KRISTIN M. O'BRIEN and BRUCE D. SIDELL, School of Marine Sciences, University of Maine, Orono, Maine 04469
Channicthyidae, a family of antarctic fishes, are unique among all vertebrates because they lack the oxygen-carrying protein, hemoglobin. Much is known about the cardiovascular adapations of these fish that allow them to survive despite the loss of hemoglobin:
These adjustments in the cardiovascular system allow a large volume of blood to be pumped through the circulatory system at high flow against only a small peripheral resistance; this configuration maintains oxygen delivery to working muscles.
Not only do Channicthyids (icefishes) lack hemoglobin, but some species within this family also lack myoglobin in their oxidative muscle tissue (Sidell 1997). Myoglobin is the intracellular oxygen-binding protein that is important for the storage and transport of oxygen within muscle cells (Wittenberg and Wittenberg 1989). Johnston and Harrison (1987) studied hearts from icefish and determined that modifications in the architecture of cardiac myocytes may enhance oxygen delivery to the mitochondria in those species that lack myoglobin. They compared the ultrastructure of ventricles between the hemoglobinless Chaenocephalus aceratus and the red-blooded Notothenia neglecta . Hearts of C. aceratus had greater mitochondrial surface and volume densities compared to the hearts of N. neglecta . Johnston and Harrison concluded that elevated mitochondrial density in myocytes compensated for the loss of myoglobin, because increased mitochondrial surface and volume densities decrease the diffusion distance of oxygen between the lumen of the heart and the mitochondrial membrane. However, these two species also differ in the presence and absence of hemoglobin, and therefore, the observed increase in mitochondrial volume and surface densities could also reflect the loss of hemoglobin in C. aceratus .
The purpose of our study was to differentiate between cardiac myocyte modifications that result from the loss of hemoglobin from those that result from the loss of myoglobin. Three species of notothenioids were examined in this study:
We examined differences in the architecture of the heart that correlate with the loss of myoglobin by comparing hearts of C. aceratus and C. rastrospinosus. We determined differences in ultrastructure that correlate with the loss of hemoglobin by comparing hearts of C. rastrospinosus and G. gibberifrons. All three species are closely related and are sedentary, benthic fishes, so any differences observed in the hearts are likely due to differences in the expression of hemoproteins.
Fishes were collected with an otter trawl in Dallman Bay and transported to Palmer Station where they were maintained in running sea water. Fishes were killed by a sharp blow to the head and heart ventricles were excised and perfused with an ice-cold ringer solution. Ventricles were then slowly perfused with an ice-cold fixative solution of 3 percent glutaraldehyde, 0.1 mol (mol) sodium cacodylate, 0.11 mol sucrose and 2 millimole (mmol) calcium chloride. Ventricles were stored in fixative at 4°C for 8 hours and then transferred to a fixative solution of 1 percent glutaraldehyde, 4 percent formaldehyde, 0.1 mol sodium cacodylate, 0.11 mol sucrose, and 2 mmol calcium chloride. Tissues were transported to our laboratory at the University of Maine, postfixed in 1 percent osmium tertroxide, and embedded in resin. Tissue blocks were thin-sectioned (approximately 80 nanometers) and placed on 400 mesh copper grids. Sections were stained with 2 percent uranyl acetate followed by 0.5 percent lead citrate. Tissue sections were viewed and photographed using a Phillips CM 10 transmission electron microscope. Micrographs were projected onto a digitizing tablet and ultrastructural parameters were quantified using point-counting methods (Weibel 1979). Parameters measured included mitochondrial volume density, mitochondrial surface density, mitochondrial cristae surface density, and myofibril volume density.
Mitochondrial volume density in hearts of C. rastrospinosus is greater than that found for G. gibberifrons (p=0.06) (figure). There is also a significant difference in the mitochondrial surface density between the two species (table). Thus, loss of hemoglobin correlates with an increase in the number and volume of mitochondria per myocyte. This increase, however, is not as great as that which results from the loss of myoglobin.
Mitochondria occupy nearly 37 percent of cell volume in the hearts of C. aceratus . This volume is significantly greater than the mitochondrial volume density in hearts of C. rastrospinosus or G. gibberifrons (table) and suggests that loss of myoglobin accounts for the large increase in mitochondrial volume density, not the loss of hemoglobin.
The electron transport machinery is located on the inner mitochondrial membrane, and thus, density of inner mitochondrial membrane reflects capacity for oxidative phosphorylation. Among the species of antarctic fishes that we examined, surface density of inner mitochondrial membrane per mitochondrial volume varied inversely with the percentage of cell volume displaced by mitochondria. The result is that mitochondrial cristae density per volume tissue remains fairly constant. This finding is consistent with our measurements of the activity of cytochrome oxidase (CO), the terminal electron acceptor in the electron transport chain. The activity of CO per gram wet weight of ventricle tissue is equal among the three species (O'Brien and Sidell unpublished data). Thus, aerobic metabolic capacity per gram heart muscle may be equal between the three species, despite differential expression of hemoproteins.
Increases in mitochondrial surface and volume densities decrease the oxygen diffusion distance between the lumen of the heart and the mitochondrial membrane. This may enhance oxygen delivery and maintain aerobic metabolic capacity when oxygen-binding proteins are absent. These ultrastructural modifications are more pronounced in species that lack both hemoglobin and myoglobin, compared to species that lack only hemoglobin.
We greatly appreciate the support from the staff at Palmer Station and the masters and crew of the R/V Polar Duke . This research was supported by National Science Foundation grant OPP 94-21657 to Bruce D. Sidell.
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