Is the teleost heart oxygen limited? - Insights using "hyperoxic" incubations of contracting cardiac tissue from rainbow trout.

Considerable effort has been devoted to understanding the negative effects of reduced PO2 on cardiac function. Much less is known about the impacts of elevated PO2 (hyperoxia) on cardiac performance and energetics, especially in fishes. The fish heart is of particular interest because cardiac dependence on oxygen is extremely variable between species and the early evolution of fish occurred when atmospheric PO2 was higher than current conditions. Although extracellular PO2 is variable and normally does not exceed 21 kPa, recent evidence suggests that teleost cardiac function is stimulated at supraphysiological PO2 values. The purpose of this study was to address whether cardiac contractility and energy metabolism is responsive to elevated PO2 values in sexually-immature female rainbow trout. Isometric force development (contractility) and oxygen consumption (V̇O2) were recorded in electrically-paced ventricular preparations. Contractility and V̇O2decreased when superfusate PO2 was decreased from ~70 kPa to 57 kPa or 45 kPa. However, PO2 calculated at the preparation core was always above 18 kPa. This estimate, along with complete recovery of contractility and V̇O2 at ~70 kPa, suggests that decreases observed in cardiac performance were not due to tissue hypoxia at the lower PO2 levels. In conclusion, the heart of female rainbow trout may be oxygen-limited in vitro and this study raises new questions about the choice of appropriate PO2 for experimentation, the relevance of elevated and varying PO2 to measurements of cardiac performance, and the possible existence of an oxygen sensor within rainbow trout cardiomyocytes.

Temperature effects on aerobic scope and cardiac performance of European perch (Perca fluviatilis).

Several recent studies have highlighted how impaired cardiac performance at high temperatures and in hypoxia may compromise the capacity for oxygen transport. Thus, at high temperatures impaired cardiac capacity is proposed to reduce oxygen transport to a degree that lowers aerobic scope and compromises thermal tolerance (the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis). To investigate this hypothesis, we measured aerobic and cardiac performance of a eurythermal freshwater teleost, the European perch (Perca fluviatilis). Rates of oxygen consumption were measured during rest and activity at temperatures between 5°C and 27°C, and we evaluated cardiac function by in vivo measurements of heart rate and in vitro studies to determine contractility of myocardial strips. Aerobic scope increased progressively from 5°C to 21°C, after which it levelled off. Heart rate showed a similar response. We found little difference between resting and active heart rate at high temperature suggesting that increased cardiac scope during activity is primarily related to changes in stroke volume. To examine the effects of temperature on cardiac capacity, we measured isometric force development in electrically paced myocardial preparations during different combinations of temperature, pacing frequency, oxygenation and adrenergic stimulation. The force-frequency product increased markedly upon adrenergic stimulation at 21 and 27°C (with higher effects at 21°C) and the cardiac preparations were highly sensitive to hypoxia. These findings suggest that at (critically) high temperatures, cardiac output may diminish due to a decreased effect of adrenergic stimulation and that this effect may be further exacerbated if the heart becomes hypoxic. Hence cardiac limitations may contribute to the inability to increase aerobic scope at high temperatures in the European perch (Perca fluviatilis).

Improved cardiac filling facilitates the postprandial elevation of stroke volume in Python regius.

To accommodate the pronounced metabolic response to digestion, pythons increase heart rate and elevate stroke volume, where the latter has been ascribed to a massive and fast cardiac hypertrophy. However, numerous recent studies show that heart mass rarely increases, even upon ingestion of large meals, and we therefore explored the possibility that a rise in mean circulatory filling pressure (MCFP) serves to elevate venous pressure and cardiac filling during digestion. To this end, we measured blood flows and pressures in anaesthetized Python regius The anaesthetized snakes exhibited the archetypal tachycardia as well as a rise in both venous pressure and MCFP that fully account for the approximate doubling of stroke volume. There was no rise in blood volume and the elevated MCFP must therefore stem from increased vascular tone, possibly by means of increased sympathetic tone on the veins. Furthermore, although both venous pressure and MCFP increased during volume loading, there was no evidence that postprandial hearts were endowed with an additional capacity to elevate stroke volume. In vitro measurements of force development of paced ventricular strips also failed to reveal signs of increased contractility, but the postprandial hearts had higher activities of cytochrome oxidase and pyruvate kinase, which probably serves to sustain the rise in cardiac work during digestion.

The effects of hypoxic bradycardia and extracellular HCO3(-)/CO2 on hypoxic performance in the eel heart.

During hypoxia, fishes exhibit a characteristic hypoxic bradycardia, the functional significance of which remains debated. Here, we investigated the hypothesis that hypoxic bradycardia primarily safeguards cardiac performance. In preparations from the European eel (Anguilla anguilla), a decrease in stimulation frequency from 40 to 15 beats min(-1), which replicates hypoxic bradycardia in vivo, vastly improved cardiac performance during hypoxia in vitro. As eels display dramatic shifts in extracellular HCO3(-)/CO2, we further investigated the effect this has upon hypoxic cardiac performance. Elevations from 10 mmol l(-1) HCO3(-)/1% CO2 to 40 mmol l(-1) HCO3(-)/4% CO2 had few effects on performance; however, further, but still physiologically relevant, increases to 70 mmol l(-1) HCO3(-)/7% CO2 compromised hypoxia tolerance. We revealed a four-way interaction between HCO3(-)/CO2, contraction frequency, hypoxia and performance over time, whereby the benefit of hypoxic bradycardia was most prolonged at 10 mmol l(-1) HCO3(-)/1% CO2. Together, our data suggest that hypoxic bradycardia greatly benefits cardiac performance, but its significance may be context specific.

Anoxia and Acidosis Tolerance of the Heart in an Air-Breathing Fish (Pangasianodon hypophthalmus).

Air breathing has evolved repeatedly in fishes and may protect the heart during stress. We investigated myocardial performance in the air-breathing catfish Pangasianodon hypophthalmus, a species that can withstand prolonged exposure to severe hypoxia and acidosis. Isometric ventricular preparations were exposed to anoxia, lactic acidosis, hypercapnic acidosis, and combinations of these treatments. Ventricular preparations were remarkably tolerant to anoxia, exhibiting an inotropic reduction of only 40%, which fully recovered during reoxygenation. Myocardial anoxia tolerance was unaffected by physiologically relevant elevations of bicarbonate concentration, in contrast to previous results in other fishes. Both lactic acidosis (5 mM; pH 7.10) and hypercapnic acidosis (10% CO2; pH 6.70) elicited a biphasic response, with an initial and transient decrease in force followed by overcompensation above control values. Spongy myocardial preparations were significantly more tolerant to hypercapnic acidosis than compact myocardial preparations. While ventricular preparations were tolerant to the isolated effects of anoxia and acidosis, their combination severely impaired myocardial performance and contraction kinetics. This suggests that air breathing may be a particularly important myocardial oxygen source during combined anoxia and acidosis, which may occur during exercise or environmental stress.

Purinoceptors exert negative inotropic effects on the heart in all major groups of reptiles.

The few and fragmentary studies on purinergic regulation of the reptile heart have reached equivocal conclusions. Indeed, unlike fish, amphibians, and mammals, it has been suggested that the turtle heart lacks purinoceptors. Here, we study the effect of adenosine and ATP on isolated heart strips from three species of reptiles: the red-eared slider (Trachemys scripta), the ball python (Python regius) and the spectacled caiman (Caiman crocodilus). Both adenosine and ATP markedly decreased contractility in atria from all three species. This was attenuated by theophylline, suggesting that the response is mediated by P1 receptors. Ventricles were less sensitive, although high concentrations of the adenyl compounds evoked decreases in contractility. Our study suggests that cardiac purinoceptors are ubiquitous across reptiles, and may play an important and underappreciated role in reptile cardiovascular physiology.

Limited effects of exogenous glucose during severe hypoxia and a lack of hypoxia-stimulated glucose uptake in isolated rainbow trout cardiac muscle.

We examined whether exogenous glucose affects contractile performance of electrically paced ventricle strips from rainbow trout under conditions known to alter cardiomyocyte performance, ion regulation and energy demands. Physiological levels of d-glucose did not influence twitch force development for aerobic preparations (1) paced at 0.5 or 1.1 Hz, (2) at 15 or 23°C, (3) receiving adrenergic stimulation or (4) during reoxygenation with or without adrenaline after severe hypoxia. Contractile responses to ryanodine, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum, were also not affected by exogenous glucose. However, glucose did attenuate the fall in twitch force during severe hypoxia. Glucose uptake was assayed in non-contracting ventricle strips using 2-[(3)H] deoxy-d-glucose (2-DG) under aerobic and hypoxic conditions, at different incubation temperatures and with different inhibitors. Based upon a lack of saturation of 2-DG uptake and incomplete inhibition of uptake by cytochalasin B and d-glucose, 2-DG uptake was mediated by a combination of facilitated transport and simple diffusion. Hypoxia stimulated lactate efflux sixfold to sevenfold with glucose present, but did not increase 2-DG uptake or reduce lactate efflux in the presence of cytochalasin B. Increasing temperature (14 to 24°C) also did not increase 2-DG uptake, but decreasing temperature (14 to 4°C) reduced 2-DG uptake by 45%. In conclusion, exogenous glucose improves mechanical performance under hypoxia but not under any of the aerobic conditions applied. The extracellular concentration of glucose and cold temperature appear to determine and limit cardiomyocyte glucose uptake, respectively, and together may help define a metabolic strategy that relies predominantly on intracellular energy stores.

Myoglobin-dependent O2 consumption of the hypoxic trout heart.

Myoglobin (Mb) plays a well-established role in facilitated O2 diffusion to sustain mitochondrial O2 consumption during hypoxia in the mammalian heart. To better understand the function of Mb in the fish heart, we have measured the effects of adding 20% carbon monoxide (CO), which inhibits Mb function, compared to inert 20% N2 on the O2 consumption and twitch force in hypoxic rainbow trout (Oncorhynchus mykiss) ventricle ring preparations. Results showed that O2 consumption was significantly reduced upon addition of CO, whereas twitch force was not affected. Control experiments at 40% CO did not decrease O2 consumption further, showing that CO was not inhibiting cytochrome c oxidase in the mitochondria. Because myocardial O2 consumption can be depressed by endogenous nitric oxide (NO) in the trout myocardium and because Mb is a scavenger of NO, CO inhibition experiments were also made in the presence of the NO synthase inhibitor, asymmetric dimethylarginine (ADMA). O2 consumption decreased to a similar extent upon CO addition, regardless of NO synthase inhibition, indicating that under hypoxic conditions Mb-dependent NO scavenging plays a minor role. Taken together, these results show that O2 consumption of the hypoxic rainbow trout heart is dependent on the function of Mb as intracellular O2 carrier.

Cardiovascular anatomy and cardiac function in the air-breathing swamp eel (Monopterus albus).

Monopterus albus, a swamp eel inhabiting the freshwaters of South East Asia, relies on an extensive vascularisation of the buccal cavity, pharynx and anterior oesophagus for gas exchange, while the gills are much reduced. In the present study we describe the macro-circulation in the cephalic region and the vascularisation of the buccal cavity of M. albus using vascular fillings and micro-computed tomography (µCT). We also show that M. albus has the capacity to use the buccal cavity for aquatic gas exchange, being able to maintain normal arterial blood gas composition, blood pressure, heart rate and cardiac output throughout 10h of forced submergence. M. albus therefore can be characterised as a facultative air-breather. Because M. albus aestivates for many months in moist mud during the dry season we characterised in vivo cardiovascular function during exposure to anoxia as well as the effects of anoxia on in vitro contractility of strip preparations from atria and ventricle. Both studies revealed a low anoxia tolerance, rendering it unlikely that M. albus can survive prolonged exposure to anoxia.

Roles of nitric oxide, nitrite and myoglobin on myocardial efficiency in trout (Oncorhynchus mykiss) and goldfish (Carassius auratus): implications for hypoxia tolerance.

The roles of nitric oxide synthase activity (NOS), nitrite and myoglobin (Mb) in the regulation of myocardial function during hypoxia were examined in trout and goldfish, a hypoxia-intolerant and hypoxia-tolerant species, respectively. We measured the effect of NOS inhibition, adrenaline and nitrite on the O(2) consumption rate and isometric twitch force development in electrically paced ventricular preparations during hypoxia, and measured O(2) affinity and nitrite reductase activity of the purified heart Mbs of both species. Upon hypoxia (9% O(2)), O(2) consumption and developed force decreased in both trout and goldfish myocardium, with trout showing a significant increase in the O(2) utilization efficiency, i.e. the ratio of twitch force to O(2) consumption, suggesting an increased anaerobic metabolism. NOS inhibition enhanced myocardial O(2) consumption and decreased efficiency, indicating that mitochondrial respiration is under a tone of NOS-produced NO. When trout myocardial twitch force and O(2) consumption are enhanced by adrenaline, this NO tone disappears. Consistent with its conversion to NO, nitrite reduced O(2) consumption and increased myocardial efficiency in trout but not in goldfish. Such a difference correlates with the lower O(2) affinity measured for trout Mb that would increase the fraction of deoxygenated heme available to catalyze the reduction of nitrite to NO. Whereas low-affinity trout Mb would favor O(2) diffusion within cardiomyocytes at high in vivo O(2) tensions, goldfish Mb having higher O(2) affinity and higher nitrite reductase activity appears better suited to facilitate O(2) diffusion and nitrite reduction in the heart during severe hypoxia, a condition particularly well tolerated by this species.

Nitric oxide increases myocardial efficiency in the hypoxia-tolerant turtle Trachemys scripta.

Nitric oxide (NO) may influence cardiac mechanical performance relative to O2 consumption by depressing respiration rate and by affecting the excitation-contraction coupling. Such effects of NO should be particularly important during hypoxia in species such as the hypoxia-tolerant turtle Trachemys scripta. In heart ventricle preparations from this species, the ratio of twitch force to O2 consumption increased by approximately 15% during full oxygenation and by approximately 60% during hypoxia in the presence of added L-arginine [the substrate for nitric oxide synthase (NOS)]. This effect was primarily due to a decrease in O2 consumption and may represent an increase in the twitch force obtained per ATP and/or in the ATP obtained per O2. Lactate production during hypoxia did not differ between preparations treated with either L-arginine or asymmetric dimethylarginine (ADMA), an inhibitor of NOS, suggesting that NO does not elicit a compensatory increase in anaerobic metabolism. ADMA did not reverse the effects of L-arginine on O2 consumption significantly, although pre-treatment with ADMA abolished the effect of L-arginine, consistent with the competitive binding of L-arginine and ADMA to NOS. Histochemical studies using the fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) revealed NO production in the presence of added L-arginine. In conclusion, NO may augment heart contractility obtained per O2 by deceasing O2 consumption without affecting either lactate production or developed force. This effect was particularly pronounced under O2 deficiency and may therefore contribute towards preserving cardiac function and to the overall excellent hypoxic tolerance of the turtle.

Histamine induces postprandial tachycardia through a direct effect on cardiac H2-receptors in pythons.

The intrinsic heart rate of most vertebrates studied, including humans, is elevated during digestion, suggesting that a nonadrenergic-noncholinergic factor contributes to the postprandial tachycardia. The regulating factor, however, remains elusive and difficult to identify. Pythons can ingest very large meals, and digestion is associated with a marked rise in metabolism that is sustained for several days. The metabolic rise causes more than a doubling of heart rate and a fourfold rise in cardiac output. This makes the python an interesting model to investigate the postprandial tachycardia. We measured blood pressure and heart rate in fasting Python regius, and at 24 and 48 h after ingestion of a meal amounting to 25% of body wt. Digestion caused heart rate to increase from 25 to 56 min, whereas blood pressure was unchanged. The postprandial rise in heart rate was partially due to a doubling of intrinsic heart rate. The H(2)-antagonist did not affect heart rate of fasting snakes but decreased heart rate by 15-20 min at 24 h into digestion, whereas it had no effects at 48 h. Thus, the histaminergic tone on the heart rose from none to 30% at 24 h but vanished after 48 h. In anesthetized snakes, histamine caused a systemic vasodilatation and a marked increase in heart rate and cardiac output mediated through a direct effect on H(2)- receptors. Our study strongly indicates that histamine regulates heart rate during the initial phase of digestion in pythons. This study describes a novel regulation of the vertebrate heart.

Tribute to P. L. Lutz: cardiac performance and cardiovascular regulation during anoxia/hypoxia in freshwater turtles.

Freshwater turtles overwintering in ice-covered ponds in North America may be exposed to prolonged anoxia, and survive this hostile environment by metabolic depression. Here, we review their cardiovascular function and regulation, with particular emphasis on the factors limiting cardiac performance. The pronounced anoxia tolerance of the turtle heart is based on the ability to match energy consumption with the low anaerobic ATP production during anoxia. Together with a well-developed temporal and spatial energy buffering by creatine kinase, this allows for cellular energy charge to remain high during anoxia. Furthermore, the turtle heart is well adapted to handle the adverse effects of free phosphate arising when phosphocreatine stores are used. Anoxia causes tenfold reductions in heart rate and blood flows that match the metabolic depression, and blood pressure is largely maintained through increased systemic vascular resistance. Depression of the heart rate is not driven by the autonomic nervous system and seems to arise from direct effects of oxygen lack and the associated hyperkalaemia and acidosis on the cardiac pacemaker. These intra- and extracellular changes also affect cardiac contractility, and both acidosis and hyperkalaemia severely depress cardiac contractility. However, increased levels of adrenaline and calcium may, at least partially, salvage cardiac function under prolonged periods of anoxia.

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes.

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.

Contractile properties of the functionally divided python heart: two sides of the same matter.

The heart of Python regius is functionally divided so that systemic blood pressure is much higher than pulmonary pressure (6.6+/-1.0 and 0.7+/-0.1 kPa, respectively). The present study shows that force production of cardiac strips from the cavum arteriosum and cavum pulmonale exhibits similar force production when stimulated in vitro. The high systemic blood pressure is caused, therefore, by a thicker ventricular wall surrounding the cavum arteriosum rather than differences in the intrinsic properties of the cardiac tissues. Similarly, there were no differences between the contractile properties of right and left atria. Force production was similar in atria and ventricle but the atria contracted and relaxed much faster than the ventricle. Graded hypoxia markedly reduced twitch force of all four cardiac tissues, and this was most pronounced when PO(2) was below 40 kPa. In contrast, the four cardiac tissues were insensitive to acidosis during normoxia although acidosis increased the sensitivity to hypoxia. Adrenergic stimulation increased twitch force of all cardiac tissues, while cholinergic stimulation only affected the atria and reduced twitch force markedly. In spite of the different oxygen availability of the two sides of the heart, the biochemical and functional properties are alike and the differences may instead be overcome by the coronary blood supply.

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells.

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca(2+) activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca(2+) activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.

The role of the sarcoplasmic reticulum in the generation of high heart rates and blood pressures in reptiles.

The functional significance of the sarcoplasmic reticulum (SR) in the generation of high heart rates and blood pressures was investigated in four species of reptile; the turtle, Trachemys scripta; the python, Python regius, the tegu lizard, Tupinanvis merianae, and the varanid lizard, Varanus exanthematicus. Force-frequency trials and imposed pauses were performed on ventricular and atrial tissue from each species with and without the SR inhibitor ryanodine, and in the absence and presence of adrenaline. In all species, an imposed pause of 1 or 5 min caused a post-rest decay of force, and a negative force-frequency response was observed in all species within their in vivo frequency range of heart rates. These relationships were not affected by either ryanodine or adrenaline. In ventricular strips from varanid lizards and pythons, ryanodine caused significant reductions in twitch force within their physiologically relevant frequency range. In atrial tissue from the tegu and varanid lizards, SR inhibition reduced twitch force across the whole of their physiological frequency range. In contrast, in the more sedentary species, the turtle and the python, SR inhibition only decreased twitch force at stimulation frequencies above maximal in vivo heart rates. Adrenaline caused an increase in twitch force in all species studied. In ventricular tissue, this positive inotropic effect was sufficient to overcome the negative effects of ryanodine. In atrial tissue however, adrenaline could only ameliorate the negative effects of ryanodine at the lower pacing frequencies. Our results indicate that reptiles recruit Ca2+ from the SR for force development in a frequency and tissue dependent manner. This is discussed in the context of the development of high reptilian heart rates and blood pressures.

Extracellular determinants of cardiac contractility in the cold anoxic turtle.

Painted turtles (Chrysemys picta) survive months of anoxic submergence, which is associated with large changes in the extracellular milieu where pH falls by 1, while extracellular K+, Ca++, and adrenaline levels all increase massively. While the effect of each of these changes in the extracellular environment on the heart has been previously characterized in isolation, little is known about their interactions and combined effects. Here we examine the isolated and combined effects of hyperkalemia, acidosis, hypercalcemia, high adrenergic stimulation, and anoxia on twitch force during isometric contractions in isolated ventricular strip preparations from turtles. Experiments were performed on turtles that had been previously acclimated to warm (25 degrees C), cold (5 degrees C), or cold anoxia (submerged in anoxic water at 5 degrees C). The differences between acclimation groups suggest that cold acclimation, but not anoxic acclimation per se, results in a downregulation of processes in the excitation-contraction coupling. Hyperkalemia (10 mmol L(-1) K+) exerted a strong negative inotropic effect and caused irregular contractions; the effect was most pronounced at low temperature (57%-97% reductions in twitch force). Anoxia reduced twitch force at both temperatures (14%-38%), while acidosis reduced force only at 5 degrees C (15%-50%). Adrenergic stimulation (10 micromol L(-1)) increased twitch force by 5%-19%, but increasing extracellular [Ca++] from 2 to 6 mmol L(-1) had only small effects. When all treatments were combined with anoxia, twitch force was higher at 5 degrees C than at 25 degrees C, whereas in normoxia twitch force was higher at 25 degrees C. We propose that hyperkalemia may account for a large part of the depressed cardiac contractility during long-term anoxic submergence.

Effects of hibernation on mitochondrial regulation and metabolic capacities in myocardium of painted turtle (Chrysemys picta).

Painted turtles hibernating during winter may endure long-term exposure to low temperature and anoxia. These two conditions may affect the aerobic capacity of a tissue and might be of particular importance to the cardiac muscle normally highly reliant on aerobic energy production. The present study addressed how hibernation affects respiratory characteristics of mitochondria in situ and the metabolic pattern of turtle myocardium. Painted turtles were acclimated to control (25 degrees C), cold (5 degrees C) normoxic and cold anoxic conditions. In saponin-skinned myocardial fibres, cold acclimation increased mitochondrial respiratory capacity and decreased apparent ADP-affinity. Concomitant anoxia did not affect this. Creatine increased the apparent ADP-affinity to similar values in the three acclimation groups, suggesting a functional coupling of creatine kinase to mitochondrial respiration. As to the metabolic pattern, cold acclimation decreased glycolytic capacity in terms of pyruvate kinase activity and increased lactate dehydrogenase (LHD) activity. Concomitant anoxia counteracted the cold-induced decrease in pyruvate kinase activity and increased creatine kinase activity. In conclusion, cold acclimation seems to increase aerobic and decrease anaerobic energy production capacity in painted turtle myocardium. Importantly, anoxia does not affect the mitochondrial functional integrity but seems to increase the capacity for anaerobic energy production and energy buffering.

Force development, energy state and ATP production of cardiac muscle from turtles and trout during normoxia and severe hypoxia.

The effects of hypoxia on energy economy of cardiac muscle were compared between the hypoxia-tolerant freshwater turtle at 20 degrees C and the hypoxia-sensitive rainbow trout at 15 degrees C. Isolated ventricular preparations were left either at rest or stimulated at 30 min(-1) to develop isometric twitch force. Under oxygenated conditions, twitch force and oxygen consumption were similar for the two species. Overall metabolism was reduced during severe hypoxia in both resting and stimulated preparations and under these conditions most of the ATP production was derived from anaerobic metabolism. During hypoxia, a metabolic depression of approximately 2/3 occurred for non-contractile processes in both turtle and trout preparations. During hypoxia, lactate production and residual oxygen consumption were similar in turtle and trout. Cellular energy state and phosphorylation potential decreased during severe hypoxia in both species and this reduction was more severe in preparations stimulated to contraction. However, in turtle ventricular preparations the energy state and phosphorylation potential stabilised at higher levels than in trout, and turtle preparations also maintained a higher twitch force throughout the hypoxic period. Moreover, twitch force relative to total ATP hydrolysis was markedly increased during hypoxia in turtle while this ratio was unchanged for trout. The main findings of this study are: (1) cellular energy liberation and the energy demand of non-contractile processes decreased to similar levels in hypoxic turtle and trout myocardium; (2) turtle myocardium maintained a substantially higher cellular energy state and twitch force development than trout myocardium during hypoxia and (3) the ratio of twitch force to ATP hydrolysis increased during hypoxia in turtle but was unchanged in trout. It is possible that this superior economy of the contracting turtle myocardium contributes to the remarkable hypoxia tolerance of freshwater turtles.