Download Cardiac ultrastructure, metabolism and O2

1287
The Journal of Experimental Biology 203, 1287–1297 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JEB2493
THE INTERPLAY AMONG CARDIAC ULTRASTRUCTURE, METABOLISM AND THE
EXPRESSION OF OXYGEN-BINDING PROTEINS IN ANTARCTIC FISHES
KRISTIN M. O’BRIEN AND BRUCE D. SIDELL*
School of Marine Sciences, University of Maine, 5741 Libby Hall, Orono, ME 04469-5741, USA and Department of
Biological Sciences, University of Maine, 5751 Murray Hall, Orono, ME 04469-5751, USA
*Author for correspondence (e-mail: [email protected])
Accepted 8 February; published on WWW 23 March 2000
Summary
We examined heart ventricle from three species of
similar. Despite significant ultrastructural differences,
Antarctic fishes that vary in their expression of oxygenoxidative capacities, estimated from measurements of
binding proteins to investigate how some of these fishes
maximal activities per gram of tissue of enzymes from
maintain cardiac function despite the loss of hemoglobin
aerobic metabolic pathways, are similar among the three
(Hb)
and/or
myoglobin
(Mb).
We
quantified
species. The combination of ultrastructural and enzymatic
ultrastructural features and enzymatic indices of metabolic
data indicates that there are differences in the density of
capacity in cardiac muscle from Gobionotothen
electron transport chain proteins within the inner
gibberifrons, which expresses both Hb and Mb,
mitochondrial membrane; proteins are less densely packed
Chionodraco rastrospinosus, which lacks Hb but expresses
within the cristae of hearts from Chaenocephalus aceratus
Mb, and Chaenocephalus aceratus, which lacks both Hb
than in the other two species. High mitochondrial densities
and Mb. The most striking difference in cellular
within hearts from species that lack oxygen-binding
architecture of the heart among these species is the
proteins may help maintain oxygen flux by decreasing the
diffusion distance between the ventricular lumen and
percentage of cell volume occupied by mitochondria,
mitochondrial membrane. Also, high mitochondrial
Vv(mit,f), which is greatest in Chaenocephalus aceratus
densities result in a high intracellular lipid content, which
(36.53±2.07), intermediate in Chionodraco rastrospinosus
may enhance oxygen diffusion because of the higher
(20.10±0.74) and lowest in G. gibberifrons (15.87±0.74).
solubility of oxygen in lipid compared with cytoplasm.
There are also differences in mitochondrial morphologies
These results indicate that features of cardiac myocyte
among the three species. The surface area of inner
architecture in species lacking oxygen-binding proteins
mitochondrial membrane per volume of mitochondria,
may maintain oxygen flux, ensuring that aerobic metabolic
Sv(imm,mit), varies inversely with mitochondrial volume
capacity is not diminished and that cardiac function is
density so that Sv(imm,mit) is greatest in G. gibberifrons
maintained.
(29.63±1.62 µm−1), lower in Chionodraco rastrospinosus
(21.52±0.69 µm−1) and smallest in Chaenocephalus aceratus
(20.04±0.79 µm−1). The surface area of mitochondrial
Key words: heart, cardiac muscle, metabolism, haemoglobin,
myoglobin, oxygen-binding protein, icefish, Antarctic fish,
cristae per gram of tissue, however, is greater in
Gobionotothen
gibberifrons,
Chionodraco
rastrospinosus,
Chaenocephalus aceratus than in G. gibberifrons and
Chaenocephalus aceratus.
Chionodraco rastrospinosus, whose surface areas are
Introduction
Antarctic icefishes (Channicthyidae) are one of six families
within the suborder Nototheniodei that dominates both species
number and biomass of fishes in the Southern Ocean (Eastman,
1993). Icefishes are unique among all vertebrates because as
adults they lack the oxygen-binding protein hemoglobin (Hb).
Because these fishes lack Hb, the oxygen-carrying capacity of
their blood is only one-tenth of that of red-blooded teleosts
(Ruud, 1954).
Channichthyids possess many unusual cardiovascular
features that appear to compensate for the loss of circulating
Hb. Their large heart-to-body mass ratio contributes to a mass-
specific cardiac output that is 4–5 times greater than that of
red-blooded teleosts (Hemmingsen et al., 1972). Blood
volumes in icefish are 2–4 times greater than those of redblooded teleosts, and they possess unusually large-diameter
capillaries that minimize the peripheral resistance against
which the heart must work (Hemmingsen and Douglas, 1970;
Fitch et al., 1984). In combination, these cardiovascular
characteristics provide a large blood volume that is circulated
through the body at high flow to maintain oxygen delivery to
working muscles.
The consensus has been that hearts from icefish also lack
1288 K. M. O’BRIEN AND B. D. SIDELL
myoglobin (Mb), the oxygen storage and transport protein
found in oxidative muscle (Hamoir and Geradin-Otthiers,
1980; Wittenberg and Wittenberg, 1989). Recent findings,
however, have revealed the presence of this protein in heart
ventricles of several species of icefishes (Sidell et al., 1997;
Moylan and Sidell, 2000). Our laboratory has recently
examined hearts from 13 of the 15 known species of
channicthyid icefishes, and we have determined that
myoglobin is expressed in eight of these species (Moylan and
Sidell, 2000). During the evolution of the icefish family,
expression of myoglobin has been lost through at least four
independent mutational events, based upon patterns of
myoglobin protein expression and phylogeny (Moylan and
Sidell, 2000). The widely dispersed pattern of presence and
absence of myoglobin within the channicthyid family initially
suggested that the protein might not be functional at their cold
body temperatures. Several recent studies, however, indicate
that Mb is indeed functional in these fishes.
Kinetic analyses reveal that Mbs from icefish and other
teleosts display faster rates of oxygen binding and dissociation
at cold temperature than mammalian Mbs (Cashon et al.,
1997). Experiments with isolated, perfused hearts from
icefishes demonstrate that selective poisoning of Mb results in
loss of mechanical performance by hearts that express the
protein, but not in hearts that lack Mb (Acierno et al., 1997).
These perfused heart experiments also show that hearts from
species that naturally lack Mb are capable of meeting greater
pressure/work challenges than hearts from icefish that express
Mb in which the protein has been poisoned. These results
strongly indicate that Mb is functional when present and that,
to maintain cardiac function, the ultrastructural and/or
metabolic characteristics of hearts lacking Mb have been
modified to compensate for loss of the protein.
Johnston and Harrison (1987) compared the ultrastructure
of the heart ventricle between a myoglobinless icefish,
Chaenocephalus aceratus, and a red-blooded nototheniid,
Notothenia neglecta. They determined that hearts from
Chaenocephalus aceratus had significantly higher
mitochondrial densities than those from N. neglecta and
hypothesized that these high densities might enhance
intracellular oxygen diffusion in hearts of species lacking Mb.
Chaenocephalus aceratus and N. neglecta, however, differ in
their expression of both oxygen-binding proteins, Hb and Mb.
Thus, whether architectural differences observed between
these hearts are correlated with the loss of Mb or Hb expression
was not definitively resolved.
A recent description of the pattern of both Mb and Hb
expression among Antarctic notothenioid fishes now permits
us to differentiate between structural and metabolic
characteristics that are specifically correlated with the loss of
Mb and those correlated with the loss of Hb. We examined
heart ventricle in two species of channicthyid icefishes,
Chaenocephalus aceratus (−Hb/−Mb) and Chionodraco
rastrospinosus (−Hb/+Mb), and a closely related red-blooded
nototheniid Gobionotothen gibberifrons (+Hb/+Mb). The
ultrastructure of cardiac muscles was examined using electron
microscopy, and cellular structures were quantified using
stereological techniques. We also measured the maximal
activities of key enzymes from several metabolic pathways as
indices of the metabolic capacities of the tissues. Because all
three species are phylogenetically closely related and
ecotypically similarly sluggish, demersal fishes, we are
confident that differences observed in cardiac muscle can be
attributed to differences in the expression of oxygen-binding
proteins rather than to lifestyle or genetic distance.
Materials and methods
Gobionotothen gibberifrons, Chionodraco rastrospinosus
and Chaenocephalus aceratus were captured using an otter
trawl deployed from the R/V Polar Duke in Dallmann Bay
(64°N, 62°W) at approximately 150 m depth during the austral
autumn of 1991, 1993, 1995 and 1997 and the winter of 1996.
Animals were maintained in shipboard circulating seawater
tanks and transported to the US Antarctic Research Station,
Palmer Station, on Anvers Island. Here, they were transferred
to the Palmer Station aquarium and maintained unfed in
covered and circulating seawater tanks at 0±0.5 °C.
Tissue preparation for electron microscopy
Fishes were killed by a sharp blow to the head. The hearts
were quickly excised and placed in an ice-cold solution
(260 mmol l−1 NaCl, 2.5 mmol l−1 MgCl2, 5.0 mmol l−1 KCl,
2.5 mmol l−1 NaHCO3, 5.0 mmol l−1 NaH2PO4, pH 8.0) and
allowed to contract for several minutes to clear them of blood.
They were then placed in an ice-cold fixative solution (3 %
glutaraldehyde, 0.1 mol l−1 sodium cacodylate, 0.11 mol l−1
sucrose and 2 mmol l−1 CaCl2, pH 7.4) and perfused with
fixative retrogradely through the bulbous arteriosus using a
peristaltic pump. The pump was fitted with small-diameter
rubber tubing and secured within the bulbous arteriosus using
surgical silk. Hearts were perfused for 1 min at a flow rate of
15 ml min−1 and then for 30 min at a flow rate of 9 ml min−1.
They were then stored in fixative at 4 °C for 8–10 h, with a
change of fixative after the initial 4–6 h. Hearts were then
transferred into Trumps buffer (1 % glutaraldehyde, 4 %
formaldehyde, 0.1 mol l−1 sodium cacodylate, 0.11 mol l−1
sucrose and 2 mmol l−1 CaCl2, pH 7.4) and stored at 4 °C until
they were transported to our laboratory at the University of
Maine.
Ventricles were cut in half lengthwise, and a transmural
section spanning from epicardium to endocardium was excised
from the center of one half of each heart. Each transmural
section was then subdivided into three regions: blocks closest
to the epicardium (V1), blocks within the myocardium (V2)
and blocks nearest the endocardium (V3). Blocks were rinsed
briefly in an ice-cold solution (0.1 mol l−1 sodium cacodylate,
7 % sucrose, 2 mmol l−1 CaCl2, pH 7.4), and then rinsed for
30 min and stored overnight at 4 °C in the solution. Blocks
were post-fixed in an ice-cold solution of 1 % osmium
tetroxide, 0.1 mol l−1 sodium cacodylate, 7 % sucrose and
2 mmol l−1 CaCl2, pH 7.4, for 1.5 h, briefly rinsed in reagent-
Cardiac ultrastructure, metabolism and O2-binding proteins 1289
grade water, dehydrated through a series of increasing
concentrations of ethanol (70 %, 95 %, 100 %) and cleared with
propylene oxide. Blocks were stored overnight at room
temperature in a mixture of propylene oxide:resin (2:1) with
the lids slightly ajar to allow the propylene oxide to evaporate
slowly. Blocks were then infiltrated with a mixture of Epon
and Araldite resin for 1 h under vacuum, with a change of resin
after the initial 30 min, and cured at 60 °C for 48 h. Hearts were
fixed on site at Palmer Station during March and April 1995
and post-fixed at the University of Maine between June and
July 1995.
Stereology
Initially, blocks from each of the three regions of ventricle
described above were sampled in two animals from each of the
three species. Ultrastructural variables were quantified to
determine the amount of variation in these variables within the
ventricle. Because no variation was detected, an additional four
blocks, one per individual, were randomly chosen from each
of the three species so that a total of 10 blocks per species were
analyzed from six individuals.
Blocks were first thick-sectioned (1.5 µm) with an LB4
microtome to verify the integrity of the tissue. Sections were
stained with 1 % Toluidine Blue in 1 % sodium borate for 30 s
on a warm plate. Blocks were then trimmed and thin-sectioned
using a diamond knife and Sorvall MT2-B ultramicrotome.
Sections were collected on 400 mesh copper grids and stained
with 2 % uranyl acetate followed by 0.5 % lead citrate.
Sections were viewed with a Philips CM-10 transmission
electron microscope equipped with a tilting goniometer stage.
The stage was adjusted to 0 ° each time, ensuring that the beam
was consistently perpendicular to the grid. Ten micrographs
were taken at a magnification of 5200× for quantifying
mitochondrial surface and volume densities and myofibril
volume densities. Ten to twelve micrographs were taken at a
magnification of 39 000× for measuring mitochondrial cristae
surface densities. Micrographs were taken using the aligned
systematic quadrats subsampling method (Cruz-Orive and
Weibel, 1981). Individual mitochondria with the most clearly
defined inner mitochondrial membrane were chosen for
micrographs from within each randomly chosen field of view at
a magnification of 39 000×. Calibration grids were photographed
at each magnification to calculate final magnifications.
Mitochondrial and myofibril volume densities were
quantified using point-counting methods; mitochondrial
surface densities were measured using the line-intercept
technique (Weibel, 1979). Micrographs were projected onto a
Summagraphics II digitizing tablet at a final magnification of
13 400×. Images were overlaid with a square lattice test pattern
with spacing equal to 1.34 µm on projected micrographs. Care
was taken to exclude epithelial cells, endothelial cells, blood
cells, extracellular matrix and luminal spaces from the
measurements.
Calculation of inner mitochondrial membrane densities
Mitochondrial cristae surface densities were quantified at a
final magnification of 96 000× using the line-intercept method
(Weibel, 1979). Micrographs of G. gibberifrons mitochondria
were printed for best resolution of mitochondrial inner
membrane. Regions of the mitochondria with cristae shown
clearly in cross section were outlined in red wax pencil, and
only these areas were used for calculating cristae surface
densities (Smith and Page, 1976). A subset of micrographs of
Chionodraco rastrospinosus and Chaenocephalus aceratus
mitochondria were also printed, and cristae surface density was
quantified from both prints and micrographs projected onto a
Summagraphics II digitizing tablet (N=2 per species). Because
there was no significant difference between measurements
made from projected micrographs and prints in these two
species (P=0.92), cristae surface densities were quantified
using projected micrographs for the remaining individuals.
Micrographs from all three species were overlaid with a square
lattice test pattern (d=0.08 µm) for analysis.
Enzymology
Tissue preparation
Animals were killed and hearts extracted as described
above. Assays requiring fresh tissue, for hexokinase (HK),
phosphofructokinase (PFK), cytochrome oxidase (CO) and
carnitine palmitoyltransferase-I (CPT-I), were performed
immediately. For all other assays, pyruvate kinase (PK), lactate
dehydrogenase (LDH), citrate synthase (CS) and 3hydroxyacyl CoA dehydrogenase (HOAD), tissues were
quickly frozen in liquid nitrogen, stored at −70 °C and shipped
on dry ice to our laboratory at the University of Maine, where
they were stored at −70 °C.
For all enzymes other than CO and CPT-I (see below),
tissues were homogenized in a 10 % w/v ice-cold buffer
(40 mmol l−1 Hepes, 1 mmol l−1 EDTA, 2 mmol l−1 MgCl2,
pH 7.8 at 1 °C). Dithiothreitol (DTT; 2 mmol l−1) was added to
the buffer for the PFK, LDH and HK assays. Tissue was
homogenized by hand using a ground-glass homogenizer.
Homogenates were further reduced by brief (3–5 s) treatment
with a Tekmar Tissuemizer and finally homogenized to
completion by hand using a ground-glass homogenizer.
All assays were performed in triplicate at 1±0.5 °C using a
Perkin Elmer Lambda 6 spectrophotometer. Temperature was
maintained using a refrigerated, circulating water bath attached
to the spectrophotometer. Background activity was measured
in the absence of initiating substrate. Assay conditions are
described in detail below. Maximal activities were determined
by measuring the rate of oxidation or reduction of pyridine
nucleotides at 340 nm for 5 min, except when noted otherwise
below.
Phosphofructokinase (EC 2.7.1.11). The methodology
employed was slightly modified from that described by Opie
and Newsholme (1967) and Read et al. (1977). The final
reaction mixture contained 7 mmol l−1 MgCl2, 200 mmol l−1
KCl, 1 mmol l−1 KCN, 2 mmol l−1 AMP, 0.15 mmol l−1 NADH,
2 mmol l−1 ATP, 4 mmol l−1 fructose 6-phosphate (F6P),
2 units ml−1 aldolase, 10 units ml−1 triosephosphate isomerase,
2 units ml−1 glycerol-3-phosphate dehydrogenase, 75 mmol l−1
1290 K. M. O’BRIEN AND B. D. SIDELL
triethanolamine, pH 8.4 at 1 °C. Reactions were initiated by the
addition of a mixture of ATP and F6P.
Lactate dehydrogenase (EC 1.1.1.27). The procedure for
this assay was that described by Hansen and Sidell (1983). The
final reaction mixture contained 2.5 mmol l−1 pyruvate,
0.15 mmol l−1 NADH, 1 mmol l−1 KCN, 50 mmol l−1 imidazole,
pH 7.7 at 1 °C. Reactions were initiated by the addition of
pyruvate.
Pyruvate kinase (EC 2.7.1.40). The method used for this
assay was that described by Hansen and Sidell (1983). The
final reaction mixture contained 150 mmol l−1 KCl, 1 mmol l−1
KCN, 10 mmol l−1 MgSO4, 0.15 mmol l−1 NADH, 5 mmol l−1
ADP, 2.5 mmol l−1 phosphoenolpyruvate (PEP), 10 units ml−1
LDH, 50 mmol l−1 imidazole, pH 7.1 at 1 °C. Reactions were
initiated by the addition of PEP.
3-Hydroxyacyl CoA dehydrogenase (EC 1.1.1.35). The
protocol for this assay was that originally described by
Beenakkers et al. (1967) as modified by Hansen and Sidell
(1983). The final reaction mixture contained 1 mmol l−1 EDTA,
1 mmol l−1 KCN, 0.15 mmol l−1 NADH, 0.1 mmol l−1
acetoacetyl CoA, 50 mmol l−1 imidazole, pH 7.7 at 1 °C.
Reactions were initiated by the addition of acetoacetyl CoA.
Citrate synthase (EC 4.1.3.7). For this assay, we used a
modification of the protocol originally described by Srere et al.
(1963). The final reaction mixture contained 0.25 mmol l−1
5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.4 mmol l−1
acetyl CoA, 0.5 mmol l−1 oxaloacetate, 75 mmol l−1 Tris-HCl,
pH 8.2 at 1 °C. The reaction was initiated by the addition of
oxaloacetate. The progress of the reaction was monitored by
following the production of the reduced anion of DTNB at
412 nm.
Hexokinase (EC 2.7.1.1). This assay was modified from that
described by Zammit and Newsholme (1976). The final
reaction mixture contained 7.5 mmol l−1 MgCl2, 0.8 mmol l−1
EDTA, 1.5 mmol l−1 KCl, 0.4 mmol l−1 NADP, 2.5 mmol l−1
ATP, 10.0 mmol l−1 creatine phosphate, 1.0 mmol l−1 α-Dglucose, 0.9 units ml−1 creatine phosphokinase, 0.7 units ml−1
glucose-6-phosphate dehydrogenase, 75 mmol l−1 Tris-HCl,
pH 7.6 at 1 °C. Reactions were initiated by the addition of
glucose.
Cytochrome oxidase (EC 1.9.3.1). The method of Wharton
and Tzagoloff (1967) was used to measure activity. Tissue was
homogenized in 50 mmol l−1 K2HPO4/KH2PO4, 0.05 % Triton
X-100, pH 7.5. The assay medium consisted of 10 mmol l−1
K2HPO4/KH2PO4, 0.65 % (w/v) reduced (Fe2+) cytochrome c
and 0.93 mmol l−1 K3Fe(CN)6. The reaction was initiated by
the addition of enzyme. Maximal activities were measured by
following the oxidation of reduced cytochrome c at 550 nm.
Carnitine palmitoyltransferase-I (EC 2.3.1.21). Maximal
activities of CPT-I were measured in intact isolated
mitochondria (Rodnick and Sidell, 1994). Tissue was
homogenized in 10 % (w/v) of ice-cold 40 mmol l−1 Hepes,
10 mmol l−1 EDTA, 5 mmol l−1 MgCl2, 150 mmol l−1 KCl,
35 mmol l−1 sucrose, and 0.5 % bovine serum albumin (BSA),
pH 7.27 at 1 °C, using a Duall ground-glass homogenizer. A
sample of the crude homogenate was reserved for measuring
total CPT-I activity. The homogenate was centrifuged at 270 g
for 10 min. The supernatant was collected and centrifuged at
270 g. The supernatant was again collected and centrifuged at
15 000 g for 20 min. The mitochondrial pellet was gently
resuspended in homogenization buffer (minus BSA) and
centrifuged at 15 000 g for 20 min. The resultant pellet was
gently resuspended in homogenization buffer lacking BSA to
give a final concentration of approximately 5 µg protein µl−1. A
sample of the mitochondrial suspension was frozen at −70 °C
for later protein determination using the bicinchoninic acid
method (Smith et al., 1985).
The final assay medium consisted of 1.0 mmol l−1 EGTA,
220 mmol l−1 sucrose, 40 mmol l−1 KCl, 0.13 % BSA,
0.1 mmol l−1 DTNB, 40 µmol l−1 palmitoleoyl-CoA, 1 mmol l−1
carnitine, 20 mmol l−1 Hepes, pH 8.0 at 1 °C. Activity was
simultaneously measured in six cuvettes. Malonyl-CoA, a
known inhibitor of CPT-I, was added to three of the six
cuvettes to a final concentration of 10 µmol l−1. Reactions were
initiated by the addition of carnitine. Maximum activity was
measured by following the production of the reduced anion of
DTNB at 412 nm. Maximal activities of CPT-I were estimated
as the fraction of total activity inhibited in the presence of
malonyl-CoA.
Statistical analyses
Data from transmural sections (V1–V3) were pooled for
each individual. Mitochondrial and myofibril volume densities
were transformed using an arcsin transformation. Comparisons
among the three species of each stereological variable and of
the maximal activity of each enzyme were analysed using an
analysis of variance (ANOVA) with a post-hoc Fisher’s leastsignificant-difference test.
Values are presented as means ± S.E.M.
Results
Stereology
The most striking differences in ultrastructure of the heart
among the three species are differences in mitochondrial
densities (Fig. 1). Mitochondrial volume densities are highest
in Chaenocephalus aceratus, which lacks both Hb and Mb,
intermediate in Chionodraco rastrospinosus, which lacks Hb
but expresses Mb, and lowest in G. gibberifrons, which
expresses both Hb and Mb. Mitochondrial surface densities
parallel this trend, with surface densities being highest in
Chaenocephalus aceratus, intermediate in Chionodraco
rastrospinosus and lowest in G. gibberifrons (Table 1).
The mitochondria are structurally different among the three
species. Mitochondrial cristae surface densities, Sv(imm,mit),
are higher in G. gibberifrons than in Chaenocephalus
aceratus and Chionodraco rastrospinosus. Cristae surface
densities tend to be higher in Chionodraco rastrospinosus
than in Chaenocephalus aceratus, although there is no
statistically significant difference between the two (P=0.36).
The surface-to-volume ratio of mitochondria varies
among the three species, being higher in G. gibberifrons
Cardiac ultrastructure, metabolism and O2-binding proteins 1291
Fig. 1. Electron micrographs of heart ventricle from three species of
Antarctic fishes that vary in their expression of hemoglobin (Hb) and
cardiac myoglobin (Mb). Mitochondrial surface and volume
densities are significantly different among the three species (P⭐0.05)
and are correlated with the expression of oxygen-binding proteins.
(A) Gobionotothen gibberifrons (+Hb/+Mb); (B) Chionodraco
rastrospinosus (−Hb/+Mb); (C) Chaenocephalus aceratus
(−Hb/−Mb). f, myofibrils; m, mitochondrion. Scale bars, 2 µm.
Table 1. Ultrastructural characteristics of cardiac myocytes
from three species of Antarctic fishes that vary in expression
of oxygen-binding proteins
Vv(mit,f)
(%)
Sv(mit,f)
(µm−1)
Vv(myf,f)
(%)
Sv(imm,mit)
(µm−1)
Sv(imm,v)
(m2 g−1)
Gobionotothen
gibberifrons
(+Hb/+Mb)
Chionodraco
rastrospinosus
(−Hb/+Mb)
Chaenocephalus
aceratus
(−Hb/−Mb)
15.87±0.74a
20.10±0.74b
36.53±2.07c
1.19±0.05a
1.34±0.05b
1.63±0.05c
40.12±0.91a
24.50±1.26b
25.07±1.64b
29.63±1.62a
21.52±0.69b
20.04±0.79b
4.46±0.31a
4.11±0.24a
6.91±0.39b
All measurements are made with cardiac myocytes as the
reference area, unless otherwise noted.
Vv(mit,f), volume density of mitochondria; Sv(mit,f), surface
density of mitochondria; Vv(myf,f), volume density of myofibrils;
Sv(imm,mit), surface density of inner-mitochondrial membranes
per volume mitochondria; Sv(imm,v), surface density of innermitochondrial membranes per gram heart ventricle, calculated using
a value for muscle density of 1.055 g cm−3 (Webb, 1990).
Values are means ± S.E.M.; N=6 for each species.
Superscripts a, b and c denote significant differences among the
three species (P⭐0.05).
(7.55±0.26 µm−1) than in Chionodraco rastrospinosus
(6.75±0.40 µm−1)
and
Chaenocephalus
aceratus
(4.52±0.27 µm−1). Thus, the species that expresses both Hb
and Mb has small mitochondria with densely packed cristae.
The species that expresses Mb, but not Hb, has slightly larger
mitochondria and more loosely packed cristae, and the
species that lacks both Hb and Mb has very large
mitochondria with a low cristae surface density (Fig. 2).
Mitochondrial cristae surface density per gram of tissue, a
generally accepted indicator of aerobic metabolic capacity, is
higher in Chaenocephalus aceratus (6.91±0.39 m2 g−1) than in
G. gibberifrons (4.46±0.31 m2 g−1) and Chionodraco
rastrospinosus (4.11±0.24 m2 g−1) (Table 1). Myofibrillar
volume densities are higher in G. gibberifrons than in
Chaenocephalus aceratus and Chionodraco rastrospinosus,
which may reflect a greater capacity for power output in hearts
of G. gibberifrons per volume of tissue than in the other species
(Table 1).
Transmural sections of cardiac tissue from each of the three
species were subdivided into three regions, and cellular
variables were quantified within each region to determine
whether they varied among different areas of the heart. These
analyses indicate that all the cellular structures measured are
distributed homogeneously within the cardiac muscle (data not
shown).
1292 K. M. O’BRIEN AND B. D. SIDELL
Metabolic characteristics
Despite significant ultrastructural differences among the
three species, differences in mass-specific metabolic indices
(per gram of tissue) are minimal. We measured the maximal
activity per gram wet mass of several enzymes from different
metabolic pathways. Values expressed in this fashion allow us
to compare inherent metabolic capacities of cardiac muscle
tissue among the different species, despite their differences in
heart-to-body-mass ratios. The maximal mass-specific activity
of cytochrome oxidase (CO), an indicator of aerobic metabolic
capacity, is similar among the three species (Table 2). Because
Chaenocephalus aceratus has a significantly higher cristae
surface density per gram of tissue than G. gibberifrons and
Chionodraco rastrospinosus, this result suggests that electron
transport elements may not be as densely packed within the
mitochondrial inner membrane of Chaenocephalus aceratus as
in the other two species.
The maximal activity of hexokinase (HK) is generally
considered a good indicator of the capacity for aerobically
oxidizing glucose (Crabtree and Newsholme, 1972a). Massspecific HK activity is similar among the three species
(Table 2). Since the maximal activities of HK, CO and citrate
synthase (CS), another aerobically poised enzyme, are similar
among the three species, it appears that the absence of oxygenbinding proteins does not compromise the aerobic metabolic
capacity of each gram of heart tissue.
CPT-I catalyses a critical step in the translocation of longchain fatty acids into mitochondria and reflects the capacity for
fatty acid oxidation (Crabtree and Newsholme, 1972b). Massspecific CPT-I activity is higher in the two species that lack
oxygen-binding proteins (Chaenocephalus aceratus and
Chionodraco rastrospinosus) than in G. gibberifrons. These
data also indirectly imply that overall aerobic metabolic
capacity may not be compromised in the channicthyids
(Table 2).
Anaerobic metabolic capacity indexed by the maximal
mass-specific activity of PFK, a key enzyme in the glycolytic
pathway, is greatest in hearts of G. gibberifrons among the
species examined (Table 2). Thus, despite the loss of
expression of Hb and/or Mb, the hearts of channicthyids do not
appear to have a greater reliance on anerobic glycolysis to fuel
muscular work compared with red-blooded species.
Fig. 2. Electron micrographs of mitochondria from cardiac muscle of
three species of Antarctic fishes. Mitochondria differ in both the
density of the inner mitochondrial membrane and the surface-tovolume ratio among the three species. (A) Gobionotothen
gibberifrons; (B) Chionodraco rastrospinosus; (C) Chaenocephalus
aceratus. Scale bars, 0.5 µm.
Organismal capacities for cardiac work
Chionodraco rastrospinosus have a larger heart-to-bodymass ratio (4.105±0.140 g ventricle kg−1 body mass, N=27)
than the other two species examined (Chaenocephalus
aceratus 3.255±0.084 g ventricle kg−1 body mass, N=30; G.
gibberifrons, 0.715±0.016 g ventricle kg−1 body mass, N=30)
(means ± S.E.M.). Expressing maximal enzyme activites per
100 g body mass accounts for these differences and may
provide insight about the total metabolic capacity of hearts in
vivo and the organismal capacity for cardiac work. When
enzymatic activities are expressed in this fashion, Chionodraco
rastrospinosus and Chaenocephalus aceratus have the highest
capacity for cardiac aerobic metabolism, as indicated by the
Cardiac ultrastructure, metabolism and O2-binding proteins 1293
Table 2. Maximal activities of enzymes from heart ventricle from three species of Antarctic fishes
Enzyme activity (µmol min−1 g−1)
Cytochrome oxidase, CO
Citrate synthase, CS
Hexokinase, HK
Phosphofructokinase, PFK
Lactate dehydrogenase, LDH
Pyruvate kinase, PK
3-Hydroxy CoA dehydrogenase, HOAD
Carnitine palmitoyl transferase-I, CPT-I
Gobionotothen gibberifrons
(+Hb/+Mb)
Chionodraco rastrospinosus
(−Hb/+Mb)
Chaenocephalus aceratus
(−Hb/−Mb)
18.81±2.04a
13.16±0.50a
1.56±0.09a
2.10±0.10*
43.29±3.16a
15.66±0.72a
2.17±0.08a
63.91±5.37a
18.71±1.55a
11.29±0.32a
1.26±0.05a
0.31±0.09a
95.52±4.07b
14.21±0.48a
2.31±0.20a
114.87±4.71b
17.97±0.79a
12.33±0.55a
1.47±0.10a
1.07±0.04b
95.96±6.38b
10.51±0.69b
3.05±0.08b
91.07±3.35c
Activities were measured at 1±0.5 °C and are expressed as µmoles of product formed per minute per gram wet mass of tissue.
Values are means ± S.E.M.
Superscripts a, b and c denote significant differences among the three species (P⭐0.10).
N=6 except for HK assayed in hearts from Chaenocephalus aceratus, in which N=9.
*Data from Crockett and Sidell (1990).
highest activities of CO, CS and HK (Table 3). Chionodraco
rastrospinosus has the greatest capacity for fatty acid oxidation
(CPT-I). The capacity for anaerobic glycolysis, as reflected in
the maximal activity of PFK, is higher in Chaenocephalus
aceratus than in Chionodraco rastrospinosus and G.
gibberifrons. Thus, the total metabolic capacity of heart
ventricular muscle is greatest in the Channicthyidae, despite
their lack of oxygen-binding proteins.
Ultrastructural variables expressed per 100 g body mass
indicate that Chionodraco rastrospinosus and Chaenocephalus
aceratus have higher mitochondrial volumes and surface areas
than G. gibberifrons. Mitochondrial cristae surface areas
Table 3. Organismal capacity for cardiac metabolism in three
species of Antarctic fishes
Enzyme activity (µmol min−1 100 g−1 body mass)
CO
CS
HK
PFK
LDH
PK
HOAD
CPT-I
Gobionotothen
gibberifrons
(+Hb/+Mb)
Chionodraco
rastrospinosus
(−Hb/+Mb)
Chaenocephalus
aceratus
(−Hb/−Mb)
1.35±0.15a
0.94±0.04a
0.11±0.01a
0.15*
3.10±0.23a
1.12±0.05a
0.15±0.01a
4.57±0.38a
7.68±0.64b
4.63±0.13b
0.52±0.02b
0.13±0.04a
39.21±1.67b
5.83±0.20b
0.95±0.08b
47.15±1.93b
5.85±0.26c
4.01±0.18c
0.48±0.03b
0.35±0.01b
31.23±2.08c
3.42±0.22c
0.99±0.03b
29.64±1.09c
Enzyme activities are expressed as µmoles of product formed per
minute per 100 g body mass, and were assayed at 1±0.5 °C.
Data are means ± S.E.M.
Superscripts a, b and c denote significant differences among the
three species (P⭐0.10).
N=6 except HK measured in hearts from Chaenocephalus
aceratus, in which N=9.
*Data from Crockett and Sidell (1990).
Enzyme abbreviations are explained in Table 2.
expressed per 100 g body mass are also highest in Chionodraco
rastrospinosus and Chaenocephalus aceratus, as are myofibril
volumes (Table 4). These ultrastructural differences correlate
with the greater aerobic metabolic capacities per 100 g body
mass of the channicthyids.
Discussion
Our results show a clear correlation between the
evolutionary loss of oxygen-binding proteins and substantial
differences in the cellular architecture of heart ventricles in
Antarctic fishes. The heart of Chaenocephalus aceratus,
which lacks both Hb and Mb, has a considerably higher
density of mitochondria (37 %) than the heart of Chionodraco
rastrospinosus (20 %), which lacks Hb, but whose heart does
express Mb. Mitochondrial densities are lowest in hearts from
G. gibberifrons (16 %), which expresses both oxygen-binding
proteins. By comparing the hearts of Chionodraco
rastrospinosus and G. gibberifrons, we can isolate features
of the heart correlated with the loss of Hb, and by comparing
the hearts of Chaenocephalus aceratus and Chionodraco
rastrospinosus, we can examine characteristics correlated
specifically with the loss of Mb. Exploiting these
comparisons, we conclude that the loss of Hb alone is
correlated with only a modest increase in mitochondrial
volume density (4 % of cell volume), while the loss of Mb
expression correlates with a more substantial increase in the
fraction of cell volume occupied by mitochondria (17 % of
cell volume). No species of Antarctic fish has been identified
that expresses Hb and lacks cardiac Mb. Consequently, we
cannot determine whether loss of Mb, in the presence of Hb,
would result in a similar expansion of mitochondrial density.
Thus, we cannot rule out the possibility that ultrastructural
alterations in the heart of Chaenocephalus aceratus may be
due to the combined effects of the loss of both Hb and Mb
that may be greater in magnitude than the additive effects of
losing either protein separately.
1294 K. M. O’BRIEN AND B. D. SIDELL
Table 4. Organismal characteristics of cardiac ultrastructure of three species of Antarctic fishes
Ultrastructural variable
expressed per 100 g body mass
Volume of mitochondria
(mm3)
Surface area of mitochondria
(m2)
Volume of myofibrils
(mm3)
Surface area of inner-mitochondrial
membrane (m2)
Gobionotothen gibberifrons
(+Hb/+Mb)
Chionodraco rastrospinosus
(−Hb/+Mb)
Chaenocephalus aceratus
(−Hb/−Mb)
10.76±0.50a
78.21±2.88b
112.72±6.39c
0.081±0.003a
0.523±0.018b
0.502±0.015b
27.19±0.62a
95.33±4.89b
77.34±5.07c
0.319±0.022a
1.69±0.10b
2.25±0.13c
See Results section for the values of heart:body mass ratios used in calculations.
The density of muscle is 1.055 g cm−3 (see Table 1).
Values are ± S.E.M.; N=6 for each species.
Superscripts a, b and c denote significant differences among the three species (P⭐0.05).
The role of high mitochondrial densities in maintaining
oxygen diffusion
Oxygen diffusion (δO∑/δt) through muscular tissue is
described by the one-dimensional diffusion equation (Mahler
et al., 1985):
δO∑/δt = DO∑ × αO∑ × A × (PO∑/X) ,
where DO∑ is the diffusion coefficient for oxygen, αO∑ is the
solubility constant for oxygen, A is the area through which
diffusion takes place, PO∑ is the partial pressure gradient across
the diffusion pathlength X, and t is time.
During periods of intense activity, blood PO∑ levels may
decline more precipitously in hearts of icefish than in redblooded fishes because of the diminished oxygen-carrying
capacity of their blood (Ruud, 1954). Oxygen delivery to
mitochondria may be further constrained in species that lack
Mb, which serves both to facilitate oxygen diffusion and as an
intracellular reservoir of oxygen.
The architecture of cardiac myocytes in channicthyids may
compensate for the loss of respiratory proteins and contribute to
maintaining oxygen diffusion to mitochondria. All three species
studied have a type I heart, lacking a coronary circulation (Davie
and Farrell, 1991). Oxygen utilized by respiring mitochondria
must diffuse from the mixed-venous blood present in the lumen
of the heart. Because mitochondria are randomly distributed
within the ventricle of each species, as mitochondrial density
increases, the diffusion distance between the cell surface and the
mitochondrial membrane decreases, effectively reducing X in
the equation above (K. M. O’Brien and B. D. Sidell, unpublished
results). In addition, there may be differences among the three
species in the degree of trabeculation of the spongy myocardium.
The mean diffusion distance between the ventricular lumen and
the mitochondrial membrane may be reduced in hearts from
species lacking oxygen-binding proteins if they are more highly
trabeculated than hearts from fishes that express one or both of
these proteins. We are currently testing this hypothesis using a
stereologically based model developed for quantifying mean
oxygen diffusion distance within hearts from each of the three
species.
High mitochondrial densities within tissues also provide a
network of lipid-rich intracellular membranes that may act as
conduits for oxygen movement. Oxygen is more than four
times more soluble in non-polar solvents than in water (Battino
et al., 1968). The resultant higher solubility constant (αO∑) of
oxygen within lipid-rich membranes compared with aqueous
cytoplasm may enhance the rate of transcellular oxygen
diffusion in mitochondria-rich cells. The importance of
intracellular lipids in enhancing oxygen movement has been
recognized in other fishes (Egginton and Sidell, 1989;
Londraville and Sidell, 1990). Oxidative skeletal muscles from
striped bass accumulate high densities of intracellular lipid
droplets in response to cold-temperature acclimation (Egginton
and Sidell, 1989). Subsequent measurements showed that these
increases in the density of intracellular lipid droplets result in
a significant increase in the solubility constant of oxygen,
leading to an enhanced intracellular rate of oxygen diffusion
(Desaulniers et al., 1996). Although increases in membrane
densities were not accounted for in this study, several others
have highlighted the potential importance of intracellular
membranes in oxygen transport.
Longmuir (1980) reported that oxygen is transported more
rapidly between blood and mitochondria along channels of
high solubility than through the aqueous cytoplasm. He
hypothesized that the endoplasmic reticulum accounted for
these ‘channels’ of oxygen movement. Mitochondrial
membranes may serve as similar conduits for oxygen diffusion.
The properties of the lipid bilayer of a membrane appear to
determine its effectiveness in transporting oxygen.
Experiments on isolated mitochondrial and plasma membranes
from bullfrog heart tissue show that mitochondrial membranes
are less viscous than plasma membranes, resulting in a higher
diffusion coefficient for oxygen and an enhanced rate of
oxygen diffusion. The lower viscosity of mitochondrial
membranes compared with plasma membranes is due to an
increased proportion of unsaturated acyl chains within the
constituent phospholipids of the mitochondrial bilayers
(Koyama et al., 1990).
The organization of intracellular membranes may be as
Cardiac ultrastructure, metabolism and O2-binding proteins 1295
important as lipid composition in determining their
effectiveness as pathways for oxygen transport. Mitochondria
forming a continuous reticulum within a cell may provide the
best conduit for oxygen diffusion because the pathway for
oxygen diffusion is continuous (Dutta and Popel, 1995). This
would result in a higher diffusive flux compared with that in a
cell containing mitochondria separated by aqueous cytoplasm,
requiring oxygen to diffuse across a heterogeneous path of
both lipid and cytoplasm. The efficiency of oxygen transport
within a membranous network may explain why hearts from
Chaenocephalus aceratus possess such a high density of
large mitochondria. These enlarged mitochondria enable a
juxtaposition of the outer mitochondrial membranes. Because
the outer mitochondrial membranes are less protein-dense than
the inner membrane cristae, they will provide the best
membranous pathway for oxygen diffusion and may
compensate for the absence of Mb.
Under some conditions, intracellular lipids may be more
effective than myoglobin at transporting oxygen. More oxygen
is found dissolved in lipid droplets within the skeletal muscle
of cold-acclimated striped bass than bound to myoglobin
(Desaulniers et al., 1996). There are also notable differences in
the behavior of oxygen dissolved in lipid compared with
oxygen bound as a ligand to Mb. Oxygen present within
intracellular lipid is able to move freely from regions of high
PO∑ to regions of low PO∑. In contrast, oxygen bound to Mb
dissociates from the protein only at very low PO∑ levels.
Therefore, lipid may be more critical than Mb for ensuring
adequate oxygen delivery within tissue at normal activity
levels, and Mb may play a backup role, releasing oxygen only
during strenuous activity (Sidell, 1998).
Differences in mitochondrial morphology
Mitochondrial volume density is normally indicative of the
oxidative capacity of a tissue: high mitochondrial density
typically reflects high metabolic demand. Hummingbirds have
the highest mass-specific metabolic rates among vertebrates
and also possess nearly the highest mitochondrial densities
found in muscle (37 %) (Suarez et al., 1991). Thus, it may be
somewhat surprising to observe comparable mitochondrial
densities in the heart of Chaenocephalus aceratus, which lacks
Hb and Mb and is a sluggish, demersal species. Closer
examination of significant differences in the architecture of
mitochondria among the three species, however, may explain
this apparent anomaly.
Mitochondrial cristae density is also usually positively
correlated with respiration rate and oxidative capacity
(Schwerzmann et al., 1989). Cristae surface densities vary
among the three species and are inversely proportional to
mitochondrial volume densities. Cristae are more densely
packed within the mitochondria of hearts of G. gibberifrons
than in those of Chionodraco rastrospinosus and
Chaenocephalus aceratus. The lower cristae surface densities
within mitochondria suggest that icefish might have a lower
oxidative capacity than the red-blooded species. However,
when the densities of inner-mitochondrial membranes are
calculated per gram of tissue, the heart from Chaenocephalus
aceratus has a significantly greater cristae surface area than
those from the other two species, whose cristae areas are
equivalent. These results suggest that the oxidative capacity
per gram of ventricle from the myoglobinless icefish
Chaenocephalus aceratus might be greater than that of the
heart from the two species that express oxygen-binding
proteins.
To gain a better insight into the aerobic capacity of hearts
from all three species, we also measured the maximal activites
of several aerobically poised enzymes. The activity of CO is
usually proportional to respiration rate and to the surface
density of inner-mitochondrial membrane per gram of tissue.
Our results show that the maximal activity of cytochrome c
oxidase (CO) per gram of tissue is equivalent among hearts
from all three species. The apparent mismatch between cristae
surface density per gram of tissue and CO activity within the
heart of Chaenocephalus aceratus may be reconciled if the
electron transport elements are less densely packed within the
inner mitochondrial membranes than in the other two species.
Alternatively, there may be differences in the catalytic rate
constant (kcat) of CO among the three species. Because all three
species are closely related in phylogeny, however, it seems
unlikely that they would express markedly different variants of
CO. This does not, however, rule out differences in the lipid
composition of the mitochondrial membranes among these
fishes that may also affect the catalytic capacity of CO.
Metabolic capacity
In addition to the activity of CO, the maximal activities of
other enzymes from aerobic pathways (HK, CS) are also
equivalent on a mass-specific basis among hearts from the
three species, indicating that aerobic metabolic capacity is not
diminished in the absence of oxygen-binding proteins. Similar
results were reported by Driedzic and Stewart (1982), who
found no differences in the maximal activities of CO, CS and
HK between hearts from the Atlantic ocean pout Macrozoarces
americanus, which lacks Mb, and the sea raven Hemitripterus
americanus, which expresses the protein. No information is
available for these species to evaluate whether ultrastructural
differences in cardiac muscle between the species might
maintain oxygen delivery to the mitochondria and aerobic
metabolic rates.
The activities of enzymes from pathways of fatty acid
oxidation (CPT-I, HOAD) are greatest in hearts from
channicthyids. In addition, hearts from G. gibberifrons have a
higher activity of PFK compared with icefishes. These data
provide further evidence not only that aerobic metabolic
capacity is not compromised in species lacking oxygenbinding proteins but also that hearts from these species do not
appear to rely more on anerobic pathways to fuel heart work
than those of their red-blooded relatives.
In summary, the metabolic characteristics of the three
species examined were remarkably similar despite differences
in the expression of oxygen-binding proteins. We did,
however, observe striking differences in cellular architecture
1296 K. M. O’BRIEN AND B. D. SIDELL
correlated with the expression of oxygen-binding proteins. The
high densities of mitochondria within hearts of species that
lack Hb and/or Mb may contribute to maintaining oxygen flux
to mitochondria by two mechanisms. First, high mitochondrial
densities shorten the diffusion distance between the lumen
of the heart and the mitochondrial membrane. Second, the
membranous network created by large mitochondrial densities
provides a favorable pathway for oxygen movement because
of the higher solubility of oxygen in lipid than in cytoplasm.
Structural alterations in the cardiac myocytes of Antarctic
fishes that lack oxygen-binding proteins may therefore
overcome potential reductions in oxygen diffusion rates so that
aerobic metabolic capacities are equivalent to those of fishes
that express hemoglobin and/or myoglobin.
We greatly appreciate the excellent support from the
personnel at the US Antarctic research station, Palmer Station,
and the masters and crew of R/V Polar Duke. Funding for this
study was provided by US National Science Foundation
Grants OPP 92-20775 and OPP 94-21657 to B.D.S.
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