Published papers

Aim To identify the physiological role of the acid‐base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. Methods We used: (i) super‐resolution microscopy to determine the subcellular location of sAC protein; (ii) live‐cell imaging of RBC intracellular pH (pH i ) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid‐base regulation; (iii) spectrophotometric measurements of haemoglobin–oxygen (Hb‐O 2 ) binding in steady‐state conditions; and (iv) during simulated arterial‐venous transit, to determine the role of sAC in systemic O 2 transport. Results Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pH i regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pH i recovery after an acid‐base disturbance. RBC pH i recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO 3 − ‐dependent sAC signaling. Inhibition of sAC decreased Hb‐O 2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O 2 binding. During in vitro simulations of arterial‐venous transit, sAC inhibition decreased the amount of O 2 that is unloaded by ~11%. Conclusion sAC represents a novel acid‐base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pH i and blood O 2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by β-adrenergic sodium-proton exchangers (β-NHE) that protect pH-sensitive haemoglobin–oxygen (Hb–O2) binding during an acidosis. In an open system that mimics the gills, β-NHE activity increased Hb–O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of β-NHE activity on Hb–O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by β-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

In the developing embryos of egg-laying vertebrates, O 2 flux takes place across a fixed surface area of the eggshell and the chorioallantoic membrane. In the case of crocodilians, the developing embryo may experience a decrease in O 2 flux when the nest becomes hypoxic, which may cause compensatory adjustments in blood O 2 transport. However, whether the switch from embryonic to adult hemoglobin isoforms (isoHbs) plays some role in these adjustments is unknown. Here, we provide a detailed characterization of the developmental switch of isoHb synthesis in the American alligator, Alligator mississippiensis. We examined the in vitro functional properties and subunit composition of purified alligator isoHbs expressed during embryonic developmental stages in normoxia and hypoxia (10% O 2 ). We found distinct patterns of isoHb expression in alligator embryos at different stages of development, but these patterns were not affected by hypoxia. Specifically, alligator embryos expressed two main isoHbs: HbI, prevalent at early developmental stages, with a high O 2 affinity and high ATP sensitivity, and HbII, prevalent at later stages and identical to the adult protein, with a low O 2 affinity and high CO 2 sensitivity. These results indicate that whole blood O 2 affinity is mainly regulated by ATP in the early embryo and by CO 2 and bicarbonate from the late embryo until adult life, but the developmental regulation of isoHb expression is not affected by hypoxia exposure.

Approximately 36,000 units of red blood cells (RBCs) are used every day in the U.S. and there is a great challenge for hospitals to maintain a reliable supply, given the 42-day expiration period from the blood donation date. For many years, research has been conducted to develop ex vivo storage solutions that limit RBC lysis and maintain a high survival rate of the transfused cells. However, little attention is directed towards potential fractionation methods to remove unwanted cell debris or aged blood cells from stored RBC units prior to transfusion, which could not only expand the ex vivo shelf life of RBC units but also avoid adverse events in transfused patients. Such fractionation methods could also limit the number of transfusions required for treating certain pathologies, such as sickle cell disease (SCD). In this work, magnetic fractionation is studied as a potential technology to fractionate functional and healthy RBCs from aged or sickle cells. It has been reported that during ex vivo RBC storage, RBCs lose hemoglobin (Hb) and lipid content via formation of Hb-containing exosomes. Given the magnetic character of deoxygenated- or met-Hb, in this work, we propose the use of a quadrupole magnetic sorter (QMS) to fractionate RBCs based on their Hb content from both healthy stored blood and SCD blood. In our QMS, a cylindrical microchannel placed inside the center of the quadrupolar magnets is subjected to high magnetic fields and constant field gradients (286 T/m), which causes the deflection of the paramagnetic, Hb-enriched, and functional RBCs from their original path and their collection into a different outlet. Our results demonstrated that although we could obtain a significant difference in the magnetic mobility of the sorted fractions (corresponding to a difference in more than 1 pg of Hb per cell), there exists a tradeoff between throughput and purity. Therefore, this technology when optimized could be used to expand the ex vivo shelf life of RBC units and avoid adverse events in transfused individuals or SCD patients requiring blood exchange therapy.

Significance In diving birds like penguins, physiologic considerations suggest that increased hemoglobin (Hb)-O 2 affinity may improve pulmonary O 2 extraction and enhance dive capacity. We integrated experimental tests on whole-blood and native Hbs of penguins with protein engineering experiments on reconstructed ancestral Hbs. The experiments involving ancestral protein resurrection enabled us to test for evolved changes in Hb function in the stem lineage of penguins after divergence from their closest nondiving relatives. We demonstrate that penguins evolved an increased Hb-O 2 affinity in conjunction with a greatly augmented Bohr effect (i.e., reduction in Hb-O 2 affinity at low pH) that should maximize pulmonary O 2 extraction without compromising O 2 delivery at systemic capillaries. Dive capacities of air-breathing vertebrates are dictated by onboard O 2 stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O 2 transport. It has been hypothesized that increased hemoglobin (Hb)-O 2 affinity improves pulmonary O 2 extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O 2 affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O 2 affinity at low pH). Although an increased Hb-O 2 affinity reduces the gradient for O 2 diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O 2 unloading in acidified tissues. We suggest that the evolved increase in Hb-O 2 affinity in combination with the augmented Bohr effect maximizes both O 2 extraction from the lungs and O 2 unloading from the blood, allowing penguins to fully utilize their onboard O 2 stores and maximize underwater foraging time.

Fish in coastal ecosystems can be exposed to acute variations in CO2 of between 0.2 and 1 kPa CO2 (2000–10,000 µatm). Coping with this environmental challenge will depend on the ability to rapidly compensate for the internal acid–base disturbance caused by sudden exposure to high environmental CO2 (blood and tissue acidosis); however, studies about the speed of acid–base regulatory responses in marine fish are scarce. We observed that upon sudden exposure to ∼1 kPa CO2, European sea bass (Dicentrarchus labrax) completely regulate erythrocyte intracellular pH within ∼40 min, thus restoring haemoglobin–O2 affinity to pre-exposure levels. Moreover, blood pH returned to normal levels within ∼2 h, which is one of the fastest acid–base recoveries documented in any fish. This was achieved via a large upregulation of net acid excretion and accumulation of HCO3− in blood, which increased from ∼4 to ∼22 mmol l−1. While the abundance and intracellular localisation of gill Na+/K+-ATPase (NKA) and Na+/H+ exchanger 3 (NHE3) remained unchanged, the apical surface area of acid-excreting gill ionocytes doubled. This constitutes a novel mechanism for rapidly increasing acid excretion during sudden blood acidosis. Rapid acid–base regulation was completely prevented when the same high CO2 exposure occurred in seawater with experimentally reduced HCO3− and pH, probably because reduced environmental pH inhibited gill H+ excretion via NHE3. The rapid and robust acid–base regulatory responses identified will enable European sea bass to maintain physiological performance during large and sudden CO2 fluctuations that naturally occur in coastal environments.

We indirectly assessed if altered transarcolemmal Ca 2+ flux accompanies the decreased cardiac activity displayed by Trachemys scripta with anoxia exposure and cold acclimation. Turtles were first acclimated to 21°C or 5°C and held under normoxic (21N; 5N) or anoxic conditions (21A; 5A). We then compared the response of intrinsic heart rate ( f H ) and maximal developed force of spontaneously contracting right atria ( F max,RA ), and maximal developed force of isometrically-contracting ventricular strips ( F max,V ), to Ni 2+ (0.1 – 10 mM), which respectively blocks T-type Ca 2+ channels, L-type Ca 2+ channels and the Na + -Ca 2+ -exchanger at the low, intermediate and high concentrations employed. Dose-response curves were established in simulated in vivo normoxic (Sim Norm) or simulated in vivo anoxic extracellular conditions (21A and 5A preparations). Ni 2+ decreased intrinsic f H, F max,RA and F max,V of 21N tissues in a concentration-dependent manner, but the responses were blunted in 21A tissues in Sim Norm. Similarly, dose-response curves for F max,RA and F max,V of 5N tissues were right-shifted, whereas anoxia exposure at 5°C did not further alter the responses. The influence of Sim Anx was acclimation temperature-, cardiac chamber- and contractile parameter-dependent. Combined, the findings suggest that: (1) reduced transarcolemmal Ca 2+ flux in the cardiac pacemaker is a potential mechanism underlying the slowed intrinsic f H of anoxic turtles at 21°C, but not 5°C, (2) a downregulation of transarcolemmal Ca 2+ flux may aid cardiac anoxia survival at 21°C and prime the turtle myocardium for winter anoxia and (3) confirm that altered extracellular conditions with anoxia exposure can modify turtle cardiac transarcolemmal Ca 2+ flux. Keywords: anoxia, temperature, cardiac pacemaker, cardiac regulation, ion channels, myocardial contractile properties, red-eared slider

White seabass ( Atractoscion nobilis) increasingly experience periods of low oxygen (O 2; hypoxia) and high carbon dioxide (CO 2, hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O 2 carrier in the blood and in many teleost fishes Hb-O 2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (β-NHEs). We hypothesized that RBC β-NHEs in white seabass are an important mechanism that can protect the blood O 2 -carrying capacity during hypoxia and hypercapnia. We determined the O 2 -binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of β-NHE activity on Hb-O 2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O 2 affinity (Po 2 at half-saturation; P 50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient −0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed β-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of β-NHE activity. The activation of RBC β-NHEs increased Hb-O 2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. β-NHE activity in white seabass has great potential to protect arterial O 2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors.

Oxygen (O2) and carbon dioxide (CO2) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O2 binding even at high O2 tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O2 delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O2 offloading following a physiologically relevant CO2-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O2 offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O2 offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O2 offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O2 delivery in red drum.

Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and β-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical ‘missing link’ through which the modern tetramer evolved—a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and β-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein’s structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures. Experimental analysis of reconstructed ancestral globins reveals that haemoglobin’s complex tetrameric structure and oxygen-binding functions evolved by simple genetic and biophysical mechanisms.

Catfish Ictalurus spp. are subjected to stressful conditions during harvest, which may be linked to fillet coloration and quality. Poor water quality in ponds, socks or hauling tanks, as well as handling stress, have been suggested to cause red fillets in catfish; however, chronic exposure has not resulted in red fillets. Short‐term occurrences of extreme poor water quality, particularly low dissolved oxygen, high carbon dioxide and high temperature, may occur in ponds or during harvest. Therefore, market‐sized Channel Catfish Ictalurus punctatus were acutely exposed (12 hr) to one of the three water quality treatments while confined during a simulated socking procedure and evaluated for stress responses by means of change in blood parameters and fillet quality. In fish subjected to the extreme treatment, hematocrit, plasma cortisol, glucose and lactate levels increased, with 22% mortality, indicating highly stressful conditions. In fish subjected to moderate and typical (control) treatments, cortisol increased but a lack of change or decrease in glucose and lactate indicated minimal anaerobic metabolism. Only one red fillet was produced by the extreme treatment and two by the typical treatment; therefore, the results suggest red fillets are not a product of poor water quality compounded by handling during harvest.

Global environmental change is increasing hypoxia in aquatic ecosystems. During hypoxic events, bacterial respiration causes an increase in carbon dioxide (CO2) while oxygen (O2) declines. This is rarely accounted for when assessing hypoxia tolerances of aquatic organisms. We investigated the impact of environmentally realistic increases in CO2 on responses to hypoxia in European sea bass (Dicentrarchus labrax). We conducted a critical oxygen (O2crit) test, a common measure of hypoxia tolerance, using two treatments in which O2 levels were reduced with constant ambient CO2 levels (~530 µatm), or with reciprocal increases in CO2 (rising to ~2,500 µatm). We also assessed blood acid-base chemistry and haemoglobin-O2 binding affinity of sea bass in hypoxic conditions with ambient (~650 μatm) or raised CO2 (~1770 μatm) levels. Sea bass exhibited greater hypoxia tolerance (~20% reduced O2crit), associated with increased haemoglobin-O2 affinity (~32% fall in P50) of red blood cells, when exposed to reciprocal changes in O2 and CO2. This indicates that rising CO2 which accompanies environmental hypoxia facilitates increased O2 uptake by the blood in low O2 conditions, enhancing hypoxia tolerance. We recommend that when impacts of hypoxia on aquatic organisms are assessed, due consideration is given to associated environmental increases in CO2.

Globins are a classical model system for the studies of protein evolution and function. Recent studies have shown that – besides the well-known haemoglobin and myoglobin – additional globin-types occur in vertebrates that serve different functions. Globin E (GbE) was originally identified as an eye-specific protein of birds that is distantly related to myoglobin. GbE is also present in turtles and the coelacanth but appeared to have been lost in other vertebrates. Here, we show that GbE additionally occurs in lungfish, the closest living relatives of the tetrapods. Each lungfish species harbours multiple (≥5) GbE gene copies. Surprisingly, GbE is exclusively and highly expressed in oocytes, with mRNA levels that exceed that of myoglobin in the heart. Thus, GbE is the first known oocyte-specific globin in vertebrates. No GbE transcripts were found in the ovary or egg transcriptomes of other vertebrates, suggesting a lungfish-specific function. Spectroscopic analysis and kinetic studies of recombinant GbE1 of the South American lungfish Lepidosiren paradoxa revealed a typical pentacoordinate globin with myoglobin-like O2-binding kinetics, indicating similar functions. Our findings suggest that the multiple copies of GbE evolved to enhance O2-supply in the developing embryo of lungfish, analogous to the embryonic and fetal haemoglobins of other vertebrates. In evolution, GbE must have changed its expression site from oocytes to eyes, or vice versa.

A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb–O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the β-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked β-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the β-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb–O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed’s relatively short duration of residency at high altitude.

When different species experience similar selection pressures, the probability of evolving similar adaptive solutions may be influenced by legacies of evolutionary history, such as lineage-specific changes in genetic background. Here we test for adaptive convergence in hemoglobin (Hb) function among high-altitude passerine birds that are native to the Qinghai-Tibet Plateau, and we examine whether convergent increases in Hb–O 2 affinity have a similar molecular basis in different species. We documented that high-altitude parid and aegithalid species from the Qinghai-Tibet Plateau have evolved derived increases in Hb–O 2 affinity in comparison with their closest lowland relatives in East Asia. However, convergent increases in Hb–O 2 affinity and convergence in underlying functional mechanisms were seldom attributable to the same amino acid substitutions in different species. Using ancestral protein resurrection and site-directed mutagenesis, we experimentally confirmed two cases in which parallel substitutions contributed to convergent increases in Hb–O 2 affinity in codistributed high-altitude species. In one case involving the ground tit ( Parus humilis ) and gray-crested tit ( Lophophanes dichrous ), parallel amino acid replacements with affinity-enhancing effects were attributable to nonsynonymous substitutions at a CpG dinucleotide, suggesting a possible role for mutation bias in promoting recurrent changes at the same site. Overall, most altitude-related changes in Hb function were caused by divergent amino acid substitutions, and a select few were caused by parallel substitutions that produced similar phenotypic effects on the divergent genetic backgrounds of different species.