Summary
Many fish species respond to low temperature by inducing mitochondrial biogenesis, reflected in an increase in activity of the mitochondrial enzyme cytochrome c oxidase (COX). COX is composed of 13 subunits, three encoded by mitochondrial (mt)DNA and 10 encoded by nuclear genes. We used real-time PCR to measure mRNA levels for the 10 nuclear-encoded genes that are highly expressed in muscle. We measured mRNA levels in white muscle of three minnow species, each at two temperatures: zebrafish (Danio rerio) acclimated to 11 and 30°C, goldfish (Carassius auratus) acclimated to 4 and 35°C, and northern redbelly dace (Chrosomus eos) collected in winter and summer. We hypothesized that temperature-induced changes in COX activity would be paralleled by COX nuclear-encoded subunit transcript abundance. However, we found mRNA for COX subunits showed pronounced differences in thermal responses. Though zebrafish COX activity did not change in the cold, the transcript levels of four subunits decreased significantly (COX5A1, 60% decrease; COX6A2, 70% decrease; COX6C, 50% decrease; COX7B, 55% decrease). Treatments induced changes in COX activity in both dace (2.9 times in winter fish) and goldfish (2.5 times in cold fish), but the response in transcript levels was highly variable. Some subunits failed to increase in one (goldfish COX7A2, dace COX6A2) or both (COX7B, COX6B2) species. Other transcripts increased 1.7–100 times. The most cold-responsive subunits were COX4-1 (7 and 21.3 times higher in dace and goldfish, respectively), COX5A1 (13.9 and 5 times higher), COX6B1 (6 and 10 times higher), COX6C (11 and 4 times higher) and COX7C (13.3 and 100 times higher). The subunits that most closely paralleled COX increases in the cold were COX5B2(dace 2.5 times, goldfish 1.7 times) and COX6A2 (dace 4.1 times, goldfish 1.7 times). Collectively, these studies suggest that COX gene expression is not tightly coordinated during cold-induced mitochondrial remodelling in fish muscle. Further, they caution against arguments about the importance of transcriptional regulation based on measurement of mRNA levels of select subunits of multimeric proteins.
Methodology
Animals and tissue collection
Zebrafish (Danio rerio, Hamilton) were obtained from a local pet store in Kingston, ON (Pet Paradise). They were held in the Animal Care aquatic facility at Queen's University in a 140 l aquarium with dechlorinated water at 26°C for at least 2 weeks to acclimate before any experimental manipulations began. The fish were fed daily (Tetramin Tropical Crisps, Tetra, Germany) ad libitum and were kept under a 12 h:12 h light:dark photoperiod. Zebrafish were randomly separated into 80 l tanks in groups of 20 and the temperature was adjusted by approximately 1°C daily until reaching a final temperature of either 11 or 30°C. Fish were then held at this temperature for at least 3 weeks. We did not conduct a time course that would enable us to demonstrate that the fish had reached a fully acclimated state.
Dace (northern redbelly dace, Chrosomus eos, Cope) were captured from the Queen's University Biological Station in the outflow of Beaver Marsh using minnow traps. This is a shallow waterway where temperatures vary spatially and daily. The winter samples were caught in April 2009 just after ice-out, with 4–6°C water temperature at the time of capture. Summer fish were caught in late August 2009, when water temperature at the time of capture was 25–27°C.
Goldfish (Carrasius auratus, Linnaeus) were acclimated to 4 and 35°C, as described in a previous study (LeMoine et al., 2008). COX enzyme activity has previously been reported for these treatment groups (LeMoine et al., 2008). Archived cDNA from that study was used for the RNA analyses in the present study.
Dace were killed by severing the spinal column; zebrafish and goldfish were killed in a solution of 0.4 g l–1 tricaine methanesulphonate and 0.8 g l–1 sodium bicarbonate, then decapitated. Fish were subsequently dissected and white muscle tissue was flash frozen in liquid nitrogen, powdered under liquid nitrogen and stored at –80°C until needed.
COX enzyme assays
Samples (∼50–150 mg) of the powdered white muscle were homogenized on ice with glass homogenizers in 20 volumes of ice-cold extraction buffer (25 mmol l–1 potassium phosphate, 1 mmol l–1 EDTA, 0.6 mmol l–1 lauryl maltoside). Homogenates were diluted 10× in assay buffer (25 mmol l–1 potassium phosphate, 0.6 mmol l–1 lauryl maltoside) and added to a well of a 96-well plate containing assay buffer and reduced 0.05 mmol l–1 cytochrome c at 25°C. Absorbance (550 nm) was followed for 3 min in a Molecular Devices Spectromax spectrophotometer (Sunnyvale, CA, USA).
Reduced cytochrome c was prepared by dissolving solid horse heart cytochrome c in 10 mmol l–1 Tris pH 8.0 and reducing it with an equal mass of ascorbic acid. The mixture was placed in dialysis tubing (5000 Mr cut-off) and transferred to 4 l (200–400 volumes) of Tris buffer (10 mmol l–1 at pH 8.0) at 4°C. After 8–12 h, the tubing was transferred to fresh Tris buffer (3 times). The reduced cytochrome c was stored in aliquots at –80°C.