Cardiac Ultrastructure, Metabolism and O2-Binding Proteins

Home , Chaenocephalus aceratus

The Journal of Experimental Biology 203, 1287Ð1297 (2000) 1287 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 oxygen- oxidative 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 percentage of cell volume occupied by mitochondria, diffusion distance between the ventricular lumen and Vv(mit,f), which is greatest in Chaenocephalus aceratus mitochondrial membrane. Also, high mitochondrial (36.53±2.07), intermediate in Chionodraco rastrospinosus densities result in a high intracellular lipid content, which (20.10±0.74) and lowest in G. gibberifrons (15.87±0.74). may enhance oxygen diffusion because of the higher There are also differences in mitochondrial morphologies solubility of oxygen in lipid compared with cytoplasm. among the three species. The surface area of inner These results indicate that features of cardiac myocyte mitochondrial membrane per volume of mitochondria, architecture in species lacking oxygen-binding proteins Sv(imm,mit), varies inversely with mitochondrial volume may maintain oxygen flux, ensuring that aerobic metabolic density so that Sv(imm,mit) is greatest in G. gibberifrons capacity is not diminished and that cardiac function is (29.63±1.62 µm−1), lower in Chionodraco rastrospinosus maintained. (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, cristae per gram of tissue, however, is greater in myoglobin, oxygen-binding protein, icefish, Antarctic fish, Chaenocephalus aceratus than in G. gibberifrons and Gobionotothen gibberifrons, Chionodraco rastrospinosus, Chionodraco rastrospinosus, whose surface areas are Chaenocephalus aceratus.

Introduction Antarctic icefishes (Channicthyidae) are one of six families specific cardiac output that is 4–5 times greater than that of within the suborder Nototheniodei that dominates both species red-blooded teleosts (Hemmingsen et al., 1972). Blood number and biomass of fishes in the Southern Ocean (Eastman, volumes in icefish are 2–4 times greater than those of red- 1993). Icefishes are unique among all vertebrates because as blooded teleosts, and they possess unusually large-diameter adults they lack the oxygen-binding protein hemoglobin (Hb). capillaries that minimize the peripheral resistance against Because these fishes lack Hb, the oxygen-carrying capacity of which the heart must work (Hemmingsen and Douglas, 1970; their blood is only one-tenth of that of red-blooded teleosts Fitch et al., 1984). In combination, these cardiovascular (Ruud, 1954). characteristics provide a large blood volume that is circulated Channichthyids possess many unusual cardiovascular through the body at high flow to maintain oxygen delivery to features that appear to compensate for the loss of circulating working muscles. Hb. Their large heart-to-body mass ratio contributes to a mass- 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 microscopy, and cellular structures were quantified using found in oxidative muscle (Hamoir and Geradin-Otthiers, stereological techniques. We also measured the maximal 1980; Wittenberg and Wittenberg, 1989). Recent findings, activities of key enzymes from several metabolic pathways as however, have revealed the presence of this protein in heart indices of the metabolic capacities of the tissues. Because all ventricles of several species of icefishes (Sidell et al., 1997; three species are phylogenetically closely related and Moylan and Sidell, 2000). Our laboratory has recently ecotypically similarly sluggish, demersal fishes, we are examined hearts from 13 of the 15 known species of confident that differences observed in cardiac muscle can be channicthyid icefishes, and we have determined that attributed to differences in the expression of oxygen-binding myoglobin is expressed in eight of these species (Moylan and proteins rather than to lifestyle or genetic distance. 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 Materials and methods myoglobin protein expression and phylogeny (Moylan and Gobionotothen gibberifrons, Chionodraco rastrospinosus Sidell, 2000). The widely dispersed pattern of presence and and Chaenocephalus aceratus were captured using an otter absence of myoglobin within the channicthyid family initially trawl deployed from the R/V Polar Duke in Dallmann Bay suggested that the protein might not be functional at their cold (64¡N, 62¡W) at approximately 150 m depth during the austral body temperatures. Several recent studies, however, indicate autumn of 1991, 1993, 1995 and 1997 and the winter of 1996. that Mb is indeed functional in these fishes. Animals were maintained in shipboard circulating seawater Kinetic analyses reveal that Mbs from icefish and other tanks and transported to the US Antarctic Research Station, teleosts display faster rates of oxygen binding and dissociation Palmer Station, on Anvers Island. Here, they were transferred at cold temperature than mammalian Mbs (Cashon et al., to the Palmer Station aquarium and maintained unfed in 1997). Experiments with isolated, perfused hearts from covered and circulating seawater tanks at 0±0.5 ¡C. icefishes demonstrate that selective poisoning of Mb results in loss of mechanical performance by hearts that express the Tissue preparation for electron microscopy protein, but not in hearts that lack Mb (Acierno et al., 1997). Fishes were killed by a sharp blow to the head. The hearts These perfused heart experiments also show that hearts from were quickly excised and placed in an ice-cold solution −1 −1 −1 species that naturally lack Mb are capable of meeting greater (260 mmol l NaCl, 2.5 mmol l MgCl2, 5.0 mmol l KCl, −1 −1 pressure/work challenges than hearts from icefish that express 2.5 mmol l NaHCO3, 5.0 mmol l NaH2PO4, pH 8.0) and Mb in which the protein has been poisoned. These results allowed to contract for several minutes to clear them of blood. strongly indicate that Mb is functional when present and that, They were then placed in an ice-cold fixative solution (3 % to maintain cardiac function, the ultrastructural and/or glutaraldehyde, 0.1 mol l−1 sodium cacodylate, 0.11 mol l−1 −1 metabolic characteristics of hearts lacking Mb have been sucrose and 2 mmol l CaCl2, pH 7.4) and perfused with modified to compensate for loss of the protein. fixative retrogradely through the bulbous arteriosus using a Johnston and Harrison (1987) compared the ultrastructure peristaltic pump. The pump was fitted with small-diameter of the heart ventricle between a myoglobinless icefish, rubber tubing and secured within the bulbous arteriosus using Chaenocephalus aceratus, and a red-blooded nototheniid, surgical silk. Hearts were perfused for 1 min at a flow rate of Notothenia neglecta. They determined that hearts from 15 ml min−1 and then for 30 min at a flow rate of 9 ml min−1. Chaenocephalus aceratus had significantly higher They were then stored in fixative at 4 ¡C for 8Ð10 h, with a mitochondrial densities than those from N. neglecta and change of fixative after the initial 4–6 h. Hearts were then hypothesized that these high densities might enhance transferred into Trumps buffer (1 % glutaraldehyde, 4 % intracellular oxygen diffusion in hearts of species lacking Mb. formaldehyde, 0.1 mol l−1 sodium cacodylate, 0.11 mol l−1 −1 Chaenocephalus aceratus and N. neglecta, however, differ in sucrose and 2 mmol l CaCl2, pH 7.4) and stored at 4 ¡C until their expression of both oxygen-binding proteins, Hb and Mb. they were transported to our laboratory at the University of Thus, whether architectural differences observed between Maine. these hearts are correlated with the loss of Mb or Hb expression Ventricles were cut in half lengthwise, and a transmural was not definitively resolved. section spanning from epicardium to endocardium was excised A recent description of the pattern of both Mb and Hb from the center of one half of each heart. Each transmural expression among Antarctic notothenioid fishes now permits section was then subdivided into three regions: blocks closest us to differentiate between structural and metabolic to the epicardium (V1), blocks within the myocardium (V2) characteristics that are specifically correlated with the loss of and blocks nearest the endocardium (V3). Blocks were rinsed Mb and those correlated with the loss of Hb. We examined briefly in an ice-cold solution (0.1 mol l−1 sodium cacodylate, −1 heart ventricle in two species of channicthyid icefishes, 7 % sucrose, 2 mmol l CaCl2, pH 7.4), and then rinsed for Chaenocephalus aceratus (−Hb/−Mb) and Chionodraco 30 min and stored overnight at 4 ¡C in the solution. Blocks rastrospinosus (−Hb/+Mb), and a closely related red-blooded were post-fixed in an ice-cold solution of 1 % osmium nototheniid Gobionotothen gibberifrons (+Hb/+Mb). The tetroxide, 0.1 mol l−1 sodium cacodylate, 7 % sucrose and −1 ultrastructure of cardiac muscles was examined using electron 2 mmol l 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 final magnification of 96 000× using the line-intercept method concentrations of ethanol (70 %, 95 %, 100 %) and cleared with (Weibel, 1979). Micrographs of G. gibberifrons mitochondria propylene oxide. Blocks were stored overnight at room were printed for best resolution of mitochondrial inner temperature in a mixture of propylene oxide:resin (2:1) with membrane. Regions of the mitochondria with cristae shown the lids slightly ajar to allow the propylene oxide to evaporate clearly in cross section were outlined in red wax pencil, and slowly. Blocks were then infiltrated with a mixture of Epon only these areas were used for calculating cristae surface and Araldite resin for 1 h under vacuum, with a change of resin densities (Smith and Page, 1976). A subset of micrographs of after the initial 30 min, and cured at 60 ¡C for 48 h. Hearts were Chionodraco rastrospinosus and Chaenocephalus aceratus fixed on site at Palmer Station during March and April 1995 mitochondria were also printed, and cristae surface density was and post-fixed at the University of Maine between June and quantified from both prints and micrographs projected onto a July 1995. Summagraphics II digitizing tablet (N=2 per species). Because there was no significant difference between measurements Stereology made from projected micrographs and prints in these two Initially, blocks from each of the three regions of ventricle species (P=0.92), cristae surface densities were quantified described above were sampled in two animals from each of the using projected micrographs for the remaining individuals. three species. Ultrastructural variables were quantified to Micrographs from all three species were overlaid with a square determine the amount of variation in these variables within the lattice test pattern (d=0.08 µm) for analysis. ventricle. Because no variation was detected, an additional four blocks, one per individual, were randomly chosen from each Enzymology of the three species so that a total of 10 blocks per species were Tissue preparation analyzed from six individuals. Animals were killed and hearts extracted as described Blocks were first thick-sectioned (1.5 µm) with an LB4 above. Assays requiring fresh tissue, for hexokinase (HK), microtome to verify the integrity of the tissue. Sections were phosphofructokinase (PFK), cytochrome oxidase (CO) and stained with 1 % Toluidine Blue in 1 % sodium borate for 30 s carnitine palmitoyltransferase-I (CPT-I), were performed on a warm plate. Blocks were then trimmed and thin-sectioned immediately. For all other assays, pyruvate kinase (PK), lactate using a diamond knife and Sorvall MT2-B ultramicrotome. dehydrogenase (LDH), citrate synthase (CS) and 3- Sections were collected on 400 mesh copper grids and stained hydroxyacyl CoA dehydrogenase (HOAD), tissues were with 2 % uranyl acetate followed by 0.5 % lead citrate. quickly frozen in liquid nitrogen, stored at −70 ¡C and shipped Sections were viewed with a Philips CM-10 transmission on dry ice to our laboratory at the University of Maine, where electron microscope equipped with a tilting goniometer stage. they were stored at −70 ¡C. The stage was adjusted to 0 ¡ each time, ensuring that the beam For all enzymes other than CO and CPT-I (see below), was consistently perpendicular to the grid. Ten micrographs tissues were homogenized in a 10 % w/v ice-cold buffer −1 −1 −1 were taken at a magnification of 5200× for quantifying (40 mmol l Hepes, 1 mmol l EDTA, 2 mmol l MgCl2, mitochondrial surface and volume densities and myofibril pH 7.8 at 1 ¡C). Dithiothreitol (DTT; 2 mmol l−1) was added to volume densities. Ten to twelve micrographs were taken at a the buffer for the PFK, LDH and HK assays. Tissue was magnification of 39 000× for measuring mitochondrial cristae homogenized by hand using a ground-glass homogenizer. surface densities. Micrographs were taken using the aligned Homogenates were further reduced by brief (3Ð5 s) treatment systematic quadrats subsampling method (Cruz-Orive and with a Tekmar Tissuemizer and finally homogenized to Weibel, 1981). Individual mitochondria with the most clearly completion by hand using a ground-glass homogenizer. defined inner mitochondrial membrane were chosen for All assays were performed in triplicate at 1±0.5 ¡C using a micrographs from within each randomly chosen field of view at Perkin Elmer Lambda 6 spectrophotometer. Temperature was a magnification of 39 000×. Calibration grids were photographed maintained using a refrigerated, circulating water bath attached at each magnification to calculate final magnifications. to the spectrophotometer. Background activity was measured Mitochondrial and myofibril volume densities were in the absence of initiating substrate. Assay conditions are quantified using point-counting methods; mitochondrial described in detail below. Maximal activities were determined surface densities were measured using the line-intercept by measuring the rate of oxidation or reduction of pyridine technique (Weibel, 1979). Micrographs were projected onto a nucleotides at 340 nm for 5 min, except when noted otherwise Summagraphics II digitizing tablet at a final magnification of below. 13 400×. Images were overlaid with a square lattice test pattern Phosphofructokinase (EC 2.7.1.11). The methodology with spacing equal to 1.34 µm on projected micrographs. Care employed was slightly modified from that described by Opie was taken to exclude epithelial cells, endothelial cells, blood and Newsholme (1967) and Read et al. (1977). The final −1 −1 cells, extracellular matrix and luminal spaces from the reaction mixture contained 7 mmol l MgCl2, 200 mmol l measurements. 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), Calculation of inner mitochondrial membrane densities 2 units ml−1 aldolase, 10 units ml−1 triosephosphate isomerase, Mitochondrial cristae surface densities were quantified at a 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 total CPT-I activity. The homogenate was centrifuged at 270 g addition of a mixture of ATP and F6P. for 10 min. The supernatant was collected and centrifuged at Lactate dehydrogenase (EC 1.1.1.27). The procedure for 270 g. The supernatant was again collected and centrifuged at this assay was that described by Hansen and Sidell (1983). The 15 000 g for 20 min. The mitochondrial pellet was gently final reaction mixture contained 2.5 mmol l−1 pyruvate, resuspended in homogenization buffer (minus BSA) and 0.15 mmol l−1 NADH, 1 mmol l−1 KCN, 50 mmol l−1 imidazole, centrifuged at 15 000 g for 20 min. The resultant pellet was pH 7.7 at 1 ¡C. Reactions were initiated by the addition of gently resuspended in homogenization buffer lacking BSA to pyruvate. give a final concentration of approximately 5 µg protein µl−1. A Pyruvate kinase (EC 2.7.1.40). The method used for this sample of the mitochondrial suspension was frozen at −70 ¡C assay was that described by Hansen and Sidell (1983). The for later protein determination using the bicinchoninic acid final reaction mixture contained 150 mmol l−1 KCl, 1 mmol l−1 method (Smith et al., 1985). −1 −1 −1 −1 KCN, 10 mmol l MgSO4, 0.15 mmol l NADH, 5 mmol l The final assay medium consisted of 1.0 mmol l EGTA, ADP, 2.5 mmol l−1 phosphoenolpyruvate (PEP), 10 units ml−1 220 mmol l−1 sucrose, 40 mmol l−1 KCl, 0.13 % BSA, LDH, 50 mmol l−1 imidazole, pH 7.1 at 1 ¡C. Reactions were 0.1 mmol l−1 DTNB, 40 µmol l−1 palmitoleoyl-CoA, 1 mmol l−1 initiated by the addition of PEP. carnitine, 20 mmol l−1 Hepes, pH 8.0 at 1 ¡C. Activity was 3-Hydroxyacyl CoA dehydrogenase (EC 1.1.1.35). The simultaneously measured in six cuvettes. Malonyl-CoA, a protocol for this assay was that originally described by known inhibitor of CPT-I, was added to three of the six Beenakkers et al. (1967) as modified by Hansen and Sidell cuvettes to a final concentration of 10 µmol l−1. Reactions were (1983). The final reaction mixture contained 1 mmol l−1 EDTA, initiated by the addition of carnitine. Maximum activity was 1 mmol l−1 KCN, 0.15 mmol l−1 NADH, 0.1 mmol l−1 measured by following the production of the reduced anion of acetoacetyl CoA, 50 mmol l−1 imidazole, pH 7.7 at 1 ¡C. DTNB at 412 nm. Maximal activities of CPT-I were estimated Reactions were initiated by the addition of acetoacetyl CoA. as the fraction of total activity inhibited in the presence of Citrate synthase (EC 4.1.3.7). For this assay, we used a malonyl-CoA. modification of the protocol originally described by Srere et al. (1963). The final reaction mixture contained 0.25 mmol l−1 Statistical analyses 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 0.4 mmol l−1 Data from transmural sections (V1ÐV3) were pooled for acetyl CoA, 0.5 mmol l−1 oxaloacetate, 75 mmol l−1 Tris-HCl, each individual. Mitochondrial and myofibril volume densities pH 8.2 at 1 ¡C. The reaction was initiated by the addition of were transformed using an arcsin transformation. Comparisons oxaloacetate. The progress of the reaction was monitored by among the three species of each stereological variable and of following the production of the reduced anion of DTNB at the maximal activity of each enzyme were analysed using an 412 nm. analysis of variance (ANOVA) with a post-hoc Fisher’s least- Hexokinase (EC 2.7.1.1). This assay was modified from that significant-difference test. described by Zammit and Newsholme (1976). The final Values are presented as means ± S.E.M. −1 −1 reaction mixture contained 7.5 mmol l MgCl2, 0.8 mmol l 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-D- Results glucose, 0.9 units ml−1 creatine phosphokinase, 0.7 units ml−1 Stereology glucose-6-phosphate dehydrogenase, 75 mmol l−1 Tris-HCl, The most striking differences in ultrastructure of the heart pH 7.6 at 1 ¡C. Reactions were initiated by the addition of among the three species are differences in mitochondrial glucose. densities (Fig. 1). Mitochondrial volume densities are highest Cytochrome oxidase (EC 1.9.3.1). The method of Wharton in Chaenocephalus aceratus, which lacks both Hb and Mb, and Tzagoloff (1967) was used to measure activity. Tissue was intermediate in Chionodraco rastrospinosus, which lacks Hb −1 homogenized in 50 mmol l K2HPO4/KH2PO4, 0.05 % Triton but expresses Mb, and lowest in G. gibberifrons, which X-100, pH 7.5. The assay medium consisted of 10 mmol l−1 expresses both Hb and Mb. Mitochondrial surface densities 2+ K2HPO4/KH2PO4, 0.65 % (w/v) reduced (Fe ) cytochrome c parallel this trend, with surface densities being highest in −1 and 0.93 mmol l K3Fe(CN)6. The reaction was initiated by Chaenocephalus aceratus, intermediate in Chionodraco the addition of enzyme. Maximal activities were measured by rastrospinosus and lowest in G. gibberifrons (Table 1). following the oxidation of reduced cytochrome c at 550 nm. The mitochondria are structurally different among the three Carnitine palmitoyltransferase-I (EC 2.3.1.21). Maximal species. Mitochondrial cristae surface densities, Sv(imm,mit), activities of CPT-I were measured in intact isolated are higher in G. gibberifrons than in Chaenocephalus mitochondria (Rodnick and Sidell, 1994). Tissue was aceratus and Chionodraco rastrospinosus. Cristae surface homogenized in 10 % (w/v) of ice-cold 40 mmol l−1 Hepes, densities tend to be higher in Chionodraco rastrospinosus −1 −1 −1 10 mmol l EDTA, 5 mmol l MgCl2, 150 mmol l KCl, than in Chaenocephalus aceratus, although there is no 35 mmol l−1 sucrose, and 0.5 % bovine serum albumin (BSA), statistically significant difference between the two (P=0.36). pH 7.27 at 1 ¡C, using a Duall ground-glass homogenizer. A The surface-to-volume ratio of mitochondria varies sample of the crude homogenate was reserved for measuring 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 (Pр0.05) 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 Gobionotothen Chionodraco Chaenocephalus gibberifrons rastrospinosus aceratus (+Hb/+Mb) (−Hb/+Mb) (−Hb/−Mb) a b c Vv(mit,f) 15.87±0.74 20.10±0.74 36.53±2.07 (%) a b c Sv(mit,f) 1.19±0.05 1.34±0.05 1.63±0.05 (µm−1) a b b Vv(myf,f) 40.12±0.91 24.50±1.26 25.07±1.64 (%) a b b Sv(imm,mit) 29.63±1.62 21.52±0.69 20.04±0.79 (µm−1) a a b Sv(imm,v) 4.46±0.31 4.11±0.24 6.91±0.39 (m2 g−1)

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 inner- mitochondrial 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 (Pр0.05).

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 (7.55±0.26 µm−1) than in Chionodraco rastrospinosus species were subdivided into three regions, and cellular (6.75±0.40 µm−1) and Chaenocephalus aceratus variables were quantified within each region to determine (4.52±0.27 µm−1). Thus, the species that expresses both Hb whether they varied among different areas of the heart. These and Mb has small mitochondria with densely packed cristae. analyses indicate that all the cellular structures measured are The species that expresses Mb, but not Hb, has slightly larger distributed homogeneously within the cardiac muscle (data not mitochondria and more loosely packed cristae, and the 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). Mass- specific 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 oxygen- binding proteins does not compromise the aerobic metabolic capacity of each gram of heart tissue. CPT-I catalyses a critical step in the translocation of long- chain fatty acids into mitochondria and reflects the capacity for fatty acid oxidation (Crabtree and Newsholme, 1972b). Mass- specific 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. Organismal capacities for cardiac work Chionodraco rastrospinosus have a larger heart-to-body- mass 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) Fig. 2. Electron micrographs of mitochondria from cardiac muscle of (means ± S.E.M.). Expressing maximal enzyme activites per three species of Antarctic fishes. Mitochondria differ in both the 100 g body mass accounts for these differences and may density of the inner mitochondrial membrane and the surface-to- provide insight about the total metabolic capacity of hearts in volume ratio among the three species. (A) Gobionotothen vivo and the organismal capacity for cardiac work. When gibberifrons; (B) Chionodraco rastrospinosus; (C) Chaenocephalus aceratus. Scale bars, 0.5 µm. 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) Gobionotothen gibberifrons Chionodraco rastrospinosus Chaenocephalus aceratus (+Hb/+Mb) (−Hb/+Mb) (−Hb/−Mb) Cytochrome oxidase, CO 18.81±2.04a 18.71±1.55a 17.97±0.79a Citrate synthase, CS 13.16±0.50a 11.29±0.32a 12.33±0.55a Hexokinase, HK 1.56±0.09a 1.26±0.05a 1.47±0.10a Phosphofructokinase, PFK 2.10±0.10* 0.31±0.09a 1.07±0.04b Lactate dehydrogenase, LDH 43.29±3.16a 95.52±4.07b 95.96±6.38b Pyruvate kinase, PK 15.66±0.72a 14.21±0.48a 10.51±0.69b 3-Hydroxy CoA dehydrogenase, HOAD 2.17±0.08a 2.31±0.20a 3.05±0.08b Carnitine palmitoyl transferase-I, CPT-I 63.91±5.37a 114.87±4.71b 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 (Pр0.10). 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 expressed per 100 g body mass are also highest in Chionodraco rastrospinosus has the greatest capacity for fatty acid oxidation rastrospinosus and Chaenocephalus aceratus, as are myofibril (CPT-I). The capacity for anaerobic glycolysis, as reflected in volumes (Table 4). These ultrastructural differences correlate the maximal activity of PFK, is higher in Chaenocephalus with the greater aerobic metabolic capacities per 100 g body aceratus than in Chionodraco rastrospinosus and G. mass of the channicthyids. gibberifrons. Thus, the total metabolic capacity of heart ventricular muscle is greatest in the Channicthyidae, despite their lack of oxygen-binding proteins. Discussion Ultrastructural variables expressed per 100 g body mass Our results show a clear correlation between the indicate that Chionodraco rastrospinosus and Chaenocephalus evolutionary loss of oxygen-binding proteins and substantial aceratus have higher mitochondrial volumes and surface areas differences in the cellular architecture of heart ventricles in than G. gibberifrons. Mitochondrial cristae surface areas Antarctic fishes. The heart of Chaenocephalus aceratus, which lacks both Hb and Mb, has a considerably higher Table 3. Organismal capacity for cardiac metabolism in three density of mitochondria (37 %) than the heart of Chionodraco species of Antarctic fishes rastrospinosus (20 %), which lacks Hb, but whose heart does express Mb. Mitochondrial densities are lowest in hearts from µ −1 −1 Enzyme activity ( mol min 100 g body mass) G. gibberifrons (16 %), which expresses both oxygen-binding Gobionotothen Chionodraco Chaenocephalus proteins. By comparing the hearts of Chionodraco gibberifrons rastrospinosus aceratus rastrospinosus and G. gibberifrons, we can isolate features (+Hb/+Mb) (−Hb/+Mb) (−Hb/−Mb) of the heart correlated with the loss of Hb, and by comparing CO 1.35±0.15a 7.68±0.64b 5.85±0.26c the hearts of Chaenocephalus aceratus and Chionodraco CS 0.94±0.04a 4.63±0.13b 4.01±0.18c rastrospinosus, we can examine characteristics correlated HK 0.11±0.01a 0.52±0.02b 0.48±0.03b specifically with the loss of Mb. Exploiting these PFK 0.15* 0.13±0.04a 0.35±0.01b comparisons, we conclude that the loss of Hb alone is LDH 3.10±0.23a 39.21±1.67b 31.23±2.08c correlated with only a modest increase in mitochondrial a b c PK 1.12±0.05 5.83±0.20 3.42±0.22 volume density (4 % of cell volume), while the loss of Mb a b b HOAD 0.15±0.01 0.95±0.08 0.99±0.03 expression correlates with a more substantial increase in the CPT-I 4.57±0.38a 47.15±1.93b 29.64±1.09c fraction of cell volume occupied by mitochondria (17 % of Enzyme activities are expressed as µmoles of product formed per cell volume). No species of Antarctic fish has been identified minute per 100 g body mass, and were assayed at 1±0.5 °C. that expresses Hb and lacks cardiac Mb. Consequently, we Data are means ± S.E.M. cannot determine whether loss of Mb, in the presence of Hb, Superscripts a, b and c denote significant differences among the would result in a similar expansion of mitochondrial density. three species (Pр0.10). Thus, we cannot rule out the possibility that ultrastructural N=6 except HK measured in hearts from Chaenocephalus alterations in the heart of Chaenocephalus aceratus may be aceratus, in which N=9. due to the combined effects of the loss of both Hb and Mb *Data from Crockett and Sidell (1990). that may be greater in magnitude than the additive effects of Enzyme abbreviations are explained in Table 2. 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 Gobionotothen gibberifrons Chionodraco rastrospinosus Chaenocephalus aceratus expressed per 100 g body mass (+Hb/+Mb) (−Hb/+Mb) (−Hb/−Mb) Volume of mitochondria 10.76±0.50a 78.21±2.88b 112.72±6.39c (mm3) Surface area of mitochondria 0.081±0.003a 0.523±0.018b 0.502±0.015b (m2) Volume of myoﬁbrils 27.19±0.62a 95.33±4.89b 77.34±5.07c (mm3) Surface area of inner-mitochondrial 0.319±0.022a 1.69±0.10b 2.25±0.13c membrane (m2)

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 (Pр0.05).

The role of high mitochondrial densities in maintaining High mitochondrial densities within tissues also provide a oxygen diffusion network of lipid-rich intracellular membranes that may act as Oxygen diffusion (δO∑/δt) through muscular tissue is conduits for oxygen movement. Oxygen is more than four described by the one-dimensional diffusion equation (Mahler times more soluble in non-polar solvents than in water (Battino et al., 1985): et al., 1968). The resultant higher solubility constant (αO∑) of oxygen within lipid-rich membranes compared with aqueous δ ∑/δt = D ∑ ×α ∑ × A × (P ∑/X), O O O O cytoplasm may enhance the rate of transcellular oxygen where DO∑ is the diffusion coefficient for oxygen, αO∑ is the diffusion in mitochondria-rich cells. The importance of solubility constant for oxygen, A is the area through which intracellular lipids in enhancing oxygen movement has been diffusion takes place, PO∑ is the partial pressure gradient across recognized in other fishes (Egginton and Sidell, 1989; the diffusion pathlength X, and t is time. Londraville and Sidell, 1990). Oxidative skeletal muscles from During periods of intense activity, blood PO∑ levels may striped bass accumulate high densities of intracellular lipid decline more precipitously in hearts of icefish than in red- droplets in response to cold-temperature acclimation (Egginton blooded fishes because of the diminished oxygen-carrying and Sidell, 1989). Subsequent measurements showed that these capacity of their blood (Ruud, 1954). Oxygen delivery to increases in the density of intracellular lipid droplets result in mitochondria may be further constrained in species that lack a significant increase in the solubility constant of oxygen, Mb, which serves both to facilitate oxygen diffusion and as an leading to an enhanced intracellular rate of oxygen diffusion intracellular reservoir of oxygen. (Desaulniers et al., 1996). Although increases in membrane The architecture of cardiac myocytes in channicthyids may densities were not accounted for in this study, several others compensate for the loss of respiratory proteins and contribute to have highlighted the potential importance of intracellular maintaining oxygen diffusion to mitochondria. All three species membranes in oxygen transport. studied have a type I heart, lacking a coronary circulation (Davie Longmuir (1980) reported that oxygen is transported more and Farrell, 1991). Oxygen utilized by respiring mitochondria rapidly between blood and mitochondria along channels of must diffuse from the mixed-venous blood present in the lumen high solubility than through the aqueous cytoplasm. He of the heart. Because mitochondria are randomly distributed hypothesized that the endoplasmic reticulum accounted for within the ventricle of each species, as mitochondrial density these ‘channels’ of oxygen movement. Mitochondrial increases, the diffusion distance between the cell surface and the membranes may serve as similar conduits for oxygen diffusion. mitochondrial membrane decreases, effectively reducing X in The properties of the lipid bilayer of a membrane appear to the equation above (K. M. O’Brien and B. D. Sidell, unpublished determine its effectiveness in transporting oxygen. results). In addition, there may be differences among the three Experiments on isolated mitochondrial and plasma membranes species in the degree of trabeculation of the spongy myocardium. from bullfrog heart tissue show that mitochondrial membranes The mean diffusion distance between the ventricular lumen and are less viscous than plasma membranes, resulting in a higher the mitochondrial membrane may be reduced in hearts from diffusion coefficient for oxygen and an enhanced rate of species lacking oxygen-binding proteins if they are more highly oxygen diffusion. The lower viscosity of mitochondrial trabeculated than hearts from fishes that express one or both of membranes compared with plasma membranes is due to an these proteins. We are currently testing this hypothesis using a increased proportion of unsaturated acyl chains within the stereologically based model developed for quantifying mean constituent phospholipids of the mitochondrial bilayers oxygen diffusion distance within hearts from each of the three (Koyama et al., 1990). species. The organization of intracellular membranes may be as Cardiac ultrastructure, metabolism and O2-binding proteins 1295 important as lipid composition in determining their calculated per gram of tissue, the heart from Chaenocephalus effectiveness as pathways for oxygen transport. Mitochondria aceratus has a significantly greater cristae surface area than forming a continuous reticulum within a cell may provide the those from the other two species, whose cristae areas are best conduit for oxygen diffusion because the pathway for equivalent. These results suggest that the oxidative capacity oxygen diffusion is continuous (Dutta and Popel, 1995). This per gram of ventricle from the myoglobinless icefish would result in a higher diffusive flux compared with that in a Chaenocephalus aceratus might be greater than that of the cell containing mitochondria separated by aqueous cytoplasm, heart from the two species that express oxygen-binding requiring oxygen to diffuse across a heterogeneous path of proteins. both lipid and cytoplasm. The efficiency of oxygen transport To gain a better insight into the aerobic capacity of hearts within a membranous network may explain why hearts from from all three species, we also measured the maximal activites Chaenocephalus aceratus possess such a high density of of several aerobically poised enzymes. The activity of CO is large mitochondria. These enlarged mitochondria enable a usually proportional to respiration rate and to the surface juxtaposition of the outer mitochondrial membranes. Because density of inner-mitochondrial membrane per gram of tissue. the outer mitochondrial membranes are less protein-dense than Our results show that the maximal activity of cytochrome c the inner membrane cristae, they will provide the best oxidase (CO) per gram of tissue is equivalent among hearts membranous pathway for oxygen diffusion and may from all three species. The apparent mismatch between cristae compensate for the absence of Mb. surface density per gram of tissue and CO activity within the Under some conditions, intracellular lipids may be more heart of Chaenocephalus aceratus may be reconciled if the effective than myoglobin at transporting oxygen. More oxygen electron transport elements are less densely packed within the is found dissolved in lipid droplets within the skeletal muscle inner mitochondrial membranes than in the other two species. of cold-acclimated striped bass than bound to myoglobin Alternatively, there may be differences in the catalytic rate (Desaulniers et al., 1996). There are also notable differences in constant (kcat) of CO among the three species. Because all three the behavior of oxygen dissolved in lipid compared with species are closely related in phylogeny, however, it seems oxygen bound as a ligand to Mb. Oxygen present within unlikely that they would express markedly different variants of intracellular lipid is able to move freely from regions of high CO. This does not, however, rule out differences in the lipid PO∑ to regions of low PO∑. In contrast, oxygen bound to Mb composition of the mitochondrial membranes among these dissociates from the protein only at very low PO∑ levels. fishes that may also affect the catalytic capacity of CO. Therefore, lipid may be more critical than Mb for ensuring adequate oxygen delivery within tissue at normal activity Metabolic capacity levels, and Mb may play a backup role, releasing oxygen only In addition to the activity of CO, the maximal activities of during strenuous activity (Sidell, 1998). other enzymes from aerobic pathways (HK, CS) are also equivalent on a mass-specific basis among hearts from the Differences in mitochondrial morphology three species, indicating that aerobic metabolic capacity is not Mitochondrial volume density is normally indicative of the diminished in the absence of oxygen-binding proteins. Similar oxidative capacity of a tissue: high mitochondrial density results were reported by Driedzic and Stewart (1982), who typically reflects high metabolic demand. Hummingbirds have found no differences in the maximal activities of CO, CS and the highest mass-specific metabolic rates among vertebrates HK between hearts from the Atlantic ocean pout Macrozoarces and also possess nearly the highest mitochondrial densities americanus, which lacks Mb, and the sea raven Hemitripterus found in muscle (37 %) (Suarez et al., 1991). Thus, it may be americanus, which expresses the protein. No information is somewhat surprising to observe comparable mitochondrial available for these species to evaluate whether ultrastructural densities in the heart of Chaenocephalus aceratus, which lacks differences in cardiac muscle between the species might Hb and Mb and is a sluggish, demersal species. Closer maintain oxygen delivery to the mitochondria and aerobic examination of significant differences in the architecture of metabolic rates. mitochondria among the three species, however, may explain The activities of enzymes from pathways of fatty acid this apparent anomaly. oxidation (CPT-I, HOAD) are greatest in hearts from Mitochondrial cristae density is also usually positively channicthyids. In addition, hearts from G. gibberifrons have a correlated with respiration rate and oxidative capacity higher activity of PFK compared with icefishes. These data (Schwerzmann et al., 1989). Cristae surface densities vary provide further evidence not only that aerobic metabolic among the three species and are inversely proportional to capacity is not compromised in species lacking oxygen- mitochondrial volume densities. Cristae are more densely binding proteins but also that hearts from these species do not packed within the mitochondria of hearts of G. gibberifrons appear to rely more on anerobic pathways to fuel heart work than in those of Chionodraco rastrospinosus and than those of their red-blooded relatives. Chaenocephalus aceratus. The lower cristae surface densities In summary, the metabolic characteristics of the three within mitochondria suggest that icefish might have a lower species examined were remarkably similar despite differences oxidative capacity than the red-blooded species. However, in the expression of oxygen-binding proteins. We did, when the densities of inner-mitochondrial membranes are however, observe striking differences in cellular architecture 1296 K. M. O’BRIEN AND B. D. SIDELL correlated with the expression of oxygen-binding proteins. 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