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First published online August 8, 2008
Journal of Experimental Biology 211, 2638-2646 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018598
High mitochondrial densities in the hearts of Antarctic icefishes are maintained by an increase in mitochondrial size rather than mitochondrial biogenesis
University of Alaska, Fairbanks, Institute of Arctic Biology, PO Box 757000, Fairbanks, AK 99775, USA
* Author for correspondence (e-mail: ffko{at}uaf.edu)
Accepted 21 May 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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coactivator-1
(PGC-1) and nuclear respiratory factor 1 (NRF-1)
stimulate mitochondrial biogenesis and maintain mitochondrial density in
muscle tissues. We hypothesized that these factors would also maintain
mitochondrial density in the hearts of Antarctic notothenioids. The percent
cell volume occupied by mitochondria is significantly lower in hearts of the
red-blooded notothenioid Notothenia coriiceps (18.18±0.69%) in
comparison with those of the icefish Chaenocephalus aceratus
(36.53±2.07%), which lacks both Hb and cardiac Mb. Mitochondrial
densities are not different between hearts of N. coriiceps and
Chionodraco rastrospinosus, which lacks Hb, but whose heart expresses
Mb. Despite differences in mitochondrial volume density between hearts of
N. coriiceps and C. aceratus, the levels of transcripts of
the genes encoding PGC-1, NRF-1 and citrate synthase, and the copy
number of mitochondrial DNA do not differ. Our results indicate that the high
mitochondrial densities in hearts of C. aceratus may result from an
increase in organelle size. The surface-to-volume ratio of mitochondria from
N. coriiceps is 1.9-fold greater than that of mitochondria from
C. aceratus. In addition, the levels of PGC-1 correlate with
mitochondrial density in muscle tissues of notothenioids possessing
mitochondria of similar size and morphology. Finally, the levels of
PGC-1 are 4.6-fold higher in the aerobic pectoral adductor muscle in
comparison with the glycolytic skeletal muscle of N. coriiceps. The
potential physiological significance of an increase in mitochondrial size in
hearts of Antarctic icefishes is discussed.
Key words: mitochondria, Antarctic fish, muscle, Notothenia coriiceps, Chaenocephalus aceratus, Chionodraco rastrospinosus
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Some 40 years later, close examination of heart ventricles from species within
the channicthyiid family determined that six of the 16 members do not express
the intracellular oxygen-binding protein myoglobin (Mb)
(Grove et al., 2004;
Moylan and Sidell, 2000;
Sidell et al., 1997). Since
then, many studies have revealed how these unique animals thrive despite the
loss of, what were once thought to be, essential oxygen-binding proteins.
The loss of Hb is associated with several modifications in the
cardiovascular system of icefishes. These animals have enlarged hearts in
comparison with those of red-blooded teleosts
(Hemmingsen et al., 1972;
Johnston and Harrison, 1987)
and circulate a blood volume equivalent to 7.6% of their body mass, which is
significantly greater than red-blooded teleosts, with blood volumes only
2–3% of body mass (Hemmingsen and
Douglas, 1970). The work of the heart is minimized by large-bore
capillaries, with cross-sectional areas approximately two-times larger than
those of red-blooded teleosts (Fitch et
al., 1984). Nevertheless, icefishes still expend more cardiac
energy per unit time than red-blooded notothenioids, indicating that, although
the loss of circulating red blood cells reduces blood viscosity, this strategy
is not energetically favorable (Sidell and
O'Brien, 2006). Oxygen delivery is also enhanced in some highly
aerobic tissues, such as retinae, by an increase in vascular density
(Wujcik et al., 2007). In
summary, these adaptations ensure adequate tissue oxygenation in icefishes
possessing a 9- to 10-times lower oxygen-carrying capacity compared with that
of red-blooded notothenioids (Ruud,
1954).
One of the more paradoxical adaptations to the loss of Hb and Mb expression
is an increase in mitochondrial volume density in heart ventricular tissue.
Typically, mitochondrial volume density correlates with aerobic metabolic
capacity (Hoppeler and Lindstedt,
1985). However, icefishes do not conform to this paradigm. There
is an interesting relationship between the expression of Hb and Mb and the
percentage cell volume occupied by mitochondria in heart ventricles.
Mitochondria comprise 16% of the cell volume in hearts of species that express
both Hb and Mb (O'Brien and Sidell,
2000). The percentage cell volume occupied by mitochondria
increases to 20% in hearts of icefishes expressing Mb, and, in icefishes that
lack both Hb and Mb, mitochondria occupy a stunning 36% of the cell volume
(O'Brien and Sidell, 2000).
Surprisingly, high mitochondrial densities in icefish hearts do not enhance
aerobic metabolic capacity. The activity per gram wet mass of tissue of
aerobically poised enzymes is similar between species that vary in expression
of Hb and Mb (Johnston and Harrison,
1987; O'Brien and Sidell,
2000). Although the high densities of mitochondria in icefish
hearts do not increase the aerobic metabolic capacity, they may provide a
favorable avenue for intracellular oxygen diffusion and compensate for the
lack of oxygen-binding proteins (O'Brien
and Sidell, 2000; Sidell,
1998).
Although the impressive remodeling of cardiac myocytes in response to the
loss of oxygen-binding proteins has been well established, the molecular
underpinnings maintaining the high mitochondrial densities in hearts of
icefishes are unknown. In mammals, mitochondrial proliferation is driven by
the transcriptional coactivator peroxisome proliferator-activated receptor
coactivator-1 (PGC-1, encoded by the PPARGC1A
gene). Together with nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2),
PGC-1 transactivates the expression of nuclear-encoded genes involved
in mitochondrial oxidative phosphorylation
(Puigserver and Spiegelman,
2003). PGC-1 and NRFs also stimulate the expression of
mitochondrial transcription factor A (TFAM), which translocates to the
mitochondrion and activates the transcription and replication of the
mitochondrial genome (Kang and Hamasaki,
2005).
We hypothesized that high mitochondrial densities in the hearts of
icefishes would be maintained by the same mitochondrial biogenic pathway
observed in mammals. Mitochondrial volume density was quantified in hearts of
the red-blooded notothenioid N. coriiceps using stereological
point-counting methods. Values were compared with previous measurements made
of mitochondrial densities in the hearts of C. aceratus
(–Hb/–Mb) and C. rastrospinosus (–Hb/+Mb)
(O'Brien and Sidell, 2000). We
also measured the expression of PGC-1 and NRF-1 in heart ventricles of
these three species, as well as two additional indices of mitochondrial
density: the expression of the aerobic metabolic gene encoding citrate
synthase, and the copy number of mitochondrial DNA (mtDNA). Our results
indicate that the high mitochondrial densities in Mb-deficient icefish hearts
are not maintained through a canonical mitochondrial biogenic pathway.
Instead, they appear to be brought about through an expansion of the outer
mitochondrial membrane that occurs independently of an increase in
mitochondrial protein expression and replication of mtDNA.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Animals were anesthetized in 1:7500 (w/v) MS-222 (tricane methanesulfonate) and then killed by brain pithing. Heart, aerobic pectoral adductor muscle and glycolytic skeletal muscle were quickly excised, frozen in liquid nitrogen and stored at –80°C. Portions of each tissue type were also diced into 1-mm-sized blocks on an ice-cold stage, immersed in nine volumes of RNAlater (Ambion, Austin, TX, USA), stored overnight at 4°C and frozen at –80°C.
Preparation of heart ventricles from N. coriiceps for electron microscopy
Hearts were excised from N. coriiceps and placed into an ice-cold
isotonic Ringer solution (260 mmoll–1 NaCl, 2.5
mmoll–1 MgCl2, 5mmoll–1 KCl,
2.5mmoll–1 NaHCO3, 5mmoll–1
NaH2PO4, pH8.0) in which they were allowed to contract
several times to clear blood from the heart lumen. Hearts were then
transferred into an ice-cold fixative solution (3% glutaraldehyde, 100
mmoll–1 sodium cacodylate, 110 mmoll–1
sucrose, 2 mmoll–1 CaCl2, pH 7.4) and perfused
through the atria at a rate of approximately 1 ml min–1 for
10 min. Tissues were placed in fresh fixative and stored at 4°C for
4–6 hours and then transferred again into fresh fixative for an
additional 2–4 hours. Hearts were stored in Trumps buffer (1%
glutaraldehyde, 4% formaldehyde, 0.1 moll–1 sodium
cacodylate, 0.11moll–1 sucrose, 2mmoll–1
CaCl2, pH7.4) at 4°C and transported back to the University of
Alaska, Fairbanks for further processing.
Transmural sections spanning from the epicardium to the endocardium were excised from the heart ventricles. Epicardial tissue was removed, and the remaining endomyocardial tissue was cut into 1-mm-sized blocks. The blocks were rinsed overnight at 4°C in 0.1moll–1 sodium cacodylate, 0.11moll–1 sucrose, 2mmoll–1 CaCl2, pH 7.4. The following day, blocks were post-fixed in an ice-cold solution of 1% osmium tetroxide, 0.1moll–1 sodium cacodylate, 0.11 moll–1 sucrose, 2 mmoll–1 CaCl2, pH 7.4, for 2h. The blocks were rinsed briefly three times with distilled water and then dehydrated through a series of acetone washes. The blocks were embedded in a mixture of Epon and Araldite resin [24% Eponate, 15% Araldite, 58% DDSA, 3% BDMA (v/v)] by first incubating for 30 min in a 2:1 acetone:resin mixture, followed by 30 min in a 1:1 acetone:resin mixture and then placed in a 1:2 acetone:resin mixture overnight, with caps slightly ajar to allow the acetone to evaporate slowly. The following day, the blocks were transferred into fresh resin, placed under vacuum for 30 min, transferred to fresh resin and placed under vacuum at 60°C for 30 min. The blocks were cured in resin for 24–36h at 60°C.
Tissue blocks were sectioned using a Sorvall MT2 ultramicrotome and placed
on 200-mesh copper grids. Sections were stained with 2% uranyl acetate,
followed by 0.5% lead citrate and viewed with a JEOL JEM-1200EX transmission
electron microscope equipped with a digital camera. Between seven and 10
micrographs were obtained from one section per individual (four individuals in
total) for measuring mitochondrial volume and surface densities using the
aligned-systematic-quadrats-subsampling method
(Cruz-Orive and Weibel, 1981).
Micrographs were taken at a magnification of x7500 and viewed with Adobe
Photoshop 6.0 at a final magnification of x13100. Images were overlaid
with a digital square lattice test pattern, with spacing equal to 0.53µm on
the micrograph. The mitochondrial volume density was quantified using
point-counting methods, and the mitochondrial surface density was measured
using the line-intercept technique
(Weibel, 1979). The
extracellular matrix, lumenal spaces and epithelial cells were excluded from
measurements.
Isolation of RNA and DNA
Total RNA was isolated using either TRIZOL reagent (Invitrogen, Life
Technologies, Carlsbad, CA, USA) or an RNeasy Fibrous Tissue Mini Kit (Qiagen,
Valencia, CA, USA). DNA was isolated using the DNeasy Tissue Kit (Qiagen). The
concentration and quality of nucleic acids were assessed with a Nanodrop
ND-1000 spectrophotometer or a PerkinElmer Lambda 25 UV/VIS spectrometer. Only
samples with a 260nm to 280nm ratio of 1.8–2.0 and a 260 nm to 230nm
ratio of 1.6–2.0 were used for further analysis. The integrity of the
RNA was verified by visualizing samples on a 1% agarose gel stained with
ethidium bromide. RNA was stored at –80°C and DNA at
–20°C.
Sequencing genes of interest for qRT-PCR
Partial cDNA sequences from the genes of interest were obtained to design
gene-specific primers for quantitative real-time PCR (qRT-PCR). Homologous
amino acid sequences from at least four species were aligned, and degenerate
primers were designed using CODEHOP
(http://bioinformatics.weizmann.ac.il/blocks/codehop.html)
(Table 1). When possible,
primers were selected to amplify regions that contained two or more splice
sites within homologous sequences.
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Complementary DNA was synthesized using either Superscript III reverse transcriptase (Invitrogen) or MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA, USA) and random hexamers.
Sequences of interest were amplified using an iCycler (Bio-Rad Laboratories, Hercules, CA, USA) programmed with a touchdown protocol with annealing temperatures between 55°C and 65°C. PCR products were separated on a 2% agarose gel and visualized with ethidium bromide. Products corresponding to the expected size were excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen). Products were cloned using a TOPO TA Cloning Kit (Invitrogen). Transformed Escherichia coli were identified by blue–white screening on LB-agar plates (15mgml–1 agar, 10mgml–1 Bacto-tryptone, 5 mg ml–1 NaCl, 5 mg ml–1 yeast extract, 1 mg ml–1 glucose, 12.5 µg ml–1 ampicillin) supplemented with 0.2 mg ml–1 X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Transformed E. coli were grown in 5 ml LB media (10 mg ml–1 Bacto-tryptone, 5 mg ml–1 NaCl, 5 mg ml–1 yeast extract, 1 mg ml–1 glucose) supplemented with 0.25 mg ml–1 ampicillin overnight in an OLS200 shaking water bath (Grant Instruments, Cambridge, UK) at 200 r.p.m. and 37°C. Plasmids were isolated using the QIAprep Miniprep kit (Qiagen), labeled for sequencing with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and purified with Centri-Sep columns (Princeton Seperations, Adelphia, NJ, USA) packed with Sephadex G-50 gel (Sigma-Aldrich, St Louis, MO, USA). Genes of interest were sequenced using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).
The 5' region of the gene encoding NRF-1 was obtained by rapid amplification of cDNA ends (RACE) PCR using the BD SMART RACE cDNA Amplification Kit (BD Biosciences, Sparks, MD, USA). Gene-specific primers for amplifying both the 5' and 3' ends of the gene were designed from sequence obtained with degenerate primers. PCR reactions were carried out using an iCycler (Bio-Rad Laboratories) programmed with a touchdown protocol and annealing temperatures between 68°C and 70°C. The RACE products were cloned and sequenced as described above.
Quantitative real-time PCR
RNA was treated twice with DNase I to remove genomic DNA – once for
25 min at 25°C and a second time for 20 min at 25°C. Gene-specific
primers were designed from partial cDNA sequences using Primer Express
Software v2.0 (Applied Biosystems). Primers were designed within regions of
cDNA that are conserved between all three species of interest
(Table 2) and in all cases,
except citrate synthase, such that at least one primer from each set spanned a
splice site to ensure that genomic DNA was not amplified.
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Quantitative RT-PCR was carried out with an ABI PRISM 7900HT Sequence
Detection System with Power SYBR Green PCR Master Mix (Applied Biosystems).
Template cDNA of all target and housekeeping genes, except 18S rRNA, was
diluted by a factor of 20 to a final concentration of 1 ng
µl–1. Complementary DNA used for measuring the expression
of 18S rRNA was diluted by a factor of 2000 to a final concentration of 0.01
ng µl–1. This was done to compensate for the greater
abundance of 18S rRNA compared with that of the target genes. DNA used for
measuring the ratio of mitochondrial-to-nuclear DNA was diluted to a final
concentration of 5 ng µl–1. Complementary DNA or genomic
DNA was pooled from all individuals and serially diluted to generate a
standard curve to determine the reaction efficiency. The reactions were
carried out in triplicate, and two types of controls were used. The first
control lacked template cDNA (or DNA for measurements of mitochondrial DNA
copy number), and the second (for measuring gene expression) contained RNA in
which the reverse transcriptase was omitted during cDNA synthesis. Both of
these controls ensured that genomic or contaminating DNA was not amplified.
Dissociation curves were analyzed to verify that only a single product was
amplified in each reaction mixture. The expression of target genes was
normalized to the expression of 18S rRNA using the comparative critical
threshold (
Ct) method corrected for primer efficiency
(Pfaffl, 2001). The expression
level of all target genes was expressed relative to levels in N.
coriiceps, except for measurements in oxidative and glycolytic muscle of
N. coriiceps. In these experiments, the expression of target genes in
oxidative muscle was expressed relative to the levels in glycolytic muscle.
The ratio of mitochondrial-to-nuclear DNA in all species was normalized to the
ratio in N. coriiceps.
Evaluation of housekeeping genes
The expression of three genes (encoding EF-1A, TBP, 18S rRNA) was evaluated
as potential housekeeping genes (HKGs) for normalizing the expression of the
target genes. The expression level of these genes was measured using qRT-PCR
in the heart ventricle of the three species of interest. The results were
analyzed using the Excel-based program BestKeeper (Pfaffl, 2004).
BestKeeper uses a combination of descriptive statistics and regression analysis of Ct values to identify the most stably expressed HKGs. The Ct values measured from all individuals are pooled for each gene. Genes with Ct values having standard deviations greater than 1 are considered unstable. Two types of linear regression analysis are also performed. First, a regression analysis is carried out between all possible combinations of all genes. Second, the average Ct values of all genes with s.d. of <1 from each individual are pooled to create the bestkeeper index. A linear regression is then carried out between the bestkeeper index and each gene. Suitable HKGs are those with the highest correlation coefficient (r) when compared with the bestkeeper index and a s.d. of <1.
Statistics
Significant differences in gene expression were determined using a
Student's t-test or an ANOVA followed by a post-hoc Tukey's
Honestly Significantly Different (HSD) test. Differences were considered
significant at P<0.05. The results are presented as the means
± s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Mitochondrial densities are twofold lower in hearts of N. coriiceps
compared with hearts of C. aceratus
(Table 3). However, there is no
significant difference in the percentage cell volume occupied by mitochondria
between N. coriiceps and C. rastrospinosus (P=0.11)
(Table 3).
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Analysis of housekeeping genes
The Excel-based software tool BestKeeper
(Pfaffl et al., 2004) was used
to analyze the expression of three commonly used HKGs for studies of gene
expression. These HKGs included the TATA-box-binding protein (TBP), elongation
factor 1A (EF-1A) and 18S ribosomal RNA (18S)
(de Kok et al., 2005;
McClelland et al., 2006;
Olsvik et al., 2005). The
expression of TBP is highly variable (s.d.>1), indicating that this gene is
not a suitable HKG for measurements of gene expression in hearts of
notothenioids (Table 4).
Measurements of the expression of the genes encoding both 18S and EF-1A (with
s.d. <1) are correlated with the bestkeeper index
(Table 4), and thus both are
appropriate HKGs. We normalized the expression of all target genes to the
expression of 18S rRNA. Normalization to EF-1A gave similar results (data not
shown).
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Expression of mitochondrial biogenic genes
PGC-1 and NRF-1 play central roles in regulating the density of
mitochondria in a variety of mammalian tissue types
(Puigserver and Spiegelman,
2003). Despite significant differences in mitochondrial volume
densities between hearts of the red-blooded N. coriiceps and the
icefish C. aceratus, there is no significant difference in the
expression of either PGC-1 or NRF-1
(Fig. 1). The amount of RNA per
gram of tissue is equivalent between the hearts of C. aceratus, C.
rastrospinosus and N. coriiceps
(Table 5). Therefore, the
relative expression of PGC-1 and NRF-1 per gram of tissue is also
equivalent between the hearts of these three species.
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Mitochondrial DNA copy number
The copy number of mtDNA typically correlates with mitochondrial volume
density (Wu et al., 1999). We
quantified the mtDNA copy number relative to the abundance of nuclear DNA
(nDNA) using qRT-PCR. The copy numbers of the mitochondrially encoded genes
NADH dehydrogenase subunit 2 (ND2) and 16S rRNA (16S) were compared with the
copy number of the nuclear gene encoding citrate synthase (CS). No significant
differences were detected in the ratio of mtDNA-to-nDNA between the heart
ventricles of N. coriiceps, C. rastrospinosus and C.
aceratus (Fig. 2). In
addition, the amount of DNA per gram of tissue does not vary between the
hearts of the three species (Table
5). As a result, there is no significant difference in the copy
number of mtDNA per gram wet mass of tissue.
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and NRF-1 regulate mitochondrial biogenesis in
notothenioids, we measured the expression of these factors in the pectoral
adductor and glycolytic skeletal muscles of N. coriiceps, which
differ in mitochondrial density but not in mitochondrial morphology. The
expression level of PGC-1 is 4.6-fold higher in pectoral adductor
muscle compared with glycolytic skeletal muscle in N. coriiceps
(Fig. 4A). The expression level
of NRF-1 tends to be higher in pectoral adductor muscle, compared with
glycolytic skeletal muscle, although this difference is not significant
(Fig. 4B; P=0.12).
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The amount of RNA per gram wet mass of muscle does not vary between
oxidative (0.36±0.08 mg g–1) and glycolytic (0.28 mg
g–1) muscles (P>0.05). As a result, the
expression of PGC-1 per gram of tissue is significantly higher in
oxidative muscle compared with glycolytic muscle of N. coriiceps.
Mitochondrial size
Mitochondria from hearts of C. aceratus are larger than those from
N. coriiceps. The mitochondrial surface-to-volume ratio is
4.52±0.27µm–1 in C. aceratus
(O'Brien and Sidell, 2000),
compared with 8.69±0.40 µm–1 in hearts of N.
coriiceps (P<0.05).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Mitochondrial volume densities are significantly higher in hearts of icefishes lacking Mb compared with red-blooded notothenioids
Consistent with previous studies, we determined that the percentage of cell
volume occupied by mitochondria is significantly lower in the hearts of the
red-blooded notothenioid N. coriiceps compared with the icefish
C. aceratus, which does not express Hb or cardiac Mb
(Table 3). This difference has
been observed not only in the heart ventricle tissue between red- and
white-blooded notothenioids but also in the aerobic pectoral adductor muscle
(Johnston, 1987;
O'Brien and Sidell, 2000;
O'Brien et al., 2003).
We did not find significant differences in mitochondrial volume densities
between the hearts of N. coriiceps and the icefish C.
rastrospinosus, which does express cardiac Mb. This is in contrast to a
previous study (O'Brien and Sidell,
2000) that determined that mitochondrial densities are 4% higher
in hearts of C. rastrospinosus compared with the red-blooded
Gobionotothen gibberifrons. N. coriiceps are more active compared
with G. gibberifrons, which may account for the higher mitochondrial
density in hearts of N. coriiceps. Overall, these data suggest that
the loss of both Hb and Mb leads to a much larger expansion (18–20%) of
mitochondrial volume density in icefish hearts compared with the expansion
upon loss of Hb alone (
4%).
The high mitochondrial densities in hearts of C. aceratus are not maintained by a conventional pathway of mitochondrial biogenesis
PGC-1 and NRF-1 play pivotal roles in controlling mitochondrial
density in mammalian muscle tissues (Evans
and Scarpulla, 1990; Lehman et
al., 2000; Puigserver et al.,
1998; Wu et al.,
1999). These factors stimulate the expression of genes required
for increasing mitochondrial density in response to an increase in ATP demand,
such as exercise or cold temperature (Baar,
2004; Wu et al.,
1999). In addition, the levels of PGC-1 and NRF-1 correlate
with the oxidative capacities of tissues, suggesting that they are required
for maintaining steady-state levels of mitochondrial densities
(Lin et al., 2002;
Puigserver et al., 1998;
Terada and Tabata, 2004).
Surprisingly, despite the twofold difference in mitochondrial volume density
between hearts of N. coriiceps and C. aceratus, we do not
detect a significant difference in the expression of PGC-1 or NRF-1 in
these animals.
The lack of difference between the two fish in their expression of
PGC-1 and NRF-1 suggests three possibilities. First, these factors may
be regulated by post-transcriptional processes. Previous work has shown that
phosphorylation of serine and threonine residues by p38 MAP kinase, as well as
deacetylation at multiple sites by the NAD-dependent deacetylase SIRT1,
regulates the DNA binding activity of PGC-1
(Gerhart-Hines et al., 2007;
Puigserver et al., 2001).
Second, PGC-1 and NRF-1 may not regulate mitochondrial biogenesis in
notothenioids. Little is known about the molecular mechanisms of mitochondrial
biogenesis in fish. A recent study in goldfish indicates that another member
of the PGC-1 family of transcriptional co-activators, PGC-1β, may be more
important than PGC-1 in controlling mitochondrial biogenesis in fish
(Lemoine et al., 2008).
Finally, the lack of difference in PGC-1 and NRF-1 between hearts of
N. coriiceps and C. aceratus may indicate that the
expression of mitochondrial proteins does not differ between these two
species. The expression of mitochondrial proteins and replication of mtDNA are
clearly regulated by PGC-1 in mammals. However, it is not known whether
PGC-1 also controls mitochondrial membrane biogenesis
(Scarpulla, 2006). If not, the
high mitochondrial densities in the hearts of Mb-deficient icefishes may arise
through a novel, PGC-1-independent pathway, in which membrane, but not
protein, biosynthesis is elevated. Previous work, as well as our own
observations, supports this hypothesis. The surface-to-volume ratio of
mitochondria from hearts of C. aceratus is significantly lower
compared with that of mitochondria from hearts of the red-blooded species
G. gibberifrons (O'Brien and
Sidell, 2000) and N. coriiceps
(Fig. 5).
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and NRF-1 do not maintain
mitochondrial density in Antarctic notothenioids, we measured the expression
of these factors in oxidative pectoral adductor muscle and glycolytic skeletal
muscle from N. coriiceps. These two tissues differ in mitochondrial
density but not mitochondrial morphology. Mitochondria occupy 34.3% of the
cell volume in pectoral adductor muscle of N. coriiceps compared with
only 1.4% in glycolytic skeletal muscle fibers
(Johnston, 1989). In addition,
the maximal activity of aerobically poised enzymes is typically 10-fold
greater in oxidative muscle tissue compared with glycolytic muscle of
notothenioids (Crockett and Sidell,
1990). We found that the levels of PGC-1 correlate with
mitochondrial densities in muscle tissues of Antarctic fishes when differences
in organelle ultrastructure are eliminated. The expression of PGC-1 is
4.6-fold higher in pectoral adductor muscle compared with glycolytic skeletal
muscle of N. coriiceps. The levels of NRF-1 tend to be higher in
oxidative muscle compared with glycolytic muscle, although this difference is
not significant.
Together, these results indicate that the differences in mitochondrial
density between hearts of C. aceratus and N. coriiceps are
not maintained by PGC-1 and NRF-1. To confirm this, we measured the
expression of two additional components of the mitochondrial biogenic pathway:
mtDNA copy number and expression of CS. PGC-1, together with NRF-1 and
NRF-2, activates the expression of the mitochondrial transcription factor
TFAM, which controls both the transcription and replication of the
mitochondrial genome (Virbasius and
Scarpulla, 1994). Consistent with our measurements of PGC-1
and NRF-1, there is no significant difference in the copy number of mtDNA
between hearts of N. coriiceps and C. aceratus. In addition,
there is no difference in the expression of CS between the hearts of these
animals. This agrees with previous measurements, which found no difference in
the activity of CS pergram of tissue between hearts of the red-blooded
notothenioid G. gibberifrons and the icefish C. aceratus
(O'Brien and Sidell,
2000).
These results, along with ultrastructural analysis of mitochondria, strongly suggest that the high mitochondrial densities in hearts of C. aceratus are not brought about through mitochondrial biogenesis. The unique architecture of mitochondria in hearts of C. aceratus suggests that each mitochondrion is enlarged through a proliferation of the outer mitochondrial membrane without a corresponding increase in inner membrane surface density, protein synthesis or mtDNA replication. To our knowledge, this is the only example in any organism in which an increase in mitochondrial density is brought about in this fashion.
Nothing is known about how mitochondrial membrane synthesis is integrated
into mitochondrial biogenesis. However, our results suggest that PGC-1,
considered the `master regulator of mitochondrial biogenesis', may not control
this process (Kelly and Scarpulla,
2004; Scarpulla,
2006). Unlike mtDNA and most mitochondrial proteins, which are
localized solely to the mitochondrion, mitochondrial membrane phospholipids
are part of a general pool of phospholipids synthesized in the endoplasmic
reticulum (ER) (Daum and Vance,
1997). From the ER, they are directed to multiple locations within
the cell. Only cardiolipin is synthesized exclusively within mitochondria,
where it is localized within the inner membrane and at contact points between
the inner and outer membranes (Ardail et
al., 1990; Chen et al.,
2006). Currently, little is known about how the ER directs
phospholipids to specific cellular compartments. Because the ER synthesizes
the majority of membrane phospholipids, it must be capable of regulating
phospholipid biosynthesis independently of mitochondrial biogenesis. In
addition, during mitochondrial biogenesis, the ER must direct newly
synthesized phospholipids specifically to the mitochondrion to maintain a
constant ratio between mitochondrial proteins and phospholipids. How these
dual functions are accomplished is unclear. However, it appears that, in the
hearts of Mb-deficient icefish, the former pathway is upregulated.
Potential advantages to an increase in mitochondrial size in the hearts of icefish lacking Mb
The unusual ultrastructure of mitochondria in the hearts of C.
aceratus raises the issue of whether there is a physiological advantage
to mitochondrial remodeling in the hearts of fishes that lack Hb and Mb. The
enlargement of mitochondria in the hearts of icefishes results in two
alterations to the architecture of cardiac myocytes. First, it leads to a
higher surface density of mitochondrial membranes per g ventricular tissue
relative to those of red-blooded fish species. Second, the surface-to-volume
ratio of individual mitochondria is decreased relative to that of red-blooded
notothenioids. Each of these modifications might have a distinct advantage to
the cardiovascular system of icefishes.
As previous studies have suggested, the increase in surface density of
mitochondrial membranes in icefishes may enhance oxygen delivery (i.e.
O'Brien and Sidell, 2000).
Oxygen is more than four times more soluble in nonpolar solvents than it is in
water (Battino et al., 1968).
As a result, the hydrocarbon core of mitochondrial membranes serves as a
storehouse for oxygen, as well as acting as an effective conduit for the
diffusion of oxygen (Desaulniers et al.,
1996; Sidell,
1998). In addition, the proliferation of mitochondrial membranes
decreases the diffusion distance for oxygen between the lumen of the heart and
mitochondria within cardiac myocytes
(O'Brien et al., 2000).
Enhanced oxygen delivery is one important advantage of the unusual
mitochondrial morphology in the hearts of C. aceratus, but there may
be other advantages. Mitochondria are complex organelles containing more than
1500 proteins and are involved in a broad array of cellular functions
(McDonald and Van Eyk, 2003).
In addition to producing ATP through oxidative phosphorylation, mitochondria
also store calcium, regulate apoptosis, produce nitric oxide and oxygen
radicals and are sites for the biosynthesis of amino acids and heme
(Scheffler, 1999). One or more
of these processes might be altered by changes in mitochondrial morphology.
For example, oxidative phosphorylation, apoptosis and proton leakage are all
affected by mitochondrial architecture
(Bach et al., 2003;
Brand et al., 1994;
Brooks et al., 2007;
Mannella, 2006). Future
studies should address not only the molecular mechanisms that bring about
changes in mitochondrial structure in response to the loss of Hb and Mb but
also the physiological impact of these modifications.
The potential role of NO in mediating high mitochondrial densities in the hearts of Antarctic icefishes
The stimulus inducing mitochondrial membrane proliferation in the hearts of
Mb-deficient icefish is unknown but appears to be related to the expression of
oxygen-binding proteins. Nitric oxide (NO) is a potent signaling molecule that
may play a role in this pathway (Sidell
and O'Brien, 2006). Two lines of evidence support this hypothesis.
First, the hearts of both red- and white-blooded Antarctic notothenioids
possess the enzyme nitric oxide synthase, which produces NO
(Amelio et al., 2006). Second,
NO levels are tightly regulated by both Hb and Mb, which are potent NO
dioxygenases that metabolize NO to nitrate
(Gardner, 2005). Thus, the
hearts of icefishes have the capacity to produce NO but not metabolize it,
leading to elevated levels of NO compared with those of red-blooded species.
Consistent with this, recent work has found higher levels of the metabolic
byproducts of NO – nitrite and nitrate – in the blood plasma of
icefishes compared with red-blooded notothenioids (K. Borley and B. Sidell,
personal communication).
Observations of the pectoral adductor muscle of C. rastrospinosus
provide further evidence that mitochondrial membrane biosynthesis is sensitive
to the presence of oxygen-binding proteins. The pectoral adductor muscles of
all notothenioids examined to date do not express Mb
(Sidell et al., 1997).
Mitochondria in the pectoral adductor muscle of C. rastrospinosus,
lacking both Hb and Mb, are enlarged compared with those in the heart
ventricle, where Mb is expressed (O'Brien
and Sidell, 2000; O'Brien et
al., 2003). In fact, mitochondria in C. rastrospinosus
pectoral muscles look strikingly similar to those found in the pectoral
adductor and heart ventricle of C. aceratus, which lack Hb and Mb
(O'Brien et al., 2003). This
suggests that mitochondrial morphology is not a genetically fixed trait in
Antarctic icefish but, rather, is influenced directly by the expression of Hb
and Mb or indirectly by NO.
Although previous studies in mammals have determined that NO stimulates
PGC-1 expression and mitochondrial biogenesis
(Nisoli et al., 2003;
Nisoli et al., 2004), our data
indicate that this pathway does not maintain high densities of mitochondria in
the hearts of Antarctic icefishes. The mitochondrial biogenic pathway may be
refractory to NO in icefishes if levels of NO-sensitive intermediates, such as
guanylyl cyclase, are expressed at a lower level compared to red-blooded
species. Nevertheless, NO may induce the expression of genes governing the
biosynthesis of mitochondrial membranes, and future studies will address this
question.
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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