In the face of hypoxia: myoglobin expression under hypoxic conditions in cultured Weddell seal skeletal muscle cells
Date
2012
Authors
De Miranda, Michael Anthony, author
Kanatous, Shane B., advisor
Florant, Gregory, committee member
Earley, Scott, committee member
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Abstract
The hallmark adaptation to breath-hold diving in Weddell seals (Leptonychotes weddellii) is enhanced concentrations of myoglobin in their skeletal muscles. Myoglobin is a cytoplasmic hemoprotein that stores oxygen for use in aerobic metabolism throughout the dive duration. In addition, throughout the duration of the dive, Weddell seals rely on oxygen stored in myoglobin to sustain aerobic metabolism in which lipid is the primary contributor of acetyl CoA for the citric acid cycle. Together, enhanced myoglobin concentrations and a lipid-based aerobic metabolism represent some of the unique adaptations to diving found in skeletal muscle of Weddell seals. This thesis presents data that suggests cultured Weddell seal skeletal muscle cells inherently possess adaptations to diving such as increased myoglobin concentrations, and rely on lipids to fuel aerobic metabolism. I developed the optimum culture media for this unique primary cell line based on myoblast confluence, myoblast growth rates, myotube counts, and myotube widths. Once the culture media was established, I then determined the de novo expression of myoglobin under normoxic and hypoxic oxygen conditions and the metabolic profile of the myotubes under each oxygen condition. I found that the optimum culture media for the Weddell seal primary skeletal muscle cells high glucose Dulbecco's modified eagles media (DMEM) supplemented with a lipid mixture at a final concentration of 2.5%, based on myoblast confluence, myotube counts, and myotube widths. I also determined that the Weddell seal skeletal muscle cells increased myoglobin under hypoxia, to levels greater than a C2C12 control cell line, which is in direct contrast to previous studies using terrestrial mouse models. While the Weddell seal cells increased myoglobin, the metabolic enzymes responded similarly to the control cell line under both oxygen conditions. In addition, I found that increasing the concentration of lipid in the culture media increased myoglobin under normoxic conditions. To our knowledge, these studies represent the first successful isolation and culture of primary skeletal muscle cells from a diving mammal and the first metabolic profile and myoglobin expression measurements under varying oxygen conditions. This unique primary cell line and my preliminary data will enable future researchers to investigate the molecular regulation of the unique adaptations in seal skeletal muscle and unravel the elusive regulatory pathways of myoglobin expression in diving mammals. Understanding the regulatory mechanisms of an oxygen storage protein will have profound impacts on various human diseases that include tissue hypoxia and ischemia.
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Subject
cell culture
Weddell seal
hypoxia
lipid
myoglobin
skeletal muscle