significantly reduced the rate of resynthesis of glycogen, thus giving rise to the

Transcription

1 Quarterly Journal of Experimental Physiology (1977) 62, THE EFFECT OF HYPOXIA ON MUSCLE GLYCOGEN RESYNTHESIS IN MAN. By J. S. MILLEDGE, D. HALLIDAY, C. POPE, P. WARD, M. P. WARD and E. S. WILLIAMS. From the Divisions of Anaesthesia, Clinical Investigation and Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ. (Receivedfor publication 1st October 1976) The effect of hypoxia and muscle fibre type on the resynthesis of muscle glycogen was studied in three healthy subjects by means of needle biopsies. Biopsies were taken before and after exercise and after 5 h recovery. In control experiments subjects breathed air during the recovery phase whereas in the test experiments they breathed 10% oxygen in nitrogen during this time. Diet was controlled throughout the experiments and provided an excess of carbohydrate. Biopsies were assayed for total muscle glycogen and were studied histochemically, using microdensitometry on PAS stained sections as a measure of glycogen in individual fibres. Exercise depleted muscle glycogen to a mean of 26 % of control values. After 5 h recovery the mean values had risen to 62 % breathing air and 51 % on 10% oxygen breathing but this difference was not significant. In biopsies taken after 5 h recovery breathing either air or 10% oxygen, there was a marked contrast in PAS staining between muscle fibre Type I and II, the Type II fibres being more heavily stained. This difference was confirmed by microdensitometry. The contrast was increased by hypoxia in all subjects but this effect did not reach levels of statistical significance. It is concluded that under these experimental conditions the overall resynthesis of muscle glycogen is not significantly affected by hypoxia. It is shown that Type II fibres resynthesise significantly more rapidly than Type I under both normoxic and hypoxic conditions. Hypoxia may enhance this difference in rate of resynthesis. Patients with respiratory diseases resulting in chronic hypoxia, or healthy men at high altitude, often complain of muscle fatigue as much as breathlessness. Muscle glycogen is depleted by exercise and Bergstrom, Hermansen, Hultman and Saltin [1967] showed that the duration of exercise terminated by fatigue correlated wellwith the initial level of muscle glycogen in their subjects. Recovery from fatigue is associated with resynthesis of muscle glycogen and it is the experience of climbers at high altitude that they seem not to recover in the usual way from fatiguing exercise. The resynthesis of glycogen requires energy from ATP derived from oxidative phosphorylation and ATP levels are reduced in rats at altitude [Cipriano and Pace, 1973]. We wondered, therefore, if hypoxia significantly reduced the rate of resynthesis of glycogen, thus giving rise to the symptom of fatigue. The advent of the technique of needle biopsy of muscle [Bergstrom, 1962] made it possible to test this hypothesis. Following work by Gollnick, Piehl and Saltin [1974] and Gollnick, Karlson, Piehl and Saltin [1974] on the differential depletion of glycogen in different muscle fibre types during exercise, and by Piehl [1974] on differential refilling of muscle fibres after exercise, we also wished to study the differential rate of resynthesis of glyzogen in these two muscle fibre types, under normoxic and hypoxic conditions. 237

2 238 Milledge et al. METHODS Subjects. Three subjects carried out the full protocol of test and control experiments. They were aged 45, 49 and 52 and weighed 73, 70 and 69 kg respectively. All subjects gave their informed consent to the procedures. The subjects were all hospital doctors and though all took some form of exercise, mainly hill walking and squash, they were not in any form of regular athletic training. Experimental. Muscle biopsies were obtained from M. vastus lateralis by the percutaneous needle biopsy technique of Bergstrom [1962]. Two biopsies were taken each time, one for histology and one for biochemistry. The biopsy for biochemistry was passed rapidly through saline and lightly blotted with filter paper to remove blood, then weighed each min for 3 min. The decreasing weights due to drying over the 3 min were plotted and extrapolated back to time zero when the biopsy was actually taken. The specimen was then frozen in liquid nitrogen and thereafter stored at - 20 C until analysed. The total time for biopsy extraction to freezing was not more than 5 min. The biopsies were analysed for glycogen by the method of Hultman [1966] with the following modifications. The muscle biopsy tissue was not homogenized but digested in 2 ml of 30 % KOH in sealed centrifuge tubes, in a boiling water bath for 20 min, shaking occasionally. Duplicate aliquots of this solution of 0 5 or 0-25 ml were taken for each sample, and combined with 5 0 ml ethanol, vortexed and left for glycogen precipitation to occur at 4 C overnight. After centrifugation, the precipitate was then hydrolysed with 0-2 ml 6N H2S04 in sealed tubes in the boiling water bath for 45 min. In our hands we found that this increased volume of acid gave a more complete hydrolysis of glycogen. The following orthotoluidine reaction was as described by Hultman [1966]. Rabbit liver glycogen (Sigma Type III) was dissolved in distilled water, digested, precipitated, and hydrolysed together with the muscle samples whose glycogen content was obtained by comparison with this standard. All standards were put up in duplicate, forming a series from 12-5 pg.mlh to 200 pg.ml- 1. Muscle samples were analysed at 2 dilutions, each in duplicate. Histology. Each specimen was orientated transversely under a dissecting microscope attached to a cork disc with OCT embedding medium (Ames Tissue Tek) and snap frozen in isopentane cooled in liquid nitrogen within 5 min of being taken. Cryostat sections 9,um thick were cut in cross-section and stained for glycogen using the periodic acid-schiff (PAS) reaction [Pearse, 1968]. Adjacent sections were stained to demonstrate the activity of myofibrillar ATPase at ph 9.5 [Hayashi and Freiman, 1966], so as to distinguish ATPase positive Type II from ATPase negative Type I fibres. Densitometry of the PAS stained sections was performed using a Kodak Wratten 74 green filter ( ,c) corresponding to the absorption band of PAS stain, and a photometer (Melico pm2 colour analyser) with an aperture of 3 mm. In reading 'whole biopsy' sections (approx. 50 fibres) the microscope slide was placed in a photographic enlarger and the photometer cell positioned in the enlarged image. In reading individual fibres the slide was projected on to a screen using a Leitz S.M. Projector. The enlargement was such that individual fibres had an image of greater than 5 mm diameter. Fibres were identified as Type I or II from a print of an ATPase stained adjacent section. Their optical density was then measured and recorded; 50 fibres of each type were measured from each biopsy. The photometer was zeroed for each section by being placed in the projected field adjacent to the biopsy section, thus eliminating the effect of variation in slide and cover glass thickness. Care was taken to count groups of fibres with approximately equal numbers of each type in one area, so that any variation in light intensity across the field would not bias the result. Fibres at the edge of the section were avoided as these often stain unevenly. Procedure Each subject performed one test and one control experiment, which were identical except for the gas breathed during recovery. The diet for the day of the experiment was controlled from breakfast until the end of the experiment at about It consisted mainly of pre-

3 Hypoxia and muscle glycogen resynthesis 239 packaged foods whose composition was known and in which a high proportion of the energy could be taken in a liquid form for convenience when wearing a mask. The diet provided 10,032 kj, 70 % of which were from carbohydrates. Subjects came to the laboratory having had the diet breakfast. The first muscle biopsy was taken, then the subject exercised for 2 h by running round a track and then for the last 20 min by running up and down stairs in a 9 storey building. Subjects paced themselves at the maximum rate they could continue for 2 h. This resulted in pulse rates of 145 to 165 min- I representing about 80 to 90 % of the work rate for a pulse of 170 min -1. The second muscle biopsy was then taken and a high energy drink and biscuit given. The subject then rested in a chair or on a bed for 5 h. In the test experiment the subject breathed 10 % oxygen in nitrogen during this 5 h period, via an RAF mask and Ruben respiratory valve, giving a P1o2 equivalent of about 6,000 m altitude. Further food and drink were given at fixed intervals during this time. In the control experiment air was breathed during this period. A third biopsy was taken at the end of the 5 h recovery period. RESULTS Experiments with recovery under normoxic and hypoxic conditions were carried out on three subjects giving complete sets of data. On a further two experiments, one normoxic and one hypoxic on two of the subjects, histological but not biochemical data were obtained. The results of the biochemical estimations of muscle glycogen for the six experiments are shown in Fig. 1. The mean value for control biopsies was 122 g of glycogen per 100 g of muscle wet weight, with a range It will be seen from Fig. 1 that there was a marked reduction in glycogen after exercise to a mean of 26 %. of control value. The results of the third biopsy are more scattered. After both hypoxic and normoxic recovery periods there is an increase in glycogen. Two out of three subjects showed depressed resynthesis on hypoxia compared with normoxia but the third subject showed the reverse. The mean values after recovery are 620% and 51 % of control values after normoxia and hypoxia respectively. This difference is not significant. Histologically, the PAS stained sections showed a marked reduction in overall density of stain in the second biopsy as compared with the control section. The third biopsy showed a return of PAS staining. The density of PAS staining was quantitated by densitometry of an area of the slide containing fibres. The results are shown in Fig. 2. The same pattern is seen as in the biochemical results, viz a reduction in density after exercise to 32% of control density and increase to 680% and 58 % of control after normoxic and hypoxic recovery periods. Again, there is no difference between rate of resynthesis with normoxia or hypoxia. There was close correlation between the biochemical and histological results (r = 0 85, degrees offreedom = 19 andp < 0-001). On microscopic examination of the sections it was seen that, whereas in the control biopsies (Biopsy 1) all fibres were well stained, and in post exercise biopsies (Biopsy 2) all fibres were depleted of glycogen, in the recovery biopsies (Biopsy 3) there was a contrast of staining between fibres. By comparing adjacent sections stained with ATPase it was found that the pattern of PAS staining followed closely the fibre typing revealed by ATPase staining, the

4 Glycogen gi100g wet wt. 1.6 Sub MPW Sub JSM Sub ESW st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Biopsy Number FIG. 1. Muscle glycogen concentration in biopsies for each subject. Normoxic recovery period, 0; hypoxic recovery period, *. OD unitsx SubMPW Sub JSM SubESW ~~~~~~~~ st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Biopsy Number Optical density of whole section of muscle biopsies. For 1st and 2nd biopsies test and control data combined, mean and range are indicated, for subjects MPW and JSM n = 6 and for ESW n = 4 for these points. For 3rd biopsies normoxic recovery period, 0; hypoxic recovery period, *. FIG. 2.

5 Hypoxia and muscle glycogen resynthesis 241 ATPase positive type II fibres being more densely stained. Further, it was our impression that this contrast was more marked after hypoxia than after normoxia in the recovery period. We have measured these differences by microdensitometry and the results are shown in Table I and Figure 3. In Table I the mean optical density (OD Unit x 100) readings for 50 fibres of each type in the biopsies for all three subjects are shown. It will be seen that both fibre types became markedly less dense after exercise and more dense on recovery. An -I OD unitsxloo 20 Sub MPW Sub JSM Sub ESW st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd Biopsy Number FIG. 3. Optical density difference of Type II minus Type I fibres (A OD II-I) for each muscle biopsy, from means of 50 fibres of each type counted. Symbols as in Fig. 2. The variation between comparable measurements, i.e. measurements on the same fibre type from the same subject at the same stage in the experiment, obtained from different experiments was pooled over subjects and biopsies to give estimates of the error due to experimental technique and within subject biological variation. The standard deviations for Type I and Type II fibres were 3-61 and 6-62 respectively, each based on 12 degrees of freedom. The means over experiments, and differences between them, could then be given appropriate standard errors.

6 242 Milledge et al. Testing the densities at the 2nd and 3rd biopsies against the first, we can draw the following conclusions: (a) The density is in all cases reduced at the 2nd biopsy (P < 0-001). (b) For Type I fibres the density has not returned to normal at the third biopsy' after hypoxia or normoxia, except in subject ESW after hypoxia, where the difference between the density at the 3rd and 1st biopsies was not quite significant at the 5% level, but in all other cases there remained a significant difference (P < 0O01). TABLE I 1st Biopsy 2nd Hypoxia 3rd Normoxia Muscle fibre type I II I II I II I II Subject MPW: Expt Mean s.e.m Subject JSM: Expt Mean s.e.m Subject ESW: Expt Mean s.e.m Table of results of optical density measurements on individual muscle fibres. Mean results of 50 fibres of each type measured in each biopsy. OD units x 100 Standard error of means (s.e.m.) based on individual fibre measurements. (c) For Type II fibres the density has not quite returned to the initial levels at biopsy 3, but the differences are not significant for JSM and ESW. The Type II fibre densities of MPW are still significantly lower than at the 1st biopsy, but the difference was less than for Type I fibres. The differential effect of fibre type is shown in Fig. 3, where the difference in mean OD between the fibre types (A OD Type II-I) is plotted for each biopsy. It will be seen that in all biopsies, Type II fibres have a greater density than Type I. After exercise with glycogen depletion the difference is less. On recovery both fibre types became more dense, Type II increasing more rapidly than Type I, so the difference again became apparent for both normoxic and hypoxic recovery. The A OD Type II-I was greater after hypoxic than after normoxic recovery in all three subjects although this difference did not reach a significant level.

7 Hypoxia and muscle glycogen resynthesis 243 DISCUSSION The experimental protocol was designed to follow closely that of Piehl [1974] so that results would be comparable with hers. The rate of glycogen resynthesis is dependent on the degree of depletion so that particular attention was paid to the duration and rate of exercise. Considerable motivation is necessary to achieve the degree of effort needed to deplete the glycogen stores of large muscle groups and it was decided that this would not be possible on a bicycle ergometer because of boredom, whereas outdoor running on a track, usually in company with another subject, was less tedious. Subjects did, in fact, maintain quite high exercise rates as judged from their heart rates, i.e. about 80-90% of their W170 or 68-87% of their maximum work rate. Depletion of glycogen to 26% of control values was achieved in these year old subjects, compared with 18 % in Piehl's 25 year old subjects. The recovery period of 5 h was also chosen, partly so as to be able to compare our results with Piehl's and also because this is about the limit of comfort for wearing a mask continuously. The rate of resynthesis is still high at this time and therefore differences in rate should be appreciable. However, since this is the case, it also follows that the scatter of results due to biological variation or slight differences in timing of sampling are likely to be large. The results of glycogen assay do not prove the hypothesis that hypoxia of this level significantly depresses glycogen resynthesis. In our first subject there was a marked depression, but another subject showed little effect and the third showed more rapid resynthesis on hypoxia than on normoxia. That these scattered results were due to real biological variation and not analytical errors was borne out by a reasonably close correlation of individual results between biochemical and microdensitometry measurements. After stopping exercise the post-exercise biopsy was taken and then 10% oxygen breathing started. It is possible that this time lag of about 20 min when glycogen resynthesis would have been at its peak rate might have been important in obscuring real differences, though Karlsson and Saltin [1971] suggest there is no significant increase in muscle glycogen until after 60 min of stopping exercise. The scatter in results at 5 h recovery also obscures differences. A greater scatter is to be expected during rapid synthesis and Piehl had a greater range of results at this point than at aily other of her 7 points. This scattering is also enhanced by the fact that the different fibre types have at this point very different glycogen contents. A sample containing more than average Type II fibres will have a higher total glycogen than a sample with a greater proportion of Type I fibres, whereas in the first biopsy before exercise, where the glycogen contents of fibres are very similar, differing proportions of fibre types would not affect results. Another source of error is the possibility of regional variation in glycogen content within the muscle mass due, for instance, to regional variation in blood flow. Again, this might only be important during rapid resynthesis or depletion of glycogen. Such variation would not be detected by duplicate samples from the same needle track.

8 244 Milledge et al. The evidence from densitometry of whole sections (Fig. 2) gives a similar overall picture of the effect of hypoxia on glycogen resynthesis-that is that there is very little effect under the conditions of this experiment. The measurement of optical density of individual muscle fibres in PAS stained sections allows us to make observations on the differential depletion and refilling of fibres of each type as defined by ATPase staining (at ph 9 5). This measure is most reliable when expressed as a difference of the OD Type II minus Type I fibres within one section (as in Fig. 3). The actual OD measurements are given in the table but caution is needed in making comparison of OD between sections, where errors due to variation in section thickness and staining may be present. However, care was taken to standardise techniques and between experiments variation was quite small. It will be seen that exercise depleted glycogen in both muscle fibre types (Table I). There was no consistent trend as to which type of fibre was most depleted. This is in accordance with the work of Gollnick et al. [1974] who showed that for exercise of this intensity, slow twitch fibres (Type I) were first depleted followed by fast twitch fibres (Type II) as work continued. On recovery it was found that Type II fibres were markedly more densely stained than Type I fibres. Type I fibres were significantly less dense in the recovery than control biopsy, whereas Type II fibre density had returned almost to control values. This was true for both hypoxic and normoxic recovery periods. This result is in accordance with Piehl [1974] who used a subjective rating, assessing each fibre to be dark, moderate, light or negatively stained and found a marked contrast in staining at 5 h after stopping exercise. This may be due to greater activity of glycogen synthetase activity in Type II fibres as suggested by Piehl [1974]. The effect of hypoxia on the rate ofresynthesis appears to be that of enhancing the difference between fibre types. In all three subjects A OD Type II- I was greater after hypoxic than normoxic recovery, although this difference did not reach the level of statistical significance. Thus our results suggest that hypoxia depresses glycogen synthesis in Type I fibres but not in Type II fibres. Gollnick et al. [1974] suggested that in the hypoxic situation of stronger muscular contraction, reliance was placed on Type II fibres. There remains the question of the association of hypoxia and muscle fatigue. Exercising under hypoxic conditions is very fatiguing, mainly due to the sensation of dyspnoea, and it is difficult to decide if there is an added increase in muscle fatigue as well. In this study, where hypoxia was limited to the recovery phase and dyspnoea was not noticable, it was the subjects' strong impression that they were more fatigued following the hypoxic than the control experiments. If this is a reliable impression, and of course these experiments could not be carried out blind, it would lend support to the suggestion of Edwards [1975] that depletion of energy stores in one fibre type, in this case Type I fibres, might be responsible for the sensation of muscle fatigue.

9 Hypoxia and muscle glycogen resynthesis 245 ACKNOWLEDGMENTS We wish to acknowledge the advice of Dr. R. H. T. Edwards of the Royal Postgraduate Medical School, Hammersmith, in planning the study and preparing the paper; of Miss S. Chinn of the Division of Computing and Statistics in the analysis of the results, and the help of Mr. R. Bowlby of Medical Illustration Department and Mr. G. Slavin of the Pathology Department of Northwick Park Hospital in developing the method of microdensitometry used in the study. We also wish to thank Miss V. Hainsworth of the Dietetics Department for help in planning and supplying the diet used in the study. REFERENCES BERGSTROM, J. (1962). Muscle electrolytes in man. Scandinavian Journal of Clinical and Laboratory Investigation. Suppl. 68. BERGSTROM, J., HERMANSEN, L., HULTMAN, E. and SALTIN, B. (1967). Diet, muscle glycogen and physical performance. ActaPhysiologica Scandinavica, 71, CIPRIANO, L. F. and PACE, N. (1973). Glycolytic intermediates and adenosine phosphates in rat liver at high altitude (3,800 m). American Journal ofphysiology, 225, EDWARDS, R. H. T. (1975). Muscle fatigue. Postgraduate Medical Journal, 51, GOLLNICK, P. D., KARLSSON, J., PIEHL, K. and SALTIN, B. (1974). Selective glycogen depletion in skeletal muscle fibres of man following sustained contractions. Journal ofphysiology, 241, GOLLNICK, P. D., PIEHL, K. and SALTIN, B. (1974). Selective glycogen depletion patterns in human muscle fibres after exercise of varying intensity and at varying peddling rates. Journal ofphysiology, 241, HAYASHI, M. and FREIMAN, D. G. (1966). An improved method of fixation for formalinsensitive enzymes with special reference to myosin adenosine triphosphatase. Journal of Histochemistry and Cytochemistry, 14, HULTMAN, E. (1966). Muscle glycogen in man determined in needle biopsy specimens. Scandinavian Journal of Clinical andlaboratory Investigation, 19, KARLSSON, J. and SALTIN, B. (1971). Oxygen deficit and muscle metabolites in intermittent exercise. Scandinavian Journal of Clinical andlaboratory Investigation, 26, PEARSE, A. G. E. (1968). Histochemistry, theoretical and applied. 3rd Ed. Vol. I, Churchill, London. PIEHL, K. (1974). Time course for refilling of glycogen stores in human muscle fibres following exercise-induced glycogen depletion. Acta Physiologica Scandinavica, 90,