Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource.
Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing glutathione levels and the NAD salvage pathway in increasing the reducing capacity of cold-adapted cells.
Endothelial adaptation to mild-moderate hypothermia down-regulates anabolic processes and increases the reducing capacity of cells to enhance their resistance to oxidation and injury associated with 0°C storage and rewarming. Inducing these characteristics in a clinical setting could potentially limit the damaging effects of energy insufficiency due to ischemia and prevent the disruption of integrated metabolism at low temperatures.