Evidence of Cold Induced Cytoskeletal Arrest in Hepatocytes of the Western Painted Turtle

Abstract

The western painted turtle (Chrysemys picta belli) overwinters in ice-covered water bodies and can survive without oxygen (anoxia) for months, during which time metabolic rate decreases so that ATP demand does not exceed anoxic ATP supply. In order to characterize the contribution of the cytoskeleton to overwintering metabolic depression in non-excitable turtle cells, we have optimized a hepatocyte cell culture system. Actin and tubulin cycling are large consumers of cellular ATP pools, and we hypothesized that their cycling activity, and consequently capacity for structural rearrangement, would be downregulated by overwintering conditions. Turtles were acclimated to either 22°C or 4°C for at least 20 days and isolated hepatocytes were plated on either fibronectin or polylysine at varying temperatures, or in cyanide containing media, which acted as a chemical proxy for anoxia. Low incubation temperature, but not cyanide, prevented hepatocytes from forming stable attachment to fibronectin, suggesting inhibition of cell spreading and arrest of actin structural rearrangement by low temperature. Attachment to polylysine, a predominately passive and electrostatic phenomenon, was not inhibited by low temperature or cyanide. Hepatocytes isolated from 4°C acclimated turtles were 30% smaller in 2D area than those of 22°C acclimated turtles. Three-hour cyanide exposure of hepatocytes from 22°C acclimated turtles also resulted in a 30% shrinkage of 2D area, indicating that temperature depression and anoxia may have overlapping inhibitory effects on cell spreading and underlying cytoskeletal turnover. Previous research has shown that cyanide treatment causes reduction of hepatocyte mitochondrial membrane potential. Ongoing work is demonstrating that, regardless of whole animal temperature acclimation, low temperature exposure results in decreased mitochondrial membrane potential as well as increased cytosolic calcium, signaling which may contribute to inhibition of energetically costly actin rearrangement.