INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

TRANSSARCOLEMMAL CA²⁺ efflux via Na⁺/Ca²⁺ exchange is essential for cardiac muscle relaxation. On a beat-to-beat basis, the Na⁺/Ca²⁺ exchanger is believed to extrude the same quantity of Ca²⁺ that enters through L-type Ca²⁺ channels during systole. While support for this notion has been derived from a variety of experimental approaches (reviewed in Refs. 3, 39, and 41), several recent studies have called this quantitative relationship into question. Specifically, the accepted 3:1 stoichiometry of Na⁺/Ca²⁺ exchange has recently been challenged in a study (9) where a 4:1, or variable, stoichiometry was proposed. Should this prove to be correct, electrophysiological estimates of Ca²⁺ efflux via Na⁺/Ca²⁺ exchange, and its relationship to Ca²⁺ influx through L-type Ca²⁺ channels, would be reduced (e.g., Ref. 5). Furthermore, an increasing number of reports (1, 6) have suggested that the parallel Ca²⁺ efflux pathway, sarcolemmal Ca²⁺-ATPase, may contribute more significantly to Ca²⁺ removal than has been previously considered. Irrespective of these quantitative discrepancies, however, it is unequivocally established that Na⁺/Ca²⁺ exchange serves a major role in controlling intracellular Ca²⁺ levels.

Abnormalities in intracellular Ca²⁺ homeostasis have been implicated in numerous models of cardiac injury or disease (7, 31, 32), including those occurring in humans. Moreover, the Na⁺/Ca²⁺ exchanger may play a prominent role in this process (8, 11, 12, 18, 31, 37, 40). For instance, in several models of cardiac hypertrophy and/or heart failure, the Na⁺/Ca²⁺ exchanger has been shown to be elevated at the transcript, protein, and activity levels (8, 25, 40, 45, 51). Increased activity of the Na⁺/Ca²⁺ exchanger may also contribute to arrhythmogenesis (12, 40) in that Na⁺/Ca²⁺ exchange constitutes a major component of the transient inward current associated with delayed afterdepolarizations or oscillatory afterpotentials (7, 27, 43). However, other investigators have either failed to detect changes (23, 44) or have reported decreases (53) in Na⁺/Ca²⁺ exchanger levels during heart failure. In general, predicting the consequences of alterations in Na⁺/Ca²⁺ exchanger levels and/or activity has not been straightforward owing to the fact that this transporter can operate to support both Ca²⁺ entry and efflux.

A major difficulty that has hindered investigation of the Na⁺/Ca²⁺ exchange function is the dearth of pharmacological agents that can selectively modulate the activity of this transporter. However, in 1996, a novel Na⁺/Ca²⁺ exchange inhibitor, KB-R7943 (formerly No. 7943), was introduced as a specific inhibitor of the reverse (i.e., Ca²⁺ influx) mode of Na⁺/Ca²⁺ exchange. In the original two reports (21, 50), preferential inhibition of reverse Na⁺/Ca²⁺ exchange was documented with similar potencies in the low micromolar range (i.e., 0.3-2.4 µM). In comparison, inhibition of forward (i.e., Ca²⁺ efflux) mode exchange activity by KB-R7943 required 10-50 times higher concentrations. Competition between KB-R7943 and extracellular Ca²⁺ was reported by one group (50), whereas inhibition was found to be noncompetitive with respect to the transported ions by the other group (21).

Since the introduction of KB-R7943, several controversial aspects have emerged regarding its specificity, transport mode selectivity, and mechanism of action, and considerably lower inhibitory potencies of KB-R7943 have been obtained from different assay systems. For example, in one study (26), 30 µM KB-R7943 produced relatively weak inhibition of Na⁺/Ca²⁺ exchange activity (i.e., 18-45%) when assessed in BHK cells or membrane vesicles expressing either of the unique exchanger isoforms NCX1, NCX2, or NCX3 and was relatively equipotent in its action. In contrast, others (20) have identified isoform-specific differences in KB-R7943-mediated inhibition of exchange activity with similar potencies to the original reports. In a recent electrophysiological study (22) assessing Na⁺/Ca²⁺ exchange currents under ionic conditions allowing bidirectional transport, no differences in the selectivity of KB-R7943 for a particular transport mode were detected. Clearly, there is little consensus regarding how KB-R7943 exerts its inhibitory effects on Na⁺/Ca²⁺ exchange activity.

Since 1996, a number of laboratories have employed KB-R7943 to investigate the role of Na⁺/Ca²⁺ exchange in mediating cellular damage in various models of cardiac injury. For example, KB-R7943 was found to offer prophylaxis against ouabain-induced arrhythmias (48), Ca²⁺ paradox (21), metabolic inhibition (36), and reoxygenation injury (33) in guinea pig cardiac muscle. On the other hand, conflicting data have appeared in studies using anesthetized rats subjected to 5 min of ischemia and 10 min of reperfusion. One group (34) documented a dose-dependent reduction in the incidence and duration of ventricular fibrillation (VF), whereas no significant influence of KB-R7943 was observed by another group (28). Despite these discrepancies, the role of Na⁺/Ca²⁺ exchange in studies of ischemia-reperfusion injury is well documented (18, 31), and the increasing interest in the salutary properties of KB-R7943 with respect to cardiac damage necessitates a more detailed description of its inhibitory actions on Na⁺/Ca²⁺ exchange activity.

In the present study, we used the giant excised patch-clamp technique to investigate the electrophysiological effects of KB-R7943 on Na⁺/Ca²⁺ exchange activity for the cloned cardiac Na⁺/Ca²⁺ exchanger NCX1.1 expressed in Xenopus laevis oocytes. A variety of protocols were employed in an effort to distinguish between the effects of KB-R7943 on the transport and regulatory properties of NCX1.1. We then investigated the effects of KB-R7943 in Langendorff-perfused whole rabbit hearts subjected to ischemia-reperfusion injury. In this model system, the incidence of arrhythmias associated with ischemia-reperfusion is 75%. A near-complete suppression of arrhythmias was associated with KB-R7943 application, indicating a critical role for reverse Na⁺/Ca²⁺ exchange in this process.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of Xenopus laevis oocytes. X. laevis were anesthetized in 250 mg/l ethyl p-aminobenzoate (Sigma) in deionized ice water for 30 min. Oocytes were surgically removed and washed in solution A, which contained (in mM) 88 NaCl, 15 HEPES, 2.4 NaHCO₃, 1.0 KCl, and 0.82 MgSO₄; pH 7.6 at room temperature (RT). The follicles were teased apart, and the oocytes were transferred to 5 ml of solution A containing ~3,500 U/ml collagenase (type II, Worthington) and incubated at RT for 45-60 min with gentle agitation. The oocytes were then washed several times in solution B, which contained (in mM) 88 NaCl, 15 HEPES, 2.4 NaHCO₃, 1.0 KCl, 0.82 MgSO₄, 0.41 mM CaCl₂, 0.3 mM Ca(NO₃)₂, and 1 mg/ml BSA (Fraction V, Sigma), pH 7.6 at RT, and transferred to 5 ml of 100 mM K₂HPO₄, pH 6.5 at RT, containing 1 mg/ml BSA. After incubation at RT for 12 min with gentle agitation, the oocytes were washed in solution B at RT. Defolliculated stage V-VI oocytes were selected and incubated at 18°C in solution B (without BSA) until injection the following day.

Synthesis of NCX1.1 cRNA. Complementary DNA encoding NCX1.1, residing in pBluescript II SK(+) plasmids (Stratagene), was linearized with HindIII (New England Biolabs), and cRNA was synthesized using T3 mMessage mMachine in vitro transcription kits (Ambion) according to the manufacturer's instructions. After injection with ~5 ng cRNA encoding NCX1.1, oocytes were maintained at 18°C in solution B without BSA (see Preparation of X. laevis oocytes). Electrophysiological measurements were obtained from days 3-6 postinjection.

Measurement of Na+/Ca²⁺ exchange activity. Na⁺/Ca²⁺ exchange current measurements were obtained using the giant excised patch-clamp technique as described previously (17, 46, 46). Borosilicate glass pipettes were pulled and polished to a final diameter of 20-30 µm and coated with a Parafilm-mineral oil mixture to enhance patch stability and reduce electrical noise. The vitellin layer was removed by dissection, and oocytes were placed in a solution containing (in mM) 100 KOH, 100 MES, 20 HEPES, 5 EGTA, and 5-10 MgCl₂; pH 7.0 at RT (with MES). Gigaohm seals were formed by suction, and membrane patches (inside-out configuration) were excised by progressive movements of the pipette tip. Rapid solution changes (~200 ms) were accomplished using a computer-controlled 20-channel solution switcher. Axon Instruments hardware (Axopatch 200a) and software (Axotape) were used for data acquisition and analysis, and Origin software was used for curve fitting (e.g., determination of IC₅₀ values) and statistical analyses. Unless indicated otherwise, a holding potential of 0 mV was employed for current measurements. For outward (i.e., reverse) Na⁺/Ca²⁺ exchange current measurements, the pipette (i.e., extracellular) solutions contained (in mM) 100 N-methyl-D-glucamine-MES, 30 HEPES, 30 tetraethylammonium (TEA) hydroxide, 16 sulfamic acid, 8.0 CaCO₃, 6 KOH, 0.25 ouabain, 0.1 niflumic acid, and 0.1 flufenamic acid; pH 7.0 at RT (with MES). Outward currents were elicited by switching from Li⁺- to Na⁺-based bath solutions containing (in mM) 100 [Na + Li] aspartate, 20 CsOH, 20 MOPS, 20 TEA hydroxide, 10 EGTA, 0-9.91 CaCO₃, and 1.0-1.5 Mg(OH)₂; pH 7.0 at 30°C (with MES or LiOH). Total Mg²⁺ and Ca²⁺ were adjusted to yield free concentrations of 1.0 mM and 0-30 µM, respectively, using MAXC software (2). For inward (i.e., forward) Na⁺/Ca²⁺ exchange current measurements, the pipettes contained (in mM) 100 sodium MES, 20 CsOH, 20 TEA hydroxide, 10 EGTA, 10 HEPES, 8 sulfamic acid, 4 Mg(OH)₂, 0.25 ouabain, 0.1 niflumic acid, and 0.1 flufenamic acid; pH 7.0 at RT (with MES). Inward currents were activated by switching between the Ca²⁺-free and Ca²⁺-containing Li⁺-based bath solutions described above. For combined inward-outward current measurements, the pipettes contained (in mM) 100 sodium MES, 20 CsOH, 20 HEPES, 20 TEA hydroxide, 4 sulfamic acid, 2 CaCO₃, 0.25 ouabain, 0.1 niflumic acid, and 0.1 flufenamic acid; pH 7.0 at RT (with MES). Outward and inward currents were activated using the same solutions as those for initiating pure outward and pure inward currents described above. In RESULTS, only the Na⁺ and Ca²⁺ concentrations of experimental solutions are described for brevity. To obtain current-voltage (I-V) relationships, pipette voltage was stepped from a holding potential (V_H) of 0 mV to various potentials (for 20 ms) in 10-mV steps, with a return to V_H between steps. Leak subtracted I-V covered the range of transmembrane potentials from 100 to +60 mV. All experiments were conducted at 30 ± 1°C. A 20 mM stock solution of KB-R7943 (generously supplied by Nippon Organon) was prepared with DMSO. The final concentration of DMSO never exceeded 0.1% (for 20 µM KB-R7943) and was typically much lower. KB-R7943 was applied to the cytoplasmic surface of the patch for all giant excised patch-clamp experiments.

Langendorff-perfused whole rabbit heart preparation. The methods are described in detail elsewhere (4). Male New Zealand White rabbits (2.5-3.0 kg) were anesthetized with isoflurane (5% in 2 l/min O₂) and heparinized (500 IU). The heart was excised, mounted on a Langendorff apparatus, and perfused with Tyrode solution (20 ml/min) containing (in mM) 115 NaCl, 28 NaHCO₃, 20 D-glucose, 4 KCl, 2 CaCl₂, 0.7 MgCl₂, and 0.5 NaH₂PO₄. The solution was bubbled with 95% O₂-5% CO₂ (37 ± 0.5°C). The right atrium was excised, and the atrioventricular node was crushed to allow pacing of the heart at 2 Hz (120 beats/min). Left ventricular developed pressure (LVDP) and end-diastolic pressure (EDP) were measured using an intraventricular latex balloon attached to a pressure transducer. The balloon was inflated with water to an EDP of ~5 mmHg. Volume-conducted electrocardiograms (leads I, II, and III) were recorded by immersing the heart in a circular acrylic bath (12 cm) filled with Tyrode solution at 37°C (~275-ml volume). The bath was fitted with three 4-mm Ag-AgCl electrodes placed in a simulated Einthoven triangle with the heart at the center. The solution around the heart was bubbled with 95% O₂-5% CO₂ during the control and reperfusion periods and with 95% N₂-5% CO₂ during ischemia.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of KB-R7943 on outward Na+/Ca²⁺ exchange currents. Figure 1A illustrates the inhibitory effects of KB-R7943 on outward (i.e., reverse) Na⁺/Ca²⁺ exchange currents. The representative current tracings were obtained from an oocyte membrane patch expressing the cloned canine cardiac exchanger NCX1.1. Outward currents were activated by applying 100 mM Na⁺ to the cytoplasmic surface of the patch. Transported Ca²⁺ in the pipette (i.e., extracellular side) was 8 mM. Regulatory Ca²⁺ (1 µM), required for activation of the exchanger, was present on the cytoplasmic side of the patch throughout the recordings. In the control recording, Na⁺/Ca²⁺ exchange current peaks and then inactivates to a steady-state level via the Na⁺-dependent (or I₁) inactivation process (13, 15). Application of KB-R7943 to the cytoplasmic surface of the patch led to a dose-dependent inhibition of both peak and steady-state outward Na⁺/Ca²⁺ exchange currents. The concentration dependency of this effect is illustrated in Fig. 1B. The inhibitory potency (IC₅₀) of KB-R7943 was calculated as 2.8 ± 1.0 and 0.6 ± 0.1 µM for peak and steady-state outward currents, respectively (means ± SD). Therefore, KB-R7943 inhibits outward Na⁺/Ca²⁺ exchange currents (corresponding to Ca²⁺ entry) and significantly inhibits the steady-state component of this current preferentially (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   The inhibitory effects of KB-R7943 on outward Na+/Ca2+ exchange activity. A: representative outward NCX1.1-mediated exchange currents activated by applying 100 mM Na+ to the cytoplasmic surface of the patch [intracellular Na+ (Na)] in the continuous presence of 1 µM regulatory Ca2+ [intracellular Ca2+ (Ca)] The pipette (i.e., extracellular) Ca2+ concentration was constant at 8 mM. The three overlapping recordings show outward currents obtained before KB-R7943 application (control) and after the addition of 3 and 20 µM KB-R7943. The inhibitory potency (i.e., IC50) of KB-R7943 to inhibit peak and steady-state outward currents was determined from the pooled data shown in B. Percentages of inhibition derived from 3-17 patches (as indicated) are shown. Data obtained in the presence of KB-R7943 were normalized according to the peak or steady-state outward current level obtained in the same patch in the absence of KB-R7943.

Effects of KB-R7943 on inward Na+/Ca²⁺ exchange currents. The representative recordings shown in Fig. 2 illustrate the effects of KB-R7943 on inward Na⁺/Ca²⁺ exchange currents (i.e., forward mode corresponding to Ca²⁺ efflux). Here, pipette solutions contained 100 mM Na⁺, and inward currents were activated by applying a 10 µM Ca²⁺-containing solution to the cytoplasmic surface of the patch. Inward currents do not exhibit the slow inactivation properties seen for outward currents, and a relatively square current waveform is observed (15, 30). Compared with its potency to inhibit outward currents, KB-R7943 was far less effective at inhibiting inward Na⁺/Ca²⁺ exchange currents. For example, at 10 µM KB-R7943, only 13.5 ± 1.3% inhibition was observed (4 determinations from 3 patches). Even at 20 µM KB-R7943, the highest concentration examined, inhibition was <20% (data not shown), and we therefore did not attempt to estimate an IC₅₀. At the concentration that we subsequently employed to assess the cardioprotective effects of KB-R7943 (i.e., 3 µM), this agent can be considered as a preferential inhibitor of the reverse transport mode of Na⁺/Ca²⁺ exchange.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The inhibitory effects of KB-R7943 on inward Na+/Ca2+ exchange activity. Representative inward NCX1.1-mediated exchange current traces were obtained in the absence (control) and presence of 10 µM KB-R7943. Inward currents were activated by rapidly applying 10 µM Ca2+ to the cytoplasmic surface of the patch. The pipette solution contained 100 mM extracellular Na+ (Na) Similar data were obtained with three patches.

Effects of KB-R7943 on combined inward-outward Na+/Ca²⁺ exchange currents. Figure 3 shows a representative recording in which both inward and outward currents were activated in the same membrane patch. In this case, the pipette (i.e., extracellular) solution contained 100 mM Na⁺ plus 2 mM Ca²⁺. Outward currents were initiated by rapid application of 100 mM Na⁺ to the cytoplasmic surface of the patch with regulatory Ca²⁺ present at 1 µM before and during the current recording. Inward currents, on the other hand, were activated by rapid application of a Li⁺-based 10 µM Ca²⁺-containing solution to the cytoplasmic surface. During the control period of recording, both outward and inward currents exhibited their usual characteristics, that is, outward currents decayed gradually, whereas inward currents were square in appearance (Figs. 1 and 2). Here, 10 µM KB-R7943 application on the cytoplasmic surface led to a profound inhibition of outward Na⁺/Ca²⁺ exchange currents. In contrast, inward currents were only modestly inhibited (i.e., 20%). Note that under this protocol, both inward and outward currents are mediated by the same population of exchangers in the same patch and that the extracellular ionic conditions are identical for both inward and outward current recordings. These data demonstrate a clear selectivity of KB-R7943 to inhibit outward (i.e., reverse) Na⁺/Ca²⁺ exchange currents compared with inward (i.e., forward) currents under these specific ionic conditions.

View larger version (8K): [in this window] [in a new window]   Fig. 3.   The inhibitory effects of KB-R7943 on combined inward-outward Na+/Ca2+ exchange activity. A representative NCX1.1-mediated exchange current recording is shown where outward and inward current measurements were acquired from the same patch. Here, pipette solutions contained 100 mM Na+ and 2 mM Ca2+. Outward currents were initiated by rapid application of 100 mM Na+ in the presence of 1 µM regulatory Ca2+ (as described in Fig. 1) to the cytoplasmic surface of the patch. Inward currents were generated by switching from 100 mM Li+ and 1 µM Ca2+ to 100 mM Li+- and 10 µM Ca2+-containing solutions on the cytoplasmic surface of the patch. The first two current transients (i.e., outward followed by inward) are control recordings, whereas the second two transients were obtained in the presence of 10 µM KB-R7943.

Interaction of KB-R7943 with Na+/Ca²⁺ exchange ionic regulatory mechanisms. In Fig. 4, the inhibitory effects of KB-R7943 are shown on representative outward Na⁺/Ca²⁺ exchange currents at two concentrations of regulatory Ca²⁺. We postulated that KB-R7943 could interfere with activation of the Na⁺/Ca²⁺ exchanger through competitive interactions involving its Ca²⁺ regulatory mechanism. Normally, a progressive activation of outward Na⁺/Ca²⁺ exchange currents is observed by increasing the concentration of regulatory Ca²⁺ on the cytoplasmic side of the membrane patch [dissociation constant (K_d) ~0.3 µM] (13, 14). In contrast, this behavior is not observed for inward Na⁺/Ca²⁺ exchange currents because the regulatory Ca²⁺-binding site is largely saturated before activation of any appreciable amount of inward current (K_d ~7 µM) (30). Therefore, an enticing possibility was that KB-R7943 could interfere with Ca²⁺-mediated activation of outward Na⁺/Ca²⁺ exchange currents, a process occurring at low concentrations of regulatory Ca²⁺ (e.g., 1 µM) where competition between Ca²⁺ and KB-R7943 could be pronounced. Conversely, inward currents might be less affected by KB-R7943 because higher Ca²⁺ concentrations are required to activate these (e.g., 10 µM Ca²⁺) and competition could be less effective. However, we observed marked inhibition of outward steady-state currents irrespective of the concentration of regulatory Ca²⁺ present (Fig. 4). Thus the transport mode selectivity of KB-R7943 does not appear to reside in competitive interactions with the Ca²⁺ regulatory mechanism.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Effects of regulatory Ca2+ concentration on KB-R7943 (KB)-mediated inhibition of outward Na+/Ca2+ exchange activity. Effects of 3 µM KB-R7943 application on outward NCX1.1-mediated exchange currents obtained at 1 µM (A) and 10 µM (B) regulatory Ca2+ are shown. Regulatory Ca2+ was applied and outward currents were activated as described in Fig. 1 until steady-state levels of exchange activity were attained. KB-R7943 was then applied for the indicated period and allowed to wash out. Pipette solutions contained 8 mM Ca2+. Data are representative of recordings from three patches at 1 µM Ca2+ and two patches at 10 µM Ca2+.

Effects of KB-R7943 on deregulated Na+/Ca²⁺ exchange currents. Figure 5 summarizes the effects of KB-R7943 on outward Na⁺/Ca²⁺ currents obtained from an excised patch before (Fig. 5A) and after (Fig. 5B) limited proteolytic treatment with -chymotrypsin (1 mg/ml) for 1 min. Proteolysis of the cytoplasmic surface of the patch deregulates the exchanger such that Na⁺-dependent and Ca²⁺-dependent regulations are no longer evident (13). After -chymotrypsin digestion, the exchanger appears to be fully activated, allowing a direct assessment of the effects of KB-R7943 on ion transport without the potentially confounding influences of intact ionic regulatory mechanisms. We observed that substantial KB-R7943-mediated inhibition of outward Na⁺/Ca²⁺ exchange current was still evident after this treatment. In six determinations from three patches, 20 µM KB-R7943 inhibited the -chymotrypsin-treated exchanger by 76 ± 2%, whereas inhibition exceeded 90% for regulated patches. Thus the majority of the inhibitory effects of KB-R7943 on outward currents do not require that the ionic regulatory mechanisms of the exchanger be intact. Although the precise mechanism of exchanger deregulation via proteolysis is unknown, these results indicate that inhibition of outward exchange activity by KB-R7943 occurs primarily through direct effects on the transport mechanism.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effects of -chymotrypsin treatment on KB-R7943-mediated inhibition of outward Na+/Ca2+ exchange activity. Representative outward NCX1.1-mediated current recordings were obtained in the absence (control) and presence of 20 µM KB-R7943 before (A) and after (B) limited proteolysis of the cytoplasmic surface of the patch with -chymotrypsin (1 mg/ml) for 1 min. Currents were activated in the presence of 1 µM regulatory Ca2+ as described in Fig. 1. Pipette solutions contained 8 mM Ca2+. Comparable data were obtained from three patches in each group (A and B).

Assessment of competitive interactions. Earlier studies (49, 50) have reported that inhibition of Na⁺/Ca²⁺ exchange activity by KB-R7943 occurs via competitive interactions with extracellular Ca²⁺. We utilized two protocols to examine this possibility further. In the first case, we determined the I-V relationships of exchange activity in -chymotrypsin-treated patches in the absence and presence of 20 µM KB-R7943. Figure 6A illustrates a typical current trace used to obtain an I-V relationship, and the results are presented in Fig. 6B. Note that KB-R7943 application leads to a significant depression of the slope of the I-V relationship for outward Na⁺/Ca²⁺ exchange currents (P < 0.05). However, simply scaling the I-V relationship obtained in the presence of 20 µM KB-R7943 (i.e., KB scaled) yields a virtually superimposable relationship to that obtained in its absence (i.e., control). This result is most compatible with the notion that KB-R7943 reduces the population of active exchangers rather than altering a basic transport property of the exchange process (28). Moreover, this result is difficult to reconcile with competitive interactions between KB-R7943 and extracellular Ca²⁺. Lowering extracellular Ca²⁺, essentially equivalent to competitive inhibition, leads to a progressive loss of voltage dependence in the I-V relationship (29). This occurs because Ca²⁺ transport becomes rate limiting and the majority of electrogenicity appears to reside in the Na⁺ transport partial reaction (16). Achieving the levels of inhibition observed with KB-R7943 through competition with extracellular Ca²⁺ would be equivalent to reducing pipette Ca²⁺ to submillimolar levels. In this case, the voltage dependence of the I-V relationships would flatten considerably (i.e., become more voltage independent) and would not be scaleable back to the control relationships (29). In contrast, the alterations in the I-V relationships produced by KB-R7943 (Fig. 6B) are similar to those observed during Na⁺-dependent inactivation, where the fraction of active exchangers is reduced (15).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of KB-R7943 on the current-voltage (I-V) relationships of outward Na+/Ca2+ exchange activity after limited proteolysis with -chymotrypsin. Representative outward NCX1.1-mediated exchange activity after treatment with -chymotrypsin (as described in Fig. 5) is illustrated in A. Current was activated in the presence of 1 µM regulatory Ca2+ as described in Fig. 1. Pipette solutions contained 8 mM Ca2+. KB-R7943 (20 µM) was applied for the period indicated and then allowed to wash out. I-V recordings (evident as spikes) were obtained before current activation (point a), during control outward currents (point b), and during KB-R7943 application (point c). The leak-subtracted I-V relationships [i.e., b  a (control) and c  a (KB-R7943)] are shown in B. The dashed line was obtained by scaling the I-V relationship obtained in the presence of KB-R7943 (i.e., c  a). Comparable data were obtained from two additional patches.

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View larger version (13K): [in this window] [in a new window]   Fig. 7.   The effects of KB-R7943 on the Na+ dependence of peak outward Na+/Ca2+ exchange activity. The relationship between peak outward NCX1.1-mediated exchange currents and cytoplasmic Na+ concentration ([Na+]i) in the absence (control) and presence of 3 µM KB-R7943 is presented. Data were normalized to the peak outward current values obtained at 100 mM Na+ in the absence or presence of KB-R7943. Outward currents were activated, and the levels of regulatory and pipette Ca2+ were as described in Fig. 1. The number of patches used to acquire each data point is indicated above (control) or below (KB-R7943) the curves.

Effects of KB-R7943 on ischemia-reperfusion-induced arrhythmias. The effects of KB-R7943 were examined on Langendorff-perfused whole rabbit hearts subjected to a 30-min global ischemic period followed by 45 min of reperfusion. The pooled data are presented in Fig. 8. The parameters evaluated were the incidence of ventricular tachycardia (VT) and VF (Fig. 8A), EDP (Fig. 8B), and LVDP (Fig. 8C). In control hearts (n = 12) subjected to this protocol, the incidence of VT and VF was 75 and 50%, respectively, during ischemia. Upon reperfusion, the incidence of VT and VF was 75%. The application of 3 µM KB-R7943 10 min before the ischemic episode completely abolished VT and VF during the ischemic period and significantly reduced the incidence of VT and VF to 12.5% during reperfusion (P < 0.05, n = 8). No significant differences were observed between control and KB-R7943-treated hearts with respect to EDP (Fig. 8B) or LVDP during ischemia (Fig. 8C), although KB-R7943 produced a significant negative inotropic effect before the ischemic insult (control LVDP was 81 ± 7 mmHg versus 58 ± 4 mmHg after KB-R7943 treatment). Overall, KB-R7943 offered significant protection against the electrophysiological abnormalities associated with ischemia-reperfusion injury in this preparation.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Effects of 3 µM KB-R7943 pretreatment on arrhythmogenesis during ischemia-reperfusion in the whole rabbit heart. A: incidence of ventricular tachycardia (VT) and fibrillation (VF) elicited during a 30-min ischemic period followed by 45 min of reperfusion. B and C: changes in end-diastolic pressure (EDP; B) and left ventricular developed pressure (LVDP; C) elicited during ischemia-reperfusion in control versus KB-R7943-treated hearts, respectively. *Significant differences between control and KB-R7943-treated hearts, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We examined the inhibitory properties of KB-R7943 on the cloned cardiac Na⁺/Ca²⁺ exchanger NCX1.1 using the giant excised patch-clamp technique. Our data demonstrate a clear selectivity for inhibition of outward (i.e., reverse) Na⁺/Ca²⁺ exchange currents by this agent under the ionic conditions that we employed. The majority of KB-R7943-mediated inhibition manifests through direct effects on the Na⁺/Ca²⁺ exchange transport mechanism, although some interaction with the ionic regulatory machinery of the exchanger was evident. We examined the cardioprotective effects of KB-R7943 on an ischemia-reperfusion model of cardiac injury and observed near-complete elimination of the electrophysiological derangements associated with this intervention. These data strengthen the notion that KB-R7943 selectively targets reverse Na⁺/Ca²⁺ exchange and that this process plays an important role in cardiac arrhythmogenesis.

Inhibitory mechanism of KB-R7943. Our study was designed to further our understanding of the inhibitory mechanism of KB-R7943. Specifically, our intention was to address several of the controversial aspects concerning the mechanism of action of KB-R7943 that have emerged since this agent was introduced in 1996. The widespread and increasing utilization of KB-R7943 as a selective inhibitor of the Na⁺/Ca²⁺ exchanger necessitates a more comprehensive understanding of its inhibitory effects. The giant excised patch-clamp technique, in conjunction with heterologous expression systems, provides several advantages for studies of this nature. First, this technique permits a nearly unequivocal assignment of measured currents to the Na⁺/Ca²⁺ exchanger. Second, we can investigate unique transport modes of the exchanger in isolation as well as characterize the roles of its identified ionic regulatory mechanisms in the inhibitory process. Information of this type is less readily or not obtainable using intact cellular or vesicular preparations where overall ion flux rates are normally measured. Finally, the giant patch-clamp technique permits greater resolution of the kinetic details of Na⁺/Ca²⁺ exchange currents compared with ⁴⁵Ca²⁺ flux-based approaches. Notably, however, mimicking the exact intracellular milieu and environment is not possible with this technique and extrapolation to results obtained from intact cells must be done cautiously.

Antiarrhythmic effects of KB-R7943. We examined the antiarrhythmic effects of KB-R7943 in an ischemia-reperfusion model of cardiac injury using Langendorff-perfused rabbit hearts. This model exhibits a high incidence of VT and VF (>50%) during both the ischemic and reperfusion epochs (4) and is well suited to assess the anti- or proarrhythmic profile of new agents or interventions. For example, ischemic preconditioning was found to significantly suppress ischemia-induced but not reperfusion-induced arrhythmias (4) in this model. Here, we demonstrate that pretreatment of hearts with KB-R7943 led to a significant reduction in the incidence of VT and VF during both the 30-min global ischemic period and the subsequent reperfusion, which may promote arrhythmogenesis. To date, KB-R7943 is the only agent found to suppress both ischemia- and reperfusion-induced arrhythmias in this isolated rabbit heart model.