Differential expression of mitochondria-encoded genes in a hibernating mammal
Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6
* Author for correspondence (e-mail: kenneth_storey{at}carleton.ca )
Accepted 13 March 2002
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Summary |
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Key words: Spermophilus tridecemlineatus, hibernation, ischemia, kidney, cDNA library
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Introduction |
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Protein phosphorylation is believed to be a primary agent by which
hibernating animals make `acute' metabolic adjustments
(Boyer and Barnes, 1999;
Storey, 2000
). Many of the
enzymes involved in ATP production (e.g. pyruvate dehydrogenase,
phosphofructokinase) (Brooks and Storey,
1992
; Storey,
1997
) as well as ATP consumption (Na/K-ATPase)
(Bennis et al., 1995
;
MacDonald and Storey, 1999
)
are reversibly inhibited by protein phosphorylation during hibernation.
Increasingly, however, the importance of differential expression of genes in
the expression and maintenance of the hibernation phenotype has been
demonstrated (Srere et al.,
1992
). Several genes have been identified that are preferentially
or increasingly expressed in the tissues of hibernating mammals. These include
2-macroglobulin (Srere et al.,
1992
) in the liver, pyruvate dehydrogenase kinase isozyme 4,
pancreatic lipase and mitochondrial-encoded genes in the heart
(Andrews et al., 1998
), and a
variety of genes in other tissues (Boyer
and Barnes, 1999
). The protein products of these genes affect a
variety of adaptive changes in the tissues and organs of hibernating animals
that allow them to survive this extreme physiological state.
A 100-fold decrease in heart rate reduces the mean arterial pressure by 60%
in hibernating ground squirrels (Anderson
et al., 1990; Harlow and
Braun, 1995
). This dramatically reduces the rate of glomerular
microfiltration by kidney and therefore the rate of urine production. Because
of the greatly reduced or `trickle' blood flow during hibernation it is likely
that the kidney and other organs become profoundly ischemic
(Frerichs et al., 1998
).
Infrequent arousals during hibernation cause a rapid warming and reperfusion
of the kidney, and the immediate restoration of blood filtration and urine
production (Zancanaro et al.,
1999
). These physiological extremes endured by the kidney during
hibernation are associated with significant adjustments to metabolic rate and
glomular ultrastructure (Harlow and Braun,
1995
). It is likely that this adaptation is in part, the result of
differential gene expression and it is our contention that the hibernating
kidney is an excellent model for natural resistance to ischemia and
hypothermia.
In the present study we investigated the role of differential gene expression in supporting hibernation in the thirteen-lined ground squirrel, Spermophilus tridecemlineatus. The differential screening of a cDNA library constructed from the kidney of hibernating squirrels revealed a gene that is upregulated during hibernation. This gene was identified as the mitochondrial-encoded cytochrome c oxidase subunit 1 (Cox1). Northern and western blot analysis of kidney and other tissues revealed significant and tissue-specific changes in mRNA and protein levels of mitochondrial encoded and nuclear encoded subunits of the electron transport chain during hibernation.
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Materials and methods |
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RNA preparation and cDNA library construction
All materials and solutions used for RNA isolation were treated with 0.1%
v/v diethylpyrocarbonate (DEPC) and subsequently autoclaved. Total RNA was
isolated from tissues of hibernating and euthermic ground squirrels using
Trizol solution (Gibco-BRL), following the manufacturer's protocol.
Poly(A)+ RNA was isolated from total RNA using an
oligo(dT)-cellulose column (Quiagen, Valencia, CA, USA). The concentration of
the poly(A)+ RNA was determined spectrophotometrically at 260 nm.
Poly(A)+ RNA from the kidneys of hibernating animals was used to
construct a cDNA library using cDNA library and Uni-ZAP unidirectional cloning
kits from Stratagene (La Jolla, CA, USA), following the manufacturer's
instructions.
Differential screening of the cDNA library
32P-labeled single-stranded cDNA probes were synthesized from
poly(A)+ RNA isolated from kidney of hibernating and euthermic
control animals. Into an autoclaved, DEPC-treated, 1.5 ml microfuge tube, 2
µl poly(A)+ RNA template (about 1 µg) and 6 µl DEPC
ddH2O were added. The mixture was heated at 65°C for 5 min and
then 5 µl of 5x1st strand buffer (Gibco-BRL), 1.5 µl
dNTPs without dCTP (5 mmoll-1 for each nucleotide), 1 µl oligo
dT primer (200 ng µl-1, NEB, Beverly, MA, USA), 1 µl RNasin
(5 U µl-1, Promega, Madison, WI, USA) and 2.5 µl
dithiothreitol (0.1 moll-1, Gibco-BRL) were added. The reaction was
mixed well and the primers were allowed to anneal to the poly(A)+
RNA at room temperature for 10 min. Following this, 1 µl M-MLV reverse
transcriptase (200 U µl-1, Gibco-BRL), and 5 µl
[-32P]dCTP (3000 Ci mmol-1, 110 TBq
mol-1; Amersham) were added to the reaction, and the mixture was
incubated at 37°C for 1 h. The RNA was degraded by adding 1 µl EDTA
(0.5 moll-1), 1 µl sodium dodecyl sulfate (10% w/v), and 3 µl
NaOH (3 moll-1) to the reaction and the mixture was incubated at
68°C for 30 min. Next, the probe was cooled to room temperature followed
by the addition of 10 µl Tris-HCl (1 moll-1, pH 7.4) and 3 µl
HCl (2 moll-1). Finally, the probe was passed through a Sephadex
G-50 column equilibrated in TE buffer, pH 8 (10 mmoll-1 Tris-HCl, 1
mmoll-1 EDTA, made with DEPC-treated water) and then brought to a
final volume of 500 µl with TE buffer; a 2 µl portion was removed for
scintillation counting. Approximately 106 disints min-1
of probe per ml of hybridization solution was used for hybridization of the
plaque lifts.
For primary screening, approximately 35 000 plaques per plate were grown on
10 agar plates. Two lifts were made from each plate using nylon membranes
(Amersham). The membranes were UV-crosslinked, and allowed to air dry. The
lifts were then hybridized with 32P-labeled, single-stranded cDNA
probes made from kidney of either hibernating or euthermic animals, in a
hybridization incubator (LAB-Line Instruments) using Denhardt's hybridization
solution with 50% formamide (Fahlman et
al., 2000). Plaques showing a stronger signal with the probe from
hibernated kidney compared with euthermic kidney were retrieved and subjected
to two more rounds of screening to confirm the stronger signal and purify the
clones. After tertiary screening, purified clones in Bluescript plasmid
vectors were rescued by in vivo excision using Exassist as the helper
phage.
Northern hybridization analysis
Northern hybridization was used to confirm the upregulation of the putative
clones in the kidney of hibernating ground squirrels and to determine
transcript abundance in other organs (heart, brown adipose, skeletal muscle).
Total RNA was isolated from tissue samples using Trizol (Gibco-BRL), separated
in a formaldehyde-agarose gel using 16µg of total RNA per lane, and then
blotted onto a Nytran membrane by capillary action. The quality of total RNA
was assessed by the identification of well-defined 18S and 28S ribosomal
bands. The DNA inserts from the tertiary screened clones were cut from the
plasmid vector using BamHI and XhoI and separated on a 1 %
agarose gel run in 40 mmoll-1 Tris-acetate, 2 mmoll-1
EDTA, pH 8.5. The inserts were purified and labeled with 32P using
a random primer procedure. Northern blots were hybridized at 44°C, using
labeled probes (8x106 disints min-1 per 10 ml
hybridization solution) and a sodium phosphate hybridization solution. After
hybridization the blots were washed with increasing stringency and then placed
into autoradiography cassettes with X-ray film (X-Omat, AR, Kodak). Prior to
hybridization the northern blots were stained with Methylene Blue (0.03 % w/v)
in ddH2O to visualize ribosomal RNA bands, and destained in
ddH2O overnight.
Transcript levels were quantified by scanning the X-ray autoradiogram using a Scan Jet 3C scanner with DeskScan II V2.2 program (Hewlett Packard) and an Imagequant V3.22 program (Innovative Optical Systems Research). The ribosomal bands of the stained northern blots were quantified and these values were used to evaluate any differences in loading between lanes. RNA transcript sizes were estimated from a plot of RNA molecular mass (Gibco-BRL standards) versus migration distance in the formaldehyde gel.
DNA sequencing and analysis
Isolated clones were sequenced by Canadian Molecular Research Services
(CMRS) Inc. (Ottawa, Ontario, Canada) using an automated DNA sequencing
procedure, and a translation program (EditSeq, DNASTAR, Inc.) was used to
define the putative protein sequence. The nucleotide sequence and the six
possible open reading frames for each clone were loaded into a Blast program
at NCBI for a similarity search in GenBank.
Western blotting
Samples of frozen tissues from euthermic and hibernating ground squirrels
were crushed under liquid nitrogen, weighed and then homogenized at 1:3 w/v
dilution in Buffer A (1 % SDS, 1 % 2-mercaptoethanol, 50 mmoll-1
Tris, pH 6.8, 1 mmoll-1 EDTA, 10 mmoll-1 sucrose).
Soluble protein content was measured with the Bio-Rad (Hercules, CA, USA)
Coomassie Blue dye binding reagent using a microplate reader. An equal amount
of protein from each sample was then boiled in 2x SDS-PAGE loading
buffer, centrifuged for 1 min at 8200g and loaded onto two identical
12 % SDS-PAGE minigels run at 150 V for 45 min. Bio-Rad kaleidoscope
prestained markers were used to track the progress of the proteins through the
gel. Protein samples, size-fractionated on 12 % SDS-PAGE gels, were
transferred to a PVDF membrane by electroblotting. These membranes were
blocked and incubated with a 1:1000 dilution of a mouse monoclonal antibody
against either subunit 1 or subunit 4 of bovine cytochrome c oxidase
(Molecular Probes, Eugene, OR, USA). Blots were then incubated with a
horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody
(Molecular Probes) and developed with colorimetric immunoblot-staining
reagents (Bio-Rad).
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Results |
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DNA sequence analysis of clone 9.1
Sequencing of clone 9.1 produced a 1604nt cDNA (GenBank Accession No.
AF330007) that was identified as encoding the gene (Cox1) for the
mitochondrial-encoded subunit 1 of cytochrome c oxidase (COI).
Analysis of the putative amino acid sequence of ground squirrel COI showed
that it encoded 508 of the 513 amino acids of the full sequence with an intact
C terminus. A comparison of the deduced amino acid sequence of the COI protein
with those from other taxa revealed a 90.5% amino acid identity with human and
98% with COI from the Eurasian red squirrel
(Reyes et al., 2000).
CoxI and CoxIV expression in the kidney
A plasmid containing the cDNA of mouse cytochrome c oxidase
subunit IV (IMAGE ID# 535611), a nuclear encoded subunit of the protein, was
purchased from Research Genetics (Huntsville, AL, USA). This cDNA insert was
32P-labelled and used to probe a northern blot of kidney mRNA from
euthermic and hibernating squirrels. The CoxIV probe bound to a band
of approx. 670nt (Fig. 1B) that
is identical in size in most species described
(Carter and Avadhani, 1991).
The relative levels of Cox1 mRNA increased 2.04±0.13-fold
(mean ± S.E.M., N=3) in kidney from hibernators compared with
controls, whereas the levels of Cox4 mRNA were relatively unchanged
(Fig. 1C). Western blot
analysis revealed a 2.4±0.2-fold (mean ± S.E.M., N=3)
increase in the amount of immunoreactive Cox 1 protein (COI)
(Fig. 1A) and a small (approx.
12%) decrease in the amount of immunoreactive Cox 4 protein (COIV)
(Fig. 1B) in hibernating
versus euthermic kidney (Fig.
1C).
Tissue-specific expression of mitochondrial and nuclear encoded
genes
The vertebrate mitochondrial genome is transcribed as a large polycystronic
transcript that is subsequently processed into distinct mRNA, tRNA and rRNAs
(Clayton, 1991). The mRNAs tend
to encode the core subunits of the proteins of the mitochondrial electron
transport chain whereas the rRNAs and tRNAs constitute the RNA component of
the mitochondrial translational machinery. Mitochondrial transcription
proceeds, therefore, in an `all or none' fashion, meaning if one transcript
increases they all increase (Clayton,
1991
). Because of the dichotomy of response to hibernation by the
mitochondrial encoded Cox1 and the nuclear encoded Cox4
gene, we decided to examine the transcript levels of an additional
mitochondrial encoded and nuclear encoded gene in parallel studies. Northern
blot analysis of Cox1, Cox4 and a previously identified ground
squirrel ATPase 6/8 bicistronic mRNA (GenBank Accession no. AF362073)
(D. S. Hittel and K. B. Storey, unpublished) and ATP
(I.M.A.G.E. ID# 3385828), a nuclear encoded subunit of the mitochondrial ATP
synthase, are shown in Fig. 2A.
During hibernation, Cox1 transcript levels increased
2.0±0.13-fold in kidney, 4.51±0.5-fold in brown adipose tissue
(BAT), 4.22±0.2-fold in heart and decreased slightly (18%) in skeletal
muscle. Transcript levels of ATP6/8 also increased
(1.98±0.2-fold) in kidney, BAT (3.97±0.3-fold) and heart
(3.98±0.4-fold) of hibernators but did not change in skeletal muscle
(Fig. 2B). The transcript
levels of the nuclear encoded Cox4 and ATPa did not change
significantly in any of the hibernating tissues examined.
|
Cold-adapted animals
The transcript levels of CoxI were appreciably lower in
cold-adapted animals compared to kidneys from euthermic controls
(Fig. 3A). Although the
functional significance of this observation is not known, it correlates with
the inability of these animals to enter torpor.
|
Mitochondrial polycistronic precursors
Northern blot analysis of CoxI expression revealed the presence of
an approx. 3 kb band in hibernating kidney and BAT
(Fig. 3B), which probably
represents an unprocessed mitochondrial RNA precursor. The presence of these
precursors has been previously noted in anoxic turtle heart
(Cai and Storey, 1996) and
ischemic kidney (Van Itallie et al.,
1993
), where they are believed to indicate an increase in the
transcription of the mitochondrial genome or the suppression of mitochondrial
RNA processing machinery.
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Discussion |
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The kidney is one of the most frequently transplanted organs, yet ischemic
damage caused before and during transplantation surgery is still a major cause
of post-operative morbidity and mortality
(Zancanaro et al., 1999;
Green, 2000
). The biochemical
mechanisms used by hibernating mammals to endure these physiological extremes
could be applied to the storage and preservation of organs destined for
transplantation.
Cytochrome c oxidase (COX) is a 13-subunit complex spanning the
inner mitochondrial membrane and responsible for the terminal reduction of
dioxygen to water in the electron transport chain
(Gnaiger et al., 1998;
Napiwotzki and Kadenbach,
1998
). Given this essential role in the electron transport chain,
and its sensitivity to ischemia/reperfusion (IR) damage
(Van Itallie et al., 1993
;
Montagna et al., 1998
;
Cochrane et al., 1999
), it is
not surprising that expression levels of cytochrome c oxidase
subunits should respond to stress. An increase in the expression of the
mitochondrial genome is known to occur in response to a wide variety of
oxidative and cold stresses (Immaculada et
al., 1993
; Storey,
2000
). Research in our laboratory has demonstrated an increase in
the transcript levels of Cox1 and other mitochondrial encoded genes
in hibernating ground squirrels (Fahlman
et al., 2000
) and anoxic turtles
(Cai and Storey, 1996
). This
indicates perhaps, a common mitochondrial response to anoxia and/or cold
stress.
The mitochondria-encoded gene Cox1 was recovered from the
differential screening of a cDNA library made from kidney of hibernating
S. tridecemlineatus. This gene encodes the large transmembrane,
oxygen binding subunit of cytochrome c oxidase (Complex IV)
(Poyton, 1999). The three core
catalytic units COX I, II and III are trans-membrane proteins encoded by the
mitochondrial genome, while the remaining 10 subunits are nuclear encoded and
expressed in a tissue-specific manner
(Lenka et al., 1998
). The
expression of nuclear and mitochondrial subunits of the mitochondrial
respiratory chain is thought to be highly coordinated. We therefore
investigated the expression of a nuclear encoded subunit of cytochrome
c oxidase, CoxIV, which is believed to regulate COX activity
according to the extramitochondrial ATP/ADP ratio
(Napiwotzki and Kadenbach,
1998
). The increased levels of CoxI mRNA and COI protein
(Fig. 1A) in the hibernating
kidney were concomitant with a slight decrease in the amount of immunoreactive
COIV protein (Fig. 1B),
suggesting perhaps a shift in the subunit ratio (normally 1:1) of the enzyme.
Increasing the amount of COI protein may prevent or limit the damage caused to
the cytochrome c oxidase complex by ischemia and cold.
The vertebrate mitochondrial genome is composed of 13 tightly packed open
reading frames (ORFs) and two ribosomal RNAs that are punctuated by tRNA
clusters (Clayton, 1991).
Transcription of the mitochondrial genome produces several large polycistronic
RNAs, which are subsequently processed into distinct coding and non-coding
molecules. The stability and subsequent translation of the protein-coding
mRNAs is regulated in a tissue-specific manner, yet poorly understood
(Clayton, 1991
). Two
commercially available mouse (Mus musculus) mitochondrial cDNAs were
used to probe northern blots of BAT, kidney, heart and skeletal muscle from
euthermic (active) and hibernating ground squirrels
(Fig. 2A). Transcript levels of
mitochondria-encoded genes were significantly increased (>fourfold) in
hibernating BAT and heart, slightly elevated (>twofold) in kidney, and
unchanged or slightly decreased in skeletal muscle. Previous studies have also
demonstrated an increase in expression of selected mitochondrial transcripts
in BAT during cold exposure and in the hearts of hibernating ground squirrels.
Brown adipose tissue depots flank the major organs and arteries of hibernating
mammals (Wang and Lee, 1996
).
The mitochondria in BAT are highly uncoupled and this allows them to generate
heat by the futile cycling of protons across the inner mitochondrial membrane
(Boyer and Barnes, 1999
). The
increased expression of the mitochondrial genome in hibernating BAT is
believed to enhance the electron transport and thus, the thermogenic capacity
of this tissue. Interestingly, the upregulation of the mitochondrial genome in
BAT does not increase the levels of all 13 protein products
(Clayton, 1991
). Specifically,
the levels of ATP synthase in BAT mitochondria are quite low, since the
primary role of BAT is to generate heat by futile proton cycling. This, in
contrast to the relatively high levels of ATPase 6/8 and
ATP
mRNA in BAT (Fig.
2A), suggests a high degree of post-transcriptional control
(Tvrdik et al., 1992
). The
increased expression of the mitochondrial genome in hibernating hearts, along
with other adaptive changes (Fahlman et
al., 2000
), may contribute to the continued functioning of the
heart at extremely low body temperatures. The upregulation of mitochondrial
transcripts has been observed in freezing and anoxia-tolerant species
investigated in our laboratory (Cai and
Storey, 1996
). The transcript levels of nuclear encoded
mitochondrial genes did not change in any of the hibernating organs studied
(Fig. 2).
The increased expression of mitochondrial encoded but not nuclear encoded genes in ground squirrel kidney, BAT and heart suggests a global mitochondrial response associated with the expression of the hibernation phenotype. Interestingly, a significant decrease in the transcript level of Cox1 was observed in the kidney of cold-adapted ground squirrels (Fig. 3A). This lack of upregulation of the expression of the mitochondrial genome may be one of the factors that prevents these animals from entering hibernation.
We propose that kidneys of hibernating ground squirrels `anticipate' the
metabolic lesions known to occur at complex IV during ischemia
(Montagna et al., 1998) by
overproducing those subunits that are particularly sensitive to
ischemia/reperfusion (IR) damage. In so doing they may limit the damage to
complex IV that would have otherwise compromised the viability of the organ
and the animal. In animal models of renal IR damage, there is an increase in
the expression of mitochondrial encoded genes, presumably to repair some
damage that has been done to the organ
(Van Itallie et al., 1993
).
Despite this effort, complex IV activity drops significantly after the IR
insult and the organ suffers significant, sometimes fatal injury. This same
study demonstrated an increase in the amount of CoxII mRNA as well
unprocessed mitochondrial RNA precursors in response to renal IR. The presence
of these precursors was also shown in hibernating tissues where the mRNA
levels of CoxI increases (Fig.
3B). The presence of these precursors indicates either that there
is a breakdown in the mitochondrial RNA processing machinery, or that there is
an increase in the expression of the mitochondrial genome
(Van Itallie et al.,
1993
).
Ischemic preconditioning is a technique initially developed to enhance the
preservation time of transplanted hearts, but has since been extended to other
organs including the liver, muscle and, more recently, the kidney
(Cochrane et al., 1999). A
preconditioned organ is made briefly ischemic to ameliorate the damage caused
by a subsequent extended period of ischemia. Prior to entering hibernation,
ground squirrels experience `test drops' during which they briefly depress
their metabolic rate (blood pressure, breathing, heart rate, body temperature)
and then rapidly return to euthermy (Wang
and Lee, 1996
). These test drops may represent an innate subacute
ischemic preconditioning program, which prepares hibernator organs for an
extended bout of torpor using pre-existing stress response pathways (one of
which is increased mitochondrial expression) to prevent widespread cellular
damage.
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Acknowledgments |
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