Role of action potential configuration and the contribution of Ca2+ and K+ currents to isoprenaline-induced changes in canine ventricular cells

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9-anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current during canine ventricular action potential

Abstract Background Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (ICl(Ca)). This study aims to explore which blocker is preferable to study ICl(Ca). Methods Whole-cell voltage-clamp technique was used to record ICa,L, IKs, IKr and IK1, while action potentials were measured using sharp microelectrodes. Results DIDS- (0.2 mM) and 9-AC- (0.5 mM) sensitive currents were identical in voltage-clamp conditions, regardless of intracellular Ca2+ buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization (Vmax) but DIDS caused marked reduction of Vmax. Both inhibitors reduced the magnitude of phase-1 but at slow stimulation rates this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1- 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 1 mM 9-AC had no additional actions upon perfusion after 0.5 mM. The half-effective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of ICa,L, IKs, IKr and IK1 were not changed by 0.5 mM 9-AC. Conclusions These results suggest that DIDS is equally useful to study ICl(Ca) during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of ICl(Ca) due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (ICa,L, IKr, IKs, IK1), however, ICa,L tracings can be contaminated with ICl(Ca) when measured in voltage-clamp condition

Contribution of ion currents to beat-to-beat variability of action potential duration in canine ventricular myocytes

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca2+ concentration ([Ca2+]i) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca2+]i. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD (I Ca, I Ks, and I Kr), while it is increased by I Na and I to. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action

Effects of adrenergic activation to the action potentials and ionic currents of cardiac cells

Összefoglalás Bevezetés Az izoproterenolnak (ISO) a kamrai szívizomsejtek ionáramaira, az akciós potenciálra (AP) gyakorolt hatásai nem kellően tisztázottak. Ezért vizsgáltuk az ISO módosító hatását az akciós potenciál konfigurációjára, az L-típusú kalciumáramra (ICa,L), valamint a késői egyenirányító áram lassú (IKs) és gyors (IKr) komponensére. Kitértünk a változások kinetikájának és frekvenciafüggésének vizsgálatára is. Anyagok és módszerek Az ICa, IKs, és IKr mérésekhez hagyományos feszültség-clamp technika egészsejtes konfigurációját, valamint akciós potenciál feszültség-clamp méréseket végeztünk. Az AP méréseket nagy ellenállású mikroelektródákkal végeztük, izolált kutya kamrai sejteken. Főbb eredmények Az ISO epikardiális és midmiokardiális sejteken az akciós potenciálok reverzibilis rövidülését okozta a plató emelésével együtt. Ezek a változások az IKs és ICa amplitúdóinak többszörös növekedésével jártak, az IKr mérsékelt fokú stimulálása mellett. Endokardiális sejteken az ISO nem okozott AP-rövidülést. Az IKr előzetes gátlása nem módosította az ISO hatását, de IKs blokkolásakor az akciós potenciál ISO hatására megnyúlt. A kalciumcsatornák előzetes gátlása mind az akciós potenciál rövidülését, mind a plató emelését megakadályozta. Megfigyeltük, hogy az ingerlési frekvencia csökkentésével az ISO nagyobb amplitúdóváltozást okozott az ICa és az IKs esetén. Az ISO okozta platónövekedés és az ICa növekedése hamarabb következett be, mint az akciós potenciál rövidülése, az IKr, vagy az IKs stimulálása. β blokkolókkal végzett kísérleteink során megállapítottuk, hogy a β1 receptorok gátlása az ISO okozta rövidülést nyújtássá változtatta, a platóváltozás latenciája pedig csökkent. Ezzel ellentétben, a β2 receptorok gátlása fokozta az AP rövidülését, és növelte a plató változásának latenciáját. Mindkét receptortípus egyidejű blokkolása meggátolta az ISO hatásának kifejlődését. A foszfodiészterázok gátlása csökkentette az időbeli különbséget a platópotenciál és az AP rövidülés megjelenése között. Összefoglalás Az ISO hatásai kutya kamrai sejteken nagyban függenek az akciós potenciál konfigurációjától, és az ISO-indukálta IKs és nem az IKr lehet a felelős az akciós potenciálok rövidüléséért. Az ISO az IKr, illetve az IKs változtatásánál gyorsabban, rövidebb latenciával növelte a kalciumáramot, a jelenségért a különböző adrenerg útvonalak, illetve a kompartmentalizáció lehet felelős. Summary Background and purpose Little is known about the role of action potential morphology in the isoproterenol (ISO) induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca2+ current (ICa), slow component (IKs) and fast component (IKr) of the delayed rectifier K+ current were studied and compared in a frequency-dependent manner, along with the timing of activation of each current. Experimental approach Whole cell configuration of the patch-clamp technique in either conventional voltage clamp or action potential voltage clamp modes were used to monitor ICa, IKs, and IKr, while action potentials were recorded using sharp microelectrodes. Results In epicardial and midmyocardial cells ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. These effects were associated with multifold enhancement of ICa and IKs and moderate stimulation of IKr. ISO-induced action potential shortening was absent in endocardial cells. IKr blockade failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of IKs blocker. Both action potential shortening and elevation of the plateau were prevented by pretreatment with ICa blocker. Both ICa and IKs currents increased with increasing the cycle length. The ISO-induced plateau shift and ICa increase developed earlier than the shortening of APD and stimulation of IKs and IKr. Blockade of β1 adrenoceptors converted the ISO-induced shortening of APD to lengthening, decreased the latency of the plateau shift. In contrast, blockade of β2 receptors augmented the APD-shortening effect and increased the latency of plateau shift. All effects of ISO were prevented by simultaneous blockade of both receptor types. Inhibition of phosphodiesterases decreased the differences observed in the turn on of the ISO induced plateau shift and APD shortening. Conclusion The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of IKs – but not IKr – may be responsible for the shortening of action potentials. ISO-induced activation of ICa is turned on faster than the stimulation of IKs and IKr in canine ventricular cells due to the involvement of different adrenergic pathways and compartmentalization.N