Responses of the in vitro turtle brain to visual and auditory stimuli during severe hypoxia.

North American pond turtles (Emydidae) are renowned for their ability to survive extreme hypoxia and anoxia, which enables several species to overwinter in ice-locked, anoxic freshwater ponds and bogs for months. Centrally important for surviving these conditions is a profound metabolic suppression, which enables ATP demands to be met entirely with glycolysis. To better understand whether anoxia limits special sensory functions, we recorded evoked potentials in a reduced brain preparation, in vitro, that was perfused with severely hypoxic artificial cerebral spinal fluid (aCSF). For recordings of visual responses, an LED was flashed onto retinal eyecups while evoked potentials were recorded from the retina or the optic tectum. For recordings of auditory responses, a piezomotor-controlled glass actuator displaced the tympanic membrane while evoked potentials were recorded from the cochlear nuclei. We found that visual responses decreased when perfused with hypoxic perfusate (aCSF PO2<4.0 kPa). In contrast, the evoked response within the cochlear nuclei was unattenuated. These data provide further support that pond turtles have a limited ability to sense visual information in their environment even while moderately hypoxic, but that auditory input may become a principal avenue of sensory perception during extreme diving in this species such as occurs during anoxic submergence.

Skeletal muscle histidine-containing dipeptide contents are increased in freshwater turtles (C. picta bellii) with cold-acclimation.

Freshwater turtles found in higher latitudes can experience extreme challenges to acid-base homeostasis while overwintering, due to a combination of cold temperatures along with the potential for environmental hypoxia. Histidine-containing dipeptides (HCDs; carnosine, anserine and balenine) may facilitate pH regulation in response to these challenges, through their role as pH buffers. We measured the HCD content of three tissues (liver, cardiac and skeletal muscle) from the anoxia-tolerant painted turtle (C. picta bellii) acclimated to either 3 or 20 °C. HCDs were detected in all tissues, with the highest content shown in the skeletal muscle. Turtles acclimated to 3 °C had more HCD in their skeletal muscle than those acclimated to 20 °C (carnosine = 20.8 ± 4.5 vs 12.5 ± 5.9 mmol·kg DM-1; ES = 1.59 (95%CI: 0.16-3.00), P = 0.013). The higher HCD content shown in the skeletal muscle of the cold-acclimated turtles suggests a role in acid-base regulation in response to physiological challenges associated with living in the cold, with the increase possibly related to the temperature sensitivity of carnosine's dissociation constant.

Cold acclimation induces life stage-specific responses in the cardiac proteome of western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance.

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3°C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. As protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold acclimation would evoke preparatory changes in protein expression to support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20°C (warm) or 3°C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis. The relative abundance of 1316 identified proteins was compared between temperatures and developmental stages. The effect of cold acclimation on the cardiac proteome was only evident in the context of an interaction with life stage, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope with age that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of the life stage- and temperature-induced changes to the cardiac proteome, including reduced Complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stage-specific anoxia tolerance in turtles.

Palaeophysiology of pH regulation in tetrapods.

The involvement of mineralized tissues in acid-base homeostasis was likely important in the evolution of terrestrial vertebrates. Extant reptiles encounter hypercapnia when submerged in water, but early tetrapods may have experienced hypercapnia on land due to their inefficient mode of lung ventilation (likely buccal pumping, as in extant amphibians). Extant amphibians rely on cutaneous carbon dioxide elimination on land, but early tetrapods were considerably larger forms, with an unfavourable surface area to volume ratio for such activity, and evidence of a thick integument. Consequently, they would have been at risk of acidosis on land, while many of them retained internal gills and would not have had a problem eliminating carbon dioxide in water. In extant tetrapods, dermal bone can function to buffer the blood during acidosis by releasing calcium and magnesium carbonates. This review explores the possible mechanisms of acid-base regulation in tetrapod evolution, focusing on heavily armoured, basal tetrapods of the Permo-Carboniferous, especially the physiological challenges associated with the transition to air-breathing, body size and the adoption of active lifestyles. We also consider the possible functions of dermal armour in later tetrapods, such as Triassic archosaurs, inferring palaeophysiology from both fossil record evidence and phylogenetic patterns, and propose a new hypothesis relating the archosaurian origins of the four-chambered heart and high systemic blood pressures to the perfusion of the osteoderms. This article is part of the theme issue 'Vertebrate palaeophysiology'.

Development-specific transcriptomic profiling suggests new mechanisms for anoxic survival in the ventricle of overwintering turtles.

Oxygen deprivation swiftly damages tissues in most animals, yet some species show remarkable abilities to tolerate little or even no oxygen. Painted turtles exhibit a development-dependent tolerance that allows adults to survive anoxia approximately four times longer than hatchlings: adults survive ∼170 days and hatchlings survive ∼40 days at 3°C. We hypothesized that this difference is related to development-dependent differences in ventricular gene expression. Using a comparative ontogenetic approach, we examined whole transcriptomic changes before, during and 5 days after a 20-day bout of anoxic submergence at 3°C. Ontogeny accounted for more gene expression differences than treatment (anoxia or recovery): 1175 versus 237 genes, respectively. Of the 237 differences, 93 could confer protection against anoxia and reperfusion injury, 68 could be injurious and 20 may be constitutively protective. Most striking during anoxia was the main expression pattern of all 76 annotated ribosomal protein (R-protein) mRNAs, which decreased in anoxia-tolerant adults, but increased in anoxia-sensitive hatchlings, suggesting adult-specific regulation of translational suppression. These genes, along with 60 others that decreased their levels in adults and either increased or remained unchanged in hatchlings, implicate antagonistic pleiotropy as a mechanism to resolve the long-standing question about why hatchling painted turtles overwinter in terrestrial nests, rather than emerge and overwinter in water during their first year. In summary, developmental differences in the transcriptome of the turtle ventricle revealed potentially protective mechanisms that contribute to extraordinary adult-specific anoxia tolerance, and provide a unique perspective on differences between the anoxia-induced molecular responses of anoxia-tolerant and anoxia-sensitive phenotypes within a species.

Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense.

Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

Heterogeneous bioapatite carbonation in western painted turtles is unchanged after anoxia.

Adsorbed and structurally incorporated carbonate in bioapatite, the primary mineral phase of bone, is observed across vertebrates, typically at 2-8 wt%, and supports critical physiological and biochemical functions. Several turtle species contain elevated bone-associated carbonate, a property linked to pH buffering and overwintering survival. Prior studies of turtle bone utilized bulk analyses, which do not provide spatial resolution of carbonate. Using Raman spectroscopy, the goals of this study were to: (1) quantify and spatially resolve carbonate heterogeneity within the turtle shell; (2) determine if cortical and trabecular bone contain distinct carbonate concentrations; and (3) assess if simulated overwintering conditions result in decreased bioapatite carbonation. Here, we demonstrate the potential for Raman spectroscopic analysis to spatially resolve bioapatite carbonation, using the western painted turtle as a model species. Carbonate concentration was highly variable within cortical and trabecular bone, based on calibrated Raman spot analyses and mapping, suggesting heterogeneous carbonate distribution among crystallites. Mean carbonate concentration did not significantly differ between cortical and trabecular bone, which indicates random distribution of crystallites with elevated and depleted carbonate. Carbonate concentrations (range: 5-22 wt%) were not significantly different in overwintering and control animals, deviating from previous bulk analyses. In reconciling bulk and Raman analyses, two hypotheses explain how overwintering turtles potentially access carbonate: (1) mobilization of mineral-associated, surface components of bone crystallites; and (2) selective, dispersed crystallite dissolution. Elevated bioapatite carbonate in the western painted turtle, averaging 11.8 wt%, represents the highest carbonation observed in vertebrates, and is one physiological trait that facilitates overwintering survival.

Changes in the material properties of the shell during simulated aquatic hibernation in the anoxia-tolerant painted turtle.

Western painted turtles (Chrysemys picta bellii) tolerate anoxic submergence longer than any other tetrapod, surviving more than 170 days at 3°C. This ability is due, in part, to the shell and skeleton simultaneously releasing calcium and magnesium carbonates, and sequestering lactate and H+ to prevent lethal decreases in body fluid pH. We evaluated the effects of anoxic submergence at 3°C on various material properties of painted turtle bone after 60, 130 and 167-170 days, and compared them with those of normoxic turtles held at the same temperature for the same time periods. To assess changes in the mechanical properties, beams (4×25 mm) were milled from the plastron and broken in a three-point flexural test. Bone mineral density, CO2 concentration (a measure of total bone HCO3-/CO32-) and elemental composition were measured using microcomputed tomography, HCO3-/CO32- titration and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Tissue mineral density of the sampled bone beams was not significantly altered by 167-170 days of aquatic overwintering in anoxic or normoxic water, but bone CO2 and Mg were depleted in anoxic compared with normoxic turtles. At this time point, the plastron beams from anoxic turtles yielded at stresses that were significantly smaller and strains that were significantly greater than the plastron beams of normoxic turtles. When data from anoxic and normoxic turtles were pooled, plastron beams had a diminished elastic modulus after 167-170 days compared with those of control turtles sampled on day 1, indicating an effect of prolonged housing of the turtles in 3°C water without access to basking sites. There were no changes in the mechanical properties of the plastron beams at any of the earlier time points in either group. We conclude that anoxic hibernation can weaken the painted turtle's plastron, but likely only after durations that exceed what it might naturally experience. The duration of aquatic overwintering, regardless of oxygenation state, is likely to be an important factor determining the mechanical properties of the turtle shell during spring emergence.

Small Non-coding RNA Expression and Vertebrate Anoxia Tolerance.

Background: Extreme anoxia tolerance requires a metabolic depression whose modulation could involve small non-coding RNAs (small ncRNAs), which are specific, rapid, and reversible regulators of gene expression. A previous study of small ncRNA expression in embryos of the annual killifish Austrofundulus limnaeus, the most anoxia-tolerant vertebrate known, revealed a specific expression pattern of small ncRNAs that could play important roles in anoxia tolerance. Here, we conduct a comparative study on the presence and expression of small ncRNAs in the most anoxia-tolerant representatives of several major vertebrate lineages, to investigate the evolution of and mechanisms supporting extreme anoxia tolerance. The epaulette shark (Hemiscyllium ocellatum), crucian carp (Carassius carassius), western painted turtle (Chrysemys picta bellii), and leopard frog (Rana pipiens) were exposed to anoxia and recovery, and small ncRNAs were sequenced from the brain (one of the most anoxia-sensitive tissues) prior to, during, and following exposure to anoxia. Results: Small ncRNA profiles were broadly conserved among species under normoxic conditions, and these expression patterns were largely conserved during exposure to anoxia. In contrast, differentially expressed genes are mostly unique to each species, suggesting that each species may have evolved distinct small ncRNA expression patterns in response to anoxia. Mitochondria-derived small ncRNAs (mitosRNAs) which have a robust response to anoxia in A. limnaeus embryos, were identified in the other anoxia tolerant vertebrates here but did not display a similarly robust response to anoxia. Conclusion: These findings support an overall stabilization of the small ncRNA transcriptome during exposure to anoxic insults, but also suggest that multiple small ncRNA expression pathways may support anoxia tolerance, as no conserved small ncRNA response was identified among the anoxia-tolerant vertebrates studied. This may reflect divergent strategies to achieve the same endpoint: anoxia tolerance. However, it may also indicate that there are multiple cellular pathways that can trigger the same cellular and physiological survival processes, including hypometabolism.

The effects of pH and Pi on tension and Ca2+ sensitivity of ventricular myofilaments from the anoxia-tolerant painted turtle.

We aimed to determine how increases in intracellular H+ and inorganic phosphate (Pi) to levels observed during anoxic submergence affect contractility in ventricular muscle of the anoxia-tolerant Western painted turtle, Chrysemys picta bellii Skinned multicellular preparations were exposed to six treatments with physiologically relevant levels of pH (7.4, 7.0, 6.6) and Pi (3 and 8 mmol l-1). Each preparation was tested in a range of calcium concentrations (pCa 9.0-4.5) to determine the pCa-tension relationship for each treatment. Acidosis significantly decreased contractility by decreasing Ca2+ sensitivity (pCa50) and tension development (P<0.001). Increasing [Pi] also decreased contractility by decreasing tension development at every pH level (P<0.001) but, alone, did not affect Ca2+ sensitivity (P=0.689). Simultaneous increases in [H+] and [Pi] interacted to attenuate the decreased tension development and Ca2+ sensitivity (P<0.001), possibly reflecting a decreased sensitivity to Pi when it is present as the dihydrogen phosphate form, which increases as pH decreases. Compared with that of mammals, the ventricle of turtles exhibits higher Ca2+ sensitivity, which is consistent with previous studies of ectothermic vertebrates.

The metabolic consequences of repeated anoxic stress in the western painted turtle, Chrysemys picta bellii.

The painted turtle is known for its extreme tolerance to anoxia, but it is unknown whether previous experience with anoxic stress might alter physiological performance during or following a test bout of anoxia. Repeatedly subjecting 25°C-acclimated painted turtles to 2h of anoxic stress every other day for 19days (10 submergence bouts total) caused resting levels of liver glycogen to decrease by 17% and liver citrate synthase (CS) and cytochrome oxidase (COX) activities to increase by 33% and 112%, respectively. When the repeatedly submerged turtles were studied during a subsequent anoxic stress test, liver COX and CS activities decreased during anoxia to the same levels of naïve turtles, which were unchanged, and remained there throughout metabolic recovery. There were no effects of the repeated anoxia treatment on any of the other measured variables, which included lactate dehydrogenase and phosphofructokinase activities in liver, skeletal muscle, and ventricle, blood acid-base status, hemoglobin, hematocrit and plasma ion (Na, K, Ca, Mg, Cl) and metabolite concentrations (lactate, glucose, free-fatty acids), before, during, or after the anoxic stress test. We conclude that although painted turtles can show a physiological reaction to repeated anoxic stress, the changes appear to have no measurable effect on anaerobic physiological performance or ability to recover from anoxia.

Transcriptomic Responses of the Heart and Brain to Anoxia in the Western Painted Turtle.

Painted turtles are the most anoxia-tolerant tetrapods known, capable of surviving without oxygen for more than four months at 3°C and 30 hours at 20°C. To investigate the transcriptomic basis of this ability, we used RNA-seq to quantify mRNA expression in the painted turtle ventricle and telencephalon after 24 hours of anoxia at 19°C. Reads were obtained from 22,174 different genes, 13,236 of which were compared statistically between treatments for each tissue. Total tissue RNA contents decreased by 16% in telencephalon and 53% in ventricle. The telencephalon and ventricle showed ≥ 2x expression (increased expression) in 19 and 23 genes, respectively, while only four genes in ventricle showed ≤ 0.5x changes (decreased expression). When treatment effects were compared between anoxic and normoxic conditions in the two tissue types, 31 genes were increased (≥ 2x change) and 2 were decreased (≤ 0.5x change). Most of the effected genes were immediate early genes and transcription factors that regulate cellular growth and development; changes that would seem to promote transcriptional, translational, and metabolic arrest. No genes related to ion channels, synaptic transmission, cardiac contractility or excitation-contraction coupling changed. The generalized expression pattern in telencephalon and across tissues, but not in ventricle, correlated with the predicted metabolic cost of transcription, with the shortest genes and those with the fewest exons showing the largest increases in expression.

The calcium stored in the sarcoplasmic reticulum acts as a safety mechanism in rainbow trout heart.

Cardiomyocyte contraction depends on rapid changes in intracellular Ca(2+). In mammals, Ca(2+) influx as L-type Ca(2+) current (ICa) triggers the release of Ca(2+) from sarcoplasmic reticulum (SR) and Ca(2+)-induced Ca(2+) release (CICR) is critical for excitation-contraction coupling. In fish, the relative contribution of external and internal Ca(2+) is unclear. Here, we characterized the role of ICa to trigger SR Ca(2+) release in rainbow trout ventricular myocytes using ICa regulation by Ca(2+) as an index of CICR. ICa was recorded with a slow (EGTA) or fast (BAPTA) Ca(2+) chelator in control and isoproterenol conditions. In the absence of ß-adrenergic stimulation, the rate of ICa inactivation was not significantly different in EGTA and BAPTA (27.1 ± 1.8 vs. 30.3 ± 2.4 ms), whereas with isoproterenol (1 µM), inactivation was significantly faster with EGTA (11.6 ± 1.7 vs. 27.3 ± 1.6 ms). When barium was the charge carrier, inactivation was significantly slower in both conditions (61.9 ± 6.1 vs. 68.0 ± 8.7 ms, control, isoproterenol). Quantification revealed that without isoproterenol, only 39% of ICa inactivation was due to Ca(2+), while with isoproterenol, inactivation was Ca(2+)-dependent (∼65%) and highly reliant on SR Ca(2+) (∼46%). Thus, SR Ca(2+) is not released in basal conditions, and ICa is the main trigger of contraction, whereas during a stress response, SR Ca(2+) is an important source of cytosolic Ca(2+). This was not attributed to differences in SR Ca(2+) load because caffeine-induced transients were not different in both conditions. Therefore, Ca(2+) stored in SR of trout cardiomyocytes may act as a safety mechanism, allowing greater contraction when higher contractility is required, such as stress or exercise.

Role of GLUT1 in regulation of reactive oxygen species.

In skeletal muscle cells, GLUT1 is responsible for a large portion of basal uptake of glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would decrease GLUT1-mediated transport would cause increased reactive oxygen species (ROS) levels in L6 myoblasts, while conditions that would increase GLUT1-mediated transport would result in decreased ROS levels. We found that the GLUT1 inhibitors fasentin and phloretin increased the ROS levels induced by antimycin A and the superoxide generator pyrogallol. However, indinavir, which inhibits GLUT4 but not GLUT1, had no effect on ROS levels. Ataxia telangiectasia mutated (ATM) inhibitors and activators, previously shown to inhibit and augment GLUT1-mediated transport, increased and decreased ROS levels, respectively. Mutation of an ATM target site on GLUT1 (GLUT1-S490A) increased ROS levels and prevented the ROS-lowering effect of the ATM activator doxorubicin. In contrast, expression of GLUT1-S490D lowered ROS levels during challenge with pyrogallol, prevented an increase in ROS when ATM was inhibited, and prevented the pyrogallol-induced decrease in insulin signaling and insulin-stimulated glucose transport. Taken together, the data suggest that GLUT1 plays a role in regulation of ROS and could contribute to maintenance of insulin action in the presence of ROS.

The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage.

BACKGROUND: We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing. RESULTS: Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented. CONCLUSIONS: Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

Anesthetic protection of neurons injured by hypothermia and rewarming: roles of intracellular Ca2+ and excitotoxicity.

BACKGROUND: Mild hypothermia is neuroprotective after cerebral ischemia but surgery involving profound hypothermia (PH, temperature less than 18°C) is associated with neurologic complications. Rewarming (RW) from PH injures hippocampal neurons by glutamate excitotoxicity, N-methyl-D-aspartate receptors, and intracellular calcium. Because neurons are protected from hypoxia-ischemia by anesthetic agents that inhibit N-methyl-D-aspartic acid receptors, we tested whether anesthetics protect neurons from damage caused by PH/RW. METHODS: Organotypic cultures of rat hippocampus were used to model PH/RW injury, with hypothermia at 4°C followed by RW to 37°C and assessment of cell death 1 or 24 h later. Cell death and intracellular Ca were assessed with fluorescent dye imaging and histology. Anesthetic agents were present in the culture media during PH and RW or only RW. RESULTS: Injury to hippocampal CA1, CA3, and dentate neurons after PH and RW involved cell swelling, cell rupture, and adenosine triphosphate (ATP) loss; this injury was similar for 4 through 10 h of PH. Isoflurane (1% and 2%), sevoflurane (3%) and xenon (60%) reduced cell loss but propofol (3 µM) and pentobarbital (100 µM) did not. Isoflurane protection involved reduction in N-methyl-D-aspartate receptor-mediated Ca influx during RW but did not involve γ-amino butyric acid receptors or KATP channels. However, cell death increased over the next day. CONCLUSION: Anesthetic protection of neurons rewarmed from 4°C involves suppression of N-methyl-D-aspartate receptor-mediated Ca overload in neurons undergoing ATP loss and excitotoxicity. Unlike during hypoxia/ischemia, anesthetic agents acting predominantly on γ-aminobutyric acid receptors do not protect against PH/RW. The durability of anesthetic protection against cold injury may be limited.

Dermal bone in early tetrapods: a palaeophysiological hypothesis of adaptation for terrestrial acidosis.

The dermal bone sculpture of early, basal tetrapods of the Permo-Carboniferous is unlike the bone surface of any living vertebrate, and its function has long been obscure. Drawing from physiological studies of extant tetrapods, where dermal bone or other calcified tissues aid in regulating acid-base balance relating to hypercapnia (excess blood carbon dioxide) and/or lactate acidosis, we propose a similar function for these sculptured dermal bones in early tetrapods. Unlike the condition in modern reptiles, which experience hypercapnia when submerged in water, these animals would have experienced hypercapnia on land, owing to likely inefficient means of eliminating carbon dioxide. The different patterns of dermal bone sculpture in these tetrapods largely correlates with levels of terrestriality: sculpture is reduced or lost in stem amniotes that likely had the more efficient lung ventilation mode of costal aspiration, and in small-sized stem amphibians that would have been able to use the skin for gas exchange.

The cellular force-frequency response in ventricular myocytes from the varanid lizard, Varanus exanthematicus.

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus, we measured sarcomere and cell shortening, intracellular Ca(2+) ([Ca(2+)](i)), action potentials (APs), and K(+) currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20-22 degrees C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca(2+)](i), a decrease in [Ca(2+)](i) transient amplitude, and a seven-fold increase in diastolic [Ca(2+)](i). The time constant for the decay of the Ca(2+) transient (tau) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier (I(Kr)) or the transient outward (I(to)) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca(2+) transient coupled with an increase in diastolic Ca(2+), which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.