Long (6- to 9-mo) bouts of estivation in green-striped burrowing frogs lead to 28% atrophy of cruralis oxidative fibers (P < 0.05) and some impairment of in vitro gastrocnemius endurance (P < 0.05) but no significant deficit in maximal twitch force production. These data suggest the preferential atrophy of oxidative fibers at a rate slower than, but comparable to, laboratory disuse models. We tested the hypothesis that the frog limits atrophy by modulating oxidative stress. We assayed various proteins at the transcript level and verified these results for antioxidant enzymes at the biochemical level. Transcript data for NADH ubiquinone oxidoreductase subunit 1 (71% downregulated, P < 0.05) and ATP synthase (67% downregulated, P < 0.05) are consistent with mitochondrial quiescence and reduced oxidant production. Meanwhile, uncoupling protein type 2 transcription (P = 0.31), which is thought to reduce mitochondrial leakage of reactive oxygen species, was maintained. Total antioxidant defense of water-soluble (22.3 ± 1.7 and 23.8 ± 1.5 μM/μg total protein in control and estivator, respectively, P = 0.53) and membrane-bound proteins (31.5 ± 1.9 and 42.1 ± 7.3 μM/μg total protein in control and estivator, respectively, P = 0.18) was maintained, equivalent to a bolstering of defense relative to oxygen insult. This probably decelerates muscle atrophy by preventing accumulation of oxidative damage in static protein reserves. Transcripts of the mitochondrially encoded antioxidant superoxide dismutase type 2 (67% downregulated, P < 0.05) paralleled mitochondrial activity, whereas nuclear-encoded catalase and glutathione peroxidase were maintained at control values (P = 0.42 and P = 0.231), suggesting a dissonance between mitochondrial and nuclear antioxidant expression. Pyruvate dehydrogenase kinase 4 transcription was fourfold lower in estivators (P = 0.11), implying that, in contrast to mammalian hibernators, this enzyme does not drive the combustion of lipids that helps spare hypometabolic muscle.
- disuse atrophy
- gene expression
the green-striped burrowing frog Cyclorana alboguttata survives extended droughts by burrowing underground, shedding a waterproof cocoon (33), and undergoing a deep metabolic depression called estivation (29). In this capacity, its hindlimbs are fully immobilized, and it has no external food supply. C. alboguttata can withstand short (3-mo) bouts of estivation without suffering the losses in muscle contractile performance and mass (13) normally associated with disuse (23) and starvation (12).
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].
Green-striped burrowing frogs, C. alboguttata (10–25 g body wt), were captured from flooded roadsides after summer rains in southeastern and mideastern Queensland, Australia, as described previously (25). They were assigned to one of three treatment groups (controls, 6-mo estivators, and 9-mo estivators), with treatments matched as closely as possible for gender and body mass. Control frogs (n = 6) were housed in individual plastic boxes (27 × 14 × 12 cm) containing wet paper toweling and were watered and fed ad libitum. To induce estivation, two separate groups of frogs (both n = 6) were placed into individual plastic containers filled with wet clay (obtained from the locality of animal capture) that was allowed to dry naturally over a period of several days. These frogs burrowed almost immediately and quickly became entombed in a hardened clay block. After 6 mo (for cruralis RNA, cruralis histology, and gracilis biochemistry) or 9 mo (for gastrocnemius contractile properties), we extracted estivating frogs by breaking open the soil block across the burrow line.
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.
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 ×250 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.
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.
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 ×33 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.
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.
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.
Gene amplification, cloning, and sequencing.
Genes of interest were amplified using standard PCR in up to 60-μl reactions using neat, 1:2 or 1:5 cDNA. The stringency of the reaction was adjusted for the degenerate primers by lowering the annealing temperature from 60°C to 50°C. The amplicons were identified by size after 1.8% agarose gel electrophoresis, gel extracted (Qiagen gel extraction kit), and resuspended in 30 μl of ultrapure water.
The cleaned amplicons were ligated into pGEMTeasy (Promega) vectors and electroporated into 40 μl of DH5-α electrocompetent Escherichia coli (200 Ω, 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.
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.
RT-PCR amplification mixtures (10 μl) contained 1 μl of cDNA (1:25 dilution), 2× SYBR Green I Master Mix buffer (5 μl; Applied Biosystems), and 600 nM forward and reverse primer. Reactions were run on a sequence detector (PRISM 7900HT, Applied Biosystems) using cycling parameters defined by the manufacturer.
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.
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).
Six months of estivation had no significant impact on total cruralis muscle area (P = 0.309) or pooled average fiber cross-sectional area (P = 0.093). However, when considered in isolation, the cross-sectional area of the oxidative fibers significantly declined by 28% from 3,370 ± 800 μm2 in the control frogs to 2,450 ± 200 μm2 (P < 0.05) in the estivators, indicating muscle atrophy. In contrast, the glycolytic fibers showed no significant reduction, although the 22% reduction (9,500 ± 1,700 and 7,400 ± 1,210 μm2) came very close to statistical significance (P = 0.055). The histological data thus indicate a preferential atrophy of the oxidative fibers after estivation, but the overall gross anatomy of the muscle was relatively well preserved (Figs. 1 and 2).
The total antioxidant power of water-soluble and membrane-bound proteins was assessed in the gracilis muscle of control and 6-mo-estivating C. alboguttata. Water-soluble antioxidants in 6-mo estivators (23.8 ± 1.5 μM/μg) matched levels in control frogs (22.3 ± 1.7 μM/μg, P = 0.532), as did membrane-bound antioxidants, which averaged 42.1 ± 7.3 and 31.5 ± 1.9 μM/μg in estivating and control C. alboguttata, respectively (P = 0.18). These data indicate maintenance of summed antioxidant potential in muscle tissue of C. alboguttata after 6 mo of estivation. To normalize to oxygen insult, we assumed a conservative reduction in oxygen consumption of 70%, to 30% of control values; Storey (29) reported 70–90% metabolic depression. Thus the total antioxidant power for water-soluble antioxidants was upregulated ∼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 × 100/30 = 79.3 μM/μg.
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).
No amphibian has been fully sequenced. Xenopus tropicalis and Xenopus laevis, the two best-annotated amphibian species, are members of an ancient lineage, the Archaebatrachia. The Hyloidea, which includes Cycloraniids, are believed to have diverged from this lineage ∼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).
The absolute mRNA transcript number of the seven genes was determined in control and 6-mo-estivating frogs from a fixed amount (2.5 μg) of total RNA. Estivation for 6 mo led to a reorganization of this small subset of the cruralis transcriptome (Figs. 4 and 5). Antioxidant defense gene expression was modulated by 6 mo of estivation, but the effect of SOD2 differed from that of CAT and GPX4. Mitochondrial SOD2 transcription was downregulated 67% (P < 0.05), whereas CAT and GPX4 gene expression was maintained at close to control values (P = 0.423 and P = 0.251; Fig. 4).
The gene for substrate combustion, PDK4, was downregulated 73% after 6 mo of estivation, but this did not reach statistical significance (P = 0.112), probably because of the wide individual variation in the control group. The genes for the mitochondrially encoded enzymes ND1 and ATP synthase were significantly and similarly downregulated by 71% (P < 0.05) and 67% (P < 0.05), respectively. In contrast, the transcripts of the nuclear-encoded mitochondrial protein UCP2 were maintained close to control values (P = 0.312; Fig. 5).
Estivation in Cycloranid frogs is characterized by a whole animal metabolic depression. Metabolic depression protects against muscle atrophy for at least two reasons. First, because lipid is combusted before muscle, hypometabolism functionally extends lipid stores and delays the need to combust muscle protein. Second, because a fixed proportion of oxygen respired is converted to reactive oxygen species (ROS), lowered aerobic flux will lead to absolute reductions in ROS consistent with metabolic rate (10) (with the assumption of constant uncoupling), thereby reducing atrophy associated with oxidative damage.
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.
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.
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.
The experimental work was supported jointly by a Commonwealth Scientific and Industrial Research Organization postdoctoral fellowship to N. J. Hudson and an Australian Research Council grant to C. E. Franklin.
We thank Amanda Niehaus for frog collection and animal husbandry, Chris Elvin for advice on degenerate primer design, Roger Pearson for advice on preparing the biochemical samples, and Yong Hong Wang and Brian Dalrymple for interesting discussions. Drewe Ferguson and Bill Barendse provided comments on an early draft of the manuscript.
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.
- Copyright © 2006 the American Physiological Society