for 48 h in a stretched position (22). We did not determine MRF levels between 0.5 and 6 h, because our goal was to determine a time at which MRFs were elevated in stretch-overloaded muscle of adult animals and then to find whether the same increases occurred in muscles of older animals. We extended our experiments to time points beyond 6 h to ensure that the attenuated response we found in Pat muscle from aged and senescent quails was not simply a lag in the response time of the older muscle.

Studies conducted on regenerating muscle suggest that MRF mRNA increases occur in satellite cell nuclei, because the time of MRF increases coincides with the initiation of satellite cell proliferation (11, 15). Our data obtained from 72-h stretch-overloaded muscles agree with this suggestion. These muscles had elevated levels of DNA, indicating that satellite cells were proliferating, and MRF mRNA levels were also increased at this time point. However, Jacobs-El et al. (14) suggested that myonuclei express elevated levels of MRF mRNA 1) on the basis of observations that MRF increases were detected as early as 2 h after stimulation and 2) because Myf5 and MRF4 in situ hybridization signals in stimulated rat muscles spread into the cytoplasm of some mature fibers. Our data also support the suggestion of an elevation of MRF mRNA in the myonuclei, because these levels were increased after just 6 h of stretch overload, and it has been shown that satellite cells in quail ALD muscle are not activated until after 24 h of stretch overload (37). In addition, a previous study in our laboratory showed that 3-14 days of stretch overload did not result in satellite cell proliferation in quail Pat muscle (19). Therefore, the increases in MRF mRNA we observed in quail Pat muscle were likely derived from myofiber nuclei. Additional studies are necessary to localize MRF transcript increases, i.e., myofiber nuclei vs. satellite cell nuclei, especially after periods of stretch overload that induce satellite cell proliferation.

Aging. Only two previous studies have reported MRF expression in muscles from aged animals (27, 29). Musaro et al. (29) showed that basal mRNA levels of MyoD, myogenin, and Myf5, but not MRF4, were higher in hindlimb muscles of aged than adult mice. In agreement with this, we found higher basal levels of MyoD mRNA in Pat muscles from senescent than adult quails. Additionally, we found that basal MRF4 mRNA levels were elevated in Pat muscles from older quails. Marsh et al. (27) also reported elevated levels of MyoD, myogenin, and MRF4 mRNA with aging. In the present study, basal levels of MyoD and MRF4 in muscles from middle-aged and aged animals were intermediate to those from adult and senescent animals, indicating that the increases occur gradually during aging. It is puzzling why MRF mRNA basal levels are higher in muscles of older animals, because basal rates of protein synthesis are lower in muscles of aged animals than younger animals (24). One possible explanation for the higher basal MRF mRNA levels in muscle of older animals may be the loss of alpha -motor neurons that occurs with aging (3). It has been demonstrated that MyoD and myogenin transcripts are elevated in denervated muscle (16, 35) and that the extent of the elevation depends on the severity of the denervation (16). Muscle of aged animals may be considered mildly denervated, and this could influence basal expression of the MRFs.

In this study and in the studies by Musaro et al. (29) and Marsh et al. (27), only mRNA levels of MRFs were investigated in muscles of older animals and not the levels of active MRF proteins. Although Musaro et al. showed accumulation of myogenin in the nuclei of muscle from aged mice by immunohistochemistry, it is not known whether the protein was active. Thus it remains unknown whether MRF proteins in muscle from aged animals bind DNA and are functional in regulating specific skeletal muscle genes. If, indeed, hypertrophy-stimulated increases in MRF mRNA levels that we have shown correspond to increases in active MRF proteins, then several skeletal muscle-specific genes are likely to be upregulated. The upregulation of these genes would result in an increase in the production of muscle-specific proteins that would support hypertrophy of the muscle. Although it was nonquantitative, we used RT-PCR to detect increases in desmin, muscle creatine kinase, and AChR mRNA expression, three products whose genes are regulated by MRFs, in stretch-overloaded muscles from adult and senescent quails. In support of the suggestion that MRFs may be involved in the diminished hypertrophic response of older fast-twitch muscle, we showed that hypertrophy-stimulated increases in MRF-regulated genes are lower in Pat muscle from senescent animals than in Pat muscle from younger, adult animals. In contrast, the ALD muscle, which showed no age-related differences in hypertrophy-stimulated MRF increases, also showed no age-related differences in hypertrophy-stimulated desmin, muscle creatine kinase, or AChR transcript increases.

In conclusion, we have shown that a hypertrophy stimulus causes elevations in MRF expression in muscle from adult, middle-aged, aged, and senescent quails. However, the elevation is attenuated in fast-twitch muscles from older quails. This attenuation may be part of the reason that muscles from aged animals do not hypertrophy as much as muscles from younger animals.

    ACKNOWLEDGEMENTS

We thank Dr. Charles Emerson for providing the qmf1, qmf2, and qmf3 cDNA, Dr. Duane Hinton for assistance with the subcloning, and Paul Llobet for assistance with the DNA analyses.

    FOOTNOTES

This research was supported by National Institute on Aging Grant AG-10871.

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. §1734 solely to indicate this fact.

Address for reprint requests: S. E. Alway, Dept. of Anatomy, MDC #6, 12901 Bruce B. Downs Bl., University of South Florida, Tampa, FL 33612.

Received 4 February 1998; accepted in final form 31 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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