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Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia.

Seal and human endothelial cells exposed to 1% O₂for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure.

We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

 

ABSTRACT The hypothalamic–pituitary–adrenal (HPA) axis controls the release of glucocorticoids, which regulate immune and inflammatory function by modulating cytokines, white blood cells and oxidative stress via glucocorticoid receptor (GR) signaling. Although the response to HPA activation is well characterized in many species, little is known about the impacts of HPA activation during extreme physiological conditions. Hence, we challenged 18 simultaneously fasting and developing elephant seal pups with daily intramuscular injections of adrenocorticotropin (ACTH), a GR antagonist (RU486), or a combination of the two (ACTH+RU486) for 4 days. We collected blood at baseline, 2 h and 4 days after the beginning of treatment. ACTH and ACTH+RU486 elevated serum aldosterone and cortisol at 2 h, with effects diminishing at 4 days. RU486 alone induced a compensatory increase in aldosterone, but not cortisol, at 4 days. ACTH decreased neutrophils at 2 h, while decreasing lymphocytes and increasing the neutrophil:lymphocyte ratio at 4 days. These effects were abolished by RU486. Despite alterations in white blood cells, there was no effect of ACTH or RU486 on transforming growth factor-β or interleukin-6 levels; however, both cytokines decreased with the 4 day fasting progression. Similarly, ACTH did not impact protein oxidation, lipid peroxidation or antioxidant enzymes, but plasma isoprostanes and catalase activity decreased while glutathione peroxidase increased with fasting progression. These data demonstrate differential acute (2 h) and chronic (4 days) modulatory effects of HPA activation on white blood cells and that the chronic effect is mediated, at least in part, by GR. These results also underscore elephant seals' extraordinary resistance to oxidative stress derived from repeated HPA activation. 

The Weddell seal (Leptonychotes weddellii) thrives in its extreme Antarctic environment. We generated the Weddell seal genome assembly and a high-quality annotation to investigate genome-wide evolutionary pressures that underlie its phenotype and to study genes implicated in hypoxia tolerance and a lipid-based metabolism. Genome-wide analyses included gene family expansion/contraction, positive selection, and diverged sequence (acceleration) compared to other placental mammals, identifying selection in coding and non-coding sequence in five pathways that may shape cardiovascular phenotype. Lipid metabolism as well as hypoxia genes contained more accelerated regions in the Weddell seal compared to genomic background. Top-significant genes wereSUMO2andEP300; both regulate hypoxia inducible factor signaling. Liver expression of four genes with the strongest acceleration signals differ between Weddell seals and a terrestrial mammal, sheep. We also report a high-density lipoprotein-like particle in Weddell seal serum not present in other mammals, including the shallow-diving harbor seal.

 

Synopsis Marine mammals exhibit some of the most dramatic physiological adaptations in their clade and offer unparalleled insights into the mechanisms driving convergent evolution on relatively short time scales. Some of these adaptations, such as extreme tolerance to hypoxia and prolonged food deprivation, are uncommon among most terrestrial mammals and challenge established metabolic principles of supply and demand balance. Non-targeted omics studies are starting to uncover the genetic foundations of such adaptations, but tools for testing functional significance in these animals are currently lacking. Cellular modeling with primary cells represents a powerful approach for elucidating the molecular etiology of physiological adaptation, a critical step in accelerating genome-to-phenome studies in organisms in which transgenesis is impossible (e.g., large-bodied, long-lived, fully aquatic, federally protected species). Gene perturbation studies in primary cells can directly evaluate whether specific mutations, gene loss, or duplication confer functional advantages such as hypoxia or stress tolerance in marine mammals. Here, we summarize how genetic and pharmacological manipulation approaches in primary cells have advanced mechanistic investigations in other non-traditional mammalian species, and highlight the need for such investigations in marine mammals. We also provide key considerations for isolating, culturing, and conducting experiments with marine mammal cells under conditions that mimic in vivo states. We propose that primary cell culture is a critical tool for conducting functional mechanistic studies (e.g., gene knockdown, over-expression, or editing) that can provide the missing link between genome- and organismal-level understanding of physiological adaptations in marine mammals. 

Nitric oxide (NO) is a potent vasodilator, which improves perfusion and oxygen delivery during tissue hypoxia in terrestrial animals. The vertebrate dive response involves vasoconstriction in select tissues, which persists despite profound hypoxia. Using tissues collected from Weddell seals at necropsy, we investigated whether vasoconstriction is aided by downregulation of local hypoxia signaling mechanisms. We focused on NO–soluble guanylyl cyclase (GC)-cGMP signaling, a well-known vasodilatory transduction pathway. Seals have a lower GC protein abundance, activity, and capacity to respond to NO stimulation than do terrestrial mammals. In seal lung homogenates, GC produced less cGMP (20.1 ± 3.7 pmol·mg protein−1·min−1) than the lungs of dogs (−80 ± 144 pmol·mg protein−1·min−1 less than seals), sheep (−472 ± 96), rats (−664 ± 104) or mice (−1,160 ± 104, P < 0.0001). Amino acid sequences of the GC enzyme α-subunits differed between seals and terrestrial mammals, potentially affecting their structure and function. Vasoconstriction in diving Weddell seals is not consistent across tissues; perfusion is maintained in the brain and heart but decreased in other organs such as the kidney. A NO donor increased median GC activity 49.5-fold in the seal brain but only 27.4-fold in the kidney, consistent with the priority of cerebral perfusion during diving. Nos3 expression was high in the seal brain, which could improve NO production and vasodilatory potential. Conversely, Pde5a expression was high in the seal renal artery, which may increase cGMP breakdown and vasoconstriction in the kidney. Taken together, the results of this study suggest that alterations in the NO-cGMP pathway facilitate the diving response.