Marine mammals exhibit numerous physiological adaptations that supported their return to the ocean. At the broadest scale, these adaptations modulate organismal energetics. Chief among these are modifications to oxygen storage and utilization, as well as metabolic adaptations that enable spatial and temporal separation of foraging from energetically intensive life history stages. All marine mammals forage during apneas, necessitating a tolerance for declining blood oxygen tensions. Unlike cetaceans, pinnipeds remain partially land-dependent and are, therefore, regularly separated from their foraging grounds. The northern elephant seal (Mirounga angustirostris) represents a tractable model for the study of extreme hypoxia and fasting tolerance in marine mammals, and a large body of research on whole organismal responses to diving and fasting exists in this species. Still, technological constraints prohibit investigations of the dynamic cellular mechanisms critical to diving and fasting in these animals.
This dissertation identifies novel molecular and cellular mechanisms regulating hypoxia and fasting tolerance in a deep-diving marine mammal, the northern elephant seal. Chapter 1 summarizes the current understanding of intrinsic hypoxia tolerance in marine mammals, as well as the role of the energetically intensive postweaning fast in elephant seals. This chapter ultimately identifies three areas for which molecular contributions to whole organismal phenotypes are poorly defined: (1) real-time responses of the seal vasculature to hypoxia; (2) seal-specific regulation of the molecular pathways contributing to oxidative stress resistance; and (3) the role of intrinsic cellular versus systemic signals in muscle cell energetics during prolonged fasting. Chapters 2-4 address areas 1-3 by developing manipulable, proliferative cell culture systems to evaluate real-time responses to hypoxia and oxidative stress and identify cell type-specific signatures of fasting tolerance.
Chapter 2 evaluates the role of the canonical hypoxia signaling pathway in the seal arterial endothelial cell response to hypoxia. These experiments combine global transcriptional data with functional assays that identified blunted angiogenic signaling and increased glutathione synthesis as key factors in the response to hypoxia. Chapter 3 interrogates the seal endothelial cell response to reoxygenation after hypoxia and considers whether a chemical oxidant treatment recapitulates this response. Reoxygenation did not substantially alter coordinated gene expression patterns in seal cells, but chemical oxidant exposure upregulated the expression of iron-handling genes in the ferroptosis pathway and downregulated genes involved in the synthesis and metabolism of sulfur-containing amino acids. Together, these chapters utilize a previously inaccessible tissue system to identify mechanisms of intrinsic hypoxia and oxidative stress tolerance in a deep-diving mammal.
Chapter 4 examines the role of cell-autonomous versus circulating factors in seal skeletal muscle myoblast energetics. These experiments utilize cells and serum derived from elephant seal pups at early and late stages of the postweaning fast. Respirometry experiments identified both cell-autonomous and serum-borne shifts toward glycolytic ATP production with fasting progression, suggesting that metabolic regulation is substrate-specific and regulated by both intrinsic and extrinsic factors in these cells.
Together, these studies expand our understanding of marine mammal physiology within the context of a natural tolerance to energetic constraints. This work highlights the utility of primary cell culture models for connecting cellular-level changes to whole organismal function. In Chapter 5, I summarize the results and their contributions to the field.
