Transient regulation of c-fos, alpha B-crystallin, and hsp70 in muscle during recovery from contractile activity

P. Darrell Neufer1, George A. Ordway2, and R. Sanders Williams1

1 Department of Internal Medicine and 2 Department of Physiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-8573

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Endurance exercise training increases the oxidative capacity of skeletal muscles, reflecting the induction of genes encoding enzymes of intermediary metabolism. To test the hypothesis that changes in gene expression may be triggered specifically during recovery from contractile activity, we quantified c-fos, alpha B-crystallin, 70-kDa heat shock protein (hsp70), myoglobin, and citrate synthase RNA in rabbit tibialis anterior muscle during recovery from intermittent (8 h/day), low-frequency (10 Hz) motor nerve stimulation. Recovery from a single 8-h bout of stimulation was characterized by large (>10-fold) transient increases in c-fos, alpha B-crystallin, and hsp70 mRNA. Similar changes were noted during recovery after 7 or 14 days of stimulation (8 h/day). Myoglobin and citrate synthase mRNA were also induced during recovery, but the changes were of lesser magnitude (2- to 2.5-fold) and were observed only following repeated bouts of muscle activity (7th or 14th day) that promoted sustained (>24 h) increases in these transcripts. These findings indicate that recovery from exercise is associated with specific transient changes in the expression of immediate early and stress protein genes, suggesting that the products of these genes may have specific roles in the remodeling process evoked by repeated bouts of contractile activity.

exercise training; 70-kDa heat shock protein; myoglobin; citrate synthase; stress proteins

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

REGULARLY PERFORMED exercise, in addition to being an important component of preventative medicine, is beneficial in the treatment of diabetes, hyperlipidemia, hypertension, and other disorders (8). Many of the benefits derived from exercise training can be attributed to adaptations occurring specifically within skeletal muscle. However, despite extensive characterization of the training-induced changes in muscle substrate utilization, mitochondrial content, capillary density, and enzyme/contractile protein profiles (7, 16, 29, 33, 35, 36), surprisingly little information is available concerning the molecular events responsible for triggering and maintaining the adaptive process. For example, although it is clear that long-term adaptations require changes in gene expression, it is not known when the molecular stimulus to adapt is sensed by the myofiber (during or after exercise), how long the stimulus persists, or how repeated bouts of exercise produce incremental effects that ultimately characterize the trained state.

Direct evidence for regulation of gene expression specifically during recovery from exercise has recently been described for the GLUT-4 glucose transporter and hexokinase II genes (25, 27, 28). Transcription rate of the GLUT-4 gene in red skeletal muscle of rats was found to be increased by ~1.8-fold 3 h after exercise, a response that was not evident after 30 min or 24 h of recovery (25). Transient increases in hexokinase II mRNA and protein levels have also been found in gastrocnemius/plantaris muscles of rats during 24 h of recovery from exercise (28), a response that appears, at least in part, to be mediated by transcriptional activation of the hexokinase II gene both during and after exercise (27). These findings have led to the hypothesis that endurance training-induced adaptations in skeletal muscle may result from the cumulative effects of transient changes in gene expression induced during recovery from each exercise bout (36).

Increases in contractile activity also elicit a marked induction (>10-fold) of a number of stress-related genes, including the c-fos, c-jun, and egr-1 immediate early genes, the 70-kDa heat shock protein (hsp70) and hsp60 heat shock genes, and the alpha B-crystallin small-molecular-weight heat shock gene (23, 24, 26). Although it remains to be determined whether the products of these genes are required for downstream adaptive events in skeletal muscle, their established roles as transcription factors and chaperone proteins in other well-defined systems prompted us, in the present study, to address the hypothesis that the expression of these genes may be altered, not only during muscle exercise (23, 24, 26) but also during recovery from contractile activity. We observed rapid, striking, and transient increases in c-fos, alpha B-crystallin, and hsp70 mRNA concentrations in rabbit tibialis anterior (TA) muscle specifically during recovery from 8 h of low-frequency motor nerve stimulation performed for 1, 7, or 14 days. These findings, therefore, provide evidence that the adaptive responses of skeletal muscle to intermittent contractile activity may be mediated, at least in part, by transient changes in gene expression during recovery.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Adult New Zealand White rabbits (n = 61) weighing ~3.0 kg were purchased from Myrtles Rabbitry (Thompson Station, TN). All radiolabeled compounds were purchased from Amersham. All restriction enzymes and other chemicals were of molecular biology grade and purchased from either Promega, Life Technologies, or Sigma.

Animal surgery and stimulation protocol. Rabbits were anesthetized by isoflurane inhalation. Under aseptic conditions, electrodes were surgically placed in one hindlimb on either side of the common peroneal nerve that innervates the TA and extensor digitorum longus muscles of the lower leg. Leads from the electrodes were attached to a microstimulator embedded in epoxy medium (gas sterilized) and secured beneath the abdominal skin. Microstimulators (32) were manufactured using a CMOS low-power, low-voltage LMC555CM timer (Hamilton Hallmark), power switched via a low-power, low-voltage CMOS D flipflop (Hamilton Hallmark), wired as a toggle flipflop, deriving clock input from a Hex CMSO logic inverter with a Schmitt trigger input (Hamilton Hallmark), which in turn derives its input from a surface-mount NPN phototransistor (OPR5500, Optek) that permitted noninvasive activation/deactivation of the stimulators. The microstimulators were powered by a 3.0-V lithium battery and delivered 1-ms square wave pulses at a frequency of ~8 Hz. Rabbits were stimulated for 8 h/day for either 1, 7, or 14 consecutive days. When the final 8-h stimulation period was completed, the animals were killed either immediately or after 1, 2, 4, 8, 16, or 24 h of rest. At the time of death, the rabbits were anesthetized with pentobarbital sodium (50 mg/kg, intravenous) and the stimulated TA muscle was surgically removed, dissected free of connective tissue, frozen in liquid nitrogen, and stored at -70°C. TA muscles from noninstrumented naive rabbits served as controls. All protocols were reviewed and approved by the Institutional Animal Care and Research Advisory Committee and were conducted in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" [Department of Health and Human Services Publication No. (NIH) 85-23, Revised 1985].

RNA isolation and Northern blot analysis. TA muscle samples were powdered in liquid nitrogen using a precooled (-70°C) mortar and pestle. Total RNA was isolated from ~200 mg of powdered muscle by the guanidinium thiocyanate-phenol-chloroform extraction method (10) with the addition of an LiCl solubilization step (30). Final RNA pellets were resuspended in deionized formamide, and concentrations were determined spectrophotometrically (260 nm). Total RNA (15 µg) was denatured and size fractionated in duplicate by gel electrophoresis in 1.2% agarose gels containing 2.0 M formaldehyde. To assess the quality and amount of RNA between sample lanes, the 28S and 18S ribosomal bands were visualized by ethidium bromide staining of the gel using a charge-coupled device camera under ultraviolet transillumination (Eagle Eye, Stratagene). The RNA was electroblotted (Genie electrophoretic blotter, Idea Scientific) to Hybond N (Amersham), cross-linked (Stratalinker, Stratagene), and prehybridized at 42°C for 4 h in a solution of 50% deionized formamide, 4× SSC (1× SSC = 150 mM sodium chloride, 15 mM sodium citrate), 5× Denhardt's solution (50× Denhardt's = 0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll), 0.1 mg/ml yeast tRNA, 50 mM sodium phosphate (pH 7.0), 0.5 mg/ml sodium pyrophosphate, and 1% sodium dodecyl sulfate (SDS). Hybridizations were carried out overnight at 42°C using the appropriate radiolabeled cDNA probe at 1-3 × 106 counts · min-1 · ml-1. Details concerning the source and preparation of the different cDNA probes used in the present study have been previously given (4, 23, 24, 26, 34). All cDNA probes were labeled with [alpha -32P]dATP (3,000 Ci/mmol) by random priming. After overnight hybridization, the membranes were washed for 30 min in 0.1× SSC-0.1% SDS at room temperature, followed by 10 min at 50°C, and subjected to autoradiography (2-24 h) using Kodak SAR-5 film with intensifying screens. Each membrane was exposed to film for a minimum of three different durations to ensure that nonsaturating signals were obtained. Signal intensity was quantified by densitometric scanning (Arcus II scanner, AGFA) and image analysis software (Molecular Analyst, Bio-Rad), normalized to 28S rRNA (from ethidium bromide-stained gels) to account for slight differences in loading between samples, and expressed relative to 0-h recovery data (immediately after stimulation).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

As an initial attempt to determine whether the adaptive response of skeletal muscle to daily periods of endurance activity may be associated with specific changes in gene expression occurring during recovery from contractile activity, we induced the TA muscle of rabbits, via continuous stimulation of the motor nerve, to contract for 8 h/day for 14 consecutive days. Northern blot analysis revealed a striking transient increase in c-fos and hsp70 mRNA concentrations, specifically during recovery from the final stimulation period (Fig. 1). Transcript levels for both genes were elevated within 2 h after the cessation of stimulation, continued to increase after 4 h, but returned to control levels by 24 h of recovery. Recovery also appeared to be associated with increases in myoglobin and citrate synthase mRNA content; however, these changes were of much smaller magnitude in the three sets of rabbits that completed the 14-day protocol.


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Fig. 1.   Representative Northern analysis from 1 of 2 experiments showing c-fos, 70-kDa heat shock protein (hsp70), myoglobin, and citrate synthase (CS) mRNA content in rabbit tibialis anterior (TA) muscle during recovery from the 14th day of 8 h/day of contractile activity elicited by continuous low-frequency electrical stimulation of the motor nerve. CTL, TA muscle obtained from unstimulated animals. RNA (15 µg) was separated, transferred to nitrocellulose, and sequentially hybridized with cDNA probes as described in MATERIALS AND METHODS. Ethidium bromide staining of 28S and 18S rRNA bands before transfer is also shown (bottom), demonstrating integrity and relative loading of RNA.

To determine whether a single bout of contractile activity is sufficient to induce specific changes in gene expression during recovery, we performed Northern blot analysis on TA muscle obtained from naive rabbits subjected to 8 h of motor nerve stimulation followed by 0-24 h of recovery. Similar to recovery from 14 days of intermittent stimulation, both c-fos and hsp70 mRNA levels increased dramatically during recovery (Fig. 2). Peak expression of both transcripts, however, occurred after only 2 h of recovery and then declined rapidly, reaching control levels within 8 h. Interestingly, recovery from 8 h of stimulation also induced a marked increase in an unidentified higher-molecular-weight transcript that cross-hybridized with the c-fos cDNA probe. Gradual increases in wash stringency to 65°C progressively eliminated hybridization of the upper band, whereas hybridization to c-fos was maintained (data not shown), suggesting that the higher-molecular-weight band represents a unique mRNA species and is not simply a longer form of the c-fos mRNA.


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Fig. 2.   Representative Northern analysis from 1 of 3 experiments showing c-fos, alpha B-crystallin (alpha BC), hsp70, myoglobin, and CS mRNA content in rabbit TA muscle during recovery from a single 8-h bout of continuous motor nerve stimulation. US, TA muscle obtained from unstimulated rabbits. ?, An unidentified band that cross-hybridized with the c-fos cDNA probe during recovery from acute stimulation only. Ethidium bromide staining of 28S and 18S rRNA bands is shown (bottom), demonstrating integrity and relative loading of RNA.

Recovery from 8 h of stimulation was also associated with a dramatic increase in the expression of alpha B-crystallin mRNA, a small-molecular-weight heat shock gene that is also markedly induced within the first 24 h of continuous motor nerve stimulation (24). alpha B-crystallin mRNA levels increased rapidly during recovery and, in contrast to the c-fos and hsp70 transcripts, remained elevated throughout the entire 24-h recovery period (Fig. 2). It is important to emphasize that, as with c-fos and hsp70, alpha B-crystallin mRNA levels were not increased after 8 h of stimulation, indicating that the induction of these genes was specific for the recovery period. Transcript levels for myoglobin and citrate synthase were not significantly altered during recovery from a single 8-h stimulation bout.

In an effort to further characterize the gene-regulatory events that may be occurring during the recovery phase following muscle activity, we followed the expression pattern of the same set of genes in four complete sets of rabbits stimulated for 8 h/day for 7 consecutive days. Dramatic inductions (>10-fold) of c-fos, alpha B-crystallin, and hsp70 mRNAs again were associated specifically with the first 2-4 h of recovery from contractile activity (Figs. 3 amd 4). Similar to levels after both 1 and 14 days of stimulation, c-fos and hsp70 transcript levels rapidly returned to baseline, whereas alpha B-crystallin mRNA remained elevated during the entire 24-h recovery period. Recovery from stimulation also appeared to be associated with a transient increase in myoglobin mRNA content (Figs. 3 and 4), although these changes were relatively modest (~2.5-fold) and, thus, made it difficult to separate the potential responses during recovery from the generalized effect of 7 days of intermittent stimulation (4.6-fold increase in myoglobin mRNA vs. unstimulated controls). Citrate synthase mRNA levels were also elevated in the TA muscle of all rabbits stimulated for 7 days (2.3-fold increase vs. unstimulated controls), with no specific response evident during recovery.


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Fig. 3.   Representative Northern analysis from 1 of 4 experiments showing mRNA content of the indicated genes in rabbit TA muscle during recovery from the 7th day of 8 h/day of continuous motor nerve stimulation. Ethidium bromide staining of 28S and 18S rRNA bands is shown (bottom), demonstrating integrity and relative loading of RNA.


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Fig. 4.   Quantification of c-fos, alpha B-crystallin, hsp70, myoglobin, and CS mRNA concentrations during recovery from the 7th day of 8 h/day of continuous motor nerve stimulation. A summary of densitometric analysis (n = 4/group) of Northern data represented in Fig. 3 is shown. Total mRNA/mg tissue for each transcript was calculated, after adjusting for slight variations in loading via scanning of ethidium bromide images, from total RNA yield/mg tissue. To facilitate comparison of the specific response during recovery, all data were expressed as increase in change relative to the 8-h stimulated TA (time 0, set to 1.0).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The findings from the present study demonstrate that recovery from intermittent contractile activity is associated with transient increases in the expression of three potentially important genes: c-fos, an immediate early gene, hsp70, the inducible member of the 70-kDa HSP family, and alpha B-crystallin, a small-molecular-weight HSP. Transcript levels for each of these genes increased by at least 10-fold within 2-4 h after the cessation of contractile activity. The c-fos and hsp70 mRNA levels returned to near control levels, whereas alpha B-crystallin mRNA remained elevated by approximately sixfold after 24 h of recovery.

The rapid and dramatic induction of these genes specifically during recovery from contractile activity is of particular interest given the cellular functions of these proteins. The c-Fos protein and members of the Jun and activating transcription factor/adenosine 3',5'-cyclic monophosphate response element binding protein families of nuclear proteins collectively comprise the mammalian transcription factor AP-1 (12, 31). Heterodimer formation among these nuclear proteins generates a diverse array of protein complexes with similar DNA-binding specificity but distinct transcriptional control properties. In addition, transcriptional activation by Fos-Jun heterodimer complexes is specifically regulated by phosphorylation and redox state (1, 2), two signaling mechanisms that may be operative in skeletal myofibers (36). The hsp70 class of proteins serves as molecular chaperones, facilitating the folding and intracellular compartmentalization of nascent proteins as well as stabilizing newly denatured proteins (11, 14). alpha B-crystallin, although originally isolated as a specialized protein of the ocular lens (37), has recently been identified as a tissue-specific HSP (18, 21, 22). Thus, collectively, the functions of these proteins, as established in other well-defined biological systems, are consistent with their participation in adaptive remodeling processes within skeletal myofibers, specifically, activation of gene transcription (c-Fos) and stabilization of nascent proteins (hsp70 and alpha B-crystallin). Further work will be required to establish the precise roles of these gene products during the adaptive response to intermittent contractile activity.

In contrast to the c-fos and hsp70 genes, which are expressed at low or undetectable levels under unstressed conditions, alpha B-crystallin is constitutively expressed at high levels in tissues rich in mitochondria, including type I and IIa skeletal myofibers, heart, specific regions of the kidney, and the "ragged red fibers" associated with skeletal muscle mitochondrial myopathies (5, 6, 13, 19, 20). The tissue-restricted expression of alpha B-crystallin and the similar time frame of induction of alpha B-crystallin with c-fos and hsp70 observed in the present study suggest that expression of all three genes during recovery from contractile activity is myofiber specific and does not stem from other cell types present within muscle tissue (e.g., neural, capillary endothelial, fibroblast cells).

It is interesting to note that c-fos, alpha B-crystallin, and hsp70 are also induced in response to stimulation delivered continuously for 24 h/day but with much different time courses; c-fos, as well as two other immediate early genes (c-jun and egr-1), are markedly induced within 4 h after the onset of stimulation (23). However, after 8 h of stimulation, expression of these immediate early genes is no longer evident, suggesting that, in the present study, the daily induction of c-fos expression during the first 2-4 h of recovery from stimulation may have been preceded by transient inductions of the c-fos gene during the 8-h stimulation periods. In contrast to c-fos, alpha B-crystallin and hsp70 transcript levels are not consistently elevated during the first 8 h of stimulation but are elevated by at least fivefold after 24 h of continuous stimulation (24, 26). The fact that alpha B-crystallin and hsp70 were rapidly and dramatically induced during the first 2-4 h of recovery from stimulation in the present study raises the possibility that the signals triggering the adaptive changes in gene expression may be facilitated by recovery and delayed by continued contractile activity.

It is important to emphasize that skeletal muscle is composed of a nonhomogenous mix of fiber types with distinct metabolic and contractile properties. In the chronic nerve stimulation model, although stimulation is delivered to the peroneal nerve that innervates all fibers of the rabbit TA muscle, rapid declines in force output (50-60% within 15 min) suggest that there is a rapid loss in the number of fibers maintaining contractile activity (9, 17). The fiber-type distribution of the rabbit TA muscle (~50% type IId fast-twitch glycolytic, ~45% type I fast-twitch oxidative, and ~5% type I slow-twitch oxidative) (3, 15) strongly implies that only subpopulations of fibers, presumably type I and IIa, are able to maintain contractile activity during an 8-h stimulation period. Interestingly, the initial induction of both alpha B-crystallin and hsp70 during the first 24 h of continuous stimulation occurs specifically within the oxidative type I and IIa myofibers and is not evident in type IId fibers until 21 days after the onset of stimulation, suggesting that the fiber-specific induction of alpha B-crystallin and hsp70 may mark those fibers that have initiated an adaptive response (23, 25). Although not directly addressed in the present study, it is likely that the striking transient induction of the c-fos, alpha B-crystallin, and hsp70 genes observed during recovery from daily bouts of contractile activity may also represent fiber-type-specific regulation of these genes.

Additional evidence that recovery represents a period in which gene regulatory events are triggered during exercise training comes from recent work that has focused on genes that encode for proteins of intermediary metabolism. After 7 days of strenuous treadmill training (40 min/day, 8% grade, 32 m/min performed twice/day), transcription rate of the GLUT-4 glucose transporter gene in red skeletal muscle of rats was found to be no different from untrained rats 30 min after exercise, increased by 1.8-fold 3 h after exercise, but returned to control levels within 24 h after exercise (25). Similar transient increases in transcription of both the GLUT-4 and citrate synthase genes were also found during recovery from a single bout of treadmill exercise. O'Doherty et al. (27, 28) have reported that hexokinase II gene transcription, mRNA content, and enzyme activity are transiently increased in the gastrocnemius/plantaris muscle group of rats during 24 h of recovery from acute treadmill exercise. Moreover, the magnitude of hexokinase II induction was found to be directly related to the duration of exercise (28), suggesting that the intensity/duration of the contractile activity is an important determinant of the adaptive response during recovery.

How can transient changes in gene expression following exercise account for long-term, training-induced adaptations within skeletal muscle? The answer is likely to be found in the kinetics of protein synthesis and degradation. The time required to generate a specific change in a given gene product will be determined by the half-life (turnover rate) of the product of the rate-limiting step. When the stimulus to adapt is intermittent, as encountered during exercise training (3-7 days/wk), the net long-term change will reflect the cumulative effects of intermittent transient changes in expression of that gene product. Gene products with relatively long half-lives (mitochondrial proteins, contractile proteins) will show a small net increase from one training session to the next, whereas gene products with relatively short half-lives (immediate early genes) will not accumulate between training sessions. In the present study, although no consistent change during recovery was detected, average myoglobin and citrate synthase mRNA levels increased by ~2- to 4.5-fold after 7 days of intermittent stimulation, suggesting that the half-life for each of these transcripts is on the order of several days rather than hours. Further efforts to define the temporal sequence of the adaptive response to exercise training, perhaps through single fiber-type analysis, are likely to shed light on the molecular mechanisms regulating this physiologically important process.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert surgical and technical assistance of Jie Liu and Donita Crippens.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-07360 and HL-06296.

Present address for P. D. Neufer: John B. Pierce Laboratory, Yale University, 290 Congress Avenue, New Haven, CT 06519.

Address reprint requests to: R. S. Williams, Molecular Cardiology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8573.

Received 6 June 1997; accepted in final form 15 October 1997.

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Abstract
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Materials & Methods
Results
Discussion
References

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