50 years of comparative biochemistry: The legacy of Peter Hochachka.

Peter Hochachka was an early pioneer in the field of comparative biochemistry. He passed away in 2002 after 4 decades of research in the discipline. To celebrate his contributions and to coincide with what would have been his 80th birthday, a group of his former students organized a symposium that ran as a satellite to the 2017 Canadian Society of Zoologists annual meeting in Winnipeg, Manitoba (Canada). This Special Issue of CBP brings together manuscripts from symposium attendees and other authors who recognize the role Peter played in the evolution of the discipline. In this article, the symposium organizers and guest editors look back on his career, celebrating his many contributions to research, acknowledging his role in training of generations of graduate students and post-doctoral fellows in comparative biochemistry and physiology.

Comparative biochemistry of cytochrome c oxidase in animals.

Cytochrome c oxidase (COX), the terminal enzyme of the electron transport system, is central to aerobic metabolism of animals. Many aspects of its structure and function are highly conserved, yet, paradoxically, it is also an important model for studying the evolution of the metabolic phenotype. In this review, part of a special issue honouring Peter Hochachka, we consider the biology of COX from the perspective of comparative and evolutionary biochemistry. The approach is to consider what is known about the enzyme in the context of conventional biochemistry, but focus on how evolutionary researchers have used this background to explore the role of the enzyme in biochemical adaptation of animals. In synthesizing the conventional and evolutionary biochemistry, we hope to identify synergies and future research opportunities. COX represents a rare opportunity for researchers to design studies that span the breadth of biology: molecular genetics, protein biochemistry, enzymology, metabolic physiology, organismal performance, evolutionary biology, and phylogeography.

Sensing and responding to energetic stress: The role of the AMPK-PGC1α-NRF1 axis in control of mitochondrial biogenesis in fish.

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro. In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α.

Evolution of the oxygen sensitivity of cytochrome c oxidase subunit 4.

Vertebrates possess two paralogs of cytochrome c oxidase (COX) subunit 4: a ubiquitous COX4-1 and a hypoxia-linked COX4-2. Mammalian COX4-2 is thought to have a role in relation to fine-tuning metabolism in low oxygen levels, conferred through both structural differences in the subunit protein structure and regulatory differences in the gene. We sought to elucidate the pervasiveness of this feature across vertebrates. The ratio of COX4-2/4-1 mRNA is generally low in mammals, but this ratio was higher in fish and reptiles, particularly turtles. The COX4-2 gene appeared unresponsive to low oxygen in nonmammalian models (zebrafish, goldfish, tilapia, anoles, and turtles) and fish cell lines. Reporter genes constructed from the amphibian and reptile homologues of the mammalian oxygen-responsive elements and hypoxia-responsive elements did not respond to low oxygen. Unlike the rodent ortholog, the promoter of goldfish COX4-2 did not respond to hypoxia or anoxia. The protein sequences of the COX4-2 peptide showed that the disulfide bridge seen in human and rodent orthologs would be precluded in other mammalian lineages and lower vertebrates, all of which lack the requisite pair of cysteines. The coordinating ligands of the ATP-binding site are largely conserved across mammals and reptiles, but in Xenopus and fish, sequence variations may disrupt the ability of the protein to bind ATP at this site. Collectively, these results suggest that many of the genetic and structural features of COX4-2 that impart responsiveness and benefits in hypoxia may be restricted to the Euarchontoglires lineage that includes primates, lagomorphs, and rodents.

Exploring the consequences of mitochondrial differences arising through hybridization of sunfish.

Previous studies have shown evidence of genomic incompatibility and mitochondrial enzyme dysfunction in hybrids of bluegill (Lepomis macrochirus Rafinesque) and pumpkinseed (Lepomis gibbosus Linnaeus) sunfish (Davies et al., 2012 Physiol. Biochem. Zool. 85, 321-331). We assessed if these differences in mitochondria had an impact on metabolic processes that depend on mitochondrial function, specifically hypoxia tolerance and recovery from burst exercise. Bluegill, pumpkinseed, and their hybrids showed no difference in the critical oxygen tension (Pcrit) and no differences in tissue metabolites measured after exposure to 10% O2 for 30min. In contrast, loss of equilibrium (LOE) measurements showed that hybrids had reduced hypoxia tolerance and lacked the size-dependence in hypoxia tolerance seen in the parental species. However, we found no evidence of systematic differences in metabolite levels in fish after LOE. Furthermore, there were abundant glycogen reserves at the point of loss of equilibrium. The three genotypes did not differ in metabolite status at rest, showed an equal disruption at exhaustion, and similar metabolic profiles throughout recovery. Thus, we found no evidence of a mitochondria dysfunction in hybrids, and mitochondrial differences and oxidative metabolism did not explain the variation in hypoxia tolerance seen in the hybrid and two parental species.

Hybridization in sunfish influences the muscle metabolic phenotype.

Hybridization has the potential to exert pleiotropic effects on metabolism. Effects on mitochondrial enzymes may arise through incompatibilities in nuclear- and mitochondrial-encoded subunits of the enzyme complexes of oxidative phosphorylation. We explored the metabolic phenotype of bluegill (Lepomis macrochirus), pumpkinseed (Lepomis gibbosus), and their unidirectional F(1) hybrids (male bluegill × female pumpkinseed). In hybrids, glycolytic enzyme activities were indistinguishable from (aldolase, pyruvate kinase) or intermediate to (lactate dehydrogenase, phosphoglucoisomerase) parentals, but complex IV activities aligned with pumpkinseed, both 30% lower than bluegill. In isolated mitochondria, the specific activities of complexes I, II, and V were indistinguishable between groups. However, both complex III and IV showed indications of depressed activities in hybrid mitochondria, though no effects on mitochondrial state 3 or state 4 respiration were apparent. The patterns in complex IV activities were due to differences in enzyme content rather than enzyme V(max); immunoblots comparing complex IV content with catalytic activity were indistinguishable between groups. The sequence differences in complex IV catalytic subunits (CO1, CO2, CO3) were minor in nature; however, the mtDNA-encoded subunit of complex III (cytochrome b) showed eight differences between bluegill and pumpkinseed, several of which could have structural consequences to the multimeric enzyme, contributing to the depressed complex III catalytic activity in hybrids.

Origins of interspecies variation in mammalian muscle metabolic enzymes.

Do the transcriptional mechanisms that control an individual's mitochondrial content, PGC1α (peroxisome proliferator-activated receptor γ coactivator-1α) and NRF1 (nuclear respiratory factor-1), also cause differences between species? We explored the determinants of cytochrome c oxidase (COX) activities in muscles from 12 rodents differing 1,000-fold in mass. Hindlimb muscles differed in scaling patterns from isometric (soleus, gastrocnemius) to allometric (tibialis anterior, scaling coefficient = -0.16). Consideration of myonuclear domain reduced the differences within species, but interspecies differences remained. For tibialis anterior, there was no significant scaling relationship in mRNA/g for COX4-1, PGC1α, or NRF1, yet COX4-1 mRNA/g was a good predictor of COX activity (r(2) = 0.55), PGC1α and NRF1 mRNA correlated with each other (r(2) = 0.42), and both could predict COX4-1 mRNA (r(2) = 0.48 and 0.52) and COX activity (r(2) = 0.55 and 0.49). This paradox was resolved by multivariate analysis, which explained 90% of interspecies variation, about equally partitioned between mass effects and PGC1α (or NRF1) mRNA levels, independent of mass. To explore the determinants of PGC1α mRNA, we analyzed 52 mammalian PGC1α proximal promoters and found no size dependence in regulatory element distribution. Likewise, the activity of PGC1α promoter reporter genes from 30 mammals showed no significant relationship with body mass. Collectively, these studies suggest that not all muscles scale equivalently, but for those that show allometric scaling, transcriptional regulation of the master regulators, PGC1α and NRF1, does not account for scaling patterns, though it does contribute to interspecies differences in COX activities independent of mass.

Evolutionary affinity of billfishes (Xiphiidae and Istiophoridae) and flatfishes (Plueronectiformes): Independent and trans-subordinal origins of endothermy in teleost fishes.

Billfishes (Scombroidei) and tunas (Scombridae), both considered part of the suborder Scombroidei, have long been studied by biologists largely because of their remarkable physiological and anatomical muscular adaptations associated with regional endothermy and continuous swimming. These attributes, combined with analyses of other morphological and molecular data, have led to a general perception that tunas and billfishes are close relatives, though this hypothesis has been vigorously debated. Using Bayesian phylogenetic analysis of nine mitochondrial and three nuclear loci (>7000bp), we show that billfishes are only distantly related to tunas, but rather share strong evolutionary affinities with flatfishes (Pleuronectiformes) and jacks (Carangidae). This phylogenetic relationship is striking because of the marked variation in phenotype and niche across these trans-ordinal groups of fishes. Billfishes and flatfishes have each evolved radically divergent morphological and physiological features: elongated bills and extraocular heater organs in billfishes, and cranial asymmetry with complete eye migration during ontogenetic development in flatfishes. Despite this divergence, we identify synapomorphies consistent with the hypothesis of a common billfish/flatfish/jack ancestor.

Evolution of the nuclear-encoded cytochrome oxidase subunits in vertebrates.

Vertebrate mitochondrial cytochrome c oxidase (COX) possesses 10 nuclear-encoded subunits. Six subunits have paralogs in mammals, but the origins and distribution of isoforms among vertebrates have not been analyzed. We used Bayesian phylogenetic analysis to interpret the origins of each subunit, inferring the roles of gene and genome duplications. The paralogous ancestries of five genes were identical throughout the major vertebrate taxa: no paralogs of COX6c and COX7c, two paralogs of COX4 and COX6a, and three paralogs of COX7a. Two genes had an extra copy in teleosts (COX5a, COX5b), and three genes had additional copies in mammals (COX6b, COX7b, COX8). Focusing on early vertebrates, we examined structural divergence and explored transcriptional profiles across zebrafish tissues. Quantitative transcript profiles revealed dramatic differences in transcript abundance for different subunits. COX7b and COX4 transcripts were typically present at very low levels, whereas COX5a and COX8 were in vast excess in all tissues. For genes with paralogs, two general patterns emerged. For COX5a and COX8, there was ubiquitous expression of one paralog, with the other paralog in lower abundance in all tissues. COX4 and COX6a shared a distinct expression pattern, with one paralog dominant in brain and gills and the other in muscles. The isoform profiles in combination with phylogenetic analyses show that vertebrate COX isoform patterns are consistent with the hypothesis that early whole genome duplications in basal vertebrates governed the isoform repertoire in modern fish and tetrapods, though more recent lineage-specific gene/genome duplications also play a role in select subunits.

Hypoxia and the metabolic phenotype of prostate cancer cells.

Many cancer cells have an unusual ability to grow in hypoxia, but the origins of this metabolic phenotype remain unclear. We compared the metabolic phenotypes of three common prostate cancer cell models (LNCaP, DU145, PC3), assessing energy metabolism, metabolic gene expression, and the response to various culture contexts (in vitro and xenografts). LNCaP cells had a more oxidative phenotype than PC3 and DU145 cells based upon respiration, lactate production, [ATP], metabolic gene expression, and sensitivity of these parameters to hypoxia. PC3 and DU145 cells possessed similar Complex II and mtDNA levels, but lower Complex III and IV activities, and were unresponsive to dinitrophenol or dichloroacetate, suggesting that their glycolytic phenotype is due to mitochondrial dysfunction rather than regulation. High passage under normoxia converted LNCaP from oxidative to glycolytic cells (based on respiration and lactate production), and altered metabolic gene expression. Though LNCaP-derived cells differed from the parental line in mitochondrial enzyme activities, none differed in mitochondrial content (assessed as cardiolipin levels). When LNCaP-derived cells were grown as xenografts in immunodeficient mice, there were elements of a hypoxic response (e.g., elevated VEGF mRNA) but line-specific changes in expression of select glycolytic, mitochondrial and fatty acid metabolic genes. Low oxygen in vitro did not influence the mRNA levels of SREBP axis, nor did it significantly alter triglyceride production in any of the cell lines suggesting that the pathway of de novo fatty acid synthesis is not directly upregulated by hypoxic conditions. Collectively, these studies demonstrate important differences in the metabolism of these prostate cancer models. Such metabolic differences would have important ramifications for therapeutic strategies involving metabolic targets.

Control of mitochondrial biogenesis during myogenesis.

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and Sol8). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2alpha increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPARalpha) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-gamma coactivator-1alpha (PGC1alpha), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1alpha-related coactivator and PGC1beta was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter ( approximately 2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity.

Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes.

We investigated if seasonal changes in rainbow trout muscle energetics arise in response to seasonal changes in erythrocyte properties. We assessed if skeletal muscle mitochondrial enzymes changed (1) acutely in response to changes in erythrocyte abundance, or (2) seasonally when we altered the age profile of erythrocytes. Rainbow trout were treated with pheynylhydrazine, causing a 75% reduction in hematocrit within 4 days. After erythropoiesis had returned hematocrit to normal, treated and control fish were subjected to a seasonal cold acclimation regime to assess the impact of erythrocyte age on skeletal muscle remodeling. Anemia (i.e. phenylhydrazine treatment) did not alter the specific activities (U g(-1) tissue) of mitochondrial enzymes in white or red muscle. Anemic pretreatment did not alter the normal pattern of cold-induced mitochondrial proliferation in skeletal muscle, suggesting erythrocyte age was not an important influence on seasonal remodeling of muscle. Anemia and cold acclimation both induced a 25-30% increase in relative ventricular mass. The increase in relative ventricular mass with phenylhydrazine treatment was accompanied by a 35% increase in DNA content (mg DNA per ventricle), suggesting an increase in number of cells. In contrast, the increase in ventricular mass with cold temperature acclimation occurred without a change in DNA content (mg DNA per ventricle), suggesting an increase in cell size. Despite the major increases in relative ventricular mass, neither anemia nor seasonal acclimation had a major influence on the specific activities of a suite of mitochondrial enzymes in heart. Collectively, these studies argue against a role for erythrocyte dynamics in seasonal adaptive remodeling of skeletal muscle energetics.

Leptin and the control of respiratory gene expression in muscle.

Leptin plays a central role in the regulation of fatty acid homeostasis, promoting lipid storage in adipose tissue and fatty acid oxidation in peripheral tissues. Loss of leptin signaling leads to accumulation of lipids in muscle and loss of insulin sensitivity secondary to obesity. In this study, we examined the direct and indirect effects of leptin signaling on mitochondrial enzymes including those essential for peripheral fatty acid oxidation. We assessed the impact of leptin using the JCR:LA-cp rat, which lacks functional leptin receptors. The activities of marker mitochondrial enzymes citrate synthase (CS) and cytochrome oxidase (COX) were similar between wild-type (+/?) and corpulent (cp/cp) rats. In contrast, several tissues showed variations in the fatty acid oxidizing enzymes carnitine palmitoyltransferase II (CPT II), long-chain acyl-CoA dehydrogenase (LCAD) and 3-hydroxyacyl-CoA dehydrogenase (HOAD). It was not clear if these changes were due to loss of leptin signaling or to insulin insensitivity. Consequently, we examined the effects of leptin on cultured C(2)C(12) and Sol8 cells. Leptin (3 days at 0, 0.2, or 2.0 nM) had no direct effect on the activities of CS, COX, or fatty acid oxidizing enzymes. Leptin treatment did not affect luciferase-based reporter genes under the control of transcription factors involved in mitochondrial biogenesis (nuclear respiratory factor-1 (NRF-1), nuclear respiratory factor-2 (NRF-2)) or fatty acid enzyme expression (peroxisome proliferator-activated receptors (PPARs)). These studies suggest that leptin exerts only indirect effects on mitochondrial gene expression in muscle, possibly arising from insulin resistance.

Fiber-type differences in muscle mitochondrial profiles.

Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2 production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences ( approximately 50%) in stoichiometries of selected enzymes of the Krebs cycle, beta-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.

Origins and consequences of mitochondrial decline in nucleated erythrocytes.

Cellular aging in nucleated erythrocytes from lower vertebrates is accompanied by losses in mitochondria but it remains unclear (i) how these losses accrue (ii) if these changes alter energetics and (iii) whether such changes increase the propensity for apoptosis. We addressed these questions using trout erythrocytes that were separated into age classes using inherent differences in buoyant density. The oldest cells showed a profound decline in mtDNA transcripts, due to reductions in both transcription (90% decline in total RNA) and mtDNA copy number (35%). No alterations in the ratio of 16S rRNA to COX I mRNA were detected, nor was there an accumulation of unprocessed mtDNA transcripts. While older cells had reduced basal respiration, there were no changes in mitochondrial enzymes stoichiometries, tissue ATP levels or dinitrophenol-induced (maximal) respiration rates. Apoptosis could not be induced in either whole blood, young or old erythrocytes by pro-oxidants, mitochondrial inhibitors or staurosporine. In contrast, cyclosporin A (CsA) caused caspase 3 activation, DNA laddering and LDH leakage, but only in young cells. Both CsA and a combination of azide, oligomycin and dinitrophenol cause mitochondrial depolarization and caspase 9 activation, but only CsA induced caspase 3 and apoptosis. Caspase inhibitor studies support the conclusion that mitochondrial changes may accompany CsA-induced cell death, but are not essential in its progression. While pifithrin failed to induce cell death, it enhanced the effects of CsA, implicating a role for p53. Collectively, these studies suggest that the mitochondrial changes with aging do not compromise cellular function, although trout erythrocytes can initiate apoptosis by non-mitochondrial pathways.

Characterization of erythrocyte carbonic anhydrase in an ancient fish, the longnose gar ( Lepisosteus osseus).

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.

Bioenergetic remodeling of heart during treatment of spontaneously hypertensive rats with enalapril.

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in <10 days). Despite changes in LV mass, specific activities of bioenergetic (cytochrome-c oxidase, citrate synthase, lactate dehydrogenase) and reactive oxygen species (ROS) (total cellular superoxide dismutase) enzymes were actively maintained within relatively narrow ranges regardless of treatment duration, organismal age, or transmural region. Although enalapril led to parallel declines in mitochondrial enzyme content and ventricular mass, total ventricular mtDNA content was unaffected. Altered enzymatic content occurred without significant changes in relevant mRNA and protein levels. Transcript levels of gene products involved in mtDNA maintenance (Tfam), mitochondrial protein degradation (LON protease), fusion (fuzzy onion homolog), and fission (dynamin-like protein, synaptojanin-2alpha) were also unchanged. In contrast, enalapril-mediated ventricular and mitochondrial remodeling was accompanied by a twofold increase in specific activity of catalase, an indicator of oxidative stress, suggesting that rapid cardiac adaptation is accompanied by tight regulation of mitochondrial enzyme activities and increased ROS production.

Anoxia induces thermotolerance in the locust flight system.

Heat shock and anoxia are environmental stresses that are known to trigger similar cellular responses. In this study, we used the locust to examine stress cross-tolerance by investigating the consequences of a prior anoxic stress on the effects of a subsequent high-temperature stress. Anoxic stress and heat shock induced thermotolerance by increasing the ability of intact locusts to survive normally lethal temperatures. To determine whether induced thermotolerance observed in the intact animal was correlated with electrophysiological changes, we measured whole-cell K(+) currents and action potentials from locust neurons. K(+) currents recorded from thoracic neuron somata were reduced after anoxic stress and decreased with increases in temperature. Prior anoxic stress and heat shock increased the upper temperature limit for generation of an action potential during a subsequent heat stress. Although anoxia induced thermotolerance in the locust flight system, a prior heat shock did not protect locusts from a subsequent anoxic stress. To determine whether changes in bioenergetic status were implicated in whole-animal cross-tolerance, phosphagen levels and rates of mitochondrial respiration were assayed. Heat shock alone had no effect on bioenergetic status. Prior heat shock allowed rapid recovery after normally lethal heat stress but afforded no protection after a subsequent anoxic stress. Heat shock also afforded no protection against disruption of bioenergetic status after a subsequent exercise stress. These metabolite studies are consistent with the electrophysiological data that demonstrate that a prior exposure to anoxia can have protective effects against high-temperature stress but that heat shock does not induce tolerance to anoxia.

Evolutionary and physiological variation in cardiac troponin C in relation to thermal strategies of fish.

Striated muscle contraction is initiated when troponin C (TnC) binds Ca(2+), which activates actinomyosin ATPase. We investigated (i) the variation between cardiac TnC (cTnC) primary structure within teleost fish and (ii) the pattern of TnC expression in response to temperature acclimation. There were few differences between rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares), yellow perch (Perca flavescens), goldfish (Carassius auratus), white sucker (Catostomus commersoni), and icefish (Chaenocephalus aceratus) in cTnC amino acid sequence. No variation existed in the regulatory Ca(2+)-binding site (site 2). The site 3 and 4 substitutions were limited to residues not directly involved in Ca(2+) coordination. Fish cTnC primary structure was highly conserved between species (93%-98%) and collectively divergent from the highly conserved sequence seen in birds and mammals. Northern blots and polymerase chain reaction showed that thermal acclimation of trout (3 degrees, 18 degrees C) did not alter the TnC isoform pattern. While cardiac and white muscle had the expected isoforms-cTnC and fast troponin C (fTnC), respectively-red muscle unexpectedly expressed primarily ftnC. Cold acclimation did not alter myofibrillar ATPase Ca(2+) sensitivity, but maximal velocity increased by 60%. We found no evidence that TnC variants, arising between species or in response to thermal acclimation, play a major role in mitigating the effects of temperature on contractility of the adult fish heart.