-~--~---~1
~
Comp.
Physiol.
147-156
13
Journal
c
(1980\
of
Comparative
Physiology.
B
(" by Springer-Verlag
1980
Endurance Exercise Training in the Fast and Slow Muscles
of a Teleost Fish (Pollachius virens)
Ian A. Johnston
and Thomas
W. Moon*
Department of Physiology, University of St.Andrews. St.An'tlrews. Fife. Great Britain
Accepted
October
I. 1979
Summary. I. The recruitment of muscle fibre types
has been investigated in the coalfish ( Pollachius
virens) using electromyography. Red trunk muscles
were active at all swimming
speeds examined
(0.25-3.61engths/s). Interestingly, white fibres were
recruited at 0.8-2.0 lengths/s providing evidence that
this muscle type is also used during sustained activity.
2. The effect of endurance exercise training on
muscle fibre size and enzymes of energy metabolism
has also been investigated. Fish were exercised continuously at 2.1 lengths/s for a period of three weeks
in an experimental swimming chamber. This swimming speed represents a significant increase in work
load relative to non-exercised fish as evidenced by
muscle fibre hyperttophy and an increase in creatine
kinase activities in both red (184%) and white (260%)
muscles.
3. Glycogen storage levels increased to a greater
extent in red ( + 520%) than white ( +200%) muscles.
Phosphofructokinase activity was eight times higher
in the red muscle of exercised fish. In contrast, there
was only a small increase in citrate synthetase
( + 30% ) and no change in either hexokinase or cytochrome oxidase activities in the red muscle of
trained fish.
4. Increased hydroxy acyl CoA dehydrogenase activities in both muscle types indicate an enhanced
capacity for fatty acid catabolism with training.
5. White muscle phosphofructokinase
activities
were not significantly different in trained and untrained fish. It is likely that the maximum potential
of white muscle for anaerobic glycogenolysis is already sufficient to meet all its energy requirements
at this swimming speed.
6. The results suggest that the capacity of coalfish
red muscle to do aerobic work remains essentially
Permanent.
1 Ottawa
address:
Ontario
Department
Canada
KIN
of Biology
9B4
niversitv
of Ot
unchanged by endurance exercise training and that
any increase in the ability to produce A TP must be
met anaerobicallv.
Introduction
Endurance exercise training has been studied in numerous mammalian species including the mouse
(Salminen et al., 1977), rat (Baldwin et al., 1973), pig
(F ogd-J orgensen and Hyldgaard-J ensen, 1975), horse
(Guy and Snow, 1977) and man (Saltin et al., 1968 ;
Gollnick et al., 1973). The results obtained are dependent both on the nature of the training regime and
the intensity of the exercise(Fitts et al., 1975; Baldwin
et al., 1977). However, in general training programmes involving running or cycling at submaximal
work loads ( ~ 60% maximal oxygen consumption)
are associated with large increases in the aerobic capacities of skeletal muscle. Thus numerous studies
have shown an increase in the activities of tricarboxylic
acid cycle enzymes (Holloszy et al. , 1970; Baldwin
et al. , 1973; Holloszy and Booth, 1976) and in the
number and size of muscle mitochondria with training
(Gollnick et al., 1973). In contrast, muscle glycolytic
enzyme activities change little unless the training regime includes a significant component of sprint exercise (Baldwin et al., 1973; Guy and Snow, 1977). An
interesting exception is hexokinase which shows large
exercise-induced increases in activity (Holloszy et al.,
1970; Baldwin et al., 1973). Hexokinase is unique
among glycolytic enzymes in that its activity in vertebrate muscles is directly related to pigmentation and
respiratory capacity (Burleigh and Schimke, 1969 ;
Crabtree and Newsholme, 1972). Other adaptations
in mammalian muscle contributing to improved endurance following training, include an increased ability
to oxidize fatty acids (Mole et al., 1971) and higher
levels of glvcogen storage ( Guv and Snow, 1977).
0340-7616/80/0135/0147/S02.00
48
I.A.
In contrast to the above results, a recent study
on the iguanid lizard Sceloporus occidentalis
failed
to show any change in either oxygen consumption,
muscle enzyme activities or endurance following
exercise training (Gleeson, 1979). It is of interest to,extend
these studies to other groups of ectotherms.
Previous
studies on exercise training in fish have been restricted
to changes in gross chemical composition
(Davison
and Goldspink.
1977) and fibre hypertrophy
(Walker ,
1971 : Walker and Pull, 1973). In the present study
coalfish
( Pollachius
t:irens) were subject to three
weeks continuous exercise at 2.1 lengths/s. The results
indicate that the aerobic capacity of the muscles remains essentially unchanged
by endurance
training
and that any increase in the ability to produce A TP
must be met anaerobically.
The differences
in the
response of ectotherms
and mammals to endurance
training is briefly discussed.
\laterials
Johnston
.w.
am
Moon:
Exercise
Training
in
Fish
Muscle
r
~
~
"
1~
I
~
I~;
+
lcl
al
Fig. I. The exercise chamber employed in the training experiments.
The inner perspex cylinder (25 cm diameter x 150 cm length) was
contained in a tank constructed out of 314" marine plywood coated
with araldite paint and contained in an outerbox fill~d with II/2"
of plastic foam. 111.3 hp variable speed el.ectric motor; p, plastic
5 blade propellor; s.baffles to reduce turbulence; 1\'. water tight
shaft bearing: he, glass heat exchanger containing refrigerated ethylene glycol: <:f. activated charcoal filter: a, aeration, The arrows
inside the chamber indicate direction of water flow
and Methods
Fi~h
Coalflsh I Pollachi,ls rirens) were caught by rod and line during
September to November in St. Andrews harbour. Fish of mean
length 17.2 :t 0.2 cm (N = 34) were acclimated for several weeks
.(0 a temperature of 10 °C in tanks of recirculated. filtered seawater.
The) \\ere fed daily on a diet of chopped mussels and fish flesh.
Trainil1l!
The design of the swimming chamber employed is shown in Fig. ] .
The rate of water flow was calculated using a calibrated Miniflow
\"elocity probe [George Kent (Stroud) Ltd.] and by introducing
dye particles through a syringe and measuring the time taken for
the particles to travel through a known length of the chamber.
Water temperature was maintained at 10 :to.5 °C using a Grants
Instruments Thermocirculatorjcooler
in conjunction with a heatexchanger. Fish were introduced into the chamber al1d ihe water
speed was gradually increased o\"er one \\eek until the final swimming speed of 2.1 lengthsjs ( 56 cmjs) \\"as reached. Training was
continuous for a further period of three \\"eeks except for 35 minutes
each day when the \\"ater flo\\" \\"as turned off to allo\\" for feeding.
Fish were fed to excess 011a similar diet 10 controls. Approximately
15°;0 of the fish started on the training exercise \\"ere unable to
complete the full programme. A total or 20 fish trained ror three
\\"eeks at ~.I lengthsjs \\"ere used in these experiments.
Fig. 2. Diagramatic
the coalfish
muscle.
representation
myotome:
Insertion
illustrated.
of
of the
R, red muscle;
e.m.g.
Wd (deep white
electrodes
muscle).
main
P. pink
into
muscle
muscle;
the
Ws (superficial
types
in
Ul. white
",hite
muscle
white
i~
muscle)
'.!1l'aph
Coalfish \\ere lightly anaesthetised in a so)ufion of MS 222 (Sandoz
Ltd.). Hooked 36 s.\\".g.insufated copper \\"ire electromyogram electrodes \\"ere bared of insulation 1 mm from the tip and inserted
in pairs into the red and white muscle regions (Fig. 2). The electrodes \\"ere auached securely to the fish by se\\"ing them to the
base of the dorsal fin rays using surgical thread. Extraceflular
potentials \,ere measured using an a-c coupled differentia] amplitier (Isle\,orth Electronics Ltd.) \\"ith a I!ain of 1.000 and a ]0 KH7
filter. and displayed oh a storage oscilloscope. Recording sites
\\ere mapped at.the end of the experiments by dissecting out the
probe tracts.
Hislochemislr)'
Small
cubes
size were
of red arid
rapidly
white
dissected
mus,les
from
the
approximately
lateral
musculature
2,5 mmJ
in
(Fig,
2).
i~A. Johnston and
W
xercise
Moon
Training
in
149
ish Muscle
Samples were mounted on cryostat chucks in OTC embedding
compound (Ames Co, Inc) and rapidly frozen by immersion in
2-methyl butane cooled to its melting point in liquid nitrogen
( -159 °C), Blocks were placed in a refrigerated cabinet at -20 °C
for one hour after which frozen sections were cut at 10 ~ thickness
and mounted on coverslips, Staining was for succinic dehydrogenase, glycogen and myofibrillar
A TPase as previously described
(Johnston and Lucking,
1978), Sections were magnified and
projected onto a sheet of cartridge paper of uniform thickness.
A total of 200 fibre outlines were traced at random from each of
six non-exercised and six trained fish, Red fibres have high glycogen
levels, high succinic dehydrogenase activities and low myofibrillar
A TPase activities which are labile at alkaline pH (Johnston et al"
1974). In contrast, white fibres have low glycogen and succinic
dehydrogenase levels and high alkaline stable myofibrillar A TPase
activities (Johnston et al" 1974; Patterson et al., 1975).
G/l'coJ!en
Determination
Quick freezing techniques were employed to arrest metabolism
and minimise post-mortem changes in muscle glycogen in. both
control and trained fish. Coalfish were killed by a blow to the
head and the last third of the trunk immediately dropped into
liquid nitrogen ( -159 °C). Samples were allowed to thaw to
-10 °C and red and white muscles were dissected whilst still frozen.
After excision, muscle samples were stored in liquid nitrogen until
analysed. Glycogen was determined in the tissues by an anthrone
method as previously described (Johnston and Goldspink, 1973).
bisphosphate. 0.4 mM NADP. 0.3% bovine serum albumin. 0.5%
purified muscle glycogen. I mM AMP and excess phosphogluco.
mutase and glucose-6-phospl\ate
He.'1:okinase. Hexokinase was assayed using an A TP regenerating
system in a medium containing 85 mM Tris-HCI pH 7.5. 8 mM
MgCI2, 0.8 mM EDT A. I mM glucose. 2.5 mM A TP .0.4 mM
NADP. 10 mM phosphoryl creatine. 100 ~g creatine phosphokinase. and 100 ~Ig glucose-6-phosphate
dehydrogenase. Control
assays contained the above medium with glucose omitted.
Phosphofructokin(/.1'e. Phosphofructokinase was assayed spectophotometrically in a medium of 50 mM Tris-HCl pH 7.5, 4.5 mM
fructose-6-phosphate,
3 mM A TP, 25 mM KC1, 6 mM MgCI2.
0.15 mM N AD H and excess aldolase. triose phosphate isomerase
and glycerolphosphate
dehydrogenase.
Cilrale S)'/1lhela.~e. Citrate synthetase was assayed in a medium
of 100 mM Tris-HCI. 0.5 mM oxaloacetic acid. 0.3 mM acetyl
CoA, 0.1 mM 5-5'dithiobis-2-nitrobenzoic
acid in 40 mM phosphate, pH 8,0. The reaction was started by addition of oxaloacetic
acid and the increase in extinction at 412 nm wavelength monitored.
C}.tochrome
following
O.,ida.\'e.
phate
buffer
using
EmM(red-ox)=
3-0H
Ac.\-I CoA
pH
Cytochrome
the oxidation
was assayed
Measurements of En=lme Actirit.l
dehydrogenase.
7.6,
5 mM
oxidase
of reduced
pH 7.6 at
550 nm.
activity
cytochrome
Enzyme
was assayed
c in 50 mM
activity
was
by
phos-
calculated
19.1.
Deh)"drogenase.
in a medium
5-acetoacetyl
3-OH
containing
N-acetyl
acyl
CoA
110 mM
dehydrogenase
phosphate
cysteamine,
and
butTer,
0.15 mM
NADH.
Red muscle was rapidly dissected from both sides of ihe entire
length of the myotome. White muscle samples of about 2 9 were
dissected from the dorsal trunk muscle mid-way between the second
and third dorsal finIS (Fig. 2}. Coalfish also contain a narrow zone
of oxidative fast muscle fibres (pink muscle) situated between the
red and white muscle regions. Care was taken to avoid contamination of red and white muscle samples with pink fibres. Muscle
was minced with scissors and homogenised at 0 °C with an. U1.traturrax' homogeniser for three periods of 25 s with cooling in 5 volumes of preparation medium.
The following media were used for individual enzymes. Hexokinase (EC 2.7.2.2), phosphofructokinase
(EC 2.7.1.11 ), cytochrome oxidase (EC 1.9.3.1), and citrate synthetase (EC 4.1.3.7}:
50 mm Tris-HCI, 5 mM EDT A, 2 mM MgCI2, I mM dithioerythritol pH 7.5. Phosphorylase (EC 2.4.1.1): 100 mM phosphate buffer
pH 6.8, 20 mM NaF, I mM EDTA, 0.5 mg/ml bovine serum albumin, 10 mM dithiothreitol.
Creatine kinase (EC 2.7.32), Mg2-,
Ca2+-myofibrillar
ATPase: 0.25 M sucrose, 15 mM Tris-HCI,
pH 7.0.
Homogenates were centrifuged at 600 9 for 20 min and with
the exception of myofibrillar A TPase, enzyme activities were determined in the supernatant. Myofibrils were prepared from the 600 9
pellet by differential centrifugation as previously described (Johnston et al., 1972).
Measurements of enzyme activity were performed at 15 °C
with appropriate enzyme and reagent blanks. Preliminary experiments were carried out to determine the optimal conditions of
substrate ion concentration,
and pH for each of the enzymes
under study. Assay procedures for individual enzymes were as
follows :
Phosphorylase.
rection
I mM
Phosphorylase
in the following
EDTA.
9 mM
was assayed
medium:
MgCI2,
SO mM
10 mM
NaF.
in the physiological
phosphate
0.2 mg/ml
buffer
pH
di7.0,
fructose-I.6-
Creatine Phosphokinase. Creatine phosphokinase was determined
as previously described in a medium containing 50 mM Tris-HCI.
pH 7.14.5 mM ADP. 10 mM cysteine and 5 mM phosphoryl creatine (Johnston et al.. 1977).
"\{).ofibrillar
A TPase. Myofibrillar
A TPase was assayed as described previously in a medium of 40 mM Tris-HCI pH 75, 5 mM
MgCI2, 3.5 mM ATP, 0.1 mM CaCI2 at an ionic strength of l=
0.12 ~ (adjusted with KCI) (Johnston et al., 1972).
Results
Fibre Recruitment
Electromyograms were recorded from the superficial
red muscle at all swimming
speeds examined
(0.25-3.64 lengthsfs) (Fig. 3A, D, E). At the lower
range of swimming speeds, potentials from the red
muscle were somewhat. irregular
and about
300-400 JlV peak to peak (Fig. 3 A). At higher speeds
red muscle electromyograms became more synchronous and increased in frequency and amplitude
(500-600 JlV) (Fig. 3 E). Larger spikes of up to
1 200 JlV amplitude were also recorded from the red
muscle even at low speeds ( < 2 lengths/s) and were
associated with changes in position of the fish in the
swimming chamber (Fig. 3 C). This activity presum-
A.
150
Johnston
and
T.W.
Moon
xercise Training
in Fish Muscle
potentials. approximately 1 m V peak to peak. were
also recorded from the white muscle when the fish
accelerated against the current flow (Fig. 3 H). Although the results indicated that different areas of
white muscle were recruited as swimming speed increased no attempt was made to map out zones of
preferential recruitment. More extensive investigations on larger fish than those used in the current
study are clearly required to establish this information. However. the results indicate that both red and
white muscle fibres are active at the speed employed
(2.llengths/s) in the current training experiment.
B
D
E
Fibre Si=e
Fig. 3. A Extracellular potentials recorded from the superficial red
muscle at 0.6 lengths/s. B A record from electrodes placed in the
deep white muscle at the same swimming speed as A. The threshold
for recruitment of deep white fibres was around 1.8-2.0 lengths/s.
C Electromyograms recorded from the superficial red muscle (same
experiment as A J.hile the fish was changing position within
the swimming chamber (water current equivalent to 0.6 lengths/s).
D Electromyograms recorded from the superficial red muscle at
3.5 lengths/s. E Typical record of electrical activity recorded from
superficial red muscle at 2.1 lengths/s (the swimming speed used
in the training experiments). F Electromyograms
recorded from
the deep white muscle (W d) of the same fish as 3£ at 2.1 lengths/s.
G Electrical activity recorded from electrodes placed in more superficial white muscle (Ws Fig. 2) (in this case 0.75-1 cm from the
lateral red muscle) at around I length/s. H Spike like activity
recorded from the superficial white muscle at 1.5 lengths/s whilst
the fish was changing position within the swimming chamber.
Note; In records A-H the horizontal bar represents Is and the
vertical bar 1 mv
ably represents potentials recorded from the underlying fast pink or white muscle fibres. No electrical
activity
was recorded from electrodes inserted into
the deep white muscle (Wd. Fig. 2. bottom) at swimming speeds below 1.9-2.0 lengths/s (Fig. 3 B). Electromyograms
recorded from this deep white muscle
at the threshold speed for their recruitment
(Fig. 3 F)
were
similar
in
amplitude
and
appearance
(500-600 ~IV) to those recorded from the red muscle
at the same swimming
speed (Fig. 3 E). In contrast.
electromyogram
electrodes placed in more superficial
white muscle (Ws. Fig. 2) were acti.ve over a similar
range of speeds to the red fibres (Fig. 3 Q). Potentials
recorded from the superficial
white muscle at low
swimming
speeds (0.8-1.5Iengths/s)
were similar in
appearance to those recorded from the red muscle
although
of smaller
amplitude
(50-150
~V).
Larger
The distribution of fibre size in the red and white
muscles of exercised and non-exercised fish is shown
in Figs. 4 and 5. Since the distributions are skewed
in each case mean values have not been calculated.
However. it can be seen that there is significant hypertrophy of both fibre types. For example, in non-exercised fish, fibres with areas smaller than 200 J.lm2 constitute 9% of the total compared to 26% in the exercised
group (Fig. 4).
A more marked increase in fibre size was observed
in the white muscle (Fig. 5). White fibres larger than
4000 J.lm2constituted only 1% of the total in control
fish compared to 22% following training (P<0.001)
(Fig. 5).
Gl)'cogen
Follol\'ing
Storage and Enzyme
Actit.'ity
Let.'els
Training
The water content of both red (76.4 j: 0.7 to 79.4 j: 0.8)
and white muscles (78.8 j: 0.5 to 80.3 j: 0.3) increased
following three weeks endurance exercise training
(P < 0.05). Enzyme activities are therefore expressed
in terms of units of activity per 9 dry weight of muscle.
Glycogen phosphorylase 'activities were similar in
both muscle types in non-exercised coalfish (Fig. 6).
Activities increased by around 30% in both red and
white muscle following training (P < 0.05). In contrast, glycogen concentration was 1.5 times higher in
red than in white muscles of control fish (Fig. 6).
Training increased glycogen storage levels more in
red ( + 520% ) than white
muscles ( + 200% )
(P<0.001).
Hexokinase activities were four times higher in
red than white muscles in non-exercised fish
(P<O.OOI). No significant differences in hexokinase
activities were observed with exercise (Fig. 7). Phosphofructokinase activities, a. key regulatory enzyme
in glycolysis, were 3.6 times higher in the white than
r.A Johnston and T.W. Moon: E~ercise Trainin.in
Fish Muscle
I~I
~
30
CON1ROl
>i
f
u.
..
t200
0
2OWh
10
~Pn
>1200
4000
FIbre Area
> 4000
Fibre Area
~Fbe
Flb18 A..
Fla.4. F~quency distribution histogram (% occurrence) of red
muscle fibre area (~m].) for nonexerciscd (controll and trained
(2.1 lenatbs/s for 3 weeks) coalfish (means :t S.E. of six fish)
FiR.5. Frequency dis(ribu(ion h"loJfam (~/. occurrencel of \\hil..
muscle fibre area (Jim:) for nonelercised (conlroll lAnd tr..in~
(~.I lenR(hs/s for thRe weeks) coalfish (means.1:S.E. of ti\... fi"hl
40
2
3
~
2
20
Alee
20
10
~
CMtrol Trained
PHOSPHORYLAse
~
TI8i'Ied
GLYCOGEN
Caltroi
Trained
~XOKIN~
CaltlO
Tr8Nd
PHOSPH()FRLK:TOKINASE
(dark shadin8) and white (li8h1 shading) muscles of control and
trained coalnsh (means + S,E.. six fish)
AI.7.
Hexokinue
and phosphofnM:tokinase acti\itie~ llIm...
les.gdry wt-1.min-l)
in the red\(dark ShadiDI) and \\.hit.: Ilifhl
§hadina) muscles of coDlrol and trained toalfish (mean~ = S.I;.
nine fish)
red muscle of tank rested coalfish (P < 0.0 I ). Interestingly, following endurance training phosphofructokinase activities increased almost 8 times in the red muscle (P< 0.01) but remained unchanged in white muscle
(FiQ. 7).
Enzymes of oxidative metabolism, cytochromc oxidase and citrate synthetase, had around seven tin,~,
the activity in red compared to white muscles. TI,l.'rl.'
was a small increase ( + 30%) in citrate syntl,.:tasl.'
Jlctivit.v in red hut nnt white muscles follo\\i.,.a tr:lin.
Fli. ..GI)'cosen
concentration (ma'l dry ~1- ') and alycoaen
phosphorylase activities (I1moles .g dry wt- I , min -I) in the red
A. lohnstonand
1~?
30
~o
?0
'0
rITRr.T~
~YT()~HR()MF
Tr"tnAd
OXIOASE
r:nnt,
~-n14
rr,,;nprl
ArV;
r.nA
014
509::t 27 I.1moles/min. 9 dry wt, + 184% ) and white
muscles (744 ::t 62 to 2689 ::t 260 I.1moles/min .9 dry wt,
+ 260% ) following
training.
3
nB
in Fish Muscle
Fig.8. Citrate synthetase. cytochrome oxidase and
3.OH acyl CoA dehydrogenase activities in the red
(dark shading) and white (light shading) muscles
of control and trained coalfish (means :t S. E., ten
fish). Unitsumoles.gdrvwt-J
min-i
~nnlrtil
~VNT~FTA~~
Exercise Traininl!
~
10
~
Control Trained
T.W. Moon:
?
ru..,,"
inn
02
Control
Tr"jnprl
MYOFIBRILLAR ATPase
Control
Trained
CREATINE KINASE
Fig.9. Mg2-Ca2+ myofibrillar
adenosine triphosphatase (~moles
P; released. mg protein- 1.min -1) and creatine kinase (~moles.gdry wt-l.min-1)
activities in the red (dark shading) and
white (light shading) muscles of control and trained coal fish (means
+SF
"ix fi"h)
ing (P < 0.05) (Fig. 8). However, cytochrome oxidase
activities were not significantly changed in either muscle type by the training programme. Activities of 3OH acyl CoA dehydrogenase in non-exercised fish
were 32 times higher in red than white muscles
(P < 0.001 ). 3-OH acyl CoA dehydrogenase activities
increased by more than 3 times in white muscle
(0.066 j: 0.008 to 0.22 j: 0.015 I-Imoles/min .g dry wt)
following three weeks continuous exercise. There was
also a small increase ( + 36%) in the activities of this
enzyme in red muscle following training.
Mg2+Ca2+ myofibrillar
ATPase activities are
thought to parallel the unloaded speeds of shortening
for a wide range of vertebrate muscle types (Bi1ri1ny,
1967). A TPase activities are 2 times higher in white
than red muscle and are not affected by endurance
exercise training (Fig. 9). In contrast creatine kinase,
which is responsible for maintaining the immediate
energy supply for contraction by rephosphorylating
ADP from muscle phosphoryl creatine stores, increased significantlv
in both red (1448 j: 89 to
The arrangement of muscle fibres in the coalfish myotome is typical of many teleost fish (Fig. 2). In general, three main fibre types can be distinguished although, as with other species, various sub-populations
have been identified histochemically (Bone, 1966; Patterson et al., 1975; Kryvi and Totland, 1977; Hulbert
and Moon, 1978).
The bulk of the swimming muscle consists of multiply innervated white fibres (Patterson and Goldspink, 1972). These have a high Mg2 + Ca 2+ myofibrillar A TPase activity and a myosin sub-unit composition characteristic of fast muscles (J ohnston et al.,
1972; Focant et al., 1976; Huriaux and Focant, 1977).
Typically white fibres have low concentrations of
myoglobin, relatively few mitochondria and capillaries
and are dependent on anaerobic glycogenolysis for
their energy supply (Patterson and Goldspink, 1972 ;
Black et al., 1966; Johnston, 1977; Mosse, 1978). An
important function of white muscle is to provide the
power for short bursts of acceleration. Many teleosts
can achieve speeds of up to 26 lengths/s during feeding and escaping from predators although this level
of power output can only be maintained for a few
seconds (Wardle, 1975).
Short periods of burst speed result in the utilisation of a significant proportion of muscle glycogen
stores and the build up of a large lactate load (Black
et al., 1966). This necessitates an extended period of
recovery of up to 18 h to re-establish preactivity states
(Black et al., 1962).
Pink fibres occur as a minor component in the
coalfish myotome (2-4% ; Fig. 2). They may be distinguished histochemically from other fibre types by
their alkaline stable (nH 10.4) mvofibrillar
ATPase
IA
.Inhnstnn
anrl
W
\A""n
xercise
Training
in Fish
\fuscle
activity (Johnston et al.. 1974). In the carp. pink muscle has been shown to have a myosin light chain
composition
similar to white fibres and a mvofibrillar
A TPase activity and aerobic capacity inter;ediate
between red and white muscle (Johnston et al.. 1977).
These fibres are probably analogous to the oxidative
fast twitch
fibres of other vertebrates
(see Close.
1972). Since pure samples of pink muscle cannot be
dissected from coa1fish of the size used in the present
study they were not considered further.
The remaining
10% of the muscle mass consists
of red fibres arranged in a superficial
strip adjacent
to the lateral line system (Fig. 2). Red muscle has
a lower Mg2+Ca2+
myofibrillar
A TPase activity than
white muscle (Fig. 8) and a much higher aerobic capacity (Fig. 6). Patterson and Goldspink
( 1972) found
that the percentage of the fibre volume occupied by
mitochondria
in coalfish was 25% and 1°,/0 for the
red and white muscles respectively. Numerous studies
have demonstrated
that fish red fibres are the first
to be recruited as swimming
speed increases ( Bone.
1966; Rayner and Keenan.
1967; Hudson,
1973;
Johnston
et al.. 1977 ; Bone et al.. 1978; and our
Fig. 3). It is now firmly established that red muscle
provides the power for slow speed swimming utilising
a variety of metabolic substrates (see Bilinski.
1974).
Fish red fibres are multiply
innervated and are probably activated by junction
potentials
since a number
of electrophysiological
studies have sho\vn them to
be generall)' incapable of generating a propagated action potential
(1akeuchi.
1959; Hidaka and Toida.
1969; Kuba. 1969; Stanfield.
1972). It seems likely
that fish red fibres are analogous to the true slow
or tonic fibres of other vertebrates (Kuffier and Vaughan- Williams.
1953 ; Burke
and Ginsborg.
1956 ;
Hesse. 1970).
Elasmobranchs,
dipnoi and certain teleosts have
focally rather than multiply
innervated
white fibres
(Bone, 1964, 1966; Bone et al.. 1978 ). In these species
red muscle appears to provide all the power necessary
for sustained locomotory
effort (Bone. 1975). However, the white fibres of most teleosts are multi-terminally innervated as are the red fibres. In a study of
Cottus white muscle Hudson ( 1969) provided evidence
that these fibres were capable of two distinct electrical
responses, namely distributed junction
potentials and
propagated all-or-none
spike potentials.
In the present study deep white fibres were recruited at speeds in excess of 1.9-2.0 lengths/s. a level
of activity which the fish is capable of maintaining
indefinitely
(Fig. 3). A number of studies with other
species including
carp (Johnston
et al.. 1977; Bone
et al., 1978) and rainbow trout (Hudson.
1973; Bone
et al., 1978) have also concluded
that white fibres
are recruited for sustained activity,
InterestinQlv.
ex-
tracellular potentials recorded from white muscle at
low swimming speeds in the present study were similar
in appearance and amplitude to those recorded from
red muscle (Fig. 4). Much larger potentials, approximately I mV peak to peak, were also recorded from
the white
muscle at low
swimming
speeds
(0.8-3 lengths/s) and were associated with changes in
position within the swimming chamber. Similar observations have been made by Bone et al. (1978) on their
study of carp and rainbow trout.
Hudson (1969) observed that Cottus fast fibres
can give two types of mechanical response on stimulation, (a) a fast twitch and, (b) in the absence of a
spike potential
a graded mechanical response
produced by summation of junction potentials. It is
tempting to speculate that it is the latter type of activity which is responsible for white muscle activation
during sustained locomotion. The use of relatively
inefficient anaerobic metabolism for the production
of A TP under these conditions seems paradoxical. A
number of authors have suggested that such species
might keep in overall aerobic balance by transferring
lactate from the white muscle to other tissues for
subsequent metabolism (Bone, 1975). Indeed, both
liver and gills oxidize lactate at high rates, through
the oxidative decarboxylation of pyruvate (Bilinski and
Jonas, 1972). Other metabolic fates of lactate might
include the provision of precursors for gluconeogenesis in peripheral tissues.'
Various authors have suggested that red muscle
has a role in the synthesis of glucose from \vhite
muscle lactate (Braekkan, 1956; Wittenberger. 197.~:
Wittenberger et al., 1975). However., the recent report
that key gluconeogenic enzymes including glucose-6phosphatase, pyruvate carboxylase and phosphoenol
pyruvate carboxylase are either absent or present at
extremely low levels of activity in plaice red muscle
argues against the general applicability of this suggestion (Johnston and Moon, 1979). Whatever the ultimate metabolic fate of lactate produced by white muscle, anaerobic glycolysis would appear to remain an
energetically inefficient strategy for producing A TP
for sustained activity. At the swimming speed
employed in the current training experiments both
red and white muscle fibres are active. Three weeks
exercise at 2.1 lengths/s is sufficient
stimulus to pro'
duce a significant hypertrophy of both red and white
fibres (Fig. 5). The level of hypertrophy ofwhite fibres
is significantly greater than that found by Walker
(1971) in the same species subjected to 42 days continuous exercise training (see also Walker and Pull.
1973).
In addition to fibre hypertrophy the exercise regime employed was found to result in adaptive changl:s
in enzyme activitv levels in both muscle tvpes. For
154
example. creatine kinase the enzyme responsible for
resynthesis of A TP from phosphoryl creatine stores
increased in activity 184% and 260% in red and white
muscles respectively (Fig. 9). In general. increases in
the activity of creatine kinase in mammals subjected
to prolonged exercise only occur in training regimes
including a significant component of sprint exercise
(Guy and Snow, 1977). No change was found in the
activity of Mg2+Ca2+ myofibrillar
ATPase with
training (Fig. 6).
A striking result in the present study was the large
increase in glycogen storage levels in both red
( + 520% ) and white ( + 200% ) muscles (Fig. 7). Similar increases in glycogen content with training have
shown in the brown trout (Davison and Goldspink,
1977) as well as in various mammalian species (Gollnick et al.. 1973; Fitts et al., 1975; Guy and Snow,
1977). The hormonal mechanisms responsible for enhanced glycogen storage in fish muscle are poorly
understood (see Fontaine, 1975). Glycogen depletion
is thought to correlate with muscle fatigue over a
range of locomotory activities in both fish (Black
et al., 1966) and mammals (Ahlborg et al., 1967). Increased glycogen"storage might be expected to improve endurance following training in fish. Indeed,
Hammond and Hickman (1966) have shown an increase in the maximum sustainable swimming speed
of trout following training. Other factors which have
been implicated in improved performance include an
increased tolerance to lactate accumulation (Hochachka, 1961).
A major difference between the response of mammals and coalfish to endurance exercise training was
observed with respect to the aerobic capacities of the
muscle fibres. In mammalian red skeletal muscle
fibres endurance exercise training is associated with
a large increase in the activities of hexokinase and
tricarboxylic acid cycle enzymes' indicating an enhanced glucose utilisation and increased capacity for
aerobic metabolism respectively (see Holloszy and
Booth. 1976). In contrast, in the coalfish there was
no significant increase in cytochrome oxidase (Fig. 6)
or hexokinase (Fig. 7) and only a small increase in
citrate synthetase activity ( + 30% ) in either red or
white fibres following training (Fig. 8). There was,
however. a small increase in red muscle 3OH-acyl
CoA dehydrogenase activity ( + 36% ) indicating an
increased capacity for fatty acid catabolism. A more
marked increase in the activities of this enzyme
occurred in the white muscle ( + 30% ) although the
activities were only 3% of that found in red muscle.
An increased ability to utilise fat as an energy source
may be expected to cause a decreased rate of glycogen
utilisation and hence higher endurance following
training.
I.A. johnston
and T.W. Moon:
Exercise Training
in Fish Mus(
In general, endurance training results in little
change in glycolytic enzyme activities in mammalian
skeletal muscle (Baldwin et al., 1973; Fogd-Jorgensen
and Hyldgaard-Jensen, 1975). This contrasts with the
findings for coalfish where phosphofructokinase activities increased approximately eight fold in red muscle (Fig. 7). The activity of an enzyme under different
physiological conditions in vivo will depend on such
factors as substrate availability, the concentration of
allosteric modulators and local H+ concentrations
at the active site. However, determinations of enzyme
activity under optimal conditions in vitro do give
a first order estimate of maximum catalytic potential.
Indeed, measurements of phosphofructokinase activity, a rate limiting enzyme in glycolysis, have been
used to give an estimate of the maximum glycolytic
flux in various vertebrate muscles (Crabtree and Newsholme, 1972; Newsholme and Start, 1973). The present results are therefore consistent with an enhanced
capacity of the red fibres to perform anaerobic work
following training. In contrast to red muscle, white
muscle phosphofructokinase activities are unaffected
by training (Fig. 7). This result is perhaps not surprising since white muscle is known to have the anaerobic
potential to supply the energy for burst swimming
at far greater levels of activity than those employed
in the current training programme. Indeed, enforced
exercise at 2.1 lengths/s would have the effect of' filtering' out high speed locomotory activity.
In conclusion, the present study indicates that the
capacity of coalfish red muscle to do aerobic work
remains essentially unchanged by endurance training
under these conditions, and that any increase in the
ability to produce A TP must be met anaerobically.
It is interesting to speculate on this difference in the
response to endurance 1raining between fish and
mammals.
Resting levels of oxygen uptake in mammals are
comparable to the maximal rates measured in ectotherms (Bennett, 1978). Thus in animals with comparable efficiencies of locomotion (e.g. species of lizards
and mammals) the scope for sustained aerobic activity
is always significantly less in ectotherms than homeotherms (Bennett and Licht, 1972; Bennett and Dawson, 1976; Bennett, 1978). In general, therefore, ectotherms show a higher dependence on anaerobic metabolism during activity and this is reflected in the organisation and design of the locomotory musculature.
For example, the bulk of fish Swimming muscle
(80-95%) is composed of white fibres. During high
speed swimming it has been calculated that goldfish
derive almost 80% of their energy requirements anaerobically even at levels of effort which can be maintained for several hours (Smit et al., 1972). Thus in
general, fish have relatively low aerobic capacities
A. ./ohnston
and T.W.
and show a high
Moon:
Exercise
dependence
Train i 11.\1in
on anaerobic
,h
metabo-
lism even during sustained activitv.
An interesting
exception are certain fast-swimmi;g
oceanic species
such as tuna, where red muscle and brain temperatures are maintained
at around 29 "C bv a vascular
counter-current
heat exchanger over a \vide range of
ambient
temperatures
(Ste~ens
These species have comparable
155
Muscle
and Neill.
1978).
metabolic
rates to
mammals and it might be expected that they would
show somewhat different responses to training. However, for the majority
of fish it may well be that
the capacity of the respiratory
systems places a relatively low limit on the proportion
of aerobic r~d fibres
in the myotome. .
We are grateful for the expert research assistance of M r T. Edmunds and Mr J. Murdoch. This \\ork\,as
supported in part
by a grant from the Natural En,ironmenhll
Research Council.
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