Diabetes mellitus attenuates the repolarization reserve in mammalian heart

Objective: In diabetes mellitus several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models changes in these parameters were reported, but no such data are available in other mammalian species including the dog. The present study was designed to analyse the effects of experimental type I diabetes on ventricular repolarization and its underlying transmembrane ionic currents and channel proteins in canine hearts. Methods and results: Diabetes was induced by a single injection of alloxan, a subgroup of dogs received insulin substitution. After the development of diabetes (8 weeks) electrophysiological studies were performed using conventional microelectrodes, whole cell voltage clamp, and ECG. Expression of ion channel proteins was evaluated by Western blotting. The QT(c) interval and the ventricular action potential duration in diabetic dogs Were moderately prolonged. This was accompanied by significant reduction in the density of the transient outward K+ current (I-to) and the slow delayed rectifier K+ current (I-Ks), to 54.6% and 69.3% of control, respectively. No differences were observed in the density of the inward rectifier K+ current (I-K1), rapid delayed rectifier K+ current (I-Kr), and L-type Ca2+ current (I-Ca). Western blot analysis revealed a reduced expression of Kv4.3 and MinK (to 25 +/- 21% and 48 +/- 15% of control, respectively) in diabetic dogs, while other channel proteins were unchanged (HERG, MiRP1, alpha(1c)) or increased (Kv1.4, KChIP2, KvLQT1). Insulin substitution fully prevented the diabetes-induced changes in I-Ks, KvLQT1 and MinK, however, the changes in I-to, Kv4.3, and Kv1.4 were only partially diminished by insulin. Conclusion: It is concluded that type I diabetes mellitus, although only moderately, lengthens ventricular repolarization, attenuates the repolarization reserve by decreasing I-to and I-Ks currents, and thereby may markedly enhance the risk of sudden cardiac death

Restricting excessive cardiac action potential and QT prolongation: a vital role for IKs in human ventricular muscle

Background - Although pharmacological block of the slow, delayed rectifier potassium current (I-Ks) by chromanol 293B, L-735,821, or HMR-1556 produces little effect on action potential duration (APD) in isolated rabbit and dog ventricular myocytes, the effect of IKs block on normal human ventricular muscle APD is not known. Therefore, studies were conducted to elucidate the role of IKs in normal human ventricular muscle and in preparations in which both repolarization reserve was attenuated and sympathetic activation was increased by exogenous dofetilide and adrenaline. Methods and Results - Preparations were obtained from undiseased organ donors. Action potentials were measured in ventricular trabeculae and papillary muscles using conventional microelectrode techniques; membrane currents were measured in ventricular myocytes using voltage-clamp techniques. Chromanol 293B (10 mu mol/L), L-735,821 (100 nmol/L), and HMR-1556 (100 nmol/L and 1 mu mol/L) produced a < 12-ms change in APD while pacing at cycle lengths ranging from 300 to 5000 ms, whereas the I-Kr blockers sotalol and E-4031 markedly lengthened APD. In voltage-clamp experiments, L- 735,821 and chromanol 293B each blocked IKs in the presence of E-4031 to block IKr. The E-4031-sensitive current (I-Kr) at the end of a 150-ms-long test pulse to 30 mV was 32.9 +/- 6.7 pA (n = 8); the L-735,821-sensitive current (I-Ks) magnitude was 17.8 +/- 2.94 pA (n = 10). During a longer 500-ms test pulse, IKr was not substantially changed (33.6 +/- 6.1 pA; n = 8), and I-Ks was significantly increased (49.6 +/- 7.24 pA; n = 10). On application of an "action potential-like" test pulse, I-Kr increased as voltage became more negative, whereas I-Ks remained small throughout all phases of the action potential - like test pulse. In experiments in which APD was first lengthened by 50 nmol/L dofetilide and sympathetic activation was increased by 1 mu mol/L adrenaline, 1 mu mol/L HMR-1556 significantly increased APD by 14.7 +/- 3.2% (P < 0.05; n = 3). Conclusions - Pharmacological IKs block in the absence of sympathetic stimulation plays little role in increasing normal human ventricular muscle APD. However, when human ventricular muscle repolarization reserve is attenuated, IKs plays an increasingly important role in limiting action potential prolongation

Antiaritmiás és proaritmiás mechanizmusok elemzése = Analysis of antiarrhythmic and proarrhythmic mechanism

Szívizomsejtben a K+-áramok antiaritmiás ''repolarizációs tartalékot'' tartanak fenn: Az IKr, IKs és IK1 akár egyikének gátlása súlyos proaritmiás repolarizáció megnyúlást okoz és letális kamrafibrillációhoz vezethet. - Alloxán-diabeteses kutyán az Ito és IKs csökkenése ugyanilyen hatású, amit a Kv4.3 és Mink csatorna fehérje expresszió egyidejű gátoltsága kisér. - Szívbillyentyű átültetés céljából használt, egészséges donor szivekből izolált humán kamrai miocitákon az IKs csökkenése szintén növeli a proaritmia és a hirtelen szívhalál rizikóját. - A K+-csatorna alegység expresszió mértékének sorrendje: KvLQT1 és ERG1-nél: ember>nyúl>tengerimalad, Mink-nél: tengerimalac>ember>nyúl. - Az INa+/Ca2+ csereáram gátlása kutyán antiaritmiás hatású, csökkenti az utódepolarizációt és a triggerelt aktivitást. - A leghatékonyabb új antiaritmikumok ''többszörös (tedisamil) ill. ''hibrid'' (azimilid, terikalant) ioncsatorna-gátlók. A pitvarszelektiv antiaritmiás szerek hatásmódja: az IKAch, IKur és IK1 izolált blokkolása. - A prekondicionálás antiaritmiás hatásában kutyán a NO trigger és mediátor, prosztaciklin- és szabadgyök-képződés viszont nem vesz részt ebben. | In the myocardial cell, there is a 'repolarization reserve' maintained by K+ currents: inhibition any of the IKr, IKs and IK1 results in severe proarrhythmic prolongation of repolarization and may cause lethal ventricular fibrillation. - The decrease of IKs and also Ito exhibits the same effect in alloxan-diabetic dogs which is accompanied by concomitant retardation of the expression of the K+ channel proteins Kv4.3 and Mink. - In undiseased human ventricular myocites obtained from donors for valve transplant surgery diminution of IKs also increases the risk of proarrhythmia and suddenc cardiac death. The extent of the order of expression of K+ channel subunits: human>rabbit>guinea pig with KvLQT1 and ERG1, and guinea pig>human>rabbit with Mink. - Inhibition of the INa+/Ca2+ exchange current is antiarrhythmic in dogs; it decreases the afterdepolarization and triggered activity. - The most efficacious new antiarrhythmics are ''multiple'' (tedisamil) or 'hybrid' (azimilid, terikalant) ion-channel blockers. - The mode of action of atrial-selective antiarrhythmics: isolated block of IKAch or IKur or IKs. - In the antiarrhythmic action of preconditioning in dogs nitrogen oxide acts as trigger and mediator, whereas formation of prostacyclin and free radicals are not involved in this effect

Diclofenac Prolongs Repolarization in Ventricular Muscle with Impaired Repolarization Reserve

Background: The aim of the present work was to characterize the electrophysiological effects of the non-steroidal anti- inflammatory drug diclofenac and to study the possible proarrhythmic potency of the drug in ventricular muscle. Methods: Ion currents were recorded using voltage clamp technique in canine single ventricular cells and action potentials were obtained from canine ventricular preparations using microelectrodes. The proarrhythmic potency of the drug was investigated in an anaesthetized rabbit proarrhythmia model. Results: Action potentials were slightly lengthened in ventricular muscle but were shortened in Purkinje fibers by diclofenac (20 mM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl 2 application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 mg/kg) significantly lengthened the QT c interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QT c . Diclofenac alone did not induce Torsades de Pointes ventricular tachycardia (TdP) while TdP incidence following dofetilide was 20%. However, the combination of diclofenac and dofetilide significantly increased TdP incidence (62%). In single ventricular cells diclofenac (30 mM) decreased the amplitude of rapid (I Kr ) and slow (I Ks ) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (I Ca ) was slightly diminished, but the transient outward (I to ) and inward rectifier (I K1 ) potassium currents were not influenced. Conclusions: Diclofenac at therapeutic concentrations and even at high dose does not prolong repolarization markedly and does not increase the risk of arrhythmia in normal heart. However, high dose diclofenac treatment may lengthen repolarization and enhance proarrhythmic risk in hearts with reduced repolarization reserve

A szívritmuszavarok és a myocardiális repolarizáció mechanizmusainak vizsgálata; antiaritmiás és proaritmiás gyógyszerhatások elemzése = Study of the mechanism of cardiac arrhythmias and repolarization, antiarrhythmic and proarrhythmic drug action

A kardiovaszkuláris betegségek és azon belül is az életet veszélyeztető kamrai és pitvari aritmiák a fő halálozási okok közé tartoznak a fejlett ipari országokban, de Magyarországon is. Ezzel összhangban, a jelen kutatási projekt is a különböző életet veszélyeztető aritmiák megelőzésének a lehetőségét, és a különböző gyógyszerek antiaritmiás és proaritmiás hatásainak a kutatását tűzte ki célul. Vizsgálataink során megállapítottuk, hogy kísérletes diabetes mellitusban mind kutyán, mind nyúlon az IKs áram és következményesen a repolarizációs rezerv csökkenése következik be amely emberben vélhetően hozzájárulhat e betegségben észlelt hirtelen szívhalál kockázat növekedéséhez. Molekuláris biológiai vizsgálatokban sikerült feltérképeznünk az emberi szívizom különféle ioncsatornáinak denzitását is. A proaritmiás gyógyszerhatások elemzésére újszerű módszert dolgoztunk ki, amelynek gyógyszerbiztonsági klinikai jelentősége és hasznosulása várható. A projekt teljesítése során további új ismereteket szereztünk a Na+/Ca2+ cseremechanizmus (NCX) repolarizációban betöltött szerepét illetően. In vivo kutya kísérletekben vizsgáltuk a peroxynitrit és gap junction csatornák szerepét az ischaemiás prekondicionálásban. Ezek az eredmények várhatóan hozzájárulnak egyrészt a szívizomzat élettani, kórélettani (aritmia mechanizmusok) ismereteinek a gyarapításában, másrészt új és biztonságos antiaritmiás terápiák kifejlesztéséhez. | Cardiovascular diseases, including life threatening ventricular and supraventricular arrhythmias, are the leading causes of mortality in industrialized countries and also in Hungary. In harmony with this, the major goal of the project was to investigate the mechanisms involved in cardiac repolarization and in antiarrhythmic and proarrhythmic drug actions. Representing important findings during the project, we established that in experimental diabetes mellitus the IKs potassium current is down-regulated resulting in the attenuation of repolarization reserve which may contribute to the increased proarrhythmic risk of diabetic patients. Using molecular biological methods we have analyzed the transmembrane ion channel densities of the human heart. To assess proarrhyhtmic drug side effects we developed a novel method which can be expected to contribute to better prediction of proarrhythmic risk in both preclinical and clinical safety pharmacology investigations. During the project, we have gained further insights regarding the role of NCX in the cardiac repolarization process. In in vivo studies we have investigated the possible role of peroxynitrite and gap junctions in ischaemic preconditioning. These results can be expected to help to better understand the physiology and pathophysiology of cardiac muscle, and arrhythmias, and should significantly contribute to the development of safer and more effective antiarrhythmic treatment modalities

Ionic mechanisms limiting cardiac repolarization-reserve in humans compared to dogs.

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

Cellular electrophysiological effect of terikalant in the dog heart

The cellular mechanism of action of terikalant, an investigational antiarrhythmic agent known to block the inward rectifier and other potassium currents, has not yet been fully clarified. The aim of the present study was therefore to analyse the in vitro electrophysiological effects of terikalant in canine isolated ventricular muscle and Purkinje fibers by applying the standard microelectrode technique. The effects of terikalant on the duration of action potential at a stimulation cycle length of 1000 ms and on the maximum upstroke velocity of the action potential in right ventricular papillary muscle were examined at 1, 2.5, 10, and 20 mu M concentrations. Terikalant significantly prolonged the action potential duration measured both at 50% and 90% of repolarization in concentration-dependent manner. The maximum upstroke velocity of the action potential was unaffected at 1 and 2.5 mu M concentrations. However, this parameter was significantly reduced at 10 and 20 mu M concentrations of terikalant. In Purkinje fibers terikalant (2.5 mu M) also produced a marked action potential lengthening effect. Frequency dependence (cycle length of 300-5000 ms) of the action potential lengthening effect of terikalant was studied at a concentration of 2.5 mu M. Prolongation of the duration of action potential occurred in a reverse frequency-dependent manner both in papillary muscle and Purkinje fibers, with a more pronounced frequency-dependence observed in Purkinje fibers. The onset kinetics of the terikalant (10 mu M) induced block of the maximum upstroke velocity of the action potential was rapid (0.6 +/- 0.1 beat(-1) n=6) like that of Class I/B antiarrhythmics, and the offset (recovery) kinetics of the drug (2956 696 ms, n=6) best resembled that of Class I/A antiarrhythmic drugs. It was concluded that terikalant, unlike pure Class III antiarrhythmic drugs, has combined mode of action by lengthening repolarization and blocking the inward sodium current in a use-dependent manner

Theoretical Possibilities for the Development of Novel Antiarrhythmic Drugs

One possible mechanism of action of the available K-channel blocking agents used to treat arrhythmias is to selectively inhibit the HERG + MIRP channels, which carry the rapid delayed rectifier outward potassium current (I-Kr). These antiarrhythmics, like sotalol, dofetilide and ibutilide, have been classified as Class III antiarrhythmics. However, in addition to their beneficial effect, they substantially lengthen ventricular repolarization in a reverse-rate dependent manner. This latter effect, in certain situations, can result in life-threatening polymorphic ventricular tachycardia (torsades de pointes). Selective blockers (chromanol 29313, HMR-1556, L-735,821) of the KvLQT1 + minK channel, which carriy the slow delayed rectifier potassium current GO, were also considered to treat arrhythmias, including atrial fibrillation (AF). However, I-Ks activates slowly and at a more positive voltage than the plateau of the action potential, therefore it remains uncertain how inhibition of this current would result in a therapeutically meaningful repolarization lengthening. The transient outward potassium current (I-to), which flows through the Kv 4.3 and Kv 4.2 channels, is relatively large in the atrial cells, which suggests that inhibition of this current may cause substantial prolongation of repolarization predominantly in the atria. Although it was reported that some antiarrhythmic drugs (quinidine, disopyramide, flecainide, propafenone, tedisamil) inhibit I-to, no specific blockers for I-to are currently available. Similarly, no specific inhibitors for the Kir 2.1, 2.2, 2.3 channels, which carry the inward rectifier potassium current (I-kl), have been developed making difficult to judge the possible beneficial effects of such drugs in both ventricular arrhythmias and AF. Recently, a specific potassium channel (Kv 1.5 channel) has been described in human atrium, which carries the ultrarapid, delayed rectifier potassium current (I-Kur). The presence of this current has not been observed in the ventricular muscle, which raises the possibility that by specific inhibition of this channel, atrial repolarization can be lengthened without similar effect in the ventricle. Therefore, AF could be terminated and torsades de pointes arrhythmia avoided. Several compounds were reported to inhibit I-Kur (flecainide, tedisamil, perhexiline, quinidine, ambasilide, AVE 0 118), but none of them can be considered as specific for Kv 1.5 channels. Similarly to Kv 1.5 channels, acetylcholine activated potassium channels carry repolarizing current (I-KAch) in the atria and not in the ventricle during normal vagal tone and after parasympathetic activation. Specific blockers Of I-KAch can, therefore, also be a possible candidate to treat AF without imposing proarrhythmic risk on the ventricle. At present several compounds (amiodarone, dronedarone, aprindine, pirmenol, SD 3212) were shown to inhibit I-KAch, but none of them proved to be selective. Further research is needed to develop specific K-channel blockers, such as I-Kur and I-KAch inhibitors, and to establish their possible therapeutic value