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Repolarizing potassium currents in working myocardium of Japanese quail: Novel translational model for cardiac electrophysiology
Abstract
Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito – the later was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 μmol l⁻¹), HMR 1556 (30 μmol l⁻¹) and 4-aminopyridine (3 mmol l⁻¹), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents, in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.
... The anatomical separation of left and right ventricles in birds and mammals allows the elevation of systemic pressure significantly above pulmonary pressure thereby providing the necessary convection for highly aerobic tissues, whilst avoiding the rupture of thin respiratory surfaces [12][13][14]. The presence of a specialized conduction system [15] and a compact atrial and ventricular wall architecture is important for the fast atrioventricular conduction and rapid ventricular repolarization [16,17] of avian and mammalian hearts compared with those of ectothermic vertebrates (independent of temperature) [14,15,18]. Indeed, the convergent evolution of rapid/ early repolarization in birds (zebrafinch) and mammals (mouse) is undoubtably important for achieving the fast heart rates typical of endotherms [17]. ...
... Rather than being definitive, the paper highlights how little we know about cellular Ca 2+ dynamics in the bird heart and why research in this area is important to understand the role of myocyte architecture in the evolution of the vertebrate heart. Figure 2. Images of freshly isolated ventricular myocytes from (a) Japanese quail Coturnix japonica as light microscope image (top) [32] and an immunofluorescent image with sarcomeres delineated with a green probe to α-actinin and nucleus in red (bottom) [16], (b) varanid lizard Varanus exanthematicus light microscope image, arrow is pointing to sarcomeric striations (top) and confocal image with the sarcolemmal membrane visible in red (bottom) [30], (c) yellow-bellied turtle Trachemys scripta scripta light microscope image (top) and confocal image with the sarcolemmal membrane visible in red (bottom) [29]. Photomicrograph image of a finch (d) and rat (e) cardiomyoctyte used with permission from [36]. ...
... Excitation-contraction coupling in all vertebrate myocytes proceeds from the action potential. Atrial and ventricular action potential waveform and the corresponding repolarizing currents (I Kr , I Ks , I to ) have recently been characterized together for the first time in an adult bird (Japanese quail) [16]. Resting heart rates for these birds range between 318 and 530 beats min −1 [45,46] which is comparable to small rodents (mice/rats) and clearly depends on rapid/ early ventricular repolarization [17]. ...
Bird cardiomyocytes are long, thin and lack transverse (t)-tubules, which is akin to the cardiomyocyte morphology of ectothermic non-avian reptiles, who are typified by low maximum heart rates and low pressure development. However, birds can achieve greater contractile rates and developed pressures than mammals, whose wide cardiomyocytes contain a dense t-tubular network allowing for uniform excitation–contraction coupling and strong contractile force. To address this apparent paradox, this paper functionally links recent electrophysiological studies on bird cardiomyocytes with decades of ultrastructure measurements. It shows that it is the strong transsarcolemmal Ca ²⁺ influx via the L-type Ca ²⁺ current ( I CaL ) and the high gain of Ca ²⁺ -induced Ca ²⁺ release from the sarcoplasmic reticulum (SR), coupled with an internal SR Ca ²⁺ release relay system, that facilitates the strong fast contractions in the long thin bird cardiomyocytes, without the need for t-tubules. The maintenance of an elongated myocyte morphology following the post-hatch transition from ectothermy to endothermy in birds is discussed in relation to cardiac load, myocyte ploidy, and cardiac regeneration potential in adult cardiomyocytes. Overall, the paper shows how little we know about cellular Ca ²⁺ dynamics in the bird heart and suggests how increased research efforts in this area would provide vital information in our quest to understand the role of myocyte architecture in the evolution of the vertebrate heart.
This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. Please see glossary at the end of the paper for definitions of specialized terms.
... At the next stage of the study, we investigated the effect of WSF of oil on the main ionic currents in navaga cardiomyocytes, paying special attention to the rapid delayed rectifier potassium current I Kr , the background inward rectifier current I K1 , and the calcium current I Ca , the ratio of the contribution of which determines the repolarization rate and the duration of the AP in fish cardiomyocytes. Another repolarizing current, the slow delayed rectifier current I Ks , characteristic of cardiomyocytes of mammals, birds [16], and some other vertebrates [17], is absent in navaga [18]. ...
... Since I Kr is the main repolarizing current in fish cardiomyocytes [17], its sensitivity to WSF of oil was assessed not only in ventricular but also in atrial myocytes. The WSF of oil in all three tested concentrations caused a statistically significant decrease in the I Kr amplitude (Fig. 2). ...
... For ventricular repolarization of pythons to be similar to that of mammals, our study suggests that at least three conditions have to be met. First, there should be evolutionary conservation of the electrophysiological processes, and our RNA sequencing demonstrates a substantial evolutionary conservation on the transcript level, including of the major ion-handling channels, in agreement with previous studies (Olson, 2006;Castoe et al., 2013;Duan et al., 2017;Filatova et al., 2021;Offerhaus et al., 2021). Second, catecholamines should augment differences in repolarization time between regions within the ventricle, and we find this to be the case in pythons. ...
Ectothermic vertebrates experience daily changes in body temperature, and anecdotal observations suggest these changes affect ventricular repolarization such that the T-wave in the ECG changes polarity. Mammals, in contrast, can maintain stable body temperatures, and their ventricular repolarization is strongly modulated by changes in heart rate and by sympathetic nervous system activity. The aim of this study was to assess the role of body temperature, heart rate, and circulating catecholamines on local repolarization gradients in the ectothermic ball python (Python regius). We recorded body-surface electrocardiograms and performed open-chest high-resolution epicardial mapping while increasing body temperature in five pythons, in all of which there was a change in T-wave polarity. However, the vector of repolarization differed between individuals, and only a subset of leads revealed T-wave polarity change. RNA sequencing revealed regional differences related to adrenergic signaling. In one denervated and Ringer’s solution–perfused heart, heating and elevated heart rates did not induce change in T-wave polarity, whereas noradrenaline did. Accordingly, electrocardiograms in eight awake pythons receiving intra-arterial infusion of the β-adrenergic receptor agonists adrenaline and isoproterenol revealed T-wave inversion in most individuals. Conversely, blocking the β-adrenergic receptors using propranolol prevented T-wave change during heating. Our findings indicate that changes in ventricular repolarization in ball pythons are caused by increased tone of the sympathetic nervous system, not by changes in temperature. Therefore, ventricular repolarization in both pythons and mammals is modulated by evolutionary conserved mechanisms involving catecholaminergic stimulation.
... Our data show that I Kur and I to are involved in early repolarization in zebra finches as well since administration of 4-AP had similarly effects on RT20 and the J-wave. A similar effect of 4-AP on early repolarization has been show in isolated cardiomyocytes of Japanese quail (Filatova et al., 2021). This suggests that birds and mammals with high heart rates share a similar mechanism of early repolarization. ...
High heart rates are a feature of small endothermic-or warm-blooded-mammals and birds. In small mammals, the QT interval is short, and local ventricular recordings reveal early repolarization that coincides with the J-wave on the ECG, a positive deflection following the QRS complex. Early repolarization contributes to short QT-intervals thereby enabling brief cardiac cycles and high heart rates. We therefore hypothesized high hearts rates associate with early repolarization and J-waves on the ECG of endothermic birds. We tested this hypothesis by comparing isolated hearts of zebra finches and mice and recorded pseudo-ECGs and optical action potentials (zebra finch, n = 8; mouse, n = 8). In both species, heart rate exceeded 300 beats per min, and total ventricular activation was fast (QRS < 10 ms). Ventricular activation progressed from the left to the right ventricle in zebra finch, whereas it progressed from apex-to-base in mouse. In both species, the early repolarization front followed the activation front, causing a positive J-wave in the pseudo-ECG. Inhibition of early repolarization by 4-aminopyridine reduced J-wave amplitude in both species. Action potential duration was similar between ventricles in zebra finch, whereas in mouse the left ventricular action potential was longer. Accordingly, late repolarization had opposite directions in zebra finch (left-right) and mouse (right-left). This caused a similar direction for the zebra finch J-wave and T-wave, whereas in the mouse they were discordant. Our findings demonstrate that early repolarization and the associated J-wave may have evolved by convergence in association with high heart rates.
Atrioventricular (AV) accessory pathways (APs) provide additional electrical connections between the atria and ventricles, resulting in severe electrical disturbances. It is generally accepted that APs originate in the altered annulus fibrosus maturation in the late prenatal and perinatal period. However, current experimental methods cannot address their development in specific locations around the annulus fibrosus due to the inaccessibility of late fetal hearts for electrophysiological investigation under physiological conditions. In this study, we describe an approach for optical mapping of the retrogradely perfused chick heart in the last third of the incubation period. This system showed stability for electrophysiological measurement for several hours. This feature allowed analysis of the number and functionality of the APs separately in each clinically relevant position. Under physiological conditions, we also recorded the shortening of the AV delay with annulus fibrosus maturation and analyzed ventricular activation patterns after conduction through APs at specific locations. We observed a gradual regression of AP with an area-specific rate (left-sided APs disappeared first). The results also revealed a sudden drop in the number of active APs between ED16 and ED18. Accessory myocardial AV connections were histologically documented in all positions around the annulus fibrosus even after hatching. The fact that no electrically active AP was present at this stage highlights the necessity of electrophysiological evaluation of accessory atrioventricular connections in studying AP formation.
The orderly contraction of the vertebrate heart is determined by generation and propagation of cardiac action potentials(APs). APs are generated by the integrated activity of time- and voltage-dependent ionic channels which carry inward Na⁺ and Ca²⁺ currents, and outward K⁺ currents. This review compares atrial and ventricular APs and underlying ion currents between different taxa of vertebrates. We have collected literature data and attempted to find common electrophysiological features for two or more vertebrate groups, show differences between taxa and cardiac chambers, and indicate gaps in the existing data. Although electrical excitability of the heart in all vertebrates is based on the same superfamily of channels, there is a vast variability of AP waveforms between atrial and ventricular myocytes, between different species of the same vertebrate class and between endothermic and ectothermic animals. The wide variability of AP shapes is related to species-specific differences in animal size, heart rate, stage of ontogenetic development, excitation-contraction coupling, temperature and oxygen availability. Some of the differences between taxa are related to evolutionary development of genomes, which appear e.g. in the expression of different Na⁺ and K⁺ channel orthologues in cardiomyocytes of vertebrates. There is a wonderful variability of AP shapes and underlying ion currents with which electrical excitability of vertebrate heart can be generated depending on the intrinsic and extrinsic conditions of animal body. This multitude of ionic mechanisms provides excellent material for studying the mechanisms with which function of the vertebrate heart can adapt or acclimate to prevailing physiological and environmental conditions.
The Publisher regrets that this article is an accidental duplication of an article that has already been published in Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 255, 2021, 110593, https://doi.org/10.1016/j.cbpb.2021.110593. The duplicate article has therefore been withdrawn.
The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
This review serves as an introduction to a Special Issue of Comparative Biochemistry and Physiology, focused on using non-human models to study biomedical physiology. The concept of a model differs across disciplines. For example, several models are used primarily to gain an understanding of specific human pathologies and disease states, whereas other models may be focused on gaining insight into developmental or evolutionary mechanisms. It is often the case that animals initially used to gain knowledge of some unique biochemical or physiological process finds foothold in the biomedical community and becomes an established model. The choice of a particular model for biomedical research is an ongoing process and model validation must keep pace with existing and emerging technologies. While the importance of non-mammalian models, such as Caenorhabditis elegans, Drosophila melanogaster, Danio rerio and Xenopus laevis, is well known, we also seek to bring attention to emerging alternative models of both invertebrates and vertebrates, which are less established but of interest to the comparative biochemistry and physiology community.
Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resemble those of reptiles. It has been suggested that birds use intracellular Ca ²⁺ stored within the sarcoplasmic reticulum (SR) to power contractile function but neither SR Ca ²⁺ content nor the cross-talk between channels underlying Ca ²⁺ -induced Ca ²⁺ -release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca ²⁺ storage and refilling capacities of the SR and the degree of transsarcolemmal and intracellular Ca ²⁺ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail ( Coturnix japonica ). A transsarcolemmal Ca ²⁺ current was detectable both in quail atrial and ventricular myocytes and was mediated only by L-type Ca ²⁺ channels. The peak density of I Ca was larger in ventricular cells than in atrial and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca ²⁺ content of quail myocardium was also larger than that reported for mammals and reached 750.6±128.2 µmol l ⁻¹ in atrial cells and 423.3±47.2 µmol l ⁻¹ in ventricular cells at 24⁰C. We observed SR-Ca ²⁺ -dependent inactivation of I Ca in ventricular myocytes indicating cross-talk between sarcolemmal Ca ²⁺ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.
Background:
Despite recent improvements in the burden of cardiovascular disease (CVD) in the UK, deaths from CVD are relatively high compared with other high-income countries. An estimated 7 million people in the UK are living with CVD, and the healthcare cost is approximately £11 billion annually. In more than 90% of cases, the risk of a first heart attack is thought to be related to modifiable risk factors including smoking, poor diet, lipidemia, high blood pressure, inactivity, obesity and excess alcohol consumption. The aim of the study is to synthesise evidence for the comparative effectiveness and cost-effectiveness of different interventions for the primary prevention of CVD.
Methods:
We will systematically search databases (for example, MEDLINE (Ovid), Embase (Ovid), Cochrane Library) and the reference lists of previous systematic reviews for randomised controlled trials that assess the effectiveness and cost-effectiveness of any form of intervention aimed at adult populations for the primary prevention of CVD, including but not limited to lipid lowering medications, blood pressure lowering medications, antiplatelet agents, nutritional supplements, dietary interventions, health promotion programmes, physical activity interventions or structural and policy interventions. Interventions may or may not be targeted at high-risk groups. Publications from any year will be considered for inclusion. The primary outcome will be all cause mortality. Secondary outcomes will be cardiovascular diseases related mortality, major cardiovascular events, coronary heart disease, incremental costs per quality-adjusted life years gained. If data permits, we will use network meta-analysis to compare and rank effectiveness of different interventions, and test effect modification of intervention effectiveness using subgroup analyses and meta-regression analyses.
Discussion:
The results will be important for policymakers when making decisions between multiple possible alternative strategies to prevent CVD. Compared to results from existing multiple separate pairwise meta-analyses, this overarching synthesis of all relevant work will enhance decision-making. The findings will be crucial to inform evidence-based priorities and guidelines for policies and planning prevention strategies of CVD.
Systematic review registration:
PROSPERO CRD42019123940.
Background:
Cardiovascular diseases are the dominant cause of morbidity and mortality worldwide. In 2008, cardiovascular deaths represented 30% of all global deaths, with more than 80% of deaths in developing countries. There is increase in incidence of hypertension, atherosclerosis, stroke, chronic heart failure and atrial fibrillation. The objective of this study is to find the pattern of cardiovascular diseases among patients admitted in cardiac ward.
Methods:
A retrospective analysis of records of admitted patients from 1st January 2016 - 30th December 2017 was conducted in Cardiology ward of Manipal Teaching Hospital, Pokhara, Nepal. A total of 2268 patients' data were taken from the ward register. The data was collected and analyzed with descriptive and inferential statistics using SPSS 18.0 version during the period from 1st June to 30th August 2018.
Results:
The age ranged between 12 to 95 years with a mean (SD) is 61.23(16.34 years). More than half (51%) of the patients were female gender. Forty-six percentages of the patients were in the age group of 56 - 75 years. More than 1/3rd (35%) were diagnosed with Ischemic heart disease (IHD) followed by hypertension (22.8%) and arrhythmia(13.4%). There was a significant association between age and gender with the pattern of cardiovascular disease.
Conclusions:
Ischemic heart disease, hypertension and arrhythmia were the major causes of hospitalization. Preventive measures at community and personal level focusing on the risk factors management of ischemic heart disease will alleviate the burden of ischemic heart disease.
Metabolic syndrome, diabetes, and ischemic heart disease are among the leading causes of death and disability in Western countries. Diabetic cardiomyopathy is responsible for the most severe signs and symptoms. An important strategy for reducing the incidence of cardiovascular disease is regular exercise. Remote ischemic conditioning has some similarity with exercise and can be induced by short periods of ischemia and reperfusion of a limb, and it can be performed in people who cannot exercise. There is abundant evidence that exercise is beneficial in diabetes and ischemic heart disease, but there is a need to elucidate the specific cardiovascular effects of emerging and unconventional forms of exercise in people with diabetes. In addition, remote ischemic conditioning may be considered among the options to induce beneficial effects in these patients. The characteristics and interactions of diabetes and ischemic heart disease, and the known effects of exercise and remote ischemic conditioning in the presence of metabolic syndrome and diabetes, are analyzed in this brief review.
A detailed description of the electrophysiological features of cardiomyocytes in the various contractile chambers of the vertebrate heart is essential to understand the evolution of cardiac electrical activity, yet very little is known about reptiles. The present study characterizes major ionic currents (INa, ICaL, IKr, IK1 and IKACh) and action potential (AP) configuration in cardiomyocytes from the ventricle, the right atrium and the sinus venosus (SV) of Burmese pythons (Python molurus) using sharp microelectrode and patch clamp recordings. Special attention was given to SV, since it consists of myocardial cells and appears to contribute to right atrial filling in snakes. We demonstrate that most of the SV in pythons has a stable resting potential of − 82.3 ± 2.6 mV (n = 9) and lacks pacemaker activity. AP duration at 50% repolarization was similar in cells from SV and atria (350.2 ± 8.7 and 330.4 ± 17.2 ms, respectively; n = 7), but shorter than ventricular APs (557.6 ± 19.2 ms, n = 5) at 30 °C. The densities of ionic currents, however, differed substantially between atrial and SV cells, where the latter had much lower densities of INa, ICaL and IKr than atrial and ventricular myocytes. IK1 in ventricle was ninefold greater than in atrial cells and 23-fold greater than in myocytes from SV. However, IKACh was absent in ventricular cells, while it was equally large in atrial and SV myocytes. Consistent with this observation, APs of atrium and SV, but not ventricle, were greatly shortened upon addition of acetylcholine (10⁻⁶ M). Thus, snake SV, right atrium and ventricle have distinct patterns of ionic currents, but the resulting electrical activity is similar in atrium and SV.
Mammals and birds acquired high performance hearts and endothermy during their independent evolution from amniotes with many sauropsid features. A literature review shows that the variation in atrial morphology is greater in mammals than in ectothermic sauropsids. We therefore hypothesized that the transition from ectothermy to endothermy was associated with greater variation in cardiac structure. We tested the hypothesis in 14 orders of birds by assessing the variation in 15 cardiac structures by macroscopic inspection and histology, with an emphasis on the atria as they have multiple features that lend themselves to quantification. We found bird hearts to have multiple features in common with ectothermic sauropsids (synapomorphies), such as the presence of three sinus horns. Convergent features were shared with crocodylians and mammals, such as the cranial offset of the left atrioventricular junction. Other convergent features, like the compact organization of the atrial walls, were shared with mammals only. Pacemaker myocardium, identified by Isl1 expression, was anatomically node‐like (Mallard), thickened (Chicken), or indistinct (Lesser redpoll, Jackdaw). Some features were distinctly avian, (autapomorphies) including the presence of a left atrial antechamber and the ventral merger of the left and right atrial auricles, which was found in some species of parrots and passerines. Most features, however, exhibited little variation. For instance, there were always three systemic veins and two pulmonary veins, whereas among mammals there are 2–3 and 1–7, respectively. Our findings suggest that the transition to high cardiac performance does not necessarily lead to a greater variation in cardiac structure.
The striking seasonal difference in sensitivity of frog cardiac muscle to acetylcholine or stimulation of parasympathetic nervous fibers has been noted almost a century ago, although its electrophysiological basis has never been revealed. The present study compares the effects of the muscarinic agonist carbamylcholine chloride (CCh 10⁻⁸–10⁻⁵ M) on electrical activity in isolated atrial and ventricular preparations from dormant frogs (Rana temporaria) caught in January (winter-acclimatized, WA) and from active frogs caught in July (summer-acclimatized, SA). Seasonal differences in the density of potassium acetylcholine-dependent current (IKACh) were also studied in atrial and ventricular myocytes from both summer and winter groups. In atrial myocardium, CCh produced concentration-dependent shortening of action potentials (APs). CCh concentration producing a 50% reduction of AP duration was lower in WA (1.03 × 10⁻⁷ M) than SA atria (2.7 × 10⁻⁷ M). 10⁻⁶ M CCh induced drastic reduction of AP amplitude rendering the tissue unexcitable in both WA or SA atrial preparations. Ventricular preparations showed greater seasonal difference in CCh sensitivity. While 10⁻⁶ M induced inexcitability in 50% of tested WA preparations, in SA preparations even 10⁻⁵ M CCh was without effect. This striking difference between cholinergic effects in SA and WA frog ventricle could be explained by seasonal changes in the IKACh density. The density of IKACh induced by 10⁻⁵ M CCh and measured at 0 mV was 14.4 ± 3.45 pA/pF in WA, but only 1.5 ± 0.4 pA/pF in SA atrial cells. In ventricular cells, the respective values were 2.61 ± 0.56 and 0.71 ± 0.09 pA/pF. Thus, hibernating winter frog has a much greater electrophysiological cholinergic response than active summer frog due to up-regulation of IKACh.
Rate-dependent repolarization (RDR) of action potential (AP) in cardiomyocyte plays a critical role in the genesis of arrhythmias and RDR in atrium has been linked with atrial fibrillation. However, detailed studies focusing on the role of RDR in rabbit atrium are scant. In this study, atrial cells were isolated from rabbit heart and rate-dependent property was explored in single atrial cell to elucidate the underlying mechanism. Our results indicated that rate-dependent prolongation was evident at the action potential duration at 20% (APD20) and 50% (APD50) repolarization but not at 90% repolarization (APD90) under control condition. Using transient outward potassium current (Ito) inhibitor 4-Aminopyridine (4-AP, 2 mM) effectively eliminated the changes in APD20 and APD50, and unmasked the rate-dependent reduction of APD90 which could be diminished by further adding L-type calcium current (ICaL) inhibitor nifedipine (30 μM). However, using the selective late sodium current (INaL) inhibitor GS-458967 (GS967, 1 μM) caused minimal effect on APD90 of atrial cells both in the absence and presence of 4-AP. In consistence with results from APs, Ito and ICaL displayed significant rate-dependent reduction because of their slow reactivation kinetics. In addition, the magnitude of INaL in rabbit atrium was so small that its rate-dependent changes were negligible. In conclusion, our study demonstrated that Ito and ICaL mediate RDR of AP in rabbit atrium, while minimal effect of INaL was seen.
Diadenosine polyphosphates (Ap(n)As) are endogenously produced molecules which have been identified in various tissues of mammalian organism, including myocardium. Ap(n)As contribute to the blood clotting and are also widely accepted as regulators of blood vascular tone. Physiological role of Ap(n)As in cardiac muscle has not been completely elucidated. The present study aimed to investigate the effects of diadenosine tetra- (Ap4A) and penta- (Ap5A) polyphosphates on contractile function and action potential (AP) waveform in rat supraventricular and ventricular myocardium. We have also demonstrated the effects of A4pA and Ap5A in myocardial sleeves of pulmonary veins (PVs), which play a crucial role in genesis of atrial fibrillation. APs were recorded with glass microelectrodes in multicellular myocardial preparations. Contractile activity was measured in isolated Langendorff-perfused rat hearts. Both Ap4A and Ap5A significantly reduced contractility of isolated Langendorff-perfused heart and produced significant reduction of AP duration in left and right auricle, interatrial septum, and especially in right ventricular wall myocardium. Ap(n)As also shortened APs in rat pulmonary veins and therefore may be considered as potential proarrhythmic factors. Cardiotropic effects of Ap4A and Ap5A were strongly antagonized by selective blockers of P2 purine receptors suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), while P1 blocker DPCPX was not effective. We conclude that Ap(n)As may be considered as new class of endogenous cardioinhibitory compounds. P2 purine receptors play the central role in mediation of Ap4A and Ap5A inhibitory effects on electrical and contractile activity in different regions of the rat heart.
Previous histological studies showed that in addition to a sinus node, an atrioventricular (AV) node, an AV bundle, left and right bundle branches, birds also possess a right AV-Purkinje ring that is located in the atrial sheet of the right muscular AV-valve along all its base length. The functionality of the AV-Purkinje ring is unknown. In this work, we studied the topology of pacemaker myocytes in the atrial side of the isolated chicken spontaneously contracting right muscular AV-valve using the method of microelectrode mapping of action potentials. We show that AV-cells having the ability to show pacemaking reside in the right muscular AV-valve. Pacemaker action potentials were exclusively recorded close to the base of the valve along its whole length from dorsal to the ventral attachment to the interventricular septum. These action potentials have much slower rate of depolarization, lower amplitude, and higher diastolic depolarization than action potentials of Purkinje (conducting) cells. We conclude the right AV-valve has a ring bundle of pacemaker cells (but not Purkinje cells) in the adult chicken heart. J. Morphol., 2015. © 2015 Wiley Periodicals, Inc.
The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.
Effects of phase delay shifts (PDS) of light in combination with moderately increased salt intake (SL) (2%) or time restriction of food (FR) during the light-time (passive phase) on heart rate (HR), blood pressure (BP) and locomotor activity (LA) in radiotelemetry-measured rats were evaluated. PDS decreased amplitude and spectral power of circadian oscillations of HR, BP and LA. Moderately increased SL did not interfere with the circadian rhythmicity of HR, BP or LA. A prominent decrease in amplitude and spectral power of circadian oscillations was observed if food was available during the lighttime. Combination of PDS with FR split cardiovascular and behavioural parameters. In conclusion, food availability during the light-time in combination with PDS decreased amplitude and spectral power of circadian oscillations of BP, HR and LA more than PDS only. Different response of cardiovascular and behavioural parameters to photic and non-photic stimuli can have consequences for shift workers.
Fetal cardiac growth in mammalian models occurs primarily by cell proliferation (hyperplasia). However, most cardiomyocytes lose the ability to proliferate close to term and heart growth continues by increasing cell size (hypertrophy). In mammals, the thyroid hormone triiodothyronine (T3) is an important driver of this process. Chicken cardiomyocytes, however, keep their proliferating ability long after hatching but little information is available on the mechanisms controlling cell growth and myocyte maturation in the chicken heart. Our aim was to study the role of T3 on proliferation and differentiation of embryonic chicken cardiomyocytes (ECCM), enzymatically isolated from 19-day-old embryos and to compare the effects to those of insulin-like growth factor-1 (IGF-1) and phenylephrine (PE). Hyperplasia was measured using a proliferation assay (MTS) and hypertrophy/multinucleation was analyzed morphologically by phalloidin staining of F-actin and nuclear staining with DAPI. We show that IGF-1 induces a significant increase in ECCM proliferation (30%) which is absent with T3 and PE. PE induced both hypertrophy (61%) and multinucleation (41%) but IGF-1 or T3 did not. In conclusion, we show that T3 does not induce maturation or proliferation of cardiomyocytes, while IGF-1 induces cardiomyocyte proliferation and PE induces maturation of cardiomyocytes.
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.
Freshwater fishes of north-temperate latitudes adjust electrical excitability of the heart to seasonal temperature changes by changing expression levels of ion channel isoforms. However, little is known about thermal responses of action potential (AP) in the hearts of marine polar fishes. To this end, we examined cardiac AP in the atrial myocardium of the Arctic navaga cod (Eleginus navaga) from the White Sea (Russia) acclimatized to winter (March) and summer (September) seasons. Acute increases in temperature from 4 to 10 °C were associated with increases in heart rate, maximum velocity of AP upstroke and negative resting membrane potential, while duration of AP was shortened in both winter-acclimatized and summer-acclimatized navaga hearts. In winter, there was a compensatory shortening (41.1 %) of atrial AP duration and this was associated with a strong increase in transcript expression of Erg K+ channels, known to produce the rapid component of the delayed rectifier K+ current, I
Kr. Smaller increases were found in the expression of Kir2.1 channels that produce the inward rectifier K+ current, I
K1. These findings indicate that the heart of navaga cod has a good acclimatory capacity in electrical excitation of cardiac myocytes, which enables cardiac function in the cold-eurythermal waters of the subarctic White Sea.
Circulating leptin levels are elevated in obesity and hyperleptinaemia has been postulated to be an independent risk factor for the development of cardiovascular diseases. Although many studies have been published on the mechanisms involved in the effects of leptin on cardiac function and pathological remodeling, scarce information is currently available analyzing the influence of prolonged leptin treatment on ionic cardiac channels remodeling in adult ventricular myocytes. Enzymatically isolated adult rat ventricular myocytes were treated with leptin or vehicle for 48h. Real-Time RT-PCR were used to analyze mRNA expression of Kir2.1, Cav1.2, Cav 3.1, Kv4.2 and Kv4.3 α-subunits and KChIP2 auxiliary subunit. The fast transient outward potassium channels (Itof) α-subunits Kv4.2, Kv4.3 and KChIP2 were analyzed by Western-blot. The fast transient outward potassium current and the action potentials were recorded in isolated myocytes by the whole-cell patch-clamp technique. Leptin treatment induced an up-regulation of Kv4.2, Kv4.3 and KChIP2 subunits mRNA expression. However, transcriptional levels of Kir2.1, Cav1.2, or Cav3.1 α-subunit channels were unmodified by leptin. Protein expression levels of Kv4.2, Kv4.3 and KChIP2 subunits were also increased by leptin. The electrophysiological study showed that leptin increases the fast transient outward potassium current amplitudes and densities shortening action potential duration. In addition, leptin activated Akt signaling in cardiomyocytes and this mechanism was involved in the effect of leptin on Itof channels. In conclusión, leptin increases both the expression and function of Itof channels in adult ventricular myocytes and this mechanism involves Akt signaling. Altogether these data suggest that leptin could exert beneficial or detrimental effects depending on the initial ventricular myocyte repolarizing reserve.
Birds and mammals both developed high performance hearts from a heart that must have been reptile-like and the hearts of extant reptiles have an unmatched variability in design. Yet, studies on cardiac development in reptiles are largely old and further studies are much needed as reptiles are starting to become used in molecular studies. We studied the growth of cardiac compartments and changes in morphology principally in the model organism corn snake (Pantherophis guttatus), but also in the genotyped anole (Anolis carolinenis and A. sagrei) and the Philippine sailfin lizard (Hydrosaurus pustulatus). Structures and chambers of the formed heart were traced back in development and annotated in interactive 3D pdfs. In the corn snake, we found that the ventricle and atria grow exponentially, whereas the myocardial volumes of the atrioventricular canal and the muscular outflow tract are stable. Ventricular development occurs, as in other amniotes, by an early growth at the outer curvature and later, and in parallel, by incorporation of the muscular outflow tract. With the exception of the late completion of the atrial septum, the adult design of the squamate heart is essentially reached halfway through development. This design strongly resembles the developing hearts of human, mouse and chicken around the time of initial ventricular septation. Subsequent to this stage, and in contrast to the squamates, hearts of endothermic vertebrates completely septate their ventricles, develop an insulating atrioventricular plane, shift and expand their atrioventricular canal toward the right and incorporate the systemic and pulmonary venous myocardium into the atria.
The endothermic state of mammals and birds requires high heart rates to accommodate the high rates of oxygen consumption. These high heart rates are driven by very similar conduction systems consisting of an atrioventricular node that slows the electrical impulse and a His-Purkinje system that efficiently activates the ventricular chambers. While ectothermic vertebrates have similar contraction patterns, they do not possess anatomical evidence for a conduction system. This lack amongst extant ectotherms is surprising because mammals and birds evolved independently from reptile-like ancestors. Using conserved genetic markers, we found that the conduction system design of lizard (Anolis carolinensis and A. sagrei), frog (Xenopus laevis) and zebrafish (Danio rerio) adults is strikingly similar to that of embryos of mammals (mouse Mus musculus, and man) and chicken (Gallus gallus). Thus, in ectothermic adults, the slow conducting atrioventricular canal muscle is present, no fibrous insulating plane is formed, and the spongy ventricle serves the dual purpose of conduction and contraction. Optical mapping showed base-to-apex activation of the ventricles of the ectothermic animals, similar to the activation pattern of mammalian and avian embryonic ventricles and to the His-Purkinje systems of the formed hearts. Mammalian and avian ventricles uniquely develop thick compact walls and septum and, hence, form a discrete ventricular conduction system from the embryonic spongy ventricle. Our study uncovers the evolutionary building plan of heart and indicates that the building blocks of the conduction system of adult ectothermic vertebrates and embryos of endotherms are similar.
Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al. in Environ Health Perspect 108:177-200, 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real-time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8 ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens.
The aim of this review is to present the properties of the slow component of the delayed rectifier potassium current (IKs) in the human ventricle. The review gives a detailed description of the physiology, molecular biology and pharmacology of the IKs current, including kinetic properties, genetic structures, agonists and antagonists. The authors also present the role of the IKs current in the human cardiac repolarization focusing on several pathophysiological situations, such as the LQT syndrome and the Torsade de Pointes arrhythmia.
To assess the role of the slow delayed rectifier potassium current (IKs) in the β-adrenergic modulation of heart rate, we experimentally determined the effect of β adrenergic stimulation on IKs and used the thus obtained data in computer simulations of SA nodal pacemaker activity, employing the mathematical model of a primary rabbit SA node pacemaker cell by Kurata and coworkers. Incorporation of our experimental findings into the SA nodal cell model resulted in a 12 ms decrease in cycle length. This decrease in cycle length is similar to the 13 ms decrease observed upon incorporation of our experimental data on the effect of β-adrenergic stimulation on the hyperpolarization-activated funny current' (If), also known as 'pacemaker current'. We conclude that IKs is an important contributor to the β-adrenergic modulation of heart rate.
G. Y. Oudit, Z. Kassiri, R. Sah, R. J. Ramirez, C. Zobel and P. H. Backx. The Molecular Physiology of the Cardiac Transient Outward Potassium Current (Ito) in Normal and Diseased Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 851–872. The Ca2+-independent transient outward potassium current (Ito) plays an important role in early repolarization of the cardiac action potential. Itohas been clearly demonstrated in myocytes from different cardiac regions and species. Two kinetic variants of cardiac Itohave been identified: fast Ito, called Ito,f, and slow Ito, called Ito,s. Recent findings suggest that Ito,fis formed by assembly of Kv4.2and/or Kv4.3alpha pore-forming voltage-gated subunits while Ito,sis comprised of Kv1.4and possibly Kv1.7subunits. In addition, several regulatory subunits and pathways modulating the level and biophysical properties of cardiac Itohave been identified. Experimental findings and data from computer modeling of cardiac action potentials have conclusively established an important physiological role of Itoin rodents, with its role in large mammals being less well defined due to complex interplay between a multitude of cardiac ionic currents. A central and consistent electrophysiological change in cardiac disease is the reduction in Itodensity with a loss of heterogeneity of Itoexpression and associated action potential prolongation. Alterations of Itoin rodent cardiac disease have been linked to repolarization abnormalities and alterations in intracellular Ca2+homeostasis, while in larger mammals the link with functional changes is far less certain. We review the current literature on the molecular basis for cardiac Itoand the functional consequences of changes in Itothat occur in cardiovascular disease.
Evolutionary origin and physiological significance of the tetrodotoxin (TTX) resistance of the vertebrate cardiac Na(+) current (I(Na)) is still unresolved. To this end, TTX sensitivity of the cardiac I(Na) was examined in cardiac myocytes of a cyclostome (lamprey), three teleost fishes (crucian carp, burbot and rainbow trout), a clawed frog, a snake (viper) and a bird (quail). In lamprey, teleost fishes, frog and bird the cardiac I(Na) was highly TTX-sensitive with EC(50)-values between 1.4 and 6.6 nmol·L(-1). In the snake heart, about 80% of the I(Na) was TTX-resistant with EC(50) value of 0.65 μmol·L(-1), the rest being TTX-sensitive (EC(50) = 0.5 nmol·L(-1)). Although TTX-resistance of the cardiac I(Na) appears to be limited to mammals and reptiles, the presence of TTX-resistant isoform of Na(+) channel in the lamprey heart suggest an early evolutionary origin of the TTX-resistance, perhaps in the common ancestor of all vertebrates.
Explanations for arrhythmia mechanisms at the cellular level are usually based on experiments in nonhuman myocytes. However, subtle electrophysiological differences between species may lead to different rhythmic or arrhythmic cellular behaviors and drug response given the nonlinear and highly interactive cellular system. Using detailed and quantitatively accurate mathematical models for human, dog, and guinea pig ventricular action potentials (APs), we simulated and compared cell electrophysiology mechanisms and response to drugs. Under basal conditions (absence of β-adrenergic stimulation), Na(+)/K(+)-ATPase changes secondary to Na(+) accumulation determined AP rate dependence for human and dog but not for guinea pig where slow delayed rectifier current (I(Ks)) was the major rate-dependent current. AP prolongation with reduction of rapid delayed rectifier current (I(Kr)) and I(Ks) (due to mutations or drugs) showed strong species dependence in simulations, as in experiments. For humans, AP prolongation was 80% following I(Kr) block. It was 30% for dog and 20% for guinea pig. Under basal conditions, I(Ks) block was of no consequence for human and dog, but for guinea pig, AP prolongation after I(Ks) block was severe. However, with β-adrenergic stimulation, I(Ks) played an important role in all species, particularly in AP shortening at fast rate. Quantitative comparison of AP repolarization, rate-dependence mechanisms, and drug response in human, dog, and guinea pig revealed major species differences (e.g., susceptibility to arrhythmogenic early afterdepolarizations). Extrapolation from animal to human electrophysiology and drug response requires great caution.
The effects of K(+)-channel blockers on the action potential duration of the myocardium were examined in isolated right ventricles from the 7 - 10-day-old, 11 - 13-day-old, and 14 - 20-day-old embryo and 1 - 7-day-old hatched chicks. E-4031 significantly prolonged action potential duration at all developmental stages examined; the prolongation was largest in the 11 - 13-day-old embryo and was accompanied by early after-depolarizations. Chromanol 293B showed smaller prolongation at all stages examined. Terfenadine prolonged action potential duration in the 11 - 13-day-old embryo, but not in other stages. Thus, the chick ventricular myocardium changes its repolarization properties during development.
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.
Both intracellular and surface electrodes were employed to record electrical activity from embryonic chick hearts between the ages of 3 and 20 days. Cells from the sinus venosus, sinoatrial (SA) valves, atrium, atrioventricular (AV) ring, and ventricle were localized and characterized on the basis of shape, amplitude, rise time, and duration of transmembrane potentials. The differences in transmembrane potentials from these various regions provided the basis for a hypothesis concerned with the distribution of pacemaker potentiality and one related to the origin of the His-Purkinje system. Action potentials recorded along the entire embryonic AV ring were comparable to those of the adult rabbit AV nodal cells in both configuration and sequence of activation and were thus categorized into three functional regions (AN, N, NH). Histological sections of 7 and 14 day hearts demonstrated muscular continuity between the right atrium and ventricle across the muscular AV valve.
The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K(+) channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na(+) channels, L-type Ca(2+) channels contribute to the plateau phase and I(Kr) channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca(2+) current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca(2+) channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype.
Birds have evolved a tightly controlled cardiovascular system that meets the rigorous energetic demands of flight. The bird heart has a functional anatomy similar to that of the mammalian heart with four chambers and an extensive coronary circulation. The electrical conducing system of the avian heart includes a sinoatrial node, atrioventricular (AV) node, AV Purkinje ring, and the bundle branches. The vascular system is composed of arteries, capillaries, and veins and includes a pair of ductus arteriosi as embryos. Control of cardiovascular function includes both cardiac and the vasculature limbs. Peripheral vasculature reactivity is regulated by local mechanical autoregulation, humoral factors, and the autonomic nervous system altering vascular resistance. Humoral factors that regulate vascular resistance include pH, CO2, nitric oxide, angiotensin II, and circulating catecholamines. Both sympathetic and parasympathetic inputs have important roles in regulating the vasculature and the heart function. Sympathetic control of the cardiac function increases both contractile force and heart rate. Parasympathetic control affects cardiac contractile force and heart rate at the pacemaker and conducting tissues. Cardiac output is regulated by chemoreflexes and baroreflexes. All of these regulatory pathways mature late in embryonic life, providing fine control by the time of hatch. Finally, the avian cardiovascular system has evolved to function effectively during long migration flights, some at high altitude and during diving.
Rat telemetry is widely used for biomedical research purposes and is used routinely in early pre-clinical drug development to screen for the potential cardiovascular risk of candidate drugs. Historically, these studies have been conducted in individually housed conditions which can impact significantly on an animal's welfare. Here we present data from a survey of pharmaceutical companies and contract research organisations to define current industry practices relating to the housing of rats during telemetry studies and to expand and complement a similar project in non-rodents. Results of the survey showed that 75% of respondents socially house rats on non-recording days of telemetry studies, whereas on recording days only 46% of respondents socially house the animals. When social housing is used on rat telemetry studies, rats are usually housed with an unrecorded companion animal. We also present and compare data from a telemetry study in standard individually ventilated cages (IVCs) with a study using new double-decker IVCs, both conducted using a companion animal approach. Telemetry signals were successfully collected from the double-decker IVCs without a loss of signal quality whilst offering a more spacious environment that allowed the animals to exhibit natural behaviours including full upright posture. Cardiovascular responses following pharmacological intervention with verapamil were similar when assessed in the standard and double-decker cages. Power analysis was conducted on pooled data from the studies in socially housed animals with preliminary results showing the power of detection of drug-induced effects is equivalent to previously published data in individually housed rats. This illustrates that telemetry recordings can be made from rats in socially housed conditions within standard or larger double-decker cages for the for the collection of cardiovascular telemetry data.
The electrocardiogram (ECG) reveals that heart chamber activation and repolarization are much faster in mammals and birds compared to ectothermic vertebrates of similar size. Temperature, however, affects electrophysiology of the heart and most data from ectotherms are determined at body temperatures lower than those of mammals and birds. The present manuscript is a review of the effects of temperature on intervals in the ECG of ectothermic and endothermic vertebrates rather than a hypothesis-testing original research article. However, the conclusions are supported by the inclusion of original data (Iguana iguana, N = 4; Python regius, N = 5; Alligator mississippiensis, N = 4). Most comparisons were of animals of approximately 1 kg. Compared to mammals and birds, the reptiles at 35-37 °C had 4 fold lower heart rates, 2 fold slower atrial and ventricular conduction (longer P- and QRS-wave durations), and 4 fold longer PR intervals (atrioventricular delay) and QT intervals (total ventricular repolarization). We conclude that the faster chamber activation in endotherms cannot be explained by temperature alone. Based on histology, we show that endotherms have a more compact myocardial architecture. In mammals, disorganization of the compact wall by fibrosis associates with conduction slowing and we suggest the compact tissue architecture allows for faster chamber activation. The short cardiac cycle that characterizes mammals and birds, however, is predominantly accommodated by shortening of the atrioventricular delay and the QT interval, which is so long in a 1 kg iguana that it compares to that of an elephant.
Background:
In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms.
Objective:
To study whether/how chronic in vivo Ang II administration remodels IKs in atrial and ventricular myocytes.
Methods:
We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4-6 weeks. We used patch clamp, immunofluorescence/confocal microscopy and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms.
Results:
We confirmed the pathological state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, while the latter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II-treated animals, the L-type Ca channel current was increased contributing to AP plateau elevation and AP duration prolongation.
Conclusion:
IKs is differentially modulated by chronic in vivo Ang II administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.
Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.
Temperature sensitivity of electrical excitability is a potential limiting factor for high temperature tolerance of ectotherms. The present study examines whether heat resistance of electrical excitability of cardiac myocytes is modified by seasonal thermal acclimatization in roach (Rutilus rutilus), a eurythermal teleost species. To this end, temperature dependencies of ventricular action potentials (APs), and atrial and ventricular K⁺ currents were measured from winter-acclimatized (WiR) and summer-acclimatized (SuR) roach. Under patch-clamp recording conditions, ventricular APs could be triggered over a wide range of temperatures (4–43 °C) with prominent changes in resting membrane potential (RMP), AP duration and amplitude. In general, APs of SuR were slightly more tolerant to high temperatures than those of WiR, e.g. the break point temperature (TBP) of RMP was 37.6 ± 0.4 °C in WiR and 41 ± 1 °C in SuR (p < 0.05). Of the two major cardiac K⁺ currents, the inward rectifier K⁺ current (IK1) was particularly heat resistant in both SuR (TBP 39.4 ± 0.4 °C) and WiR (TBP 40.0 ± 0.4 °C) ventricular myocytes. The delayed rectifier K⁺ current (IKr) was not as heat resistant as IK1. Surprisingly, IKr of WiR tolerated heat better (TBP 31.9 ± 0.8 °C) than IKr of SuR (TBP 24.1 ± 0.5 °C) (p < 0.05). IKr (Erg2) channel transcripts of both atrial and ventricular myocytes were up-regulated in WiR. IK1 (Kir2) channel transcripts were not affected by seasonal acclimatization, although ventricular IK1 current was up-regulated in summer. Collectively, these findings show that thermal tolerance limits of K⁺ currents in isolated myocytes between seasonally acclimatized roach are much less pronounced than the heat sensitivity of ECG variables in intact fish.
Objective: Heart failure in patients and in animal models is associated with action potential prolongation of the ventricular myocytes. Changes in several membrane currents have been already demonstrated to underlie this prolongation. However, information on the two components (IKr and IKs) of the delayed rectifier potassium current (IK) in rapid pacing induced heart failure is lacking. Methods and results: Action potentials and whole-cell currents, IK, Ito1, IK1, and ICa-L were recorded in apical myocytes of left ventricle from 10 rabbits subjected to left ventricular pacing at 350–380 beats/min for 3–4 weeks and 10 controls with sham operation. Action potential duration at 90% repolarization (APD90) was prolonged in myocytes from failing hearts compared to controls at both cycle lengths of 333 and 1000 ms. Both E-4031-sensitive and -resistant components of IK (IKr, IKs) in myocytes from failing hearts were significantly less than those of control hearts; tail current densities of IKr and IKs following depolarization to +50 mV were 0.62±0.05 vs. 0.96±0.12 pA/pF (P<0.05), and 0.27±0.08 vs. 0.52±0.08 pA/pF (P<0.05), respectively. There was no significant difference between control and failing myocytes in the voltage- and time-dependence of activation of total IK, IKr and IKs. The peak of L-type Ca2+ current (ICa-L) was significantly reduced in myocytes from failing hearts (at +10 mV, −9.29±0.52 vs. −12.28±1.63 pA/pF, P<0.05), as was the Ca2+-independent transient outward current (Ito1; at +40 mV, 4.8±0.9 vs. 9.6±1.3 pA/pF, P<0.05). Steady state I–V curve for IK1 was similar in myocytes from failing and control hearts. Conclusions: Decrease of IK (both IKr and IKs) in addition to reduced Ito1, may underly action potential prolongation at physiological cycle length and thereby contribute to arrhythmogenesis in heart failure.
The ultra-rapid delayed rectifier K+ current (IKur) plays an important role in early cardiac action potential repolarisation in a number of species. Although the rabbit heart has proven useful for studying cardiac repolarisation, the role of IKur in rabbit heart is less well known, as is the potential contribution of this ion channel to drug-induced changes in repolarisation. The aim of this study was to investigate the effects of 4-aminopyridine (4-AP) and 2-isopropyl-5-methylcyclohexyl diphenylphosphine oxide (DPO-1) on action potential duration (APD) in isolated rabbit atria and ventricular papillary muscles. Intracellular action potentials were recorded at 36–37°C and APD was measured at 20, 50 and 90% repolarisation (APD20, 50 and 90). Exposure to 4-AP at 50 μM for 30 min caused a significant prolongation in APD20 of 29.0±3.3% and 39.4±9.4% in left atria and right ventricular papillary muscles, respectively (stimulated at 1 Hz). In contrast, late repolarisation was less affected; increases in APD90 were 8.6±2.8% and 18.7±5.0% for atria and papillary muscles, respectively. The prolongation was reverse frequency dependent (0.2–1 Hz). Similarly, application of DPO-1 at 0.3 μM for 30 min significantly prolonged APD20 by 21.9±8.8% and 45.8±23.0% in atria and papillary muscles, respectively. APD90 was less affected, with increases of 10.8±2.2 and 25.2±13.2% in atria and papillary muscles, respectively. In conclusion, it is likely that IKur may play a functional role in repolarisation in rabbit in both atria and ventricle.
It has been suggested that deficient protein trafficking to the cell membrane is the dominant mechanism associated with type 2 Long QT syndrome (LQT2) caused by Kv11.1 potassium channel missense mutations, and that for many mutations the trafficking defect can be corrected pharmacologically. However, this inference was based on expression of a small number of Kv11.1 mutations. We performed a comprehensive analysis of 167 LQT2-linked missense mutations in four Kv11.1 structural domains and found that deficient protein trafficking is the dominant mechanism for all domains except for the distal carboxy-terminus. Also, most pore mutations-in contrast to intracellular domain mutations-were found to have severe dominant-negative effects when co-expressed with wild-type subunits. Finally, pharmacological correction of the trafficking defect in homomeric mutant channels was possible for mutations within all structural domains. However, pharmacological correction is dramatically improved for pore mutants when co-expressed with wild-type subunits to form heteromeric channels.
Effects of taurine on the delayed rectifier K(+) channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca(2+) concentrations ([Ca]i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K(+) current (IK) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]i was pCa 10, IK at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K(+) channel, an effect dependent on [Ca]i and capable of regulating APD.
A number of drugs currently in clinical use cause unwanted prolongation of the QT interval. This condition occasionally evolves to fatal, polymorphic ventricular dysrhythmias. A posteriori, many of these drugs, at clinically relevant concentrations, have been shown to block the ionic current carried by the HERG channel, a major player in the repolarization process by which the heart recovers its resting state. This article describes an experimental strategy designed to reveal possible mechanisms by which drugs may delay ventricular repolarization. This strategy is designed to determine the ability of these compounds to block K+ conductance in HERG channels. In view of the difficulties involved in studying such channels in their natural location, they are expressed in mammalian cells and are studied under whole-cell patch clamp configuration. For the novel drugs that are candidates to further development, cardiac safety examination should be extended to other major ion channels of human myocardium. The activity of such channels can be recorded by patch clamp techniques in myocytes disaggregated from atrial tissue specimens excised during elective cardiac surgery. The experimental conditions adopted for these experiments should replicate, as closely as possible, the physiological environment embracing the native channels. The effects of various compounds on the HERG channel are reported and a method for calculating cardiac safety indices is described and applied to terfenadine and cetirizine. These indices are useful tools for deciding whether a candidate drug deserves to enter the development pathway. In conclusion, patch clamp studies in cloned and native human heart ion channels can provide fundamental information concerning the cardiac safety profile of novel drugs intended for use in humans.
Early cardiac development involves the formation of a heart tube, looping of the tube and formation of chambers. These processes are highly similar among all vertebrates, which suggest the existence of evolutionary conservation of the building plan of the heart. From the jawless lampreys to man, T-box transcription factors like Tbx5 and Tbx20 are fundamental for heart formation, whereas Tbx2 and Tbx3 repress chamber formation on the sinu-atrial and atrioventricular borders. Also, electrocardiograms from different vertebrates are alike, even though the fish heart only has two chambers whereas the mammalian heart has four chambers divided by septa and in addition has much higher heart rates. We conclude that most features of the high-performance hearts of mammals and birds can be traced back to less developed traits in the hearts of ectothermic vertebrates. This article is part of a Special Issue entitled: Cardiomyocyte biology: Cardiac pathways of differentiation, metabolism and contraction.
• The transient outward K+ current (Ito) is a major repolarizing ionic current in ventricular myocytes of several mammals. Recently it has been found that its magnitude depends on the origin of the myocyte and is regulated by a number of physiological and pathophysiological signals. • The relationship between the magnitude of Ito, action potential duration (APD) and Ca2+ influx (QCa) was studied in rat left ventricular myocytes of endo- and epicardial origin using whole-cell recordings and the action potential voltage-clamp method. • Under control conditions, in response to a depolarizing voltage step to +40 mV, Ito averaged 12.1 ± 2.6 pA pF−1 in endocardial ( n= 11 ) and 24.0 ± 2.6 pA pF−1 in epicardial myocytes ( n= 12 ; P 90 (90 % repolarization) was twice as long in endocardial myocytes, whereas QCa inversely depended on the magnitude of Ito. L-type Ca2+ current density was similar in myocytes from both regions. • To determine the effects of controlled reductions of Ito on QCa, recordings were repeated in the presence of increasing concentrations of the Ito inhibitor 4-aminopyridine. • Inhibition of Ito by as little as 20 % more than doubled QCa in epicardial myocytes, whereas it had only a minor effect on QCa in myocytes of endocardial origin. Further inhibition of Ito led to a progressive increase in QCa in epicardial myocytes; at 90 % inhibition of Ito, QCa was four times larger than the control value. • We conclude that moderate changes in the magnitude of Ito strongly affect QCa primarily in epicardial regions. An alteration of Ito might therefore allow for a regional regulation of contractility during physiological and pathophysiological adaptations.
In the mammalian heart, Ca2+-independent, depolarization-activated potassium (K+) currents contribute importantly to shaping the waveforms of action potentials, and several distinct types of voltage-gated K+ currents that subserve this role have been characterized. In most cardiac cells, transient outward currents, Ito,f and/or Ito,s, and several components of delayed reactivation, including IKr, IKs, IKur and IK,slow, are expressed. Nevertheless, there are species, as well as cell-type and regional, differences in the expression patterns of these currents, and these differences are manifested as variations in action potential waveforms. A large number of voltage-gated K+ channel pore-forming (α) and accessory (β, minK, MiRP) subunits have been cloned from or shown to be expressed in heart, and a variety of experimental approaches are being exploited in vitro and in vivo to define the relationship(s) between these subunits and functional voltage-gated cardiac K+ channels. Considerable progress has been made in defining these relationships recently, and it is now clear that distinct molecular entities underlie the various electrophysiologically distinct repolarizing K+ currents (i.e. Ito,f, Ito,s, IKr, IKs, IKur, IK,slow, etc.) in myocyardial cells.
Hypoxia is known to stimulate vascular growth by up-regulating vascular endothelial growth factor (VEGF), but little is known about the function of hypoxia in the development of the coronary vasculature, and the relationship between hypoxia and VEGF in this event. To test the effects of hypoxia and VEGF on coronary vasculogenesis/angiogenesis in the developing heart, ventricles from 6-day-old quail embryos were cultured on three-dimensional collagen gels. After 2 days of growth in normal medium and 1 day of starvation in low serum medium (0.5% fetal bovine serum), the heart explants were further cultured under various oxygen levels for another 24, 48, and 72 hr. Angioblasts and endothelial cells, which migrated out from the heart explants, were identified by QH1 antibody using immunofluorescence and confocal microscopy. In the normoxic culture environment, the endothelial cells began to proliferate and migrate out from the heart explants after 3 days of growth; they formed tubes mainly after another 72 hr. In contrast, this vascular growth was accelerated under hypoxic conditions, as evidenced by increased tube formation with significant differences observed at 48 hr. On the other hand, hyperoxia delayed this process. Reverse transcription-polymerase chain reaction results indicated that VEGF (including VEGF122, VEGF166, and VEGF190) was up-regulated in the heart explants under hypoxia and down-regulated under hyperoxia. VEGF neutralizing antibody added to the culture medium partially blocked this vascular growth. We conclude from this study that hypoxia can stimulate or up-regulate coronary vasculogenesis/angiogenesis and that VEGF signaling plays a major role in this event. Dev Dyn 1999;216:28–36. © 1999 Wiley-Liss, Inc.
Chromanol HMR 1556 [(3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylmethanesulfonamide], a novel inhibitor of the slow component of the delayed outward current in heart muscle cells (IKs), has been characterized in several in-vitro systems. mRNA encoding for the human protein minK was injected into Xenopus oocytes, leading to the expression of IKs channels. HMR 1556 inhibited this current half-maximally at a concentration of 120 nmol/l (IC50). Expression of the K+ channels Herg, Kv1.5, Kv1.3 and Kir2.1, and also the cationic current HCN2, were blocked little or not at all by 10 mol/l HMR 1556. In isolated ventricular myocytes from the guinea pig the whole-cell patch-clamp method revealed inhibition of the IKs current with an IC50 of 34 nmol/l. Other current components, like IKr and IK1, were only slightly blocked at an HMR 1556 concentration of 10 mol/l, whereas 10 mol/l HMR 1556 inhibited the transient outward current Ito and the sustained outward current Isus in rat ventricular myocytes by 25% and 36%, respectively. The L-type Ca2+ channel in guinea pig cardiomyocytes was blocked by 10 mol/l HMR 1556 by 31%. Guinea pig right papillary muscles were investigated by the micropuncture technique at various pacing rates. In the frequency range of 0.5-7 Hz HMR 1556 (1 mol/l) caused a prolongation of the action potential duration at 90% repolarization (APD90) by 19%-27%. In the presence of isoproterenol (10 mol/l) the prolongation of the APD90 was more pronounced at low pacing rates (47% at 0.5 Hz and 35% at 1 Hz, compared with 25% at 7 Hz). The monophasic action potential was recorded in Langendorff-perfused guinea pig hearts. In spontaneously beating preparations, HMR 1556, at 0.1 mol/l and 1 mol/l, prolonged the MAPD90 by 3% and 10%, respectively, with no further prolongation at 10 mol/l. The prolongation was much greater at low pacing rates [25% at 100 beats per min (bpm) and 13% at 150 bpm] than at fast pacing rates (9% at 350 bpm). The left ventricular pressure LVPmax was not affected at 1 mol/l HMR 1556, but it decreased by 15% at 10 mol/l. Other parameters, like the heart rate and coronary flow, were only slightly decreased at 1 mol/l HMR 1556. In conclusion, HMR 1556 is a potent and selective inhibitor of the IKs current in guinea pig ventricular myocytes. The prolongation of the action potential duration is maintained at fast pacing rates.
To obtain information conceming the time course and instantaneous distribution of
the excitatory process of the normal human healt, studies were made on isolated human hearts from seven individuals who died from various cerebral conditions, but who had no history of cardiac disease. Measurements were made from as many as 870 intramural terminals.
In the isolated human hearts three endocardial areas were synchronously excited
0 to 5 msec after the start of the left ventricular activity potential. These areas increased rapidly in si ze dUl'ing the next 5 to 10 msec and became confluent in 15 to 20 msec. The left ventricular areas Rrst excited were (1) high on the anterior paraseptal wall just below the attachment of the mitral valve; (2) central on the left surface of the interventricular septum and (3) posterior paraseptal about one third of the distance from apex to base. The last part of the left ventricle to be activated usually was the posterobasal area. Endocardial activation of the right ventricle was found to start near the insertion of the anterior papillary muscle 5 to 10 msec af ter onset of the left ventricular cavity potential. Septal activation started in the micldle third of the left side of the interventricular septurn, somewhat anteriorly, and at the lower third at the junction of the septum and posterior wall. The epicardial excitation pattem reflected the movements of the intramural excitation wave. Conduction velocity was determined in one heart by an appropriate stimulation technic. Atrial excitation, explored in two hearts, spread more or less according to concentric isochronic lines.
Control studies, carried out on Rve canine hearts, disclosed that the pattem of
ventricular excitation did not change af ter isolation and perfusion. However, total
excitation was completed earlier in the isolated heart, and conduction velocity increased. Careful mapping illustrations are presented.