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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
1Commonwealth Scientific and Industrial Research Organization Livestock Industries and 2School of Integrative Biology, University of Queensland, Brisbane, Queensland, Australia
Submitted 31 May 2005 ; accepted in final form 19 October 2005
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ABSTRACT |
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disuse atrophy; antioxidant; mitochondria; gene expression
Proximally, these losses in mammalian atrophy models are the result of a negative protein balance due to a relative reduction in protein synthesis (30) and a relative increase in protein degradation (6). The ultimate reasons reflect the plastic response of muscle to its contractile history or an energy balance that cannot be met by food and endogenous fat reserves. The ability of C. alboguttata to rapidly emerge from their subterranean burrows suggests that they have certain physiological modifications that limit these effects, thereby permitting opportunistic feeding and breeding when the summer rains come.
Some ability to inhibit muscle atrophy appears to be a relatively common phenomenon in dormant animals, as exemplified by hibernating bears (11), hamsters (32), and squirrels (28), as well as estivating frogs. However, the mechanisms underpinning this phenotype are largely unknown. Metabolic depression per se likely has a protective role (14), in part by delaying the need to mobilize muscle protein. Additionally, in the specific case of C. alboguttata, spontaneous release of the neurotransmitter acetycholine is maintained at iliofibularis neuromuscular junctions (15), permitting ongoing communication between muscle and nerve, despite an absence of contraction.
In this study, we aimed to further characterize the impact of estivation on muscle structure in extended (6- to 9-mo) bouts of estivation, which more closely approximate the field situation. To this end, we have assayed in vitro muscle performance and muscle fiber composition/morphometry. In addition, we aimed to gain a better understanding of the physiological mechanisms underpinning the inhibition of muscle atrophy by assessing gene expression of several candidate proteins. We used quantitative PCR to test three main hypotheses. First, we examined antioxidant enzymes [glutathione peroxidase 4 (GPX4), catalase (CAT), and superoxide dismutase 2 (SOD2)], because an accumulation of oxidative damage contributes to protein degradation in atrophying mammalian muscle (16, 18–20). We also verified the antioxidant defense transcript data at the biochemical level. Second, we predicted that muscle protein would be spared as a major fuel source. To this end, we tested pyruvate dehydrogenase kinase 4 (PDK4), the biochemical switch indicating a preferential combustion of lipids in hibernating mammals (5). Third, we hypothesized that estivating frogs would increase metabolic efficiency, inasmuch as their food supply is unpredictable and sporadic. To this end, we tested the expression of genes fundamental to mitochondrial energy metabolism and efficiency [uncoupling protein (UCP) type 2, NADH ubiquinone oxidoreductase subunit 1 (ND1), and ATP synthase].
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METHODS |
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The different muscles were selected for various practical reasons. At its distal end, gastrocnemius has a bunch of connective tissue, which provides an excellent site for suturing, making it ideal for in vitro contractile studies. The cruralis is the largest single muscle in the hindlimb; therefore, it was used for RNA extraction, inasmuch as RNA yield can limit the validity of expression analysis. The contralateral cruralis was used for histology to help interpret the gene expression data. The gracilis, which can be identified and excised easily, was chosen for biochemistry. All frogs were euthanized by double pithing in compliance with the University of Queensland ethics guidelines. The gastrocnemius, cruralis, and gracilis were excised within 15 min.
Histology.
Cruralis slices (5 mm) were dissected from control and 6-mo-estivating frogs, mounted in Tissue-Tek OCT compound, and plunged into precooled 2-methylbutane (–150°C) for 
30 s. The frozen blocks were removed and wrapped in aluminum foil to prevent desiccation and stored at –80°C in an airtight container for sectioning.
The frozen muscle blocks were mounted and serially sectioned (10 µm) in a cryostat (model HM505, Microm) at –20°C. The sections were melted directly onto glass slides, air dried for 3 min, and returned to the cryostat chamber for storage in an airtight container before staining. Slides were removed from the cryostat chamber and stained immediately for succinate dehydrogenase to differentiate between oxidative and glycolytic muscle fibers following a protocol described elsewhere (26). Sections were incubated in the staining medium in airtight, light-proof containers for 
2 h, at which time each slide was dehydrated and mounted with 8-cyclopentyl-1,3-dipropylxanthine. Each section was photographed at x250 magnification with a digital camera mounted on an Olympus compound microscope. Images were analyzed with SigmaScan software to examine fiber cross-sectional area (measuring 50 fibers per muscle, chosen at random with a grid overlay and a random number generator) and distribution.
Antioxidant biochemistry.
The gracilis muscle was roughly sliced, inserted into a cryotube, snap frozen in liquid nitrogen, and temporarily stored at –80°C. A bead beater (BIO101 FastPrep FP120) was used to homogenize 0.2 g of tissue in 1,000 µl of PBS in two 30-s pulses. Between pulses, the sample was kept on ice for 2 min. The resultant slurry was pipetted into a fresh Eppendorf tube and spun at 3,000 g in a tabletop microcentrifuge at 4°C for 3 min. The supernatant was divided into aliquots, stored at –80°C, and subsequently used for analysis of water-soluble proteins. The pellet was briefly washed in PBS, resuspended and incubated in a mild detergent (1% CHAPS), and respun at 3,000 g. The supernatant was divided into aliquots, stored at –80°C, and subsequently used for analysis of membrane-bound proteins.
The antioxidant potential of water-soluble and membrane-bound proteins was assessed using a commercially available colorimetric kit (Total Antioxidant Power; Oxford Biomedical Research) following the manufacturer's instructions. The colorimetric reading was assayed on a multiplate colorimetric spectrophotometer at 490 nm. Total protein content was determined using a commercially available spectrophotometric kit (BSA protein assay; Sigma) assayed at 562 nm. The antioxidant power results are expressed per microgram of total protein.
Muscle mechanics. The gastrocnemius muscle was attached to a force transducer (model FE301; Grass). The gastrocnemius was specified, because it has a bundle of connective tissue that allows convenient suturing. It was tied at the distal end with silk suture material and pinned directly through the knee joint at the proximal end. The muscle was bathed in circulating Ringer solution (pH 7.4) at 24 ± 1°C. The transducer was connected to a MacLab data acquisition system, and the signal was amplified using a Bridge Amp (AD Instruments, Castle Hill, Australia), from which it was directed to a MacLab 4e computer running Chart software (version 3.5). A micromanipulator was used to set the muscle at its optimal length, and contractions were elicited with electrical stimuli. Twitch (8-V single pulse, 3-ms duration) and tetanus (8 V, 20 pulses at 90 Hz for 6 ms) responses were elicited every minute until the response fell to <50% of the maximum tetanus. The time difference between stimulus and response and half-rise and half-decay times were also determined for the maximal twitch response.
The middle of the belly of the contralateral gastrocnemius was mounted on a stub and rapidly frozen at –40°C in methylbutane suspended in liquid nitrogen. Sections (10 µm) were cut in a cryostat at –20°C, mounted on a slide with Kaiser's glycerol, and stained with eosin. Several sections were observed under a dissection microscope at x33 magnification and photographed using an Olympus DP10 camera. The cross-sectional area of the largest section was calculated using Videoscan (IBM) software and used to size correct the force production data.
RNA extraction.
A standard phenol-chloroform protocol was used to extract total RNA from the cruralis of control and 6-mo-estivating frogs (n = 6). The cruralis was chosen for practical reasons: in anurans, it is a large muscle capable of providing a suitable yield of RNA. Briefly, the frozen cruralis muscle tissue was double wrapped in industrial-strength aluminum foil and pulverized with a hammer. The muscle powder (200 mg) was homogenized in 5 ml of Trizol (Invitrogen) reagent using an Ultraturex (Labortechnik, Staufen, Germany) probe and then centrifuged (4°C) with 1 ml of chloroform. The RNA was precipitated by centrifugation with 2.5 ml of isopropanol. The pellet was washed with ethanol, resuspended in 150 µl of ultrapure water, and stored at –80°C. RNA yield and quality were assayed by spectrometry (i.e., the ratio of absorbance at 260 nm to absorbance at 280 nm; Eppendorf Biophotometer) and visually by agarose gel (1.8%) electrophoresis. By this method, 200 mg of cruralis tissue typically yielded 150 µg of total RNA.
cDNA synthesis. A fixed amount of total RNA (2.5 µg) per individual was used in each cDNA reaction. All RNA samples exhibited strong 16S signals. The RNA was treated with DNase (Ambion) to eliminate genomic DNA and then reverse transcribed into cDNA following the Superscript III (Invitrogen) protocol (with random hexamers). Contaminating RNA was eliminated by incubation of the product with 0.5 µl of RNase H for 20 min at 37°C. The samples were PCR cleaned (Qiagen on-column digestion; Hilden, Germany) and resuspended in 50 µl of ultrapure water.
Cross-species primer design. To identify the closest amphibian matches, the human reference protein sequence for each candidate gene was used as a target to identify similar sequences in GenBank using the basic local alignment search tool (BLAST) BLASTp. Functional homology to the human reference sequence was assigned if the BLAST hit score was >100. Degenerate primers were designed on the basis of conserved motifs in the corresponding protein sequences from three organisms (typically 1 or 2 amphibians and a representative from another group, e.g., a fish or a mammal; Table 1), which were SLOW aligned using EclustalW on ANGIS using the default settings. By cross-referencing to the original nucleotide sequences and only designing alternatives for the bases that varied, we were able to establish consensus sequences with low (<70) degeneracies.
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The cleaned amplicons were ligated into pGEMTeasy (Promega) vectors and electroporated into 40 µl of DH5-
electrocompetent Escherichia coli (200 
, 25 µF, 1.7 kV).
Ampicillin-resistant colonies were screened for the presence of the insert by diagnostic PCR (M13 forward and reverse primers) and agarose gel electrophoresis.
Plasmid DNA was isolated from positive colonies using the Big Dye 3.1 protocol (Applied Biosystems). Gel separations were performed by the Australian Genome Research Facility.
Quantitative PCR. The cloned C. alboguttata sequences were used as the template to design specific 180- to 205-bp amplicons for quantitative PCR (Table 2). All nondegenerate primer design was performed using Primer Express 2.0 software, with acceptance of the default settings and optimization for quantitative PCR.
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Each assay (in quadruplicate) included a no-template control. All PCR efficiencies were >95%, and all the assays produced unique dissociation curves. Sequence Detection Software (version 2.0, Applied Biosystems) results were exported as tab-delimited text files and imported into Microsoft Excel for further analysis. The expression of each gene was quantified absolutely by comparison with standard curves generated from plasmid serial dilutions.
Statistical analysis. The two treatments were compared by Student's t-test. If data failed the assumption of normality, they were compared using the nonparametric Mann-Whitney U-test. The histological data were compared using analysis of covariance with animal length as the covariate. In all cases, P < 0.05 was considered significant. All statistical analysis was performed on Sigmastat software (version 2.0).
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RESULTS |
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3.5-fold: 79.3 and 22.3 µM/µg in estivators and controls, respectively. This normalization was calculated by multiplying the estivator data by 100/30; i.e., 23.8 x 100/30 = 79.3 µM/µg. Muscle mechanics. The isometric contractile properties of the gastrocnemius muscle were determined for control and 9-mo-estivating C. alboguttata. The mass of the gastrocnemius (g) relative to snout-to-vent length (cm) was 0.0516 ± 0.03 and 0.0443 ± 0.09 for controls and 9-mo estivators, respectively (P = 0.459). The absolute twitch force produced by the gastrocnemius (mN/mm2) normalized to the snout-to-vent length (cm) was 318 ± 29 and 270 ± 51 for controls and estivators, respectively (P = 0.431); for tetanus it was 656 ± 38 and 524 ± 84, respectively (P = 0.186).
Equally, when these data were reexpressed relative to gastrocnemius cross-sectional area, the isometric twitch properties (maximal force production, half-rise and half-decay times, and latency) of the 9-mo estivators immediately after release from their burrows did not differ significantly from the those of the control group (see Table 4). The isometric tetanic properties of the gastrocnemius (maximal force production and tetanic-to-twitch ratio) were also maintained at control levels (see Table 4). However, the rate of fatigue of the gastrocnemius was significantly greater in the 9-mo estivators than in the controls, indicative of a loss in endurance after lengthy bouts of estivation (Fig. 3).
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152,000,000 years ago (2), which seems to be close to the upper limit at which reasonable heterologous hybridization can occur (24). Consequently, degenerate primers were designed to amplify >200 bp of the C. alboguttata sequence (Table 1). The nucleotide sequence returned from C. alboguttata genes cloned in this study showed the highest similarity to amphibian and reptilian sequences and was typically 80% similar to X. laevis and X. tropicalis (Table 3).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In the present work, we have investigated the reorganization of metabolism in estivation as indicated by a small subset of mRNA transcripts. This confirms previous work that dormancy in vertebrates is associated with a reprogrammed transcriptome (8, 27). In our experimental design, we have used three C. alboguttata hindlimb muscles: gracilis, cruralis, and gastrocnemius. We have elected to discuss the in vitro performance, biochemistry, and gene expression results from these muscles as if they apply interchangeably. However, we cannot completely exclude the possibility that the muscles undergo different structural and functional responses to estivation, such as those that might result from variation in fiber composition and loading history. In addition, it is conceivable, although in our opinion unlikely, that the gastrocnemius contractile data might have been significantly different had the muscles been sampled at 6 mo (consistent with the biochemistry and histology) instead of 9 mo.
Histology and muscle performance. In C. alboguttata cruralis muscle, the glycolytic fibers were relatively unaffected by 6 mo of estivation, but the oxidative fibers were significantly reduced in cross-sectional area (P < 0.05). Nevertheless, the reduction in glycolytic fiber area did approach significance, and if the sample size had been larger or estivation length had been increased, it might have reached significance. Fundamentally, the impact of 6 mo of estivation on C. alboguttata muscle appears to be more consistent with mammalian models of disuse (where oxidative fibers preferentially atrophy) than starvation (where glycolytic fibers tend to atrophy).
The present findings that the raw force data (normalized to frog length, not muscle cross-sectional area) tend to be lower after estivation (although not significantly with this sample size and estivation length) probably reflect the atrophy of individual fibers as documented by the histological data. Consequently, there may be some impact of 6 mo of estivation on whole animal locomotor performance. On the other hand, the muscle area-corrected contractile properties of the gastrocnemius are largely unchanged after 9 mo of estivation (Table 4), which is broadly in agreement with previous work on brief (3-mo) bouts of estivation in C. alboguttata (24) and hibernation in hamsters, Mesocricetus auratus (48). They indicate that, even after extended bouts of estivation, the functional capacity of the remaining actin, myosin, sarcoplasmic reticulum, and myofibrillar organelles remain largely unimpaired.
However, the significant reduction in endurance capabilities of the gastrocnemius (Fig. 3; P < 0.05) and the fact that the decline in tetanic force production (Table 4) approaches significance (P = 0.087) indicate that at 6–9 mo, contractile evidence for degenerative changes has accumulated in C. alboguttata. This evidence supports the histological data that the oxidative fibers are most affected. Nevertheless, the overall disuse response is fundamentally weaker than in clinical situations in mammals, where significant atrophy can occur in as little as 4 days of limb immobilization (25). Consequently, C. alboguttata represents a pertinent system to identify biochemical and gene expression correlates for decelerated muscle atrophy.
Antioxidant defense. The raw absolute "total antioxidant power" data are consistent with the activities of specific muscle antioxidants observed in the estivating spadefoot toad Scaphiosus couchii, in which absolute values of glutathione transferase, glutathione reductase, GPX4, and CAT were maintained during a short (2-mo) period of estivation (10). However, relative to a diminished oxygen insult, total antioxidant defense is effectively elevated threefold in estivating C. alboguttata.
Muscle disuse atrophy in mammals is associated with an accumulation of oxidative damage (16, 18–20) due in part to transcription of antioxidant enzymes in mammalian species in response to muscular activity. For example, unloaded mammalian muscle contains 54% less CAT (21), which is thought to contribute to ROS-induced muscle atrophy. Furthermore, experimental application of antioxidants such as vitamin E decelerates muscle atrophy in immobilized rat limbs (17). It is of interest, therefore, that, in a muscle system specifically adapted to disuse followed by rapid remobilization, relative CAT defense is endogenously upregulated. This points to CAT as an interesting candidate for the therapeutic control of muscle disuse atrophy in vertebrates.
The transcript data for control C. alboguttata show that different antioxidant enzymes vary widely in abundance in muscle tissue: GPX4 is abundant, mitochondrial SOD2 is rare, and CAT is intermediate.
The contrasting response within C. alboguttata of SOD2 vs. CAT and GPX4 transcription is of particular interest. CAT and GPX4 are representative of nuclear-encoded antioxidants, whereas SOD2 is mitochondrially encoded. A likely explanation is that as SOD2 specifically protects mitochondria from oxidative damage, it simply tracks the aerobic activity of the mitochondrion itself. Oxygen consumption is reduced 70% in estivation, and SOD2 defense almost exactly parallels this change.
Fuel selection. Estivating amphibians, similar to hibernating mammals, preferentially combust lipids during dormancy (29), consistent with the sparing of lean muscle mass. In hibernating mammals, as represented by the 13-lined ground squirrels Spermophilus tridecemlineatus, the combustion of lipids is controlled at the biochemical level by a fivefold upregulation of PDK4 enzyme activity (5).
The present study shows a 71% downregulation in PDK4 transcripts in 6-mo estivators, although this does not reach statistical significance because of high interindividual variation. This variation may be a product of inadequate sample size (n = 6) or a genuine biological effect relating to 1) gender [female frogs generally tend to deposit significantly more lipid than male frogs (3)] or 2) the overall size range, which may impose allometric scaling considerations on substrate use. Nevertheless, the response of frog PDK4 to estivation contrasts markedly with that of hibernating mammals and suggests that estivators, as represented by C. alboguttata, use an alternative biochemical switch point.
Energy metabolism and ROS production.
Usually, a fixed proportion (5%) of respired oxygen is converted to ROS (1). By assessing ND1 and UCP2 transcripts, we were interested to see whether mitochondrial remodeling suggested a reduction (<5%) in the relative proportion of ROS produced. The maintenance of UCP2 transcripts at control levels suggests that the energetically costly process of uncoupling is maintained during estivation. The results for ND1, which is used as an indicator of electron transport activity and the generation of the mitochondrial proton gradient (taken together with the ATP synthase data), are consistent with a marked downregulation in electron transport activity and mitochondrial quiescence.
In addition, the similarity in the expression patterns of ND1 and ATP synthase relating to mitochondrial activity and SOD2 relating to mitochondrial antioxidant defense is noteworthy, inasmuch as they are encoded by the mitochondrial genome.
The ND1, ATP synthase, and UCP2 data are consistent with the respiratory data available for overwintering hypoxic European common frogs, Rana temporaria. During hibernation under ice-covered ponds, R. temporaria exhibit no change in proton conductance but do show decreased electron transport activity (43). These results suggest common mechanisms of metabolic depression in amphibia, whether it be terrestrial estivation at 25°C or aquatic hibernation at 0°C. Given that hibernating and estivating frogs rely on finite endogenous lipid resources, reducing mitochondrial uncoupling might extend dormancy, but this is not the case.
An explanation may be that low UCP2 expression correlates with high levels of ROS production (7). The approximate maintenance of UCP2 expression may reflect a compromise to keep ROS production to a minimum, without wasting too much fuel on proton gradient dissipation. On the other hand, previous work in endotherms suggests that electron transport enzymes and UCP expression might vary between mammals and amphibia. For example, ND2, another subunit in complex I of the respiratory chain, exhibits a fourfold upregulation in the hibernating ground squirrel, Spermophilus lateralis (9). Similarly, in torpid hummingbirds (31) and hibernating arctic ground squirrels, Spermophilus parryii (4), the UCP3 muscle transcripts respond by upregulating 1.5- to 3.4- and 3-fold, respectively. UCP futile cycling generates heat, consistent with the rewarming bouts central to mammalian hibernation physiology, at cold (close to 0°C) ambient temperatures. In contrast, C. alboguttata is ectothermic and hypometabolic at 25°C in the laboratory. These contrasting thermal requirements may dominate the expression patterns of UCPs in organisms from such different taxa.
The maintenance of UCP2 compared with the depression in ND1 and ATP synthase expression could also impact the fundamental aerobic physiology of the mitochondrial respiratory chain, such as delayed muscle phosphocreatine resynthesis after exercise (22). These alterations may contribute to the present performance data showing a 32% reduction in endurance of 9-mo-estivating muscle under repeated isometric contraction (P < 0.05).
In conclusion, oxidative fibers atrophy during extended bouts of estivation in C. alboguttata, as assayed histologically and by in vitro contractile performance. This change is more similar to mammalian disuse than starvation is but much less pronounced. Relative to oxygen insult, antioxidant defense is bolstered, but this seems to be a reflection of the maintenance of nuclear-encoded antioxidants (CAT and GPX4), rather than mitochondrially encoded antioxidants (SOD2). Consequently, atrophy associated with oxidative damage to vulnerable, static, estivating skeletal muscle seems to be kept to a minimum by 1) a combination of a reduction in total oxygen insult (lowering absolute production of ROS after a coordinated downregulation of ND1 and ATP synthase); 2) a maintenance of UCP2 transcription (keeping the relative production of ROS to 5%), despite the associated energetic cost; and 3) a modulation of specific antioxidant enzymes. Additionally, although lipid reserves are presumably exhausted before protein is combusted in C. alboguttata, the biochemical switch point appears to differ from that in hibernating mammals. Although there are commonalities between estivation and hibernation at the molecular genetic, biochemical, and physiological levels, this study highlights some fundamental differences; some of these differences may be purely taxonomic in origin (PDK4), whereas others (UCP2) may reflect considerations of thermal biology.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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