Effects Of Temperature And Thermal Acclimation On Contractile Properties And Metabolism Of Skeletal Muscle In The Flounder (Platichthys Flesus L.)

In some cyprinid fish the effects on swimming performance of an acute decrease in temperature are partially offset by several weeks’ acclimation to low temperature (Hazel & Prosser, 1974; Smit, van den Berg & Kijn-den Hartog, 1974). The mechanisms underlying this are complex and include changes in excitationcontraction coupling (Penney & Goldspink, 1980), contractile properties (Johnston, Davison & Goldspink, 1975; Sidell, 1980; Johnston, Sidell & Driedzic, 1985) and metabolic characteristics (Sidell, 1980; Johnston, 1982; Jones & Sidell, 1982).

In common carp acclimation from 23 to 7°C results in a two-fold increase in muscle unloaded contraction velocity and maximum isometric tension development at low temperatures (Johnston et al. 1985). Measurements of enzyme activities (Sidell, 1980; Johnston et al. 1985) and mitochondrial volume densities (Johnston, 1982) in a carp and goldfish suggest that adaptations in contractile performance are matched by concomitant increases in the potential for aerobic ATP production and increases in the capacity to utilize fatty acids as fuels (Johnston et al. 1985).

Similar metabolic changes have been observed with other teleost fish following temperature acclimation experiments, including mummichogs (Fundulus hetero-clitus), chain pickerel (Esox niger) and striped bass (Morone saxatilis) (Jones & Sidell, 1982; Kleckner & Sidell, 1985; Moerland & Sidell, 1985). However, it is something of a paradox that in none of these cases is there any evidence for changes in the properties of the contractile proteins with acclimation (Sidell, Johnston, Moerland & Goldspink, 1983; Sidell & Johnston, 1985).

The flounder (Platichthys flesus L.) is a common coastal European fish living from 55 m to the tide line, and also penetrating into rivers and lakes in communication with the sea. It is a relatively eurythermal species, tolerating temperatures from 0 to 25 °C (Duthie & Houlihan, 1982). In the present study, acclimation of flounders to temperatures towards the extreme of their natural range did not alter contractile properties. Instead changes in the curvature of the force-velocity relationship are described which may serve to stabilize power output at lower temperatures.

A group of 12 flounders (Platichthysflesus L.) (303 ± 38g body weight, 25·9±l·2 standard length; mean ± S.E.) were acclimated in tanks of filtered sea water to either 5°C or 23°C for 5–9 weeks. They were subject to a 12h light: 12h dark photoperiod and fed daily on live ragworms and lugworms.

Strips of white muscle were isolated from ventral myotomes 3–5 (counting from the head). Small bundles of fibres were transferred to a cooled (0°C) dissection stage and single fibres isolated under silicone oil (BDH MS 550) using a binocular microscope (×35).

Single fibre segments were chemically skinned after mounting on the apparatus (see below) in a 1 % solution of Brij 58 (polyoxyethylene 20 cetyl ether) dissolved in relaxing solution (in mmol l⁻¹): imidazole-HCl, 20; EGTA (ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid), 10; phosphocreatine, 10; ATP, 5; MgCl₂, 6-8; KC1, 110; and 20 units ml⁻¹ crystalline creatine phosphokinase. The pH of solutions was adjusted to 7·2 at 20°C (with HC1) and allowed to vary freely with temperature such that it closely followed the ApH/AT relationship of the imidazole buffer (pH was 7·58 at 0°C and 7·11 at 25°C).

Activating solution was made by addition of CaCl₂(1 moll⁻¹ volumetric solution) to relaxing solution to give a final concentration of 10–12 mmol l⁻¹. Free ion concentrations were calculated with the aid of an iterative computer programme (based on Fabiato & Fabiato, 1979) using values for the apparent dissociation constant KCa.EGTA of 3·865×10⁶ (7·1 at 22°C). Corrections were made for changes in temperature as described by Godt & Lindley (1982). Activating solution had the following composition at pH7·2 and 20°C; pMg 3·01, pMgATP 2·29, pCa 3·37, ionic strength 0·18 mol l⁻¹ at 0°C. The free Ca²⁺ concentration of solutions was found to be maximally activating at all temperatures.

Single fibre segments (∼2mm length, 90 pm diameter) were mounted between stainless steel hooks using Plexiglas/acetone glue (Altringham & Johnston, 1982). Muscle fibres were immersed in the first of three water-jacketed baths (temperature control ±0·2°C) and skinned for 15–20 min in detergent. Sarcomere length of relaxed fibres was determined by laser diffraction and set to 2·3 μm. Fibre length and diameter were measured in situ using a graticule and high-power microscope. Force was measured with a silicon beam strain gauge (AME 801, Horten, Norway) with a resonant frequency of 3 kHz including fibre and hook, sensitivity was 0·5 mN V⁻¹ and baseline stability was 5 mV h⁻¹.

Unloaded contraction velocity (V_slack) was determined for maximally activated fibres using the slack-test method (Edman, 1979). Rapid (∼1 ms) releases to abolish tension were given using a servo-system based on a loud-speaker coil (Johnston & Sidell, 1984). The time to take up the slack was recorded using a storage oscilloscope. Between each release the fibre was re-extended (∼20ms) to its original length. Unloaded contraction velocity was determined from a least-squares fit of a plot of the applied length change versus the time taken to redevelop tension (Fig. 1).

where P₀ = maximum isometric tension, P = load, V = contraction velocity and a and b are constants.

The entire red muscle strip from the ventral myotomes was dissected, minced and approximately 0-5 g taken for enzyme analyses. White muscle was sampled from an adjacent site to that used for mechanics experiments.

Tissue was homogenized in a ground glass homogenizer with ice-cold extraction buffer (1:10 w/v) containing 100 mmol I⁻¹ Tris (hydroxymethyl) aminomethane-HC1 (Tris-HCl), lmmoll⁻¹ EDTA, 0·5mmoll⁻¹ dithioerythritol (DTE), pH7·4. DTE was omitted for determinations of carnitine palmitoyl transferase. Maximal activities of the following enzymes: hexokinase, (EC 2.7.1.1.); 6-phosphofructo-kinase, (EC 2.7.1.11); lactate dehydrogenase, (EC 1.1.1.27); cytochrome c oxidase (EC 1.9.3.1.) and carnitine palmitoyl transferase (EC 2.3.1.23) were determined spectrophotometrically (15±0·2°C) at saturating substrate and cofactor concentrations. Controls were performed in the absence of substrate and any ‘background’ activity subtracted from the measured reaction rate. Details of the assay conditions used are given in Johnston et al. (1985).

Measurements of P₀, V_slack and V_max were made on 2–3 fibres per fish at each of the temperatures studied. Results obtained for fish acclimated to either 5 °C or 23 °C were compared using a one-way analysis of variance.

No significant difference was found in the mechanical properties (maximum isometric tension, unloaded contraction velocity, force-velocity characteristics) of muscle fibres isolated from flounders acclimated to either 5 °C or 23 °C (Figs 1,2). Muscle fibres in the flounder relaxed completely following 2–3 min activations at temperatures up to 25°C.

Maximum isometric tension development (P₀) increased from around 140 kN m⁻² at 0°C to 210kNm⁻² at 25°C (Q₁₀ = 1·2; Fig. 3). Measurements of unloaded contraction velocity extrapolated from the force-velocity relationship were found to be in close agreement with those determined by the slack-test method (Fig. 4). (muscle lengths s⁻¹; L₀S⁻¹) increased from 1·6 at 0°C to 5·8 at 25°C. A plot of log V_maxversus temperature (not shown) was found to be linear over the entire range, equivalent to a Q₁₀ of around 2·0.

The force-velocity relationship becomes progressively more curved with decreasing temperature (higher values of a/P₀) (Fig. 5). Hill’s constant a/P₀ is 0·27 at 0°C, 0·12 at 10°C and 0·08 at 25°C (P<0·01). For a given load power output increases as the P-V curve becomes less curved. The net result is that for flounder changes in the curvature of the P-V relationship partially compensate for the reduction in contraction speed as temperature is decreased. Thus contraction velocity at loads for maximum power output is much less temperature dependent (Q₁₀ = 1·3) than for V_max, being equivalent to 0·4L₀ S⁻¹ at 0°C, 0·54LQS⁻¹ at 10°C and 0·63 L₀S⁻¹ at 25°C. Maximum power output has a similar low thermal dependence (Q₁₀ = 1·2) (Fig. 5).

The activities of some enzymes of intermediary metabolism in red and white myotomal muscles are shown in Fig. 6. Marker enzyme activities of mitochondrial metabolism (cytochrome oxidase), aerobic glucose utilization (hexokinase) and fatty acid oxidation (carnitine palmitoyl transferase) are 1·6 to 2·8 times higher in muscles of cold-acclimated than warm-acclimated fish. Glycolytic enzyme activities (6-phosphofructokinase, lactate dehydrogenase) are not significantly altered by temperature acclimation in either muscle type.

Temperature has a direct effect on a number of the extrinsic (fibre recruitment patterns, hormone titres) and intrinsic factors determining muscle performance (properties of myosin crossbridges, excitation-contraction coupling mechanisms, metabolism etc.). The extent to which the temperature dependence of individual parameters is important in establishing activity patterns is unclear. Skinned muscle fibres provide a useful preparation for investigating the effects of temperature on the mechanical properties of contractile proteins in isolation from nerve and membrane effects (see Johnston, 1985a).

Myosins from polar fishes are able to generate relatively high isometric tensions at low temperatures (Johnston & Harrison, 1985) but are unstable at high temperatures (Johnston et al. 1975). For example, skinned fibres isolated from the Antarctic icefish, Chaenocephalus aceratus, contract spontaneously in relaxing solution ([Ca²⁺] = 10⁻⁹moll⁻¹) when the temperature is raised to 22°C. At temperatures above 10 °C they fail to relax completely following short activations at maximal [Ca²⁺] resulting in a Ca²⁺-insensitive force component (Johnston, 1985b). The development of residual tension is associated with proportional increases in instantaneous stiffness and very low contraction velocities suggesting the formation of abnormal crossbridge linkages (Johnston & Altringham, 1985). In contrast, skinned fibres from tropical fish develop very low maximal tensions at 0°C but do not show the development of residual tension at temperatures up to 30°C (Johnston & Brill, 1984; Johnston & Altringham, 1985). In carp, contractile properties are modified by temperature acclimation: residual tension development is pronounced at high temperatures in fibres isolated from 7 °C-but not 23 °C-acclimated individuals (Johnston et al. 1985). The contractile properties of flounder skinned fibres are independent of acclimation temperature (Figs 3, 4). Maximum isometric tension at 0°C is 2–3 times that for homologous fibre types in tropical species but only 55 % of those for Antarctic fish (Johnston & Harrison, 1985). In contrast to fibres from many coldwater teleosts (Johnston & Altringham, 1985) those from flounder relax completely following short activations at temperatures up to 25 °C. The contractile proteins have properties that appear to represent a compromise between those optimal for the extremes of its thermal range. This would obviate the need for changes in the proteins with acclimation and may represent a general evolutionary strategy for fish that can tolerate sudden changes in body temperatures. Support for this idea comes from studies of the ATPase activity and Ca⁺-sensitivity of myofibrillar suspensions from muscles of the mummichog, Fundulus heteroclitus. This species inhabits coastal saltmarshes and during summer tidal cycles may experience rapid temperature changes between 15 °C and 30 °C. In common with flounder, there are no changes in the characteristics of myofibrillar proteins with temperature acclimation and the ATPase has a low thermal dependence over the range 12–35°C (Sidell et al. 1983).

The changes in the curvature of the P-V relationship with temperature found for flounder muscle fibres (Fig. 5) may provide an intrinsic mechanism which serves to stabilize power output at different temperatures. A decrease in temperature from 20 to 0°C would result in a 75 % decrease in unloaded contraction velocity but only a 31 % fall in maximum power output (Figs 3, 5). A decrease in Hill’s constant a/P₀ with increasing temperature has also been observed for skinned fibres from icefish and marlin but over a narrower range of temperatures (Johnston & Altringham, 1985). Interspecific variations in the shape of the force-velocity curve should therefore be considered along with maximum tension development and contraction speed in assessing temperature adaptation of fish myosins.

It seems likely that changes in central patterns of muscle fibre recruitment may also play a role in stabilizing locomotory performance following acute changes in body temperature. For example, in carp the threshold speed at which fast motor units are recruited decreases as the temperature is lowered from 20 to 10°C (Rome,Loughna & Goldspink, 1984). This suggests that more muscle fibres are recruited in order to swim at the same speed following a step decrease in temperature.

In flounder, following a period of cold-acclimation, the activities of enzymes associated with aerobic ATP turnover and fatty acid catabolism increase (Fig. 6). There is evidence from a variety of other species that the mechanism underlying this metabolic restructuring is an increase in the volume density of mitochondria (Johnston, 1982; Sidell, 1983; Tyler & Sidell, 1984). Sidell (1977) investigated the rate of turnover of cytochrome c in the green sunfish Lepomis cyanellus and found that a reduction in body temperature resulted in a smaller decrease in its rate of degradation than synthesis, leading to a net increase in protein concentration, presumably reflecting changes in mitochondrial turnover. Expansion of aerobic metabolism by such a mechanism may require several weeks (Sidell, Wilson, Hazel & Prosser, 1973). Determinations of optimal enzyme activities provide information on maximal metabolic potentials of tissues but do not tell us anything about the fluxes of substrates under different physiological states. The flux of carbon through metabolic pathways is thought to be integrated and controlled via modulation of the activity of regulatory enzymes by H⁺, adenylates, substrates, products and other metabolites (Newsholme & Start, 1973). One possibility is that the concentrations of these modulators change following an acute drop in body temperature in such a way as partially to offset the direct effects of temperature on enzyme reaction rate (Walesby & Johnston, 1980; Walsh & Somero, 1982). This may serve to reduce the temperature dependence of the ATP-generating pathways providing an immediate temperature compensation mechanism to parallel that described for the contractile proteins (Fig. 5).

This work was supported by project grant GR3/4936 from the Natural Environment Research Council. Alele Wokoma is grateful to the Federal Government of Nigeria for a research studentship.