Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs

Cardiac repolarization, through which heart-cells return to their resting state after having fired, is a delicate process, susceptible to disruption by common drugs and clinical conditions. Animal models, particularly the dog, are often used to study repolarization properties and responses to drugs, with the assumption that such findings are relevant to humans. However, little is known about the applicability of findings in animals to man. Here, we studied the contribution of various ion-currents to cardiac repolarization in canine and human ventricle. Humans showed much greater repolarization-impairing effects of drugs blocking the rapid delayed-rectifier current IKr than dogs, because of lower repolarization-reserve contributions from two other important repolarizing currents (the inward-rectifier IK1 and slow delayed-rectifier IKs). Our findings clarify differences in cardiac repolarization-processes among species, highlighting the importance of caution when extrapolating results from animal models to man. Abstract The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50–100 nmol l−1 dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l−1 BaCl2) and IKs block (1 μmol l−1 HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ∼30% larger and ∼29% smaller in human, and Na+–Ca2+ exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKsα-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human–canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease. Many drugs can affect transmembrane K+ currents and thereby cause therapeutically useful (Honhloser & Woosley, 1994; Brendorp et al. 2001) or harmful (Surawicz, 1989; El-Sherif, 1992) effects. Blocking cardiac K+ channels prolongs repolarization and refractoriness, producing Class III antiarrhythmic effects both in ventricles and atria (Sing & Vaughan-Williams, 1970). Excessive lengthening of repolarization may induce life-threatening ventricular tachyarrhythmias like torsades de pointes (Hondeghem & Snyders, 1990; El-Sherif, 1992). Predicting the risk of such serious side effects is a major challenge in cardiac safety pharmacology. Torsade-risk estimation is hampered by a lack of easily usable methods and by incomplete understanding of the repolarization process in both experimental animals and humans. Repolarization is controlled by two major inward currents (Na+ and Ca2+) and four major outward K+ currents (rapid and slow delayed-rectifier (IKr and IKs), transient-outward (Ito) and inward-rectifier (IK1) currents), as well as other less well-characterized currents, electrogenic pumps and exchangers (Nerbonne & Kass, 2005). According to the concept of ‘repolarization reserve’ (Roden, 1998), normal repolarization is accomplished by multiple different potassium channels providing a strong safety reserve for repolarization. Thus, in normal cardiac tissue the pharmacological block or impairment of a single type of potassium channel does not necessarily lead to marked QT interval prolongation. However, in situations where the density of one or more types of potassium channels is decreased by congenital disorders or remodelling, inhibition of other potassium channels may lead to unexpectedly augmented action potential duration (APD) prolongation resulting in proarrhythmic reactions. In genetic channelopathies certain potassium channels, which normally contribute to repolarization, can attenuate the capability of the heart to repolarize (Biliczki et al. 2002; Jost et al. 2005). Transmembrane ion currents flow through channel complexes composed of α- and β-subunit proteins including ERG (encoded by KCNH2), minK (KCNE1), MiRP1–4 (KCNE25), KvLQT1 (KCNQ1), Kv4.3 (KCND3), Kv1.4 (KCNA4), KChIP2 (KCNIP2) and Kir2.1–2.4 (KCNJ2, KCNJ12, KCNJ4, KCNJ14). These proteins are abundantly expressed in mammalian hearts, but their relative contributions vary considerably among species (Varróet al. 2000; Zicha et al. 2003). Differential K+ current expression causes interspecies differences in the response to K+ channel blocking drugs, affecting predictive value for their effects in humans (Nerbonne & Kass, 2005). Despite the very common use of the dog in evaluating long-QT risk in man, there is little quantitative information available about the relative responses of human versus canine hearts to QT-prolonging interventions or regarding underlying differences in ionic currents. Here, we compared the contribution of three particularly important K+ currents, IKr, IK1 and IKs, to repolarization in dog and human hearts, studied the molecular basis of differences observed, and analysed their importance with a mathematical model. For methodological details, please see Supplemental Methods. Patients Hearts were obtained from organ donors whose non-diseased hearts were explanted to obtain pulmonary and aortic valves for transplant surgery. Before cardiac explantation, organ donors did not receive medication apart from dobutamine, furosemide, and plasma expanders. The investigations conformed to the principles of the Declaration of Helsinki. Experimental protocols were approved by the University of Szeged and National Scientific and Research Ethical Review Boards (Nos. 51-57/1997OEj and 4991-0/2010-1018EKU (339/PI/010)). After explantation, each heart was perfused with cardioplegic solution (for contents see Online Data Supplement) and kept cold (4–6°C) for 2–4 h prior to dissection. Animals All experiments complied with the Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23, revised 1985). The protocols were approved by the Review Board of the Department of Animal Health and Food Control of the Ministry of Agriculture and Rural Development, Hungary (XII./01031/000/2008 and XIII./1211/2012). Adult mongrel dogs of either sex weighing 8–16 kg were anaesthetized with pentobarbital (30 mg kg−1 i.v.). Hearts were removed through right lateral thoracotomies and rinsed in modified Locke's solution containing (mmol l−1): Na+ 140, K+ 4, Ca2+ 1.0, Mg2+ 1.0, Cl− 126, HCO3− 25 and glucose 11; pH 7.35–7.45, 95% O2-5% CO2, 37°C. Action potentials (APs) were recorded in right ventricular trabeculae and papillary muscle preparations (<2 mm diameter), from 15 non-diseased human donor hearts (9 male and 6 female, age = 44.6 and 25 dogs, with as in (Varróet al. 2000; et al. 2002; Jost et al. 2005). cardiomyocytes were from the ventricular of non-diseased human donor hearts male and female, age = and dog hearts with (Varróet al. 2000; et al. 2002; Jost et al. 2005). Experimental cardiomyocytes were in a the of and and to The and protocols Supplemental were as for K+ currents (Varróet al. 2000; et al. 2002; Jost et al. and for Ca2+ current and Na+–Ca2+ current et al. et al. 2005). quantitative ventricular were obtained from human male and female, age = and dog hearts, and in was isolated with the was with was a with and were to were for each Data was with the for differences. proteins were obtained from the used for were in and The was in containing After h were and at was by the and in for proteins were in with and with minK and or were with or to was with and analysed with All were relative to the for KvLQT1 and minK, for dog and human female, age = ventricular ventricular cardiomyocytes were with were with and for h with with containing at with the for h at was by h with Control were with were obtained with and were in with each three to were and were and from are expressed as was by and with as were for was recorded with from a potential of and IK1 was significantly larger in dog than human cardiomyocytes outward current density at was greater in dog versus human potassium current (IK1) in human and dog ventricular cardiomyocytes IK1 in a human and a dog ventricular IK1 IK1 density at and dog versus of IKr and IKs are in IKr are in and IKs in of IKr are in the and IKs in the IKr current at after did not significantly species In IKs current at after was about 4.5-fold larger in dog versus human and IKs in human and dog ventricular cardiomyocytes A and IKr from a human and a dog ventricular IKr current and IKs from a human and a dog ventricular IKs current of and the of IKr and IKs the cardiac action we compared the of the and currents These were obtained by right ventricular human and canine action potentials recorded with the IK1 current the AP was larger in dog than in human the IKr current was similar The IKs the action potential was very and not different the two species currents recorded with action potential obtained by normal human or canine ventricular action potentials with a in a papillary muscle and currents obtained by currents by action potential in the of the from current in the prior to the in human and dog ventricular of IKr and IKs currents in the at which current were are for each current and The and kinetics of IKr and IKs at the of and potentials are in The IKs kinetics of human and dog are similar and IKr at and relevant to current the resting of a rapid with a of not significantly different human and dog. more the kinetics more The were similar at for human and dog. to the was at more the was greater in dog. The of the and kinetics of human and canine IKr and IKs of IKr and IKs were by with from to to potentials from to the were to The of currents as a of the duration of the were well by single the of IKs kinetics was by IKs with to from a potential of the were for to potentials from to and the of the current was by a single the of IKr kinetics was by IKr with to from a potential of the were for to potentials from to and the of the current was by a The the of slow and of the results for of the is in the right The right the relative of the and slow at different in dog and human ventricular The contribution of IKr and IKs to repolarization was by blocking currents with (10 μmol nmol and (1 μmol respectively. that μmol l−1 of IK1 affecting IKr, IKs and Ito (Biliczki et al. In human ventricular inhibition of IK1 AP duration by prolongation in dog human, In inhibition of IKr greater prolongation in humans compared to the dog The response was to differences in effects and not as by similar species IKs block did not significantly in either studied of IK1 (10 IKr nmol l−1 dofetilide) or IKs (1 μmol l−1 block with techniques in canine and human right papillary and after with or for and effects are each action potential AP duration at 90% of repolarization (APD90) each of and studied the role of IK1 and IKs differences in to the larger by IKr block in human versus canine cardiomyocytes. were to in the or of μmol l−1 to IK1 or to block IKs The in to by the effect of the with repolarization reserve the in the of the effect with IK1 the contribution of IK1 to repolarization In human increased by in the of versus in the of The relative from prolongation with IK1 to prolongation with IK1 removed a in IKr blocking effect with IK1 For dog increased by 25 in the of versus in the of indicating a 56% in IKr blocking effect with IK1 This a larger contribution of IK1 to repolarization reserve in the dog versus man. For IKs increased by in the of IKs block in humans, versus in the of of to the of the IKs In the dog, by in the of versus in indicating a to of Thus, the larger IKs of canine to greater repolarization reserve versus humans. of and inhibition in human and dog ventricular muscle preparations at to μmol l−1 nmol l−1 and μmol l−1 nmol l−1 in human and dog ventricular muscle. show differences at to μmol l−1 nmol l−1 and μmol l−1 nmol l−1 in human and dog ventricular muscle. show differences the potential molecular basis for the observed differences in IK1 and IKs was for underlying IKr and expression for are in mRNA was more in the dog than the mRNA for and in the The IKr was expressed in canine and human expression was not significantly different human and dog but the the protein minK was more expressed in dog. of for KvLQT1 and minK proteins are in are in In with Kir2.1 was significantly in canine than human hearts, was in humans. ERG was as two larger molecular to and and two smaller to and was less in human were not significantly different from The very similar expression of in with and is with for a particularly important role of in IKr et al. and with a study of with heart et al. were in dog hearts, KvLQT1 was greater in of IKr and and mRNA levels of ERG and in ventricular human and dog and of for ERG and in human and dog ventricular isolated cardiomyocytes with for human versus canine are in showed significantly for canine and was but significantly greater for dog. In ERG was for the two species KvLQT1 was but significantly greater for human but in with the was much greater for dog versus Supplemental for for IKr and and in ventricular cardiomyocytes of human and dog cardiomyocytes. of human and dog ventricular cardiomyocytes are at the for various in human versus dog cardiomyocytes. are for ERG and KvLQT1 and minK of and for dog versus were for each for The and to repolarization reserve to smaller IKs and IK1 expression in human hearts as the basis for their larger response to IKr the potential role of other ionic current we compared other currents canine and human recorded as the and current from was smaller in human versus dog by from was ∼30% larger in human kinetics of Ito and ICa currents were not different in from human and dog ventricle. (10 current was not significantly different species and Ito density obtained with the and for human versus dog. for Ito are and as current and and as current and as density obtained with the for human dog. for ICa are and as current and and as current and as was to current and after of (10 to currents from dog and human are inward and outward current density the contribution of ionic current to repolarization reserve in human versus canine hearts, we the canine ventricular AP model & the current densities in the dog model to the observed differences in humans, to obtain Supplemental Supplemental the resulting APD90 at in the dog model was versus human to at dog human IKr block increased APD90 by in the human AP model versus in the dog model with experimental findings IKr inhibition increased human APD90 by in the of IK1 indicating a in IKr blocking effect with the IK1 contribution to repolarization reserve For the canine model IKr block increased APD90 by in the of IK1 indicating a in IKr blocking effect when IK1 is This is with experimental suggesting a larger contribution of IK1 to repolarization reserve in the dog. IKr block human APD90 by in the of IKs of to the of IKs contribution to repolarization For the dog AP model IKr block by in the of IKs indicating a to of the repolarization reserve effect of Thus, the model the importance of larger IKs to greater repolarization reserve in we used the model to the contributions of and Ito differences. Supplemental the by IKr inhibition in canine and human The effect of IKr inhibition in the human model was with or Ito modified to canine APD90 in the human model resulting from IKr inhibition were by canine Ito in the human model. canine the human model the IKr blocking effect canine to the larger repolarization reserve in the dog the These results that and Ito differences not contribute to the repolarization reserve in the dog. the contribution of ionic current to repolarization reserve in human versus canine hearts, we the in a with the more human ventricular AP model et al. see Supplemental the resulting APD90 at in the canine and human models were and experimental APD90 at dog human IKr block increased APD90 by in the human versus in the dog with experimental findings the human ionic model IKr block increased by in the of IK1 versus in the of IK1 These results a in IKr blocking effect with IK1 For the dog ionic model IKr block increased by in the of IK1 versus in the of IK1 indicating a in IKr blocking effect when IK1 was This the experimental indicating a larger contribution of IK1 to repolarization reserve in the dog compared to man. IKr block increased by in the of IKs block in the human model versus in the of IKs of to the of IKs contribution to repolarization In the dog ionic model IKr block by in the of IKs versus in indicating a to of the repolarization reserve effect of Thus, the model the importance of larger IKs to greater repolarization reserve in we used modelling to the contributions of and Ito and that contribute to the differences in IKr blocking effects human and dog of effect of and inhibition human and dog ventricular muscle by the canine ventricular AP model human at IK1 block IKr block and for dog human at IKs block IKr block and for dog In we that IKr inhibition causes greater prolongation in human versus canine ventricular indicating repolarization reserve in man. current measurements showed larger IK1 and IKs densities in canine versus human hearts and with larger repolarization reserve in canine hearts to IK1 and IKs suggested that the ionic current differences are to differences in mRNA expression of underlying compared experimental non-diseased human donor hearts and canine is a potential in relative the humans and dogs that tissue which were to other than by of the that both study and not and differences in ion channel subunit protein expression and current densities the of findings to the heart were to measurements in of canine and human hearts to and densities were from the ventricular but were recorded from right ventricular This was both for from ventricular tissue were to obtain and more to by and to from each human heart by available to the information obtained from each human because measurements were by the and of human donor tissue and because of the for after tissue results in ventricular were with AP from right ventricular indicating that at for two the are Our important expression differences in channel mRNA expression human and dog ventricle. Kir2.1 expression was about greater in the dog than human, but and levels were in In human hearts, we mRNA expression with that of the subunit underlying IK1 & 2005). protein expression in human was by Kir2.1 currents strong inward inward is incomplete and is less et al. In the of IK1 in dog that for Kir2.1 channels, but in human a of Kir2.1 and properties et al. to mRNA showed in human compared to dog tissue expression of was not A higher expression in humans the of different channel subunit in human tissue versus dog. This two to the kinetics of channels compared to channels of the relative contribution of IKr to the repolarization reserve is to higher in humans et al. & subunit affect of to IKr channels, as higher were obtained for channels as compared to channels et al. not in the of IKr in humans versus dogs and was not different Thus, relative expression may not relative subunit expression in ion little information is available about the molecular basis of repolarization among prolongation and to IKr blocking drugs with the and the and dogs et al. particularly and particularly IKs et al. This results from expression in the KvLQT1 expression in et al. 2003). expression we observed for human versus dog in the with dogs having much larger minK, but smaller expression than humans, with considerably larger IKs & observed larger IK1 and IKs, with similar IKr, for dog compared to the other to and Kv4.3 current densities and of the channels et al. 2003). other currents in to IKs, such as IKr and Ito by the lower minK expression in human ventricles we in prolongation in human versus dog in response to IKr similar IKr, is a that may important the that the properties of K+ channels in dog of humans (Varróet al. 2000; Jost et al. the reserve to repolarization-delaying drugs may the two the clinical potential of drugs with blocking properties experiments in dogs, and dogs in to However, there is greater of currents in human and dog, and heart human and dog than human and et al. 2001) and the similar relative of versus dog K+ currents in the study & to the human versus dog results in the the of the and model more QT prolongation by drugs is a major and are to in Our findings important for the of IK1 block or not necessarily lead to QT prolongation in humans, in the dog, but a of repolarization reserve (Roden, et al. 2002; & IK1 channel to ion channel or may not significantly human QT but QT prolongation and life-threatening torsades de pointes in the of repolarization The study to the of the molecular and ionic determinants of repolarization reserve in the human and the to determinants with of animal species used as a model for human cardiac Our results through a of channel that may by different including and et al. & et al. 2002; et al. The channels not & and were not studied in their contribution to IK1 important species and differences in relative expression of proteins et al. et al. 2002; & 2005). The densities of IK1 and of proteins in atria versus ventricles et al. 2002; & 2005). In the we ventricular tissue in but in human et al. and dog et al. proteins not channels, but can show et al. of Kir2.1 and proteins IK1 channels with lower than Kir2.1 et al. et al. 2005). the mRNA expression of Kir2.1 and in human was the dog, channels may more in the human than in the dog to the lower IK1 density that we observed in humans. to a role for channels in human IK1 et al. 2003). All of human were in cardioplegic solution to In in which we canine heart in cardioplegic solution and recorded ionic currents and we did not effects of cardioplegic with prior to heart explantation, a in cardiac effects of the properties of explanted The effects of pharmacological canine vary among different For the larger in canine ventricular with K+ channel & & than we observed in the The are to to differences in experimental conditions. For is important that species responses are a single than observed for one species in one with for species in a different The Na+–Ca2+ current current was and as This because that other ionic currents. However, for the of the we other ionic currents Na+ and Ca2+ currents, with to the experimental by et al. which is a relative used for current et al. The is and Na+ and K+ and and is not well in dog and human cardiomyocytes et al. we experimental regarding we a contribution of species in the of currents to repolarization reserve IKs is larger in dog than there was that the is to the that the normal very little at potentials for which there is a in IKs to The density of IKs in canine versus human heart to at in to expression of minK in the dog. However, there is a the a greater expression in the dog and the results greater expression in canine cardiomyocytes. In minK were for greater IKs in the dog, kinetics the species, which differences in minK may in the species differences in IKs, other are and in ventricular cardiomyocytes repolarization reserve compared to dog. The response similar IKr to lower IK1 and IKs densities in human The underlying molecular basis to expression of and minK human and canine These results that the by IK1 and IKs repolarization is in humans, humans susceptible to repolarization impairment from IKr blocking Animal models are used to study cardiac and pharmacological Our findings the importance of caution when extrapolating results from animal models to man, from species as similar in ionic current as and of the and of the and for All approved the of the This was by from the Scientific Research and the National for Research and the National and by the and the of the for Health Care Research Research and and the of The for with the Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental Supplemental 6 The is not for the or of information by the than to the for the