Péter Biliczki

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

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 (IKs) 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 &mgr;mol/L), L-735,821 (100 nmol/L), and HMR-1556 (100 nmol/L and 1 &mgr;mol/L) produced a <12-ms change in APD while pacing at cycle lengths ranging from 300 to 5000 ms, whereas the IKr 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 (IKr) 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 (IKs) 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 IKs was significantly increased (49.6±7.24 pA; n=10). On application of an “action potential–like” test pulse, IKr increased as voltage became more negative, whereas IKs 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 &mgr;mol/L adrenaline, 1 &mgr;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.

Interaction of different potassium channels in cardiac repolarization in dog ventricular preparations: role of repolarization reserve.

The aim of this study was to investigate the possible role of the interaction of different potassium channels in dog ventricular muscle, by applying the conventional microelectrode and whole cell patch‐clamp techniques at 37°C. Complete block of IKr by 1 μM dofetilide lengthened action potential duration (APD) by 45.6±3.6% at 0.2 Hz (n=13). Chromanol 293B applied alone at 10 μM (a concentration which selectively blocks IKs) did not markedly lengthen APD (<7%), but when repolarization had already been prolonged by complete IKr block with 1 μM dofetilide, inhibition of IKs with 10 μM chromanol 293B substantially delayed repolarization by 38.5±8.2% at 0.2 Hz (n=6). BaCl2, at a concentration of 10 μM which blocks IKl without affecting other currents, lengthened APD by 33.0±3.1% (n=11), but when IKr was blocked with 1 μM dofetilide, 10 μM BaCl2 produced a more excessive rate dependent lengthening in APD, frequently (in three out of seven preparations) initiating early afterdepolarizations. These findings indicate that if only one type of potassium channels is inhibited in dog ventricular muscle, excessive APD lengthening is not likely to occur. Dog ventricular myocytes seem to repolarize with a strong safety margin (‘repolarization reserve’). However, when this normal ‘repolarization reserve’ is attenuated, otherwise minimal or moderate potassium current inhibition can result in excessive and potentially proarrhythmic prolongation of the ventricular APD. Therefore, application of drugs which are able to block more than one type of potassium channel is probably more hazardous than the use of a specific inhibitor of one given sort of potassium channel, and when simultaneous blockade of several kinds of potassium channel may be presumed, a detailed study is needed to define the determinants of ‘repolarization reserve’.

Selective inhibition of sodium-calcium exchanger by SEA-0400 decreases early and delayed afterdepolarization in canine heart

The sodium–calcium exchanger (NCX) was considered to play an important role in arrhythmogenesis under certain conditions such as heart failure or calcium overload. In the present study, the effect of SEA‐0400, a selective inhibitor of the NCX, was investigated on early and delayed afterdepolarizations in canine ventricular papillary muscles and Purkinje fibres by applying conventional microelectrode techniques at 37°C. The amplitude of both early and delayed afterdepolarizations was markedly decreased by 1 μM SEA‐0400 from 26.6±2.5 to 14.8±1.8 mV (n=9, P<0.05) and from 12.5±1.7 to 5.9±1.4 mV (n=3, P<0.05), respectively. In enzymatically isolated canine ventricular myocytes, SEA‐0400 did not change significantly the L‐type calcium current and the intracellular calcium transient, studied using the whole‐cell configuration of the patch‐clamp technique and Fura‐2 ratiometric fluorometry. It is concluded that, through the reduction of calcium overload, specific inhibition of the NCX current by SEA‐0400 may abolish triggered arrhythmias.

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 plus 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 293B, HMR-1556, L-735,821) of the KvLQT1 plus minK channel, which carriy the slow delayed rectifier potassium current (I(Ks)), 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 0118), 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.

ORM‐10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart

At present there are no small molecule inhibitors that show strong selectivity for the Na+/Ca2+ exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM‐10103, a new NCX inhibitor, on the NCX and L‐type Ca2+ currents and on the formation of early and delayed afterdepolarizations.

Effect of a neuroprotective drug, eliprodil on cardiac repolarisation: importance of the decreased repolarisation reserve in the development of proarrhythmic risk

The aim of this study was to analyse the effects of eliprodil, a noncardiac drug with neuroprotective properties, on the cardiac repolarisation under in vitro circumstances, under normal conditions and after the attenuation of the ‘repolarisation reserve’ by blocking the inward rectifier potassium current (IK1) current with BaCl2. In canine right ventricular papillary muscle by applying the conventional microelectrode technique, under normal conditions, eliprodil (1 μM) produced a moderate reverse rate‐dependent prolongation of the action potential duration (7.4±1.5, 8.9±2.1 and 9.9±1.8% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=9). This effect was augmented in preparations where IK1 was previously blocked by BaCl2 (10 μM). BaCl2 alone lengthened APD in a reverse frequency‐dependent manner (7.0±1.3, 14.2±1.6 and 28.1±2.1% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). When eliprodil (1 μM) was administered to these preparations, the drug induced a marked further lengthening relative to the APD values measured after the administration of BaCl2 (12.5±1.0, 17.6±1.5 and 20.5±0.9% at cycle lengths of 300, 1000 and 5000 ms, respectively; n=8). In the normal Langendorff‐perfused rabbit heart, eliprodil (1 μM) produced a significant QTc prolongation at 1 Hz stimulation frequency (12.7±1.8%, n=9). After the attenuation of the ‘repolarisation reserve’ by the IK1 blocker BaCl2 (10 μM), the eliprodil‐evoked QTc prolongation was greatly enhanced (28.5±7.9%, n=6). In two out of six Langendorff preparations, this QTc lengthening degenerated into torsade de pointes ventricular tachycardia. Eliprodil significantly decreased the amplitude of rapid component of the delayed rectifier potassium current (IKr), but slow component (IKs), transient outward current (Ito) and IK1 were not considerably affected by the drug when measured in dog ventricular myocytes by applying the whole‐cell configuration of the patch‐clamp technique. The results indicate that eliprodil, under normal conditions, moderately lengthens cardiac repolarisation by inhibition of IKr. However, after the attenuation of the normal ‘repolarisation reserve’, this drug can induce marked QT interval prolongation, which may result in proarrhythmic action.

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 µM). The maximum upstroke velocity was decreased in both preparations. Larger repolarization prolongation was observed when repolarization reserve was impaired by previous BaCl2 application. Diclofenac (3 mg/kg) did not prolong while dofetilide (25 µg/kg) significantly lengthened the QTc interval in anaesthetized rabbits. The addition of diclofenac following reduction of repolarization reserve by dofetilide further prolonged QTc. 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 µM) decreased the amplitude of rapid (IKr) and slow (IKs) delayed rectifier currents thereby attenuating repolarization reserve. L-type calcium current (ICa) was slightly diminished, but the transient outward (Ito) and inward rectifier (IK1) 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.