The hypoxia response pathway in the Antarctic fish Notothenia coriiceps is functional despite a poly Q/E insertion mutation in HIF-1α.

Antarctic notothenioid fishes, inhabiting the oxygen-rich Southern Ocean, possess a polyglutamine and glutamic acid (poly Q/E) insertion mutation in the master transcriptional regulator of oxygen homeostasis, hypoxia- inducible factor-1α (HIF-1α). To determine if this mutation impairs the ability of HIF-1 to regulate gene expression in response to hypoxia, we exposed Notothenia coriiceps, with a poly Q/E insertion mutation in HIF-1α that is 9 amino acids long, to hypoxia (2.3 mg L-1 O2) or normoxia (10 mg L -1 O2) for 12 h. Heart ventricles, brain, liver, and gill tissue were harvested and changes in gene expression quantified using RNA sequencing. Levels of glycogen and lactate were also quantified to determine if anaerobic metabolism increases in response to hypoxia. Exposure to hypoxia resulted in 818 unique differentially expressed genes (DEGs) in liver tissue of N. coriiceps. Many hypoxic genes were induced, including ones involved in the MAP kinase and FoxO pathways, glycolytic metabolism, and vascular remodeling. In contrast, there were fewer than 104 unique DEGs in each of the other tissues sampled. Lactate levels significantly increased in liver in response to hypoxia, indicating that anaerobic metabolism increases in response to hypoxia in this tissue. Overall, our results indicate that the hypoxia response pathway is functional in N. coriiceps despite a poly Q/E mutation in HIF-1α, and confirm that Antarctic fishes are capable of altering gene expression in response to hypoxia.

Characterization of the hypoxia-inducible factor-1 pathway in hearts of Antarctic notothenioid fishes.

The ability of Antarctic notothenioid fishes to mount a robust molecular response to hypoxia is largely unknown. The transcription factor, hypoxia-inducible factor-1 (HIF-1), a heterodimer of HIF-1α and HIF-1ß subunits, is the master regulator of oxygen homeostasis in most metazoans. We sought to determine if, in the hearts of Antarctic notothenioids, HIF-1 is activated and functional in response to either an acute heat stress or hypoxia. The red-blooded Notothenia coriiceps and the hemoglobinless icefish, Chaenocephalus aceratus, were exposed to their critical thermal maximum (CTMAX) or hypoxia (5.0 ± 0.3 mg of O2 L-1) for 2 h. Additionally, N. coriiceps was exposed to 2.3 ± 0.3 mg of O2 L-1 for 12 h, and red-blooded Gobionotothen gibberifrons was exposed to both levels of hypoxia. Levels of HIF-1α were quantified in nuclei isolated from heart ventricles using western blotting. Transcript levels of genes involved in anaerobic metabolism, and known to be regulated by HIF-1, were quantified by real-time PCR, and lactate levels were measured in heart ventricles. Protein levels of HIF-1α increase in nuclei of hearts of N. coriiceps and C. aceratus in response to exposure to CTMAX and in hearts of N. coriiceps exposed to severe hypoxia, yet mRNA levels of anaerobic metabolic genes do not increase in any species, nor do lactate levels increase, suggesting that HIF-1 does not stimulate metabolic remodeling in hearts of notothenioids under these conditions. Together, these data suggest that Antarctic notothenioids may be vulnerable to hypoxic events, which are likely to increase with climate warming.

Hypoxia-Inducible Factor-1α in Antarctic notothenioids contains a polyglutamine and glutamic acid insert that varies in length with phylogeny.

The long evolution of the Antarctic perciform suborder of Notothenioidei in the icy, oxygen-rich waters of the Southern Ocean may have reduced selective pressure to maintain a hypoxic response. To test this hypothesis, cDNA of the key transcriptional regulator of hypoxic genes, hypoxia-inducible factor-1α (HIF-1α), was sequenced in heart ventricles of the red-blooded notothenioid, Notothenia coriiceps, and the hemoglobinless icefish, Chaenocephalus aceratus. HIF-1α cDNA is 4500 base pairs (bp) long and encodes 755 amino acids in N. coriiceps, and in C. aceratus, HIF-1α is 3576 bp long and encodes 779 amino acids. All functional domains of HIF-1α are highly conserved compared to other teleosts, but HIF-1α contains a polyglutamine/glutamic acid (polyQ/E) insert 9 amino acids long in N. coriiceps and 34 amino acids long in C. aceratus. Sequencing of this region in four additional species, representing three families of notothenioids, revealed that the length of the polyQ/E insert varies with phylogeny. Icefishes, the crown family of notothenioids, contain the longest polyQ/E inserts, ranging between16 and 34 amino acids long, whereas the basal, cold-temperate notothenioid, Eleginops maclovinus, contains a polyQ/E insert only 4 amino acids long. PolyQ/E inserts may affect dimerization of HIF-1α and HIF-1ß, HIF-1 translocation into the nucleus and/or DNA binding.