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Respiratory capacity of developing chick red and white skeletal muscle
Camp. Biochem. Physiol. Vol. 75A. No. 3, pp. 491.-495,1983 Printed in Great Britain
0
0300-96q9!83 $3.00 + 0.00 - , 1983 Pergamon Press Ltd
RESPIRATORY CAPACITY OF DEVELOPING CHICK RED AND WHITE SKELETAL MUSCLE WILLIAM
Exercise
S. BARNES
and
SCOTTM.
HASSON
Physiology Laboratory, School of Health, Physical Education and Recreation, University of Northern Colorado, Greeley. CO X063 I, USA (Rrceiced
13 October
1982)
Abstract-i.
Oxygen consumption, cytochrome oxidase and succinoxidase activity was measured in samples of leg and breast muscle from chick embryos ranging in age from 11to 19 days. 2. Respiratory parameters increased significantly in both muscle groups during embryonic life, 3. By the later stages of incubation, leg and breast muscles differed significantly in cytochrome and succinoxidase activity. 4. Oxygen uptake between leg and breast muscles did not differ si&ni~~antly during later development. 5. The results suggest at least a partial pre-natal differentiation of skeletal muscle in the domestic fowl.
INTRODUCTION
Adult skeletal muscle is a heterogeneous tissue, composed of different fiber types, of which at least two are recognized as white (type II, anaerobic) and red (type I, aerobic) (Dubowitz, 1965). The factors contributing to the mechanisms by which these fibers acquire their adult characteristics are not yet well understood. It has been hypothesized that differentiation into distinct fiber types takes place from a common pool of identical fibers (Dubowitz. 1968). Germino et (I/. (1965) and Cosmos et al. (1965) have suggested that initially, all fibers are red and that di~erentiation into more specialized white fibers takes place as the muscle matures. The extent to which differentiation occurs pre- or post-natally seems to depend upon the animal being investigated. For example, cat (Nystrom, 1966, 1968) pig (Cooper et ~1.. 1970) mouse and rat (Dubowitz, 1965) muscle appears to be undifferentiated at birth, while monkey (Beatty et al., I967), guinea pig (Dubowitz, 1965), sheep (Ashmore et al.. 1972) and human (Duhowitz, 1965) is fully differentiated. In other species (e.g. rabbit, hamster, and goat), the degree of differenti~~tion at birth is incomplete (Beckett & Bourne, 1958; Dubowitz. 1965). Investigations of embryonic and new-born chick muscle (Ashmore & Doerr, 1971) suggest the possibility that all red fibers are formed pre-natally in a relatively short period of time and that fibers formed during the later stages of myogenesis are always white. Support for this notion is provided by Bass et ul. (1970) who have shown that glycolytic metabolism in embryonic chick skeletal muscle is apparently very limited, with aerobic metabolism predominating. Post-natal aerobic enzyme activity changes very little or decreases during Iater development. while postnatal increases in glycolytic enzyme activity are pronounced. This perhaps indicates a major change in metabolism taking place shortly before hatching and during the first few weeks of age. The pre- and post-natal ontogeny of several metabolic enzyme systems have been investigated for skeletal muscle of the common domestic fowl. These include: creatine phosphokinase (Reporter et al., 1963). 491
adenylate kinase (Kendrick-Jones & Perry, 1967), phosphorylase (Cosmos, 1966) hexokinase, lactate dehydrogenase, citrate synthase (Bass ~1 LII.. 1970; Dawson & Kaplan, 1965) r-glycerolphosphate dehydrogenase (Dawson & Kaplan, 1965) and succinate dehydrogenase (Germino rt al., 1965). Two oxidative enzyme complexes, cytochrome oxidase and succinoxidase, are frequently measured to indicate the functional capacity of the respiratory chain in skeletal muscle (Holloszy, 1967). No reports could be located which investigated the development of these enzymes during myogenesis. Reports have been published on the respiratory capacity of the entire chick embryo during development (Albaum & Worley, 1942; Albaum et rri., 1946). however, only limited information seems to be available on individual tissues during embryogenesis. In the present investigation. an ilt vitro study has been made of the oxygen consumptioll, cytochrome oxidase and succinoxidase activities of red and white skeletal muscle fiber groups from embryonic chickens. MATERIALS
AND
METHODS
Fertilized eggs from the Grey Leghorn strain of fowl (Guiius dornesricus) were incubated at 37 C. 70”, humidity for lengths of time varying from I I to 19 days, The eggs were turned dally and their development followed by the candling technique. There was some variability among individual embryos in a given batch of eggs incubated for the same length of time and under identical conditions. Therefore, upon removal, embryos were staged according to the criteria of Hamburger & Hamilton (1951). Embryos which differed too greatly from the expected age-related development were disgarded. At 48 hr intervals, beginning on the I I th day of incubation and extending through the 19th day, embryos were sacrificed by decapitation and samples of leg and breast muscle removed for study. The breast samples included the combined pectoralis major and minor and the leg samples included all the muscles of the thigh. A superficial examination of the skeletal muscle of the fowl demonstrates that two different types of whole muscle are present. red (e.g. leg muscles) and white (e.g. breast muscles). Histochemical reactions with v,arious muscles from adult chickens has shown breast muscle to be primar-
WILLIAM S. RARMS and SCOTT M. HASSON
492
ily composed of white (type II) fibers (Cosmos ct it!., 1965; Cosmos. 1966). The combined leg muscles consist more of a mixture of red (type I), white and intermediate (type II) fibers (Cosmos t’t ~1.. 196.5). Although the properties and distributation of tiber types in the leg muscles of the fowl have not been fully characterized. the red (type I) fiber type seems to predominate (Aulie & Grav. 1979). The reciprocity of glycolytic and mitochondrial enzymes is well demonstrated in these tissues. Attempts to dissect individual embryonic muscles, with the exception of the pectoralis, proved difficult and unreliable. After decapitation of the embryo. portions of the skeletal system with the muscles attached were rapidly dissected out. The muscle tissue was separated from the bone, stripped of fat and connective tissue and chilled in ice-cold Hank’s BSS. The samples were blotted. weighed, chopped into a fine mince with scissors and homogenized in a glass Potter-Elvehjem homo_ecnizer. The tubes were immersed in ice water during this operation. Homogenization was completed with 20 complete passes of the tuhc. Investigation of the metabolic development of the muscle iu oco required 4 -6 embryos be pooled for each analysis. Homogenates for assays were prepared m 0.001 M potassium phosphate buffer, pH 7.4. Each tube contained 1g of muscle per IO ml. Succinate oxidase and cytochrome oxidase activities were measured m~lnometric~llIy at 3ll‘C as described by Potter (1964). Oxygen uptake was measured in triplicate at two different concentrations. In estimating cytochrome oxidase activity. the oxygen uptakes were corrected for ascorbate autoxidation according to the method of Oscai & Mole (1975). Tissue respiration was measured in a Gilson differential respirometer, at 37’C. with air as the gas phase. Three to six cmhryos were obtained. Slips of breast and/or leg muscle were dissected free and quickly immersed in a vigorously oxygenated (95”,, 02-5’:,, CO,), physiologically balanced saline solution (BSS) containing (in mM): NaCI, 135.0: NaHCO,. 15.0; Na,HPO&. 1.0; KCI. 5.0: CaCI,, 2.0: M&I,, 1.0; glucose. I 1.0(Hofmann, lY76). The tissue was allowed to equilibrate for 1 hr at 20-73 C and then minced with scissors until fragments approx 1mm3 in size were obtained. These fragments were washed twice in fresh BSS and resuspended in 15ml Warburg flasks. The center well of each Bask contained approx 0.2 ml of 15”: KOH and a pleated filter paper wick. Each Hask contained approx 200 mg of tissue and 3.0 ml of BSS. The final pH of the mixture was adjusted to 7.4. Oxygen uptake was measured in triplicate and expressed as microliters of 0, consumed per mmute per gram (dry). STP. Dry weights of the tissues in each flask were determined at the end of the incubation period by drying the tissue to a constant weight at 100-l IO’C. Cytochrome c (type III) was obtained from Sigma
of development investigated, cytochrome oxidase activity was significantly higher in the red muscle as compared to the white muscle.
Figure 2 shows the activity of succinoxidase in red and white muscle, plotted against the age of the embryo. In the red muscle. enzyme activity decreased (17‘>;,)between days 11 and 15 and then increased sharply (532) between days I5 and 19. In contrast, enzyme activity in the white muscle increased slightly (4”:;) between days 1I and 15 :md continued to increase, more rapidly (17’?,,),between days 15 and 19. The level of succinoxidase activity was significantly higher in red tnuscle after I I days of incubation. By the 13th day, this difference no longer existed and the enzyme activity levels in the two muscle groups were comparable. Between days I5 and 19, enzyme activity in the red muscle rose more sharply than in the white and once again significant differences in total enzyme activity existed. To demonstrate the relationship of cytochrome oxidase and succinoxidase activities during development of red and white muscle, the ratio of oxygen uptakes through the two systems wxs calculated. Figure 3 shows the ratio plotted against the age of the embryo. As can be seen, the rate of development of cyto-
l
: II
DAYSlbF INClYBATIOli’
Fig. I. The development of cytochrome leg (red) and breast (white) muscles of chick embryos. Values are means ri: SE cance (P < 0.01) between leg and breast
oxidase systems in I I to I day old [* denotes signifisamples] (n = 6).
Chemical Co.
RESLiLTS l
Figure I shows the results from experiments with homogenates of white (breast) and red (leg) muscle from chick embryos at various stages of development. The total enzyme activity is expressed as microliters of oxygen consumed per gram of muscle (wet wt) per minute of incubation. The developmental pattern of cytochrome oxidase activity appeared to be quite similar for both red and white muscle tissue. Between I I and I5 days of incubation, enzyme activity in both muscle types remained fairly constant. Between days 15 and 19, there was a marked increase in activity for both red (920/,) and white (83‘?0,)muscfe. At each stage
ill II
DAYS OF INCUBATION
Fig. 2. The development of succinoxidase systems in leg (red) and breast (white) muscles of I l&t9 day old chick embryos. Values are means 2 SE [* denotes significance ‘^ ^^., . (Y < U.tfIJ between le& and breast samplesj (n = 6)
Respiratory capacity of developing chick red and white skeletal muscle
Fig. 3. The relationship in the development of cytochrome oxidase to succinoxidase activity in leg (red) and breast (white) muscles of chick embryos between I I and 19 days of incubation.
DAYS OF INCUBATION Fig. 4. Oxygen consumption by minced muscle nrenarations of leg (red) and-breast (white) muscles of il.-i9 day old chick embryos. Values are means rt SE f* denotes si&ificance (P < 0.01) between leg and breast- samples] fn = 6).
chrome oxidase activity and succinoxidase activity is similar between red and white muscle. The ratio of these activities remains fairly constant from day 11 to day 1.5, at which time, cytochrome oxidase activity seems to increase dispro~rtionately in both muscle types. Up to day 17, the rate of cytochrome oxidase activity, relative to succinoxidase activity, is consistently higher in red muscle. At day 19, the activity ratios of the two enzymes is essentially the same.
Figure 4 shows the oxygen consllmption by minced preparations of red and white muscle at various stages of development. Oxygen uptake, expressed as microliters of oxygen consumed per gram of tissue (dry wt) per minute, progressively increased in both red and white muscle between days I 1 and 19. The increases were 166 and 77% for red and white muscle, respectively. Only on the 1I th day of incubation were the oxygen consumptions of the two muscle types significantly different. At this stage of development, the white muscle had larger capacity to use oxygen than the red. As development progressed, both muscle types seemed to acquire similar respiratory capacities.
493
DISCUSSION The results of this investigation suggest that, as development progresses, there is a positive relationship between the increasing oxygen uptakes observed in both muscle types and corresponding increases in oxidative enzyme activity. However, individual differences in enzyme activity between the two types of muscle do not appear to manifest themselves in terms of distinctive metabolic rates. One possible explanation for this paradox is that other components of the oxidative system have simply not kept up with the development of the cytochrome and succinate oxidases and consequently, impose limitations on oxygen consumption. Along these lines, Bass et uI. (1970) have found that red and white muscles from newly hatched chicks do not differ significantly in their concentrations of another “oxidative” enzyme; citrate synthase. Another possibility is that the oxygen consumption m~dsurements were insensitive to these differences. A number of factors are known to influence the resting oxygen consumption of muscle preparations (Stainsby & Lambert, 1979). It appears that oxygen uptake values obtained from a variety of muscle preparations may reflect not only the inherent metabolic capacity of the individual muscles investigated, but aiso a variety of methodological differences. Biochemical deveiopm~nt in muscle involves the development of enzyme systems adequate to convert metabolic fuels into ATP at a rate sufficient to allow continued contractile activity. What constitutes adequacy depends upon the specific contractile characteristics of the muscles involved. It might be expected that red muscle. which is thought to be better adapted to long-duration activity, would differ considerably in this respect from white muscle. which is thought to be involved primarily with explosive, short-duration bursts of activity. In general, adult red muscle contains approximately five times the cytochrome oxidase (Lemberg & Barrett, 1973) and twice the succinoxidase (Lawrie, 1952) of white muscle. Furthermore, in some species, the metabolic rates of red muscle are as much as six times those of white muscle (Gordon, 1968). With regard to the present in~~estigation, differentiation of the activity patterns of these enzyme systems proceeds during pre-natal development and results in muscle groups with distinctive metabolic profiles. The absolute values for enzyme activity reported here for embryonic muscle tissue are well below those typically reported for more adult muscle (Aulie & Grav. 1979). Clearly, the muscle is still quite immature. Nevertheless. by the later stages of embryogenesis, there appears to be a clear-cut subdivision of muscle into two fiber types, which correspond in cytochrome oxidase and succinoxidase activity to the red and white fibers of mature muscle. Differences in contractile properties have been detected in the muscles of newly hatched chickens (Syrovy et al., 1971). This functional differentiation must be the result of fetal growth and development. The present findings suggest the possibility that metabolic differentiatton occurring prenatally may contribute to distinct postnatal function. The factors controlling growth and metabolic differentiation in muscle are still unknown. Although the
WILLIAM S. BARNES and SCOTT M. HASSON
494 motor
pattern
of :I given m~~scle has been assumed to b~ocllernic~~l and functional properties (Salmons & Sreter, 1976; Gordon & Vrbova, 1975): myogenetic as well as hormonal factors could be involved (B~~idw~n rr tri.. 1978: Nougues & Bacou, 1977). Kendrick-Jones & Perry (1967) have demonstrated that a high correlation exists between the rise in activity of certain enzymes and the onset of activity of skeletal muscle. This suggests that the use of the muscle might be the stimulus for the rise in specific activities of these enzymes. tt seems unlikely that the progressive increases in the activity of the oxidizing enzymes investigated in this study or the differences in znzyme activity observed between red and white muscle could be explained on the basis of increased motor activity. In fact, the environment, iu OPO, appears to become more restrictive and confining as development progresses. Moreover, animals which are quite inlmature at birth and incapable of much movement. seem to be the animals most influenced by postnatal fullction~il consider~lt~ons. Enzyme activities in these animals do not approach adult values until some time pr>sr prrrruf~t. Animals born at a more advanced stage of development and capable of independent existence. like the chick, are already rapidly approaching adult values at birth. While the activities of cytochrome oxidase and succinoxidase reported here do not reach adult values. they are clearly increasing rapidly and the rates of increase are distinctly different for red and white muscle. It may well be. that trophic nerve effects contribute to prenatal differentiation of red and white muscle in the chicken, regard&s of whether the conduction of motor nerve impulses or mechanical work of the muscle arc involved. Muscles from I I day old chick embryos. investigated in this study, were probably already innervated since ~nllcrv~~ti~~lof at least some fibers has been reported to occur as early as 6-7 days ir7 ot’o (Drachman. 1967) and endplates can be Iocalized in muscles of about ii) day old chick embryos (Gutmztnn c’i &, 1969). There appear to be specific neural factors that regulate gene expression in the muscle fiber that may be demonstratable even in the absence of dcmonstratable neuromuscular connections (Guth. 1971). For example, elimination of conducted nerve impulses by various experirne~lt~~~ procedures does not reproduce fully the well known cKects of dener~~~~tion, provided that the motor nerve remain ~ln~~tornic~~l~yand flinctionaily connected to the muscle (Drachman. 1967). Furthermore, implanting a nerve from one type of muscle into another. while leaving the original innervation intact, does not result in formation of functionally effective neuromuscular junctions (Guth. I97 I ). Nevertheless, the implanted nerve does seem to induce the conversion of a significant number of fibers from one type to another. Findings such as these, could suggest the release of a l~yp{~thetic~~itrophic substance, capable of modifying the yualitativc. phenotypic characteristics, of the muscle cell. The metabolic and biochemical differences observed between developing red and white muscle might also reflect intrinsic, myogenetic differences between the two muscle types. Supporting this notion, are the findings of Shafiq rsf ~ri. (1972), which show a differer~ti~ll response of f&t (white) and slow (red) regulate
its
muscle fibers to denervation at birth. Potentially fast fibers fail to mature when deprived of neural input, while slow fibers develop more or less normally. In any case. there are undoubtably many factors which contribute to the biochemical and biophysical specificity of the muscle fiber.
SWViMARY
1. Samples of leg (red) and breast (white) skeletal muscle were obtained from chick embryos ranging in age from I 1 days to 19 days. The cytochrome oxidase and succinoxidase activities, as well as the oxygen consumption of the samples were measured manometrically. 3. Oxygen consumption in the two muscle groups increased as development progressed but were not signi~~ntiy different from one another from the 13th to the 19th days of illcubation. 4. Both the cytochrome oxidase and succinoxidase activities of the two muscle groups increased over the observed incubation period. with the greatest increases occurring in the leg (red) muscle. 5. Leg (red) and breast (white) muscles were signifciantly di&rent in their cytochrome oxidase activity at every stage of development investigated. Succinoxidase activities in the two muscle groups differed significantly on days 1 I, 17 and 19. 6. The ratio of cytochrome oxidase activity to succinoxidasc activity increased, in both muscle groups, as development progressed. This ratio was consistently higher in the leg (red) muscle until the 19th day of incubation, when the ratios were identical for both muscle groups. 7. It is concluded that marked differences between teg (red) and breast (white) muscles of the chicken can be observed in the oxidative system during the later stages of ~~~bryologic~~l development.
REFERENCES ALBAUM H. G., NOVIKOFF A. B. & OCCUR M. (1946) The development of the cytochrome oxidase and succinoxidase systems in the chick embryo. .I. hi<)/. Cltrt~ 165.
125% f 30. AmhUM
H. G. &
cytochrome
L. G. (1942) The development of in the chick embryo. J. hi& Clref~z.
WORLEV
oxidase
t43, 697-M 700. As~MoR~ f. R. & DOERR L. (1971) Postnatal development of fiber types in normal and dystrophic skeletal muscle of the chick. Espl Ne~rol. 30, 43 l-466. ASHMORF C. R.. ROBINSON D. W.. RATTRAY P. Lyr DOIXR L. (1972) Biphasic development of muscle fibers in the fetal iamb. E.vpl. Nrtrrof. 37. 241--X5. AUL~E A. & GRAv H. J. 11979)Effect ofcold acclimation on the owidative capacity of skeletal muscles and liver in young bantam chicks. J. ~i~~~~~~~i~. ~~i~~j~~~. 62A. 33.5-338. Bhz_DwrN K. M., HOOKER A. M., CAMPBELL P. .I. & Lwrs R. E. t 1978)Enzyme changes in neonatal skeletai muscie: effect of thyroid deficiency. AN. J. Ph~sioi. 235. C97mCI 07, Bhss A., hJSCH G. & PE’rrE D. (1970) Postnatal differentiation of the enzyme activity pattern of enerav-supplvine
Respiratory capacity of developing chick red and white skeletal muscle Differentiation of red and white fibers in muscle from and infant rhesus monkeys. J. Nistochom. Cyt0chertr. 15, 93- 103. BECKETTE. B. & BOURNEG. H. (1958) Some histochemic~l observations on enzyme reactions in goat fetal cardiac and skeletal muscle and some human muscle. ,4cta Anctr. 35. 224-253. COOPERC. C., CASSENSR. G., KASTENSCHMIDT L. L. & BRISKEYE. J. (1970) Histochemical characterization of muscle differentiation. Deal Biol. 23. 169-184. C’OSMOSE. (1966) Enzymatic activity of differentiating muscle fibers. I. Development of phosphoryl~se in muscles of the domestic fowl. Deal Biol. 13, 163-I81. COSMOS E., BUTLERJ. & SCOTTR. (1965) Phosphoryiase activity in differentiating muscle fibers of the domestic fowl. J. HistoLRert~.C~rochenz. 13, 719-720. DAWSON D. M. & KAPLAKN. 0. (1965) Factors influencing the ~oncellt~dtiol~ of enzymes in various muscles. J. hiol. Chrtn. 240, 32 15-322I. DRACHMAN D. B. (1967) Is acetylcholine the trophic neuromuscular transmitter’? Archs Neuroi. 17, 206-2 17. DUBOWITZ V. (1965) Enzyme histochemistry of skeletal muscle. Parts I and II. J. Neural. Neurosurg. Psychiut. 28, 5 16524. DUBOW~TZV. (1968) Developirzy and Diseused Muscle. .4 ~js~f~~~el)jj~~~~ Srtr&. Spastics International Medical Publications and William Heineman Medical Books Ltd. GERMIN N. I., D’ALBORAH. & WAHRMANNJ. P. (1965) Succinic dehydrogenase in the development of the skeletal muscles of chicks. Acta Amt. 62, 434-444. CIORLW)?I M. S. (1968) Oxygen consumption of red and white muscles from tuna fishes. Science, Iifusli. 159. x7-90. GORDON T. & VRBOVAE. (1975) The influence of innervation on the differentiation of contractile speeds of developing chick muscles. Pflue~ers Arch. 360, 199-218. GUTII L. (1971) A review of the evidence for the neural regulation of gene expression in muscle. In Cff~~fruefi~i~~ ($ ;Wuscie C&s mtd R&ted Procrsses (Edited by POUOLSKYR. J.), pp. 189-201. Prentice-Hall, Englewood Cliffs. New Jersey. GUTMAN E.. HANZLIKOVA V. & HOLECKOVA E. (1969) De/ fetal, neonatal
velopment of fast and slow muscles of the chicken irt ciao and their latent period in tissue culture. E?cpl Cell Res.
56.33-38. HAMBURGER V. & HAMILTON H. L. (1951)
A series of normal stages in the development of the chick embryo. J. ,&f0rph.88, 49-Y:.
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HOFMANNW. W. (1976) Oxygen consumption by hum:m and rodent striated muscle irk citro. AM. J. Physiof. 230, 34-40. H~LLOZY J. 0. (1967) Biochemi~l adaptations in muscle. f. hioi. Chrtn. 242, 2278-2282. KENURICK-JONES J. & PERRYS. V. (1967) The enzymes of adenine nucleotide metabolism in developing skeletal muscle. Biochrm. J. 103, 207-214. LAWRIER. A. (1952) Biochemical differences between red and white muscles. Nuture, Land. 170, 122--l23. LEMBERC~ R. & BARRETTJ. (1973) ~~r~J~~~r~~fl~~.~. pp. 395-397. Academic Press. London. N~IJGUES J. & BACQLIF. (1977) Enzymatic activities of muscle fibers differentiated, in citrci. from pectoralis major (white) and adductor magnus (red) muscles of chick embryos. Esprrinwrriu 33, 7 14-7 16. NYSTROMB. (1966) Succinic dehydrogenase in developing cat muscles. Nutftrr. Lund. 212. 954-955. NVSTROM B. (1968) Histochemistry of developing cat muscles. Actu Neural. Scurtd. 44, 4055439. OS~AI L. B. & MOLF.P. A. (1975) Laboratory techniques involving small animals in exercise physiology research. In Luercise und Sports Scirwes Recie\vs (Edited by Wn.MOREJ. H. & KE~GH J. F.), Vol. 3, pp. 351-370. Ac;tdemic Press, New York. POMER V. R. ( 1964) Respiratory enzymes. In ,~~~~~~u~~if~~rj~ T~&niqrte,s (Edited hv UMBREITW. W.. BURRISR. Ii. & STAUFF~:R J. F.). 4th edn., pp. 162-l 65. Burgess, Minneapolis. REPORTERM. C., KONIGSBERGI. R. & STREHLERB. L. (1963) Kinetics of accumulation of creatine phosphokinase activity in developing embryonic skeletal muscle iri rino and in monolayer culture. Espi Ceil Res. 30, 410-417. SALMONS S. & SRETERF. A. (1976) Significance of impulse activity in the transformation of skeletal muscle type. Nuwr, torrd. 263, 30.-34. SHAFIQS. A.. ASIEDUS. A. & MILH~RATA. T. (1972) Effect of neonatal neurectomy on differenti~~tion of fiber types in rat skeletal muscle. Espi .~ruroi. 35, 529-540. STAINSBY W. N. & LAMBERT C. R. (1979) Determinants of oxygen uptake in skeletal muscle. In Eserciw and Sporrs Scierwes Rrviews (Edited bv HUTTON R. S. & MILLER D. I.). Vol. 7. pp. 1’5-15 I. The Franklin Institute Press, USA. SYROVYI., GUTMANN E. & MELKHNA J. (197t ) Differenti~~l response of myosin-ATPase activity and contr~l~tion properties of fast and slow rabbit muscles following denervation. Esperirnelltitr 27. 1426-I 427.
0
0300-96q9!83 $3.00 + 0.00 - , 1983 Pergamon Press Ltd
RESPIRATORY CAPACITY OF DEVELOPING CHICK RED AND WHITE SKELETAL MUSCLE WILLIAM
Exercise
S. BARNES
and
SCOTTM.
HASSON
Physiology Laboratory, School of Health, Physical Education and Recreation, University of Northern Colorado, Greeley. CO X063 I, USA (Rrceiced
13 October
1982)
Abstract-i.
Oxygen consumption, cytochrome oxidase and succinoxidase activity was measured in samples of leg and breast muscle from chick embryos ranging in age from 11to 19 days. 2. Respiratory parameters increased significantly in both muscle groups during embryonic life, 3. By the later stages of incubation, leg and breast muscles differed significantly in cytochrome and succinoxidase activity. 4. Oxygen uptake between leg and breast muscles did not differ si&ni~~antly during later development. 5. The results suggest at least a partial pre-natal differentiation of skeletal muscle in the domestic fowl.
INTRODUCTION
Adult skeletal muscle is a heterogeneous tissue, composed of different fiber types, of which at least two are recognized as white (type II, anaerobic) and red (type I, aerobic) (Dubowitz, 1965). The factors contributing to the mechanisms by which these fibers acquire their adult characteristics are not yet well understood. It has been hypothesized that differentiation into distinct fiber types takes place from a common pool of identical fibers (Dubowitz. 1968). Germino et (I/. (1965) and Cosmos et al. (1965) have suggested that initially, all fibers are red and that di~erentiation into more specialized white fibers takes place as the muscle matures. The extent to which differentiation occurs pre- or post-natally seems to depend upon the animal being investigated. For example, cat (Nystrom, 1966, 1968) pig (Cooper et ~1.. 1970) mouse and rat (Dubowitz, 1965) muscle appears to be undifferentiated at birth, while monkey (Beatty et al., I967), guinea pig (Dubowitz, 1965), sheep (Ashmore et al.. 1972) and human (Duhowitz, 1965) is fully differentiated. In other species (e.g. rabbit, hamster, and goat), the degree of differenti~~tion at birth is incomplete (Beckett & Bourne, 1958; Dubowitz. 1965). Investigations of embryonic and new-born chick muscle (Ashmore & Doerr, 1971) suggest the possibility that all red fibers are formed pre-natally in a relatively short period of time and that fibers formed during the later stages of myogenesis are always white. Support for this notion is provided by Bass et ul. (1970) who have shown that glycolytic metabolism in embryonic chick skeletal muscle is apparently very limited, with aerobic metabolism predominating. Post-natal aerobic enzyme activity changes very little or decreases during Iater development. while postnatal increases in glycolytic enzyme activity are pronounced. This perhaps indicates a major change in metabolism taking place shortly before hatching and during the first few weeks of age. The pre- and post-natal ontogeny of several metabolic enzyme systems have been investigated for skeletal muscle of the common domestic fowl. These include: creatine phosphokinase (Reporter et al., 1963). 491
adenylate kinase (Kendrick-Jones & Perry, 1967), phosphorylase (Cosmos, 1966) hexokinase, lactate dehydrogenase, citrate synthase (Bass ~1 LII.. 1970; Dawson & Kaplan, 1965) r-glycerolphosphate dehydrogenase (Dawson & Kaplan, 1965) and succinate dehydrogenase (Germino rt al., 1965). Two oxidative enzyme complexes, cytochrome oxidase and succinoxidase, are frequently measured to indicate the functional capacity of the respiratory chain in skeletal muscle (Holloszy, 1967). No reports could be located which investigated the development of these enzymes during myogenesis. Reports have been published on the respiratory capacity of the entire chick embryo during development (Albaum & Worley, 1942; Albaum et rri., 1946). however, only limited information seems to be available on individual tissues during embryogenesis. In the present investigation. an ilt vitro study has been made of the oxygen consumptioll, cytochrome oxidase and succinoxidase activities of red and white skeletal muscle fiber groups from embryonic chickens. MATERIALS
AND
METHODS
Fertilized eggs from the Grey Leghorn strain of fowl (Guiius dornesricus) were incubated at 37 C. 70”, humidity for lengths of time varying from I I to 19 days, The eggs were turned dally and their development followed by the candling technique. There was some variability among individual embryos in a given batch of eggs incubated for the same length of time and under identical conditions. Therefore, upon removal, embryos were staged according to the criteria of Hamburger & Hamilton (1951). Embryos which differed too greatly from the expected age-related development were disgarded. At 48 hr intervals, beginning on the I I th day of incubation and extending through the 19th day, embryos were sacrificed by decapitation and samples of leg and breast muscle removed for study. The breast samples included the combined pectoralis major and minor and the leg samples included all the muscles of the thigh. A superficial examination of the skeletal muscle of the fowl demonstrates that two different types of whole muscle are present. red (e.g. leg muscles) and white (e.g. breast muscles). Histochemical reactions with v,arious muscles from adult chickens has shown breast muscle to be primar-
WILLIAM S. RARMS and SCOTT M. HASSON
492
ily composed of white (type II) fibers (Cosmos ct it!., 1965; Cosmos. 1966). The combined leg muscles consist more of a mixture of red (type I), white and intermediate (type II) fibers (Cosmos t’t ~1.. 196.5). Although the properties and distributation of tiber types in the leg muscles of the fowl have not been fully characterized. the red (type I) fiber type seems to predominate (Aulie & Grav. 1979). The reciprocity of glycolytic and mitochondrial enzymes is well demonstrated in these tissues. Attempts to dissect individual embryonic muscles, with the exception of the pectoralis, proved difficult and unreliable. After decapitation of the embryo. portions of the skeletal system with the muscles attached were rapidly dissected out. The muscle tissue was separated from the bone, stripped of fat and connective tissue and chilled in ice-cold Hank’s BSS. The samples were blotted. weighed, chopped into a fine mince with scissors and homogenized in a glass Potter-Elvehjem homo_ecnizer. The tubes were immersed in ice water during this operation. Homogenization was completed with 20 complete passes of the tuhc. Investigation of the metabolic development of the muscle iu oco required 4 -6 embryos be pooled for each analysis. Homogenates for assays were prepared m 0.001 M potassium phosphate buffer, pH 7.4. Each tube contained 1g of muscle per IO ml. Succinate oxidase and cytochrome oxidase activities were measured m~lnometric~llIy at 3ll‘C as described by Potter (1964). Oxygen uptake was measured in triplicate at two different concentrations. In estimating cytochrome oxidase activity. the oxygen uptakes were corrected for ascorbate autoxidation according to the method of Oscai & Mole (1975). Tissue respiration was measured in a Gilson differential respirometer, at 37’C. with air as the gas phase. Three to six cmhryos were obtained. Slips of breast and/or leg muscle were dissected free and quickly immersed in a vigorously oxygenated (95”,, 02-5’:,, CO,), physiologically balanced saline solution (BSS) containing (in mM): NaCI, 135.0: NaHCO,. 15.0; Na,HPO&. 1.0; KCI. 5.0: CaCI,, 2.0: M&I,, 1.0; glucose. I 1.0(Hofmann, lY76). The tissue was allowed to equilibrate for 1 hr at 20-73 C and then minced with scissors until fragments approx 1mm3 in size were obtained. These fragments were washed twice in fresh BSS and resuspended in 15ml Warburg flasks. The center well of each Bask contained approx 0.2 ml of 15”: KOH and a pleated filter paper wick. Each Hask contained approx 200 mg of tissue and 3.0 ml of BSS. The final pH of the mixture was adjusted to 7.4. Oxygen uptake was measured in triplicate and expressed as microliters of 0, consumed per mmute per gram (dry). STP. Dry weights of the tissues in each flask were determined at the end of the incubation period by drying the tissue to a constant weight at 100-l IO’C. Cytochrome c (type III) was obtained from Sigma
of development investigated, cytochrome oxidase activity was significantly higher in the red muscle as compared to the white muscle.
Figure 2 shows the activity of succinoxidase in red and white muscle, plotted against the age of the embryo. In the red muscle. enzyme activity decreased (17‘>;,)between days 11 and 15 and then increased sharply (532) between days I5 and 19. In contrast, enzyme activity in the white muscle increased slightly (4”:;) between days 1I and 15 :md continued to increase, more rapidly (17’?,,),between days 15 and 19. The level of succinoxidase activity was significantly higher in red tnuscle after I I days of incubation. By the 13th day, this difference no longer existed and the enzyme activity levels in the two muscle groups were comparable. Between days I5 and 19, enzyme activity in the red muscle rose more sharply than in the white and once again significant differences in total enzyme activity existed. To demonstrate the relationship of cytochrome oxidase and succinoxidase activities during development of red and white muscle, the ratio of oxygen uptakes through the two systems wxs calculated. Figure 3 shows the ratio plotted against the age of the embryo. As can be seen, the rate of development of cyto-
l
: II
DAYSlbF INClYBATIOli’
Fig. I. The development of cytochrome leg (red) and breast (white) muscles of chick embryos. Values are means ri: SE cance (P < 0.01) between leg and breast
oxidase systems in I I to I day old [* denotes signifisamples] (n = 6).
Chemical Co.
RESLiLTS l
Figure I shows the results from experiments with homogenates of white (breast) and red (leg) muscle from chick embryos at various stages of development. The total enzyme activity is expressed as microliters of oxygen consumed per gram of muscle (wet wt) per minute of incubation. The developmental pattern of cytochrome oxidase activity appeared to be quite similar for both red and white muscle tissue. Between I I and I5 days of incubation, enzyme activity in both muscle types remained fairly constant. Between days 15 and 19, there was a marked increase in activity for both red (920/,) and white (83‘?0,)muscfe. At each stage
ill II
DAYS OF INCUBATION
Fig. 2. The development of succinoxidase systems in leg (red) and breast (white) muscles of I l&t9 day old chick embryos. Values are means 2 SE [* denotes significance ‘^ ^^., . (Y < U.tfIJ between le& and breast samplesj (n = 6)
Respiratory capacity of developing chick red and white skeletal muscle
Fig. 3. The relationship in the development of cytochrome oxidase to succinoxidase activity in leg (red) and breast (white) muscles of chick embryos between I I and 19 days of incubation.
DAYS OF INCUBATION Fig. 4. Oxygen consumption by minced muscle nrenarations of leg (red) and-breast (white) muscles of il.-i9 day old chick embryos. Values are means rt SE f* denotes si&ificance (P < 0.01) between leg and breast- samples] fn = 6).
chrome oxidase activity and succinoxidase activity is similar between red and white muscle. The ratio of these activities remains fairly constant from day 11 to day 1.5, at which time, cytochrome oxidase activity seems to increase dispro~rtionately in both muscle types. Up to day 17, the rate of cytochrome oxidase activity, relative to succinoxidase activity, is consistently higher in red muscle. At day 19, the activity ratios of the two enzymes is essentially the same.
Figure 4 shows the oxygen consllmption by minced preparations of red and white muscle at various stages of development. Oxygen uptake, expressed as microliters of oxygen consumed per gram of tissue (dry wt) per minute, progressively increased in both red and white muscle between days I 1 and 19. The increases were 166 and 77% for red and white muscle, respectively. Only on the 1I th day of incubation were the oxygen consumptions of the two muscle types significantly different. At this stage of development, the white muscle had larger capacity to use oxygen than the red. As development progressed, both muscle types seemed to acquire similar respiratory capacities.
493
DISCUSSION The results of this investigation suggest that, as development progresses, there is a positive relationship between the increasing oxygen uptakes observed in both muscle types and corresponding increases in oxidative enzyme activity. However, individual differences in enzyme activity between the two types of muscle do not appear to manifest themselves in terms of distinctive metabolic rates. One possible explanation for this paradox is that other components of the oxidative system have simply not kept up with the development of the cytochrome and succinate oxidases and consequently, impose limitations on oxygen consumption. Along these lines, Bass et uI. (1970) have found that red and white muscles from newly hatched chicks do not differ significantly in their concentrations of another “oxidative” enzyme; citrate synthase. Another possibility is that the oxygen consumption m~dsurements were insensitive to these differences. A number of factors are known to influence the resting oxygen consumption of muscle preparations (Stainsby & Lambert, 1979). It appears that oxygen uptake values obtained from a variety of muscle preparations may reflect not only the inherent metabolic capacity of the individual muscles investigated, but aiso a variety of methodological differences. Biochemical deveiopm~nt in muscle involves the development of enzyme systems adequate to convert metabolic fuels into ATP at a rate sufficient to allow continued contractile activity. What constitutes adequacy depends upon the specific contractile characteristics of the muscles involved. It might be expected that red muscle. which is thought to be better adapted to long-duration activity, would differ considerably in this respect from white muscle. which is thought to be involved primarily with explosive, short-duration bursts of activity. In general, adult red muscle contains approximately five times the cytochrome oxidase (Lemberg & Barrett, 1973) and twice the succinoxidase (Lawrie, 1952) of white muscle. Furthermore, in some species, the metabolic rates of red muscle are as much as six times those of white muscle (Gordon, 1968). With regard to the present in~~estigation, differentiation of the activity patterns of these enzyme systems proceeds during pre-natal development and results in muscle groups with distinctive metabolic profiles. The absolute values for enzyme activity reported here for embryonic muscle tissue are well below those typically reported for more adult muscle (Aulie & Grav. 1979). Clearly, the muscle is still quite immature. Nevertheless. by the later stages of embryogenesis, there appears to be a clear-cut subdivision of muscle into two fiber types, which correspond in cytochrome oxidase and succinoxidase activity to the red and white fibers of mature muscle. Differences in contractile properties have been detected in the muscles of newly hatched chickens (Syrovy et al., 1971). This functional differentiation must be the result of fetal growth and development. The present findings suggest the possibility that metabolic differentiatton occurring prenatally may contribute to distinct postnatal function. The factors controlling growth and metabolic differentiation in muscle are still unknown. Although the
WILLIAM S. BARNES and SCOTT M. HASSON
494 motor
pattern
of :I given m~~scle has been assumed to b~ocllernic~~l and functional properties (Salmons & Sreter, 1976; Gordon & Vrbova, 1975): myogenetic as well as hormonal factors could be involved (B~~idw~n rr tri.. 1978: Nougues & Bacou, 1977). Kendrick-Jones & Perry (1967) have demonstrated that a high correlation exists between the rise in activity of certain enzymes and the onset of activity of skeletal muscle. This suggests that the use of the muscle might be the stimulus for the rise in specific activities of these enzymes. tt seems unlikely that the progressive increases in the activity of the oxidizing enzymes investigated in this study or the differences in znzyme activity observed between red and white muscle could be explained on the basis of increased motor activity. In fact, the environment, iu OPO, appears to become more restrictive and confining as development progresses. Moreover, animals which are quite inlmature at birth and incapable of much movement. seem to be the animals most influenced by postnatal fullction~il consider~lt~ons. Enzyme activities in these animals do not approach adult values until some time pr>sr prrrruf~t. Animals born at a more advanced stage of development and capable of independent existence. like the chick, are already rapidly approaching adult values at birth. While the activities of cytochrome oxidase and succinoxidase reported here do not reach adult values. they are clearly increasing rapidly and the rates of increase are distinctly different for red and white muscle. It may well be. that trophic nerve effects contribute to prenatal differentiation of red and white muscle in the chicken, regard&s of whether the conduction of motor nerve impulses or mechanical work of the muscle arc involved. Muscles from I I day old chick embryos. investigated in this study, were probably already innervated since ~nllcrv~~ti~~lof at least some fibers has been reported to occur as early as 6-7 days ir7 ot’o (Drachman. 1967) and endplates can be Iocalized in muscles of about ii) day old chick embryos (Gutmztnn c’i &, 1969). There appear to be specific neural factors that regulate gene expression in the muscle fiber that may be demonstratable even in the absence of dcmonstratable neuromuscular connections (Guth. 1971). For example, elimination of conducted nerve impulses by various experirne~lt~~~ procedures does not reproduce fully the well known cKects of dener~~~~tion, provided that the motor nerve remain ~ln~~tornic~~l~yand flinctionaily connected to the muscle (Drachman. 1967). Furthermore, implanting a nerve from one type of muscle into another. while leaving the original innervation intact, does not result in formation of functionally effective neuromuscular junctions (Guth. I97 I ). Nevertheless, the implanted nerve does seem to induce the conversion of a significant number of fibers from one type to another. Findings such as these, could suggest the release of a l~yp{~thetic~~itrophic substance, capable of modifying the yualitativc. phenotypic characteristics, of the muscle cell. The metabolic and biochemical differences observed between developing red and white muscle might also reflect intrinsic, myogenetic differences between the two muscle types. Supporting this notion, are the findings of Shafiq rsf ~ri. (1972), which show a differer~ti~ll response of f&t (white) and slow (red) regulate
its
muscle fibers to denervation at birth. Potentially fast fibers fail to mature when deprived of neural input, while slow fibers develop more or less normally. In any case. there are undoubtably many factors which contribute to the biochemical and biophysical specificity of the muscle fiber.
SWViMARY
1. Samples of leg (red) and breast (white) skeletal muscle were obtained from chick embryos ranging in age from I 1 days to 19 days. The cytochrome oxidase and succinoxidase activities, as well as the oxygen consumption of the samples were measured manometrically. 3. Oxygen consumption in the two muscle groups increased as development progressed but were not signi~~ntiy different from one another from the 13th to the 19th days of illcubation. 4. Both the cytochrome oxidase and succinoxidase activities of the two muscle groups increased over the observed incubation period. with the greatest increases occurring in the leg (red) muscle. 5. Leg (red) and breast (white) muscles were signifciantly di&rent in their cytochrome oxidase activity at every stage of development investigated. Succinoxidase activities in the two muscle groups differed significantly on days 1 I, 17 and 19. 6. The ratio of cytochrome oxidase activity to succinoxidasc activity increased, in both muscle groups, as development progressed. This ratio was consistently higher in the leg (red) muscle until the 19th day of incubation, when the ratios were identical for both muscle groups. 7. It is concluded that marked differences between teg (red) and breast (white) muscles of the chicken can be observed in the oxidative system during the later stages of ~~~bryologic~~l development.
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L. G. (1942) The development of in the chick embryo. J. hi& Clref~z.
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HOFMANNW. W. (1976) Oxygen consumption by hum:m and rodent striated muscle irk citro. AM. J. Physiof. 230, 34-40. H~LLOZY J. 0. (1967) Biochemi~l adaptations in muscle. f. hioi. Chrtn. 242, 2278-2282. KENURICK-JONES J. & PERRYS. V. (1967) The enzymes of adenine nucleotide metabolism in developing skeletal muscle. Biochrm. J. 103, 207-214. LAWRIER. A. (1952) Biochemical differences between red and white muscles. Nuture, Land. 170, 122--l23. LEMBERC~ R. & BARRETTJ. (1973) ~~r~J~~~r~~fl~~.~. pp. 395-397. Academic Press. London. N~IJGUES J. & BACQLIF. (1977) Enzymatic activities of muscle fibers differentiated, in citrci. from pectoralis major (white) and adductor magnus (red) muscles of chick embryos. Esprrinwrriu 33, 7 14-7 16. NYSTROMB. (1966) Succinic dehydrogenase in developing cat muscles. Nutftrr. Lund. 212. 954-955. NVSTROM B. (1968) Histochemistry of developing cat muscles. Actu Neural. Scurtd. 44, 4055439. OS~AI L. B. & MOLF.P. A. (1975) Laboratory techniques involving small animals in exercise physiology research. In Luercise und Sports Scirwes Recie\vs (Edited by Wn.MOREJ. H. & KE~GH J. F.), Vol. 3, pp. 351-370. Ac;tdemic Press, New York. POMER V. R. ( 1964) Respiratory enzymes. In ,~~~~~~u~~if~~rj~ T~&niqrte,s (Edited hv UMBREITW. W.. BURRISR. Ii. & STAUFF~:R J. F.). 4th edn., pp. 162-l 65. Burgess, Minneapolis. REPORTERM. C., KONIGSBERGI. R. & STREHLERB. L. (1963) Kinetics of accumulation of creatine phosphokinase activity in developing embryonic skeletal muscle iri rino and in monolayer culture. Espi Ceil Res. 30, 410-417. SALMONS S. & SRETERF. A. (1976) Significance of impulse activity in the transformation of skeletal muscle type. Nuwr, torrd. 263, 30.-34. SHAFIQS. A.. ASIEDUS. A. & MILH~RATA. T. (1972) Effect of neonatal neurectomy on differenti~~tion of fiber types in rat skeletal muscle. Espi .~ruroi. 35, 529-540. STAINSBY W. N. & LAMBERT C. R. (1979) Determinants of oxygen uptake in skeletal muscle. In Eserciw and Sporrs Scierwes Rrviews (Edited bv HUTTON R. S. & MILLER D. I.). Vol. 7. pp. 1’5-15 I. The Franklin Institute Press, USA. SYROVYI., GUTMANN E. & MELKHNA J. (197t ) Differenti~~l response of myosin-ATPase activity and contr~l~tion properties of fast and slow rabbit muscles following denervation. Esperirnelltitr 27. 1426-I 427.