Gina L. J. Galli

The cardiovascular responses of the freshwater turtle Trachemys scripta to warming and cooling.

SUMMARY Seven freshwater turtles Trachemys scripta were instrumented with flow probes and cannulated for blood pressure measurements. The turtles were warmed from 24 to 34°C, and cooled down to 24°C, with and without atropine. Animals exhibited a hysteresis of heart rate and blood flow to both the pulmonary and systemic circulations, which was not cholinergically mediated. Blood pressure remained constant during both warming and cooling, while systemic resistance decreased during heating and increased during cooling, indicating a barostatic response. There was a large right-to-left (R–L) shunt during warming and cooling in untreated animals, which remained relatively constant. Atropinisation resulted in a large L–R shunt, which decreased during warming and increased during cooling. Nevertheless, heating rates were the same in untreated and atropinised animals, and cooling rates were significantly longer in atropinised animals, indicating that shunt patterns contribute little to heat exchange.

Temperature sensitivity of cardiac function in pelagic fishes with different vertical mobilities: Yellowfin tuna (thunnus albacares), bigeye tuna (Thunnus obesus), mahimahi (coryphaena hippurus), and swordfish (xiphias gladius)

We measured the temperature sensitivity, adrenergic sensitivity, and dependence on sarcoplasmic reticulum (SR) Ca2+ of ventricular muscle from pelagic fishes with different vertical mobility patterns: bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares), and mahimahi (Coryphaena hippurus) and a single specimen from swordfish (Xiphias gladius). Ventricular muscle from the bigeye tuna and mahimahi exhibited a biphasic response to an acute decrease in temperature (from 26° to 7°C); twitch force and kinetic parameters initially increased and then declined. The magnitude of this response was larger in the bigeye tuna than in the mahimahi. Under steady state conditions at 26°C, inhibition of SR Ca2+ release and reuptake with ryanodine and thapsigargin decreased twitch force and kinetic parameters, respectively, in the bigeye tuna only. However, the initial inotropy associated with decreasing temperature was abolished by SR inhibition in both the bigeye tuna and the mahimahi. Application of adrenaline completely reversed the effects of ryanodine and thapsigargin, but this effect was diminished at cold temperatures. In the yellowfin tuna, temperature and SR inhibition had minor effects on twitch force and kinetics, while adrenaline significantly increased these parameters. Limited data suggest that swordfish ventricular muscle responds to acute temperature reduction, SR inhibition, and adrenergic stimulation in a manner similar to that of bigeye tuna ventricular muscle. In aggregate, our results show that the temperature sensitivity, SR dependence, and adrenergic sensitivity of pelagic fish hearts are species specific and that these differences reflect species‐specific vertical mobility patterns.

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

SUMMARY 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.

The Sarcoplasmic Reticulum and the Evolution of the Vertebrate Heart

The sarcoplasmic reticulum (SR) is crucial for contraction and relaxation of the mammalian cardiomyocyte, but its role in other vertebrate classes is equivocal. Recent evidence suggests differences in SR function across species may have an underlying structural basis. Here, we discuss how SR recruitment relates to the structural organization of the cardiomyocyte to provide new insight into the evolution of cardiac design and function in vertebrates.

Temperature effects on Ca2+ cycling in scombrid cardiomyocytes: a phylogenetic comparison

SUMMARY Specialisations in excitation–contraction coupling may have played an important role in the evolution of endothermy and high cardiac performance in scombrid fishes. We examined aspects of Ca2+ handling in cardiomyocytes from Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). The whole-cell voltage-clamp technique was used to measure the temperature sensitivity of the L-type Ca2+ channel current (ICa), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca2+ content (ssSRload and maxSRload). Current–voltage relations, peak ICa density and charge density of ICa were greatest in mackerel and yellowfin at all temperatures tested. ICa density and kinetics were temperature sensitive in all species studied, and the magnitude of this response was not related to the thermal preference of the species. SRload was greater in atrial than in ventricular myocytes in the Pacific bluefin tuna, and in species that are more cold tolerant (bluefin tuna and mackerel). ICa and SRload were particularly small in bonito, suggesting the Na+/Ca2+ exchanger plays a more pivotal role in Ca2+ entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca2+ storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel.

Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart

SUMMARY The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival.

Direct measurements of SR free Ca reveal the mechanism underlying the transient effects of RyR potentiation under physiological conditions

Aims Most of the calcium that activates contraction is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). It is controversial whether activators of the RyR produce a maintained increase in the amplitude of the systolic Ca transient. We therefore aimed to examine the effects of activation of the RyR in large animals under conditions designed to be as physiological as possible while simultaneously measuring SR and cytoplasmic Ca. Methods and results Experiments were performed on ventricular myocytes from canine and ovine hearts. Cytoplasmic Ca was measured with fluo-3 and SR Ca with mag-fura-2. Application of caffeine resulted in a brief increase in the amplitude of the systolic Ca transient accompanied by an increase of action potential duration. These effects disappeared with a rate constant of ∼3 s−1. Similar effects were seen in cells taken from sheep in which heart failure had been induced by rapid pacing. The decrease of Ca transient amplitude was accompanied by a decrease of SR Ca content. During this phase, the maximum (end-diastolic) SR Ca content fell while the minimum systolic increased. Conclusions This study shows that, under conditions designed to be as physiological as possible, potentiation of RyR opening has no maintained effect on the systolic Ca transient. This result makes it unlikely that potentiation of the RyR has a maintained role in positive inotropy.

Ca2+ cycling in cardiomyocytes from a high-performance reptile, the varanid lizard (Varanus exanthematicus)

The varanid lizard possesses one of the largest aerobic capacities among reptiles with maximum rates of oxygen consumption that are twice that of other lizards of comparable sizes at the same temperature. To support this aerobic capacity, the varanid heart possesses morphological adaptations that allow the generation of high heart rates and blood pressures. Specializations in excitation-contraction coupling may also contribute to the varanids superior cardiovascular performance. Therefore, we investigated the electrophysiological properties of the l-type Ca2+ channel and the Na+/Ca2+ exchanger (NCX) and the contribution of the sarcoplasmic reticulum to the intracellular Ca2+ transient (Δ[Ca2+]i) in varanid lizard ventricular myocytes. Additionally, we used confocal microscopy to visualize myocytes and make morphological measurements. Lizard ventricular myocytes were found to be spindle-shaped, lack T-tubules, and were ∼190 μm in length and 5–7 μm in width and depth. Cardiomyocytes had a small cell volume (∼2 pL), leading to a large surface area-to-volume ratio (18.5), typical of ectothermic vertebrates. The voltage sensitivity of the l-type Ca2+ channel current (ICa), steady-state activation and inactivation curves, and the time taken for recovery from inactivation were also similar to those measured in other reptiles and teleosts. However, transsarcolemmal Ca2+ influx via reverse mode Na+/Ca2+ exchange current was fourfold higher than most other ectotherms. Moreover, pharmacological inhibition of the sarcoplasmic reticulum led to a 40% reduction in the Δ[Ca2+]i amplitude, and slowed the time course of decay. In aggregate, our results suggest varanids have an enhanced capacity to transport Ca2+ through the Na+/Ca2+ exchanger, and sarcoplasmic reticulum suggesting specializations in excitation-contraction coupling may provide a means to support high cardiovascular performance.

A Novel Cardiotoxic Mechanism for a Pervasive Global Pollutant

The Deepwater Horizon disaster drew global attention to the toxicity of crude oil and the potential for adverse health effects amongst marine life and spill responders in the northern Gulf of Mexico. The blowout released complex mixtures of polycyclic aromatic hydrocarbons (PAHs) into critical pelagic spawning habitats for tunas, billfishes, and other ecologically important top predators. Crude oil disrupts cardiac function and has been associated with heart malformations in developing fish. However, the precise identity of cardiotoxic PAHs, and the mechanisms underlying contractile dysfunction are not known. Here we show that phenanthrene, a PAH with a benzene 3-ring structure, is the key moiety disrupting the physiology of heart muscle cells. Phenanthrene is a ubiquitous pollutant in water and air, and the cellular targets for this compound are highly conserved across vertebrates. Our findings therefore suggest that phenanthrene may be a major worldwide cause of vertebrate cardiac dysfunction.

Ginkgolide K protects the heart against endoplasmic reticulum stress injury by activating the inositol-requiring enzyme 1α/X box-binding protein-1 pathway.

Endoplasmic reticulum (ER) stress is increasingly recognized as an important causal factor of many diseases. Targeting ER stress has now emerged as a new therapeutic strategy for treating cardiovascular diseases. Here, we investigated the effects and underlying mechanism of ginkgolide K (1,10‐dihydroxy‐3,14‐didehydroginkgolide, GK) on cardiac ER stress.