Cell
Cell Tissue
Res (1982)
222: 325-337
and
Tissue
Research
((:;)Springer-Verlag
1982
Capillarisation, oxygen diffusion distances
and mitochondrial content
of carp muscles following acclimation
to summer and winter temperatures
Ian A. Johnston
Department of Physiology, University
of St. Andrews, St. Andrews, Fife, Scotland, Great Britain
Summary. Many species of fish show a partial or complete thermal
compensation of metabolic rate on acclimation from summer to winter
temperatures. In the present study Crucian carp (Carassius carassius L.) were
acclimated for two months to either 2° Cor 28° C and the effects of temperature
acclimation on mitochondrial content and capillary supply to myotomal
muscles determined.
Mitochondria occupy 31.4% and 14.7% of slow fibre volume in 2° C- and
28° C-acclimated fish, respectively. Fast muscles of cold- but not warmacclimated fish show a marked heterogeneity in mitochondrial volume. For
example, .only 5% of fast fibres in 28° C-acclimated fish contain 5 %
mitochondria compared to 34% in 2° C-acclimated fish. The mean mitochondrial volume in fast fibres is 6.1 % and 1.6% for cold- and warm-acclimated fish,
respectively.
Increases in the mitochondrial compartment with cold acclimation were
accompanied by an increase in the capillary supply to both fast (1.4 to 2.9
capillaries/fibre) and slow (2.2 to 4.8 capillaries/fibre) muscles. The percentage
of slow fibre surface vascularised is 13.6 in 28° C-acclimated fish and 32.1 in
2° C-acclimated fish. Corresponding values for fast muscle are 2.3 and 6.6% for
warm- and cold-acclimated fish, respectively. Maximum hypothetical diffusion
distances are reduced by approximately 23-30% in the muscles of 2°-Ccompared to 28° C-acclimated fish. However, the capillary surface supplying
1 J.Ull3of mitochondria is similar at both temperatures.
Factors regulating thermal compensation of aerobic metabolism and the
plasticity of fish muscle to environmental change are briefly discussed.
Key words: Temperature acclimation -Quantitative
-Skeletal muscle -Teleosts
cytology -Capillarisation
In fish the aerobic capacity of skeletal muscle is known to be influenced by
spawning migration (Bostrom and Johansson 1972), seasonal depletion (Johnston
Send offprint requests to; Ian A. Johnston, Department
of Physiology, University
ofst.
Andrews, St.
Andrews, Fife, Scotland, Great Britain
0302-766X/82/0222/0325/$02.60
I.A. Johnston
326
1981 a), environmental oxygen tensions (Johnston and Bernard 1981) and
temperature (prosser 1973; Smit et al. 1974; Johnston and Lucking 1978; Johnston
and Maitland 1980; Sidell 1980). Of these factors the effects of temperature
acclimation has been most studied (see Hazel and Prosser 1974; Somero 1978;
Johnston 1980).
Many freshwater species experienc&differences in body temperature of 2030° C between summer and winter. A variety of metabolic responses to cold
acclimation have been documented. These include at the two extremes, complete
conservation of physiological function and torpor (precht 1958; Kanungo and
Prosser 1959; Prosser 1973).
Qualitative changes 'in the kinetic properties of enzymes of intermediary
metabolism with temperature acclimation are rare and may be restricted to fish with
duplicated gene loci (e.g. tetraploid species, such as salmonids) (Shaklee et al.
1977). Instead, the decrease in catalytic function that accompanies a lowered body
temperature is thought to be compensated for by changes in enzyme microenvironment (e. g. pH, associated phospholipids, substrate/modulator concentrations) and
increases in enzyme concentration (Hazel and Pros~er 1974; Side111977; Wilson
1977; Walesby and Johnston 1980). Thus, a number of studies have reported an
increase in metabolic rate following acclimation of fish from summer to winter
temperatutes. For example, Smit et al. (1974) acclimated goldfish (Carassius
auratus) to either 15° or 30° C. Following four to five weeks a partial thermal
compensation of oxygen consumption was observed over a whole range of
swimming speeds. Cold acclimation also resulted in a parallel increase in the oxygen
consumption of muscle homogenates (Smit et al. 1974). In the case of striped bass
(Morone saxatilis) oxygen consumption rates are higher in muscle homogenates
from 5° C- than 25° C-acclimated fish even when compared at their respective
acclimation temperatures (Jones and SideI11981).
Enhanced aerobic capacity with cold acclimation reflects both an increase in the
volume of the mitochondrial compartment (Johnston and Maitland 1980) and an
increase in the oxygen consumption of isolated mitochondria (Thillart and
Modderkolk 1978). It is unclear whether the latter results from an increased
concentration of respiratory enzymes within the mitochondrion or an altered
membrane composition causing qualitative changes in enzyme activity (Hazel
1972; Thillart and Modderkolk 1978).
Cold acclimation also results in a reduced oxygen delivery to mitochondria due
to the direct effects of temperature on diffusion rate and solvent viscosity within the
cell. Prosser (1973) has calculated that Krogh's constant for diffusion ofO2 through
muscle tissues decreases by 1.4% per oC. Thus, at 2° C oxygen diffusion rate would
be reduced by 36% compared to 28° C.
The present study investigates the relation between fibre surface and volume,
the size of the mitochondrial compartment and capillary supply to the muscles of
carp ( Carassius carassius) acclimated for two months to simulate either summer
(28° C, long day-Iength) or winter (2° C, short day-Iength) conditions.
Materials
and methods
.
Crucian carp ( Carassius carassius L.) of average weight (300 g) were acclimated for two months to either
2° r (.hnrt nhntnneriod 8 h li!Zht: 16h dark) or 28° C (long photoperiod 16h light: 8 h dark) to simulate
~
Capillarisation
of carp muscles
327
winter and summer conditions, respectively. Fish were maintained in biologically filtered, aerated fresh.
water and fed a diet of commercial fish pellets so as to maintain similar bodyweights in the two groups.
Fixation of muscles
Fish were killed by a blow to the head and transection of the spinal cord. Fixative (3 % glutaraldehyde in
0.1 M phosphate buffer, pH 7.4) was injected into the myotomes adjacent to the sample site, which was
located posterior to the dorsal fin. During this time the fish was kept on ice with the trunk bent to its
point of maximum flexure. Initial in situ fixation was for 1 h. Subsequently, small fibre bundles (25-75
fibres) were dissected from both the superficial slow fibre layer and the mid-region of the epaxial fast
muscle (see Johnston and Maitland 1980). Fibre bundles were pinned to cork strips at their resting
lengths in situ and fixed for a further 2-24 h in 3% glutaraldehyde, 0.1 M phosphate buffer, pH 7.4, at
4° C. Tissue samples were postfixed in 1% osmium tetroxide, 0.1 M phosphate, pH 7.4, dehydrated in a
series of alcohols up to 100% and embedded inAraldite resin. Orientation ofmuscle fibres in embedded
material .,I,as ascertained from examination of 1~ semithin sections stained with toluidine blue.
Ultrathin sections were mounted on formvar-coated 150mesh copper grids, double stained with uranyl
acetate and lead citrate, and viewed with a Phillips 301 electron microscope.
Quantification
of muscle capillarisation
and mitochondrial
volume
The outlines of 400 muscle fibres and adjacent blood capillaries were drawn from either low
magnification electron micrographs ( x 4,840) (slow fibres) or toluidine blue-stained semithin sections
( x 1,040) (fast fibres) by projecting respectively a 1/4plate or 35 mm negative onto cartridge paper using
a photographic enlarger. Fibre area, perimeter and capillary contact length were determined directly
using a summagraphics digitiser in conjunction with a mini-computer (Walesby andJ ohnston 1980). The
Table 1. Effects of temperature acclimation on vascularisation
muscle
and mitochondrial
content of carp slow
Parameter
Symbol/
calculation
2°C-acclimated
Mean::!: S.E.
28" C-acclimated
Mean:t S.E.
No. of fibres
A
100
100
No. of capillaries
B
480
220
Fibre area (~m2)
c
752:!: 37
705 :t 40
Fibre perimeter (11In)
D
105+- 3
93:t
Mitochondrial
5
E
31..4:!:1.3
14.7:t0.7***
No. of capillaries per fibre
F
2.2:t
Capillary
G
4.8+0.2
J33;7:!:1.5
32.1 :t f.2
13.6:!: 1.0**
21.9
42.3
0045
0.018
0.14
0.14
7.1
10.1
% Fibre
volume ( %)
contact length (~m)
surface
vascularised
H = G xtOO
D
Mean perimeter served by one
Capillary surface supplying
1111D3fibre volume
Capillary surface supplying
1111D3of mitochondria
Maximum hypothetical
difussion distance
..Statistically
D
I=-
capillary
F
G
1=-
K=~
C
E
L="1~
VB7
significant at the P<O.O1 and ...P<O.OOllevels
12.6:t
0.1**
1.0**
TA .Tohnstnn
:?SI
method of calculating other indices of capillarisation follow those adopted by Flood and co-workers (see
Flood 1979; Kryvi et al. 1980). Details of the method of calculating the various indices of capillarisation
are given in Table 1.
The fraction of fibre volume occupied by mitochondria ( % MF) was estimated from analyses of 100
electron micrographs of transverse sections ( x 3,800-7,000) using a point-grid method (WeibeI1967).
Size of grid spacing was 1.5 times the meaIl mitochondrial diameter, and a minimum of 200 points per
micrograph was analysed. The results obtained were in good agreement ( -5 %) with direct estimates of
MF using the digitiser and mini-computer.
Statistical analyses
Statistical analyses were carried out using the Student's t-test for equal sample numbers
Results
Figures 1-8 show the typical appearance of fast and slow muscle fibres from carp
acclimated to either 2° C or 28° C. The fraction of slow fibre volume occupied bv
,
--,
2
,.J
3
4
Fig. I. Semithin (111In) section of slow muscle from a 2" C-acclimated carp. Toluidine blue. Note
high density of capillaries and mitochondria. Many of the caDillaries contain red blood cells (RC
Fig.2. Semithin (ll1m) section of slow muscle from a 28° C-acclimated carp. Toluidine blue. Note the
reduced mitochondrial content (M) and number of capillaries per fibre (C) compared with Fig.l
Fig.3. Semithin (111In) section of fast muscle from a 2° C-acclimated carp. Toluidine blue. Note the wide
range of fibre sizes and relative abundance of capillaries ( C)
Fig. 4. Semithin (111In) section of fast muscle from a 2SoC-acclimated carp. Toluidine blue. Relatively
few capillaries ( C) are visible
Capillarisation
of carp muscles
329
Table 2. Effects of temperature acclimation on vascularisation
muscle
Parameter
Symbol/
calcul"t.inn
and mitochondrial
content of carp fast
2° C-acclimated
28° C-acclimated
MeanT
Mean+
S.E.
S.E.
No. offibres
A
100
100
No. of capillaries
B
290
140
Fibre area (~m2)
C
1528:t184
1258:t112
Fibre perimeter (~m)
D
139:t 7
129:t
Mitochondrial
6
E
6.1 :t 0.7
1.6:t
0.2**
No. of capillaries per fibre
F
2.9:t 0.1
1.4:t
0.1**
Capillary contact length (~m)
G
9.2+ 0.6
3.0:t
0.4.*
% Fibre
G x 100
H=
--n-
6.6:!:0.5
2.3+ 0.2**
D
I=-r;-
479
91.4
Capillary surface supplying
1 J.lm3 fibre volume
0.006
0.002
Capillary surface supplying
1 um3 of mitochondria
0.10
0.13
11
17
surface
volume ( %)
vascularised
Mean perimeter served by one
capillary
Maximum hypothetical
ciifi]..in" cii.t""",,
'. Statistically
,jA-;c
L=V~
significant at the P<O.O1 and ...P<O.OO1Ievels
mitochondria (MF) is 2.1 times higher in 2° C- than 28° C-acclimated fish
(P<0.001). Average MF in slow fibres from 2°C-acclimated fish is 31.4% with
22% of the fibres containing > 40% mitochondria by volume. This is somewhat
higher than reported previously for cold-acclimated carp' (Johnston and Maitland
1980). No obvious difference is evident in the complexity of mitochondrial cristae
from cold- and warm-acclimated fish (Figs. 5, 6).
The MF of fast fibres is 3.8 times higher in 2° C- than 28° C-acclimated carp
(P < 0.001). Fast fibres in 2° C-acclimated fish show a wide distribution ofMF with
22 and 54% of the fibres having more than 5 % and 10 % mitochondria, respectively
(Fig. 10). In contrast, only 4% of the fast fibres from 28° C-acclimated fish have a
MF > 5 %, and 34% have a MF < 1 % (Fig. 10).
Average mitochondrial volume of fast fibres is only 11% and 19% of that of
slow fibres in 2° C- and 28° C-acclimated fish, respectively (Tables 1, 2). The
abundance and complexity of cristae is greater in slow than fast fibres (Figs. 5-8).
As noted previously glycogen granules are more abundant in the muscles of coldthan warm-acclimated fish (Figs. 5-8) (Johnston and Maitland 1980).
Indices of capillarisation for fast and slow muscles are shown in Tables 1, 2.
Both the number of capillaries per fibre and the % of fibre surface vasc'ularised are 2
to 3 times higher in fish acclimated to winter than summer conditions (P<0.01).
Acclimation to 2° C is associated with a small decrease in the calculated maximum
.,
Fig.5. Low magnification electron micrograph of a slow fibre from a 2° C-acclimated fish showing the
high mitochondrial content (M) and irregular packing of myofibrils (MY). Note the presence of
numerous glycogen granules (G); capillaries (C); nucleus (N)
Fig.6. Low magnification electron micrograph of a slow fibre from a 2~o C-acclimated fish. Note the
relatively low mitochondrial content compared to Fig. 5 and the presence of a satellite cell containing
endoplasmic reticulum
Fig.7. Low magnification
electron micrograph
elongated peripheral myofibrils
Fig. 8. Low magnification
of a fast fibre from a 2° C-acclimated
(PM) and numerous mitochondria
electron micrograph
almost complete absence of mitochondria.
fish showing
(M)
of fast fibres from a 28°C-acclimated
fish. Note the
Capillarisation
of carp muscles
331
diffusion distance for oxygen in both muscle types (Tables 1, 2). In contrast, the
ratio of capillary surface to mitochondrial volume is similar at both acclimation
temperatures (Tables 1,2).
Slow fibres have a better capillary supply than fast fibres (Figs. 11-14). For
example, in 2° C-acclimated fish the capillary surface supplying 1 J.1m3of fibre
volume is 7.5 times greater in slow compared to fast fibres (Tables 1, 2).
Discussion
There is a large literature on the adaptation of mammalian skeletal muscle to
different work loads and eKercise regimes. Training regimes involving submaximal
exercise on bicycles and treadmills have demonstrated higher maximum oxygen
uptakes and increases in muscle respiratory enzyme activities, particularly in red
fibres (see Holloszy and Booth 1976; Pette 1980). Changes in the respiratory
capacity of mammalian muscles with exercise training are usually accompanied by
only small or no significant changes in capillary supply (Folkow and Halicka 1968;
Muller 1976). It seems likely that the blood supply to most mammalian limb
muscles is sufficient to meet the oxygen needs of normal activity patterns.
Histological data concerning capillary density provide only part of the information
needed to assess the perfusion of a tissue, since both blood flow and extraction
capacity of muscles can vary over wide limits (Granger and Shepherd 1973; Muller
1976).
The results in the present study demonstrate the great plasticity of carp muscle
in adapting to environmental change. The two- to three-fold increase in capillary
supply and mitochondrial volume between the muscles of summer and winter fish is
far larger than that observed with exercise training in other animals (Holloszy and
Booth 1976; Pette 1980).
Smit et al. (1974) working with a closely related species (Carassius auratus)
found that the relationship between swimming efficiency (power/net oxygen
consumption) and speed is altered by temperature acclimation. This can be
interpreted as either a change in the relative proportions of fast and slow myotomal
fibres and/or a change in the threshold speed of recruitment of fast motor units.
Experimental evidence exists for an altered distribution of fibre types with
temperature acclimation (Smit et al. 1974; Johnston and Lucking 1978; Sidell
1980). Acclimation of goldfish to either 3° Cor 31° C for three months results in an
increase in the number of slow fibres and fast oxidative glycolytic fibres in coldcompared to warm-adapted fish of the same bodyweight (Johnston and Lucking
1978). It is not yet known whether this results from a genuine transformation of
muscle phenotype or is due to the proliferation of myosatelite cells.
There have been no studies on the effects of temperature acclimation on the
pattern of fibre recruitment in fish. However, the increased spread ofMF (0.5-18 %)
(Fig. 10) in the fast muscles of cold-acclimated fish may reflect a decrease in the
threshold speed of recruitment of fast motor units.
Electromyographical studies with carp have shown fast fibres to be recruited at
relatively low-sustained swimming speeds (Johnston et al. 1977; Bone et al. 1978).
Similar results have also been obtained for other teleosts with multiply innervated
fast muscles (Hudson 1973; Johnston and Moon 1980a, b). Primitive teleosts and
elasmobranchs have focally innervated fast fibres (Bone 1964). There is some
TA. Johnston
332
40
40
summer fish
summer fish
20
20
(/)
w
[I:
m
L1.
~
{1)
UJ
a:
aI
u::
40
#
40
winter fish
20
20
, 5
9
15 25
35
45
55
% MITOCHONDRIA
1
10
3
5
7
9
11 13 15 17
% MITOCHONDRIA
Fig. 9. Histograms showing the frequency distribution of mitochondrial volume in the slow musclesof
carp acclimated to winter (2°C) and summer (28°C) conditions
Fig. 10. Histograms showing the frequency distribution of mitochondrial volume in the fast musclesof
carp acclimated to winter (2° C) and summer (28°C) conditions
evidence that in these fish fast motor units are only recruited at burst swimming
speeds (Bone 1966; Bone et al. 1978). The capillary supply and mitochondrial
volume of carp fast fibre (Tables 1, 2; Fig. 10) indicates a considerably
greater
aerobic capacity than found in the homologous
muscles in primitive
teleosts
(Egginton
and Johnston 1981), sharks (Kryvi
1977) and sturgeon (Kryvi et al.
1980). Further studies are required to determine whether temperature affects fibre
recruitment
and to establish firmly this apparent relationship
between fast muscle
innervation
and the division of labour of fish muscles during swimming.
The range of fast fibre size in the fish in the present study is from 12010,000 J.1m2.There is a considerable heterogeneity in the MF of fast fibres in 2° Cbut not 28° C-acclimated fish (Fig. 10). In general, small fibres ( < 500 J.1m2)tend to
have a higher MF and glycogen storage levels than very large fibres ( > 1,500 J.1m2)
(Johnston and Moon 1981; Johnston 1981b). This heterogeneity
in the aerobic
capacity and metabolic characteristic
of different sized fast fibres led some earlier
workers to refer to fast muscle in carp as being mosaic or composed ofmixed fibre
types (Boddeke et al. 1959). However, Altringham
and Johnston (1981) have
recently shown that branches of a given fast motor axon in cod innervate fibres with
a whole spectrum of sizes. It therefore seems improbable
that small fibres have a
distinct pattern of recruitment.
An interesting possibility is that fibres with a high
mitochondrial
volume (Fig. 10) have a role in the aerobic catabolism
of lactate
produced by anaerobic glycogenolysis in large fibres in the same or adjacent motor
units (see also Johnston and Moon 1981). Inexercised-conditioned
rainbow trout
acceleration from 0.9 to 3.5 bodylengths, S-l results in a transient increase in total
hntiv l~~t~t~ whi~h neIl k~ Il.t. 10 min and is not si!!nificantlv
different from initial
Capillarisation
of carp muscles
333
40
40
20
20
C/)
w
0:
m
[1:
C/)
w
(1:
m
u:
#
*
40
40
20
20
11
0 1 2 3 4 5 6 7 8 910
CAPILLARIES/FIBRE
12
O 1 2 3 4 5 6 7 8 9
CAPILLARIES/FIBRE
Fig. 11. Histograms showing the frequency distribution of the number of capillaries per fibre in slow
muscle of carp acclimated to winter (2° C) and summer (28° C) conditions
Fig. 12. Histograms showing the frequency distribution of the number of capillaries per fibre in fast
muscle of carp acclimated to winter (2° C) and summer (28° C) conditions
values after 60min (Wokoma and Johnston 1981). Since fast motor units are
recruited at this swimming speed in trout (Greer-Walker and Emerson 1978;
Johnston and Moon 1980a), it is likely that there is a continuous production of
lactate. As there is no net accumulation of lactate under steady state conditions, its
rate of production must equal its rate of catabolism by more aerobic tissues such as
the gills, liver, red muscle and possibly small fast fibres.
Regardless of whether there is a shift in the pattern of recruitment of fast fibres
with temperature acclimation, the general trend is for a transition to more aerobic
fibre types and a more aerobic type of muscle in cold-adapted fish. A drop in body
temperature from 28° C to 2° C would result in an approximately 80% fall in the
rate of A TP production assuming a mean QlO for aerobic metabolism ofaround 2.
The MF of fast and slow fibres increases 281% and 114 %, respectively, in 2° Ccompared to 28° C-acclimated fish. Thus, for a perfect thermal compensation of
oxygen consumption rates it might be expected that the O2 consumption per unit
mass of mitochondria would need to increase around two times in fast and four to
five times in slow muscle mitochondria. The actual figures would be less than this
for myotomal muscle due to the increased proportions of slow and fast oxidative
glycolytic fibre types (Johnston and Lucking 1978; Sidell 1980). Thillart
and Modderkolk (1978) isolated mitochondria from the fast and slow muscles of
goldfish acclimated to either 30° C, 20° Cor 5° C. They found that in 5° Ccomnared to ~O°C-acclim~ted fi~h ~t~t.~TTT( A T)P-~~t.iv~t.~d)r~~nir~t.inn ~t. ~ (';0r
:\14
I.A. Johnston
Fig. 14. Histograms showing the frequency distribution of percentage fibre vascularised in fast muscle of
carp acclimated to winter (2° C) and summer (28° C) conditions
increased 2.1 fold in slow and 1.6 fold in fast muscle mitochondria with succinate as
substrate.
Since nothing is known of the oxygen demand of the mitochondria, muscle
myoglobin content, blood flow or oxygen-carrying capacity of haemoglobin at the
respective acclimation temperatures, few quantitative conclusions can be drawn
concerning relative tissue perfusion rates at different temperatures. However, it is
interesting to note that the capillary surface supplying 1 J.lm3of mitochondria is
similar in both 2° C- and 28° C-acclimated fish. The 23-30% decrease in
hypothetical diffusion distance in cold-acclimated muscles may reflect a compensatory adaptation to the effects of a lowered body temperature on oxygen
diffusion rates.
Factors controlling aerobic capacity and capillary density in skeletal muscle are
relatively poorly understood. Many experiments with mammalian twitch fibres
have implicated the pattern of motor neurone activity as a major determinant of
muscle phenotype. For example, chronic electrical stimulation of fast glycolytic
muscles with a slow motor neurone firing pattern (10 Hz) results in a
transformation of its contractile membrane and metabolic properties toward that of
a slow muscle (see Lomo 1976; Pette 1980; Vrbova 1980). Cotter et al. (1973) found
that long-term electrical stimulation of rabbit fast muscles resulted in a 20 %
increase in the number of capillaries per fibre and a transformation toward a more
aerobic type of metabolism. The increase in capillary density with chronic
~t.imulation takes olace as earlv as four days and precedes changes in mitochondrial
Capillarisation
of carp muscles
335
content and aerobic metabolism (Brown et al. 1976). The respiratory capacity of
mammalian muscle is also modulated by thyroid hormone levels. For example,
thyroid status has been shown to influence the number and cristae development of
mitochondria in rat muscles. A number of studies have demonstrated a decreased
MF, and reduced aerobic enzyme activities in the animals of hypothyroid animals
(Winder et al. 1975; Baldwin et al. 1978; Nicol and Johnston 1981).
The endocrine or other factors regulating aerobic capacity in fish muscle are
largely unknown. For example, there is little evidence to suggest that either thyroid
hormones or glucocorticoids have a role in modifying metabolic rate during
temperature acclimation in fish. Klicka (1965) acclimated goldfish to either 5° C,
15° C, 25° Cor 30° C. He found that the pattern of thermal compensation of oxygen
consumption was not altered following either thyroidectomy, thiourea treatment,
or injection of TSH or ACTH. Similar results have been obtained with
hypophysectomised mud-minnows (Hanson and Stanley 1970). However, in this
study hypophysectomy was found to cause a reduction in oxygen consumption, but
had no effect on metabolic temperature compensation between 5 and 22° C.
Recently, Stevens (1979) has reported that temperature acclimation alters both
tail beat frequency and stride length (distance moved per tail beat) at a given
swimming speed. Such observations may reflect a change in the pattern of motor
neurone activity with temperature and hence provide a possible stimulus for
acclimatory changes in muscle properties.
Acknowledgements. This work was supported by grants from the Science Research Council and NA TO.
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Accepted September 3, 1981