SCALING OF MITOCHONDRIAL RESPIRATION IN WHITE MUSCLE FROM AN ACTIVE (Pomatomus saltatrix) AND INACTIVE (Centropristis striata)SCALING OF MITOCHONDRIAL RESPIRATION IN WHITE MUSCLE FROM AN ACTIVE (Pomatomus saltatrix) AND INACTIVE (Centropristis striata) FISH
Jessica L. Burpee
A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of Master of Science
Department of Biology and Marine Biology
University of North Carolina Wilmington 2009
Approved by:
Advisory Committee
Dr. Stephen Kinsey
Statistical Analysis...................................................................................................9
RESULTS ..........................................................................................................................10
TABLES ............................................................................................................................23
FIGURE LEGENDS..........................................................................................................25
FIGURES...........................................................................................................................27
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ABSTRACT
White muscle (WM) in many fishes grows hypertrophically, and fiber size often
increases during development from <50 μm in juveniles to >250 μm in adults. This leads
to increases in intracellular diffusion distances that may impact the scaling of metabolic
properties. We have previously analyzed the scaling with body mass of aerobic capacity
(mitochondrial volume density; b=-0.06), and the maximal rate of an aerobic process
(post-contractile phosphocreatine recovery rate; b=-0.07) in isolated fish WM. In the
present study, we examined the scaling with body mass of maximal oxygen consumption
rates of isolated mitochondria (VO2 mt) in an active, pelagic species (bluefish, Pomatomus
saltatrix) and a sedentary, benthic species (black sea bass, Centropristis striata). In
contrast to prior studies that evaluated aerobic flux in isolated tissues or whole animals,
the measurement of respiration in isolated mitochondria is not limited by the diffusive
flux of oxygen or metabolites. The mitochondrial protein content was measured, and
transmission electron microscopy was used to measure mitochondrial fractional volume
of the samples used for O2 consumption measurements and the total mitochondrial
fractional volume (TMFV) of the WM from each species. This allowed respiration to be
standardized to milligrams of protein, milliliters of mitochondria, and grams of muscle,
and in each case VO2 mt scaled independently of body mass. TMFV of the WM scaled
negatively in C. striata, but not in P. saltatrix, and was significantly higher in the active
species. The size-independence of VO2 mt conflicts with the negative allometry of aerobic
capacity and flux found in prior studies on both fishes and mammals. These data suggest
that differences in WM aerobic capacity across species are due simply to the volume
density of mitochondria and not to changes in mitochondrial function. Further, diffusion
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constraints within the muscle likely influence mitochondrial density, distribution, and
aerobic flux in intact cells, but the negative scaling typical of these processes is not
observed when the effects of diffusion are experimentally removed.
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ACKNOWLEDGEMENTS
I would like to thank my family for all of the love and support they have
surrounded me with. Thank you Mom, for your endless words of encouragement. Dad,
thank you for challenging me to never stop learning. And Ailis, thank you for being a
role model and a friend that I know I will have for life.
I would like to thank my amazing friends, who have made this journey
unforgettable. Kristin Hardy, you have been so much more than a friend to me and I am
so grateful that you are in my life. Bruce Dunbar, thank you for your relentless
enthusiasm and always being no more than a phone call away. Hillary Lane, your fervor
for life has inspired me to never give up.
I am very grateful to the faculty members who have taught me more than I could
have ever imagined. My advisor, Dr. Stephen Kinsey, thank you for all of the character
that you encouraged me to build. Dr. Richard Dillaman, thank you for sharing my
passion for mitochondria. Dr. Heather Koopman, thank you for showing me that I should
never be afraid to ask questions. Mark Gay, thank you for your remarkable patience and
knowledge.
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1. Respiration of isolated white muscle mitochondria...............................................23
2. Maximal oxygen consumption rates of white muscle mitochondria per milligram
of protein, milliliter of mitochondria, and gram of muscle....................................24
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Figure
1. Micrographs from small (upper) and large (lower) P. saltatrix (A, B) and C.
striata (C, D) taken at a magnification of × 1,800.................................................27
2. Total mitochondrial fractional volume (TMFV) from small and large P. saltatrix
and C. striata..........................................................................................................28
3. Oxygen consumption recordings from P. saltatrix (A) and C. striata (B)
mitochondrial isolates demonstrating state 3 and state 4 rates. (C) Example of
continuous state 3 rate caused by ATPase contamination where adenylate kinase
(ADK) was added to produce a state 4 rate ...........................................................29
4. TEM micrographs of mitochondria isolated from (A) P. saltatrix and (B) C.
striata taken at a magnification of × 5,600 ............................................................30
5. Scaling with body mass of VO2 mt ...........................................................................31
INTRODUCTION
During steady state contraction, muscle mitochondria provide sufficient ATP to meet
energetic demands (Taylor, 1987), and skeletal muscle mitochondria utilize >90% of the
total oxygen consumed by an organism exercising at the maximal rate of oxygen
consumption (V02max) (Taylor, 1987; Weibel, 2002). The correlation between V02max and
skeletal muscle mitochondrial function has been the focus of several comparative studies
on species differing in both body size (i.e., allometric variation) (Hoppeler and Weibel,
2000), behavior (i.e., sedentary versus active) (Somero and Childress, 1980; Weibel,
1987; Hoppeler, 1990; Weibel et al., 1991), and endurance (i.e., influence of exercise
training) (Holloszy and Booth, 1976). Such comparisons suggest that differences in
muscle V02max are primarily due to qualitative and quantitative variations in the
mitochondrial capacity for ATP production (Hoppeler et al., 1987; Schwerzmann et al.,
1989; Hochachka, 1994).
Muscle aerobic capacity is largely dependent on changes in mitochondrial
fractional volume (Vmt). Hoppeler and Weibel (2000) expressed VO2 max as the product of
Vmt and the rate of mitochondrial oxygen consumption (VO2 mt):
VO2 max = Vmt VO2 mt
In most species, VO2 mt rates based on mitochondrial volume range from 3-5 ml O2 min-1
ml-1 of mitochondria, irrespective of body mass (Hoppeler and Lindstedt, 1985; Taylor,
1987; Taylor et al., 1989). The narrow range of respiration rates is due to the highly
conserved metabolic stoichiometries of mitochondria defined by the ATP: O2 ratio (Leary
et al., 2003). Consistent with the principle of allometric scaling of metabolism with body
mass (Mb), whole animal VO2 max /M and Vmt /M in mammals scale in parallel, and VO2 mt
does not change with body size (Moyes et al., 1998; Hoppeler and Weibel, 2000; Moyes,
2003). However, mitochondrial content can vary up to fivefold inter- and
intraspecifically with no significant variation in body mass (Mathieu et al., 1981). Within
an individual, different fiber types can vary greatly in Vmt from <0.01 in fast-twitch,
glycolytic fibers to >0.45 in slow-twitch, oxidative fibers (Hochachka and Somero, 1973;
Hochachka, 1994; Suarez, 1996; Moyes and Hood, 2003). Thus, adaptive variations in
mitochondrial content have a large influence on whole animal VO2 max.
Although many studies have investigated the effects of qualitative and
quantitative variations in mitochondria over a wide range of VO2 max values, the parameters
defining aerobic metabolism have largely focused on mammals (Taylor and Weibel,
1981; Taylor, 1987). Unlike mammalian muscle, fish muscle is often separated into
distinct regions of red muscle (RM), which is specialized for sustained, aerobic activity
and white muscle (WM that is employed exclusively during burst contractions (Bone,
1966; Rayner and Keenan, 1967; Jane and Lauder, 1994). White fibers, which generally
comprise the majority (70-90%) of body mass in fishes typically have a Vmt < 0.02
(Johnston, 1981) and fuel contraction almost exclusively by anaerobic metabolism
(Moyes et al., 1992). Although fish WM fibers are dependent on endogenous fuels to
support burst contraction (Curtain et al., 1997; Dobson and Hochachka, 1987; Hochachka
and Mossey 1998; Schulte et al., 1992), recovery from contraction is aerobic. Anaerobic
burst exercise utilizes the creatine kinase (CK) reaction to buffer initial ATP demand by
catalyzing the reversible transfer of a phosphoryl group from phosphocreatine (PCr) to
ADP forming ATP:
2
During recovery from contraction in fish muscle, ATP and PCr resynthesis occurs
aerobically by mitochondrial metabolism (Curtain et al., 1997). Thus, the recovery rate
of high-energy phosphates is largely dependent on the Vmt of the muscle.
Although it may be assumed that aerobic processes of the mitochondria and whole
animal VO2 max scale in parallel, ontogenetic changes in cellular structure and organization
could cause these indices of aerobic capacity to scale independently. During growth, fish
muscle mass increases by both hyperplasia, an increase in fiber number, and hypertrophy,
an increase in fiber size (Weatherly and Gill, 1985; Gill et al., 1989; Mommsen, 2001).
Since many fishes display a several thousand-fold increase in body mass during post-
metamorphic growth and hypertrophy is often prevalent, WM fibers can reach diameters
>500µm (Battram and Johnston, 1991; Egginton et al., 2002, Johnston et al., 2003; Nyack
et al., 2007). Such extreme increases in fiber diameter may potentially affect metabolic
scaling properties due to increasing oxygen and metabolite diffusion distances within the
fiber (Kinsey and Moerland, 2002; Boyle et al., 2003; Johnson et al., 2004; Hardy et al.,
2006). Nyack et al. (2007) found that indices of aerobic capacity in black sea bass
(Centropristis striata), including mitochondrial fractional volume, cytochrome oxidase
activity, and the rate of aerobic recovery after burst contraction, scale negatively with
increasing body mass. These experiments were conducted using WM fiber bundles in a
high O2 medium. Therefore, the influence of O2 diffusion on post-contractile aerobic
recovery was negligible. However, the diffusion of metabolites such as ADP and ATP
could influence recovery rates in these experiments (Nyack et al., 2007).
The present study examined the allometric scaling of aerobic function in WM
from a benthic species, black sea bass (C. striata), and a pelagic species, bluefish
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(Pomatomus saltrix). The use of isolated mitochondrial fractions from WM allowed
investigation of muscle aerobic capacity where all influences of diffusion have been
removed. The objective of the present study was to determine the effect of allometric
scaling and behavioral differences on mitochondrial function by (1) measuring VO2 mt
(state 3) of mitochondria isolated from WM of fish over a large range in body mass, and
(2) examining differences in VO2 mt in WM from two teleost species that differ in activity
levels. We hypothesized that VO2 mt will be independent of body mass and activity level.
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All animal maintenance and experimental procedures were approved by the
University of North Carolina Wilmington (UNCW) Institutional Animal Care and Use
Committee. Black sea bass (Centropristis striata) were obtained from the UNCW
Aquaculture Facility (Wrightsville Beach, NC) and transported in aerated coolers to
aquaria located at the UNCW campus. Bluefish (Pomatomus saltrix) were collected
using seine nets and hook and line along the coast of Wrightsville Beach and Carolina
Beach, NC. All animals were maintained in aquaria containing circulating, filtered
seawater (35 ppt) at 25ºC on a 10 h: 14 h photoperiod. Fish were fed shrimp 3 times
weekly and fasted 24 h prior to experimental use. Fish were sequestered in opaque
containers with aerated seawater and euthanized using concentrated (> 250 mgl-1)
FINQUEL MS-222 (tricaine methanesulfonate, Argent Laboratories, Redmond, WA).
Euthanasia was verified by loss of equilibrium and absence of opercular movement.
Body mass and total length (TL) were recorded for each animal. Scales and skin were
immediately removed from the epaxial region of the fish extending from the dorsal fin to
the caudal peduncle.
Muscle Mitochondrial Fractional Volume
Vmt of P. saltatrix white muscle (WM) fibers was calculated from micrographs
using transmission electron microscopy (TEM) (C. striata Vmt was measured previously
by Nyack et al., 2007). Small, rectangular sections of white muscle were excised parallel
to the fiber orientation just below the dorsal fin. Individual muscle fibers were teased
apart and fixed at room temperature for 1 h in 2.5% (wt/vol) glutaraldehyde in 0.2M
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sodium cacodylate buffer (pH 7.4), then refrigerated for a minimum of 24 h. The fibers
were placed in a secondary fixative of 1% osmium tetraoxide for 2 h, dehydrated with an
ascending series (50%, 70%, 95%, 100%, 100%) of acetone, and embedded in Spurr
epoxy resin (Spurr, 1969; Electron Microscopy Sciences; Hatfield, PA, USA).
Embedded fibers were cut into 90 nm sections on a Reichert Ultracut E. Series
microtome (Leica, Inc., Vienna, Austria) using a systematic random sampling method to
ensure full representation of mitochondria throughout the muscle (Howard and Reed,
1998). Sections were mounted on Formvar-coated (0.25% Formvar in ethylene
dichloride), high transmission copper grids, and stained with 2% uranyl acetate in 50%
ethyl alcohol and Reynolds’ lead citrate (Reynolds, 1963). Sections were viewed with a
Philips CM-12 TEM (Hillsboro, OR), and micrographs were taken at a magnification of
×1,800, ×3,000, and ×5,600. Two micrographs per grid were randomly taken from five
grids for a total of 10 micrographs per fiber sample. Micrographs were developed and
digitalized using a Microtek Scanmaker 4 (Microtek Lab, Carson, CA), and the images
were analyzed in Adobe Photoshop version 7.7. Mitochondria in the micrographs were
darkened using the Photoshop Burn Tool which increases exposure of selected areas to
enhance contrast and emphasize structure. A stereological point-counting method was
applied to calculate mitochondrial volume (Howard and Reed, 1998; Nyack et al., 2007).
A point grid was superimposed on each micrograph, and all points touching extracellular
space were subtracted from the total number of points per micrograph. All points hitting
mitochondria were recorded and divided by the total number of points within the
intracellular space to determine the total mitochondrial fractional volume of the muscle.
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Mitochondrial Isolation
Approximately 40 g of WM was excised from the lateral sides of each fish
extending from the operculum to the caudal peduncle. WM samples were collected from
an individual animal when possible; however, it was necessary to combine tissue from 2-
4 small fish for a single experiment. All isolation procedures were carried out on ice.
During dissection, tissue was placed in a beaker containing 2 volumes of isolation
medium (in mmoll-1: 140 KCl, 10 EDTA, 5 MgCl2, 20 HEPES, 0.5% bovine serum
albumin (BSA), pH 7.1 at 25ºC). Tissue was transferred to a Petri dish, finely diced with
scissors, and all fluid was decanted through two layers of cheesecloth. The diced tissue
was divided into 10 portions and homogenized in 8 volumes of isolation medium using a
Potter-Elvehjem tissue grinder (Wheaton Science Products, Millville, NJ). Tissue was
dispersed by five passes with a loose fitting pestle, three passes with a medium fitting
pestle, and two passes with a tight fitting pestle. The homogenates were centrifuged at
4ºC for 5 min at 1400 g. The supernatant was poured through four layers of cheesecloth
then centrifuged for 7 min at 2600 g. Remaining supernatant was poured through eight
layers of cheesecloth and centrifuged for 10 min at 9000 g to obtain the mitochondrial
pellets. The supernatant was discarded and the resultant pellets were resuspended in 20
ml isolation medium (minus BSA) then centrifuged for 10 min at 9000 g. Mitochondrial
pellets were combined from the centrifuge tubes and resuspended in 1-2 ml isolation
medium (minus BSA).
Oxygen Consumption Measurements
Oxygen consumption rates of the isolated mitochondria were measured in a
water-jacketed bath using a Clarke type polarographic oxygen electrode (YSI Inc.,
7
Yellow Springs, OH). The oxygen monitoring system was calibrated with air saturated
respiration medium (in mmoll-1: 140 KCl, 5 Na2HPO4, 20 HEPES, 0.5% fatty acid-free
BSA, pH 7.1 at 25 º C) to 100% full scale. Approximately 2-3 mg of mitochondrial
protein was added to 2 ml of respiration medium in a magnetically stirred sample
chamber kept at 15ºC by a water circulator. Following 3 min of temperature
equilibration, the oxygen electrode was immersed in the mitochondrial solution and
oxygen uptake was traced by a chart recorder. Saturating amounts of substrate (0.1 mM
malate and 2.5 mM pyruvate) were added using a microliter syringe (Hamilton Co.,
Reno, NV), followed by the addition of 0.3 mM ADP to stimulate the maximal
mitochondrial oxygen consumption rate (state 3). Once all of the ADP was
phosphorylated, the rate of oxygen consumption declined and reached a new steady state
(state 4). Viability of the isolated mitochondria was determined by the respiratory control
ratio (RCR; state 3/state 4) (Estabrook, 1967), where a RCR > 5 was considered to reflect
well coupled mitochondria. In a few samples, myofibrillar ATPase contamination of the
mitochondrial pellet caused a nearly continuous state 3 rate. Under these circumstances,
500 units of myokinase (adenylate kinase) were added to the sample to scrub the solution
of ADP and obtain a state 4 rate.
Standardization of Mitochondrial Respiration Measurements
For all mitochondrial preparations, state 3 VO2 mt was standardized in three ways:
(1) VO2 mt per milligram of protein, (2) VO2 mt per milliliter of mitochondria, and (3) VO2 mt
per gram of muscle. Following oxygen consumption measurements, a 2 ml aliquot of the
mitochondrial suspension was removed from the sample chamber. The suspension in the
chamber was continuously stirred prior to removal to ensure homogeneity of the aliquots.
8
Samples were centrifuged for 5 min at 9000 g to re-pellet the mitochondria. Protein
content of the mitochondrial pellet was determined using the Bio-Rad Protein Assay
(Bio-Rad Laboratories, Hercules, CA), based on the Bradford method. The protein
concentration (mgml-1) and total volume of the pellet were recorded. The mitochondrial
volume of each pellet was determined from TEM micrographs. Pellets were prepared for
electron microscopy and micrographs were analyzed as described above for muscle
mitochondrial fractional volume, with the following alterations. All micrographs were
taken at ×5,600, the lowest magnification in which it was possible to identify individual,
intact mitochondria. Stereological point counting was used in which the points that
landed on empty space (between individual mitochondria) were subtracted from the total
number of reference points. The sum of points hitting mitochondria was divided by the
sum of reference points on a total of 10 micrographs per pellet. The volume of
mitochondria in each pellet was obtained by multiplying the mitochondrial fractional
volume by the total volume of the pellet. The VO2 mt per milliliter of mitochondria and the
white muscle TMFV of each species were used to calculate VO2 mt per gram of muscle.
Statistical Analysis
Means were compared using t-tests with a significance level of P < 0.05. Linear
regression analysis was used to evaluate scaling of VO2 mt with body mass using
SigmaPlot version 10.0 (SSPS, Chicago, IL).
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RESULTS
Representative TEM micrographs displaying mitochondrial distribution are
shown in Figure 1. Comparison of P. saltatrix and C. striata white muscle fibers
revealed a significant difference in TMFV between the species (Figure 2). Mean TMFV
for P. saltatrix was 0.031 ± 0.001 and Nyack et al. (2007) previously determined that
TMFV for C. striata WM was 0.012 ± 0.001. In P. saltatrix there was no significant
difference in TMFV between size classes. However, in C. striata TMFV was
significantly higher in the small fish than in the large. The TMFV scaling exponent was
determined for both species by the equation: TMFV = aMb, where M is body mass, a is a
constant, and b is the scaling exponent (Schmidt-Nielsen, 1984). The mass specific
TMFV of white muscle was independent of body mass in P. saltatrix (b = -0.01; P >
0.05) while C. striata had a significantly negative scaling exponent (b = -0.06; Nyack et
al., 2007).
Mitochondrial Oxygen Consumption
Examples of oxygen consumption recordings are shown in Figure 3. State 3
respiration was elicited by the addition of saturating amounts of pyruvate, malate, and
ADP. State 4 respiration occurred after all of the ADP in the system was phosphorylated.
State 3 and state 4 rates were used to calculate an RCR for each preparation.
Mitochondria isolated from the WM of both P. saltatrix and C. striata were well-coupled,
with RCR values > 5 (Table 1). The mean state 3 respiration rate was significantly
different between P. saltatrix and C. striata.
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The state 3 respiration rate for each mitochondrial preparation was standardized to
milligrams of protein, milliliters of mitochondria, and grams of muscle, and VO2 mt was
expressed as both ml O2 · min-1 and nmol O2 · min-1 to facilitate comparison to other
studies (Table 2). For oxygen consumption measurements, approximately 1 ml of each
mitochondrial pellet was used. The mean amount of mitochondrial protein added to the
oxygen consumption chamber was 2.6 mg. The pellets analyzed following oxygen
consumption measurements were highly enriched in intact mitochondria (Figure 4).
Stereological analysis of the mitochondrial pellet determined a mean volume of 0.67 ±
0.14 (P. saltatrix) and 0.34 ± 006 (C. striata) µl mitochondria per milliliter of suspension
in the oxygen consumption chamber. A total of 3 ml mitochondrial suspension was used
for each oxygen consumption measurement.
The maximum oxygen consumption rate per milligram of protein and per
milliliter of mitochondria was higher for C. striata than P. saltatrix, though the
differences were not significant (Table 2). The VO2 mt per gram of muscle was determined
from the TMFV (Figure 2) and the VO2 mt per volume of mitochondria for both species
(Table 2). Maximal mitochondrial oxygen consumption per gram of muscle was 2-fold
higher in P. saltatrix due to a greater TMFV than C. striata. Based on the VO2 mt per gram
of muscle, the maximum ATP-production rate of WM in P. saltatrix was 7.8 µmol ATP ·
min-1 · g-1 and C. striata was 4.6 µmol ATP · min-1 · g-1, assuming an ATP:O2 of 6 (Leary
et al., 2003).
Values presented in Table 2 were defined based on fish ranging in size from 51 to
518 g in P. saltatrix and 66 to 3,296 g in C. striata. However, the relationship between
animal mass and VO2 mt showed no scaling effect in either species despite a 10 to 50-fold
11
increase in body mass (Figure 5). Regression analysis indicated that for each species, VO2
mt measurements (per milligram of protein, per milliliter of mitochondria, and per gram of
muscle) were not significantly different across the range of body masses.
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DISCUSSION
Studies of aerobic capacity based on isolated mitochondria normally express VO2mt
as a function of protein content. The protein levels (typically assayed using the Bradford
or Biuret method) therefore serve as a proxy for the amount of mitochondria in a sample
in units of milligrams of mitochondrial protein per milliliter. These measurements
assume a highly pure mitochondrial preparation that is free of non-mitochondrial material
(Schwerzmann et al., 1986). Such preparations may be possible from mitochondria-rich
tissues such as heart (Moyes et al., 1991), liver (Schwerzmann et al., 1986), and red
muscle (RM) (Moyes et al., 1991) that possess Vmt > 0.40. However, fish WM, as used
in the present study, has a Vmt ≈ 0.02 (Johnston, 1981; Fig. 2). The low mitochondrial
density of WM requires a much larger amount of tissue (40 g in contrast to < 2 g for RM)
to yield a volume of isolated mitochondria equivalent to preparations of highly aerobic
tissues (Moyes et al., 1989). The low Vmt in fish WM and the large amount of tissue
needed for mitochondrial isolation may therefore decrease the purity of the final
mitochondrial pellet. This is reflected in the amount of protein required to achieve an
acceptable rate of oxygen consumption and a high RCR. VO2 mt measurements of fish RM
have been made using 50 to 200 µg of protein (Moyes et al., 1989, Johnston et al., 1994),
whereas WM measurements require ≥ 2 mg (Figure 3). This difference probably reflects
the fact that most of the protein in preparations from RM is derived from mitochondria,
whereas in WM non-mitochondrial protein constitutes a substantial fraction of the protein
pool. This source of experimental variation may partly explain the relatively large
differences among studies in measurements of VO2 mt for fish WM when based on
milligrams of protein. For example, Moyes et al. reported values of 77 ± 5.6 nmol O2 ·
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min-1 · mg protein-1 for trout (1992), and 104 ± 15 nmol O2 · min-1 · mg protein-1 for carp
using malate and pyruvate as substrates (1989). In contrast, the VO2 mt values for P.
saltatrix and C. striata (29.09 ± 3.79 and 24.07 ± 3.44 nmol O2 · min-1 · mg protein-1,
respectively) were somewhat lower than those for trout and carp WM, likely because of
slight differences in the isolation procedure that led to variation in sample purity
(reflected by milligrams of protein). However, these differences in absolute rates per
milligram of protein would not impact the scaling behavior of VO2 mt or the general
conclusions of this study.
By quantifying the mitochondrial fractional volume of the samples used for
oxygen consumption measurements, we were able to assess VO2 mt in terms that can be
more clearly related to the intact muscle. TEM analysis of the pellets eliminated the
potential biases associated with measuring VO2 mt per mg protein by relating the oxygen
consumption measurements directly to the volume of mitochondria in each sample. The
VO2 mt rates per milliliter of mitochondria for WM from P. saltatrix (0.95 ± 0.19 ml O2 ·
min-1 · ml mitochondria-1) and C. striata (1.49 ± 0.26 ml O2 · min-1 · ml mitochondria-1)
are similar to those observed in fast-twitch muscle from cat (1.40 ml O2 · min-1 · ml
mitochondria-1; Hoppeler et al., 1982) and mouse (2.47 ml O2 · min-1 · ml mitochondria-1;
Schwerzmann et al., 1989). Our rates also compare favorably to the narrow VO2 mt range
of 3 to 5 ml O2 · min-1 · ml mitochondria-1 that is assumed for all mammals (reviewed in
Suarez, 1996).
The VO2 mt per ml of mitochondria and the muscle TMFV can be used to calculate
the VO2 mt per gram of muscle (Hoppeler and Weibel, 2000), assuming that 1 ml of fiber is
equal to 1 g of fiber. The mean values of VO2 mt per gram of muscle for P. saltatrix (1.31
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± 0.26 µmol O2 · min-1 · g muscle-1) and C. striata (0.76 ± 0.13 µmol O2 · min-1 · g
muscle-1) were similar to rates observed in Notothenioid fishes. Johnston (1987)
examined the interspecific differences of VO2 mt from the RM of icefishes and observed
rates of 1.20 ± 0.28 µmol O2 · min-1 · g muscle-1 (Chaenocephalus aceratus) and 1.07 ±
0.23 µmol O2 · min-1 · g muscle-1 (Notothenia gibberifrons). Our rates of VO2 mt per gram
of muscle for P. saltatrix and C. striata are also consistent with the PCr recovery rates
observed by Nyack et al. (2007). These authors found that postcontractile PCr recovery
in C. striata WM fibers was approximately 1 µmol · min-1 · g muscle-1 , which
corresponds to an intracellular ATP-production rate of 1 µmol · min-1 · g muscle-1 (since
there is a 1:1 stoichiometry of PCr and ATP in the creatine kinase reaction). Assuming
an ATP:O2 of 6, the in vivo VO2 mt needed to power PCr recovery in C. striata WM is
roughly 0.17 µmol O2 · min-1 · g muscle-1, which corresponds to ~20% of the VO2 mt rate
observed in this species (0.76 µmol O2 · min-1 · g muscle-1 for C. striata). This indicates
that there is sufficient aerobic capacity (VO2 mt) to support the observed rates of whole
muscle PCr recovery while mitochondria are operating well below the maximal rate of
respiration.
Increases in VO2 mt can be attained by increasing the inner membrane surface
density of the mitochondria (Schwerzmann et al., 1986; Schwerzmann et al., 1989). A
high cristae surface density facilitates more respiratory chain enzymes to consume O2 and
catalyze the synthesis of ATP (Hoppeler et al., 1987). The lack of a significant difference
in VO2 mt across species in the present study suggests that whole animal aerobic capacity is
largely controlled by Vmt, rather than by qualitative differences in the function of
mitochondria (Hoppeler and Weibel, 2000). By investigating two behaviorally different
15
species of fish we were able to observe how variation in Vmt relates to animal aerobic
capacity. P. saltatrix is an active, pelagic species that relies on both red and white
muscle for routine locomotor activity. In contrast, C. striata is a sedentary, benthic
species that has virtually no red muscle and utilizes WM for brief burst contractions
during predator-prey interactions. The behavioral contrast of the two species is reflected
by the observed differences in WM TMFV and muscle oxygen consumption. P. saltatrix
had a 3-fold higher TMFV than C. striata (Figure 2) and a 2-fold higher VO2 mt per gram
of muscle, although the latter difference was not significant (Table 2).
The size class independence of VO2 mt for both species of fish (Figure 4) is
consistent with mammalian studies that have shown the close association between whole
animal VO2 max and skeletal muscle VO2 mt over a wide range of body masses (Weibel et al.,
2004). Although allometric scaling of VO2 max can be attributed to changes in V mt, it is
unclear what drives the structural decrease in V mt with increasing body mass, and the
mechanisms involved in metabolic scaling have been heavily debated (West et al., 1997;
Weibel, 2002; Suarez et al., 2004). What is clear, however, is that aerobic metabolism at
all levels of biological organization relies not only on catalytic capacity, but also on
convective and diffusive transport. During postmetamorphic development, WM fibers in
C. striata can reach diameters > 450 µm (Nyack et al., 2007). The increased intracellular
diffusion distances in these fibers may influence the structural design and function of the
cell. For instance, oxygen diffusive flux has been shown to cause a shift in mitochondrial
distribution in WM during hypertrophic fiber growth of fishes and crustaceans (Kinsey et
al., 2007; Nyack et al., 2007; Hardy et al., 2009). It has also been suggested that oxygen
diffusion may influence mitochondrial densities in Antarctic fishes that lack myoglobin
16
(Archer and Johnston, 1991). In addition, measurements of an aerobic process that is
dependent on diffusion (PCr recovery) in living WM of C. striata display negative
allometry (Nyack et al., 2007). This body mass and fiber size dependent decrease in
aerobic metabolic flux may be due to increasing diffusion constraints as fibers get larger.
The present study demonstrates that when aerobic flux is measured in the absence of all
diffusion constraints, these body and fiber size effects are removed, lending support to
the notion that diffusion exerts some control over mitochondrial density, distribution and
aerobic flux.
In summary, the method we used for isolating mitochondria from fish WM
yielded VO2 mt values similar to prior studies of muscle from both fishes and mammals.
Differences in VO2 mt in WM from an active and inactive species could be largely
attributed to different densities of mitochondria, rather than differences in the properties
of mitochondria. Further, VO2 mt was found to be independent of body mass in fish WM.
The size-independence of VO2 mt is at odds with the negative allometry found in prior
studies for indices of aerobic capacity and aerobic flux in fish WM. Whereas the aerobic
structure and function of intact muscle is dependent on diffusion of molecules like
oxygen and ATP, respiration measurements in isolated mitochondria represent aerobic
flux in the absence of all diffusion constraints. Thus, previously described changes in
mitochondrial density, distribution and aerobic flux with increases in muscle fiber size
and animal body mass do not appear to be associated with changes in mitochondrial
respiratory function.
LITERATURE CITED
Archer, S.D., Johnston, I.A. (1991). Density of cristae and distribution of mitochondria in the slow muscle fibers of Antarctic fish. Physiol. Zool. 64: 242-258. Battram, J.C., Johnston, I.A. (1991). Muscle growth in the Antarctic teleost, Notothenia neglecta (nybelin). Antart. Sci. 3: 29-33. Bone, Q. (1966). On the function of two types of myotomal muscle fiber in elasmobranch fish. J. Mar. Biol. Ass. U.K. 46: 321-349. Boyle, K.L., Dillaman, R.M.,Kinsey, S.T. (2003). Mitochondrial distribution and glycogen dynamics suggest diffusion constraints in muscle fibers of the blue crab, Callinectes sapidus. J. Exp. Zool. 297A: 1-16. Clarke, A., Johnston, N.M. (1999). Scaling of metabolic rate with body mass and temperature in teleost fish. J. Anim. Ecol. 68: 893-905. Egginton, S., Sktilbeck, C., Hoofd, L., Calvo, J., Johnston, I.A. (2002). Peripheral oxygen transport in skeletal muscle of Antarctic and sub-Antarctic notothenioid fish. J. Exp. Biol. 205: 769-779. Ellington, W.R. (2001). Evolution and physiological roles of phosphagen systems. Ann. Rev. Physiol. 63: 289-325. Estabrook, R. (1967). Mitochondrial respiratory control and the polarographic measurement of ADP/O ratios. Methods Enzymol. 10: 41-47. Gill, H.S., Weatherley, A.H., Lee, R., Legere, D. (1989). Histochemical characterization of myotomal muscle of five teleost species. J. Fish Biol. 34: 375-386. Hardy, K.M., Dillaman, R.M., Locke, B.R., Kinsey, S.T. (2009). A skeletal muscle model of extreme hypertrophic growth reveals the influence of diffusion on cellular design. Am. J. Physiol. (Integr. Reg. Comp.) (In Press). Hardy, K.M., Locke, B.R., Da Silva, M.D., Kinsey, S.T. (2006). A reaction-diffusion analysis of energetics in large muscle fibers secondarily evolved for aerobic locomotor function. J. Exp. Biol. 209: 3610-3620. Hochachka, P.W. (1994) Solving the common problem: matching ATP synthesis to ATP demand during exercise. Adv. Vet. Sci. Comp. Med. 38A: 41-56. Hochachka, P.W., Somero, G.N. (1973). Strategies of biochemical adaptation. Saunders, Philadelphia.
18
Holloszy, J.O., Booth, F.W. (1976). Biochemical adaptation to endurance exercise in muscle. Ann. Rev. Physiol. 38: 273-291. Hoppeler, H. (1990). The different relationship of VO2max to muscle mitochondria in humans and quadrupedal animals. Respir. Physiol. 80:137-146. Hoppeler, H., Kayar, S.R., Claassen, H., Uhlmann, E., Karas, R.H. (1987). Adaptive variation in the mammalian respiratory system in relation to energetic demand: III. Skeletal muscles: setting the demand for oxygen. Respir. Physiol. 69: 27-46. Hoppeler, H., Lindstedt, S.L. (1985). Malleability of skeletal muscle in overcoming limitations: structural elements. J. Exp. Biol. 115: 355-364. Hoppeler, H., Weibel, E.R. (2000). Structural and functional limits for oxygen supply to muscle. Acta. Physiol. Scand. 168:4 45-456. Howard, C.V., Reed, M.G. (1998). Unbiased Stereology: 3-Dimesional Measurements in Microscopy. BIOS Scientific Publishers, Oxford, UK. Jayne, B.C, Lauder, G.V. (1994). How fish use slow and fast muscle fibers: Implications for models of vertebrate muscle recruitment. J. Comp. Physiol. A. 175: 123-131. Johnson, L.K., Dillaman, R.M., Gay, D.M., Blum, J.E., Kinsey, S.T. (2004). Metabolic influences of fiber size in aerobic and anaerobic muscles of the blue crab, Callinectes sapidus. J. Exp. Biol. 207: 4045-4056. Johnston, I.A. (1981). Structure and function of fish muscles. Symp. Zool. Soc. Lond. 48: 71-113. Johnston, I.A., Fernández, D.A., Calvo, J., Vieira, V.L.A., North, A.W., Abercromby, M., Garland, T. (2003). Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective. J. Exp. Biol. 206: 2595-2609. Kinsey, S.T., Hardy, K.M., Locke, B.R. (2007). The long and winding road: influences of intracellular metabolite diffusion on cellular organization and metabolism in skeletal muscle. J. Exp. Biol. 210: 3505-3512. Kinsey, S.T., Moerland, T.S. (2002). Metabolite diffusion in giant muscle fibers of the spiny lobster, Panulirus argus. J. Exp. Biol. 205: 3377-3386. Koumans, J.T.M., Akster, H.A. (1995). Myogenic cells in the development and growth of fish. Comp. Biochem. Physiol. 110A: 3-20. Leary, S.C., Lyons, C.N., Rosenberger, A.G., Ballantyne, J.S., Stillman, J., and Moyes, C.D. (2003). Fiber-type differences in muscle mitochondrial profiles. Am. J. Physiol. (Integr. Reg. Comp.) 285: R817-R826
19
Milligan, C.L., Wood, C.M. (1986). Tissue intracellular acid-base status and the fate of lactate after exhaustive exercise in the rainbow trout. Metabolism 37: 552-556. Mathieu, O., Krauer, R., Hoppeler, H., Gehr, P., Lindstedt, S.L., Alexander, R.M., Taylor, C.R., Weibel, E.R. (1981). Design of the mammalian respiratory system. VII. Scaling mitochondrial volume in skeletal muscle to body mass. Respir. Physiol. 44: 113- 128. Mommsen, T.P. (2001). Paradigms of growth in fish. Comp. Biochem. Physiol. 129B: 207-219. Moyes, C.D. (2003). Controlling muscle mitochondrial content. J. Exp. Biol. 206: 4385- 4391. Moyes, C.D., Battersby, B.J., Leary, S.C. (1998). Regulation of muscle mitochondrial design. J. Exp. Biol. 201: 299-307. Moyes, C.D., Hood, D.A., (2003). Origins and consequences of mitochondrial variation in vertebrate muscle. Ann. Rev. Physiol. 65: 177-201. Moyes, C.D., Mathieu-Costello, O.A., Brill, R.W., Hochachka, P.W. (1991). Mitochondrial metabolism of cardiac and skeletal muscle from a fast (Katsuwonis pelamis) and a slow (Cyprinus carpio) fish. Can. J. Zool. 272: C1345-C1351. Moyes, C.D., Schulte, P.M. Hochachka, P.W. (1992). Recovery metabolism in white fish muscle: the role of the mitochondria. Am. J. Physiol. Regul. Integr. Comp. Physiol. Noda, L. The Enzymes. New York: Academic Press, 1973. Nyack, A.C., Locke, B.R., Valencia, A., Dillaman, R.M., Kinsey, S.T. (2007). Scaling of postcontractile phosphocreatine recovery in fish white muscle: effect of intracellular diffusion. Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: R2077-R2088. Rayner, M.D., Keenan, M.J. (1967). Role of red and white muscle in the swimming of the skipjack tuna. Nature 214: 392-393. Reynolds, E.S. (1963). The use of lead citrate with high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 208-212. Schwerzmann, K., Cruz-Olive, L.M., Eggman, R., Sanger, A., Weibel, E.R. (1986). Molecular architecture of the inner membrane of mitochondria from rate liver: a combined biochemical and stereological study. J. Cell. Biol. 102: 97-103. Schwerzmann, K., Hoppeler, H., Kayar, S.R., Weibel, E.R. (1989). Oxidative capacity of muscle mitochondria: correlation of physiological, biochemical, and morphometric characteristics. Proc. Nat. Acad. Sci. USA 86: 1583-1587.
20
Somero, G.N., Childress, J.J. (1980). A violation of the metabolism-size scaling paradigm: activities of glycolytic enzymes in fish muscle increase in larger size fish. Physiol. Zool. 53: 322-337. Spurr, R.A. (1969). A low viscosity epoxy resin embedding medium of electron microscopy. J. Ultra. Res. 26: 31-34. Srere, P.A. (1985). Organization of proteins within the mitochondrion. In: Organized multienzyme systems. (Welch, G.R. ed.). Academic Press, NY, pp. 1-66. Stienen, G.J.M., Kiers, J.L., Bottinelli, R., Reggiani, C. (1996). Myofibrillar ATPase activity in skinned human skeletal muscle fibres: fibre type and temperature dependence. J. Physiol. 493: 299-307. Suarez, R.K. (1996). Upper limits to mass-specific metabolic rates. A. Rev. Physiol. 58: 583-605. Suarez, R.K. (1998). Oxygen and the upper limits to animal design and performance. J. Exp. Biol. 201: 1065-1072. Suarez, R.K., Lighton, J.R.B., Brown, G.S., Mathieu-Costello, O. (1991). Mitochondrial respiration in humming bird flight muscle. Proc. Nat. Acad. Sci. USA 88: 4870-4873. Taylor, C.R. (1987). Structural and functional limits to oxidative metabolism: insights from scaling. Annu. Rev. Physiol. 155: 163-170. Taylor, C.R., Weibel, E.R. (1981). Design of the mammalian respiratory system. I. Problem and strategy. Respir. Physiol. 44: 1-10. Taylor, C.R., Weibel, E.R., Karas, R.H., Hoppeler, H. (1989). Matching structures and functions in the respiratory system. Allometric and adaptive variations in energy demand. In: Comparative Pulmonary Physiology. Current Concepts (Wood, S., ed.) Basel: Dekker, NY, pp. 27-65. Walliman, T., Wyss, M., Brdiczka, D., Nicolay, K. and Eppenberger, H.M. (1992). Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the ‘phosphocreatine circuit’ for cellular energy homeostasis. Biochem. J. 281: 21-40. Weatherly, A.H., Gill, H.S. (1985). Dynamics of increase in muscle fibers in teleosts in relation to size and growth. Experientia 41: 353-354. Weibel, E.R. (1987). Scaling of structural and functional variables in the respiratory system. Annu. Rev. Physiol. 49: 147-159. Weibel, E.R. (2002). The pitfalls of power laws. Nature 417: 131-132.
21
Weibel, E.R., Bacigalupe, L.D., Schmitt, B., Hoppeler, H. (2004). Allometric scaling of maximal aerobic rate in mammals: muscle aerobic capacity as determinant factor. Respir. Physiol. Neurobiol. 140: 115-132. Weibel, E.R., Taylor, C.R., Hoppeler, H. (1991). The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc. Nat. Acad. Sci. U.S.A. 88: 10357-10361. Weis-Fough, T. (1964). Diffusion in insect wing muscle: the most active tissue known. J. Exp. Biol. 41: 229-256. West, G.B., Brown, J.H., Enquist, B.J. (1997). A general model for the origin of allometric scaling laws in biology. Science. 276: 122-126.
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TABLES
Table 1. Respiration of isolated white muscle mitochondria
State 3 and state 4 values are means ± SE determined at 15ºC. Values in parentheses are number of preparations. State 3 rates were measured in the presence of 0.1 mM malate, 2.5 mM pyruvate, and 0.3 mM ADP. State 4 was observed after all the ADP was phosphorylated. RCR (respiratory control ratio; state 3/state 4).
P. saltatrix C. striata State 3 (% O2 · min-1) (ml O2 · min-1) State 4 (% O2 · min-1) (ml O2 · min-1) RCR
6.44 ± 0.72 1.38e-3 ± 0.15e-3 (9)* 1.14 ± 0.11 2.44e-4 ± 0.25e-4 (9) 5.76 ± 0.40
4.76 ± 0.50 1.01e-3 ± 0.42e-3 (19)* 0.70 ± 0.08 1.50e-4 ± 0.19e-4 (19) 7.61 ± 0.62
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Table 2. Maximal oxygen consumption rates of white muscle mitochondria per milligram of protein, milliliter of mitochondria, and gram of muscle
V02mt P. saltatrix C. striata ml O2 · min-1 · mg protein-1 4.82e-4 ± 0.85e-4 (9) 5.37e-4 ± 0.77e-4 (19) ml O2 · min-1 · ml mitochondria-1 0.95 ± 0.19 (9) 1.49 ± 0.26 (14) ml O2 · min-1 · g muscle-1 29.1e-3 ± 5.82e-3 (9) 17.0e-3 ± 2.89e-3 (14) nmol O2 · min-1 · mg protein-1 29.09 ± 3.79 (9) 24.07 ± 3.44 (19) nmol O2 · min-1 · ml mitochondria-1 42600 ± 8518 (9) 66790 ± 11667 (14) nmol O2 · min-1 · g muscle-1 1304.9 ± 260.9 (9) 761.8 ± 129.4 (14) Values are means ± SE. Values in parentheses are number of preparations.
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FIGURE LEGENDS
Figure 1. Micrographs from small (upper) and large (lower) P. saltatrix (A, B) and C.
striata (C, D) taken at a magnification of ×1,800. Individual mitochondria are darkly
shaded as described in methods. Scale bar 10 µm.
Figure 2. Total mitochondrial fractional volume (TMFV) from small and large P.
saltatrix and C. striata. Mean body mass for small and large P. saltatrix was 53.2 g and
438.5 g, respectively. Mean body mass for small and large C. striata was 1.9 g and
3266.4 g, respectively (Nyack et al., 2007). *TMFV is significantly different between
small and large C. striata. Values shown are means ± SE.
Figure 3. Oxygen consumption recordings from P. saltatrix (A) and C. striata (B)
mitochondrial isolates demonstrating state 3 and state 4 rates. (C) Example of continuous
state 3 rate caused by ATPase contamination where adenylate kinase (ADK) was added
to produce a state 4 rate.
Figure 4. TEM micrographs of mitochondria isolated from (A) P. saltatrix and (B) C.
striata taken at a magnification of × 5,600. The isolated mitochondria were prepared for
electron microscopy following oxygen consumption measurements. Micrographs depict
a sample that was highly enriched in healthy, intact mitochondria. Scale bar 20 µm.
Figure 5. Scaling with body mass of VO2 mt from white muscle of P. saltatrix and C.
striata. All VO2 mt rates are presented as ml O2· min -1 · unit volume or mass-1. (A) VO2 mt
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per milligram of mitochondrial protein. P. saltatrix VO2 mt = -2.73 M -0.27 (r2 = 0.14, p =
0.31); C. striata VO2 mt = -3.50 M 0.06 (r2 = 0.02, p = 0.60). (B) VO2 mt per milliliter of
mitochondria. P. saltatrix VO2 mt = -0.01 M -0.03 (r2 = 0.002, p = 0.91); C. striata VO2 mt =
0.16 M -0.03 (r2 = 0.004, p = 0.83). (C) VO2 mt per gram of muscle. P. saltatrix VO2 mt = -1.52
M -0.03 (r2 = 0.002, p = 0.91); C. striata VO2 mt = -1.62 M -0.09 (r2 = 0.04, p = 0.50). Points
represent individual mitochondrial preparations for each species.
26