Cell Tissue Res (1999) 296:531–539
© Springer-Verlag 1999
REGULAR ARTICLE
Dawn A. Lowe · Stephen E. Alway
Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation Received: 5 June 1998 / Accepted: 19 November 1998
Abstract The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ~150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after
This research was supported by NRSA Postdoctoral Fellowship AG05815 (D.A.L.) and NIH Grant AG10871 (S.E.A.) D.A. Lowe · S.E. Alway (✉) Dept. of Anatomy, College of Medicine, 12901 Bruce B. Downs Bldv. MDC #6, University of South Florida, Tampa, Florida 33612, USA e-mail:
[email protected]; Tel.: +1 813 974 9390; Fax: +1 813 974 2058
7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Key words Myogenic regulatory factors · RNA · Anterior latissimus dorsi · Skeletal muscle · Stretch-overload · Japanese quail
Introduction Myogenic regulatory factors (MRFs) are a family of skeletal-muscle-specific transcription factors that control the expression of several muscle genes. The family is currently composed of four members: MyoD, Myf5, myogenin, and MRF4. During embryogenesis, MRFs are critical for establishing the myogenic lineage and controlling terminal differentiation of myoblasts (for a review, see Ludolph and Konieczny 1995). Induction of MRF mRNA has also been reported in fully differentiated myofibers. For example, MRF mRNAs are upregulated in regenerating (Eppley et al. 1993; Füchtabauer and Westphal 1992; Grounds et al. 1992; Koishi et al. 1995) and hypertrophic (Carson and Booth 1998; Jacobs-El et al. 1995; Lowe et al. 1998) skeletal muscle. Satellite cells are myogenic precursor cells that lie dormant between the basal lamina and the plasmalemma of muscle fibers. When activated, these cells can proliferate, differentiate, and fuse to existing muscle fibers or to each other (for a review, see Schultz and McCormick 1994). Satellite cell proliferation typically begins 24–48 h following some stimulus and, during this and subsequent events, MRFs are expressed in these cells (e.g., Grounds et al. 1992). Satellite cells are activated under regenerating and hypertrophic conditions and, because MRF increases temporally coincide with satellite cell proliferation, it has often been concluded that MRF increases occur in the nuclei of satellite cells or newly formed myotubes (Füchtabauer and Westphal 1992; Grounds et al. 1992; Koishi et al. 1995; Lowe et al. 1998).
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However, MRF increases have also been observed at times before satellite cell proliferation would have been expected, i.e., at times less than 24 h after a stimulus was applied (Eppley et al. 1993; Jacobs-El et al. 1995; Lowe et al. 1998). These observations indicate that myonuclei also have increased levels of MRFs. Studies that have addressed the issue of MRF localization in differentiated fibers directly have done so by using in situ hybridization and immunohistochemistry (Eppley et al. 1993; Jacobs-El et al. 1995; Koishi et al. 1995). Results from those studies suggest that MRFs are expressed in the nuclei of mature myofibers. However, it is unknown whether the MRF-positive myonuclei observed in those studies were bona fide myonuclei or nuclei from satellite cells that had recently proliferated and fused to myofibers. We have undertaken the present studies to investigate MRF expression further in muscle with and without proliferating satellite cells. We have hypothesized that MRF expression is elevated in response to a hypertrophy stimulus whether or not satellite cell proliferation occurs. To test this hypothesis, muscle hypertrophy has been induced following muscle exposure to gamma irradiation. A dose of gamma irradiation causes DNA-strand breaks; the breaks are not lethal unless the cell enters mitosis (Denekamp and Rojas 1989). Myofiber nuclei are post-mitotic, so they are resistant to gamma irradiation. However, satellite cells have the ability to divide and thus are radiosensitive. Gamma irradiation has been used in previous studies to diminish or eliminate the proliferation of satellite cells in skeletal muscle (e.g., Phelan and Gonyea 1997; Rosenblatt and Parry 1993). Hypertrophy of skeletal muscle from aged animals is attenuated compared with that from adult animals (e.g., Carson and Alway 1996). It is unknown whether this diminished capacity to hypertrophy is attributable to dysfunctional satellite cells or to a reduced ability of myofiber nuclei to govern increased protein demands. Localizing changes in MRF expression could help delineate mechanisms responsible for the reduced growth ability of skeletal muscle from aged animals. Therefore, we have also examined hypertrophy-stimulated MRF changes in irradiated and non-irradiated muscles from aged animals. Results from our first experiments revealed that MRF expression was elevated in hypertrophy-stimulated muscles in which satellite cells had not proliferated. These data suggested that myonuclei, and possibly non-mitotic satellite cells, were important in regulating the transcription of genes in the early stages of muscle hypertrophy. Therefore, we next hypothesized that muscle hypertrophy could proceed without the proliferation of satellite cells. In contrast, earlier studies had shown that irradiated muscles did not hypertrophy (Phelan and Gonyea 1997; Rosenblatt and Parry 1992, 1993; Rosenblatt et al. 1994) and that satellite cell proliferation was necessary in order to maintain a constant DNA to cytoplasm ratio during hypertrophy (McCall et al. 1998). In our next experiments, irradiated muscles were induced to hypertro-
phy for a longer period of time (7 days) so that indices of hypertrophy could be measured.
Materials and methods Experimental design Increase in MRF mRNA in the absence of proliferating satellite cells The first experiments were designed to compare hypertrophystimulated MRF mRNA increases in muscles that contained proliferating satellite cells with muscles that were devoid of proliferating cells. Cells that had undergone proliferation were identified in muscle cross sections based upon nuclear uptake of a thymidine analog, bromodeoxyuridine (BrdU). BrdU is incorporated into DNA during mitosis and thus should be absent in nuclei of irradiated muscle. Twelve adult and 10 aged quails received 2500 Rads gamma irradiation; 9 adult and 8 aged quails received 0 Rads. Approximately 2 h after 2500 or 0 Rads of irradiation had been delivered, BrdU pellets were implanted into about half of the quails (6 adult irradiated; 4 aged irradiated; 5 adult non-irradiated; 4 aged non-irradiated). One hour later, a muscle hypertrophy stimulus, viz., stretch-overload, was applied to the left wing muscles of each quail. Muscles from the right wing served as intra-animal controls. After 3 days of stretch-overload, the anterior latissimus dorsi (ALD) muscles from both wings were excised and weighed. BrdU incorporation was analyzed in hypertrophy-stimulated and contralateral control muscles from each quail that had been implanted with a BrdU capsule. This analysis required less than 8% of the ALD muscle mass; therefore, all other measurements could be made on these same ALD muscles. Hypertrophy-stimulated and control ALD muscles from each adult and aged quail were assayed for DNA and RNA content and MRF mRNA expression. Acute muscle hypertrophy in the absence of proliferating satellite cells The next experiments were designed to determine whether muscle hypertrophy could occur without nuclear contribution from proliferating satellite cells. Nine adult quails received 2500 Rads of irradiation; 6 adult quails received 0 Rads. Two hours later, 6 of the irradiated and 4 of the non-irradiated quails were implanted with BrdU pellets. One hour later, the hypertrophy stimulus was applied to the left wing of each quail for a duration of 7 days. At the end of this period, stretch-overloaded and contralateral control ALD muscles were excised and weighed. Cell proliferation was analyzed in ALD muscles from BrdU-implanted quails as in the first experiments. Protein content was determined in stretch-overloaded and control ALD muscles from each irradiated and nonirradiated quail. Animals and experimental protocols Animals Japanese quails (Coturnix coturnix japonica) were hatched and raised at the University of South Florida as described previously (Lowe et al. 1998). Japanese quail are physically and sexually mature at 6 weeks of age and do not change in body weight or composition beyond 2 months after hatching (Marks 1978, 1993). The life span of the Japanese quail is approximately 30 months with a mortality rate of approximately 60% by 28 months of age (Woodard and Abplanalp 1971). Quails in our colony have lived as long as 34 months. In the present studies, adult quails were 3 months old, and aged quails were 30 months old. The body mass (mean+SD) of adult and aged quails when the first experiments began was 158.8+4.9 g (n=21) and 168.0+5.8 g (n=18), respec-
533 tively. The average body mass of quails used in the second experiments was 169.4+8.1 g (n=15). All experimental protocols met the guidelines set by the American Physiological Society, and the Principles of Laboratory Animal Care (NIH no. 86–23) were followed. Irradiation Satellite cell proliferation was eradicated by exposing quails to 2500 Rads gamma irradiation. Two quails at a time were placed in a plexiglas mouse cage and sandwiched between pieces of foam so that they were separated and immobilized. Irradiation was delivered in a single dose at 70 Rads/min by using a cesium-137 irradiation unit (J.L. Shepherd & Assoc.). Whole-body irradiation was used to ensure that bone-marrow-derived myogenic progenitor cells, which have recently been shown to migrate into regenerating muscle (Ferrari et al. 1998), would not confound our studies. Theoretically, whole-body irradiation would prevent all bone marrow myogenic progenitor cells from proliferating; local irradiation (irradiating only the muscles to be studied) would not have affected the majority of these cells. BrdU incorporation Time-release BrdU capsules (Innovative Research, Sarasota, Fla.) were implanted subcutaneously in the dorsal cervical region of the quails. The capsules were designed to deliver a constant dose of 22 µg BrdU/g body mass per day, which has been shown to label satellite cells without altering satellite cell mitosis in quail hypertrophic muscle (Carson and Alway 1996). Hypertrophy stimulus and muscle sampling Muscles of the left wing were induced to hypertrophy by stretchoverload as described previously (Alway et al. 1989). Muscles from unloaded right wings served as intra-animal controls. Following stretch-overload, quails were anesthetized with pentobarbital sodium (35 mg/kg i.p.), and ALD muscles from both wings were excised and weighed. For quails that had BrdU capsules implanted, approximately 1-mm-thick cross-sectional slices of control and overloaded ALD muscles were taken, and each was mounted between two pieces of non-irradiated pectoralis muscle and frozen for cryosectioning. The proximal-distal location of the slices taken were varied between animals such that the entire length of the ALD muscle was represented. This was carried out to ensure that irradiation affected the entire muscle. Non-irradiated pectoralis muscle served as a positive control for irradiated ALD muscle. The remaining ALD muscle tissue (>92% of the mass) was reweighed, frozen in liquid nitrogen, and stored at –80°C. For quail that did not have BrdU capsules implanted, ALD muscles from both wings were excised, weighed, and frozen in liquid nitrogen. Muscle histochemistry Muscle cross sections (10 µm thick) were cut in a microtome cryostat at –19°C. Three sections, 200 µm apart, from each muscle were examined for BrdU incorporation by using biotinylated monoclonal anti-BrdU antibody as described by the manufacturer (Zymed Laboratories, San Francisco, Calif.). BrdU-labeled nuclei appeared dark brown and non-BrdU-labeled nuclei were light purple from the hematoxylin counterstain. Approximately 500 nuclei per muscle were counted and classified as either fiber-associated or interstitial, depending upon whether they appeared to be associated with a myofiber. Fiber-associated nuclei were assumed to be myonuclei or satellite cell nuclei. The percent of nuclei in each category that were BrdU-labeled was also tabulated. All histological analyses were performed blind as to muscle treatment.
DNA, RNA, and protein analyses DNA and RNA were isolated from whole ALD muscles (or ALD muscle remaining after less than 8% of the mass was taken for histochemistry) by using 0.75 ml TriReagent per muscle according to the directions of the manufacturer (Molecular Research Center, Cincinnati, Ohio). DNA was solubilized in 40 mM NaOH and quantitated in duplicate by absorbance at 260 nm. RNA was solubilized in RNase-free H2O and quantitated in duplicate by absorbance at 260 nm. The protein content of ALD muscles (from the second experiments) was determined spectrophotometrically by using the BCA Protein Assay (Pierce, Rockford, Ill.) and bovine serum albumin standards. The reported DNA, RNA, and protein contents of ALD muscles were corrected for the slice of muscle taken for histochemistry (based on the assumption that the concentrations of DNA, RNA, and protein in that slice were the same as those in the remaining muscle).
RNase protection assays RNase protection assays (RPAs) were carried out to determine MRF mRNA levels in ALD muscles as described in detail previously (Lowe et al. 1998). In brief, quail cDNA clones MyoD (cc509; Charles de le Brousse and Emerson 1990), myogenin (cc527; Pownall and Emerson 1992), and Myf5 (cc528; Pownall and Emerson 1992) were subcloned to make riboprobes that could be used simultaneously in the assays. Quail MRF4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobes were made from the cloning of reverse transcription/polymerase chain reaction products. Riboprobes were transcribed using biotin-14CTP and used with 30 µg RNA in the assays (Ambion HybSpeed RPA, Austin, Tex.). Protected mRNA fragments were electrophoresed on 5% acrylamide/urea gels, transferred to nylon, detected by chemiluminescence, and quantitated by densitometry. Each MRF signal was normalized by the GAPDH signal in that sample, and the percent differences between contralateral control and stretch-overloaded ALD muscles were calculated.
Statistics Body mass, DNA and RNA contents, and hypertrophy-stimulated MRF changes were compared between irradiated and non-irradiated quails using Student’s t-tests. Myogenin increases were also compared between adult and aged quails by Student’s t-tests. Muscle mass and protein content were compared between control and stretch-overloaded muscles using paired t-tests. Percent BrdUlabeled nuclei in irradiated and non-irradiated muscles were analyzed by Mann-Whitney U statistics. Significance was set an α level of 0.05. Results are reported as the mean ± standard error.
Results Increases in MRF mRNA in the absence of proliferating satellite cells All adult and aged quails survived for 3 days post-irradiation, and food and water intake was normal. Adult irradiated quails did not undergo a significant change in body mass (-1.0±0.9%; P=0.28), nor did the non-irradiated adult quails (+1.2±1.1%; P=0.41). The average body mass of the aged irradiated quails decreased by 4.9+0.7% (P=0.001), whereas the body masses of the non-irradiated aged quails did not change during the 3 days studied (-1.5±0.8%; P=0.11).
534 Table 1 Percent of total nuclei labeled with bromodeoxyuridine in anterior latissimus dorsi muscle of non-irradiated and irradiated, adult and aged quails after 3 days of stretch-overload (mean+standard error; *significantly less than corresponding 0 Rads)
Fig. 1 DNA and RNA contents of contralateral control and 3-day stretch-overloaded anterior latissimus dorsi muscles from nonirradiated (0 Rads) and irradiated (2500 Rads), adult (A) and aged (B) quails. A 0 Rad, n=9; 2500 Rad, n=12. *Significantly different from 0 Rads, Stretch. B 0 Rad, n=8; 2500 Rad, n=10. No differences were detected between non-irradiated and irradiated muscles from aged quails
Elimination of cell proliferation In adult quails, the DNA content of irradiated stretchoverloaded muscle was less than that of non-irradiated stretch-overloaded muscle (P=0.009; Fig. 1A) but was not different from that of contralateral control muscles (P=0.66). This was a crude indication that proliferation did not occur in irradiated muscles. DNA content was not different between non-irradiated and irradiated control ALD muscles from adult quails (P=0.11), suggesting that irradiation did not alter the DNA content of non-proliferating cells. Irradiation had no effect on RNA content of control muscles from adult quails (P=0.20), but there was an effect on stretch-overloaded muscles (P=0.002; Fig. 1A). DNA and RNA contents of control and stretch-overloaded ALD muscles from aged animals were not affected by irradiation (P≥0.12; Fig. 1B). DNA content did not change with stretch-overload in non-irradiated or irradiated muscles (P≥0.72), whereas RNA content approximately doubled (P
