Ontogeny of Carbon Monoxide-Related Gene Expression in a Deep-Diving Marine Mammal

Abstract

Marine mammals such as northern elephant seals (NES) routinely experience hypoxemia and ischemia-reperfusion events to many tissues during deep dives with no apparent adverse effects. Adaptations to diving include increased antioxidants and elevated oxygen storage capacity associated with high hemoprotein content in blood and muscle. The natural turnover of heme by heme oxygenase enzymes (encoded by HMOX1 and HMOX2) produces endogenous carbon monoxide (CO), which is present at high levels in NES blood and has been shown to have cytoprotective effects in laboratory systems exposed to hypoxia. To understand how pathways associated with endogenous CO production and signaling change across ontogeny in diving mammals, we measured muscle CO and baseline expression of 17 CO-related genes in skeletal muscle and whole blood of three age classes of NES. Muscle CO levels approached those of animals exposed to high exogenous CO, increased with age, and were significantly correlated with gene expression levels. Muscle expression of genes associated with CO production and antioxidant defenses (HMOX1, BVR, GPX3, PRDX1) increased with age and was highest in adult females, while that of genes associated with protection from lipid peroxidation (GPX4, PRDX6, PRDX1, SIRT1) was highest in adult males. In contrast, muscle expression of mitochondrial biogenesis regulators (PGC1A, ESRRA, ESRRG) was highest in pups, while genes associated with inflammation (HMOX2, NRF2, IL1B) did not vary with age or sex. Blood expression of genes involved in regulation of inflammation (IL1B, NRF2, BVR, IL10) was highest in pups, while HMOX1, HMOX2 and pro-inflammatory markers (TLR4, CCL4, PRDX1, TNFA) did not vary with age. We propose that ontogenetic upregulation of baseline HMOX1 expression in skeletal muscle of NES may, in part, underlie increases in CO levels and expression of genes encoding antioxidant enzymes. HMOX2, in turn, may play a role in regulating inflammation related to ischemia and reperfusion in muscle and circulating immune cells. Our data suggest putative ontogenetic mechanisms that may enable phocid pups to transition to a deep-diving lifestyle, including high baseline expression of genes associated with mitochondrial biogenesis and immune system activation during postnatal development and increased expression of genes associated with protection from lipid peroxidation in adulthood.

Elevated antioxidant defence in the brain of deep-diving pinnipeds

“…However, the precise orchestra of protective mechanisms, including antioxidant capacity, might vary with species based on Frontiers in Physiology frontiersin.org their life history traits, with age and with sex. For instance, the muscle expression of several antioxidant genes increased with age and diving ability in northern elephant seals and hooded seals (Vázquez-Medina et al, 2011;Piotrowski et al, 2021) and some antioxidants were observed to be higher in northern elephant females than in males (Piotrowski et al, 2021).…”

“…Interestingly, while HMOX1 expression in skeletal muscle of northern elephant seals is associated with the expression of other antioxidants, correlates with age and was highest in adult females, HMOX2 expression did not vary with age or sex (Piotrowski et al, 2021). Adult females are thought to dive beyond their calculated aerobic dive limit (Hassrick et al, 2007) and thus, the observed elevated expression may be needed to protect them from a more severe risk of oxidative damage (Piotrowski et al, 2021). These results show that the precise interplay of antioxidants may vary with species age and sex.…”

“…Several studies have shown that HMOX2 gene expression is activated by oxidative stress, while hypoxia can regulate gene expression and translation (Muñoz-Sánchez and Chánez-Cárdenas, 2014 for a review). Interestingly, while HMOX1 expression in skeletal muscle of northern elephant seals is associated with the expression of other antioxidants, correlates with age and was highest in adult females, HMOX2 expression did not vary with age or sex (Piotrowski et al, 2021). Adult females are thought to dive beyond their calculated aerobic dive limit (Hassrick et al, 2007) and thus, the observed elevated expression may be needed to protect them from a more severe risk of oxidative damage (Piotrowski et al, 2021).…”

Distribution of carbon monoxide (CO) in several tissues from Atlantic bottlenose dolphins (Tursiops truncatus)

“…To date, extravascular tissue CO has been reported in tissues from healthy rodents and from deceased humans (Vreman et al, 2005;Vreman et al, 2006). Extravascular CO has also been recently reported in skeletal muscle from elephant seals which is over four times as high as that found in similar tissues from the healthy rodents and humans (Piotrowski et al, 2021). While most of the prior research has focused on measuring CO in blood, heme oxygenase proteins in tissues, and expression of heme oxygenase genes in relation to various conditions and treatments, very few studies have investigated the quantity and pattern of distribution of extravascular CO.…”

“…The extraction of CO and its quantification in tissues follows previously established methods (Byrne et al, 2022;Piotrowski et al, 2021;Vreman et al, 2005). Briefly, tissues were thawed and rinsed with chilled KH2PO4 buffer (pH 7.4) to remove any remaining excess blood and then placed into microcentrifuge tubes.…”