I. Johnston and M. Lucking: Temperature Acclimation in Fish Mu ... tiated (Johnston et al., 1974, 1977) from the other fibre types. Table I. ..... Bob Adams is grate-.
--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.
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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
