The molecular regulation of exercised-induced muscle fibre hypertrophy in the common carp: expression of MyoD, PCNA and components of the calcineurin-signalling pathway.

Growth was investigated over 16 d in juvenile common carp (Cyprinus carpio L.) held in either static water (tank rested, TR16) or exercised in a flume at 2.5-3.2 body lengths s-1 for 18 h a day (exercised, E16). Relative to the start of the experiment (TR0), the TR16 group showed a 31% increase in body mass (specific growth rate, 1.57% d-1), whereas there was no net change in the E16 group. There was, however, a significant exercise-induced hypertrophy of slow muscle fibres with average fibre cross-sectional area (FCSA) increasing by 35% in the E16 group, compared with 11% in the TR16 group. In contrast, FCSA of fast muscle fibres increased by 34% in the TR16 group compared to just 18% in the E16 group. The relative concentrations and subcellular localisation of proteins hypothesised to play a role in the regulation of muscle growth were measured. MyoD concentration was similar in the TR0, TR16 and E16 groups in both slow and fast muscle. However, there was a small (5%-10%) but statistically significant increase in nuclear localisation of MyoD in those groups showing a significant increase in FCSA over the time course of the experiment. PCNA concentration was 31% and 12% higher in the TR16 than in either the TR0 or E16 groups for slow and fast muscle, respectively. Exercise resulted in a approximately 10% increase in nuclear factor of T-cells (NFAT2) concentration in slow muscle but no change in NFAT2 localisation. Calcineurin B concentration was similar in tank rested and exercised groups. The results do not support a major role for the calcineurin-signalling pathway in the regulation of muscle hypertrophy in the common carp.

Lysosomal enzyme activities in muscle following starvation and refeeding in the saithe Pollachius virens L.

Saithe (Pollachius virens L.) were starved for 66 days at 10 degrees C and activities of aryl sulfatase, acid proteinase, beta-glucuronidase, RNAase and acid phosphatase measured in homogenates prepared from fast and slow myotomal muscles. In fed fish, hydrolase activities were generally higher in slow than fast muscles. With the exception of acid proteinase activity in slow muscle, the activities of all the lysosomal enzymes increased by 70 to 100% during starvation. In general, there was a proportionally larger increase in the hydrolase activities in fast than in slow muscle. In a second experiment, fish were starved for 74 days, and refed for up to 52 days. The increases in aryl sulfatase and acid proteinase activity produced in fast muscle with starvation were found to be rapidly reversed by refeeding. Lysosomal enzyme activities in fish sampled after 10 days refeeding were not significantly different from fed controls. Membrane fractions enriched in aryl sulfatase activity were prepared from the fast muscle of 66-day starved fish. These were capable of degrading both myosin heavy chains and actin to lower molecular weight peptides at acid (pH 5.0), but not at neutral pH. The results suggest a role for lysosomal enzymes in the breakdown of myofibrillar proteins during starvation.

Muscle atrophy during starvation in a marine teleost.

The marine telost Pollachius virens undergoes a natural starvation during the winter, and provides a reversible, non-pathological model for studying muscle wasting. In the present study fish were kept without food under laboratory conditions for up to 12 weeks. The effects of starvation on muscle fibre size, volume fractions of mitochondria and myofibrils, and capillary supply were determined. Starvation results in a preferential atrophy and degradation of fast muscle myofibrillar proteins. For example, fibre cross-sectional area decreased from 1014 to 535 micrometers 2 (p less than 0.005) and myofibrillar volume fraction from 79.0% to 56.4% (p less than 0.001) in fast fibres following 12 weeks starvation. In contrast there was little change in these parameters in slow muscle fibres. Evidence is presented that M-line and Z-disc breakdown occur as an initial stage of myofibrillar degradation. Sarcoplasmic reticulum in atrophied fibres often appeared swollen and multi-membraned lysosome-like vesicles were common. The percentage of slow fibres (44 to 64%; p less than 0.025) and fast fibers (51 to 86%; p less than 0.01) without capillary contact increased and the percentage of fibre perimeter vascularised decreased during a 12 week starvation (6.3 to 3.3% in slow fibres and 2.8 to 1.1% fast fibres). The volume fractions of mitochondria in slow fibres decreased in parallel to the decrease in capillary supply (from 34.6 to 18.6%; p less than 0.001). Mechanisms of myofibrillar degradation during muscle wasting are discussed.

Ca2+-uptake by tissue sections and biochemical characteristics of sarcoplasmic reticulum isolated from fish fast and slow muscles.

Frozen sections (10 micrometer) were cut from fast, slow and cardiac muscles of rainbow trout, frog, and rat. Rates of 45Ca2+-uptake by thin sections were compared at each animal's normal body temperature. Initial rates of Ca2+-uptake were 1.8 and 2.4 times higher, respectively, in trout and rat fast than slow muscles. In spite of a lower body temperature (10 degrees C) rates of Ca2+-uptake by trout fast muscles were 2.8 times higher than for rat Extensor digitorum longus at 37 degrees C. It is suggested that the high functional capacity of fish sarcoplasmic reticulum (SR) is related to adaptations associated with the need for rapid cycles of contraction and relaxation during high speed swimming. The biochemical characteristics of SR isolated from trout fast and slow muscles has also been investigated. The ratio of Ca2+-ATPase activity between fast and slow fibres (2:1) was similar to that obtained for Ca2+-uptake by whole muscle sections. No evidence was obtained for modulation of calcium transport by cAMP dependent protein kinases. The protein composition of highly purified trout SR was investigated by polyacrylamide gel electrophoresis. The concentration of 95 000 to 100 000 dalton (calcium pump protein) was significantly reduced in slow compared to fast muscle SR. Slow muscle SR contains a high proportion of additional protein bands of 48 000 and 31 000 molecular weight.

Quantitative analyses of ultrastructure and vascularization of the slow muscle fibres of the anchovy.

A quantitative study has been made of the ultrastructure and vascularization of slow fibres in the lateral muscles of the European anchovy (Engraulis encrasicolus). Mitochondria and myofibrils occupy 45.5 and 44.3% of total fibre volume respectively. More than 95% of all myofibrils are adjacent to mitochondria. A total of 51% of the sarcolemma is in direct contact with capillaries with a mean of 12.9 capillaries per fibre. In transverse sections anchovy slow fibres are considerably flattened (long to short axis 12:1) such that the surface to volume ratio is more than twice that of a cylindrical fibre of the same area (1115 micron2). The capillary surface required to supply 1 micron3 of mitochondria is 0.18 micron2 and the maximum distance between any capillary and mitochondrion 8 microns. T-system and sarcoplasmic reticulum occupy 0.43 and 2.7% of fibre volume respectively. Adaptations for increasing the capacity of skeletal muscle for aerobic work are discussed.

The role of myostatin and the calcineurin-signalling pathway in regulating muscle mass in response to exercise training in the rainbow trout Oncorhynchus mykiss Walbaum.

Rainbow trout Oncorhynchus mykiss Walbaum were exercised at 0.8 and 1.6 body lengths s(-1) for 18 h a day over a 30 day period. Exercise resulted in a 24-30% increase in the average cross-sectional area of fast muscle fibres relative to tank-rested controls. The concentrations of growth factors and transcription factors hypothesised to play a role in regulating exercise-induced muscle fibre hypertrophy were measured. Exercise training resulted in a minor increase in calcineurin localisation in the nucleus. However, nuclear factor of T-cells 2 (NFAT2) nuclear localisation did not follow a pattern that was consistent with NFAT2-mediated transcriptional activity and changes in calcineurin signaling. The active peptide of myostatin, a negative regulator of muscle growth in mammals, was downregulated in exercise groups relative to tank-rested controls, but only by 6-7%. It was concluded that myostatin and calcineurin signaling do not play a major role in regulating exercise-induced muscle hypertrophy in trout.

Cold adaptation in marine organisms.

Animals from polar seas exhibit numerous so called resistance adaptations that serve to maintain homeostasis at low temperature and prevent lethal freezing injury. Specialization to temperatures at or below 0 degrees C is associated with an inability to survive at temperatures above 3-8 degrees C. Polar fish synthesize various types of glycoproteins or peptides to lower the freezing point of most extracellular fluid compartments in a non-colligative manner. Antifreeze production is seasonal in boreal species and is often initiated by environmental cues other than low temperature, particularly short day lengths. Most of the adaptations that enable intertidal invertebrates to survive freezing are associated with their ability to withstand ariel exposure. Unique adaptations for freezing avoidance include the synthesis of low molecular mass ice-nucleating proteins that control and induce extracellular ice-formation. Marine poikilotherms also exhibit a range of capacity adaptations that increase the rate of some physiological processes so as to partially compensate for the effects of low temperature. However, the rate of embryonic development in a diverse range of marine organisms shows no evidence of temperature compensation. This results in a significant lengthening of the time from fertilization to hatching in polar, relative to temperate, species. Some aspects of the physiology of polar marine species, such as low metabolic and slow growth rates, probably result from a combination of low temperature and other factors such as the highly seasonal nature of food supplies. Although neuromuscular function shows a partial capacity adaptation in Antarctic fish, maximum swimming speeds are lower than for temperate and tropical species, particularly for early stages in the life history.

Temperature adaptation of enzyme function in fish muscle.

Temperature directly affects the performance of fish muscle through a variety of extrinsic and intrinsic mechanisms. A common observation is that species adapted to different temperatures over evolutionary time scales, or individuals exposed to temperature change from periods ranging from minutes to months are able to adjust muscle performance so as to partially offset the effects of temperature change. The underlying mechanisms are complex, involve a variety of levels of organization (behavioural, organismic, tissue and molecular) and probably vary with the time scale of adaptation to temperature. The present essay considers the extent to which interspecific differences in the structural and functional characteristics of muscle enzymes contribute to adjustments in contractile performance at different body temperatures.

Sustained force development: specializations and variation among the vertebrates.

The kinds of muscle fibre that are recruited for sustained force production by different vertebrates are described. Although aerobic metabolism always accounts for a significant proportion of their ATP turnover, no single characteristic such as colour, number and form of motor endplates, membrane properties, myosin isotype or contraction speed is diagnostic of such muscles. As mechanical power output increases, there is a tendency for a decrease in fatigue resistance with repetitive usage and an increase in both aerobic capacity and the fraction of energy requirements derived from glycolysis.

Muscle action during locomotion: a comparative perspective.

This essay explores how the properties of striated muscles are matched to the tasks they perform during running, swimming and flying. During exercise the major locomotory muscles undergo alternate cycles of lengthening and shortening. Force development is greatly influenced by the timing of stimulation in relation to the length-change cycle and by the nature of elastic structures connecting the muscle fibres to the skeleton. The storage and recovery of elastic strain energy by the tendons (apodema in insects) results in a considerable saving of metabolic energy. Strain is independent of locomotory frequency, body size and muscle temperature. In contrast, the frequency of cycles, and hence strain rate, generally increases with speed and is inversely proportional to body size. The maximum isometric stress (P0) striated muscles can exert is rather similar. During steady running or hopping in mammals the peak muscle stress is around one-third of P0. Behaviours such as vertical jumping impose higher stresses requiring disproportionately larger muscles and tendons, which may limit the storage of elastic strain energy. Muscles of small animals consume significantly more energy per gram than do those of large ones. This may be because they need to activate and deactivate their muscles at a higher rate to move at an equivalent speed. When differences in force production are normalised, by multiplying the energy consumed per stride by stride frequency, similar values for the mass-specific cost of locomotion are found in animals with different leg architectures, numbers of legs, skeletal type, body sizes and muscle temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis of muscle breakdown during starvation in the marine flatfish Pleuronectes platessa.

The present study describes the effects of starvation for a duration of four months on the ultrastructure of skeletal muscles from the marine flatfish (Pleuronectes platessa L.). Starvation is associated with a decrease in resting metabolic rate from 20.1 +/- 2.2 to 11.6 +/- 1.5 mg . O2/kg/h (P less than 0.05) and muscle wasting. Median fibre size fell from 700 micrometer 2 to 500 micrometer 2 in intermediate (fast oxidative) and from 1,800 micrometer 2 to 600 micrometer 2 in starved, white (fast-glycolytic) muscle fibres. In contrast, median fibre size in red (slow oxidative) muscle remained within the range 300-400 micrometer 2. The fraction of red fibre volume occupied by myofibrils (58.6%) and mitochondria (24.5%) did not change significantly with starvation. There was, however, a decrease in stored lipid 110.7% to 3.2%) and an alteration in the structure of the cristae in mitochondria from red muscle. Atrophy of white muscle fibres is associated with a decrease in both the diameter and fractional volume occupied by myofibrils (85.7% to 61.9% P less than 0.01). In a high proportion of white fibres peripheral degeneration of Z-discs is evident causing an unravelling of the thin filament lattice. It is suggested that this allows a partial decrease in myofibril diameter and hence the maintenance of contractile function in muscle from starved fish. In severely degenerating white fibres, disorganised thick and thin filaments and numerous multi-membrane lysosome-like vesicles are observed. Starvation results in an increase in the average content of mitochondria in white fibres from 2.2 to 6.7% (P less than 0.01). In fed plaice mitochondria constitute less than 1% of the volume of the white fibre in 43.5% of the fibres. The proportion of white fibres containing more than 6% mitochondria increases from 6.5% to 58% with starvation.

Comparative studies of contractile proteins from the skeletal and cardiac muscles of lower vertebrates.

The available evidence suggests that the properties of the contractile proteins from lower vertebrates are broadly similar to those found in skeletal and cardiac muscles of mammalian species. However, the proteins from ectotherms are generally more unstable on isolation.

Capillarisation, oxygen diffusion distances and mitochondrial content of carp muscles following acclimation to summer and winter temperatures.

Many species of fish show a partial or complete thermal compensation of metabolic rate on acclimation from summer to winter temperatures. In the present study Crucian carp (Carassius carassius L.) were acclimated for two months to either 2 degrees C or 28 degrees C and the effects of temperature acclimation on mitochondrial content and capillary supply to myotomal muscles determined. Mitochondria occupy 31.4% and 14.7% of slow fibre volume in 2 degrees C- and 28 degrees C-acclimated fish, respectively. Fast muscles of cold- but not warm-acclimated fish show a marked heterogeneity in mitochondrial volume. For example, only 5% of fast fibres in 28 degrees C-acclimated fish contain 5% mitochondria compared to 34% in 2 degrees C-acclimated fish. The mean mitochondrial volume in fast fibres is 6.1% and 1.6% for cold- and warm-acclimated fish, respectively. Increases in the mitochondrial compartment with cold acclimation were accompanied by an increase in the capillary supply to both fast (1.4 to 2.9 capillaries/fibre) and slow (2.2 to 4.8 capillaries/fibre) muscles. The percentage of slow fibre surface vascularised is 13.6 in 28 degrees C-acclimated fish and 32.1 in 2 degrees C-acclimated fish. Corresponding values for fast muscle are 2.3 and 6.6% for warm- and cold-acclimated fish, respectively. Maximum hypothetical diffusion distances are reduced by approximately 23-30% in the muscles of 2 degrees C-compared to 28 degrees C-acclimated fish. However, the capillary surface supplying 1 micron 3 of mitochondria is similar at both temperatures. Factors regulating thermal compensation of aerobic metabolism and the plasticity of fish muscle to environmental change are briefly discussed.

Respiratory characteristics of muscle fibres in a fish (Chaenocephalus aceratus) that lacks haem pigments.

1. Oxygen consumption, mitochondrial content and enzyme activities were determined in identified muscle fibre types of the 'haemoglobin-less' icefish Chaenocephalus aceratus Lönnberg. 2. Small bundles (2-12) of fast and slow fibres were isolated from the myotomal and superficial pectoral fin abductor muscles, respectively. At 0 degrees C the time to 50% peak force and the half-relaxation time of isometric twitches were, respectively, 18 +/- 1 and 38 +/- 4 ms for fast and 43 +/- 3 and 119 +/- 21 ms for slow muscle fibres (mean +/- S.E.). 3. Measurements of enzyme activities in homogenates suggest that phosphocreatine hydrolysis and oxidative phosphorylation are the main energy-supplying pathways in fast and slow muscles, respectively. Activities of glycolytic enzymes were relatively modest and showed no consistent differences between fibre types. 4. The relationship between oxygen consumption and mitochondria in slow muscle was also determined for a 'red-blooded' antarctic (Notothenia gibberifrons), a cold-temperate (Myoxocephalus scorpius) and a warm-temperate (Oreochromis niloticus) fish. Volume densities of mitochondria were as follows (mean +/- S.D.): C. aceratus, 0.50 +/- 0.08; N. gibberifrons, 0.30 +/- 0.10; M. scorpius, 0.23 +/- 0.05; and O. niloticus, 0.20 +/- 0.05. ADP-stimulated respiration rates were measured in isolated fibre segments. In spite of their different mitochondrial contents, slow fibres from the two antarctic fish utilized pyruvate and palmitoyl-1-carnitine at similar rates (1.0-1.2 mumol O2g-1 wet mass min-1 at 0 degrees C). This suggests that the high density of mitochondria in icefish muscle is related, in part, to diffusion limitations.

Changes in tension generation and ATPase activity in skinned muscle fibres of the carp following temperature acclimation.

Common carp (Cyprinus carpio L.) were acclimated to either 7 degrees C or 23 degrees C for 1-2 months. Skinned fibre preparations were isolated from the white myotomal muscle, and ATPase activity measured during maximal isometric contractions. At 7 degrees C, fibres from the cold acclimated fish were found to generate more force than those from warm acclimated fish (123.1 and 97.2kN m-2 respectively), and more "work" (force X time integral) was obtained for each ATP hydrolysed. ATP turnover per myosin head in fibres from cold-acclimated fish was lower than in fibres from warn-acclimated fish (1.85 and 2.84 ATP S1(-1) s-1).

Effects of phosphate on the contractile properties of fast and slow muscle fibres from an Antarctic fish.

Single fast myotomal fibres and small bundles of slow fibres (from the adductor pectoralis profundus muscle) were isolated from the Antarctic teleost Notothenia neglecta. Fibres were skinned by a brief detergent treatment. The effects of phosphate on the mechanical properties and ATPase activity of fast and slow fibres were studied. 20 mM-phosphate inhibited maximum isometric tension in slow fibres by 34%, but by only 11% in fast fibres. A half-maximal response was obtained at approximately 5 mM-phosphate. These concentrations are within the range measured in muscle, and the effect is probably of physiological significance. This species is of particular interest, since there is evidence that the energy supply to the fast muscle is largely based on phosphocreatine breakdown, which would result in large changes in intracellular phosphate concentration during exercise. The maximum contraction velocity of both fast and slow fibres was not affected by 10 mM-phosphate, nor was the ATPase activity of the slow fibres during isometric contraction. The phosphate-induced depression in tension in slow fibres was associated with a proportional decrease in stiffness. The rate of force recovery after rapid, small amplitude stretches and releases was increased by phosphate, as was the rate of rise of force during stretch activation. The results are discussed with reference to the different patterns of energy supply for contraction in muscle, and an attempt is made at explaining the data in terms of changes in cross-bridge kinetics.

Differences in temperature dependence of muscle contractile properties and myofibrillar ATPase activity in a cold-temperature fish.

Single muscle fibres were isolated from the fast myotomal muscle of the teleost Myoxocephalus scorpius L. and chemically skinned with 1% Brij. Maximum Ca2+-activated force (P0) increased from 14.5 +/- 1.1 N cm-2 at 2 degrees C to 19.1 +/- 1.8 N cm-2 at 15 degrees C (mean +/- S.E.). Maximum contraction velocity was determined by Hill's slack-test method (V0) and by extrapolation from force-velocity (P-V) relationships (Vmax). There was a linear relation between log10 V0 and temperature below 15 degrees C (Q10 = 1.9, P less than 0.01). The force-velocity characteristics of the fibres were determined at 2 degrees C and 20 degrees C. Points below 0.6 P0 on the P-V curve could be fitted by a linear form of Hill's equation. Extrapolated Vmax values were 0.55 muscle lengths s-1 (L0 s-1) at 2 degrees C and 1.54 L0 s-1 at 20 degrees C. Curvature of the P-V relationship was independent of temperature. The Mg2+, Ca2+-ATPase activity of Triton-X 100 extracted myofibrils was determined under similar ionic conditions to those used in skinned fibre experiments. (Ionic strength 0.16 mmol l-1, pMgATP 2.5). A linear relationship between log10 ATPase and temperature was only obtained below 15 degrees C (P less than 0.001). Above 15 degrees C, the Q10 for ATPase decreased significantly. The Q10(0-15 degrees C) for ATPase activity (3.9) was significantly higher than for unloaded contraction velocity. Supercontraction of isolated myofibrils to very short sarcomere lengths and differences in the mechanical constraints for crossbridge cycling between the preparations probably account for the lack of proportionality between these two parameters.

Power output and force-velocity relationship of red and white muscle fibres from the Pacific blue marlin (Makaira nigricans).

Single white fibres and small bundles (two to three) of red fibres were isolated from the trunk muscle of Pacific Blue Marlin (50-121 kg body weight). Fibres were chemically skinned with 1% Brij. Maximum Ca2+-activated force production (Po) was 57 kN m-2 for red fibres and 176 kN m-2 for white fibres at 25 degrees C. The force-velocity (P-V) characteristics of these fibres were determined at 15 and 25 degrees C. Points below 0.6 Po on the P-V curve could be fitted to a linear form of Hill's equation. The degree of curvature of the P-V curve was similar at 15 and 25 degrees C (Hill's constant a/Po = 0.24 and 0.12 for red and white fibres respectively). Extrapolated maximum contraction velocities (Vmax) were 2.5 muscle lengths s-1 (Lo S-1) (red fibres) and 5.3 Lo S-1 (white fibres) at 25 degrees C. Q10(15-25 degrees C) values for Vmax were 1.4 and 1.3 for red and white fibres respectively. Maximum power output had a similar low temperature dependence and amounted to 13 W kg-1 for red and 57 W kg-1 for white muscle at 25 degrees C. The results are briefly discussed in relation to the locomotion and ecology of marlin.