Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Muscle transcriptome adaptations with mild eccentric ergometer exercise

The muscle has a wide range of possibilities to adapt its phenotype. Repetitive submaximal concentric exercise (i.e., shortening contractions) mainly leads to adaptations of muscle oxidative metabolism and endurance while eccentric exercise (i.e., lengthening contractions) results in muscle growth and gain of muscle strength. Modified gene expression is believed to mediate these exercise-specific muscle adjustments. In the present study, early alterations of the gene expression signature were monitored by a muscle-specific microarray. Transcript profiling was performed on muscle biopsies of vastus lateralis obtained from six male subjects before and in a 24-h time course after a single bout of mild eccentric ergometer exercise. The eccentric exercise consisted of 15 min of eccentric cycling at 50% of the individual maximal concentric power output leading to muscle soreness (5.9 on a 0-10 visual analogue scale) and limited muscle damage (1.7-fold elevated creatine kinase activity). Muscle impairment was highlighted by a transient reduction in jumping height after the eccentric exercise. On the gene expression level, we observed a general early downregulation of detected transcripts, followed by a slow recovery close to the control values within the first 24 h post exercise. Only very few regulatory factors were increased. This expression signature is different from the signature of a previously published metabolic response after an intensive endurance-type concentric exercise as well as after maximal eccentric exercise. This is the first description of the time course of changes in gene expression as a consequence of a mild eccentric stimulus.

Neer Award 2007: Reversion of structural muscle changes caused by chronic rotator cuff tears using continuous musculotendinous traction. An experimental study in sheep

HYPOTHESIS: Chronic rotator cuff tears are associated with irreversible architectural muscle changes and a high rate of repair failure. The changes observed in man and their irreversibility with a single stage repair can be reproduced in sheep. It was the purpose of this experiment to test the hypothesis that slow, continuous elongation of a retracted musculotendinous unit allows reversal of the currently irreversible structural muscle changes. MATERIALS AND METHODS: The infraspinatus tendon of 12 sheep was released using a greater tuberosity osteotomy and allowed to retract for 4 months. Then, a new device was mounted on the scapular spine and used to extend the infraspinatus muscuculotendinous unit transcutaneously by 1 mm per day. Thereafter, the tendon was repaired back to the greater tuberosity. We assessed the muscular architecture using magnetic resonance imaging, macroscopic dissection, histology, and electron microscopy. Fatty infiltration (in Hounsfield units 1/4 HU) and muscular cross-sectional area (in % of the control side) were monitored with computed tomography at tendon release, initiation of elongation, repair, and at sacrifice. RESULTS: Sixteen weeks after tendon release, the mean tendon retraction was 29 +/- 6 mm (14% of original length, P = .008). In 8 sheep, elongation was achieved as planned (group I), but in 4, the elongation failed technically (group II). The mean traction time was 24 +/- 6 days with a mean traction distance of 19 +/- 4 mm. At sacrifice, the mean pennation angle in the infraspinatus of group I was not different from the control side (29.8 degrees +/-7.5 degrees vs. 30 degrees +/-6 degrees , P = .575). In group II, the pennation angle had increased from 30 degrees +/-6 degrees to 55 degrees +/-14 degrees (P = .035). There was no fatty infiltration at the time of tendon release. After retraction, there was a significant increase in fatty infiltration of the infraspinatus muscle and a decrease of its cross-sectional area to 57% of the contralateral side (P = .0001). During traction, the degree of fatty infiltration remained unchanged (36 HU to 38 HU, P = .381), and atrophy improved to a muscle square area of 78% of the contralateral side (P = .0001) in group I. In group II, an increase of fatty infiltration was measured from 36 HU to 28 HU; however, this increase was not significant (P = .144). Atrophy did not change in group II (57-55%, P = .946). At sacrifice, the remaining muscle mass was 64% in group I and 46% in group II (P = .019). DISCUSSION: Our preliminary results document, that continuous elongation of a retracted, fatty infiltrated and atrophied musculotendinous unit is technically feasible. CONCLUSION: In the sheep, continuous elongation can lead to restoration of normal muscle architecture, to partial reversal of muscle atrophy, and to arrest of the progression of fatty infiltration. LEVEL OF EVIDENCE: Basic science level 2; Prospective comparative therapeutic study.

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Protective Effect of Focal Adhesion Kinase against Skeletal Muscle Reperfusion Injury after Acute Limb Ischemia.

OBJECTIVES In cardiac muscle, ischemia reperfusion (IR) injury is attenuated by mitochondrial function, which may be upregulated by focal adhesion kinase (FAK). The aim of this study was to determine whether increased FAK levels reduced rhabdomyolysis in skeletal muscle too. MATERIAL AND METHODS In a translational in vivo experiment, rat lower limbs were subjected to 4 hours of ischemia followed by 24 or 72 hours of reperfusion. FAK expression was stimulated 7 days before (via somatic transfection with pCMV-driven FAK expression plasmid) and outcomes were measured against non-transfected and empty transfected controls. Slow oxidative (i.e., mitochondria-rich) and fast glycolytic (i.e., mitochondria-poor) type muscles were analyzed separately regarding rhabdomyolysis, apoptosis, and inflammation. Severity of IR injury was assessed using paired non-ischemic controls. RESULTS After 24 hours of reperfusion, marked rhabdomyolysis was found in non-transfected and empty plasmid-transfected fast-type glycolytic muscle, tibialis anterior. Prior transfection enhanced FAK concentration significantly (p = 0.01). Concomitantly, levels of BAX, promoting mitochondrial transition pores, were reduced sixfold (p = 0.02) together with a blunted inflammation (p = 0.01) and reduced rhabdomyolysis (p = 0.003). Slow oxidative muscle, m. soleus, reacted differently: although apoptosis was detectable after IR, rhabdomyolysis did not appear before 72 hours of reperfusion; and FAK levels were not enhanced in ischemic muscle despite transfection (p = 0.66). CONCLUSIONS IR-induced skeletal muscle rhabdomyolysis is a fiber type-specific phenomenon that appears to be modulated by mitochondria reserves. Stimulation of FAK may exploit these reserves constituting a potential therapeutic approach to reduce tissue loss following acute limb IR in fast-type muscle.

Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting

Myostatin, a member of the transforming growth factor-β superfamily, is a genetic determinant of skeletal muscle growth. Mice and cattle with inactivating mutations of myostatin have marked muscle hypertrophy. However, it is not known whether myostatin regulates skeletal muscle growth in adult men and whether increased myostatin expression contributes to wasting in chronic illness. We examined the hypothesis that myostatin expression correlates inversely with fat-free mass in humans and that increased expression of the myostatin gene is associated with weight loss in men with AIDS wasting syndrome. We therefore cloned the human myostatin gene and cDNA and examined the gene’s expression in the skeletal muscle and serum of healthy and HIV-infected men. The myostatin gene comprises three exons and two introns, maps to chromosomal region 2q33.2, has three putative transcription initiation sites, and is transcribed as a 3.1-kb mRNA species that encodes a 375-aa precursor protein. Myostatin is expressed uniquely in the human skeletal muscle as a 26-kDa mature glycoprotein (myostatin-immunoreactive protein) and secreted into the plasma. Myostatin immunoreactivity is detectable in human skeletal muscle in both type 1 and 2 fibers. The serum and intramuscular concentrations of myostatin-immunoreactive protein are increased in HIV-infected men with weight loss compared with healthy men and correlate inversely with fat-free mass index. These data support the hypothesis that myostatin is an attenuator of skeletal muscle growth in adult men and contributes to muscle wasting in HIV-infected men.

Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

High resolution ultrastructural mapping of total calcium: electron spectroscopic imaging/electron energy loss spectroscopy analysis of a physically/chemically processed nerve-muscle preparation.

We report on a procedure for tissue preparation that combines thoroughly controlled physical and chemical treatments: quick-freezing and freeze-drying followed by fixation with OsO4 vapors and embedding by direct resin infiltration. Specimens of frog cutaneous pectoris muscle thus prepared were analyzed for total calcium using electron spectroscopic imaging/electron energy loss spectroscopy (ESI/EELS) approach. The preservation of the ultrastructure was excellent, with positive K/Na ratios revealed in the fibers by x-ray microanalysis. Clear, high-resolution EELS/ESI calcium signals were recorded from the lumen of terminal cisternae of the sarcoplasmic reticulum but not from longitudinal cisternae, as expected from previous studies carried out with different techniques. In many mitochondria, calcium was below detection whereas in others it was appreciable although at variable level. Within the motor nerve terminals, synaptic vesicles as well as some cisternae of the smooth endoplasmic reticulum yielded positive signals at variance with mitochondria, that were most often below detection. Taken as a whole, the present study reveals the potential of our experimental approach to map with high spatial resolution the total calcium within individual intracellular organelles identified by their established ultrastructure, but only where the element is present at high levels.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.

Indirect coupling of phosphate release to de novo tension generation during muscle contraction.

A key question in muscle contraction is how tension generation is coupled to the chemistry of the actomyosin ATPase. Biochemical and mechanochemical experiments link tension generation to a change in structure associated with phosphate release. Length-jump and temperature-jump experiments, on the other hand, implicate phase 2slow, a significantly faster, markedly strain-sensitive kinetic process in tension generation. We use a laser temperature jump to probe the kinetics and mechanism of tension generation in skinned rabbit psoas fibers--an appropriate method since both phosphate release and phase 2slow are readily perturbed by temperature. Kinetics characteristic of the structural change associated with phosphate release are observed only when phosphate is added to fibers. When present, it causes a reduction in fiber tension; otherwise, no force is generated when it is perturbed. We therefore exclude this step from tension generation. The kinetics of de novo tension generation by the temperature-jump equivalent of phase 2slow appear unaffected by phosphate binding. We therefore propose that phosphate release is indirectly coupled to de novo tension generation via a steady-state flux through an irreversible step. We conclude that tension generation occurs in the absence of chemical change as the result of an entropy-driven transition between strongly bound crossbridges in the actomyosin-ADP state. The mechanism resembles the operation of a clock, with phosphate release providing the energy to tension the spring, and the irreversible step functions as the escapement mechanism, which is followed in turn by tension generation as the movement of the hands.

Interindividual variation of isolated muscle performance and fibre-type composition in the toad Bufo viridus

Interindividual analyses of physiological performance represent one of the most powerful tools for identifying functional positive and negative linkages between various performance traits. In this study we investigated functional linkages in the whole-gastrocnemius performance of juvenile Bufo viridis by examining interindividual variation in in vitro muscle performance and muscle fibre-type composition. We used the work-loop technique to investigate the maximum in vitro power output and fatigue resistance of the gastrocnemius muscle during repeated sets of three cycles at the cycle frequency of 5 Hz, simulating an intermittent style of locomotion. We found several significant correlations between different measures of in vitro muscle performance, including a negative correlation between maximum net power output and fatigue resistance of power, indicating functional trade-offs between these performance traits. We also investigated the extent of individual variation in the proportions of different fibre types, and tested for correlations between individual variation in muscle fibre-type composition and the previously measured isolated muscle performance. Fast glycolytic fibres represented 84.0+/-3.4% of the muscle, while the combined slow oxidative and fast oxidative-glycolytic fibres represented 16+/-3.4%. We found no significant correlations between measures of in vitro muscle performance and the proportion of different fibre types in the gastrocnemius muscle. However, despite this lack of correlation between whole-muscle performance and muscle fibre-type composition data, we suggest the functional linkages detected between different measures of in vitro muscular performance have important ecological and evolutionary consequences.

Sustained swimming performance and muscle structure are altered by thermal acclimation in male mosquitofish

Males of the eastern mosquito fish (Gambusia holbrooki) possess one of the widest reproductively active temperature ranges for any ectotherm, ranging across seasons from at least 18degreesC to 34degreesC. In this study, we tested the ability of male G. holbrooki to acclimate their sustained swimming performance following long-term exposure to 18degreesC or 30degreesC. We also investigated some of the possible physiological mechanisms associated with thermal acclimation responses in swimming performance, including changes in slow muscle fibre size and abundance and the expression of myosin heavy chains (MyHC). We found that U-crit, of 18degreesC-acclimated G. holbrooki was 20% greater at 18degreesC than 30degreesC-acclimated fish, and the Ucrit of the 30degreesC-acclimated group was more than 15% greater at 30degreesC. Slow, fast and intermediate muscle fibres were identified on the basis of their myosin ATPase staining reaction. Although the number of slow and intermediate muscle fibres was similar between groups, the total cross-sectional area of aerobic fibre types was 40% greater in 18degrees-than 30degreesC-acclimated fish, reflecting an increase in the average fibre diameter. An S58 antibody raised against chicken slow skeletal muscle myosin stained a sub-set of the slow fibres identified by myosin ATPase staining. The number of S58-positive muscle fibres was 50% greater in 30degreesC-than 18degreesC-acclimated fish, implying that different MyHCs are being expressed in cold and warm acclimated individuals. Given the sexually coercive mating system of this species, increases in the sustained swimming performance via thermal acclimation may benefit the ability of males to maintain a high rate of sneaky copulations. (C) 2004 Elsevier Ltd. All rights reserved.