Frog Muscle Research Articles

Electron microscopy and three-dimensional reconstruction of native thin filaments reveal species-specific differences in regulatory strand densities

Throughout the animal kingdom striated muscle contraction is regulated by the thin filament troponin–tropomyosin complex. Homologous regulatory components are shared among vertebrate and arthropod muscles; however, unique protein extensions and/or components characterize the latter. The Troponin T (TnT) isoforms of Drosophila indirect flight and tarantula femur muscle for example contain distinct C-terminal extensions and are ∼20% larger overall than their vertebrate counterpart. Using electron microscopy and three-dimensional helical reconstruction of native Drosophila, tarantula and frog muscle thin filaments we have identified species-specific differences in tropomyosin regulatory strand densities. The strands on the arthropod thin filaments were significantly larger in diameter than those from vertebrates, although not significantly different from each other. These findings reflect differences in the regulatory troponin–tropomyosin complex, which are likely due to the larger TnT molecules aligning and extending along much of the tropomyosin strands’ length. Such an arrangement potentially alters the physical properties of the regulatory strands and may help establish contractile characteristics unique to certain arthropod muscles.

Inferring crossbridge properties from skeletal muscle energetics

Work is generated in muscle by myosin crossbridges during their interaction with the actin filament. The energy from which the work is produced is the free energy change of ATP hydrolysis and efficiency quantifies the fraction of the energy supplied that is converted into work. The purpose of this review is to compare the efficiency of frog skeletal muscle determined from measurements of work output and either heat production or chemical breakdown with the work produced per crossbridge cycle predicted on the basis of the mechanical responses of contracting muscle to rapid length perturbations. We review the literature to establish the likely maximum crossbridge efficiency for frog skeletal muscle (0.4) and, using this value, calculate the maximum work a crossbridge can perform in a single attachment to actin (33 x 10(-21) J). To see whether this amount of work is consistent with our understanding of crossbridge mechanics, we examine measurements of the force responses of frog muscle to fast length perturbations and, taking account of filament compliance, determine the crossbridge force-extension relationship and the velocity dependences of the fraction of crossbridges attached and average crossbridge strain. These data are used in combination with a Huxley-Simmons-type model of the thermodynamics of the attached crossbridge to determine whether this type of model can adequately account for the observed muscle efficiency. Although it is apparent that there are still deficiencies in our understanding of how to accurately model some aspects of ensemble crossbridge behaviour, this comparison shows that crossbridge energetics are consistent with known crossbridge properties.

MicroRNA regulation below zero: Differential expression of miRNA-21 and miRNA-16 during freezing in wood frogs

Natural freeze tolerance depends on numerous biochemical adaptations that address the multiple stresses imposed on cells by freezing and preserves viability by suppressing energy-expensive cell functions in the frozen state. We hypothesized that microRNAs, small non-coding RNA transcripts that bind to mRNA, could act to establish rapid biological controls that aid the reorganization of metabolic priorities for freezing survival. Selected microRNA species (miR-16 and miR-21) were evaluated using RT-PCR in liver and skeletal muscle of wood frogs (Rana sylvatica) comparing controls (5°C acclimated) with animals frozen for 24h at −3°C. Levels of miR-21 increased significantly during freezing by 1.5-fold and 1.3-fold in liver and skeletal muscle, respectively. MiR-16 transcripts also rose significantly by 1.5-fold in liver of frozen frogs but fell by 50% in skeletal muscle. Protein levels of Dicer, a type III RNase that is responsible for mature microRNA processing in the cytoplasm, were unchanged in liver and decreased significantly by 50% in muscle. This data provides the first report of differential regulation of microRNA species in a freeze tolerant vertebrate and suggest a mechanism for rapid, yet reversible, gene silencing when animals transition into the frozen state.

Mechanical properties of intact single fibres from wild-type and MLC/mIgf-1 transgenic mouse muscle

The effects of overexpression of the local form of insulin like growth factor-1 (mIgf-1) on skeletal muscle were investigated by comparing the mechanical properties of single intact fibres from the flexor digitorum brevis of wild-type (WT) and (MLC/mIgf-1) transgenic mice (TG)at 21-24 degrees C. Isolated single fibres were clean enough to measure accurately the sarcomere length. The parameters investigated were: tetanic absolute and specific force, the force-velocity relationship, and the sarcomere length-tension relationship. In addition, we investigated the properties of the "static stiffness", a non-crossbridge Ca(2+)-dependent increase of fibre stiffness previously found in frog muscle. Both average cross-sectional area and tetanic force almost doubled in TG fibres, so that specific force was the same in both preparation: 312 +/- 20 and 344 +/- 34 kN m(-2) in WT and TG fibres, respectively. None of the relative force-velocity parameters was altered by Igf-1 overexpression, however, V(max) (8-10 l(0) s(-1)) was greater than previously reported in whole muscles. The sarcomere length-tension relationship was the same in TG and WT fibres showing the classical shape with a plateau region between 2.28 and 2.52 microm and a linear descending limb. The static stiffness was present in both WT and TG fibres and showed similar characteristics to that of frog skeletal muscle. In contrast to the other parameters, static stiffness in TG fibres was about 24% smaller than in WT fibres suggesting a possible effect of Igf-1 overexpression on its mechanism.

Effects of adrenaline on glycogenolysis in resting anaerobic frog muscles studied by 31P-NMR

The effects of adrenaline (also called epinephrine) on glycogenolysis in living anaerobic muscles were examined based on time-dependent changes of (31)P-NMR spectra of resting frog skeletal muscles with and without iodoacetate treatments. The phosphate-metabolite concentration and the intracellular pH determined from the NMR spectra changed with time, reflecting the advancement of various phosphate metabolic reactions coupled with residual ATPase reactions to keep the ATP concentration constant. The results could be explained semi-qualitatively as the ATP regenerative reactions, creatine kinase reaction and glycogenolysis, advanced with time showing the characteristic two phases. Thus, it was clarified for living muscles that adrenaline activates the phosphorylase step of glycogenolysis, and the adrenaline-activated glycogenolysis is further regulated at the phosphofructokinase step by PCr and also possibly by AMP. Associated with the adrenaline-activated glycogenolysis in the examined muscles, the P(i) concentration and the intracellular pH, factors affecting the muscle force, changed significantly, suggesting complicated effects of adrenaline on the muscle contractility.

Shortening-induced force depression is primarily caused by cross-bridges in strongly bound states

The steady-state isometric force following active muscle shortening is smaller than the corresponding force obtained for purely isometric contractions. This so-called residual force depression has been observed consistently for more than half a century, however its mechanism remains a matter of scientific debate. [Maréchal, G., Plaghki, L., 1979. The deficit of the isometric tetanic tension redeveloped after a release of frog muscle at a constant velocity. J. Gen. Physiol. 73, 453–467] suggested that force depression might be caused by alterations in the cross-bridge kinetics following muscle shortening, but there is no research studying force depression systematically for altered cross-bridge kinetic conditions. The purpose of this study was to investigate if force depression affects so-called weakly and strongly bound cross-bridges to the same degree. In order to achieve this aim, we modified the ratio of weakly to strongly bound cross-bridges with 2,3-butanedione monoxime (BDM) in single frog fibers. BDM inhibits the formation of strongly bound cross-bridges in a dose-dependent manner, thus the ratio of weakly to strongly bound cross-bridges could be altered in a systematic way. We found that the absolute amount of force depression was decreased by 50% while the relative amount was decreased by 12% in BDM exposed fibers compared to fibers in normal Ringer's solution. Furthermore, force depression was accompanied by a decrease in stiffness that was much greater in normal compared to BDM exposed fibers, leading to the conclusion that force depression was caused by an inhibition of cross-bridge attachment following fiber shortening and that this inhibition primarily affected cross-bridges in the strongly bound states.

Residues of Polybrominated Diphenyl Ethers in Frogs (Rana limnocharis) from a Contaminated Site, South China: Tissue Distribution, Biomagnification, and Maternal Transfer

Environmental pollutants are suspected to be a cause of global declines in amphibian populations, but few data are available on the bioaccumulation of polybrominated diphenyl ethers (PBDEs) in amphibians. To examine the tissue distribution, biomagnification potential, and maternal transfer of PBDEs in frogs, eighteen PBDE congeners were measured in the muscle, liver, and egg tissues of rice frogs (Rana limnocharis) and insects collected from an electronic waste (e-waste) recycling site in South China. PBDE levels in the frogs ranged from 0.63 to 11.6, 4.57 to 56.2, and 10.7 to 125 ng/g wet wt in the muscles, livers, and eggs, respectively. The frogs exhibited a unique congener profile, compared to those in aquatic and terrestrial species, with BDEs 99, 153, 183, 209, and 47 as the dominant congeners, intermediating between aquatic and terrestrial species. Most of the PBDE congeners in general showed higher affinity to liver than to muscle tissue. Except for BDEs 28, 47, 66, 138, and 206, the average biomagnification factors (BMFs) for all PBDE congeners were greater than 1.0, providing clear evidence of their biomagnification from insects to frogs. A parabolic relationship between log BMFs and bromine atom numbers or log Kow of PBDEs was observed, with the maximum BMF values for PBDEs with 6 bromine atoms (or at a log K(ow) of approximately 8.0). Relatively higher levels of 3-MeO-BDE 47 were found in male frogs, suggesting that male frogs in the present study might have higher metabolic capacity for PBDEs compared to female frogs. The ratio of levels in egg/female liver, indicating mother-to-egg transfer capacity, increased with increasing bromine atom numbers up to 7 and then declined as the bromine atom numbers rose. This indicated that the physicochemical properties of the congeners (e.g., K(ow), molecular sizes, and structures), resulting in different affinities to transport proteins, might impact their maternal transfer in frogs.

A 3D skeletal muscle model coupled with active contraction of muscle fibres and hyperelastic behaviour

This paper presents a three-dimensional finite element model of skeletal muscle which was developed to simulate active and passive non-linear mechanical behaviours of the muscle during lengthening or shortening under either quasi-static or dynamic condition. Constitutive relation of the muscle was determined by using a strain energy approach, while active contraction behaviour of the muscle fibre was simulated by establishing a numerical algorithm based on the concept of the Hill's three-element muscle model. The proposed numerical algorithm could be used to predict concentric, eccentric, isometric and isotonic contraction behaviours of the muscle. The proposed numerical algorithm and constitutive model for the muscle were derived and implemented into a non-linear large deformation finite element programme ABAQUS by using user-defined material subroutines. A number of scenarios have been used to demonstrate capability of the model for simulating both quasi-static and dynamic response of the muscle. Validation of the proposed model has been performed by comparing the simulated results with the experimental ones of frog gastrocenemius muscle deformation. The effects of the fusiform muscle geometry and fibre orientation on the stress and fibre stretch distributions of frog muscle during isotonic contraction have also been investigated by using the proposed model. The predictability of the present model for dynamic response of the muscle has been demonstrated by simulating the extension of a squid tentacle during a strike to catch prey.

Comparison Of Mechanical Properties Of Single Intact Fibres From Wild-type And Mlc/migf-1 Mouse Muscle

In this study we compared the mechanical properties of single intact muscle fibres of wild-type (WT) and MLC/mIgf-1 (TG) mice, in which the localized Igf-1 transgene expression sustains hypertrophy (Musaro et al., Nat. Genet. 27, 2001). The study has been focussed on “static stiffness” (SS), a non crossbridge calcium-dependent stiffness previously identified in activated frog muscle fibres (Bagni et al., Biophys. J. 82, 2002).Single intact fibres, dissected from the flexor digitorum brevis muscle, were mounted in an experimental chamber (∼23°C) between the lever arms of a force transducer and of an electromagnetic motor to apply fast stretches. Sarcomere length was measured by means of a videocamera and with laser diffraction. Tetanic tension and force-velocity relation in WT and TG mice were not significantly different, however, the maximum shortening velocity (Vmax) was faster than previously reported and comparable with frog muscle. Compared to frog fibres, the plateau of length-tension relation shifted according to the different myofilament lengths. TG fibres exhibited an increase in diameter and maximum force, but specific force was the same as for WT fibres. SS was present either in WT or in TG fibres and its time course, independent from isometric tension, was faster than in frog.A preliminary analysis suggests that the only significant mechanical difference between WT and TG fibres is in the SS properties. This may be related to a different compliance of the structure responsible of the SS that we speculated could be titin.

New X-ray Data about Myosin-binding Protein C in Frog Muscle

Frog striated muscle gives many meridional X-ray reflections at spacings greater than 400A which are still incompletely understood. Some come from C-protein, as discovered by Offer (CSH Symp.37, 83-97, 1972) and by Rome (ibid,331-339). Others may come from a “forbidden” first order myosin meridional reflection, as discussed by Malinchik and Lednev (JMRCM.13, 406-419, 1992). In both cases the reflections will be split by interference fringes from the two half A -bands. Squire has suggested that the apparent C-protein repeat of ∼ 435A (rather than 429A) in relaxed muscle may be due to interaction with actin (JMB.331, 713-724, 2003).We have studied these reflections at high resolution on the BioCAT beam line at the Argonne National Lab., in relaxed and contracting muscle. In resting muscle, two main peaks occur in the relevant region, at ∼419A and ∼442A, the latter being about 4 times more intense than the former, indicating an underlying repeat of ∼437A and an apparent interference distance of ∼8200A (and an actual one of ∼7100A). In contracting muscle, the pattern is very much weaker, and the corresponding spacings are 412A, 447A (i.e. a wider doublet), 440A underlying spacing, and 5300A apparent interference distance. Part of the change may be ascribed to a weakening of the presumed contribution from the myosin “forbidden” meridional reflection, but the apparent interference distance would now indicate an actual distance of ∼4460A, short compared to the expected value (∼5900A) for C-protein ; the long C repeat spacing (440A) is also notable.At lower angles still, the observed reflections index as the higher even orders of the sarcomere repeat, as reported by Bordas and colleagues (J. Cell. Biol.105, 1311-1318, 1987), and which shorten in contraction. But why only even orders appear is still an intriguing puzzle.

Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K(m) creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with (32)P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of (32)P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.

In Vitro Study of Mechanical and Kinetic Properties of Myosin II from Frog Skeletal Muscle

We provide for the first time the protocol for efficient extraction and conservation of myosin II from frog skeletal muscle, a methodological achievement that makes it possible to apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic characteristics in single muscle cells, where it works as an ensemble in each half-sarcomere. With the in vitro motility assay, we estimate the sliding velocity of actin on frog myosin II (VF) and its modulation by temperature (range 4-30 °C) and substrate concentration. VF is 8.88 ± 0.511 μm/s at 30.6 °C and decreases down to 1.6 ± 0.23 μm/s at 4.5 °C. The in vitro mechanical and kinetic parameters are integrated with the in situ mechanical and kinetic parameters of frog muscle myosin working in array in each half-sarcomere. By comparing VF with the shortening velocity determined in intact frog muscle fibres under different loads and their dependence on temperature (Piazzesi et al., J. Physol., 549:93, 2003), we find that VF is 40-50% less than the in situ unloaded shortening velocity (V0) at the same temperature and we determine the load that explains the reduced value of VF. With the integrated approach we can define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we clarify the relation between the rate of ADP release and the rate of detachment of myosin from actin and their temperature dependence. Supported by NIH (Grant no. 5R01AR49033) and MiUR Italy.

Crossbridge Properties During The Quick Force Recovery In Single Frog Muscle Fibers

Fast stretches (∼25 nmhs−1 amplitude and ∼400 μs duration) which induced the forced rupture of the crossbridge ensemble were applied to intact muscle fibers to investigate the actomyosin bond properties during the force recovery following a step length change (release or stretch of 2 or 4 nm amplitude). Force and sarcomere length were measured with a fast force transducer (∼50 kHz natural frequency) and a striation follower device. To reduce fiber damaging by the stretches and to reduce the influence of myofilament compliance on the measurements, experiments were made on the tetanus rise at tension of about 0.5 the maximum plateau tension. Fast stretches were applied before and at progressively increasing times (up to 20 ms) after the step length change. The rupture force of the crossbridge ensemble (Pc) and the sarcomere elongation at Pc (Lc) were measured. In contrast with the data obtained previously on the tetanus rise (Bagni et al. J. Physiol., 2005; 565). The results showed that: (1) Pc was almost independent of the tension developed by the fiber and (2), Lc was not constant but increased immediately after the release and decreased after the stretch. These changes were still present 2 ms later when the quick recovery was almost complete and disappeared completely within 15-20 ms. Data analysis suggests that: 1) crossbridge number remains almost constant during the quick force recovery; 2) crossbridge detachment by the fast stretch is preceded by the reversal of the myosin head power stroke and, 3) the extent of the power stroke can be measured by the changes in Lc occurring during the quick recovery.

A phenomenological model of the muscle as an active composite

A phenomenological model consisting of a spring, a contractive element, and a Voigt element is proposed for describing muscle functions. To refine the description of the mechanical behavior of muscle tissue, an additional, inertial element is introduced into the model. The contractive element is characterized based on the Hill equation. A technique is elaborated for identifying the parameters of the model from the experimental time-dependent length of the muscle during its isotonic contraction. The phenomenological model is used for describing the deformation of tailor's muscle of frogs under isotonic and isometric conditions.

A Reconsideration of the Link between the Energetics of Water and of ATP Hydrolysis Energy in the Power Strokes of Molecular Motors in Protein Structures

Mechanical energy from oxygen metabolism by mammalian tissues has been studied since 1837. The production of heat by mechanical work was studied by Fick in about 1860. Prior to Fick’s work, energetics were revised by Joule’s experiments which founded the First Law of Thermodynamics. Fenn in 1923/24 found that frog muscle contractions generated extra heat proportional to the amount of work done in shortening the muscle. This was fully consistent with the Joule, Helmholtz concept used for the First Law of Thermodynamics. The link between the energetics of water and ATP hydrolysis in molecular motors is recommended for reconsideration.

Effect of temperature on residual force enhancement in single skeletal muscle fibers

It is well accepted that the steady-state isometric force following active stretching of a muscle is greater than the steady-state isometric force obtained in a purely isometric contraction at the same length. This property of skeletal muscle has been called residual force enhancement (FE). Despite decades of research the mechanisms responsible for FE have remained largely unknown. Based on previous studies showing increases in FE in fibers in which cross-bridges were biased towards weakly bound states, we hypothesized that FE might be associated with a stretch-induced facilitation of transitioning from weakly to strongly bound cross-bridges. In order to test this hypothesis, single fibers ( n=11) from the lumbrical muscles of frog ( Rana pipiens) were used to determine FE at temperatures of 7 and 20 °C. At the cold temperature, cross-bridges are biased towards weakly bound states, therefore we expected FE to be greater at 7 °C compared to 20 °C. The average FE was significantly greater at 7 °C (11.5±1.1%) than at 20 °C (7.8±1.0%), as expected. The enhancement of force/stiffness was also significantly greater at the low (13.3±1.4%) compared to the high temperature (5.6±1.7%), indicating an increased conversion from weakly to strongly bound cross-bridges at the low temperature. We conclude from the results of this study that muscle preparations that are biased towards weakly bound cross-bridge states show increased FE for given stretch conditions, thereby supporting the idea that FE might be caused, in part, by a stretch-induced facilitation of the conversion of weakly to strongly bound cross-bridges.

Scanning electron microscopic observation of the sensory region in the frog muscle spindle

The equatorial sensory region of muscle spindles in the fourth toe extensor digitorum longus muscle of the adult frog was examined by scanning electron microscopy. Segments of this thin and long muscle after fixation were longitudinally cut with a razor blade and then treated with an HCl-hydrolysis method to remove connective tissues. Cells of the inner capsule extended thin and flattened cytoplasmic processes, showing a sieve-like appearance. Some specimens after a partial disruption of the inner capsule reevaluated at the fine structural level that numerous sensory terminals with varicose swellings longitudinally arranged along each intrafusal muscle fiber.

Toward a Unified Theory of Muscle Contraction. II: Predictions with the Mean-Field Approximation

The contractile behavior of a single half-sarcomere has been calculated from the lattice model with dimeric myosin and extensible filaments, using the model cycle with two working strokes, explicit Pi-release transitions and faster binding for the second head of the dimer. The mean-field approximation is used to generate independent state probabilities for myosin heads, assuming that the positional symmetry of actin filaments in the half-sarcomere is preserved. This model predicts absolute values of the active tension, stiffness and ATPase of fast fibers and their variation with shortening velocity, the phase-2 tension response to a length-release step and the transient tension rise during ramp stretching, in reasonable agreement with experimental data for frog muscle. It accounts for three observations beyond the reach of traditional models: (i) with elastically stiff myosin, a two-stroke model explains the rate of rapid tension recovery as a function of step size, (ii) slow Pi release from A.M.ADP.Pi after the first stroke generates the flat tension response observed after rapid recovery from a small release step, (iii) a discrete lattice model generates undamped oscillations in the isotonic length response to a force step, as observed when the sarcomeres are highly ordered. The discrete lattice also generates length-dependent oscillations in the tension-length curve and the tension response to ramp shortening, which may be smoothed out if lattice symmetry is broken.

Structure and Function of Skeletal Muscle in Zebrafish Early Larvae

Zebrafish muscles were examined at an early developmental stage (larvae 5–7 d). Using aluminum clips, preparations (∼1.5 mm length, 150 μm diameter) were mounted for force registration and small angle x-ray diffraction. Sarcomeres were oriented mainly in parallel with the preparation long axis. Electrical stimulation elicited fast and reproducible single twitch contractions. Length–force relations showed an optimal sarcomere length of 2.15 μm. x-ray diffraction revealed clear equatorial 1.1/1.0 reflections, showing that myofilaments are predominantly arranged along the preparation long axis. In contrast, reflections from older (2 mo) zebrafish showed two main filament orientations each at an ∼25° angle relative to the preparation long axis. Electrical stimulation of larvae muscles increased the 1.1/1.0 intensity ratio, reflecting mass transfer to thin filaments during contraction. The apparent lattice volume was 3.42 × 10−3 μm3, which is smaller than that of mammalian striated muscle and more similar to that of frog muscles. The relation between force and stimulation frequency showed fusion of responses at a comparatively high frequency (∼186 Hz), reflecting a fast muscle phenotype. Inhibition of fast myosin with N-benzyl-p-toluene sulphonamide (BTS) showed that the later phase of the tetanus was less affected than the initial peak. This suggests that, although the main contractile phenotype is fast, slow twitch fibers can contribute to sustained contraction. A fatigue stimulation protocol with repeated 220 ms/186 Hz tetani showed that tetanic force decreased to 50% at a train rate of 0.1 s−1. In conclusion, zebrafish larvae muscles can be examined in vitro using mechanical and x-ray methods. The muscles and myofilaments are mainly orientated in parallel with the larvae long axis and exhibit a significant fast contractile component. Sustained contractions can also involve a small contribution from slower muscle types.

Epigenetic silencers are enriched in dormant desert frog muscle

Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding protein rbbp4, histone deacetylase 1 and nuclear receptor co-repressor 2] using qRT-PCR. These seven genes showed a modest (1.1-3.5-fold) but coordinated upregulation in 6-month aestivating muscle. This reached significance for SIN3A and DNA cytosine-5-methyltransferase 1 in standard pair-wise comparisons (p < 0.05), and the candidates as a whole when analysed by Fisher's combined probability test (p < 0.01). These data are consistent with the hypothesis that the transcriptional silencing and metabolic depression that occurs during seasonal dormancy are associated with chromatin remodelling, and present a novel example of an environmentally induced epigenetic modification in an adult vertebrate.