Physiol. Genomics Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 31: 521-530, 2007. First published September 11, 2007; doi:10.1152/physiolgenomics.00131.2007
1094-8341/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables
Right arrow Addendum
Right arrow All Versions of this Article:
31/3/521    most recent
00131.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Crawford, F. I. J.
Right arrow Articles by Loudon, A. S. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Crawford, F. I. J.
Right arrow Articles by Loudon, A. S. I.
Received 20 June 2007; accepted in final form 5 September 2007.
Physiological Genomics 31:521-530 (2007)
1094-8341/06 $8.00 © 2007 American Physiological Society

Influence of torpor on cardiac expression of genes involved in the circadian clock and protein turnover in the Siberian hamster (Phodopus sungorus)

Fiona I. J. Crawford1, Cassandra L. Hodgkinson1, Elena Ivanova1, Larisa B. Logunova1, Gary J. Evans2, Stephan Steinlechner3 and Andrew S. I. Loudon1

1 Faculty of Life Sciences, University of Manchester, Manchester
2 PIC UK Limited, Kingston Bagpuize, Oxfordshire, United Kingdom
3 Department of Zoology, School of Veterinary Medicine, Hannover, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
The Siberian hamster exhibits the key winter adaptive strategy of daily torpor, during which metabolism and heart rate are slowed for a few hours and body temperature declines by up to 20°C, allowing substantial energetic savings. Previous studies of hibernators in which temperature drops by >30°C for many days to weeks have revealed decreased transcription and translation during hypometabolism and identified several key physiological pathways involved. Here we used a cDNA microarray to define cardiac transcript changes over the course of a daily torpor bout and return to normothermia, and we show that, in common with hibernators, a relatively small proportion of the transcriptome (<5%) exhibited altered expression over a torpor bout. Pathways exhibiting significantly altered gene expression included transcriptional regulation, RNA stability and translational control, globin regulation, and cardiomyocyte function. Remarkably, gene representatives of the entire ubiquitylation pathway were significantly altered over the torpor bout, implying a key role for cardiac protein turnover and translation during a low-temperature torpor bout. The circadian clock maintained rhythmic transcription during torpor. Quantitative PCR profiling of heart, liver, and lung and in situ hybridization studies of clock genes in the hypothalamic circadian clock in the suprachiasmatic nucleus revealed that many circadian regulated transcripts exhibited synchronous alteration in expression during arousal. Our data highlight the potential importance of genes involved in protein turnover as part of the adaptive strategy of low-temperature torpor in a seasonal mammal.

hibernation; ubiquitylation; hypometabolism; Djungarian hamster; microarray


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
THE SIBERIAN (OR DJUNGARIAN) hamster, Phodopus sungorus, is a native of southwestern Siberia and northeastern Kazakhstan and survives the extreme winters with a number of photoperiodically induced seasonal adaptations including daily torpor cycles. During a typical torpor bout, body temperature (Tb) decreases over 3–4 h to a minimum temperature of 15°C, thereby reducing heat loss to the environment. Unlike hibernation bouts, which are of several days to weeks in duration, a daily torpidator such as the Siberian hamster only exhibits a transient drop in temperature during the light phase when the animal is normally inactive. Torpor is thought to be driven by output from the circadian clock, because torpor cycles free-run in constant darkness with a period similar to that of the circadian activity cycle (35), while destruction of the suprachiasmatic nucleus (SCN) ablates torpor rhythms (34).

Little is known of the transcriptional changes that occur during torpor, but it has been hypothesized that since transcription and translation are energetically expensive processes, only proteins required during hypometabolism or arousal may be transcribed and translated at this time. While initial studies of hibernators focused on expression of a few candidate genes, the advent of high-throughput technologies such as microarrays has enabled the screening of a much larger proportion of the transcriptome. Several of these studies have focused on the heart, because this organ must exhibit special adaptations to survive prolonged periods of low temperature without suffering cardiac arrest. A recent study of the golden-mantled ground squirrel revealed that <2% of the arrayed transcriptome in the heart exhibited altered expression during torpor (43). Another study of the related thirteen-lined ground squirrel revealed that <1% of the assayed clones underwent significant changes during a hibernation bout, and included within this were genes involved in calcium handling and contractility of cardiomyocytes (4). Recently, we described (11) a seasonal adaptation in the Siberian hamster whereby ventricular myocytes isolated from short photoperiod (SP)-housed animals stored more Ca2+ in the sarcoplasmic reticulum and exhibited larger Ca2+ transient amplitudes on stimulation, leading to predicted stronger contraction of cardiomyocytes, compared with long photoperiod (LP)-adapted cardiomyocytes, implying that this axis may be targeted for transcriptional control during low-temperature bouts.

Here we describe the results of array studies in which we have compared cDNA profiles at five time points across the torpor cycle, using cardiac tissue from torpid and normothermic Siberian hamsters. Our data revealed that a relatively small subset of genes showed changes in expression during torpor, but this included alterations in a cohort of ubiquitylation pathway genes, suggesting that protein turnover and modification may be key features of daily torpor. One cardiac peptide (BNP) also showed significantly altered expression on our arrays. We also identified significantly altered expression of Bmal1, a key circadian clock gene, and subsequent studies of six different clock genes by quantitative PCR (Q-PCR) revealed consistent alterations of expression in heart, lung, and liver during a torpor bout, suggesting a general peripheral tissue response of the circadian axis during torpor. Within the SCN, we also identified torpor-associated changes in expression of two circadian clock genes (Per 1 and 2) during torpor. Our data therefore suggest that cardiac pathways involved in protein turnover may be regulated in a complex manner over the course of a torpor bout.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Male Siberian hamsters, derived from breeding colonies at the School of Veterinary Medicine, Hannover, were born in natural photoperiod and ambient temperatures during the spring/summer of 2002. On weaning, animals were individually housed at 18°C in a photoperiod that decreased stepwise (1 h/wk) to a final photoperiod of 8 h light:16 h dark. Food and water were available ad libitum throughout the experiment. All research and animal care procedures were approved by the Review Board for the Care of Animals of the district government, Hanover, Germany (no. 509.6-42502-01/497) and were performed according to international guidelines for the use of laboratory animals.

To monitor Tb with ±1°C accuracy, temperature-sensitive radio transmitters (Mini-Mitter model XM) were implanted into the peritoneal cavity under pentobarbital anesthesia. Animals were observed for outward signs of torpor including decreased activity, slow breathing rate, and decreased reaction to a stimulus (a gentle tap on the side of the cage). Hamsters that did not respond to this stimulus were tentatively labeled "torpid." Where temperature transmitters were implanted, temperature profiles were examined.

Normothermic control animals were sampled at eight time points 3 h apart, covering the entire day-night cycle (SP normothermic). Animals were sampled with respect to lights on, which is defined as zeitgeber time (ZT) 0. Torpid animals were sampled at five time points 3 h apart, starting at ZT22 and finishing at ZT10 (SP torpid). Immediately after culling, core body temperatures of all animals were taken to confirm torpor or normothermia, by inserting a digital thermometer probe 1.5 cm into the throat. Samples were taken and extracted under RNase-free conditions.

Animals were culled by CO2 inhalation. Hamsters in the dark phase were culled under dim red light (<1 lx), and all animals were enucleated postmortem to prevent possible light-induced gene induction. To ensure that expected responses to photoperiod had occurred, hamsters were weighed and pelage scored with a previously defined scale (1 = full agouti summer pelage, 6 = full white winter pelage) (16). Brain, heart, lungs, and liver were rapidly removed, frozen on dry ice, wrapped in baked foil, and stored at –80°C. Samples were transported to the UK on dry ice. Once in the UK, samples were maintained at –80°C.

RNA isolation.
Total RNA was isolated from heart, liver, and lung in TRIzol (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were assessed with an Agilent 2100 Bioanalyser in conjunction with the RNA 6000 Nano assay kit (Agilent) and RNA 6000 ladder (Ambion).

Isolation and cloning of hamster-specific sequences.
Partial sequences for target genes were isolated from cDNA (Gapdh and Dbp) or gDNA (Esrra, Ube2l3, Bnp, Anp, and Cry2) with primers (MWG Biotech or Sigma Genosys) based on regions of conserved homology between several mammalian species. These sequences have been deposited in GenBank (accession nos. EF063002–EF063008). To confirm that hamster cDNA would likely hybridize with mouse cDNA in a microarray study we compared published Siberian hamster gene sequences and those sequenced within this study to published homologous mouse sequences with BLASTn (1). These showed that there was a 91% sequence similarity between the two genomes (Supplemental Table 1).1

Microarrays.
Microarrays were kindly provided by the Human Genome Mapping Project Resource Centre (HGMPRC), Cambridge, UK. They were manufactured with a murine cDNA clone set developed by the US National Institute on Aging, which was arrayed onto glass slides by the HGMPRC. The clone set constituted 15K sequenced cDNA clones from early embryonic mouse libraries, each with an average size of 1.5 kb (25).

Each clone in the array set was spotted in duplicate, and four array sets per time point were used, resulting in eight replicates for each clone. Reciprocal labeling was incorporated such that half of each cDNA pool (comprising RNA extracted from 3–6 hearts per pool) was labeled with Cy3 and half with Cy5 to control for potential dye-related bias. Fifty-microgram aliquots of total RNA in nuclease-free water (Ambion) were heated with oligo(dT) primer at 70°C for 10 min. RNA was converted to Cy3- or Cy5-labeled cDNA in a solution containing 1x First Strand Synthesis Buffer (Invitrogen), 10 mM DTT (Invitrogen), 0.5 mM dATP, dGTP, and dCTP (Bioline), 0.2 mM dUTP (Bioline), 67 µM Cy3- or Cy5-dUTP (Amersham Biosciences), and 400 U of Superscript II reverse transcriptase (Invitrogen). The mixture was incubated at 42°C for 2 h. Unincorporated fluorescent nucleotides were removed with an AutoSeqG-50 column (Amersham Biosciences) according to the manufacturer's protocol. The remaining solution was ethanol precipitated and resuspended in nuclease-free water (Ambion). Arrays were prehybridized as described by Hegde et al. (21) and dried by centrifugation in 50-ml centrifuge tubes (Corning) at 3,000 rpm for 2 min. Probes were mixed and hybridized as described previously (21). Arrays were agitated at 42°C in low-stringency wash (1x SSC + 0.2% SDS; 5 min) and twice in high-stringency wash (0.1x SSC + 0.2% SDS; 5 min each), dried by centrifugation, and scanned with a GenePix4000A Microarray scanner with GenePix Pro software. Overall levels of Cy3 and Cy5 fluorescence were balanced, and the image of each slide was saved as a TIFF file. Array images were manually checked to ensure that all features (= spots) had been correctly identified and flagged. Fluorescence intensities of Cy5 and Cy3 were calculated for each feature and for its immediate surroundings (background).

Values for all five time points were then imported into MaxdView version 1.0.3. Data that had been flagged as "not present" (neither of the 2 pools of cDNA had hybridized to the probe sufficiently to be detected) or "bad" (data flagged as suspect by the operator, e.g., GenePix Pro had detected nonfeature fluorescence) were removed so that only spots designated "present" in 40 of 40 replicate features remained. Cy3 and Cy5 values (median minus background) for each feature were logged, and feature ratios were calculated. These log ratios were then normalized with the scatter plot smoother lowess, which corrects for intensity-dependent dye bias, and centers the data on zero (45). The ratio of fluorescence between conditions was then calculated by subtracting the normothermic value from that of the same time point in torpor.

Each transcript was represented by eight ratios per time point. With the t-test plug-in in MaxdView, Student's t-tests were performed for each transcript at all five time points to test the null hypothesis that the mean of the ratios obtained does not differ from zero. Features with ratios significantly different from zero (t ≥ 2.3646; P ≤ 0.05) for at least one time point across the torpor cycle were included in subsequent analyses. The accession number for each feature showing significant up- or downregulation was subjected to a BLASTn search, and the closest corresponding hit was noted. Each transcript was then assigned a "Molecular Function" and grouped within the Gene Ontology hierarchy.

Taqman Q-PCR.
Q-PCR was performed as previously described (5) with Siberian hamster-specific primers and probes (MWG) as listed in Supplemental Table 2. Amplification was performed as previously described but using q-PCR Mastermix Plus (Eurogentec) according to the manufacturer's instructions. PCR amplification of samples was performed alongside gene-specific standards of known concentration prepared with the primers described above. The PCR products were purified and diluted to produce a set of standards ranging from 3 x 101 to 3 x 107 double-stranded molecules. In addition to Gapdh two further transcripts (Esrra and Ube2l3) were selected as controls, based on their apparent lack of variance during torpor in our microarray study. Since all three transcripts had similar profiles but different levels of expression, values were normalized to the same scale (maximum 100,000), to calculate an amalgamated control value for each sample. The value (number of molecules) for the gene of interest was then divided by its mean control value to give its value as a proportion of control.

In situ hybridization.
In situ hybridization was performed and quantified as previously described (5).

Statistical analysis.
Differences between experimental groups were compared by analysis of variance followed by Tukey honestly significant difference test for unequal n, Kruskal-Wallis, and Mann-Whitney U-tests where appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Body weight and pelage score.
Hamsters used in this study had a median pelage score of 5, while hamsters born at the same time and housed in LP (16 h light:8 h dark) had pelage scores of 1. Body weights of SP-housed normothermic and torpid hamsters were also significantly lower than their LP-housed littermates (P < 0.001; 1-way ANOVA).

Body temperature.
Most SP-housed hamsters exhibited periodic torpor bouts interspersed with longer periods of normothermia. An example of an individual animal is shown in Fig. 1A. Torpid hamsters had significantly lower Tb values than normothermic hamsters at all time points investigated except the final time point (ZT10), when most animals had completed arousal (Mann-Whitney U-test). Mean values over the time course of torpor are shown in Fig. 1B for torpid and normothermic controls.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Core body temperature profiles of torpid and normothermic hamsters. Open bars, lights on; filled bars, lights off; arrow, time of culling. A: core body temperature profile of an individual showing torpor. B: mean data from normothermic ({lozenge}) and torpid ({blacksquare}) hamsters at times of collection. Data plotted are means ± SE for n = 3–8 animals/group. Zeitgeber time (ZT)22 is double plotted. **P ≤ 0.01.

 
Microarray results.
Of the 15,247 clones represented on the array set, 8,040 (52.7%) were detected on 40 of 40 features, and their representative transcripts were thus considered to be present in the heart at a measurable level between ZT22 and ZT10. Of these, 298 clones (3.7% of those detected), showed significant fold changes over at least one time point during torpor (P ≤ 0.05). BLAST searches revealed 24 clones with no significant similarity to any sequences in the NCBI database, 7 were homologous to chromosome sequence, with no specified gene, and the remaining 267 sequences represented transcripts from 209 different genes. Of these 209 genes, sufficient information was available to classify 184 into 17 different functional groups. The remainder were classed as having "unknown" function. A full list of the genes and their status at each time point is provided in Supplemental Table 3.

At each stage of torpor (ZT22, 1, 4, and 7), there were more transcripts downregulated than upregulated, but this was reversed on return to normothermia at ZT10 (Fig. 2A). A number of pathways showed significant alteration during the course of the torpor bout (Fig. 2). These included genes involved in ATP generation ("Cellular Metabolism" and "Transport"), iron homeostasis ("Transport"), transcription and RNA stability ("Transcription" and "Protein Biosynthesis"), and ubiquitylation and protein degradation ("Protein Catabolism") pathways. Most groups were represented by both up- and downregulated genes during torpor, with greatest change being detected on arousal at ZT7. Notably, transcripts from "circulation" and "response to stress" groups were mainly downregulated during the torpor bout. Of particular note was the ubiquitylation pathway, where components representing the entire pathway were altered during the bout (Fig. 3).


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. A: time course of changes of 209 transcripts exhibiting significantly altered expression over the torpor cycle, classified into functional groups. B: relative number of transcripts in these functional groups exhibiting altered expression in torpor.

 

Figure 3
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 3. Fold change values from microarray analyses for genes involved in the ubiquitylation pathway over the torpor bout: ubiquitin and an activating enzyme, conjugating enzyme, ubiquitin ligases, and ubiquitin-specific proteases. Data plotted are means ± SE for n = 8 technical replicates/group. *P ≤ 0.05.

 
Microarray analysis showed significant upregulation of the transcript for cardiac hormone Bnp during arousal from torpor. Subsequent Q-PCR analysis of Bnp and the related peptide hormone Anp did not show significant changes in these transcripts, although there was a trend for increased Bnp expression at ZT7 (data not shown). In addition, microarray analysis revealed that two circadian genes, Bmal1 and Rev-erb ß, showed altered expression during the time course of our experiment.

Clock gene changes over the torpor cycle.
Profiles of clock transcripts in the SCN (by in situ hybridization) are shown in Fig. 4. In the normothermic SCN, Per1 and Per2 exhibited robust diurnal rhythms, with peak expression in the mid- to late light phase, respectively. The rhythm of Per1 was ~3 h advanced compared with that of the heart (see below), while for Per2 this phase difference extended to 9–12 h. On arousal, Per1 appeared slightly elevated while Per2 was significantly suppressed relative to normothermia (P = 0.033, Tukey test). Q-PCR values for six circadian clock genes are shown for heart, liver, and lung in Figs. 5–67, respectively. In the normothermic heart, all clock gene transcripts exhibited significant rhythmic expression over the day-night cycle (P ≤ 0.003, 1-way ANOVA and Kruskal-Wallis; Fig. 5), with the exception of Cry2 (P = 0.440; 1-way ANOVA). Per1, Dbp, and Rev-erb {alpha} peaked at late photophase, in antiphase to Bmal1, while Per2 peaked later in the night. Circadian clock transcripts continued to vary in abundance during torpor, although the transcript for Bmal1 was significantly upregulated on and after arousal from torpor (P = 0.021 and 0.034 for ZT7 and ZT10, respectively, Mann-Whitney U-tests), confirming changes seen in the microarray study. Dbp was significantly depressed during arousal (P = 0.021, Mann-Whitney U-test).


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 4. Relative optical density (ROD) in situ hybridization values for Per1 and Per2 mRNA expression in the suprachiasmatic nucleus of normothermic ({lozenge}) and torpid ({blacksquare}) hamsters over a torpor bout. Open bars, lights on; filled bars, lights off. Data plotted are means ± SE for n = 3–6 animals/group. ZT22 is double plotted. Insets: normothermic [short photoperiod normothermic (SPN), left] and torpid [short photoperiod torpid (SPT), right] examples from ZT7. *P ≤ 0.05.

 

Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 5. Quantitative PCR (Q-PCR) values of clock gene mRNA in the heart of normothermic ({lozenge}) and torpid ({blacksquare}) hamsters. Open bars, lights on; filled bars, lights off. Data plotted are means ± SE for n = 3–5 animals/group. ZT22 is double plotted. *P ≤ 0.05.

 

Figure 6
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 6. Q-PCR values of clock gene mRNA in liver of normothermic ({lozenge}) and torpid ({blacksquare}) hamsters. Open bars, lights on; filled bars, lights off. Data plotted are means ± SE for n = 3–5 animals/group. ZT22 is double plotted. *P ≤ 0.05, **P ≤ 0.01.

 

Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 7. Q-PCR values of clock gene mRNA in lung of normothermic ({lozenge}) and torpid ({blacksquare}) hamsters. Open bars, lights on; filled bars, lights off. Data plotted are means ± SE for n = 3–5 animals/group. ZT22 is double plotted. *P ≤ 0.05.

 
Comparison of the temporal pattern of expression of circadian clock transcripts in heart, liver, and lung revealed a consistent flattening of the Dbp peak during torpor and increased Rev-erb {alpha} during rewarming (Figs. 5–7). Per2 appeared suppressed at ZT7 in peripheral tissues (liver, P = 0.023; heart, P = 0.090; Tukey tests) and SCN. This resulted in a phase delay in the daily rise of Per2 relative to normothermia in peripheral organs and a dip in Per2 expression in the SCN. Significant changes in Bmal1 were detected in the heart, but not in other tissues.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Our data reveal that relatively few transcripts show altered expression over the course of a low-temperature torpor bout. This observation is consistent with earlier publications using hibernating model organisms (golden-mantled and 13-lined ground squirrels; Ref. 43) and supports the proposition that transcript levels of most genes are maintained during daily torpor and hibernation (3, 32, 40). Of the few transcripts that were altered in the present study, the majority were downregulated in torpor, consistent with observations by Williams et al. (43). The greatest number of transcript changes observed here occurred during rewarming (i.e., ZT7). This may reflect a switch from key transcripts required in torpor to those essential during or after arousal, or as a response to the process of arousal: it is believed that arousal is the most challenging stage of the torpor bout for the heart, since heart rate increases before increases in Tb and oxygen demand outstrips supply at this time, resulting in a potential oxygen deficit and stress (13, 29). The increased number of transcriptional changes seen might also result from a loosening of the tight controls on transcription and RNA degradation that are thought to occur during hypometabolism. Several genes downregulated in torpor were suppressed well into the period of normothermia (ZT10), suggesting that some transcripts may be cold sensitive and hence do not accumulate until the animal has returned to normothermia. After arousal, several transcripts were significantly upregulated relative to normothermia, suggesting continued effects of torpor after arousal.

Circadian clock.
There is compelling evidence for the involvement of the hypothalamic SCN "master clock" in the maintenance of correctly timed torpor and hibernation bouts in seasonal mammals. In Siberian hamsters, torpor entry is gated by the circadian system and destruction of the SCN prevents expression of normal SP-induced torpor (34). We show here that normothermic rhythms of Per1 and Per2 mRNA expression in the SCN are also conserved during torpor. During initial stages of torpor, SCN expression of Per1 and Per2 is similar to that seen in normothermic hamsters, suggesting that decreased Tb and hypometabolism do not affect Per mRNA expression at this phase of the cycle. However, during arousal, a marked decrease in Per2 and an apparent increase in Per1 relative to normothermic values were detected. Similar observations have been made by Herwig et al. (22), who reported upregulation of Per1 at ZT7 in the SCN. Remarkably, we observed similar changes in these key clock transcripts in some peripheral organs, with lowered Per2 and elevated Per1 expression on arousal. These differences occurred concurrently in the SCN and periphery, in contrast to the normothermic situation, where the circadian rhythm of transcripts in peripheral body organs phase lags that of the SCN by 4–6 h (20, 31). This suggests that there may be global changes in expression of these transcripts throughout the body at the end of torpor, perhaps regulated directly by temperature or by common neurotransmitter or neuroendocrine-mediated pathways.

The upregulation of Bmal1 in the heart during and after arousal may be due to decreased RNA degradation or increased synthesis. Our study revealed a significant downregulation of Rev-erb ß (a suppressor of Bmal1 transcription; Refs. 18, 37) at ZT7 in the torpid heart, presumably facilitating Bmal1 upregulation. Furthermore, Hdac3, whose protein is believed to prevent transcription at the Bmal1 promoter in association with REV-ERB {alpha} (46), was downregulated in the rewarming heart, implying a net decrease in repressive activity on the Bmal1 promoter during arousal. Prolongation of Bmal1 expression at the end of torpor may affect expression of other clock genes after arousal, possibly increasing transcription of E-box-regulated genes such as Period.

We have demonstrated here that elements of the circadian clock are not temperature labile at the RNA level during torpor. It is possible that the changes in RNA expression seen on arousal may result in protein changes that would subsequently alter the period of the clock in the absence of an external reentraining stimulus. This might explain the decrease in circadian period observed in torpor-expressing Siberian hamsters compared with normothermic controls (39).

Transcription, RNA stability, and translational control.
Transcripts in the "Transcription" group were mostly downregulated during torpor, in line with the consensus that transcription is decreased in hypometabolic mammals including torpid Siberian hamsters (3, 40). A possible candidate involved may be Ews, which was significantly decreased throughout torpor, returning to normothermic levels of expression at ZT10. EWS is thought to facilitate transcription initiation and elongation (2, 26), and its downregulation may reduce cAMP-stimulated transcription. Two replication-independent histones, which coil nuclear DNA into nucleosomes [H3 histone family 3B (H3F3B) and H2A histone family, member Z (H2AFZ)], were upregulated during arousal. H2AFZ is thought to be more permissive of promoter binding than other H2A family members (33), so upregulation of this histone may facilitate increased transcription.

Rbm3 is thought to be induced at low temperatures to counteract the cold-mediated decline in protein synthesis (12). This transcript was downregulated at ZT1 during torpor and upregulated at ZT10 after arousal. Reduced expression in torpor may indicate suppression of the normal response to cold shock, preventing increased protein synthesis. The transcript encoding eukaryotic initiation factor 4E (eIF4E) was also downregulated on entry into torpor and upregulated before arousal at ZT4. eIF4E is a rate-limiting constituent of the transcription initiation complex whose activity was previously reported to be reduced in the liver of hibernating golden-mantled ground squirrels by increased activity of its repressor, 4E-binding protein 1 (41). While the mechanism of eIF4E regulation may be different in these two species, both studies imply a decreased translation from 5'-capped mRNAs during hypometabolism.

Globins.
The globins, in the "Circulation" group, were consistently downregulated during torpor, along with 2,3-bisphosphoglycerate (2,3-BPG) mutase (Bpgm). Hba-a1 and Hbb-b1 are present in adult mammals, while Hbb-y is a fetal globin with a higher affinity for oxygen. The apparent presence of embryonic globin expression in the adult Siberian hamster might be due to similarity between sequences allowing hybridization of adult transcripts to features containing embryonic cDNA. Alternatively, because {alpha}- and ß-globins are arranged in clusters, transcription of embryonic and adult genes may be activated in tandem if the normal silencing of embryonic genes is perturbed. Some aspects of neonatal physiology are believed to be used in torpor and hibernation (19). Expression of these globin RNAs and Hbb-b2 was previously discovered in the mouse lens, where they are proposed to perform roles in oxygen transport, to act as an oxygen sink, or to promote apoptosis (44). The globins might act similarly in Siberian hamsters, although because all globin transcripts are suppressed during torpor and arousal, they would be implicated in winter normothermia rather than torpor. BPGM catalyzes the synthesis of 2,3-BPG, which decreases the affinity of hemoglobin for oxygen (42). Therefore, downregulation of this transcript may help to increase oxygen transport during torpor, possibly preventing hypoxia due to decreased respiration rates during torpor.

Iron homeostasis.
The concomitant regulation of two antagonistic iron carriers during arousal implies a deficit of cellular iron at this time. Iron-regulated transporter 1 (Ireg1), which transports iron out of cells into the circulation, was downregulated at ZT7, while transferrin receptor (Tfrc), which binds and internalizes circulating transferrin-bound iron, was upregulated at the same time. Since both genes contain iron response elements (IREs) (10), it is possible that other IRE-containing genes are affected on arousal. Changes in cellular iron levels might indicate changes in heme synthesis, which has also been shown to alter peripheral clock gene expression (24).

Cardiac physiology.
A number of genes involved in cardiac physiology were significantly altered on the array. The regulatory subunit myosin light polypeptide 9 (Myl9) was upregulated soon after initiation of torpor at ZT22. Studies of hibernating golden-mantled ground squirrels have revealed that the myosin light chain isoform (Myl1 ventricular isoform) was similarly upregulated, suggesting that structural alterations in the cardiomyocyte may commonly be altered during a low-temperature bout (14), perhaps facilitating heart contraction at decreased temperatures. Expression of the gap junction gene connexin 43 (Cx43) is downregulated after arousal, and this is strikingly similar to the protein profile observed in golden hamsters after arousal from hibernation, where CX43 is upregulated in cold-adapted SP hamsters, so that when protein levels decline after arousal they fall to summer levels (36). Siberian ground squirrels also express higher levels of CX43 in winter, and this is correlated with faster conduction velocity and lower excitation threshold of the heart (15). Since conduction velocity decreases and excitation threshold increases at low temperatures, the changes in Cx43 may compensate for temperature effects during both hibernation and torpor (15, 36).

Ubiquitylation pathway and protein degradation.
A large part of the "Protein Catabolism" group was formed from transcripts associated with all stages of the ubiquitylation pathway. This is summarized in Fig. 8. Our data suggest that there are global changes in protein turnover and regulation during a torpor bout. Generally, ubiquitylation-related transcripts were downregulated during early torpor, with upregulation occurring at arousal. Reduced activity of protein degradation pathways during torpor may be advantageous because it would reduce the overall energetic costs associated with degradation, and would further serve to stabilize protein levels in the face of reduced translation.


Figure 8
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 8. Key steps involved in protein ubiquitylation with torpor-regulated genes indicated. E1, ubiquitin-activating enzyme; E2, ubiquitin conjugating enzyme; E3, ubiquitin ligase; ub, ubiquitin; prot, protein; Ubc, ubiquitin C; Ube1x, ubiquitin activating enzyme E1, Chr X; Ubce4, ubiquitin conjugating enzyme 4; Chip, Hsp70-interacting protein; Psmc3, proteasome (prosome, macropain) 26s subunit, ATPase 3; Usp, ubiquitin-specific protease.

 
Of particular interest was the regulation of ubiquitin ligases (E3s), since these are relatively specific, recognizing only a few proteins (38). Proteasome (prosome, macropain) 26S subunit, ATPase 3 (Psmc3), and Hsp70-interacting protein (Chip) are E3s downregulated during arousal from torpor. Their substrates, hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and heat shock protein 70 (HSP70), respectively (8), are both involved in stress responses and coprecipitate with heat shock protein 1{alpha} (Hspca; also detected at its highest level relative to normothermia at ZT7). The downregulation of Psmc3 mRNA could therefore lead to upregulation of the hypoxia response through stabilization of HIF-1{alpha}. HIF-1{alpha} upregulation has previously been discovered in thirteen-lined ground squirrel organs during arousal (30), and in conjunction with our data this implies that a hypoxia response may be initiated in the hypometabolic heart as it returns to normothermia. The predicted stabilization of HIF-1{alpha} might impact on expression of circadian clock transcripts since HIF-1{alpha} is closely related to the core circadian protein BMAL1 and is known to interact with and stabilize several circadian clock proteins and to initiate transcription of clock genes including Per1 and Ror{alpha} (6, 7, 9, 17, 23, 27, 28). This raises the intriguing possibility that altered HIF-1{alpha} signaling at arousal may be a causal mechanism involved in the changes in circadian clock gene transcripts we observed in this study.

Conclusion.
Our data reveal that a number of pathways previously only identified in hibernating mammals show similar alterations over a daily hypometabolic torpor bout. Furthermore, we show that relatively small numbers of transcripts have altered expression, implying that gene regulation is well buffered against the effect of temperature change in this system. Although circadian clocks are generally temperature compensated, we consistently observed altered expression of many circadian clock gene transcripts during late torpor and at arousal. A number of transcripts involved in cardiac physiology show alterations, suggesting torpor-associated plasticity within cardiomyocytes. The most striking feature of our data was the discovery that representatives from all key steps in ubiquitylation pathways showed significantly altered expression over the hypometabolic bout. This implies that fine control of protein degradation pathways may be a necessary and key adaptive feature of torpor in mammals.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC).


   DISCLOSURES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
This work was funded in part by PIC UK Ltd.


   ACKNOWLEDGMENTS
 
We thank Dr Graham Plastow (PIC UK Ltd.), Dr. Andy Hayes, Prof. Steve Oliver, Dr. Sandrine Dupré, Dr. Zeenat Atcha, and Helen Lydon for assistance during the course of this study. We also thank colleagues at the Human Genome Mapping Project Resource Center, Cambridge, UK, for kind provision of cDNA arrays.

Present addresses: F. I. J. Crawford, Unit of Cardiac Physiology, University of Manchester, 3rd Floor, Core Technology Facility, 46 Grafton St., Manchester M13 9NT, UK; C. L. Hodgkinson, Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK; E. Ivanova, Developmental Genetics, Babraham Research Campus, Babraham Institute, Babraham, Cambridge CB22 3AT, UK.


   FOOTNOTES
 
Address for reprint requests and other correspondence: A. S. I. Loudon, Univ. of Manchester, 3.614 Stopford Bldg., Oxford Road, Manchester, M13 9PT, UK (e-mail: andrew.loudon{at}manchester.ac.uk)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402, 1997.[Abstract/Free Full Text]
  2. Araya N, Hirota K, Shimamoto Y, Miyagishi M, Yoshida E, Ishida J, Kaneko S, Kaneko M, Nakajima T, Fukamizu A. Cooperative interaction of EWS with CREB-binding protein selectively activates hepatocyte nuclear factor 4-mediated transcription. J Biol Chem 278: 5427–5432, 2003.[Abstract/Free Full Text]
  3. Berriel-Diaz M, Lange M, Heldmaier G, Klingenspor M. Depression of transcription and translation during daily torpor in the Djungarian hamster (Phodopus sungorus). J Comp Physiol [B] 174: 495–502, 2004.[Medline]
  4. Brauch KM, Dhruv ND, Hanse EA, Andrews MT. Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal. Physiol Genomics 23: 227–234, 2005.[Abstract/Free Full Text]
  5. Carr AJ, Johnston JD, Semikhodskii AG, Nolan T, Cagampang FR, Stirland JA, Loudon AS. Photoperiod differentially regulates circadian oscillators in central and peripheral tissues of the Syrian hamster. Curr Biol 13: 1543–1548, 2003.[CrossRef][Web of Science][Medline]
  6. Chauvet C, Bois-Joyeux B, Berra E, Pouyssegur J, Danan JL. The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1. Biochem J 384: 79–85, 2004.[CrossRef][Web of Science][Medline]
  7. Chilov D, Hofer T, Bauer C, Wenger RH, Gassmann M. Hypoxia affects expression of circadian genes PER1 and CLOCK in mouse brain. FASEB J 15: 2613–2622, 2001.[Abstract/Free Full Text]
  8. Corn PG, McDonald ER, Herman JG, El-Deiry WS. Tat-binding protein-1, a component of the 26S proteasome, contributes to the E3 ubiquitin ligase function of the von Hippel-Lindau protein. Nat Genet 35: 229–237, 2003.[CrossRef][Web of Science][Medline]
  9. Cowden KD, Simon MC. The bHLH/PAS factor MOP3 does not participate in hypoxia responses. Biochem Biophys Res Commun 290: 1228–1236, 2002.[CrossRef][Web of Science][Medline]
  10. Crichton RR, Wilmet S, Legssyer R, Ward RJ. Molecular and cellular mechanisms of iron homeostasis and toxicity in mammalian cells. J Inorg Biochem 91: 9–18, 2002.[CrossRef][Web of Science][Medline]
  11. Dibb KM, Hagarty CL, Loudon AS, Trafford AW. Photoperiod-dependent modulation of cardiac excitation contraction coupling in the Siberian hamster. Am J Physiol Regul Integr Comp Physiol 288: R607–R614, 2005.[Abstract/Free Full Text]
  12. Dresios J, Aschrafi A, Owens GC, Vanderklish PW, Edelman GM, Mauro VP. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis. Proc Natl Acad Sci USA 102: 1865–1870, 2005.[Abstract/Free Full Text]
  13. Drew KL, Harris MB, LaManna JC, Smith MA, Zhu XW, Ma YL. Hypoxia tolerance in mammalian heterotherms. J Exp Biol 207: 3155–3162, 2004.[Abstract/Free Full Text]
  14. Fahlman A, Storey JM, Storey KB. Gene up-regulation in heart during mammalian hibernation. Cryobiology 40: 332–342, 2000.[CrossRef][Medline]
  15. Fedorov VV, Li L, Glukhov A, Shishkina I, Aliev RR, Mikheeva T, Nikolski VP, Rosenshtraukh LV, Efimov IR. Hibernator Citellus undulatus maintains safe cardiac conduction and is protected against tachyarrhythmias during extreme hypothermia: possible role of Cx43 and Cx45 up-regulation. Heart Rhythm 2: 966–975, 2005.[CrossRef][Web of Science][Medline]
  16. Figala J, Hoffmann K, Goldau G. Zur Jahresperiodik beim Dsungarischen Zwerghamster Phodopus sungorus Pallas. Oecologia (Berl) 12: 89–118, 1973.[CrossRef]
  17. Ghorbel MT, Coulson JM, Murphy D. Cross-talk between hypoxic and circadian pathways: cooperative roles for hypoxia-inducible factor 1 alpha and CLOCK in transcriptional activation of the vasopressin gene. Mol Cell Neurosci 22: 396–404, 2003.[CrossRef][Web of Science][Medline]
  18. Guillaumond F, Dardente H, Giguere V, Cermakian N. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J Biol Rhythms 20: 391–403, 2005.[Abstract/Free Full Text]
  19. Harris MB, Olsen LE, Milsom WK. The origin of mammalian heterothermy: a case for perpetual youth? In: Life in the Cold: Evolution, Mechanisms, Adaptation, and Application, edited by Barnes BM and Carey HV. Fairbanks, AK: Inkworks, 2004.
  20. Hastings MH, Field MD, Maywood ES, Weaver DR, Reppert SM. Differential regulation of mPER1 and mTIM proteins in the mouse suprachiasmatic nuclei: new insights into a core clock mechanism. J Neurosci 19: RC11, 1999.[Abstract/Free Full Text]
  21. Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, Quackenbush J. A concise guide to cDNA microarray analysis. Biotechniques 29: 548–556, 2000.[Web of Science][Medline]
  22. Herwig A, Revel F, Saboureau M, Pévet P, Steinlechner S. Daily torpor alters multiple gene expression in the suprachiasmatic nucleus and pineal gland of the Djungarian hamster (Phodopus sungorus). Chronobiol Int 23: 269–276, 2006.[CrossRef][Web of Science][Medline]
  23. Hogenesch JB, Gu YZ, Jain SJ, Bradfield CA. The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA 95: 5474–5479, 1998.[Abstract/Free Full Text]
  24. Kaasik K, Lee CC. Reciprocal regulation of haem biosynthesis and the circadian clock in mammals. Nature 430: 467–471, 2004.[CrossRef][Medline]
  25. Kargul GJ, Dudekula DB, Qian Y, Lim MK, Jaradat SA, Tanaka TS, Carter MG, Ko MSH. Verification and initial annotation of the NIA mouse 15K cDNA clone set. Nat Genet 28: 17–18, 2001.[CrossRef][Web of Science][Medline]
  26. Kim J, Pelletier J. Molecular genetics of chromosome translocations involving EWS and related family members. Physiol Genomics 1: 127–138, 1999.[Abstract/Free Full Text]
  27. Koyanagi S, Kuramoto Y, Nakagawa H, Aramaki H, Ohdo S, Soeda S, Shimeno H. A molecular mechanism regulating circadian expression of vascular endothelial growth factor in tumor cells. Cancer Res 63: 7277–7283, 2003.[Abstract/Free Full Text]
  28. Miki N, Ikuta M, Matsui T. Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1. J Biol Chem 279: 15025–15031, 2004.[Abstract/Free Full Text]
  29. Milsom WK, Zimmer MB, Harris MB. Regulation of cardiac rhythm in hibernating mammals. Comp Biochem Physiol A 124: 383–391, 1999.[CrossRef][Medline]
  30. Morin PJ, Storey KB. Cloning and expression of hypoxia-inducible factor 1 alpha from the hibernating ground squirrel, Spermophilus tridecemlineatus. Biochim Biophys Acta 1729: 32–40, 2005.[Medline]
  31. Nuesslein-Hildesheim B, O'Brien JA, Ebling FJ, Maywood ES, Hastings MH. The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the Siberian hamster encodes both daily and seasonal time. Eur J Neurosci 12: 2856–2864, 2000.[CrossRef][Web of Science][Medline]
  32. O'Hara BF, Watson FL, Srere HK, Kumar H, Wiler SW, Welch SK, Bitting L, Heller HC, Kilduff TS. Gene expression in the brain across the hibernation cycle. J Neurosci 19: 3781–3790, 1999.[Abstract/Free Full Text]
  33. Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W. Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev 12: 162–169, 2002.[CrossRef][Web of Science][Medline]
  34. Ruby NF, Zucker I. Daily torpor in the absence of the suprachiasmatic nucleus in Siberian hamsters. Am J Physiol Regul Integr Comp Physiol 263: R353–R362, 1992.[Abstract/Free Full Text]
  35. Ruf T, Heldmaier G. The impact of daily torpor on energy-requirements in the Djungarian hamster, Phodopus sungorus. Physiol Zool 65: 994–1010, 1992.
  36. Saitongdee P, Milner P, Becker DL, Knight GE, Burnstock G. Increased connexin43 gap junction protein in hamster cardiomyocytes during cold acclimatization and hibernation. Cardiovasc Res 47: 108–115, 2000.[Abstract/Free Full Text]
  37. Sato TK, Panda S, Miraglia LJ, Reyes TM, Rudic RD, McNamara P, Naik KA, Fitzgerald GA, Kay SA, Hogenesch JB. A functional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron 43: 527–537, 2004.[CrossRef][Web of Science][Medline]
  38. Schwartz DC, Hochstrasser M. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. Trends Biochem Sci 28: 321–328, 2003.[CrossRef][Web of Science][Medline]
  39. Thomas EM, Jewett ME, Zucker I. Torpor shortens the period of Siberian hamster circadian rhythms. Am J Physiol Regul Integr Comp Physiol 265: R951–R956, 1993.[Abstract/Free Full Text]
  40. van Breukelen F, Martin SL. Reversible depression of transcription during hibernation. J Comp Physiol [B] 172: 355–361, 2002.[CrossRef][Medline]
  41. van Breukelen F, Sonenberg N, Martin SL. Seasonal and state-dependent changes of eIF4E and 4E-BP1 during mammalian hibernation: implications for the control of translation during torpor. Am J Physiol Regul Integr Comp Physiol 287: R349–R353, 2004.[Abstract/Free Full Text]
  42. Wang YL, Wei ZY, Bian Q, Cheng ZJ, Wan M, Liu L, Gong WM. Crystal structure of human bisphosphoglycerate mutase. J Biol Chem 279: 39132–39138, 2004.[Abstract/Free Full Text]
  43. Williams DR, Epperson LE, Li W, Hughes MA, Taylor R, Rogers J, Martin SL, Cossins AR, Gracey AY. Seasonally hibernating phenotype assessed through transcript screening. Physiol Genomics 24: 13–22, 2005.[Abstract/Free Full Text]
  44. Wride MA, Mansergh FC, Adams S, Everitt R, Minnema SE, Rancourt DE, Evans MJ. Expression profiling and gene discovery in the mouse lens. Mol Vis 9: 360–396, 2003.[Web of Science][Medline]
  45. Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15, 2002.[Abstract/Free Full Text]
  46. Yin L, Lazar MA. The orphan nuclear receptor Rev-erb alpha recruits the N-CoR/histone deacetylase 3 corepressor to regulate the circadian Bmal1 gene. Mol Endocrinol 19: 1452–1459, 2005.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Mertens, O. Stiedl, S. Steinlechner, and M. Meyer
Cardiac dynamics during daily torpor in the Djungarian hamster (Phodopus sungorus)
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R639 - R650.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables
Right arrow Addendum
Right arrow All Versions of this Article:
31/3/521    most recent
00131.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Crawford, F. I. J.
Right arrow Articles by Loudon, A. S. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Crawford, F. I. J.
Right arrow Articles by Loudon, A. S. I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.