Physiological and Evolutionary Implications of the Pattern of Expression of Oxygen-Binding Hemoproteins in Antarctic Notothenioid Fishes

Antarctic icefish do not express hemoglobin (Hb). Icefishes possess cardiovascular modifications including increased densities of blood vessels, larger ventricles and increased blood volume compared to red-blooded relatives. In addition to delivering oxygen to tissues, Hb degrades nitric oxide (NO), a small signaling molecule. To investigate the mechanism driving development of icefish cardiovascular characteristics, I present and test the hypothesis that loss of Hb results in increased steady-state levels of NO, triggering downstream signaling pathways such as angiogenesis. I measured NO breakdown products, as a proxy for NO, and found that icefish have higher steady-state levels of NO metabolites in their plasma compared to red-blooded notothenioids. Expression of angiogenesis genes (HIF-1α, PHD2, and VEGF) did not differ significantly between red- and white-blooded notothenioids indicating that, while NO levels are higher in adult icefish, angiogenesis is not active. To investigate whether loss of Hb directly can increase NO and stimulate angiogenesis, hematocrit of red-blooded N. coriiceps was severely reduced using the hemolytic agent, phenylhydrazine HCl. Anemic fish exhibited a significantly higher concentration of NO metabolites in the plasma than did control fish, indicating an increase in NO. Expression of HIF-1α, PHD2, and VEGF mRNA was higher in anemic animals compared to control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that likely is mediated via NO signaling. In addition to lacking Hb, several species of icefish have lost ability to express myoglobin (Mb), an oxygen-binding protein expressed in the ventricle of most notothenioids. Previous studies have indicated Mb expression was lost on four separate occasions during evolution of the icefish family. Sequencing of the Mb gene from D. hunteri identified a duplicated TATA box previously proposed as the mechanism responsible for loss of Mb in C. aceratus. Sequencing of Mb from all 16 species of icefish identified the duplicated TATA box is present in all but two icefish species. The presence of the duplicated TATA box in Mb-expressing icefish suggests that the loss of Mb in C. aceratus and D. hunteri may occur by a mechanism independent of the duplicated TATA box