/Ca2+ exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca 2+ i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca 2+ i rise in conditions when [Ca 2+ ]i was augmented via activation of the late sodium current (INaL) or inhibition of the Na EXPERIMENTAL APPROACHAction potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca KEY RESULTSEnhanced INaL increased the Ca 2+ load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca 2+ ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca 2+ release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion. CONCLUSIONS AND IMPLICATIONSSelective NCX inhibition -presumably by blocking revINCX (reverse mode NCX current) -is effective against arrhythmogenesis caused by [Na]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca 2+ i handling, should be considered as a promising anti-arrhythmic therapeutic strategy.

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a b s t r a c tIn this study we evaluated the effects of selective Na þ /Ca 2 þ exchanger (NCX) inhibition by ORM-10103 on the [Ca 2 þ ] i transient (CaT), action potential (AP), and cell viability in isolated canine ventricular cardiomyocytes exposed to a simulated ischemia/reperfusion protocol performed either alone (modeling moderate low-flow ischemia) or with simultaneous strophantidine challenge (modeling more severe low-flow ischemia). CaTs were monitored using a Ca 2 þ -sensitive fluorescent dye, APs were recorded by intracellular microelectrodes, and anaerobic shifts in cellular metabolism were verified via monitoring native NADH fluorescence. Simulated ischemia increased the NADH fluorescence, reduced the amplitudes of the AP and CaT and induced membrane depolarization. APs moderately shortened, CaTs prolonged. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusioninduced increase in AP and CaT variabilities were substantially reduced, but ischemia-induced shifts in AP morphology were barely influenced. In conclusion, selective NCX inhibition by ORM-10103 is highly effective against ischemia/reperfusion induced pathologic alterations in [Ca 2 þ ] i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology.

Na/Ca exchanger (NCX) is the main Ca transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca ([Ca]). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca]. L-type Ca current (I) was not affected by 1µM ORM-10962 in the absence of SR Ca release, while I was suppressed by ORM-10962 during normal Ca cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca], suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca handling and/or the preserved inducibility of forward NCX by high [Ca]. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

Gyakran feltételezték, hogy a szív aritmogenezisében a Na + /Ca 2+ -kicserélő (NCX) fokozott aktivitásának kritikus szerepe van, de a vizsgálati eredmények ellentmondásosak voltak. Ennek logikus oka lehetett a korai NCX-gátlószerek nem kielé-gítő szelektivitása. Első, izolált kutya szívizomsejteken végzett kísérleteinkben a viszonylag szelektív SEA0400 és a jóval szelektívebb ORM-10103 alkalmazásával elemeztük a gátlás hatékonyságát az intracelluláris Ca 2+ (Ca 2+ i) háztartásban és az akciós potenciál (AP) morfológiájában kialakuló aritmogén eltolódásokkal szemben. A (Ca 2+ )i emelkedést önmagában alkalmazott (mérsékelt low fl ow) vagy strophantidin terheléssel kombinált (súlyos low fl ow) szimulált iszkémia/reperfúziós (I/R) protokollokkal hoztuk létre. Egy másik, perfundált izolált patkányszíveken végzett kísérletben regionális I/R-protokollal kiváltott extraszisztolés (ES), kamrai tachycardiás (VT) és kamrafi brillációs (VF) periódusok aritmia-diagrammokból meghatározott időtartamának és gyakoriságának összehasonlításával elemeztük az NCX, NHE és kombinált NCX+NHE-gátlás hatékonyságát, reperfúzió-indukált aritmiákkal szemben. Eredményeink szerint a szelektív, részleges NCX-gátlás -vélhető-en a revINCX gátlásával -hatékonyan gátolja a Ca 2+ i háztartás zavarai által indukált aritmogén utódepolarizációkat, illetve triggerelt aktivitást, ugyanakkor nem képes normalizálni az AP morfológiában kialakuló aritmogén eltolódásokat. Perfundált szívekben az NHE-gátlás jóval hatékonyabban gátolta a reperfúzió-indukált szubsztrát-dependens aritmiákat, ugyanakkor a kombinált gátlás nem növelte az önmagában alkalmazott NCX-gátlás antiaritmiás hatékonyságát.Is selective Na + /Ca 2+ exchanger inhibition effective against ischemia/reperfusion induced ventricular arrhyth mias? A crucial role for augmented Na + /Ca 2+ exchanger (NCX) activity in cardiac arrhythmogenesis was often suggested, however, the results of the related studies were controversial. A feasible explanation of this controversion could be the unsatisfactory selectivity of the early NCX inhibitors. In our fi rst study performed on isolated canine cardiomyocytes we evaluated the effi cacy of NCX inhibition using the relatively selective SEA0400, or a new, highly selective ORM-10103 against arrhythmogenic shifts in intracellular Ca 2+ (Ca 2+ i) handling and action potential (AP) morphology, when (Ca 2+ )i was augmented via a simulated ischemia/reperfusion (I/R) protocol performed either alone (moderate low-fl ow protocol) or with simultaneous strophantidine challenge (severe low-fl ow protocol). In the second study Langendorff perfused rat hearts were exposed to regional I/R protocol and effi cacies of NCX, NHE and combined NCX+NHE inhibition against reperfusion induced arrhythmias were compared by analyzing arrhythmia diagrams for incidence and duration of periods of repetitive extrasystoles (ES) and ventricular tachycardia (VT) or fi brillation (VF). Our results confi rm that selective, partial NCX inhibition -presumably by blocking fwdINCX -is highly effective ...

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