Biphasic Induction of Immediate Early Gene Expression Accompanies Activity- dependent Angiogenesis and Myofiber Remodeling of Rabbit Skeletal Muscle Jeffrey B. Michel,* George A. Ordway,* James A. Richardson,§ and R. Sanders Williams*II Departments of *Internal Medicine, 11 Biochemistry, *Physiology, and IPathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235 Abstract (TA)' muscle via the motor nerve causes transformation of skeletal myofibers from a fast-glycolytic to a slow-oxidative Sustained contractile activity of skeletal muscle promotes phenotype and stimulates capillary proliferation (12, 13). In angiogenesis, as well as transformation of contractile protein response to this stimulus, genes encoding proteins involved in isoforms and mitochondrial proliferation within myofibers. oxidative metabolism are induced while those encoding glyco- Since the products of immediate early genes such as c-fos, lytic enzymes are downregulated (5, 7, 10). These effects are c-jun, and egr-1 function in many signaling pathways gov- followed, in turn, by a switch to expression of slow/cardiac erning cellular responses to external stimuli, we sought to isoforms of proteins of the contractile apparatus (14). Morpho- determine whether sustained contractile activity induces logically, the fractional volume of mitochondria within myo- their expression in skeletal muscle. Low voltage electrical fibers is increased up to 10-fold, and an angiogenic response stimulation was applied to the motor nerve innervating rab- augments capillary density to a similar extent (12, 15). The bit tibialis anterior muscles for periods ranging from 45 physiological consequences of these adaptations render the mus- min to 21 d. Northern and Western analysis demonstrated cle resistant to fatigue during sustained contractile work. Quali- marked but transient inductions of c-fos, c-jun, and egr-1 tatively similar, but lesser, effects on mitochondrial content mRNA and protein within the first 24 h. Longer durations and capillary density of skeletal muscles can be produced by of stimulation were associated with a secondary and sus- endurance exercise training ( 1, 2), though the profound trans- tained rise in the abundance of c-fos, c-jun, and p88' formation of contractile protein isoforms produced by nerve protein that, surprisingly, was not accompanied by detect- stimulation is usually not observed. able changes in mRNA. Immunohistochemistry demon- The signaling pathways responsible for these pleiotropic strated c-fos immunoreactivity within myofiber and vascu- adaptations are, however, poorly understood. Intracellular mes- lar cell nuclei during both early and late phases of this sengers such as cyclic AMP and calcium, and peptide signaling response. These findings reveal a complex pattern of c-fos, molecules such as basic fibroblast growth factor are increased c-jun, and egr-1 expression in response to nerve stimulation in skeletal muscle following the onset of continuous nerve stim- and suggest that these proteins could function in regulatory ulation (8, 9, 12-17), but the events that transduce signals pathways that modify muscle phenotype. (J. Clin. Invest. arising within contracting muscles to the relevant genes are 1994. 94:277-285.) Key words: c-fos * c-jun * proto-onco- unknown. Based on their important role in controlling a wide genes * muscle * rabbits variety of cellular responses to external and internal stimuli, we reasoned that products of immediate early genes such as c-fos, Introduction c-jun and egr-J were likely to be involved as third messengers in one or more of the adaptive responses induced by neural Contractile activity is a major determinant of the morphology activity and contractile work in skeletal muscle. and functional capabilities of skeletal muscle ( 1, 2). The conse- The proto-oncogene products c-fos and c-jun form dimers quences of physical inactivity contribute to morbidity associated through leucine zipper domains to bind AP-1 sites within certain with many human diseases (3) and, conversely, habitual exer- promoters (18, 19). The egr-J gene encodes a single mRNA cise promotes salutary effects, some of which are attributable which is translated into two proteins p88el- and p82egr-l. The to adaptive responses of skeletal muscle (4). In previous studies larger form results from translational initiation at an ACG site (5-11) we have explored molecular mechanisms that control upstream of the canonical AUG site but is otherwise identical features of the adaptive responses of skeletal muscle to sus- to p82e-'. Both forms are detected in cells following treatment tained contractile activity, but the potential contributions of im- with phorbol ester though p88egr-4 is found in lower abundance mediate early genes to these processes have not been examined. relative to p82egv- (20, 21, 22). c-fos and c-jun play an im- Chronic electrical stimulation of the rabbit tibialis anterior portant role in many cell lineages to modulate cellular growth and differentiation during development, and in response to phys- iological stimuli (18, 23). The egr-J gene is activated during Address all correspondence to R. Sanders Williams, Ryburn Center differentiation of nerve and bone cells (24, 25) and appears to for Molecular Cardiology, University of Texas Southwestern Medical be required for differentiation of myeloid precursor cells to Center, NB 11.200, 5323 Harry Hines Blvd., Dallas, TX 75235-8573. macrophages (26). Immediate early genes also are implicated Received for publication 2 December 1993 and in revised form 2 in the hypertrophic response of cardiac myocytes to pressure March 1994. J. Clin. Invest. () The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: DAB, 3,3 '-diaminobenzidine; 0021-9738/94/07/0277/09 $2.00 GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NADH-DH, Volume 94, July 1994, 277-285 NADH dehydrogenase; TA, tibialis anterior. Neural Control of Immediate Early Genes in Skeletal Muscle 277 overload (27, 28) and in cellular responses to acidosis, ATP sodium pyrophosphate and 1% SDS. Hybridizations were carried out depletion, and oxidative stress (29-31). overnight in hybridization solution (prehybridization solution with lx We observed in this study that c-fos, c-jun, and egr-1 mRNA Denhardt's solution) at 420C using the appropriate cDNA probe at a and proteins are transiently induced in skeletal muscle within final concentration of 0.5-1.0 x 107 cpmlml. Probes were ethanol pre- the first 24 h after the onset of continuous motor nerve stimula- cipitated and only probes with specific activities greater than 1.0 X 109 Elevated levels of c-fos immunoreactivity were present in cpm/Iug were used. Filters were washed in 0.1 x SSC, 0.1% SDS at tion. room temperature for 30 min followed by 30 min in 0.1 x SSC, 0.1% both myofibers and vascular cells of the stimulated muscle. SDS at 55-650C. Filters were placed on film (Amersham Hyperfilm- With more prolonged stimulation up to 21 d, which is necessary MP) and exposed for 12-96 h. to induce the mitochondrial biogenesis, fiber-type transforma- c-fos mRNA was detected using a 480-bp probe from exon IV, a tion, and angiogenesis that constitute the major adaptive re- region with little homology to fos-B orfra-J, obtained by Polymerase sponses to this stimulus, we observed a second induction of Chain Reaction (PCR) using genomic rabbit DNA as the template c-fos, c-jun, and p88ev4- protein. Late accumulation of c-fos and the following primers: forward-S '-CCGAATTCGGCTfCCCAG- immunoreactivity was also present within nuclei of both myo- AGGAGATGTCT-3'; reverse- 5'-GGCTGCAGCAGCTNGGGAA- fibers and vascular cells. In contrast to the acute and transient GGAGTCAGC-3'. The product was subcloned into EcoRI and PstI induction observed during the first day, however, the subsequent sites of pGEM 4Z and the resulting plasmid was termed pGEM4.FOS. increase in c-fos, c-jun, and p88egl-' protein was sustained over Sequencing of this fragment showed > 90% identity to published rat, several weeks, and was unaccompanied by detectable levels of murine and human c-fos nucleotide sequences, and 100% identity of the predicted amino acid sequence within this region. An EcoRI fragment of mRNAs encoding these proteins by Northern analysis. These the plasmid JAC.1 (33) obtained from the American Type Culture results implicate immediate early genes in signal transduction Collection (ATCC, Rockville, MD) was used to detect c-jun mRNA. pathways triggered by neural activity and resulting in angiogen- egr-1 message was detected using a 300-bp BglIH/Rsa fragment from esis and fiber-type transformation in skeletal muscle. In addi- nucleotides 1958-2253 in the non-zinc-finger portion of the murine tion, these data suggest the existence of previously unrecognized cDNA (pB700-3.6-, a kind gift of V. Sukhatme, U. Chicago) (34). A posttranscriptional controls on expression of immediate early 1.1-kb Human Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) genes during responses to physiological inputs. cDNA (Clonetech, Palo Alto, CA) was used to detect GAPDH mRNA. All cDNA probes were labeled with (alpha-32P)dATP (6,000 Ci/ mmol) by random priming using a commercial kit (Pharmacia Fine Methods Chemicals, Piscataway, NJ). The oligonucleotide probe complementary to 28S RNA, 5 '-GGTCTAAACCCAGCTCACGTTC-3', was synthe- Animal surgery and preparation. Adult New Zealand white rabbits sized based on published sequences of murine 28S RNA. This oligonu- weighing 2.2-3.5 kg were anesthetized by isoflurane inhalation. Under cleotide was end-labeled with (gamma-32P)dATP (6,000 Ci/mmol) by sterile conditions, pulse generators were implanted and their electrodes T4 kinase. placed adjacent to the common peroneal nerve of one hindlimb ac- Western analysis. Soluble protein extracts were prepared from mus- cording to the procedure described originally by Salmons and Vrbova cle samples by homogenizing 0.4-0.5 g of frozen tissue in 2 ml phos- (32). Electrical leads were sutured in place and care was taken to phate buffered saline, (PBS, pH 7.3) followed by the addition of 2% avoid injury to the nerve.