Handbook of Experimental Pharmacology - Part 6 pdf

176 K.R. Sipido et al. will affect thecontributionof the Na/Ca exchangerto electrical activity and po- tential arrhythmogenesis. The most immediately relevant changes are altered expression of the Na/Ca exchanger and modulation of exchanger activity due to altered [Ca 2+ ] i and [Na + ] i . As recently reviewed, such changes are variable with the degree of hypertrophy, stage of decompensation and the stimulus leading to remodelling (Schillinger et al. 2003; Sipido et al. 2002; Verdonck et al. 2003b). This cautions against generalizing scenarios on the Na/Ca ex- changer in hypertrophy and heart failure. Nevertheless, from data on some of the best-studied examples of remodelling, some important aspects can be highlig hted. 4.2.1 Increased Ca 2+ Removal via NCX in End-Stage Heart Failure Inmyocytesfromhumanheartsobtained atthetimeoftransplantation, [Ca 2+ ] i transients are prolonged and Ca 2+ uptake into the SR is reduc ed (Beuckelmann etal. 1992; Hasenfuss andPieske2002; Piacentino,III etal. 2003). Thispromotes removal of Ca 2+ via the Na/Ca exchanger. Although the amount of charge extruded by the exchanger in steady state is determined by the amount of Ca 2+ en try, not by the expression/activity levels of NCX (Bridge et al. 1990; Negretti et al. 1995), the latter will determine the kinetics of the current and time course of Ca 2+ removal. Therefore, an increased inward current at the end oftheactionpotentialcanbepartoftheprolongationoftheactionpotential typically seen in human heart failure. Increased Ca 2+ removal through NCX has also been observed in some well-characterized animal models of heart failure. In the dog with tachycardia-induced heart failure, the increased Ca 2+ removal is the result of reduced SR Ca 2+ uptake and increased activity and expression of the exchanger (Hobai and O’Rourke 2000; O’Rourke et al. 1999). M odelling of the electrical activity in this dog model, however, did not clearly indicate that this will contribute to action potential prolongation (Winslow et al. 1999). The rabbit with heart failure due to combined pressure and volume overload also has increased Ca 2+ removal by the Na/Ca exchanger (Pogwizd et al. 1999). Enhanced expression and function result in larger inward and outward exchanger currents, and this is also seen in theoretical models of the NCX current during the action potential as illustrated in Fig. 7 (Pogwizd et al. 2003). The net effect on the action potential profile is therefore at present uncertain. Wha t is clear, however, is that in this model the increased inward current is particularly important in the enhanced propensity for arrhythmias triggered by DADs (Pogwizd et al. 2001). For any given release of Ca 2+ from the SR, the NCX current is larger in myocytes from the failing hearts. In combinationwith areduceddensity ofthe repolarizinginwardrectifiercurrent, the probability that spontaneous Ca 2+ release will effectively elicit an action potential is greatly enhanced. Sodium Calcium Exchange as a Target for Antiarrhythmic Therapy 177 Fig. 7a–c Alterations in the amplitude and time course of the Na/Ca exchange current shown in panel c, in a rabbit model o f heart failure (HF) with increased intracellular Na + ,as calculated from the measured changes in action potential (a), global Ca 2+ transient (b)and calculated Ca 2+ near the membrane (b, inset). Whereas the higher NCX expression simply increases the amplitude of the current (if Na + remains at 8.5 mM), the increase in Na + results in a large increase in the outward current component. (After Pogwizd et al. 2003) 4.2.2 Increased Ca 2+ Influx via NCX in Heart Failure and Hypertrophy The same rabbit model just mentioned was also studied extensively by Fiolet and co-workers. They found that [Na + ] i is increased though increased activity of the Na/H exchanger (Baartscheer et al. 2003b,c). The expected higher Ca 2+ influxviathe exchangercouldexplain thehigher diastolic Ca 2+ values that were observed andthe larger fractionalCa 2+ release,despitealowerSRCa 2+ conten t. Similar to the Pogwizd et al. studies, Baartscheer et al. reported a higher incidence of DADs, in particular under adrenergic stimulation. Inhibition of the Na/H exchanger reversed these changes, indicating that the primary event is the increase in Na + influx (Baartscheer et al. 2003c). [Na + ] i is also increased in human heart failure (Pieske et al. 2002) and increased Ca 2+ influx via the exchanger could contribute to the Ca 2+ available for contraction (Dipla et al. 1999; Piacentino et al. 2003; Weber et al. 2002). 178 K.R. Sipido et al. In heart failure in humans and the animal models mentioned abov e, the amplitude of the [Ca 2+ ] i transient is actually reduced at normal heart rates, as is the Ca 2+ loading of the SR. This is not necessarily so in compensated hy- pertrophy (Shorofsky et al. 1999). This is exemplified by the dog with chronic atrioventricular block (AVB). Bradycardia and volume overload lead to biven- tricular hypertrophy with preserved and even enhanced contractile function, at least in the first 6 weeks (Vos et al. 1998). At the cellular level, NCX activity is increased and [Na + ] i is higher (Sipido et al. 2000; Verdonck et al. 2003a). Be- cause there is no apparent reduction in SER CA function, these changes result in the SR Ca 2+ co ntent being larger, and spontaneous Ca 2+ release is more likely to occur, accompanied by large inward exchanger currents (Sipido et al. 2000). In the context of a balance of Ca 2+ fluxes during the cardiac cycle, any additional Ca 2+ influx during the action potential must r esult in increased exchanger efflux at a later time during the cycle. So even if functional empha- sis is placed on increased Ca 2+ influx due to higher [Na + ] i ,thiswillalways be accompanied by enhanced efflux and inward exchanger currents that can contribute to arrhythmogenesis. 4.2.3 Incidence of Afterdepolariza tions in Cardiac Hypertrophy and Failure The occurrence of DADs has been documentedin the above-mentioned animal models and in human heart failure. In compensated hypertrophy, this follows rather straightforwardly from an enhanced Ca 2+ loading of the myocytes. In vivo, DADs can be elicited by pacing protocols that enhance contractility, further exacerbated by increased Na + loading under ouabain (de Groot et al. 2000; Fig. 8). In the case of heart failure with reduced SR Ca 2+ loading, DADS and triggered action potentials still occur because of a lowered threshold with reduced inward rectifierents (Pogwizd et al. 2001; Pogw izd 2003), and/or because diastolicCa 2+ is elevated(Baartscheer et al.2003a). Another important element is adrenergicstimulation,which mayinduceDADswith highincidence in myocytes from the failing heart, including human (Baartscheer et al. 2003a; Pogwizd et al. 2001; Verkerk et al. 2001). In addition, Purkinje fibres may be more sensitive to the development of DADs (Boyden et al. 2000), and a hig h incidence has been reported in the dog after myocardial infarction (Boutjdir et al. 1990). As mentioned abo ve, the role of the NCX current in EADs is in providing inward current during the priming phase, with a more prominent role for the exchanger as depolarizing current during adrenergic stimulation. There are currently few experimental data that have directly linked exchanger current to EADs in hypertrophy and failure. We ha ve proposed such a role in the dog with chronic AVB (Sipido et al. 2000; Volders et al. 1998), and it could be hypothesized for the EADs observed in human failing myocytes under adrenergic stimulation (Veldkamp et al. 2001). Sodium Calcium Exchange as a Target for Antiarrhythmic Therapy 179 Fig. 8a,b Arrhythmias related to DADs in the dog with chronic AV block. a The dog with chronic AV block has increased loading of the SR (Sipido et al. 2000) and a train of 8 stim uli (S) results in an increase in DAD slope (arrow)andoneDAD-relatedtriggeredbeat(*) reco rded here in vivo with a MAP catheter (de Groot et al. 2000). b In these dogs, [Na + ] i in the isolated myocytes is enhanced (Verdonck et al. 2003a) and in vivo the heart is more sensitive to ouabain. In the presenc eof ouabain, +LV dP/dt is higher, and pacing now results in ventricular tachycardia (12 beats). In MAP, DADs are visible during VT and directly after tachycardia terminates. (Reproduced from de Groot et al. 2000) 180 K.R. Sipido et al. Alastelementisheterogeneity in exchangercurrentdensity between regions inthe heart,whichis already present at baseline (Zygmuntet al. 2000) and could be enhanced during remodelling (Sipido et al. 2000;Yoshiyama et al. 1997) thus con tributing to dispersion of repolariza tion. 4.2.4 In Vivo Evidence for Na/Ca Exchange-Mediated Arrhythmias in Heart Failure Suddendeathandpotentiallylethalarrhythmiashavebeendocumentedduring the many recent ICD studies in heart failure. Ventricular tachycardia is often initiated by ecto pic activity, but these types of ECG recordings do not allow us to distinguish between (micro) re-entry or ab n ormal focal activity underlying the extrasystoles. Yet there is evidence that triggered activity is an important mechanism underlying arrhythmias in patients with heart failure (Paulus et al. 1992; Pogwizd et al. 1992, 1995; and see review, Janse 2004). Though there is no direct evidence for the Na/Ca exchanger, in view of the discussions above, the role of the Na/Ca exchanger in afterdepolarizations is clear. 4.3 Na/Ca Exchange and Congenital Arrhythmias Currently there are no known direct associations between mutations of the Na/Ca exchanger and arrhy thmic disease. There are, however, indirect links to other congenital syndromes leading to PVT and associated with EADs and DADs. In congenital LQTS type 3, a late depolarizing Na + curren t contributes to action potential prolongation and provides the conditioning phase for EADs (Bennett et al. 1995; Clancy and Rudy 1999; Nuyens et al. 2001). In the D1790G mutation LQTS3, theoretical modelling indicates an important role for in- creased inward NCX current consequent on an increase in intracellular Ca 2+ (Wehrens et al. 2000). In LQTS4, a mutation in ankyrin results in defective targeting of the Na/Ca exchanger to the T-tubular membrane and abnormal Ca 2+ cycling (Mohler et al. 2003). In LQTS1, arrhythmias are occurring prefer- ably under adrenergic stimulation and the Na/Ca exchanger could thus be indirectly involved. Adrenergic stimulation is also the trigger for often-lethal arrhythmias in the catecholaminergic polymorphic ventricular tachycardia (CPVT) syndrome that is associated with mutations in Ca 2+ -handling proteins such as the ryanodine receptor (Laitinen et al. 2001; Priori et al. 2001) and calsequestrin (Lahat et al. 2001). Sodium Calcium Exchange as a Target for Antiarrhythmic Therapy 181 5 What Are the Expected Consequences of Na/Ca Exchange Block on Ca Handling? 5.1 General Considerations AsimpleanalysissuggeststhatinhibitorsofNCXwillhavetwoclassesofeffects on the Ca-dependent arrhythmias discussed above. There will be effects due to (1) changes of the degree of Ca 2+ (over)load of the cell and (2) changes of the membrane current and resulting arrhythmias produced by a given degree of Ca 2+ overload. We will now consider these two effects. Under normal conditions the NCX produces a net Ca 2+ efflux. Therefore, partial inhibition of the exchanger produced either by stopping a fraction of the NCX completely or stopping all of the NCX partially would be expected to increase systolic [Ca 2+ ] i un til the increased [Ca 2+ ] i compensates for the decrease of NCX sites and results in the same time-averaged Ca 2+ efflux as in control. This effect by itself will be positively inotropic. However, the tendency toloadthecellwithcalciummayalsoresultinastateofCa 2+ overload and thence arrhythmias. On the other hand, the fact that NCX has been inhibited means that each Ca 2+ wave will res ult in less Ca 2+ efflux from the cell and thus in a smaller arrhythmogenic inward current. This would suggest that a given degree of inotropy produced by NCX inhibition will be accompanied by less arrhythmogenic problems than will be the result of producing the inotropy by other means of loading the cell with calcium. In heart failure with a low level of SR Ca 2+ loading, inhibition of the Na/Ca exchanger may thus be an option. Indeed,inmyocytesfromthedogwithtachycardia-inducedcardiomyopathy, partial inhibition of the Na/Ca exchanger resulted in a positive inotropic effect, and afterdepolarizations were not observed (Hobai et al. 2004). A different situation may exist when Ca 2+ overload occurs by primary increase in Ca 2+ influx via other pathwa ys, as can occur during adrenergic stimulation. Inhibition of the Ca 2+ removal pathway may then result in an unacceptable further increase of cellular Ca 2+ . 5.2 Unidirectional Block of NCX It has been reported that the drug KB-R7943 can inhibit reverse-mode NCX more effectively than forward mode (Iwamoto et al. 1996). This would be expected to decrease Ca 2+ influx through the exchanger and this might be beneficial, e.g. during reperfusion. The relative lack of effect on forward- mode exchange would allow the exchanger to continue to pump Ca 2+ out of the cell and thereby decrease the degree of Ca 2+ overload. However, since the arrhythmias are produced by the forward mode of the exchange, any 182 K.R. Sipido et al. remaining overload would still result in arrhythmias. It is necessary, however, to be cautious about the results obtained with these drugs. This is because the reversal potential of NCX is determined by the transmembrane Na + and Ca 2+ gradients. Therefore, at least near equilibrium, it is impossible to inhibit one directio n of the pump more than the other, as this would change the direction of the net flux and thereby change the reversal potential. Ifthereispreferentialdrugbindinginthepresenceofhighinternal[Na + ], as suggested for SEA0400 in heterologous expression systems (Lee et al. 2004), such ‘selectivity’ might be an advantage. 6 Current Experience with NCX Blockers NCX blockers as antiarrhythmic agents have been tested primarily in condi- tions where the arrhythmogenic mechanism was thought to be related to Ca 2+ overload either related to the exchanger itself, as in the case of Na + overload, or through other channels, such as the repeated activation of Ca 2+ channels in atrial fibrillation. One can examine these data in two ways: first, simply as an evaluation of the efficiency of a given compound; second, as a test for clarifying the contribution of NCX to the arrhythmias. Given the fact that, so far, the specificity of the compounds is rather poor, the first objective can be addressed, but the result of the second is rather uncertain. In this part we will review the properties and selectivity o f the available compounds and the results obtained. 6.1 ‘First Generation’ of NCX Blockers A large number of agents were initially used to inhibit NCX, but these had low potency and completely lack ed selectivity. Amiloride (Siegl et al. 1984), a widely used diuretic, its deriva tives (4,5)3  ,4  -dichlorobenzamil (DCB) and 2  ,4  -dimethylbenzamil (DMB), and an antiarrhythmic drug, bepridil, with strong Na + and Ca 2+ channel-blocking properties, are typical examples (Kac- zorowski et al. 1989). These agents were reported to be effective in different experimental arrhythmia models, but their low potency and lack of selectivity made them unsuitable to use as tools to test the role of NCX in arrhythmoge- nesis. 6.2 NCX Inhibitory Peptide NCX inhibitory peptide (XIP) was developed (Chin et al. 1993; Li et al. 1991) based on the structure of NCX. It is a useful tool in patch-clamp experiments, Sodium Calcium Exchange as a Target for Antiarrhythmic Therapy 183 where it can be applied through thepatch pipette with IC 50 of the submicromo- lar range. It is an excellent tool for ev aluation of NCX function . However, since it does not cross the plasma membrane, it cannot be used in in vivo studies and has little potential therapeutic value in patients. Hobai et al. recently reported that dialysis of myocytes from failing hearts increased SR Ca 2+ loading (Hobai et al.2004; Hobai and O’Rourke 2004), which is consistent with the inotropic effect of NCX inhibition postulated above. XIP wasalsousedbytheseauthorstoprobetheNCXcurrentduringtheaction potential (see Sect. 3.1). 6.3 KB-R7943 Recently, better drugs have been developed, even if still not optimally specific (Fig. 9). KB-R7943, an isothiourea deriva te (Watano et al. 1996), has been reported as an effective blocker of NCX. In some studies it was described as a more potent reverse (Iwamoto et al. 1996) than forward mode inhibitor, but in other studies it was shown that both the forward and reverse mode of NCX was equally affected by the compound (Kimura et al. 1999; Tanaka et al. 2002). There is also great inconsistency of the reported IC 50 values for NCX block by KB-R7943, ranging from 0.3 µMto9.5µM (Iwamoto et al. 1996; Takahashi et al. 2003; Tanaka et al. 2002; Watano et al. 1996). These differences probably reflect difficulties and variety in the methodology of measuring NCX in the cardiac muscle. Also, species differences may have importance, since rat has different Ca 2+ handling and higher intracellular Na + concentration than other mammals. Fig. 9 Chemical structure of some newer NCX inhibitors 184 K.R. Sipido et al. The major problem with KB-R7943 as a useful pharmacological tool seems its poor selectivity. KB-R7943 has been fo und to inhibit fast Na + ,L-typeCa 2+ , inward rectifier and delayed K + currents in the low micr omolar range (Fig. 10; Tanaka et al. 2002). Despite this, KB-R7943 has been used in a number of cellular, mul ticellular and in vivo studies. In the in vitro studies, it was fo und to reduce Ca 2+ overload induced by glycosides (Satoh et al. 2000) and during ischaemia/reperfusion (Baczko et al. 2003; Ladilov et al. 1999), as illustrated in Fig. 11. In the latter co ndition, cardioprotective and antiarrhythmic effects could be documented in multicellular or intact heart preparations (Mukai et al. 2000; Nakamura et al. 1998; Satoh et al. 2003). Further antiarrhythmic effects were shown in Na + overload induced by glycosides or Na-channel openers (Amran et al. 2004; Satoh et al. 2003). Administered in vivo, KB-R7943 also prevented the development of atrial fibrillation evoked by rapid pacing (Miyata et al. 2002), but failed to reduce arrhythmic death in an in vivo ischaemia study (Miyamoto et al. 2002). While the multiple effects on other transmembrane ion channels men tioned previously make it hard to use these results as indications for the role of NCX in the pathophysiology, useful information comes from it. First, the additional block of Na + and Ca 2+ current may actually be an advantage for ischaemia-reperfusion related arrhythmias by decreasing the danger of Fig.10 Effects of SEA-0400 and KB-R7943 on ioniccurrents in isolatedguinea-pig ventricular myocytes. The inward and outward NCX currents are measured at −80 mV and +30 mV, respectively, Na + current at −20 mV, L-type Ca 2+ current at +10 mV, inward rectifying K + current at −60 mV and delayed rectifier K + current at +50 mV. Inhibition of current amplitudes in the presence of SEA-0400 (1 µM) and KB-R7943 (10 µM) is expressed as apercentageofthevaluesintheabsenceofcompounds.(ReproducedfromTanakaetal.2002) Sodium Calcium Exchange as a Target for Antiarrhythmic Therapy 185 Fig .11a,b Protection against Ca 2+ overload by KB-R7943. a Chemically induced hypoxia and reoxygenation-induced Ca 2+ overload in rat ventricular myocytes. [Ca 2+ ] i measurements in 15 mM [K + ] o (1) under baseline conditions, and (2) during 8 min chemically induced hypoxia, (3) followed by 8 min reoxygena tion. KB-R7943 (5 µM), was applied during reoxy- genation. *p < 0.05 vs control (15 mM [K + ] o ), n = 6 experiments, 20 and 29 cells. Reproduced from Baczko et al. (2003). b Block of strophanthidin-induced arrhythmia but not inotropy. A, Continuous recording of twitch cell shortening in a rat ventricular myocyte superfused with 50 µmol/L strophanthidin and 5 µmol/L KB-R7943 added as indicated by bars. B,Ca 2+ transients recorded during control per fusion (a), 5 min after starting strophanthidin perfu- sion (b), when arrhythmia appeared (11 min, indicated by bar; c), and 3 min after addition of KB-R7943 (d). 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