Phenylhydrazine-induced anemia causes nitric-oxide-mediated upregulation of the angiogenic pathway in Notothenia coriiceps

Abstract

SUMMARYAntarctic icefishes possess several cardiovascular characteristics that enable them to deliver oxygen adequately in the absence of hemoglobin (Hb). To gain insight into mechanisms driving development of these cardiovascular characteristics of icefish, we chemically induced severe anemia in a red-blooded notothenioid, Notothenia coriiceps. After 10 days of treatment with phenylhydrazine HCl, the hematocrit and Hb concentration of N. coriiceps decreased by >90% and >70%, respectively. Anemic fish exhibited a significantly higher concentration of nitric oxide (NO) metabolites in their plasma compared with that of control animals, indicating that corporeal levels of NO are higher in anemic animals than in control fish. The activity of nitric oxide synthase (NOS) was measured in brain, retina, pectoral muscle and ventricle of control and anemic animals. With the exception of retina, no significant differences in NOS activities were observed, indicating that the increase in plasma NO metabolites is due to loss of Hb, which normally plays a major role in the degradation of NO, and not due to an overall increase in the capacity for NO production. To determine whether loss of Hb can stimulate remodeling of the cardiovascular system, we measured expression of HIF-1a, PHD2 and VEGF mRNA in retinae of control and anemic fish. Expression of all three genes was higher in anemic animals compared with control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that probably is mediated through nitric oxide signaling.

Hemoglobin α in the blood vessel wall

“…Surgically implanted osmotic pumps were used to treat individuals with a powerful hemolytic agent that resulted in a drastic reduction in hematocrit (>90%) and HbA concentration (>70%). Levels of NO were significantly higher in anemic animals compared to the full HbA-expressing controls and were similar to the levels of NO reported for white-blooded icefishes 72,73 . Although MEJs have not been identified in these fish, it would be surprising if these anatomical structures weren’t present.…”

“…Building upon the above work, Borley and collaborators subsequently conducted a study in which they induced severe anemia in Notothenia coriiceps , an HbA-expressing notothenioid with a normal hematocrit of 35–40% 73 . Surgically implanted osmotic pumps were used to treat individuals with a powerful hemolytic agent that resulted in a drastic reduction in hematocrit (>90%) and HbA concentration (>70%).…”

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change

“…Although the cryonotothenioid radiation began~22 Ma [30,31], the stably-cold temperatures and high oxygen concentrations of the SO necessary to facilitate viable reduction in hematocrit emerged well after the initial divergence of the group (Fig 1C, S3 Fig). Therefore, we propose that reduced hematocrits and tolerance of experimental anemia in several lineages of red-blooded notothenioids evolved independently of the icefish phenotypes [5,6].…”

“…Intriguingly, several red-blooded Antarctic notothenioids survive experimentally induced anemia. Treatment of the bullhead notothen, Notothenia coriiceps, with the hemolytic agent phenylhydrazine reduces the percentage of erythrocytes in blood from 35% to 4% without lethality [5]. Similarly, the notothen, Trematomus bernacchii, survives conversion of its hemoglobin to the inactive carbonmonoxy state (95% CO-Hb) [6]; in contrast, CO-Hb exceeding 40% is lethal in humans [7].…”

Antarctic notothenioid fish: what are the future consequences of ‘losses’ and ‘gains’ acquired during long-term evolution at cold and stable temperatures?

“…Icefishes largely compensate for the lack of Hb by utilizing a suite of cardiovascular system modifications: large-bore blood vessels, which are present in high density in some tissues; large blood volumes; and large hearts containing more mitochondria than hearts of similar-sized Hbexpressing fishes (Hemmingsen and Douglas, 1970;Holeton, 1970;Hemmingsen et al, 1972;Fitch et al, 1984;O'Brien and Sidell, 2000;Wujcik, et al, 2007). There is some evidence to support the hypothesis that some of these characteristics or 'hallmark traits' of icefishes, such as enhanced vascular densities (Wujcik et al, 2007), may have originated via pathways mediated by nitric oxide (Sidell and O'Brien, 2006;Beers et al, 2010;Borley et al, 2010). Whatever their mechanism of origin, integration of all of these features allows channichthyids to circulate large blood volumes at relatively high flow rates and, importantly, without excessive pressure development.…”