The Journal of Experimental Biology 203, 1287–1297 (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 8–10 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