Free Radical Biology & Medicine, Vol. 11, pp. 239-246, 1991 Printed jn the USA. All rights reserved.

0891-5849/91 $3.00 + .00 Copyright ¢ 1991 Pergamon Press plc

Original Contribution HSP70 AND OTHER POSSIBLE HEAT SHOCK OR OXIDATIVE STRESS PROTEINS ARE INDUCED IN SKELETAL MUSCLE, HEART, AND LIVER DURING EXERCISE

DAVID C . S A L O , * t CASEY M . DONOVAN,t a n d KELVIN J. A . DAVIES*~ *Institute for Toxicology, tDepartment of Exercise Science, and ~:Department of Biochemistry, The University of Southern California, Health Sciences Campus, 1985 Zonal Avenue, HSC-PSC 614-616, Los Angeles, CA 90033, U.S.A. (Received 14 November 1990; Revised 21 February 1991; Accepted 26 February 1991) A b s t r a c t - - E x e r c i s e causes heat shock (muscle temperatures of up to 45 °C, core temperatures of up to 44 °C) and oxidative stress (generation of 0 2 - and H202), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32p]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30--60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O 2- generation with increasing temperatures. HSP70 has been reported to transport polypeptides from the nucleus to mitochondria where they are incorporated into complete mitochondrial proteins. HSP70 may, thus, be a vital link in the molecular mechanism of exercise-induced mitochondrial biogenesis. Keywords--Free radicals, Gene regulation, Gene expression, Protein synthesis, Mitochondria, Ubiquinone

INTRODUCTION

Gene expression is also altered during glucose depletion7 and oxidative stress. 8'9 Cells starved for glucose overproduce a set of proteins called the glucose-regulated proteins, o r G R P . 7 Similarly, cells exposed to low levels of oxidative stress (such as H202) exhibit protection against subsequent exposure to higher, normally lethal, oxidative stress levels, s'9 This induction of oxidative stress resistance is thought to be linked to the overexpression of genes which encode the oxidative stress proteins, or OSP. s The functions of HSP, GRP, and OSP are incompletely understood, but evidence suggests that many shock/stress proteins are enzymes that either provide immediate stress protection 1° or conduct cellular repair processes. 11 Fever, which elevates body temperatures several degrees above normal, results in the induction of HSP. 12 A physiological situation which can elevate muscle temperatures to 45°C, and core temperature to 44°C, is physical exercise. 13'14 Exercise also causes oxidative stress via increased production of oxygen radicals and other reactive oxygen species, 15'16 and sustained physi-

Cells exposed to various environmental stresses in vitro respond by synthesizing a unique set of polypeptides termed shock or stress proteins. 14 Of the many conditions which induce the synthesis of such proteins, heat shock is the most widely investigated. 1-4 Early studies demonstrated that mild temperature increases engender thermotolerance in stressed cells, 5'6 suggesting a protective function for shock/stress proteins. The mechanism of thermotolerance remains incompletely understood, but shock/stress responses are highly conserved phenomena that are observed in a wide variety of procaryotic and eucaryotic cells. ~-5 This research was supported by grant No. ES-03598 from the NIH/NIEHS to K.J.A.D. All correspondence and reprint requests should be addressed to K.J.A.D. at the following new permanent address: Prof. Kelvin J. A. Davies, Chariman, Department of Biochemistry and Molecular Biology, The Albany Medical College, New Scotland Avenue, Albany, New York 12208, U.S.A. 239

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cal activity results in the progressive depletion of glucose and glycogen stores; 17 a phenomenon which is highly correlated with fatigue. 17'18 Given all these factors, it appeared that exercise might be the ideal tool with which to study the effects of shock or stress on gene expression, and the physiological significance of shock/stress proteins. Recently there has been increasing interest in a family of 70 kDa shock/stress proteins (HSP70) which appear to play a role in protein translocation and assembly processes. 19-21 Of particular interest is the proposal that HSP70 may be required for the transport of nuclear encoded polypeptides destined for processing and assembly in mitochondria, w'21 One of the major effects of regular exercise training or conditioning is an increased mitochondrial biogenesis in which the mitochondrial content of muscle can actually double. 22'23 Since precursor polypeptides from the nucleus are required for the assembly of most complete mitochondrial proteins 24 HSP70 could provide a vital link in the mechanism of exercise-induced mitochondrial biogenesis. We, therefore, focused the attention of this investigation on the potential induction of HSP70 during exercise. MATERIALS AND METHODS

Exercise studies

Female Sprague-Dawley rats (Charles Rivers) were acclimatized to exercise on a rodent treadmill (Stanhope Scientific) during a 5-day mild walking/running protocol. Acclimatized rats were run to exhaustion (mean running time of 64.9 _ 8 min) at a constant speed of 26.8 m/min, on a grade of 10%, as previously described. 15.23.25 Animals were sacrificed at various time intervals following exercise, and hind-limb muscles, hearts, and livers were immediately recovered. Excised organs were freed of connective tissue, minced, and then homogenized (Tissuemizer, Teckmar Inc.) on ice in 25mM sucrose (pH 7.4). Homogenates were centrifuged at 10,000 × g (20 min at 4 °C) and the resulting supernatants were recentrifuged at 10,000 x g for an additional 20 min (4 °C) to produce tissue extracts. It should be noted that actomyosin is removed from skeletal and cardiac muscle extracts by the above procedures.

Protein synthesis in isolated muscles

Whole soleus, plantaris, and extensor digitorum longus (EDL) muscles were excised from hind limbs, without muscle tissue damage, by severing the tendons. Muscles were incubated in scintillation vials containing Krebs-Hanseleit buffer (pH 7.4) under an atmosphere

of 95% 0 2, 5% CO2, with constant agitation. Following exposure to either heat shock or oxidative stress, individual muscles were rinsed and proteins were pulselabeled with [3H]leucine for 2 h. After labeling muscles were again rinsed, and the [3H] label was chased by addition of buffer containing 1 mM unlabeled leucine. Muscle extracts were prepared by homogenization and centrifugation, as above.

mRNA studies

A cDNA probe for HSP 70 mRNA was obtained by restriction digest of pAT153 vectors (American Type Culture Collection, Rockville, MD) containing the pH2.3 cDNA clone for human (chromosome 6) HSP 70. 26 HindIII/BamHI restriction digests were separated into 3.3 and 2.3 Kb fragments on 1% low melting temperature agarose gels, stained with ethidium bromide. The 2.3 Kb fragment, which is the HSP 70 cDNA insert, was isolated and [32p]-labeled as below. A cDNA probe for ot-tubulin 27 was used to correct small differences in RNA loading levels. Total RNA was isolated from tissue extracts by the guanidinium isothiocyanate method of Chomczynski and Sacchi. 28 RNA (4 txg) was then electrophoresed on 1% agarose gels containing 16%, formaldehyde, and blotted onto nylon transfer membranes by Northern transfer. 29 Northern blots were hybridized with ~_32p_ labeled cDNA probes prepared according to the random primer labeling method of Feinberg and Vogelstein. 3° Membranes were hybridized overnight at 42 °C and then washed in 0.1 × SSC + 0.01% S D S . 29 Hybridized membranes were autoradiographed (Cronex x-ray film, DuPont) between two intensifying screens at - 7 0 °C.

RESULTS AND DISCUSSION

The concentrations of at least 15 proteins are increased by exercise

Denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of soluble proteins in hind-limb muscle preparations revealed significant increases in at least 15 protein bands immediately following exercise (Fig. 1). The concentrations of some polypeptides were as much as 10-fold higher in muscles from exercised animals than muscles from sedentary c o n t r o l s , and f o r m e d well d e f i n e d p e a k s in electrophoretograms (e.g., the protein with an apparent molecular mass of 106 kDa in Fig. 1A and 1B). In other cases exercise produced clear apparent increases of 3-4 fold in the concentrations of proteins whose electrophoretic peaks, unfortunately, were partially obscured by poly-

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45 kDa proteins reported in Fig. 1 and Table 1 exhibited increased synthesis during either heat shock (seven proteins) or oxidative stress (seven proteins), or both (five proteins). Similar patterns of [3H]leucine incorporation were observed in all three muscle fiber types studied: soleus, a largely slow-twitch oxidative fiber; plantaris, a largely fast-twitch oxidative fiber; and extensor digitorum longus (EDL), a largely fast-twitch glycolytic fiber. The extent of protein synthesis, however, varied somewhat with each muscle fiber type. From the results of Table 2 it would appear that a combination of heat shock and oxidative stress might explain the exercise results of Fig. 1 and Table 1; neither heat-shock nor

oxidative stress alone appear to provide sufficient explanation. Since both heat shock and oxidative stress clearly occur during exercise 13-s6 this rationale is at least tenable. Skeletal muscle HSP70 mRNA levles in exercise

A 70 kDA protein was one of only two polypeptides (70 kDA and 106 kDA) whose synthesis was increased 2-6 fold by both heat shock and oxidative stress in Table 2. The concentration of a 70 kDa protein was also apparently tripled by exercise in both muscle and liver (Table 1). These results, plus the potential role of HSP70 in exercise-induced mitochondrial biogenesis outlined in the Introduction, encouraged us to study transcription of the HSP70 gene. In isolated skeletal muscles, liver, and heart, both heat shock and oxidative stress (performed as per Table 2) resulted in an increased concentration of

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CARDIAC MUSCLE HSP70 mRNA NORTHERN BLOTS

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Fig. 3. Increased cardiac muscle levels of HSP70 mRNA following exercise. Cardiac muscle HSP70 mRNA levels were assessed, in actomyosinfree cardiac muscle extracts, by Northern blotting and scanning densitometry, as described in the legend to Fig. 2. A representative autoradiogram is shown at the top of the figure. The main portion of the figure shows the averaged results (arbitrary units) from scanning densitometry of HSP70 mRNA bands. Two distinct HSP70 mRNA species (2.9 kb and 3.3 kb) were seen to undergo differential concentration changes following exercise. In some samples a faint 2.3 kb HSP70 mRNA species was also seen following exercise (not shown).

mRNA coding for HSP70, as judged by Northern analysis (data not shown). We next studied the effect of exercise on measurable HSP70 mRNA levels in whole hind-limb muscle extracts, soleus extracts, plantaris extracts, and EDL extracts (Fig. 2). HSP70 mRNA levels in whole hind-limb extracts were elevated six fold 1 h after exercise, declined gradually, but remained slightly elevated even 5 h after exercise. A similar pattern of results was obtained with soleus, plantaris, and EDL. The results of Fig. 2 may indicate that HSP70 gene transcription is maximal between 0-2 h following exercise, and declines thereafter (assuming no change in RNAse activity). If correct, this interpretation would suggest that the protein studies of Fig. 1 and Table 1 (immediately following exercise) may actually have underestimated HSP70 translation, which would be expected to peak between 2 and 5 h postexercise. Cardiac muscle HSP70 mRNA levels in exercise

Cardiac muscle exhibits a 4-5-fold increase in metabolism during exercise, 42 and the mitochondrial den-

sity of cardiac muscle exhibits a fractional increase with exercise training. 43 As shown in Fig. 3, cardiac muscle HSP70 mRNA levels increased immediately following exercise. Two HSP70 mRNA species (2.9 kb and 3.3 kb) were observed to change with exercise in cardiac muscle (Fig. 3). Unlike skeletal muscle, however, in which several HSP70 mRNA species behaved identically, differential responses were observed in cardiac muscle. The 3.3 kb species remained at elevated levels for several hours postexercise, whereas the 2.9 kb species returned to control levels within 3 h. Differential changes in discrete HSP70 mRNA species have also been observed in neural tissues. 44

Relationship between heat shock and oxidative stress during exercise

Since both heat shock and oxidative stress appear to cause overexpression of HSP70 (Table 2) it is pertinent to ask if any mechanistic link exists between the two shocks or stresses. Indeed Loven 45 has previously proposed that the effects of heat shock may actually be mediated by increased production of active oxygen

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Fig. 4. Heat stress causes mitochondrial uncoupling and increased superoxide production. Skeletal muscle mitochondria were isolated from whole hind-limb muscles as previously described.15'23 In the left panel respiratory control ratios (RCR) were calculated as the ratio of State III oxygen consumption (10 mM succinate + 0.2 mM ADP) divided by State IV oxygen consumption (succinate + ATP) as described. 47 Superoxide (02-) generation was measured by the rate of (superoxide dismutase inhibitable) reduction of succinoylated cytochrome c. 48 This method actually probably underestimates 02- production by mitochondria (due to internal superoxide dismutase). In the right panel, percent mitochondrial uncoupling is defined as follows: [RCR at 37°C - RCR at the temperature of interest] / RCR at 37°C × 100. Percent 02- generation (right panel) is defined as a percentage of the maximal 02- production, observed at 49°C. A linear correlation of 0.98 (P < 0.01) was obtained for the relationship between % uncoupling and % O2- generation.

species. Brooks et al. 13,14 have reported that muscle and liver mitochondria undergo progressive uncoupling (loss

of respiratory control) during temperature increases from 37 °C to 45 °C (the p h y s i o l o g i c a l temperature range during exercise o f various intensities). C h a n c e et al. 46 h a v e reported that m i t o c h o n d r i a are one o f the main intracellular sources o f superoxide ( 0 2 - ) and H 2 0 2 under physiological conditions, and that u n c o u p l e d m i t o c h o n dria generate 0 2 - and H 2 0 2 at m u c h higher rates than do w e l l - c o u p l e d mitochondria. In our hands m i t o c h o n d r i a exhibited a temperatured e p e n d e n t loss o f respiratory control (i.e., uncoupling), and an increased generation o f 0 2 - , in the physiological temperature range o f 37 °C to 45 °C (Fig. 4, left). Thus, m i t o c h o n d r i a l O 2 - generation exhibits an excellent linear correlation ( r = 0 . 9 8 ) with mitochondrial uncoupling (Fig. 4, right). The generation o f O 2 - (and its dismutation to H202) has been closely linked with the u n i v a l e n t reduction o f o x y g e n by mitochondrial u b i s e m i q u i n o n e . 46 W e h a v e p r e v i o u s l y s h o w n that exercise causes an increase in an E S R detectable s e m i q u i none radical ~5 and a subsequent study, 49 as well as unpublished observations, has identified this radical species as m i t o c h o n d r i a l u b i s e m i q u i n o n e . Thus, exercise h y p e r t h e r m i a (and perhaps other forms o f heat shock) m a y cause a partial mitochondrial u n c o u p l i n g , associated with elevated u b i s e m i q u i n o n e concentrations, and increased 0 2 - generation. The 0 2 - and H 2 0 2 so f o r m e d (or a product o f their interaction) m a y be the agent(s) actually responsible for H S P 7 0 o v e r e x p r e s s i o n (and perhaps other heat shock responses). This interpre-

tation is certainly consistent with the findings that several shock/stress proteins are c o m m o n to both heat shock and oxidative stress 5° and that adaptation to mild oxidative stress confers crossresistance to heat shock. 5~ A c k n o w l e d g e m e n t s - The authors wish to thank Ms. Cynthia D.

Hunter for preparation of the typescript.

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ABBREVIATIONS

H S P - - h e a t shock protein(s) G R P - - g l u c o s e regulated protein(s) O S P - - o x i d a t i v e stress protein(s) P A G E - - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s SDS-PAGE--sodium dodecylsulfate-PAGE IEF--isoelectric focusing M . O. P. - - m a c r o x y p r o t e i n a s e E D L - - e x t e n s o r digitorum longus (muscle) O 2 - - - s u p e r o x i d e radical " O H - - h y d r o x y l radical R C R - - r e s p i r a t o r y control ratio ( m i t o c h o n d r i a l )