AJP - Regu AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 275: R905-R912, 1998;
0363-6119/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 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 Google Scholar
Google Scholar
Right arrow Articles by Battersby, B. J.
Right arrow Articles by Moyes, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Battersby, B. J.
Right arrow Articles by Moyes, C. D.
Vol. 275, Issue 3, R905-R912, September 1998

Influence of acclimation temperature on mitochondrial DNA, RNA, and enzymes in skeletal muscle

Brendan James Battersby and Christopher D. Moyes

Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Skeletal muscle fibers typically undergo modifications in their mitochondrial content, concomitant with alterations in oxidative metabolism that occur during the development of muscle fiber and in response to physiological stimuli. We examined how cold acclimation affects the mitochondrial properties of two fish skeletal muscle fiber types and how the regulators of mitochondrial content differed between tissues. After 2 mo of acclimation to either 4 or 18°C, mitochondrial enzyme activities in both red and white muscle were higher in cold-acclimated fish. No significant differences were detected between acclimation temperatures in the abundance of steady-state mitochondrial mRNA (cytochrome-c oxidase 1, subunit 6 of F0F1-ATPase), rRNA (16S), or DNA copy number. Steady-state mRNA for nuclear-encoded respiratory (adenine nucleotide translocase 1) and glycolytic genes showed high interindividual variability, particularly in the cold-acclimated fish. Although mitochondrial enzymes were 10-fold different between the two muscle types, mitochondrial DNA copy number differed only 4-fold. The relative abundance of mitochondrial mRNA and nuclear mRNA in red and white muscle reflected the differences in copy number of their respective genes. These data suggest that the response to physiological stimuli and determination of tissue-specific mitochondrial properties likely result from the regulation of nuclear-encoded genes.

metabolism; polymerase chain reaction; gene expression; rainbow trout; cytochrome-c oxidase; adenosine triphosphatase

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

MITOCHONDRIAL BIOGENESIS is a complex process required by tissues to increase their oxidative phosphorylation capacity. Development, differentiation, and energetic challenges, such as exercise training and cold exposure, lead to changes in mitochondrial content (15, 16, 27). The molecular mechanisms by which increases in mitochondria are achieved and maintained are an active area of research. Because most of the complexes in electron transport have both nuclear- and mitochondrial-encoded subunits, mitochondrial biogenesis is complicated by the need to coordinate expression of the mitochondrial and nuclear genomes. A number of transcription factors and regulatory elements [nuclear respiratory factors (NRF1 and NRF2), OXBOX, REBOX, MT1, MT3, and MT4] regulate transcription of nuclear-encoded respiratory genes (10, 14, 24, 37, 42). Shared sensitivity to specific transcription factors gives rise to functional "gene families," which is thought to facilitate coordinated gene expression during mitochondrial biogenesis (17, 29, 37). Chronic increases in metabolic rate are accompanied by severalfold increases in mRNA for a number of nuclear-encoded respiratory genes (see Ref. 16). Although these nuclear genes are generally thought to be under transcriptional control (44), increases in mRNA are also due to posttranscriptional events (1, 2). Complexes I, III, IV, and V of oxidative phosphorylation (OXPHOS) possess subunits encoded by mitochondrial DNA. Studies with mammals suggest increases in expression of these mitochondrial genes is achieved by gene amplification; increases in mitochondrial mRNA levels are due to increases in mitochondrial DNA copy number (44).

Fish provide a useful model for studying the factors that determine mitochondrial content in different fibers and physiological conditions. Fish skeletal muscle is separated into two anatomically discrete red and white fibers. The mitochondrial volume density of red muscle ranges from 25-44% depending on the species, a content similar to that of mammalian cardiac muscle (40). In contrast, white muscle contains 2-4% mitochondria, similar to the range of most mammalian skeletal muscles (18, 20, 21). As with mammals, endurance exercise leads to increases in mitochondrial enzyme activity (11). Although mitochondrial proliferation is thought to be a response to chronic elevations in metabolic rate (17), a number of fish species also increase mitochondrial content in response to cold acclimation (e.g., Refs. 9, 13). Although this mitochondrial proliferation occurs in both red and white muscle, the impact of the inherent differences in mitochondrial content between fiber types has not been explored from a regulatory perspective. The purpose of the present study was to address the molecular basis for mitochondrial differences between fiber types and with acclimation by using measurements of mitochondrial enzymes, DNA, mRNA, and rRNA and nuclear-encoded RNA.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Rainbow trout (Oncorhynchus mykiss) were obtained from Pure Springs Trout Farms (Belleville, ON, Canada) in the summer of 1995 and the fall of 1996. Fish, ~1 kg in size, were randomly divided between two flow-through tanks and kept under a 12:12-h light-dark photoperiod. The desired experimental acclimation temperatures (4 and 18°C) were gradually achieved from 10°C over a 1-wk period and held for 2 mo. After the acclimation period, samples of red axial muscle and white epaxial muscle were quickly excised and frozen in liquid nitrogen. Tissues were pulverized into a powder slurry with a mortar and pestle cooled under liquid nitrogen and stored at -86°C.

Enzymes. Powdered tissue was homogenized in 9 vol of homogenization buffer (20 mM HEPES, 1 mM EDTA, and 0.1% Triton X-100) with a Polytron tissue homogenizer (Kinematica). Samples were kept on ice during homogenization and ground with three bursts of 10 s.

Maximal enzyme activities were determined under optimal conditions by using a Beckman DU 640 spectrophotometer. Enzymes were assayed at 15°C, which was maintained by a VWR Scientific water bath. The temperature for the enzyme assays was chosen to fall within the physiological range of the experimental animals. The Q10 for mitochondrial enzymes was found to be ~2, as in previous studies (31). The protocols for each assay were based on previously described methods (31): for citrate synthase (CS), 20 mM Tris (pH 8.0), 0.2 mM DTNB, 0.3 mM acetyl-CoA, 0.05% Triton X-100, and 0.5 mM oxalacetate (omitted for control); for complex I (NADH dehydrogenase), 25 mM potassium phosphate buffer (pH 7.4), 0.05 mM dichlorophenol-indophenol, 0.1 mM NADH, and 0.01% Triton X-100; and for cytochrome-c oxidase (COX), 20 mM Tris (pH 8.0), 0.5% Tween 20, and 0.05 mM reduced cytochrome c, which was reduced by using ascorbate, dialyzed exhaustively against 20 mM Tris (pH 8.0), and frozen in aliquots at -86°C. Sample homogenates were preincubated for 6 min at 15°C with buffer and detergent before cytochrome c addition. Shorter incubations reduced activities.

DNA isolation. Total cellular DNA was isolated from powdered tissue following methods described in Ausubel et al. (3). Briefly, tissue was digested in 100 mM NaCl, 10 mM Tris (pH 8.0), 25 mM EDTA (pH 8.0), and 0.5% SDS with 0.1 mg/ml proteinase K for 18 h at 50°C. Samples were then extracted with an equal volume of phenol-chloroform-isoamyl alcohol (24:25:1), vortexed for 10 s, and then centrifuged at 3,000 g for 5 min at room temperature. DNA was precipitated overnight at 4°C with 0.5 vol of 7.5 M ammonium acetate and 2 vol of 100% ethanol. Potential RNA contamination of samples was removed by resuspending DNA in TE buffer (10 mM Tris · Cl-1 mM EDTA, pH 8.0) and incubated at 37°C for 1 h with 0.1% SDS and 1 µg/ml RNase. After RNase digestion, DNA samples were extracted again with an equal volume of phenol-chloroform-isoamyl alcohol and precipitated as described above. Samples were resuspended in TE buffer (pH 8.0) and stored at -20°C until analysis.

RNA isolation. Total RNA was recovered from frozen tissue by using an acid guanidinium thiocyanate-phenol-chloroform extraction based method (7). In short, tissue was homogenized in 10 vol of GTC buffer (4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, and 1.0% beta -mercaptoethanol) with a Polytron for 20 s. To each sample the following were added: 1 vol of 2 M sodium acetate (pH 4.0), 10 vol of buffer-saturated phenol (pH 4.3), and 2 vol of chloroform-isoamyl alcohol (49:1). After the addition of the chloroform-isoamyl alcohol mixture, samples were shaken vigorously for 15 s, placed on ice for 15 min, and subsequently centrifuged at 3,000 g for 30 min at room temperature. The upper aqueous layer was collected and precipitated with 1 vol of isopropanol overnight at -20°C. RNA was resuspended in 500 µl of GTC, reprecipitated with an equal volume of isopropanol, and then stored at -20°C until further use. Samples for Northern blot analysis were resuspended in TE buffer (pH 8) and heated briefly at 65°C to aid in resuspension. RNA was glyoxylated after triplicate quantitation (260 nm).

Construction of cDNA probes. A human 18S ribosomal probe was kindly provided by Dr. Eric Shoubridge (Montreal Neurological Institute). Other probes were constructed on site via PCR from total DNA extractions (mitochondrial mRNA species) or first-strand cDNA from total RNA extractions (nuclear mRNA species).

PCR was carried out on a programmable thermal controller (MJ Research) by using Taq polymerase (Pharmacia). Mitochondrial-encoded genes were amplified from total DNA by using primers (Ransom Hill Bioscience) which were designed based on the published sequence of rainbow trout mitochondrial DNA (Ref. 46; GenBank accession no. L29771). Universal primers for 16S rRNA were provided by Dr. S. Lougheed (Queen's University). The sequence for each primer pair is listed in Table 1. DNA was amplified in reaction buffer [10 mM Tris · HCl (pH 9.0), 1.5 mM MgCl2, and 50 mM KCl], 200 µM dNTP, 200 ng primers, and 2.5 U Taq polymerase. The PCR cycles used to amplify these mitochondrial transcripts were the following: initial denaturation at 95°C for 60 s, subsequent denaturations at 95°C for 30 s, annealing at 60°C (16S) or 55°C [COX1 and subunit 6 of F0F1-ATPase (ATP6)] for 90 s, extension at 72°C for 90 s, and final extension at 72°C for 10 min. PCR products were separated on a 1.0% agarose gel stained with 0.5 µg/ml ethidium bromide. Amplified fragments for 16S, COX1, and ATP6, corresponding to the correct size, were cloned following standard procedures (36) into pCRII (Invitrogen) and then transfected into One-Shot cells (Invitrogen). DNA sequencing by MOBIX Central Core Facility (McMaster University) of positive clones by using the T7 promoter site on pCRII, confirmed the sequence of 16S (pCM45), COX1 (pCM44), and ATP6 (pCM29).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Primer sequence designed for amplifying rainbow trout mitochondrial DNA gene products and rat ANT1

The sequences for the nuclear-encoded adenine nucleotide translocase 1 (ANT1) and muscle-type pyruvate kinase (PK) are not known in trout, so probes were made via PCR with primers based on published rat sequence by using rat gastrocnemius cDNA as a template. First-strand cDNA was generated from total rat RNA by reverse transcription (RT) by using the following protocol: a mixture of 1× avian myeloblastosis virus reverse transcriptase (AMV-RT) buffer (Promega), 40 µg total RNA, and 1 µg random hexamers (Promega). The mixture was heated for 5 min at 65°C and then cooled quickly in ice. The following was added to the above mixture to initiate RT: 1 mM dNTPs, 36 U RNA Guard (Pharmacia), and 10 U AMV-RT (Promega). The mixture was incubated at room temperature for 10 min, transferred to 37°C for 60 min, boiled for 5 min, and then cooled quickly in ice. Subsequent PCR reactions relied on this cDNA as a template. Primers (Table 1) used for ANT1 amplification were designed for a specific 390-bp region (nt 2,821-3,211) within the second exon (GenBank accession no. X61667). Templates were amplified by using the same PCR protocol described for mitochondrial probe construction except for a 57.5°C annealing temperature. A 1,406-bp region of PK was amplified by using an annealing temperature of 60°C. Amplified sequences were cloned into the pCRII vector as described above. Sequences of the clones for ANT1 (pCM26) and PK (pCM32) were confirmed DNA sequencing (MOBIX). Attempts to hybridize the resulting probe to trout RNA as well as rat gastrocnemius RNA revealed a fragment similar in size.

RNA analysis. Northern blot analyses were used to identify steady-state mitochondrial and nuclear transcription products after cold and warm acclimation (36). RNA samples were denatured by the DMSO-glyoxal method for 1 h at 50°C (3). Samples were then quickly cooled on ice and fractionated on a 1.4% agarose gel at 4 V/cm. After electrophoresis, RNA was transferred overnight by capillary transfer onto a nylon membrane (Duralon-UV membrane, Stratagene). The membrane was rinsed with 2× saline-sodium citrate (SSC) to remove any residual agarose and allowed to dry. RNA was fixed to the membrane by ultraviolet cross-linking (UV-Crosslinker, Fisher Scientific) according to the manufacturer's specifications.

All plasmids were digested with EcoR I to prepare probes for hybridization. Inserts were separated from the cut plasmid on 1% agarose gel, purified by Qiaex II gel extraction kit (Qiagen), and quantified by ethidium bromide staining. Approximately 50 ng of double-stranded insert was randomly labeled with 50 µCi of [alpha -32P]dCTP (New England Nuclear) by using Ready-to-Go-DNA Labeling Beads-dCTP (Pharmacia). Typically, probes had a specific activity of ~109 cpm/µg DNA.

Membranes were prehybridized in 25 mM K2HPO4 (pH 7.4), 5× SSC, 5× Denhardt's reagent (20 mg/ml Ficoll 400, 20 mg/ml polyvinylpyrrolidone, and 20 mg/ml BSA), 50 µg/ml denatured salmon sperm DNA, and 50% formamide for 3 h in a Hybaid minihybridization oven at 42°C. Prehybridization solution was then replaced with a hybridization solution [25 mM K2HPO4 (pH 7.4), 5× SSC, 5× Denhardt's, 50 µg/ml denatured salmon sperm DNA, 50% formamide, and 25 g/l dextran sulfate], and a radioactive probe that was left to hybridize overnight at 42°C. Membranes were washed twice with 1× SSC and 0.1% SDS for 15 min, then twice with 0.25× SSC and 0.1% SDS for 15 min. Membranes were then exposed to autoradiography film (New England Nuclear) at room temperature for 4-48 h (depending on probe). Membranes were probed sequentially (never stripped), in the order described in the respective figure legends. Densities of RNA bands were quantified on a Molecular Dynamic computer densitometer with Molecular Dynamics ImageQuant (version 3.3) software.

Quantitative competitive PCR. A quantitative competitive PCR method was established based on previous methods to measure skeletal muscle mitochondrial DNA content (33, 47). Briefly, a known concentration of competing template is amplified with the DNA of interest by using the same primer pairs, yielding fragments differing in size. The ratio of the target DNA to the competitor can give a reasonable estimate as to the amount of mitochondrial DNA present per total DNA. The competitor acts as an internal standard, eliminating potential tube-to-tube variation that can occur with PCR reactions.

A competitor template was made by removing an internal 260-bp Sac II fragment from pCM45, which contains a 603-bp fragment of trout 16S. Cut plasmid DNA was separated on a 1.0% agarose gel, purified with a Qiaex II gel extraction kit, and ligated overnight with T4 DNA ligase (Promega). Ligated plasmids were transfected into XL-1 Blue (Stratagene) following standard methods (36). Subsequent DNA sequencing of the Delta 16S plasmid (pCM46) confirmed the elimination of the 260-bp fragment from the pCM45. For purposes of the quantitative competitive (QC) PCR, new forward and reverse primers were designed (Table 1) to be specific for both wild-type (WT) trout 16S (pCM45) and Delta 16S (pCM46).

An optimal concentration of the competitor pCM46 was initially determined to avoid unequal competition with the target DNA. To establish this concentration, a constant amount of total trout DNA was amplified alongside different concentrations of competitor template. The corresponding equivalence point taken from the titration curve (data not shown) determined the competitor concentration used in all subsequent QC-PCR reactions.

Total DNA from each sample was amplified in triplicate with Taq polymerase (Pharmacia) in the following reaction mix: 1× supplied Pharmacia reaction buffer [10 mM Tris · HCl (pH 9.0), 1.5 mM MgCl2, and 50 mM KCl], 800 µM dNTP, and 220 ng primers. For the purpose of QC-PCR, 20 cycles of the following were used: initial denaturation at 95°C for 4 min, subsequent denaturation at 95°C for 45 s, annealing at 60°C for 90 s, and extension at 72°C for 60 s. PCR products were separated out electrophoretically on a 1.5% agarose gel and stained with 0.5 µg/ml of ethidium bromide to distinguish between the amplified 603-bp WT16S and 343-bp Delta 16S fragments. A Polaroid negative (type 665) was taken of the ethidium bromide-stained gel to quantify differences in amplification between the WT and Delta  fragments and subsequently analyzed with a Molecular Dynamic computer densitometer with Molecular Dynamics ImageQuant (version 3.3) software.

Determination of mitochondrial DNA copy number. For an individual sample, the amount of mitochondrial DNA determined by QC-PCR (ng) was divided by the total DNA (µg) recovered per gram of tissue from the sample. This value was then divided by Avagadro's number. The resulting value is then divided by the molecular weight of trout mitochondrial DNA (11.27 × 106) to obtain the number of mitochondrial DNA copies per gram tissue.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enzymes. Maximal enzyme activities were measured to determine whether mitochondrial content changed with cold acclimation. Temperature acclimation had similar effects on COX and CS activities; their activities in cold-acclimated fish were 72-74% higher in white muscle and 37-40% higher in red muscle (Table 2). In both tissues, complex I activities were less affected by thermal acclimation. Activities of cristae enzymes (COX1) relative to the matrix enzyme CS suggests enzymatic relationships are highly conserved across tissue types and acclimation states (Fig. 1A). In contrast, the ratio of enzyme activity to mitochondrial DNA is less conserved (Fig. 1B).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Enzyme activities of warm- and cold- acclimated rainbow trout in both red and white muscle


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1.   Relationship of mitochondrial enzymes in trout red and white muscle (RM and WM, respectively) with temperature acclimation. A: enzymes expressed relative to a unit (U; amount of enzyme required to convert 1 µmol of product to substrate in 1 min) of citrate synthase (CS; mean ± SE, n = 7 for 4°C, n = 8 for 18°C). B: mean relationship per gram of tissue of mitochondrial enzymes expressed relative to amount of mitochondrial DNA (mtDNA). COX, cytochrome-c oxidase.

Changes in RNA. Mitochondrial RNA transcripts were chosen to reflect not only different complexes in the electron transport chain (ATP6 and COX1) but also different transcript types. Transcription of mitochondrial DNA can continue until the entire strand is completed or can be terminated after 16S rRNA. In red and white muscle, no significant differences in the abundance of any mitochondrial RNA species were detected with thermal acclimation (Fig. 2).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2.   Mean (±SE) relative steady-state levels of RNA from trout red (A; cold and warm, both n = 5) and white (B; cold, n = 4; warm, n = 5) muscle after a 2-mo acclimation period at either 4 or 18°C. A single blot with all 19 samples (10 µg/lane) was used to probe for subunit 6 of F0F1-ATPase(ATP6), followed by subunit 1 of COX (COX1). A 2nd blot, also with all 19 samples (20 µg/lane), was used to probe for adenine nucleotide translocase 1 (ANT1) followed by mitochondrial 16S rRNA. Below each panel is same membrane probed for 18S mRNA. Data are presented relative to signal from samples warm fish (mean set at 1 for each signal). mRNA levels of different probes (e.g., ATP6 vs. COX1) are not directly comparable.

Because gene expression may differ between nuclear and mitochondrial genomes, the coordinate expression of these genomes was explored by monitoring ANT1, a nuclear mRNA transcript. Although there was an apparent trend toward higher ANT1 mRNA levels in cold-acclimated fish, these differences were not statistically significant (Fig. 2). This is due in part to the high variability in the steady-state levels seen during cold acclimation (Fig. 3A). The levels of ANT1 mRNA were comparable at each acclimation temperature in both tissues. Also, the length of the ANT1 transcript differed by ~200 bp between tissues (Fig. 3B).


View larger version (62K):
[in this window]
[in a new window]
  		Fig. 3.   Northern blots. A: variability in expression of steady-state ANT1 and pyruvate kinase (PK) in white muscle from cold (lanes 1-5)- and warm (lanes 6-9)-acclimated rainbow trout with 20 µg of total RNA loaded per lane. Once preliminary studies confirmed that probes hybridized to a single band, this membrane was hybridized with both probes simultaneously. Blot was subsequently probed for 18S. B: difference in molecular size between ANT1 mRNA transcripts in red and white muscle from 10 µg of total RNA loaded per lane. Molecular size was determined relative to lambda -DNA EcoR I/Hind III markers (not illustrated).

Changes in mitochondrial DNA. The QC-PCR analysis of mitochondrial DNA copy number indicates that there is no significant difference in the amount of mitochondrial DNA per total DNA in either tissue muscle after acclimation (Fig. 4A). Despite the 10-fold difference in enzyme content between red and white muscle, no significant difference was present in the amount of mitochondrial DNA expressed per total cellular DNA across tissues. However, expressing the number of mitochondrial DNA copies per gram of tissue (Fig. 4B) revealed an approximately fourfold difference between red and white muscle, regardless of the acclimation temperature.


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of temperature acclimation on mean (±SE) amount of mitochondrial DNA (mtDNA) in red and white muscle (n = 5) expressed per total DNA (A) and expressed per gram of tissue (B). * Significant difference (P < 0.05, ANOVA) between red and white muscle at respective temperatures.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of temperature on mitochondrial content. Increases in mitochondrial enzyme activity and volume density are known to occur in response to cold acclimation of fish (9, 13). In our study, the magnitude of difference between warm- and cold-acclimated rainbow trout was in the same range as that observed to occur with exercise training in the same species (11). The nature of the change in mitochondrial content with physiological stresses has many parallels with the differences observed across species. In comparing species with widely different metabolic rates, Taylor, Weibel, Hoppeler, and their colleagues report that muscles of different aerobic capacities differ primarily in mitochondrial volume density; differences in cristae content per unit mitochondrial volume are much less variable, generally falling within the range 20-40 m2/ml (see Ref. 40). This is not to suggest that intertissue and interspecies differences in cristae content do not occur but, rather, they are less frequently observed and of lesser magnitude. Among homeotherms, hummingbirds (41) and pronghorn antelope (25) possess higher than average cristae packing densities. Fish in general (20) and skipjack tuna in particular (31) demonstrate cristae densities higher than most mammals. The nearly constant relationship between CS and COX across tissues and acclimation states (Fig. 1) suggests the relationship between cristae and matrix enzymes is conserved under these conditions.

Discussion of the effects of physiological stimuli on mitochondrial content must also consider the influence of muscle fiber type. Mammalian muscle fibers, which have a mitochondrial volume density in the range of fish white muscle, are capable of undergoing severalfold changes in mitochondrial content (16). In contrast, endurance exercise training has little effect on cardiac muscle mitochondrial content (see Ref. 28). Although there are obvious differences in how endurance training would affect energetics in cardiac and skeletal muscle, the impact of the differences in mitochondrial content between fiber types must be considered. The difference in mitochondrial content between mammalian cardiac and skeletal muscles is of the same range as that between fish red and white muscle. On an absolute basis, red muscle increased CS activity by 4.1 U/g, whereas the change in white muscle was only 0.86 U/g. On a relative basis, it can be argued that the effects of acclimation were greater in white muscle (CS increased 72 vs. 40%). A high mitochondrial content in red muscle might afford protection against the energetic stress of cold acclimation, obviating major increases in mitochondrial content. Alternatively, spatial considerations may limit the increase in mitochondrial content with cold acclimation (see Ref. 40).

Effect of temperature acclimation on mitochondrial gene expression. In contrast to the changes observed with enzymes, there were no significant differences in steady-state mitochondrial RNA levels in either tissue with temperature acclimation. In studies with mammalian muscle, mitochondrial enzymes and mitochondrial RNA levels typically change in parallel (17, 45). The failure to observe this relationship in this study has several possible origins. The precision with which mitochondrial enzymes can be measured is greater than that for mRNA due to technical limitations in Northern blot densitometric quantitation. The lack of differences in mitochondrial mRNA and rRNA (Fig. 2), however, are consistent with the lack of differences in mitochondrial DNA (Fig. 4). Estimation of the effects of acclimation were also complicated by a relatively high degree of interindividual variablity in cold-acclimated animals, particularly with white muscle. This is illustrated in a blot of white muscle RNA probed for ANT1 and PK. Some cold-acclimated individuals demonstrated reciprocal relationships between ANT1 and PK mRNA (Fig. 3A). This is reminiscent of the reciprocal relationship between glycolytic and mitochondrial mRNA species observed during adaptation to hypoxia (43).

ANT is a protein found in the inner mitochondrial membrane that exchanges cytosolic ADP for ATP produced in the matrix (22). As such, this protein plays a key role in regulating oxidative phosphorylation under various physiological conditions (23). Temperature can have a dramatic effect on the kinetic control of ANT, consequently limiting ATP synthesis independent of ADP availability (35). Moreover, colder temperatures decrease the sensitivity of trout red muscle mitochondria to ADP, ultimately affecting ATP synthesis (5). A trend toward higher ANT1 mRNA levels was apparent in both red and white muscle of cold-acclimated fish (Fig. 2), although this trend was not statistically different.

ANT1 mRNA transcript length differs by ~200 bp between fish red and white muscle (Fig. 3B). It is unlikely that this is due to isoform switching, since ANT1 is the only isoform expressed in adult muscle fibers across a variety of taxa (38). There is evidence that both ANT1 and ANT2 can be expressed as two distinct length transcripts arising from different polyadenylation sites (12, 26). Such a mechanism may account for the difference in transcript length noted between red and white muscle.

Genetic control of mitochondrial content. Typically, an increase in steady-state mitochonrial RNA levels results in a parallel change in mitochondrial DNA copy number. The greatest changes in mitochondrial DNA copy number demonstrated to date, in excess of 4-fold, occur with chronic stimulation (44, 45), whereas more modest changes of 1.5- to 2-fold are exhibited during exercise training (32, 34). However, in the liver of cold-acclimated rats, a twofold increase in mitochonrial RNA can be achieved without a change in mitochondrial DNA (27). In this study, the ratio of mitochondrial to nuclear DNA did not change after thermal acclimation in either fiber type nor were there differences between red and white muscle. However, expressing mitochondrial DNA content as per gram of tissue depicts a different picture among tissues (Fig. 4B). An ~4-fold difference in mitochondrial DNA copy number was noted between tissues in contrast to a 10-fold difference in mitochondrial enzymes per gram of tissue between red and white muscle. There is no mechanistic reason why mitochondrial DNA copy number need be constant in relation to mitochondrial ultrastructural or enzymatic parameters. Muscle mitochondria exists as a reticulum, and any expression of mitochondrial parameters reflects global averages, not amounts "per mitochondrion" (see Ref. 29 for further discussion). Because transcription does not limit mitochondrial protein synthesis (2) and the mitochondrial genome is present in excess (4), mitochondrial DNA does not appear to be the main determinant of mitochondrial content. Research on human mitochondrial myopathies suggests that only a fraction of the WT mitochondrial genome (~15%) is required to express the normal aerobic phenotype of a particular muscle (6). During muscle development, mitochondrial content largely reflects the contractile properties of a particular fiber, as determined by the nucleus and innervation pattern (39). The relationship between gene dosage and transcript levels is tissue dependent. Red and white muscle possess similar ratios of mitochondrial DNA and nuclear DNA (Fig. 4A), however, the ratios of mitochondrial (e.g., 16S) to nuclear transcripts (e.g., ANT1) differs fourfold (Fig. 5, A vs. B).


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5.   Relationship of steady-state RNA (per 10 µg total RNA) in proportion to DNA abundance (per g tissue) from cold-acclimated fish tissues. A: steady-state mitochondrial 16S RNA is plotted against mtDNA copy number. B: ANT1 mRNA is plotted against total DNA content. Values are expressed as means ± SE.

The findings of the present study indicate that the relationship among mitochondrial enzymes is similar in both red and white muscle in response to temperature acclimation. Significant changes in steady-state ANT1 mRNA and mitochonrial RNA levels do not appear to be required to maintain a higher mitochondrial content after thermal acclimation. In trout, the magnitude of change in mitochondrial enzymes due to thermal acclimation may be too small to result in detectable differences in mRNA, or a high degree of variability in the response of fish to transitions in temperature may be an inherent physiological property. Mitochondrial DNA content is not tightly coupled to compensatory changes in aerobic capacity between tissues. Instead, interactions among nuclear-encoded genes appear to be primarily responsible for the distinct mitochondrial contents of fish red and white muscle.

    ACKNOWLEDGEMENTS

We thank the following people for their assistance in these studies: Dr. B. Tufts for use of fish-holding facilities, Dr. V. Friesen for access to a thermal cycler, Dr. S. Lougheed for the universal PCR primers, Dr. Eric Shoubridge for the 18S probe, and S. Leary for critical review of the manuscript.

    FOOTNOTES

This study was supported by an operating grant to C. D. Myers from National Sciences and Engineering Research Council Canada.

Present address of B. J. Battersby: Montreal Neurological Institute, 3801 University St., McGill University, Montreal, QB, Canada H3A 2B4.

Address for reprint requests: C. D. Moyes, Dept. of Biology, Queen's University, Kingston, ON, Canada K7L 3N6.

Received 28 October 1997; accepted in final form 13 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Attardi, G., A. Chomyn, M. P. King, B. Kruse, P. L. Polosa, and N. N. Murdter. Biogenesis and assembly of the mitochondrial respiratory chain: structural, genetic and pathological aspects. Biochem. Soc. Trans. 18: 509-513, 1990[Medline].

2.   Attardi, G., and G. Schatz. Biogenesis of mitochondria. Annu. Rev. Cell Biol. 4: 289-333, 1988.

3.   Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. Short Protocols in Molecular Biology. New York: Wiley, 1992.

4.   Bendich, A. J. Why do chloroplasts and mitochondria contain so many copies of their genome? Bioessays 6: 279-282, 1987[Medline].

5.   Blier, P. U., and H. E. Guderley. Mitochondrial activity in rainbow trout red muscle: the effect of temperature on the ADP-dependence of ATP synthesis. J. Exp. Biol. 176: 145-157, 1993[Abstract].

6.   Boulet, L., G. Karpati, and E. A. Shoubridge. Distribution and threshold expression of the tRNALys mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF). Am. J. Hum. Genet. 51: 1187-1200, 1992[Medline].

7.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

8.   Constable, S. H., R. J. Favier, J. A. McLane, R. D. Fell, M. Chen, and J. O. Holloszy. Energy metabolism in contracting rat skeletal muscle: adaptation to exercise training. Am. J. Physiol. 253 (Cell Physiol. 22): C316-C322, 1987[Abstract/Free Full Text].

9.   Eggington, S., and B. D. Sidell. Thermal acclimation induces adaptive changes in subcellular structure of fish skeletal muscle. Am. J. Physiol. 256 (Regulatory Integrative Comp. Physiol. 25): R1-R9, 1989[Abstract/Free Full Text].

10.   Evans, M. J., and R. C. Scarpulla. NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells. Genes Dev. 4: 1023-1034, 1990[Abstract/Free Full Text].

11.   Farrell, A. P., J. A. Johansen, and R. K. Suarez. Effects of exercise-training on cardiac performance and muscle enzymes in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 9: 303-312, 1991.

12.   Graham, B. H., K. G. Waymire, B. Cottrell, I. A. Trounce, G. R. MacGregor, and D. C. Wallace. A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator. Nat. Genet. 16: 226-234, 1997[Medline].

13.   Guderley, H. Functional significance of metabolic responses to thermal acclimation in fish muscle. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R245-R252, 1990[Abstract/Free Full Text].

14.   Haraguchi, Y., A. B. Chung, S. Neill, and D. C. Wallace. OXBOX and REBOX, overlying promoter elements of the mitochondrial F0F1-ATP beta  subunit gene. J. Biol. Chem. 269: 9330-9334, 1994[Abstract/Free Full Text].

15.   Holloszy, J. O., and E. F. Coyle. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J. Appl. Physiol. 56: 831-838, 1984[Abstract/Free Full Text].

16.   Hood, D. A., A. Balaban, M. K. Connor, E. E. Craig, M. L. Nishio, M. Rezvani, and M. Takahashi. Mitochondrial biogenesis in striated muscle. Can. J. Appl. Physiol. 19: 12-48, 1994[Medline].

17.   Hood, D. A., R. Zak, and D. Pette. Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome-c oxidase subunits. Eur. J. Biochem. 179: 275-280, 1989[Medline].

18.   Howald, H., H. Hoppeler, H. Claassen, O. Mathieu, and R. Straub. Influences of endurance training on the ultrastructural composition of the different muscle fiber types in humans. Pflügers Arch. 403: 369-376, 1985[Medline].

19.   Johnston, I. A. Structure and function of fish muscles. Symp. Zool. Soc. Lond. 48: 71-113, 1991.

20.   Johnston, I. A., J. Calvo, H. Guderley, D. Fernandez, and L. Palmer. Latitudinal variation in the abundance and oxidative capacities of muscle mitochondria in perciform fishes. J. Exp. Biol. 201: 1-12, 1997[Abstract/Free Full Text].

21.   Johnston, I., A, and T. W. Moon. Fine structure and metabolism of multiply innervated fast muscle fibers in teleost fish. Cell Tissue Res. 219: 93-109, 1981[Medline].

22.   Klingenberg, M. Structure-function of the ADP/ATP carrier. Biochem. Soc. Trans. 20: 547-550, 1992[Medline].

23.   Korzeniewski, B., and J. P. Mazat. Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies. Biochem. J. 319: 143-148, 1996.

24.   Li, K., J. A. Hodge, and D. C. Wallace. OXBOX, a positive transcriptional element of the heart-skeletal muscle ADP/ATP translocator gene. J. Biol. Chem. 265: 20585-20588, 1990[Abstract/Free Full Text].

25.   Lindstedt, S. L., J. F. Hokanson, D. J. Wells, S. D. Swain, H. Hoppeler, and V. Navarro. Running energetics in the pronghorn antelope. Nature 353: 748-750, 1991[Medline].

26.   Lundardi, J., and G. Attardi. Differential regulation of expression of the multiple ADP/ATP translocase genes in human cells. J. Biol. Chem. 266: 16534-16540, 1991[Abstract/Free Full Text].

27.   Martin, I., O. Vinas, T. Mampel, R. Iglesias, and F. Villarroya. Effects of cold environment on mitochondrial genome expression in the rat: evidence for a tissue-specific increase in the liver, independent of changes in mitochondrial gene abundance. Biochem. J. 296: 231-234, 1993.

28.   Moyes, C. D. Cardiac metabolism in high performance fish. Comp. Biochem. Physiol. A Physiol. 113: 69-75, 1996.

29.   Moyes, C. D., B. J. Battersby, and S. C. Leary. Regulation of mitochondrial design. J. Exp. Biol. 201: 299-307, 1998.

30.   Moyes, C. D., O. A. Mathieu-Costello, R. W. Brill, and P. W. Hochachka. Mitochondrial metabolism of cardiac and skeletal muscles from a fast (Kastuwonus pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 70: 1246-1253, 1992.

31.   Moyes, C. D., O. A. Mathieu-Costello, N. Tsuchiya, C. Filburn, and R. G. Hansford. Mitochondrial biogenesis during cellular differentiation. Am. J. Physiol. 272 (Cell Physiol. 41): C1345-C1351, 1997[Abstract/Free Full Text].

32.   Murakami, T., Y. Shimomura, N. Fujitsuka, N. Nakai, S. Sugiyama, T. Ozawa, M. Sokabe, S. Horai, K. Tokuyama, and M. Suzuki. Enzymatic and genetic adaptation of soleus muscle mitochondria to physical training in rats. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E388-E395, 1994[Abstract/Free Full Text].

33.   Piatak, M., K. C. Luk, B. Williams, and J. D. Lifson. Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. Biotechniques 14: 70-81, 1993[Medline].

34.   Puntschart, A., H. Claassen, K. Jstarndt, H. Hoppeler, and R. Billeter. mRNAs of enzymes involved in energy metabolism and mtDNA are increased in endurance-trained athletes. Am. J. Physiol. 269 (Cell Physiol. 38): C619-C625, 1995[Abstract/Free Full Text].

35.   Quentin, E., N. Averet, B. Guerin, and M. Rigoulet. Temperature dependence of the coupling efficiency of rat liver oxidative phosphorylation: role of adenine nucleotide translocator. Biochem. Biophys. Res. Commun. 202: 816-821, 1994[Medline].

36.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1989.

37.   Scarpulla, R. C. Nuclear respiratory factors and the pathways of nuclear-mitochondrial interaction. Trends Cardiovasc. Med. 6: 39-45, 1996.

38.   Stepien, G., A. Torroni, A. B. Chung, J. A. Hodge, and D. C. Wallace. Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation. J. Biol. Chem. 267: 14592-14597, 1992[Abstract/Free Full Text].

39.   Stockdale, F. E. Myogenic cell lineages. Dev. Biol. 154: 284-298, 1992[Medline].

40.   Suarez, R. K. Upper limits to mass-specific metabolic rates. Annu. Rev. Physiol. 58: 583-605, 1996[Medline].

41.   Suarez, R. K., J. R. B. Lighton, G. S. Brown, and O. A. Mathieu-Costello. Mitochondrial respiration in hummingbird flight muscles. Proc. Natl. Acad. Sci. USA 88: 4870-4873, 1991[Abstract/Free Full Text].

42.   Suzuki, H., Y. Hosokawa, M. Nishikimi, and T. Ozawa. Existence of common homologous elements in the transcriptional regulatory regions of human nuclear genes and mitochondrial gene for the oxidative phosphorylation system. J. Biol. Chem. 266: 2333-2338, 1991[Abstract/Free Full Text].

43.   Webster, K. A., P. Gunning, E. Hardeman, D. C. Wallace, and L. Kedes. Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts. J. Cell. Physiol. 142: 566-573, 1990[Medline].

44.   Williams, R. S. Mitochondrial gene expression in mammalian striated muscle; evidence that variation in gene dosage is the major regulatory event. J. Biol. Chem. 261: 12390-12394, 1986[Abstract/Free Full Text].

45.   Williams, R. S., S. Salmons, E. A. Newsholme, R. E. Kaufman, and J. Mellor. Regulation of nuclear and mitochondrial gene expression by contractile activity in skeletal muscle. J. Biol. Chem. 261: 376-380, 1986[Abstract/Free Full Text].

46.   Zardoya, R., A. Garrido-Pertierra, and J. M. Bautista. The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss. J. Mol. Evol. 41: 942-951, 1995[Medline].

47.   Zimmermann, K., and J. W. Mannhalter. Technical aspects of quantitative competitive PCR. Biotechniques 21: 268-279, 1996[Medline].

Am J Physiol Regul Integr Compar Physiol 275(3):R905-R912
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Physiol.Home page
G. B. McClelland, P. M. Craig, K. Dhekney, and S. Dipardo
Temperature- and exercise-induced gene expression and metabolic enzyme changes in skeletal muscle of adult zebrafish (Danio rerio)
J. Physiol., December 1, 2006; 577(2): 739 - 751.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
E. Rissanen, H. K. Tranberg, and M. Nikinmaa
Oxygen availability regulates metabolism and gene expression in trout hepatocyte cultures
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2006; 291(5): R1507 - R1515.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
M. Lucassen, N. Koschnick, L. G. Eckerle, and H.-O. Portner
Mitochondrial mechanisms of cold adaptation in cod (Gadus morhua L.) populations from different climatic zones
J. Exp. Biol., July 1, 2006; 209(13): 2462 - 2471.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. C. Dalziel, S. E. Moore, and C. D. Moyes
Mitochondrial enzyme content in the muscles of high-performance fish: evolution and variation among fiber types
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R163 - R172.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Lucassen, A. Schmidt, L. G. Eckerle, and H.-O. Portner
Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2003; 285(6): R1410 - R1420.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. C. Leary, C. N. Lyons, A. G. Rosenberger, J. S. Ballantyne, J. Stillman, and C. D. Moyes
Fiber-type differences in muscle mitochondrial profiles
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R817 - R826.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
S. Itoi, S. Kinoshita, K. Kikuchi, and S. Watabe
Changes of carp FoF1-ATPase in association with temperature acclimation
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2003; 284(1): R153 - R163.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
H. Guderley and J. St-Pierre
Going with the flow or life in the fast lane: contrasting mitochondrial responses to thermal change
J. Exp. Biol., August 1, 2002; 205(15): 2237 - 2249.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
G. P. Burness, S. C. Leary, P. W. Hochachka, and C. D. Moyes
Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study
Am J Physiol Regulatory Integrative Comp Physiol, October 1, 1999; 277(4): R1164 - R1170.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
I. Hardewig, P. L. M. van Dijk, C. D. Moyes, and H. O. Portner
Temperature-dependent expression of cytochrome-c oxidase in Antarctic and temperate fish
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 1999; 277(2): R508 - R516.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 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 Google Scholar
Google Scholar
Right arrow Articles by Battersby, B. J.
Right arrow Articles by Moyes, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Battersby, B. J.
Right arrow Articles by Moyes, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online