Insulin-like growth factor immunoreactivity increases in muscle after acute eccentric contractions

ZHEN YAN, RAY B. BIGGS, AND FRANK W. BOOTH Department of Physiology and Cell Biology, The University of Texas Medical School, Houston, Texas 77225

YAN, ZHEN, RAY B. BIGGS, AND FRANK W. BOOTH. Insulin- like growth factor immunoreactivity increases in muscle after acute eccentric contractions. J. Appl. Physiol. 74(l): 410-414, 1993.-The purpose of the study was to note whether insulin- like growth factor (IGF) immunoreactivity increased after ec- centric contractions. IGF immunoreactivity in the rat tibialis anterior muscle was measured on 5 successive days (4-5 rats/ group, n = 28) after an acute bout of 192 eccentric contractions elicited by electrical stimulation. The muscle tissue sections were immunocytochemically processed with rabbit anti-human IGF-I serum. Immunoreactivity was analyzed with videomicros- copy and computer-aided image processing. Four days after ec- centric contractions, IGF immunoreactivity was significantly higher than control [0.081 t 0.073 (SD) absorbance at 480 nm vs. 0.026 t 0.018; P < 0.051. The increases in IGF-I immunore- activity were mostly within the muscle fibers. These results suggest that an acute bout of eccentric exercise increases IGF-I immunoreactivity in rat type II muscle 4 days postexercise.

exertion; resistance exercise; skeletal muscle

ECCENTRIC RESISTANCE TRAINING results in an en- hanced muscle strength in humans (4,6,10,13) and mus- cle hypertrophy in animals (19, 21). Eccentric contrac- tion is defined as a forcible stretching or lengthening of the active muscle during cross-bridge cycling (1, 12). A single acute bout of 192 eccentric contractions increases the synthesis rates (41-65%) of total mixed protein and of myofibril protein 12-17 and 36-41 h after ending exer- cise (21). However, the mechanisms initiating and main- taining the increase in muscle protein synthesis rate after eccentric exercise are not known. In one study, a 150% increase of insulin-like growth factor- (IGF) I mRNA was found in the soleus and plantaris muscles on the 2nd day of compensatory overload-induced hyper- trophy in the rat (5). There are no reports on IGF immu- noreactivity after eccentric exercise. On the other hand, it is clear that IGF-I peptide can increase muscle protein synthesis rate. In one study, a 40% increase in protein synthesis was shown in L6 myoblasts during a 4-h expo- sure to IGF-I in tissue culture (2). In a second study, the inclusion of IGF-I peptide in the maintenance medium of cultured primary myofibers for 4-5 days produced fibers of larger diameter and with more myonuclei per fiber length than those produced in maintenance medium without IGF-I (17). These fibers had more myosin accu- mulation, higher myosin synthesis rates, and lower myo- sin degradation rates. In a third report, protein synthesis rate was increased more if muscle fibers were rhythmi- cally stretched than if they underwent no mechanical ac-

tivity at a given concentration of IGF-I in cultured myo- fibers (18). In summary, muscle protein synthesis rates are increased by eccentric contractions and by IGF-I. On the basis of these. observations, it is possible that an in- crease in IGF-I concentration could be in the signal path- way from eccentric contraction to the observed increase in muscle protein synthesis. The purpose of the present study was to test whether IGF immunoreactivity in- creased after eccentric exercise.

MATERIALS AND METHODS

Animal Care

Adult female Sprague-Dawley rats (Harlan, Indiana- polis, IN) were housed in animal quarters maintained at 21°C with a 12:12-h light-dark cycle. Water and rodent laboratory chow (Purina) were provided ad libitum. Ex- perimental protocols had been approved by the Institu- tional Animal Care and Use Committee of The Univer- sity of Texas Health Science Center at Houston.

Experimental Design

Rats were assigned randomly to either one of five post- exercise groups or a control nonexercised group, for which five subsets existed. Each subset consisted of one animal from the control group and one animal from each of the five postexercise groups. The five postexercise groups consisted of rats that were killed 1, 2, 3, 4, and 5 days after a single bout of 192 eccentric contractions. Within each subset, rats were killed on the same morning and their muscles were processed at the same time for immunocytochemistry.

Eccentric Contraction

Rats were anesthetized with pentobarbital sodium (48 mg/kg body wt). Animals were exercised with a model of nonvoluntary electrically contracted skeletal muscle (19-21). Subcutaneous platinum electrodes were posi- tioned bilaterally along the lower leg muscles of the right hindlimb. The foot was secured to a pulley-bar apparatus with the rat supported on a platform above the foot lever. The muscle contraction of the anesthetized rat was in- duced with a Grass S48 electrical stimulator with l-ms pulses at 100 Hz and 30 V with a 2.5-s train duration every 15th s for six trains. After each set of six trains (contractions), there was a rest period of 1 min (first 16 sets) or 30 s (last 16 sets). A longer rest duration was provided after every 4th set (3.5 min in the first 16 sets

410 0161-7567/93 $2.00 Copyright 0 1993 the American Physiological Society

GROWTH FACTOR IN MUSCLE HYPERTROPHY 411

TABLE 1. IGF-I immunocytoreactivity in the tibialis anterior muscle at various times after 192 eccentric contractions

Days Postexercise

Control 1 2 3 4 5

IGF-I 26-tl8 25217 43&28 42t33 81+73* 61+58 n 5 4 5 4 5 5

Values are means + SD expressed in optical density units X 10m3. n, no. of animals. IGF, insulin-like growth factor. * P < 0.05 vs. control.

and 3 min in the last 16 sets). This experimental regimen was identical to the muscle stimulation protocol de- scribed elsewhere in which an increase in protein synthe- sis rate was reported after eccentric contraction (21). Muscle stimulation causes both the anterior and poste- rior muscle compartments of the lower leg to contract, with a net plantar flexion resulting in a lengthening of the contracting tibialis anterior muscle (eccentric con- traction).

Muscle Preparation

The tibialis anterior muscle was fixed in situ as fol- lows. On the day rats were killed, they were anesthetized with pentobarbital sodium (48 mg/kg) and a laparotomy was performed. A 150 mM phosphate buffer solution (150 mM NaCl, 14 mM potassium phosphate, pH of 7.4) was infused through a catheter into the dorsal artery at a rate of 0.5 ml/min. After 2 min of infusion, the 130 mM phosphate buffer was discontinued and the fixative solu- tion [130 mM phosphate buffer (130 mM NaCl, 14 mM potassium phosphate), 4% paraformaldehyde, 0.5% glu- taraldehyde, 100 PM MgCl,, and 100 ,uM CaCl,, pH of 7.41 was infused for 15 min while the right ankle was taped at a 90’ angle. At the end of infusion, the right tibialis anterior muscle was removed from the rat and placed for 45 min in a vial containing the aforementioned fixative solution without glutaraldehyde. The fixative so- lution was replaced with phosphate-glycine solution (130 mM phosphate buffer solution with 50 mM glycine, pH 7.4) for 30 min, and the sample was washed successively with 5,10, and 20% sucrose in 130 mM phosphate buffer solutions, pH 7.4, with the time of each sucrose wash varying from 15 min to 3 h. Muscle blocks were then mounted with OCT compound embedding medium (Miles, Elkhart, IN) and stored at -2OOC. In a cryostat, -g-pm-thick sections were cut and placed onto DL-poly- lysine-coated glass slides. For each rat, five sections were placed onto a slide (3 to receive primary and secondary antibodies; 1 to receive secondary antibody only, the re- agent control; and 1 to receive Goldner’s modification of Masson’s trichrome stain) (9). Slides were stored over- night in phosphate buffer at 4°C.

Immunocytochemistry

Slides from the same subset were processed at the same time. Muscle sections on slides underwent the fol- lowing procedures at room temperature. Sections were blocked with 3% normal goat serum (NGS). The primary antibody was contained in anti-IGF-I/somatomedin C

rabbit antiserum (UB-189) (8) and was a gift of the Hor- mone Distribution Program of the National Hormone and Pituitary Program of the National Institute of Dia- betes and Digestive and Kidney Diseases (NIDDK; Bal- timore, MD). The anti-human-IGF-I rabbit antiserum used in the present study has a 0.5% cross-reactivity with IGF-II and it cross-reacts minimally with 10v6 M insulin (Hormone Distribution Program, NIDDK). The termi- nology “IGF immunoreactivity” is used in the present paper to describe the reaction product obtained with this antiserum, because a small portion of the 200% increase in IGF immunoreactivity on the 4th day after 192 eccen- tric contractions could be due to cross-reactivity with IGF-II. The antiserum was diluted l:l,OOO in 3% NGS and was placed on the muscle sections for 2 h. After un- bound IGF-I antibody was washed away, the Vectastain ABC immunoperoxidase kit (Vector Laboratories, Bur- lington, CA) was used to localize IGF-I antibody bound to antigen and as a reagent control on the other half of the muscle sections. The kit’s biotinylated goat anti-rabbit immunoglobulin G (diluted 1200 in 3% NGS) was put on all muscle sections. After a wash, sections were exposed to 3% H,O, in methanol. After the sections were washed again, the kit’s avidin-biotinylated horseradish peroxi- dase macromolecular complex was added. After the next wash, a peroxidase substrate [3-amino-9-ethylcarbazole (AEC) working solution] was put onto the muscle sec- tion. The resultant red insoluble reaction product was quantified by image processing.

Image Processing

Setup and calibration. A videomicroscope, which con- sists of a BH-2 microscope (Olympus, Lake Success, NY) and a color video camera ‘(Microimage Video Systems, World Video, Boyertown, PA), was used for image pro- cessing. Once the image was loaded, it was analyzed using Jandel Video Analysis Software (JAVA 1.3) on a per- sonal computer. The instruments were warmed up for r10 min to ensure a steady light intensity. An ~4 lens was used when loading the image with the cube selection knob at the position that allowed 80% of the light to enter the photo tube. The lens aperture was adjusted to 0.10 and the light source aperture to maximum. No filter was selected, and the position of the lens was adjusted by focusing an actual slide. Before the analysis of a slide, the videomicroscope was calibrated internally. To accom- plish this, the light intensity was adjusted so that the absolute gray level detected at the center of the image in the absence of a slide was 150 absolute units.

Analysis of the image. For each cross section of the whole muscle, the average gray level for the whole muscle cross section was determined by measuring random sites, the proportion of which represented the whole muscle cross section, i.e., if 20% of the muscle cross section were very lightly stained, then one random site was selected from the very lightly stained area; if the remaining 80% of the muscle cross section were moderately stained, then four random sites were selected in these areas (approach 1). (A random site consisted of -50 muscle fibers.) If the entire muscle cross section was evenly stained, four to eight random sites within the whole muscle cross section

GROWTH FACTOR IN MUSCLE HYPERTROPHY 413

were selected (approach 2). The mean of the random sites within each whole muscle cross section was calculated. The mean gray level for each rat was obtained from the mean gray levels of two to three whole muscle cross-sec- tion gray levels on the same slide. The mean gray level of each rat was converted to an optical density at 480 nm with use of a standard curve, which was generated as described below. Each rat’s mean optical density was corrected by the optical density of the reagent control. After analysis of all rats, the mean for each time point was obtained. In some of the slides in approaches 1 and 2, either extracellular space increased because of the eccen- tric exercise or muscle fibers shrank during either the fixing procedure or the histochemical treatment proce- dure, resulting in an increase of extracellular space and a possible increase of the image intensity within the cells. To eliminate the effect of shrinkage in these slides, we measured the average gray level of muscle bundles, in- cluding the extracellular space, instead of the average gray level in the muscle fiber.

Standard curue establishment. The standard solution was prepared by the same reaction as in the immunocyto- chemical treatment of the sections. Briefly, a reaction cocktail was made by adding 200 ~1 of 30% H,O, and’100 ,LJ of 14% AEC (in N,N-dimethylformamide) to 5 ml of Na-acetate buffer (pH 5.2). The reaction was started by adding 30 ~1 of goat anti-rabbit immunoglobulin G conju- gated with horseradish peroxidase (protein concn 13.9 mg/ml). The solution was incubated at room tempera- ture for 10 min and spun in a clinical centrifuge at 4,000 rpm for 3 min. The pellet was redissolved in 5 ml of 95% ethanol by vigorous vortex. Then the solution was fil- tered through a 0.2-pm filter to exclude any undissolved precipitate. A serial dilution was made with 95% ethanol and was used for establishing a standard curve. The gray levels of the standards were determined under the video- microscope by pipetting -200 ~1 of each standard dilu- tion to fill the well (0.177 cm deep) of a hanging drop slide, which was then covered by a glass cover slip. Imme- diately after the measurement under the videomicro- scope, the optical densities of each standard dilution were measured at 480 nm with a Beckman DU-50 spectro- photometer. (The spectrum of this solution was made using the spectrophotometer, and a peak absorbance at 480 nm was found.) A standard curve and mathematical equation were derived for converting the gray level to optical density at 480 nm. The standard curve generated by this approach was linear (r = 0.998) for the absor- bance range of the samples in the present study.

Statistics

The error mean square value from an analysis of vari- ance was used to calculate a least significant differences test for groups with unequal replication (16). P < 0.05 was designated as significant.

RESULTS

The purpose of the semiquantification of IGF-I immu- noreactivity was to objectively distinguish any differ- ences among experimental groups rather than to indicate subjectively that one group stained more intensely than another. This assay does not give absolute quantities of IGF-I peptide. The sensitivity of the assay enables detec- tion of concentration differences of AEC of the magni- tude of 0.002%. An external standard of known gray level permitted calibration of the image-processing system each day. Thus, variations due to the image processing per se were minimal.

The IGF-I immunoreactivity in the tibialis anterior muscle was -200% greater (P < 0.05) 4 days after an acute bout of eccentric contractions than the control val- ues (Table 1,. Fig. 1). Although IGF-I immunoreactivity started to increase 2 days postexercise, the value only reached the significant level 4 days postexercise. It is unlikely that variations in the repeatability of gray level measurements contributed to the large standard devia- tions (Table l), because a correlation of 0.998 existed between AEC concentration and gray level. The large standard deviations for individual group means (Table 1) could be due to any or all of the following reasons: 1) variations in the thickness of muscle sections (the micro- tome did not cut sections of consistent thickness), 2) vari- ations among days for the immunocytochemistry, be- cause each subset (a subset consists of 1 animal from each experimental group) underwent immunocytochem- istry on different days [the rate of color development for a positive control (sections of marcaine-damaged mus- cle) varied for each subset], and 3) the response to the eccentric contraction varied among animals within a day (e.g., 2 of the 5 rats within day 4 had IGF immunoreactiv- ity levels similar to the control values, while the other 3 rats had optical densities 2,3, and 20 times their control values). Others have observed nonresponders to eccen- tric exercise (R. Armstrong and G. Dudley, personal com- munication). Nevertheless the variation in IGF immuno- reactivity did not prevent the detection of a significant increase in IGF-I immunoreactivity in type II muscle 4 days after an acute bout of 192 eccentric contractions.

The eccentric contraction-mediated increase of IGF immunoreactivity was within and sometimes outside the muscle fibers (Fig. 1). However, the increase in IGF im- munoreactivity is not always consistent within the mus- cle. Because of the nature of this study, it is not possible to elucidate the origin of the increased availability of IGF-I peptide.

Some of the postexercise sections showed focal accu- mulation of inflammatory cells, which others have inter- preted to mean that the eccentric contractions caused focal damage to muscle fibers (7, 15). At this point, we cannot exclude the possibility that events associated with accumulation of inflammatory cells could play a role

414 GROWTH FACTOR IN MUSCLE HYPERTROPHY

in the increase of IGF-I immunoreactivity in the muscle postexercise.

Address for reprint requests: F. W. Booth, Dept. of Physiology and Cell Biology, The University of Texas Medical School, PO Box 20708, Houston, TX 77225.

DISCUSSION Received 15 January 1992; accepted in final form 10 August 1992.

The new information provided in the present study is a 200% increase in IGF immunoreactivity (4 days) after the 192 eccentric contractions and the large number of fibers showing the increase in IGF immunoreactivity in those muscles having an increased IGF response. A pre- vious report examined IGF-I mRNA (5), but not the im- munoreactivity of the IGF-I peptide, after continuous overload, rather than an acute eccentric exercise.

REFERENCES

1.

2.

ATHA, J. Strengthening muscle. Exercise Sports Sci. Rev. 9: l-73, 1981.

3.

The experimental model of compensatory overload-in- duced hypertrophy, which was used to obtain the in- crease in IGF-I mRNA, differs from a single acute bout of eccentric exercise both in the duration of the exercise stimulus and the amount of postexercise recovery (3). Whereas compensatory overload-induced hypertrophy is essentially a continuous exercise stimulus (2,880 min of application in 2 days) with no long period of recovery, the 192 eccentric contractions, which were employed in the present study, consist of a total of 6.4 min of contraction followed by days of recovery. Because of this difference, it is not certain that IGF immunoreactivity would be in- creased after 192 eccentric contractions. Furthermore the increased IGF-I mRNA quantities observed in com- pensatory overload-induced muscle do not definitively prove an increase in IGF immunoreactivity, because pro- tein level is a dual function of synthesis and degradation. Thus a direct measurement of an increase in IGF immu- noreactivity had not been noted postexercise in any con- tractile model in vivo.

4.

5.

6.

7.

8.

9.

10.

11.

Increases of 41-56% in mixed and myofibrillar protein synthesis rates were found as early as 12-17 h and main- tained to at least 36-41 h after 192 eccentric contractions by the tibialis anterior muscle of the rat (21). IGF immu- noreactivity was not increased at a time point (24 h after 192 eccentric contractions; present study) that is be- tween the previously reported times (12-17 h and 36-41 h after 192 eccentric contractions) (21) in which protein synthesis rates were found to be increased. However, an alternative mode by which IGF-I could play a role in the increased protein synthesis rate by 12 h, or up to 41 h after 192 eccentric contractions, is by increasing the skel- etal muscle sensitivity and/or responsiveness to IGFs. This is supported by two recent reports of increases in both sensitivity and responsiveness to IGF-I for glucose and amino acid transport in the epitrochearis muscles of rats 3.5 h after a 2-h swim (11, 14).

12.

13

14

15.

16.

17.

We thank Mildred Lai and Jason Lai for assistance in establishing immunocytochemistry in our laboratory, Dr. Adrian Sheldon for assis- tance in image processing, and Drs. Chris Kirby and Craig Stump for assistance in critique of the manuscript.

The hormone distribution program of the National Institute of Dia- betes and Digestive and Kidney Diseases provided the funding to per- mit the gift of the anti-IGF-I antiserum. Research funding was pro- vided by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393.

18.

19.

20.

21.

BALLARD, F. J., L. C. READ, G. L. FRANCIS, C. J. BAGLEY, AND J. C. WALLACE. Binding properties and biological potencies of insulin- like growth factors in L6 myocytes. Biochem. J. 233: 223-230,1986. BOOTH, F. W., AND D. B. THOMASON. Molecular and cellular adap- tation of muscle in response to exercise: perspectives of various models. Physiol. Rev. 71: 541-585, 1991. COLLIANDER, E. B., AND P. A. TESCH. Effects of eccentric and con- centric muscle actions in resistance training. Acta PhysioZ. Stand. 140: 31-39, 1990. DEVOL, D. L., P. ROTWEIN, J. L. SADOW, J. NOVAKOFSKI, AND P. J. BECHTEL. Activation of insulin-like growth factor gene expression during work-induced skeletal muscle growth. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E89-E95, 1990. DUDLEY, G. A., P. A. TESCH, B. J. MILLER, AND P. BUCHANAN. Importance of eccentric actions in performance adaptations to re- sistance training. Adv. Space Environ. Med. 62: 543-550, 1991. EVANS, W. J., AND J. G. CANNON. The metabolic effects of exercise- induced muscle damage. Exercise Sports Sci. Rev. 19: 99-125,1991. FURLANETTO, R. W., L. E. UNDERWOOD, J. J. VAN WYK, AND A. J. D’ERCOLE. Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J. CZin. In- vest. 60: 648-657, 1977. GURR, E. A Practical MunuuZ of Medical and Biological Staining. New York: Interstate, 1956, p. 110-111. H;~KKINEN, K., AND P. V. KOMI. Effect of different combined con- centric and eccentric muscle work regimens on maximal strength development. J. Hum. Mov. Stud. 7: 33-44, 1981. HENRIKSEN, E. J., L. L. LOUTERS, C. SI STUMP, AND C. M. TIPTON. Prior exercise increases stimulation of glucose transport by insu- lin-like growth factor I in rat skeletal muscle (Abstract). Med. Sci. Sports Exercise 24: S89, 1992. KNUTTGEN, H. G. Force, work, power, and exercise. Med. Sci. Sports 10: 227-228, 1978. KOMI, P. V., AND E. R. BUSKIRK. Effect of eccentric and concentric muscle conditioning on tension and electrical activity of human muscle. Ergonomics 15: 417-434, 1972. LOUTERS, L. L., E. J. HENRIKSEN, C. S. STUMP, AND C. M. TIPTON. Effect of prior exercise on insulin-like growth factor I-stimulated system A amino acid transport in skeletal muscle (Abstract). Med. Sci. Sports Exercise 24: S180, 1992. MCCULLY, K. K., AND J. A. FAULKNER. Characteristics of length- ening contractions associated with injury to skeletal muscle fibers. J. AppZ. Physiol. 61: 293-299, 1986. STEEL, R. G. D., AND J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw-Hill, 1960, p. 112-114. VANDENBURGH, H. H., P. KARLISH, J. SHANSKY, AND R. FELD- STEIN. Insulin and IGF-I induce pronounced hypertrophy of skele- tal myofibers in tissue culture. Am. J. Physiol. 260 (Cell Physiol. 29): c475-C484, 1991. VANDENBURGH, H. H., P. KARLISH, AND R. L. SOLERSSI. Insulin and insulin-like growth factor-I stimulation of skeletal myofiber growth in vitro is enhanced by mechanical activity (Abstract). J. CeZZ BioZ. 115: 22lA, 1991. WONG, T. S., AND F. W. BOOTH. Skeletal muscle enlargement with weight-lifting exercise in rats. J. AppZ. Physiol. 65: 950-954, 1988. WONG, T. S., AND F. W. BOOTH. Protein metabolism in rat gastroc- nemius muscle after stimulated chronic concentric exercise. J. AppZ. Physiol. 69: 1709-1717, 1990. WONG, T. S., AND F. W. BOOTH. Protein metabolism in rat tibialis anterior muscle after stimulated chronic eccentric exercise. J. AppZ. Physiol. 69: 1718-1724, 1990.