Temperature Acclimation in the Common Carp: Force-Velocity Characteristics and Myosin Subunit Composition of Slow Muscle Fibres

The maximum cruising speed of some freshwater fish is increased at low temperatures and decreased at high temperatures after several weeks of cold acclimation (Fry and Hart, 1948; Heap and Goldspink, 1986). The mechanisms underlying this plasticity in swimming performance are complex and include changes in the relative proportions of different muscle fibre types (Johnston and Lucking, 1978; Jones and Sidell, 1982), altered patterns of muscle fibre recruitment (Rome et al. 1985) and adaptations in the properties of membranes and nerves (Harper et al. 1989). In the goldfish (Carassius auratus L.) the ATPase activity of fast muscle myofibrils is around three times higher at 1°C in fish acclimated to 1°C than in those acclimated to 26°C (Johnston et al. 1975). Similar increases in ATPase activity with cold-acclimation occur in other cyprinids, including common carp (Cyprinus carpio L.) and roach (Rutilus rutilus L.) (Heap étal. 1985), but may not be widespread among teleosts (Walesby and Johnston, 1981; Jones and Sidell, 1982; Sidell and Johnston, 1985; Johnston and Dunn, 1987).

Skinned fibres have been used to investigate the force-velocity characteristics of fast and slow myotomal muscles in common carp acclimated to either 7 or 23°C (Johnston et al. 1985). At 7°C, maximum tension (P₀) and unloaded contraction velocity (V_max) were about 1.5–2.0 times higher in cold-than in warm-acclimated fish. Recent studies, however, have shown differences in the mechanical properties of skinned and live fish muscle fibres. In particular, skinned fibres have lower tensions and shortening speeds, making them unsuitable for quantitative calculations of muscle power output (Altringham and Johnston, 1988; Curtin and Woledge, 1988). Unfortunately, all attempts to obtain a suitable live fibre preparation from fast myotomal muscle in the carp have proved unsuccessful (see also Rome et al. 1988). However, stable intact preparations can be isolated from the superficial slow fibre region of the pectoral fin abductor superficialis (Ab.s) muscle. In the present study we have used this preparation to calculate muscle power output from the force-velocity relationship in carp acclimated to either 8 or 20°C. The effect of temperature acclimation on the subunit composition of slow muscle myosin has also been investigated.

Common carp (Cyprinus carpio L.) were obtained from Humberside Fisheries, Driffield, England. The total length and body mass of the fish studied was 31.0±2.5cm and 735±10g (mean±s.D.; N=24). Fish were held in tanks of partially recirculated filtered fresh water at either 8 or 20°C for 6-12 weeks (12 h light: 12 h dark). Trout pellets were fed daily.

Carp were killed by a blow to the head followed by decapitation and pithing. The intact pectoral fin and associated skeleton were removed from the fish, the cleithrum being severed at the level of the lateral line. The assembly was pinned out on a silicone elastomer base (Sylgard 184, Dow Corning), with the abductor superficialis muscle (Ab.s) face down. The preparation was covered with Ringer’s solution (in mmoll⁻¹): NaCl, 119.0; sodium pyruvate, 10; KC1, 2.7; MgCl₂, 1; CaCl₂, 1.8; NaHCO₃, 2.5; pH7.4 at 5°C). The other muscles of the pectoral assembly were removed and the bone reduced as far as possible without damaging the attachments of the Ab.s. The preparation was repinned via the skin and the layer of fast fibres removed from the ventral surface. Finally, the slow fibre region was pared down to a bundle of 60–100 fibres (see Fig. 1). Aluminium foil clips were attached as close to the bone as possible (200–300 μm), and the bone was further reduced. Dissection was performed on a cooled plate (8°C) and the Ringer was changed frequently.

The apparatus consisted of a Perspex chamber through which aerated Ringer was circulated at constant temperature (±0.1 °C). Force was measured using a silicon beam strain gauge (AE801, AME, Horten, Norway). Muscle length was measured and controlled via a servo motor (MFE model R4–077, Emerson Electronics, Bourne End, Bucks) and control unit built in-house (Altringham and Johnston, 1988). Sarcomere length was measured by laser diffraction and set to 2.3 μm. This resting sarcomere length was selected on the basis of preliminary experiments on the length-tension relationship of fibres. The average length of fibre bundles was measured using a binocular microscope. Fibres were stimulated via two platinum wire electrodes lying on either side, using 1.5 ms pulses at 1.2× threshold voltage. The stimulus strength and frequency required to produce maximum tetanic contractions were determined at 8 and 20°C for each preparation. Data were collected and analysed on a Nicolet 3091 digital oscilloscope, and stored on a BBC microcomputer.

The isometric properties of fibre bundles from both acclimation groups were measured at 8 and 20°C. The effects of temperature were reversible so several cycles of heating and cooling were performed on each preparation. Contraction velocity was measured at various loads using isovelocity releases (see Fig. 2A). During the plateau phase of tetanus (2 s, 25 Hz) the preparations were given an initial 2 ms release, of varying magnitudes, to lower the tension. A second, slower release was adjusted to hold the tension constant after the step (Altringham and Johnston, 1988). Force-velocity (P–V) curves were constructed by plotting velocity against the relative tension over the first 10 ms interval after release. P–V data for individual fibres were fitted to a hyperbolic-linear curve described by Marsh and Bennett (1986):

At the end of experiments preparations were frozen at their resting lengths using isopentane cooled to its melting point in liquid nitrogen (–159°C). Frozen fibre bundles were inserted into a block of liver tissue mounted on a cryostat chuck and re-frozen. Frozen sections (10μm) were cut and stained for myofibrillar ATPase and succinate dehydrogenase activity (Johnston et al. 1974). The cross-sectional area of individual muscle fibres was determined by digital plannimetry using a microscope drawing arm. The total cross-sectional area of muscle fibres, excluding connective tissue and space, was used to calculate the tension.

The myosins from seven warm-acclimated and six cold-acclimated carp were studied electrophoretically. Muscle fibre bundles (5–18 mg) were placed in a test tube containing 25 vols of 40mmoll⁻¹ NaCl, 3mmoll^−l phosphate buffer pH7.0 at 2°C and crushed with a glass rod. The buffer was removed and replaced with 25 vols of a solution containing (mmol I⁻¹): Na₄P₂O₇, 50; EGTA, 2.5; β- mercaptoethanol, 1; 50% glycerol, pH8.8 at 2°C. The sample was homogenised using a hand-held pestle, and left to extract for 60 min on ice. The myosin extract was centrifuged at 40000g for 60min at 1°C, and stored at –20°C.

Sodium pyrophosphate gels contained: 25 mmol I⁻¹ Na₄P₂O₇ (pH 8.8 at 1°C), 3.8% acrylamide, 0.2% N,N’-methylene bisacrylamide (BIS), 10% glycerol, 0.1% N,N,N’,N’-tetramethylethylenediamine (TEMED) and 0.05% ammonium persulphate. The electrode buffer, which contained 25 mmol I⁻¹ Na₄P₂O₇ (pH 8.8 at 1°C) and 10% glycerol, was recirculated at 700mlmin⁻¹. Electrophoresis was carried out at 1–2°C. Samples (20μl for analytical and 60 μl for preparative gels) were loaded and run on to the gel for 30 min at 35 V; the voltage was then increased to 100 V and run for 18 h. The myosin bands were cut out from preparative gels, rapidly stained with Coomassie Blue, and incubated for 30 min at 30°C in an equal volume of 125 mmol I⁻¹ Tris-HCl pH 6.75, 4% SDS, 5% β-mercaptoethanol, 20% glycerol and 0.004% Bromophenol Blue. The subunit composition of myosins was examined using 8 % and 15 % SDS-PAGE gels (Laemmli, 1970).

Gels were stained for 2h at 30°C in 0.1 % Coomassie Blue, 50% methanol, 7 % acetic acid, and destained in 50 % methanol, 7 % acetic acid. A rapid method was used for preparative gels. The gel was placed in the same staining solution as above until the bands became visible (about 5 min) and washed for 10 min in five changes of ultrapure water (Milli-Q). The bands were then ready for cutting out for SDS-PAGE. SDS-PAGE gels of the myosin light chains were silver-stained using the Sigma silver stain kit. Protein bands were characterised using myosin light chain markers from carp myotomal muscles (Crockford and Johnston, 1990; Johnston et al. 1990) and proteins of known relative molecular mass (14×10³– 200×10³) (Sigma). Gels were scanned at 650nm using a Shimadsu CS9000 densitometer.

Data for the two acclimation groups were compared using an unpaired t-test.

The arrangement of pectoral fin muscles in the carp is shown in Fig. 1. Isolated fibre bundles were fibre-typed using serial frozen sections stained for myofibrillar ATPase and succinate dehydrogenase (SDH) activity (Fig. 1). The preparations were mostly composed of slow fibres which have a high SDH activity and a low myofibrillar ATPase activity which was readily inactivated at pH 10.4 (Johnston et al. 1974). Some preparations also contained a small percentage of fibres which stained intensely for myofibrillar ATPase activity following alkaline preincubation (Fig. 1). These correspond to fast oxidative or ‘intermediate’ muscle fibres (Johnston et al. 1974). The percentages of fast oxidative fibres in the preparations were 3.3±3.1 for from 8°C-acclimated and 2.8±2.7 for 20°C-acclimated fish (mean±s.D.).

Isometric contractile properties showed a range of thermal sensitivities and varied with acclimation temperature (Table 1). Ræ values for maximum tetanic tension (a number analogous to Q₁₀ for non-rate variables, Bennett, 1984) were 1.10 and 1.34 for fish acclimated to 8 and 20°C, respectively. At 8°C, maximum tetanic tension was 32% higher in fibres from 8°C-than in those from 20°C-acclimated fish (P<0.01). Values for tetanic tension measured at the acclimation temperature of each group were similar (Table 1).

The times to 50% peak force (T₀._5a) and from peak force to 50% relaxation (T₀._5r) were measured (Fig. 2B; Table 1). For isometric twitches T₀._5r was much more temperature dependent than T₀._5a (Fig. 2B). Q₁₀ values were 2.53 and 2.13 for T₀._5r and 1.21 and 1.28 for T₀._5a in 8°C-and 20°C-acclimated fish, respectively. The corresponding Q₁₀ values for tetanic contractions were more similar: the Q₁₀ values for T₀._5r and T₀._5a were, respectively, 2.14 and 2.17 in 8°C-acclimated fish and 1.69 and 1.86 in 20°C-acclimated fish. For both twitches and tetani, T₀._5a was 15 % higher and T₀._5r was 20% higher at 8°C for fibres from 8°C-than for those from 20°C-acclimated fish (Table 1, P<0.01). Measured at the respective acclimation temperatures, T₀._5a was 1.6 times and T₀._5r was 1.7 times faster in 20°C-than in 8°C-acclimated fish. Thus, the rates of tension development and relaxation exhibited capacity adaptations in the region of 13 – 17% following cold acclimation.

The P – V relationship of fibres was studied at 8°C. Representative P – V curves from cold-and warm-acclimated carp are shown in Fig. 3, and all the data are summarised in Table 2. The unloaded contraction velocity (V_max) of fibres was 17% higher in 8°C-than in 20°C-acclimated fish (P<0.05). W_max/(V_maxP₀), which provides a measure of the curvature of the force-velocity relationship (Marsh and Bennett, 1986), was not affected by acclimation temperature. The calculated maximum mechanical power output was 47% higher at 8 °C in 8 °C-than in 20°C-acclimated fish, largely due to greater force production in the cold-acclimated fish (Table 2).

On sodium pyrophosphate gels myosins from the slow region of the Ab.s co-migrated with slow myotomal muscle myosin. No differences were observed in the relative mobilities of native myosins (pyrophosphate gels) or myosin heavy chains (8% SDS – PAGE gels) between cold-and warm-acclimated fish (not shown).

Electrophoretically purified native myosins were examined on 15% SDS – PAGE gels. The apparent relative molecular masses for the myosin light chains were; LCl_f, 27.5 × 10³; LC2_f, 18.8 × 10³; LC1_S, 26 × 10³; LC2_S, 19.9 × 10³. Fibre preparations from 20°C-acclimated carp contained almost exclusively slow muscle light chain isoforms (LC1_S and LC2_S) (Figs 4, 5) with traces of fast muscle myosin light chains (LCl_f and LC2_f). Preparations of Ab.s from 8°C-acclimated carp contained a much higher proportion of the two fast muscle myosin isoforms (Figs 4, 5). LC3_f was either not present on the gels or it may have co-migrated with LC2_S, since both peptides have a similar apparent relative molecular mass in carp (Crockford and Johnston, 1990).

Cold-acclimation produced modest increases in the rates of force development and relaxation in slow muscle fibres at low temperatures (Fig. 2; Table 1). Much larger changes in twitch duration have been shown to occur in fast myotomal muscle fibres. For example, Fleming et al. (1990) studied a fast myotomal nerve-muscle preparation from common carp acclimated to either 8°C or 20°C. The half-times for twitch activation and relaxation were around half as long at 8°C in cold-as in warm-acclimated fish (Fleming et al. 1990). Similarly, twitch duration for the whole pectoral fin adductor muscle of the goldfish (Carassius auratus) at 5°C was half as long in 10°C-as in 28°C-acclimated fish (Heap et al. 1987). This mixed muscle contains predominantly fast oxidative and fast glycolytic fibre types (Heap et al. 1987). Muscle fibres from cold-acclimated goldfish were found to contain a larger number of smaller-diameter myofibrils and to have a higher surface density of sarcoplasmic reticulum than fibres in warm-acclimated fish (Penney and Goldspink, 1980). These factors, together with the increase in myofibrillar ATPase activity (Johnston et al. 1975), probably account for the observed reductions in twitch duration in cold-acclimated individuals. Fleming et al. (1990) found that the pCa-tension relationship, parvalbumin content and the surface and volume density of sarcoplasmic reticulum (SR) in myotomal muscles were not altered by temperature acclimation in the common carp. In this case the faster relaxation of twitch tension in the fast myotomal muscle of cold-acclimated fish was associated with an increase in SR Ca²⁺-ATPase activity (Fleming et al. 1990; Johnston et al. 1990).

Acclimation of common carp to 8°C resulted in a perfect capacity adaptation of force production in slow fibres relative to 20 °C-acclimated fish (Table 1). Other studies have shown that maximum tension (P₀) does not vary continuously with acclimation temperature, but reaches upper and lower limits (Penney and Goldspink, 1981). For example, P₀ for fast myotomal fibres at 0°C is similar in carp acclimated to 2 – 11 °C, but declines progressively at higher acclimation temperatures (Crockford and Johnston, 1990). The volume density of myofibrils is slightly higher in the muscles of warm-than in cold-acclimated crucian carp (Johnston and Maitland, 1980). Thus, differences in myofibril density cannot explain the changes in force production with temperature acclimation. The higher P₀ values in cold-acclimated fish at low temperatures probably reflect an increase in the number of attached cross bridges in the force-generating state and/or in the force generated by each cross bridge. Both these mechanisms are thought to contribute to increases in force with acute rises in temperature (Bressler, 1981; Stephenson and Williams, 1981; Brenner, 1986).

The calculated maximum power output of slow muscle fibres was around 50 % higher at 8°C in cold-than in warm-acclimated carp (Table 2). This was due to a large increase in force production (Table 1) and a modest increase in contraction velocity (Table 2). In contrast, V_max for skinned fast muscle fibres increases by up to twofold following cold-acclimation (Johnston et al. 1985). It is therefore likely that muscle power output shows a more substantial capacity adaptation in fast than in slow muscle fibres.

The curvature of the force-velocity (P – V) relationship was not changed by temperature acclimation (Fig. 3). Langfeld et al. (1989) investigated the effects of acute temperature change on the P – V relationship of intact fast myotomal muscle fibres in the sculpin (Myoxocephalus scorpius). They found that the P – V relationship became progressively less curved at low temperatures. To quantify this effect the curves were normalised for P₀ and V_max at each temperature. The change in curvature between 8 and 1°C was sufficient to increase the relative power output by around 15% at 1°C (Langfeld et al. 1989). Although the curvature of the P – V relationship does not vary with acclimation temperature in carp, the above mechanism will serve to increase relative power output at low temperatures.

Fish myosin is composed of two heavy chains of M_r 200 × 10³ and four light chains ranging in relative molecular mass from 17 × 10³ to 29 × 10³ (Focant et al. 1981; Rowlerson et al. 1985). Force production and shortening speed in vertebrate skeletal muscle are thought to be primarily determined by myosin heavy chain composition (Reiser et al. 1985; Lannergren, 1987). The myosin heavy chains (MHC) are a multi-gene family of proteins that are expressed in a developmental stage-specific and tissue-specific manner (Richter et al. 1987). Cloning studies indicate that common carp possess at least 28 different myosin chain genes (Gerlach et al. 1990), about twice the number reported in mammals (Leinwand et al. 1983). Gerlach et al. (1990) found that a 1.7 kb DNA probe specific for fast muscle MHC chains hybridized more strongly to RNA from warm-acclimated than to that from cold-acclimated common carp. This suggests that temperature acclimation may involve changes in MHC gene expression. The alkali myosin light chains only differ in sequence at the NH₂ terminus and are thought to be produced via an alternative splicing mechanism from a single gene (Nabeshima et al. 1984). Crockford and Johnston (1990) found evidence that the composition of the alkali light chains of myosin varies with acclimation temperature. Single fast muscle fibres from cold-acclimated carp contained an extra band on isoelectric focusing gels with a pl intermediate between those of LC2_f and LC3_f and an apparent relative molecular mass of 20 × 10³. Densitométrie scans showed that the ratio of (LCl_f+LC3_f+extra band):LC2_f was approximately 1 in both cold-and warm-acclimated fish. This suggests that the extra band corresponds to an additional light chain (LC3_fc) which is only expressed in cold-acclimated fish. The total LC3_f content of fast muscle fibres was also lower in cold-than in warm-acclimated individuals (Crockford and Johnston, 1990). Studies on single fibres from rabbit plantaris muscle have shown that V_max increases with the myosin LC3 content, suggesting a modulatory role for the alkali light chains (Greaser et al. 1988). Thus, the relatively large changes in contractile properties of fast muscle fibres with temperature acclimation probably result from changes in the expression of both myosin heavy and light chains. It is well established that fast and slow myosins can be expressed in the same fibre type, and that myosin heavy and light chain genes are independently regulated (Reiser et al. 1985; Greaser et al. 1988). In the present study fibre bundles from the superior abductor muscle of 8°C-acclimated carp contained a higher content of myosin light chain isoforms normally associated with faster-contracting fibre types (Figs 4, 5). Since the percentage of fast fibres in preparations did not vary with acclimation temperature, it would appear that LClf and LC2_f are co-expressed with LC1_S and LC2_S in the slow fibres of 8°C-acclimated fish. This may contribute to the higher values of force production and V_max observed following cold-acclimation.

This work was supported by a Project Grant (GR3/6177) from the Natural Environment Research Council. KSL was in receipt of an NERC Research Studentship.