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SUMMARYCold acclimation of ectotherms results typically in enhanced oxidative capacities and lipid remodeling, changes that should increase the risk of lipid peroxidation (LPO). It is unclear whether activities of antioxidant enzymes may respond in a manner to mitigate the increased potential for LPO. The current study addresses these questions using killifish (Fundulus heteroclitus macrolepidotus) and bluegill (Lepomis macrochirus) acclimated to 5 and 25°C for 9days and 2months, respectively. Because the effects of temperature acclimation on pro-and antioxidant metabolism may be confounded by variable activity levels among temperature groups, one species (killifish) was also subjected to a 9-day exercise acclimation. Oxidative capacity of glycolytic (skeletal) muscle (indicated by the activity of cytochrome c oxidase) was elevated by 1.5-fold in killifish, following cold acclimation, but was unchanged in cardiac muscle and also unaffected by exercise acclimation in either tissue. No changes in citrate synthase activity were detected in either tissue following temperature acclimation. Enzymatic antioxidants (catalase and superoxide dismutase) of either muscle type were unaltered by temperature or exercise acclimation. Mitochondria from glycolytic muscle of cold-acclimated killifish were enriched in highly oxidizable polyunsatured fatty acids (PUFA), including diacyl phospholipids (total carbons:total double bonds) 40:8 and 44:12. Increased oxidative capacity, coupled with elevated PUFA content in mitochondria from cold-acclimated animals did not, however, impact LPO susceptibility when measured with C11-BODIPY. The apparent mismatch between oxidative capacity and enzymatic antioxidants following temperature acclimation will be addressed in future studies.

Tissues of Antarctic marine fishes are very high in lipids, predominantly triacylglycerols (TAG). In addition to conferring static lift to these swimbladderless fishes, these rich lipid stores long have been considered as an important caloric resource to the animals. We have performed in vitro measurements of the rates of oxidation of 14C-labeled carbohydrates and fatty acids by oxidative skeletal muscle and heart ventricle of an Antarctic teleost, Gobionotothm gibberifrons to assess the relative importance of these substrates to aerobic energy metabolism.Capacities for regeneration of ATP calculated from oxidation rates of these fuels clearly indicate that fatty acids are more effective substrates of energy metabolism than either glucose or lactate with both tissues. Substrate competition experiments conducted between the saturated fatty acid palmitate (16:O) and the monoenoic unsaturate oleate (18:l) comparing the oxidation rate of radiolabeled fatty acid in the presence and absence of unlabeled competitor demonstrate a clear preference of both tissue types for catabolism of the monounsaturated substrate. Measurements of maximal activity of the putative flux-generating enzyme of mitochondria1 P-oxidation, carnitine palmitoyltransferase (CPT), with a variety of fatty acyl CoA esters also show significant preference for a monoenoic fatty acyl CoA, palmitoleoyl CoA (16:l). The general pattern of results suggests that monounsaturated fatty compounds are the most readily utilized substrates for energy metabolism by oxidative muscle tissues of this Antarctic species. o 1995 Wiley-Liss, Inc

The production of reactive oxygen species is a regular feature of life in the presence of oxygen. Some reactive oxygen species possess sufficient energy to initiate lipid peroxidation in biological membranes, self-propagating reactions with the potential to damage membranes by altering their physical properties and ultimately their function. Two of the most prominent patterns of lipid restructuring in membranes of ectotherms involve contents of polyunsaturated fatty acids and ratios of the abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine. Since polyunsaturated fatty acids and phosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to lipid peroxidation. Although membranes from animals living at low body temperatures may be more prone to oxidation, the generation of reactive oxygen species and lipid peroxidation are sensitive to temperature. These scenarios raise the possibility that membrane susceptibility to lipid peroxidation is conserved at physiological temperatures. Reduced levels of polyunsaturated fatty acids and phosphatidylethanolamine may protect membranes at warm temperatures from deleterious oxidations when rates of reactive oxygen species production and lipid peroxidation are relatively high. At low temperatures, enhanced susceptibility may ensure sufficient lipid peroxidation for cellular processes that require lipid oxidation products.

SUMMARY It is unknown whether Antarctic fishes can defend themselves against oxidative stress induced by elevations in temperature. We hypothesized that Antarctic icefishes, lacking the oxygen-binding protein hemoglobin, might be more vulnerable to temperature-induced oxidative stress compared with red-blooded notothenioids because of differences in their mitochondrial properties. Mitochondria from icefishes have higher densities of phospholipids per mg of mitochondrial protein compared with red-blooded species, and these phospholipids are rich in polyunsaturated fatty acids (PUFA), which can promote the formation of reactive oxygen species (ROS). Additionally, previous studies have shown that multiple tissues in icefishes have lower levels of antioxidants compared with red-blooded species. We quantified several properties of mitochondria, including proton leak, rates of ROS production, membrane composition and susceptibility to lipid peroxidation (LPO), the activity of superoxide dismutase (SOD) and total antioxidant power (TAOP) in mitochondria isolated from hearts of icefishes and red-blooded notothenioids. Mitochondria from icefishes were more tightly coupled than those of red-blooded fishes at both 2°C and 10°C, which increased the production of ROS when the electron transport chain was disrupted. The activity of SOD and TAOP per mg of mitochondrial protein was equivalent between icefishes and red-blooded species, but TAOP normalized to mitochondrial phospholipid content was significantly lower in icefishes compared with red-blooded fishes. Additionally, membrane susceptibility to peroxidation was only detectable in icefishes at 1°C and not in red-blooded species. Together, our results suggest that the high density of mitochondrial phospholipids in hearts of icefishes may make them particularly vulnerable to oxidative stress as temperatures rise.

Studies in temperate fishes provide evidence that cardiac mitochondrial function and the capacity to fuel cardiac work contribute to thermal tolerance. Here, we tested the hypothesis that decreased cardiac aerobic metabolic capacity contributes to the lower thermal tolerance of the haemoglobinless Antarctic icefish, , compared with that of the red-blooded Antarctic species, Maximal activities of citrate synthase (CS) and lactate dehydrogenase (LDH), respiration rates of isolated mitochondria, adenylate levels and changes in mitochondrial protein expression were quantified from hearts of animals held at ambient temperature or exposed to their critical thermal maximum (CT). Compared with , activity of CS, ATP concentration and energy charge were higher in hearts of at ambient temperature and CT While state 3 mitochondrial respiration rates were not impaired by exposure to CT in either species, state 4 rates, indicative of proton leakage, increased following exposure to CT in but not The interactive effect of temperature and species resulted in an increase in antioxidants and aerobic metabolic enzymes in but not in Together, our results support the hypothesis that the lower aerobic metabolic capacity of hearts contributes to its low thermal tolerance.

We tested the hypothesis that blackfin icefish (), one of the six species in the family Channichthyidae (the icefishes) that do not express haemoglobin and myoglobin, lack regulatory cardiovascular flexibility during acute warming and activity. The experimental protocols were designed to optimize the surgical protocol and minimize stress. First, minimally invasive heart rate () measurements were made during a thermal ramp until cardiac failure in and compared with those from the closely related red-blooded black rockcod (). Then, integrative cardiovascular adjustments were more extensively studied using flow probes and intravascular catheters in during acute warming (from 0 to 8°C) at rest and after imposed activity. had a lower routine than (9 beats min versus 14 beats min) and a lower peak during acute warming (38 beats min versus 55 beats min) with a similar cardiac breakpoint temperature (13 and 14°C, respectively). Routine cardiac output () for at ∼0°C was much lower (26.6 ml min kg) than previously reported, probably because fish in the present study had a low (12 beats min) indicative of a high routine vagal tone and low stress. increased oxygen consumption during acute warming and with activity. Correspondingly, increased considerably (maximally 86.3 ml min kg), as did vascular conductance (5-fold). Thus, unlike earlier suggestions, these data provide convincing evidence that icefish can mount a well-developed cardiovascular regulation of heart rate, cardiac output and vascular conductance, and this regulatory capacity provides flexibility during acute warming.