--Journal
I.
comp.
Physiol.
If4,
111-116
(1978)
of
Comparative
Physiology.
B
(1;) by Springer-Verlag
1978
Temperature Induced Variation in the Distribution
of Different Types of Muscle Fibre in the Goldfish (Carassius auratus)
Ian
Johnston
Department
and
Margaret.
Lucking
of Physiology, University
Accepted November
of St. Andrews, St. Andrews, Fife, Scotland
18, 1977
Summary. Goldfish ( Carassius auratus L.) were acclimated to environmental temperatures of 3 °C, 18 °C
and 31 °C for a period of three months. Cytochemical
techniques were used to study the metabolism and
myofibrillar A TPase activities of individual muscle
fibres. Fish muscle is composed of three basic fibre
types each with distinct contractile and metabolic
characteristics. Cold acclimation resulted in a shift to
a more aerobic type of metabolism, particularly in the
red and pink fibres. In addition, environmental temperature was found to affect the size and relative
distribution of the different fibre types in the myotome. The total number of pink and red fibres increased significantly with cold acclimation. Mechanisms of environmentally-induced
adaptation of
muscle fibre phenotype are discussed.
In addition to changes in the metabolism and
distribution of muscle-fibre types, biochemical studies
have provided evidence for different kinetic forms of
Mg2+Ca2+ myofibrillar ATPase at different environmental temperatures. Activities
of myofibrillar
ATPase assayed at 31°C were 2-3 times higher in fish
acclimated to the higher environmental temperature.
Activation enthalpy (iJH*) of the ATPase was also
significantly reduced in the cold adapted enzyme.
Reduction of iJH * in the cold acclimated A TPase is
thought to reduce the temperature sensitivity of the
activation process thus partly compensating for the
reduced cell temperature.
Introduction
Aquatic poiki.lotherms often show a complete or
partial compensation in metabolic rate,. and locomotory activity following acclimation to different environmental temperatures (Hazel and Prosser, 1974).
For example, acclimated Atlantic salmon were found
to exhibit similar levels of spontaneous activity at
different environmental temperatures (Fry, 1967; Peterson and Anderson, 1969). Studies of swimming
behaviour in flumes and respirometers have also
shown a positive correlation between maximum
swimming speed and acclimation temperature in a
number of species (Roots and Prosser, 1962; Griffiths
and Alderdice, 1972; Smit et al., 1974). Adaptation in
muscular performance may result from changes in
neural function (Bass, 1971; Lagerspetz, 1974), contractile proteins (Johnston et al., 1975a) and muscle
metabolism (Hochachka and Hayes, 1962; Hazel and
Prosser, 1974). Temperature compensation is thought
to involve changes in the concentrations and kinetic
properties of key enzymes (Wilson, 1973; Hoch'lchka
and Somero, 1973; Hazel and Prosser, 1974), shifts in
the relative importance of different metabolic pathways (Hochachka and Hayes, 1962; Somero, 1973)
and modifications of lipid metabolism and membrane
properties (Knipprath and Mead, 1967; Dean, 1969).
In general cold adaptation is associated with a shift
to a more aerobic type of metabolism (Hazel and
Prosser, 1974). Numerous studies have reported increased activities of tricarboxylic acid cycle and electron transport chain enzymes in fish muscle adapted
to low environmental temperatures (Jankowsky and
Korn, 1965; Lehmann, 1970; Hazel, 1972).
In common with other vertebrate skeletal muscles
fish myotomes are composed of populations of fibres
consisting of three basic types differing in their patterns of innervation (Barets, 1961; Bone, 1964), contractile properties (Barets, 1961; Johnston and Tota,
1974) and metabolic characteristics (Johnston et al.,
1975a; 1977). Most studies of temperature adaptation
in fish muscle have been concdrned with white muscle
(Hazel and Prosser, 1974). However, it is likely that
the metabolic responses to changes in environmental
temperature will vary according to the metabolic
characteristics of each fibre type (Dean, .1969; Hazel
and Prosser, .1974). In the present study it was found
that acclimation temperature not only had a profound effect on the metabolism of individual fibres
but also resulted in a change in the relative distribution of different muscle fibre types.
0340-7616/78/0124/01
/$01.20
112
Material" and Method"
Fish. Common goldfish ( Carassius auratus L.) were obtained from
a local supplier during September 1976. Fish for studies of muscle
fibre type distribution averaged 5 cm in length and 1.5 9 in weight.
In order to obtain sufficient muscle for biochemical measurements
larger fish of approximately 50 9 weight were used for preparation
of myofibrils. Fish were maintained under natural daylight conditions in 100 gallon tanks of circulated filtered freshwater regulated to 3 cC, 18cC or 31 cC by thermostatically-controlled
( :to.2 °C) cooler circuits.
Initially all fish were maintained at 18 °C and the water temperature was raised or lowered gradually over a period or 2 weeks,
until the acclimation temperature was reached. Acclimation was
for a continuous period of 3 months. Fish were fed on alternate
days with commercial fish pellets. Since fish acclimated to 31°C
displayed a greater feeding activity than those at 18 °C and 3 °C,
they were fed more in order to keep the body weights of all three
groups the same.
Histochemistry. Fish were stunned by a blow to the head and killed
by decapitation. Tissue blocks were cut with a sharp scalpel
through the whole cross-section of the trunk at a point 8-10
myotomes from the tail. Preliminary studies had shown that at this
point there was minimal variation in fibre number and diameter
between adjacent myotomes. Blocks of tissue were mounted on
cryostat chucks, and embedded in OCT compound. Tissue was
rapidly frozen by immersion in isopentane cooled to its melting
point in liquid nitrogen ( -159 °C). Blocks were placed in a refrigerated cabinet at -20 °C for 1 h after which serial sections were
cut at 8-12 microns thickness and mounted on cover slips.
Sections were stained for glycogen, lipid, phosphorylase, succinic dehydrogenase (SDH) and myofibrillar
A TPase at 18 °C as
described previously (Johnston et al., 1974, 1975b; Patterson et al.,
1975). Preincubation
of sections in 18mM CaCIl 100mM 2amino-2-methyl-1-pfopanol,
pH 10.4-10:5 for short periods prior
to staining for ATPase activity allowed pink fibres to be differentiated (Johnston et al., 1974, 1977) from the other fibre types.
Measurement of Fibre Number, Distribution and Size. Serial sections stained for SDH and myofibrillar ATPase activity andpreincubated to show pink fibres were projected onto large sheets of
paper, the areas traced out and the percentage area occupied by
each fibre type calculated. Total numbers of red, and pink fibres
and also those white fibres staining for succinic dehydrogenase
activity were determined directly by counting from the projected
image. Fibre diameters of the three fibre types, distinguished on
the basis of differences in staining for oxidative, glycolytic and
myofibrillar
A TPase enzymes, were measured using a calibrated
microscope eyepiece. A total of 100 fibres were measured randomly for each of the 3 fibre types. Measurements of fibre number
and size were made on a total of 8 fish at each of the acclimation
temperatures. Data were compared using analyses of variance for
equal sample numbers.
Preparation of Myofibrils. Red muscle was carefully dissected from
the trunk musculature of the acclimated goldfish. Only the most
superficial red fibres were taken from each fish to avoid contamination with pink or white fibres. The muscle from 6 fish was
used for each preparation. Following mincing with scissors muscle
was homogenised at 0 °C with a Polytron blender for 3 x 40 s with
intermittent cooling, in 0.1 M KCI, 5 mM Tris-HCI pH 7.2. Homogenisation was monitored by microscopical examination. All subsequent operations were performed at ().--4°C. The homogenate was
centrifuged at 2000 g for 5 min and myofibrils prepared from the
residue as described by Perry and Grey (1956). Myofibrils were
finally suspended in 0.1M KCI, 5mM Tris-HCrpH7..2
ata concentration of approximately 5 mg/ml. Protein was determined by a
standardised biuret method IGornal1 et al.. 1949).
I. Johnston and M. Lucking:
Temperature
Acclimation
in Fish Mu
Assay of ATPase Activity. Mg2+Ca2+ Myofibrillar
ATPase activity w~s measured by monitoring H+ release in a pH stat in a
medium of90mM
KCI, 5mM Mg2+; 3.5mM ATP, 1tnM sodium
azide 0.1 mM CaCI2 pH 7.5. Temperature control was achieved
using a thermostated water jacket and thermistor probe (temperature control :to.01°C). ATPase,activity
was measured at various
temperatures between 0 and 35°C. Activation enthalpies were
calculated from the slopes of the corresponding Arrhenius plots.
Regression lines were tested for linearity and significant difference
bv an analvsis of variance method.
Results
In order to ch!lracterise different muscle fibre types it
is necessary to stain for a whole range of energy
stores and enzyme activities. In common with many
vertebrates, histochemical profiles of fish myotomal
muscle reveal three distinct fibre types. In goldfish,
red fibres constitute around ~5 % of the total crosssectional area except in the last two myotQmes adjacent to the tail where the proportion is around 1517% (Table I). These fibres are arranged superficially
in a wedge shape adjacent to the lateral line system
(Fig.l). Red muscle is thought to be composed of
slow twitch oxidative fibres. They stain lieayjly for
the mitochondrial
enzyme succinic dehydrogenase
and have a low myofibrillar
A TPase activity. The
bulk of the trunk musculature is white and is composed of fibres which have a high myofibrillar AT Pase activity and a low staining for oxidative enzymes (Fig.l). In many fish species there is another
fibre type situated between the red and white muscle
layers. These so-called pink fibres may be characterised histochemically by preincubating frozen sections
in 18mM
CaClz, 100mM
2-amino-2-methyl-lpropanol pH 10.4 for short periods prior to staining
for myofibrillar
ATPase activity (Johnston et al.,
1974). By selecting the appropriate conditions it is
Table I. Effect of acclimation temperature on the fibre size and
percentage area occupied by different types of muscle fibre in the
goldfish. Mean:tS.E. of8 fish
Acclimation
Fibre area( %
temperature
oc
Red fihre"
Pink
3
18
31
4.0:t0.23
4,0:t0.40
3.3:!:0.30
4.7:!:0.30
3
18
,1
13.00 ::!:OJS
12.60::!:0.12
11.69::!:0.18
fihr,,"
White fibres
5.0:!:0.60
89.1:t0.75
90.9:t0.60
92.0+0.60
17.60:t0.22
18.60+0.20
15.56:;;;0.18
23.60:tO.27
24.42:tO.26
23.74:tO.26
8.5:!: 1.04
.
I. Johnston and M. LuckinR: Temperature Acclimation in Fish Muscle
113
--~~--",,---,,_.~---"c:-~--"'"-~--~
Fig. I. a Transverse section through the myotome of a goldfish acclimated to 3 °C for three months showing the lateral line canal system
(L), red (R), pink (P) and white ( W) fibre types. Section is stained for the mitochondrial marker succinic dehydrogenase. (Magnification
x 40).b Similar section from a 3 °C acclimated fish stained for glycogen. Magnification
x 40). Insert shows another section stained for
myofibrillar A TPase activity after having been preincubated for a short period in 18 mM CaCI2, 100 mM 2-amino-2-methyl-l-propanol,
pH 10.4, to selectively show up pink fibres. c Section stained for succinic dehydrogenase from a fish acclimated to 18 °C. Note the
significantly lower staining for succinic dehydrogenase activity (Magnification
x 40). d Similarly stained section from a fish acclImated
to 31 °C. Note the greatly reduced proportion of fast oxidative pink fibres. (Magnification
x 40)
-'
possible to inactivate both red and white fibres leaving the pink fibres as the only type staining for
A TPase activity (Fig.l). Pink fibres are particularly
prominent in carp species where they occupy an area
equal or greater to that of the red muscle (Table 1). It
has recently been demonstrated that pink fibres correspond to fast twitch oxidative fibres with an intermediate myofibrillar A TPase activity to red and
white fibres (Johnston et al., 1977).
Comparison of serial sections stained for myofibrillar A TPase to localise pink fibres (see above) and
for succinic dehydrogenase activity allowed counts to
be made of total fibre number and area. In order to
compare staining intensity of tissue sections from the
different groups. sections were incubated in batches of
three (3 oC, 18 oC, 31°C) under identical conditions.
Staining for succinic dehydrogenase, phosphorylase,
and glycogen was considerably higher in the superficial red fibres of cold acclimated fish (Fig. 1).
It can be seen from Table 1 that the percentage
area occupied by each of the three fibre types is not
fixed but varies according to acclimation temperature. The area occupied by red fibres was significantly
less at the higher environmental temperature (31 OC)
(P < 0.05). Pink fibre area increased froni 5 % of the
cross-section at 31°C to 8.5% at 3°C (P<0.01). The
increased areas occupied by red and pink fibres with
cold acclimation resulted in a corresponding reduction in the white muscle (P < 0.01; Table 1). The
increase in red and pink fibre area with cold acclim:Jtinn w:!s found to he due to a substantial increase
in total fibre number, although fibre size was also
somewhat larger (Table 1). Red fibre number increased by approximately 20% in 3 oC compared to
18°C or 31°C acclimated fish (Fig. 2; P<0.01). Pink
fibre number was also found to be inversely correlated with acclimation temperature (Fig.2). The
total number of pink fibres more than doubled in fish
acclimated to 3°C compared to 31°C (P<0.01;
Fig. 2). White fibres were found to be unstained for
succinic dehydrogenase activity at the highest environmental temperature. However, in 3 oC acclimated fish, some 1500 white fibres, adjacent to the
pink muscle zone, showed some staining for oxidative
enzymes (Fig. 1, 2). It would appear that acclimation
to lower temperatures not only results in changes in
the oxidative capacity of individual fibres but is also
associated with changes in the relative numbers of
different types of muscle fibres.
In view of these qualitative changes in fibre
biochemistry it was thought to .be of interest to
examine the Mg2+Ca2+ myofibrillar ATPase activity
of the superficial red fibres from fish acclimated to
3 oC and 31°C. Evidence is presented for adaptive
changes in the properties of the A TPase (Table 2;
Fig.3). Activities of myofibrillar A TPase assayed at
31°C were 2-3 times higher in fish acclimated to the
higher environmental temperature (P < 0.01; Fig. 3).
Cold acclimation also resulted in a lower enthalpy of
activation (L1H*) of the A TPase (Table 2; P <0.01).
The A TPase activities of both hot and cold acclimated myofibrils were calcium sensitive: activities
I. Johnston and M. Lucking:
114
Temperature
Acclimation
in Fish Muscle
3000
0;
-0
E
"
c 2000
..
...
-0
Gi
u
III
"
Fig. 2. The total number of red and pink
fibres and number of white fibres showing
staining for succinic dehydrogenase activity
in the myotomes of goldfish ( Carassius
auratus) acclimated for 3 months to 3 °C,
18 °C and 31°C. Histograms represent
Mean :t S.E. of fibre counts from 8 fish
~ 1000
-a
'0
Table 2. Effect of acclimation temperature on the properties of the
Mg2 + Ca 2+ myofibrillar
A TPase (~ molesjmgjmin)
from the
assayed in the presence of 4 mM. EGT A being approximately
15% of that assayed in the presence of
superficial Ted muscle of goldfish. Results repre~ent the mean of
2 preparations pooled from the muscle of. 6 fish
trace amounts
Acclimation
temperature
Mg2+ Ca2+
A TPase
(OC)
activity
11°C:
sensitivity
EGTA
( %)
Mg2+
Mg2+
enthalpy
(1.c",,\/mnl,,\
0.27
0.63
71n
'C.'
iii
o
E
(lj
O
E
.~
-0.
In
0..
.0
86.7
R~'
12,850
18.200
,,
~
In
(lj
a
E
o
di
.2
33
34
35
36
1/T(OK)x103
Fig. 3. Arrhenius plot of Mg2+Ca2+ myofibrillar
ATPase activity
for the superficial red muscle fibres of goldfish acclimated to 3 °C
(open circles) and 31°C (solid circles). Assay conditions are given
in the text. The proportion of myosin in the myofibril was assumed
to be 54% (Benda1l. 1969)
ni~c"~~i()n
Ca2+
A TPase activity)
3
1f
(10-4 M) (Table 2).
Activation
Calcium
( 1-
of calcium
Fishmyotomes are composed of three basic types of
muscle fibre arranged in anatomically discrete regions. Red fibres contain high concentrations of
myoglobin and are rich in mitochondria (Patterson
and Goldspink, 1973; Nag, 1972). Electromyographical studies in carp have shown that these slow twitch
fibres are the first to be recruited during slow swimming (Johnston et al., 1977). The next fibre type to be
recruited with increasing swimming speed is the socalled pink fibre (Johnston et al., 1977). It has been
suggested that the recruitment of these fast oxidative
fibres is associated with sustained effort at speeds at
which the fish can no longer meet all its energy
requirements by gas exchange at the gills (Johnston
et al., 1977). Still faster cruising speeds and bursts of
activity invol.ve recruitment of fast twitch white fibres. These fibres which make up the bulk of the
myotome have very few mitochondria and rely on
anaerobic glycolysis for their energy supply (Johnston
and Goldspink, 1973; Hudson, 1973; Walker and
Pull, 1973).
Experimental studies involving goldfish swimming in exercise chambers have shown a partial
compensation in locomotory performance with temperature acclimation (Fry, 1967; Smit et al., 1974).
Interestingly, adaptation to temperatures above 10°C
Johnston and M. Lucking:
Temperature
Acclimation
in Fish Muscle
does not result in a corresponding increase in ventilation rate even though oxygen tension is lower and
activity higher at these ambient temperatures (Freeman, 1955). Indeed acclimation to higher environmental temperatures has been shown to result in a
reduced oxygen uptake (Smit et al., 1974). Ventilation
rate in fish is a compromise between the conflicting
demands of gas exchange and osmoregulation at the
gills (Randall et al., 1972; Smit et al., 1972). An elevated ventilation rate at high temperatures would
therefore result in both an increase in energy expenditure by the respiratory muscles and also increased osmotic work associated with ion transport
across the gill epithelium. It appears therefore that
acclimation to higher environmental temperatures
leads to an increased contribution of anaerobic pathways of energy production in the muscles rather than
an increase in ventilation rate (Hazel and Prosser,
1974; Smit et al., 1974). Indeed carp species have
impressive anaerobic capacities and are able to meet
all their energy demands anaerobically during prolonged periods of environmental hypoxia (Johnston,
1975). Evidence for a shift to anaerobic metabol.ism
at higher temperatures also comes from studies of
tIssue respiration (Hazel and Prosser, 1974; Dean,
1969; Smit et al., 1974). In the present study it was
also found that staining for succinic dehydrogenase
activity was considerably reduced in the fast and slow
twitch oxidative fibres of fish acclimated to higher
environmental
temperatures (Fig.1). Similar decreases in mitochondrial density, and tricarboxylic
acid cycle and electron transport chain enzyme concentrations and activities have been reported in the
white muscles of a number of species (Jankowsky and
Korn, 1965; Wilson, 1973; Lehmann, 1970; Hazel,
1972; Smit et al., 1974). Significantly the only white
fibres showing staining for SDH activity in the present study were those adjacent to the pink muscle in
fish at the lower environmental
temperatures
(Fig. 1, 2). The ratio of volume occupied by mitochondria in the red and white muscles of a closely
related species, Carassius carassius, has been shown
to be in the order of 23: 1 (Patterson and Goldspink,
1973). Increases in aerobic metabolism in cold acclimation are therefore likely to be of proportionally
greater significance in the red and pink muscle fibres.
Environmental temperature was found to affect
the relative distribution of muscle fibre types in the
myotome. Fish kept at the lowest environmental.
temperature had a significantly higher proportion of
slow and fast twitch oxidative fibres. The proportion
of pink muscle in fish acclimated to 31°C was only
5% compared to 8.5% at 3 °C (Table 1). Although
pink fibres in cold acclimated fish were sliQht]v JarQ-
115
er, most of the increase in area was accounted for by
a substantial increase in muscle fibre number (Tabl.e 1 ; Fig. 2).
In addition, evidence has been found for adaptive
changes in the thermodynamic properties of the
Mg2+Ca2+ myofibriUar ATPase with temperature
acclimation. Adaptation to 31°C resulted in a significant increase in A TPase activity compared to 3 °C
fish (Table 2; Fig.3). Activation enthalpy was also
reduced in the col.d acclimated enzyme (Table 2). This
presumably represents an adaptive advantage in reducing the temperature sensitivity of the activation
process in the cold acclimated A TPase. A good correlation between activation enthal.py (AH*) and cell
temperature has been shown for a large number of
fish species for both Mg2+Ca2+ myofibrillar ATPase
(Johnston and Goldspink, 1975; Johnston and Walesby, 1977) and pyruvate kinases from white muscle
(Somero and Low, 1976). Changes in the catalytic
efficiencies of enzymes for function at different cell
temperatures are related to structural modifications
in enzyme thermostability (Ushakov, 1964; Johnston
et al., 1973; Johnston and Walesby, 1977). Cold adapted enzymes are considerably more thermolabile at
high temperatures than those from homologoui tis.sues of tropical species (Ushakov, 1964). For .example, the half life of inactivation of white muscle
Mg2 + Ca 2+ myofibrillar A TPase at 37 °C has been
shown to vary from around 1 min in Antarctic species
to over 500 min in fish living in a hot-springs sodalake (Johnston and Walesby, 1977). Experiments involving goldfish white muscle have also shown simil.ar changes in the denaturation rates of myofibrillar
A TPase following temperature acclimation (Johnston
et al., 1975b).
Compensatory adaptations in l.ocomotory behaviour in response to temperature acclimation
would therefore appear to involve changes in the
metabolism, contractile properties and relative distribution of different muscle fibre types. Similar changes
in enzyme activity and fibre distribution, to those
that result from environmental temperature adaptation, have been reported in rodent skeletal muscles
subject to chronic endurance running (Baldwin et al.,
1972, 1973; Winder et al., 1974). Little is known
,
.
about the role of nervous, neuroendocrIne, hormonal
and local. factors in temperature adaptation in teleosts (Hazel and Prosser, 1974; Lagerspetz, 1974).
However, changes in the distribution of the types of
muscle fibres provide a straightforward means of demonstrating that temperature adaptation has occurred and provide a promising system for future investigations on the various mechanisms of temperatllr~ ~rrlim~ti()n in fi~h
.
116
This work was supported by grants from The Royal Society
Scientific Investigations Fund and The Science Research Council.
The skilled photographic assistance ofMr.
Bob Adams is gratefullv acknowled!!ed.
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Temperature
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in Fish Muscle
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