Metabolic rate increases with acclimation temperature and is associated with mitochondrial function in some tissues of threespine stickleback.

The metabolic rate (MO2) of eurythermal fishes changes in response to temperature, yet it is unclear how changes in mitochondrial function contribute to changes in MO2. We hypothesized that MO2 would increase with acclimation temperature in the threespine stickleback (Gasterosteus aculeatus) in parallel with metabolic remodeling at the cellular level but that changes in metabolism in some tissues, such as liver, would contribute more to changes in MO2 than others. Threespine stickleback were acclimated to 5, 12 and 20°C for 7 to 21 weeks. At each temperature, standard and maximum metabolic rate (SMR and MMR, respectively), and absolute aerobic scope (AAS) were quantified, along with mitochondrial respiration rates in liver, oxidative skeletal and cardiac muscles, and the maximal activity of citrate synthase (CS) and lactate dehydrogenase (LDH) in liver, and oxidative and glycolytic skeletal muscles. SMR, MMR and AAS increased with acclimation temperature, along with rates of mitochondrial phosphorylating respiration in all tissues. Low SMR and MMR at 5°C were associated with low or undetectable rates of mitochondrial complex II activity and a greater reliance on complex I activity in liver, oxidative skeletal muscle and heart. SMR was positively correlated with cytochrome c oxidase (CCO) activity in liver and oxidative muscle, but not mitochondrial proton leak, whereas MMR was positively correlated with CCO activity in liver. Overall, the results suggest that changes in MO2 in response to temperature are driven by changes in some aspects of mitochondrial function in some, but not all, tissues of threespine stickleback.

Development and Results of the First Certification Examination in the American Board of Medical Specialties Neurocritical Care Subspecialty.

This article reviews the development of the American Board of Medical Specialties subspecialty in neurocritical care (NCC) and describes the requirements for certification and the results of the first certification examination administered in October 2021. The American Board of Psychiatry and Neurology (ABPN) is the administrative board, and the sponsoring boards are the American Board of Anesthesiology (ABA), American Board of Emergency Medicine (ABEM), American Board of Internal Medicine (ABIM), and American Board of Neurological Surgery. The American Board of Medical Specialties approved the subspecialty in 2018, and the Accreditation Council for Graduate Medical Education developed and approved the training requirements in 2021. The fellowship programs are either 12 or 24 months in length and may become available in Academic Year 2022-2023. The first NCC examination was developed by a multispecialty group of subject matter experts following established test development procedures and was successfully administered to 1,011 candidates in October 2021. There were 406 (40.2%) ABIM candidates, 356 (35.2%) ABPN candidates, 208 (20.6%) ABA candidates, and 41 (4.1%) ABEM candidates. The end-of-test survey indicated that most examinees were satisfied with their test taking experience, and the .92 reliability index indicated that the test scores were reliable. An established process was also followed to set the criterion-referenced passing standard, and the resulting pass rate of 72.7% was judged to be reasonable. In summary, the combined efforts of representatives from the ABPN, ABA, ABEM, ABIM, and American Board of Neurological Surgery yielded a quality assessment instrument to identify physicians who possess the expertise required to be certified in NCC. The test development committee will continue to expand and improve the pool of test questions for the next examination, which is scheduled for October 2022.

Aerobic scope is not maintained at low temperature and is associated with cardiac aerobic capacity in the three-spined stickleback Gasterosteus aculeatus.

Metabolic thermal plasticity is central to the survival of fishes in a changing environment. The eurythermal three-spined stickleback Gasterosteus aculeatus displays thermal plasticity at the cellular level with an increase in the activity of key metabolic enzymes in response to cold acclimation. Nonetheless, it is unknown if these changes are sufficient to completely compensate for the depressive effects of cold temperature on whole organismal metabolic rate (MO2 ). The authors hypothesized that as a cold-tolerant, eurythermal fish, absolute aerobic scope (AAS), the difference between the maximum metabolic rate (MMR) and standard metabolic rate (SMR), would be maintained in G. aculeatus following acclimation to a range of temperatures that span its habitat temperatures. To test this hypothesis, G. aculeatus were acclimated to 5, 12 and 20°C for 20-32 weeks, and SMR, MMR and aerobic scope (AS) were quantified at each acclimation temperature. The maximal activity of citrate synthase (CS), a marker enzyme of aerobic metabolism, was also quantified in heart ventricles to determine if cardiac aerobic capacity is associated with AS at these temperatures. SMR increased with acclimation temperature and was significantly different among all three temperature groups. MMR was similar between animals at 5 and 12°C and between animals at 12 and 20°C but was 2.6-fold lower in fish at 5°C compared with those at 20°C, resulting in a lower AAS in fish at 5°C compared with those at 12 and 20°C. Correlated with a higher AAS in animals acclimated to 12 and 20°C was a larger relative ventricular mass and higher CS activity per 100 g body mass compared with animals at 5°C. Together, the results indicate that despite their eurythermal nature, AS is not maintained at low temperature but is associated with cardiac aerobic metabolic capacity.

Resilience of cardiac performance in Antarctic notothenioid fishes in a warming climate.

Warming in the region of the Western Antarctic Peninsula is occurring at an unprecedented rate, which may threaten the survival of Antarctic notothenioid fishes. Herein, we review studies characterizing thermal tolerance and cardiac performance in notothenioids - a group that includes both red-blooded species and the white-blooded, haemoglobinless icefishes - as well as the relevant biochemistry associated with cardiac failure during an acute temperature ramp. Because icefishes do not feed in captivity, making long-term acclimation studies unfeasible, we focus only on the responses of red-blooded notothenioids to warm acclimation. With acute warming, hearts of the white-blooded icefish Chaenocephalus aceratus display persistent arrhythmia at a lower temperature (8°C) compared with those of the red-blooded Notothenia coriiceps (14°C). When compared with the icefish, the enhanced cardiac performance of N. coriiceps during warming is associated with greater aerobic capacity, higher ATP levels, less oxidative damage and enhanced membrane integrity. Cardiac performance can be improved in N. coriiceps with warm acclimation to 5°C for 6-9 weeks, accompanied by an increase in the temperature at which cardiac failure occurs. Also, both cardiac mitochondrial and microsomal membranes are remodelled in response to warm acclimation in N. coriiceps, displaying homeoviscous adaptation. Overall, cardiac performance in N. coriiceps is malleable and resilient to warming, yet thermal tolerance and plasticity vary among different species of notothenioid fishes; disruptions to the Antarctic ecosystem driven by climate warming and other anthropogenic activities endanger the survival of notothenioids, warranting greater protection afforded by an expansion of marine protected areas.

Exploring nature's natural knockouts: in vivo cardiorespiratory performance of Antarctic fishes during acute warming.

We tested the hypothesis that blackfin icefish (Chaenocephalus aceratus), 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 (fH) measurements were made during a thermal ramp until cardiac failure in C. aceratus and compared with those from the closely related red-blooded black rockcod (Notothenia coriiceps). Then, integrative cardiovascular adjustments were more extensively studied using flow probes and intravascular catheters in C. aceratus during acute warming (from 0 to 8°C) at rest and after imposed activity. Chaenocephalus aceratus had a lower routine fH than N. coriiceps (9 beats min-1 versus 14 beats min-1) and a lower peak fH during acute warming (38 beats min-1 versus 55 beats min-1) with a similar cardiac breakpoint temperature (13 and 14°C, respectively). Routine cardiac output (Q̇) for C. aceratus at ∼0°C was much lower (26.6 ml min-1 kg-1) than previously reported, probably because fish in the present study had a low fH (12 beats min-1) indicative of a high routine vagal tone and low stress. Chaenocephalus aceratus increased oxygen consumption during acute warming and with activity. Correspondingly, Q̇ increased considerably (maximally 86.3 ml min-1 kg-1), 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.

The loss of hemoglobin and myoglobin does not minimize oxidative stress in Antarctic icefishes.

The unusual pattern of expression of hemoglobin (Hb) and myoglobin (Mb) among Antarctic notothenioid fishes provides an exceptional model system for assessing the impact of these proteins on oxidative stress. We tested the hypothesis that the lack of oxygen-binding proteins may reduce oxidative stress. Levels and activity of pro-oxidants and small-molecule and enzymatic antioxidants, and levels of oxidized lipids and proteins in the liver, oxidative skeletal muscle and heart ventricle were quantified in five species of notothenioid fishes differing in the expression of Hb and Mb. Levels of ubiquitinated proteins and rates of protein degradation by the 20S proteasome were also quantified. Although levels of oxidized proteins and lipids, ubiquitinated proteins, and antioxidants were higher in red-blooded fishes than in Hb-less icefishes in some tissues, this pattern did not persist across all tissues. Expression of Mb was not associated with oxidative damage in the heart ventricle, whereas the activity of citrate synthase and the contents of heme were positively correlated with oxidative damage in most tissues. Despite some tissue differences in levels of protein carbonyls among species, rates of degradation by the 20S proteasome were not markedly different, suggesting either alternative pathways for eliminating oxidized proteins or that redox tone varies among species. Together, our data indicate that the loss of Hb and Mb does not correspond with a clear pattern of either reduced oxidative defense or oxidative damage.

Cardiac mitochondrial metabolism may contribute to differences in thermal tolerance of red- and white-blooded Antarctic notothenioid fishes.

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, Chaenocephalus aceratus, compared with that of the red-blooded Antarctic species, Notothenia coriiceps. 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 (CTmax). Compared with C. aceratus, activity of CS, ATP concentration and energy charge were higher in hearts of N. coriiceps at ambient temperature and CTmax While state 3 mitochondrial respiration rates were not impaired by exposure to CTmax in either species, state 4 rates, indicative of proton leakage, increased following exposure to CTmax in C. aceratus but not N. coriiceps The interactive effect of temperature and species resulted in an increase in antioxidants and aerobic metabolic enzymes in N. coriiceps but not in C. aceratus Together, our results support the hypothesis that the lower aerobic metabolic capacity of C. aceratus hearts contributes to its low thermal tolerance.

Molecular drivers of mitochondrial membrane proliferation in response to cold acclimation in threespine stickleback.

Little is known about how the synthesis of mitochondrial phospholipids is integrated into mitochondrial biogenesis in fish or mammals. Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the addition of fatty acyl CoA to the sn-1 position of glycerol-3-phosphate, in what is considered the rate-limiting step in phospholipid biosynthesis. Previous studies have shown that mitochondrial volume density increases in oxidative skeletal muscle but not liver of Gasterosteus aculeatus (threespine stickleback) in response to cold acclimation. We hypothesized that maximal activity of GPAT would increase in oxidative skeletal muscle but not liver during cold acclimation, coinciding with mitochondrial biogenesis. GPAT activity was measured in liver and oxidative skeletal (pectoral adductor) muscle of threespine stickleback acclimated to 8°C or 20°C. In addition, mRNA levels of enzymes involved in phospholipid synthesis, including cytidine diphosphodiacylglycerol synthase-1 (CDS1), CDS2, GPAT1, GPAT2 and 1-acylglycerol 3-phosphate acyltransferase-2 (AGPAT2), were quantified in liver and pectoral muscle of stickleback harvested during cold acclimation. GPAT activity and transcript levels of AGPAT2 increased in response to cold acclimation in pectoral muscle but not liver. Transcript levels of GPAT1 increased in liver but not pectoral muscle. Overall our results suggest that the activity of GPAT, and possibly AGPAT as well, increase during cold acclimation and may contribute to mitochondrial phospholipid biosynthesis required for mitochondrial biogenesis.

Expansion of capacities for iron transport and sequestration reflects plasma volumes and heart mass among white-blooded notothenioid fishes.

The family Channichthyidae or "icefishes" (suborder Notothenioidei) represents the only vertebrates lacking hemoglobin (Hb) as adults. Several icefish species also do not express cardiac myoglobin (Mb). We address how levels of proteins involved in iron (Fe) processing (transport, sequestration, and export) vary among white- and red-blooded notothenioids, and whether absence of Hb and/or Mb in channichthyids is accompanied by expansion of contents of Fe-binding proteins to protect against unchaperoned Fe. Levels of transferrin (Tf), ferritin (Ft), ceruloplasmin (Cp), and non-heme Fe were quantified in plasma, serum, and/or nonhematopoietic tissues (cardiac ventricle, skeletal muscle, and liver) from species of white-blooded (Chaenocephalus aceratus, Champsocephalus gunnari, Chionodraco rastrospinosus, Pseudochaenichthys georgianus) (the first two species not expressing Mb) and red-blooded (Notothenia coriiceps, Gobionotothen gibberifrons) notothenioids. We also measured levels of ascorbate (Asc), a mediator of Fe uptake. While plasma concentrations of Tf and tissue levels of Asc are similar among species, concentrations of plasma Asc are lower in white-blooded species. Concentrations of Ft and non-heme Fe and activities of Cp are also generally reduced in icefishes compared with red-blooded notothenioids. The presence of cardiac Mb in some icefish species does not appear to influence levels of proteins involved in Fe processing. To address further the question of Fe sequestration within a physiological context, we account for well-characterized differences in blood volume and heart mass among white- and red-blooded notothenioids. We report that total contents of plasma Tf are greater, while ventricle non-heme Fe is at least at parity in white- vs. red-blooded species.

Energetic costs of protein synthesis do not differ between red- and white-blooded Antarctic notothenioid fishes.

Antarctic icefishes (Family Channichthyidae) within the suborder Notothenioidei lack the oxygen-binding protein hemoglobin (Hb), and six of the 16 species of icefishes lack myoglobin (Mb) in heart ventricle. As iron-centered proteins, Hb and Mb can promote the formation of reactive oxygen species (ROS) that damage biological macromolecules. Consistent with this, our previous studies have shown that icefishes have lower levels of oxidized proteins and lipids in oxidative muscle compared to red-blooded notothenioids. Because oxidized proteins are usually degraded by the 20S proteasome and must be resynthesized, we hypothesized that rates of protein synthesis would be lower in icefishes compared to red-blooded notothenioids, thereby reducing the energetic costs of protein synthesis and conferring a benefit to the loss of Hb and Mb. Rates of protein synthesis were quantified in hearts, and the fraction of oxygen consumption devoted to protein synthesis was measured in isolated hepatocytes and cardiomyocytes of notothenioids differing in the expression of Hb and cardiac Mb. Neither rates of protein synthesis nor the energetic costs of protein synthesis differed among species, suggesting that red-blooded species do not degrade and replace oxidatively modified proteins at a higher rate compared to icefishes but rather, persist with higher levels of oxidized proteins.

Cold acclimation increases levels of some heat shock protein and sirtuin isoforms in threespine stickleback.

Molecular chaperones [heat shock proteins (HSPs)] increase in response to rapid changes in temperatures, but long-term acclimation to cold temperature may also warrant elevations in HSPs. In fishes, cold acclimation increases mitochondrial density and oxidative stress in some tissues, which may increase demand for HSPs. We hypothesized that levels of HSPs, as well as sirtuins (SIRTs), NAD-dependent deacetylases that mediate changes in metabolism and responses to oxidative stress (including increases in HSPs), would increase during cold acclimation of threespine stickleback (Gasterosteus aculeatus). Transcript levels of hsp70, hsc70, hsp60 and hsp90-α, sirts1-4, as well as protein levels of HSP60, HSP90 and HSC70 were quantified in liver and pectoral adductor muscle of stickleback during cold acclimation from 20 °C to 8 °C. In liver, cold acclimation stimulated a transient increase in mRNA levels of hsp60 and hsc70. Transcript levels of sirt1 and sirt2 also increased in response to cold acclimation and remained elevated. In pectoral muscle, mRNA levels of hsp60, hsp90-α, hsc70 and sirt1 all transiently increased in response to cold acclimation, while levels of sirts2-4 remained constant or declined. Similar to transcript levels, protein levels of HSC70 increased in both liver and pectoral muscle. Levels of HSP90 also increased in liver after 4 weeks at 8 °C. HSP60 remained unchanged in both tissues, as did HSP90 in pectoral muscle. Our results indicate that while both HSPs and SIRTs increase in response to cold acclimation in stickleback, the response is tissue and isoform specific, likely reflecting differences in metabolism and oxidative stress.

Exposure to critical thermal maxima increases oxidative stress in hearts of white- but not red-blooded Antarctic notothenioid fishes.

Antarctic icefishes have a significantly lower critical thermal maximum (CT(max)) compared with most red-blooded notothenioid fishes. We hypothesized that the lower thermal tolerance of icefishes compared with red-blooded notothenioids may stem from a greater vulnerability to oxidative stress as temperature increases. Oxidative muscles of icefishes have high volume densities of mitochondria, rich in polyunsaturated fatty acids, which can promote the production of reactive oxygen species (ROS). Moreover, icefishes have lower levels of antioxidants compared with red-blooded species. To test our hypothesis, we measured levels of oxidized proteins and lipids, and transcript levels and maximal activities of antioxidants in heart ventricle and oxidative pectoral adductor muscle of icefishes and red-blooded notothenioids held at 0°C and exposed to their CT(max). Levels of oxidized proteins and lipids increased in heart ventricle of some icefishes but not in red-blooded species in response to warming, and not in pectoral adductor muscle of any species. Thus, increases in oxidative damage in heart ventricles may contribute to the reduced thermal tolerance of icefishes. Despite an increase in oxidative damage in hearts of icefishes, neither transcript levels nor activities of antioxidants increased, nor did they increase in any tissue of any species in response to exposure to CT(max). Rather, transcript levels of the enzyme superoxide dismutase (SOD) decreased in hearts of icefishes and the activity of SOD decreased in hearts of the red-blooded species Gobionotothen gibberifrons. These data suggest that notothenioids may have lost the ability to elevate levels of antioxidants in response to heat stress.

Warm acclimation alters antioxidant defences but not metabolic capacities in the Antarctic fish, Notothenia coriiceps.

The Southern Ocean surrounding the Western Antarctic Peninsula region is rapidly warming. Survival of members of the dominant suborder of Antarctic fishes, the Notothenioidei, will likely require thermal plasticity and adaptive capacity in key traits delimiting thermal tolerance. Herein, we have assessed the thermal plasticity of several cellular and biochemical pathways, many of which are known to be associated with thermal tolerance in notothenioids, including mitochondrial function, activities of aerobic and anaerobic enzymes, antioxidant defences, protein ubiquitination and degradation in cardiac, oxidative skeletal muscles and gill of Notothenia coriiceps warm acclimated to 4°C for 22 days or 5°C for 42 days. Levels of triacylglycerol (TAG) were measured in liver and oxidative and glycolytic skeletal muscles, and glycogen in liver and glycolytic muscle to assess changes in energy stores. Metabolic pathways displayed minimal thermal plasticity, yet antioxidant defences were lower in heart and oxidative skeletal muscles of warm-acclimated animals compared with animals held at ambient temperature. Despite higher metabolic rates at elevated temperature, energy storage depots of TAG and glycogen increase in liver and remain unchanged in muscle with warm acclimation. Overall, our studies reveal that N. coriiceps displays thermal plasticity in some key traits that may contribute to their survival as the Southern Ocean continues to warm.

Mitochondrial biogenesis in cold-bodied fishes.

Mitochondrial biogenesis is induced in response to cold temperature in many organisms. The effect is particularly pronounced in ectotherms such as fishes, where acclimation to cold temperature increases mitochondrial density. Some polar fishes also have exceptionally high densities of mitochondria. The net effect of increasing mitochondrial density is threefold. First, it increases the concentration of aerobic metabolic enzymes per gram of tissue, maintaining ATP production. Second, it elevates the density of mitochondrial membrane phospholipids, enhancing rates of intracellular oxygen diffusion. Third, it reduces the diffusion distance for oxygen and metabolites between capillaries and mitochondria. Although cold-induced mitochondrial biogenesis has been well documented in fishes, little is known about the molecular pathway governing it. In mammals, the co-transcriptional activator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is thought to coordinate the three components of mitochondrial biogenesis: the synthesis of mitochondrial proteins, the synthesis of phospholipids and the replication of mitochondrial DNA. Some components of the mitochondrial biogenic pathway are conserved between fishes and mammals, yet the pathway appears more versatile in fishes. In some tissues of cold-acclimated fishes, the synthesis of mitochondrial proteins increases in the absence of an increase in phospholipids, whereas in some polar fishes, densities of mitochondrial phospholipids increase in the absence of an increase in proteins. The ability of cold-bodied fishes to fine-tune the mitochondrial biogenic pathway may allow them to modify mitochondrial characteristics to meet the specific needs of the cell, whether it is to increase ATP production or enhance oxygen diffusion.

Oxidative stress is transient and tissue specific during cold acclimation of threespine stickleback.

Linkages between cold acclimation and oxidative stress in fishes are unclear and contradictory results have been published. We sought to determine whether oxidative stress occurs during cold acclimation of threespine stickleback (Gasterosteus aculeatus), and, if so, when it occurs and whether it varies among tissues. Fish were warm (20°C) or cold (8°C) acclimated for 9 weeks, and harvested during acclimation. Oxidative stress was assessed in oxidative and glycolytic muscles and liver by measuring levels of protein carbonyls and glutathione, and the activity and transcript levels of superoxide dismutase (SOD). Protein carbonyl levels increased in liver after 1 week at 8°C and then decreased after week 4, and remained unchanged in glycolytic and oxidative muscle. Glutathione levels increased in liver on day 3 of cold acclimation and may minimize oxidative stress later during acclimation. When measured at a common temperature, the activity of SOD increased in oxidative and glycolytic muscles on day 2 of cold acclimation, and on day 3 in liver, and remained elevated in all tissues compared with warm-acclimated animals. When measured at the acclimation temperature, the activity of SOD was significantly higher only at week 9 in oxidative muscle of cold-acclimated stickleback compared with warm-acclimated fish, and remained constant in glycolytic muscle and liver. Increased SOD activity in oxidative muscle may be required to prevent oxidative stress brought about by increased mitochondrial density. In both muscle and liver, SOD activity increased independently of an increase in transcript level, suggesting post-translational modifications regulate SOD activity.

Inter-relationship between mitochondrial function and susceptibility to oxidative stress in red- and white-blooded Antarctic notothenioid fishes.

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.

Homeoviscous adaptation occurs with thermal acclimation in biological membranes from heart and gill, but not the brain, in the Antarctic fish Notothenia coriiceps.

As temperatures continue to rise, adjustments to biological membranes will be key for maintenance of function. It is largely unknown to what extent Antarctic notothenioids possess the capacity to remodel their biological membranes in response to thermal change. In this study, physical and biochemical properties were examined in membranes prepared from gill epithelia (plasma membranes), cardiac ventricles (microsomes, mitochondria), and brains (synaptic membranes, myelin, mitochondria) from Notothenia coriiceps following acclimation to 5 °C (or held at ambient temperature, 0 °C) for a minimum of 6 weeks. Fluidity was measured between 0 and 30 °C in all membranes, and polar lipid compositions and cholesterol contents were analyzed in a subset of biological membranes from all tissues. Osmotic permeability was measured in gills at 0 and 4 °C. Gill plasma membranes, cardiac mitochondria, and cardiac microsomes displayed reduced fluidity following acclimation to 5 °C, indicating compensation for elevated temperature. In contrast, no fluidity changes with acclimation were observed in any of the membranes prepared from brain. In all membranes, adjustments to the relative abundances of major phospholipid classes, and to the extent of fatty acid unsaturation, were undetectable following thermal acclimation. However, alterations in cholesterol contents and acyl chain length, consistent with the changes in fluidity, were observed in membranes from gill and cardiac tissue. Water permeability was reduced with 5 °C acclimation in gills, indicating near-perfect homeostatic efficacy. Taken together, these results demonstrate a homeoviscous response in gill and cardiac membranes, and limited plasticity in membranes from the nervous system, in an Antarctic notothenioid.

Thermal profiles reveal stark contrasts in properties of biological membranes from heart among Antarctic notothenioid fishes which vary in expression of hemoglobin and myoglobin.

Antarctic notothenioids are noted for extreme stenothermy, yet underpinnings of their thermal limits are not fully understood. We hypothesized that properties of ventricular membranes could explain previously observed differences among notothenioids in temperature onset of cardiac arrhythmias and persistent asystole. Microsomes were prepared using ventricles from six species of notothenioids, including four species from the hemoglobin-less (Hb-) family Channichthyidae (icefishes), which also differentially express cardiac myoglobin (Mb), and two species from the (Hb+) Nototheniidae. We determined membrane fluidity and structural integrity by quantifying fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and leakage of 5(6)-carboxyfluorescein, respectively, over a temperature range from ambient (0 °C) to 20 °C. Compositions of membrane phospholipids and cholesterol contents were also quantified. Membranes from all four species of icefishes exhibited greater fluidity than membranes from the red-blooded species N. coriiceps. Thermal sensitivity of fluidity did not vary among species. The greatest thermal sensitivity to leakage occurred between 0 and 5 °C for all species, while membranes from the icefish, Chaenocephalus aceratus (Hb-/Mb-) displayed leakage that was nearly 1.5-fold greater than leakage in N. coriiceps (Hb+/Mb+). Contents of phosphatidylethanolamine (PE) were approximately 1.5-fold greater in icefishes than in red-blooded fishes, and phospholipids had a higher degree of unsaturation in icefishes than in Hb + notothenioids. Cholesterol contents were lowest in Champsocephalus gunnari (Hb-/Mb-) and highest in the two Hb+/Mb + species, G. gibberifrons and N. coriiceps. Our results reveal marked differences in membrane properties and indicate a breach in membrane fluidity and structural integrity at a lower temperature in icefishes than in red-blooded notothenioids.