Differential Response to Na+ Channel Blockade, β-Adrenergic Stimulation, and Rapid Pacing in a Cellular Model Mimicking the SCN5A and HERG Defects Present in the Long-QT Syndrome
Abstract The long-QT syndrome (LQTS) is a hereditary disorder characterized by an abnormally prolonged QT interval and by life-threatening arrhythmias. Recently, two of the genes responsible for LQTS have been identified: SCN5A, a voltage-dependent Na+ channel on chromosome 3 (LQT3), and HERG, responsible for the rapid component of the delayed rectifier current (IKr), on chromosome 7 (LQT2). We developed an in vitro model to attempt reproduction of the expected alterations in LQT3 and LQT2 patients. Guinea pig ventricular myocytes were exposed to anthopleura toxin A (anthopleurin), an inhibitor of the inactivation of the Na+ current, and to dofetilide, a selective blocker of IKr. Both interventions significantly prolonged action potential duration (APD), by 54±13 and 62±16 ms, respectively. Cells pretreated with anthopleurin significantly shortened APD in response to mexiletine, isoproterenol, and rapid pacing (from 264±38 to 226±32 ms after mexiletine, P<.001). On the contrary, cells exposed to dofetilide did not shorten the APD after mexiletine and even prolonged it after initial exposure to isoproterenol (from 280±25 to 313±20 ms, P<.001); during rapid pacing, APD was shortened but less (38±9 versus 60±11 ms, P<.05) than in anthopleurin-treated cells. This study shows that a cellular model for LQTS, based on the recent advances in molecular genetics, can provide adequate “phenotypes” of prolonged repolarization amenable to the testing of interventions of potential clinical relevance. We found differential responses to Na+ channel blockade, to β-adrenergic stimulation, and to rapid pacing according to specific pretreatment with either anthopleurin (to mimic LQT3) or dofetilide (to mimic LQT2). These different responses in myocytes bear striking similarities with the differential response to analogous interventions in LQTS patients with mutations on the SCN5A and HERG genes.
The LQTS is a hereditary disease1 2 3 associated with life-threatening ventricular tachycardias, of the “torsades de pointes” type, which may lead to sudden death of affected and untreated patients. LQTS is characterized by prolonged ventricular repolarization (prolonged QT interval), abnormal T-wave morphology, and sinus pauses that often precede the onset of the arrhythmias. Activation of the sympathetic nervous system by physical or emotional stress often precipitates the arrhythmic events.
Linkage analysis has demonstrated genetic heterogeneity, with families linked to chromosomes 11, 7, 3, and 4.4 5 6 7 Positional cloning has allowed the identification of four LQTS loci: LQT1 has been mapped to chromosome 11p15.5; LQT2, to chromosome 7q35-36; LQT3, to chromosome 3p21-24; and LQT4, to chromosome 4q25-27. The respective genes for LQT1, LQT3, and LQT2 have been identified as KVLQT1, SCN5A, and HERG. There is no information yet on the function of KVLQT1, even though its inferred partial amino acid sequence suggests that it encodes a K+ channel.8 SCN5A is a Na+ channel located on chromosome 39 10 that produces the fast INa. HERG is a human K+ channel located on chromosome 711 that produces a current likely to be IKr.12 13
The evidence that genes encoding for K+ channels are altered in LQT1 and LQT2 and that a gene encoding for a Na+ channel is altered in LQT3 establishes that LQTS is not a single disease and suggests that LQTS represents a common phenotypic expression of at least three different inherited cardiac ion channel diseases. This concept has the important implication that therapy may be differential: for LQT3, INa is the target; for LQT2, IKr is the target.
Although the precise nature of the defects in the Na+ channel and the HERG channel is not known in terms of specific alterations in channel gating, incomplete inactivation of INa and reductions in IKr are reasonable assumptions and would result in delayed repolarization. We developed an in vitro experimental preparation that might reproduce the expected alterations of INa (to mimic LQT3) and of IKr (to mimic LQT2) using pharmacological interventions known to produce changes in INa inactivation and reducing K+ current using anthopleurin14 and dofetilide,15 respectively. Our objective was to determine whether prolonging APD by delaying Na+ channel inactivation or by blocking IKr would generate a characteristic “phenotype” that could be used to assess the response to pacing, β-adrenergic stimulation, and Na+ channel blockade.
Materials and Methods
Guinea pig ventricular myocytes were obtained by enzymatic digestion via aortic retrograde perfusion of the coronary bed, by following a modified protocol previously published by Powell.16 Guinea pigs weighing 250 to 300 g were lightly anesthetized with sodium thiopental and then stunned (decerebration via cervical dislocation). After thoracotomy, the heart, including the ascending aorta, was quickly removed from the chest and placed in a cold (4°C) physiological (saline) solution. In order to reduce the oxygen consumption and lower the metabolic activity, the heart was kept in the cold solution during the subsequent surgical procedure, consisting of the removal of the connective tissue and the isolation of the ascending aorta. A silk suture was then used to bind the heart to the perfusion apparatus. A 5-minute retrograde aortic perfusion on a Langendorff apparatus, with an oxygenated (100% O2) HEPES-buffered solution (containing the following concentrations [mmol/L]: NaCl 133.5, KCl 3.4, MgCl2 1.2, NaH2PO4 1.2, HEPES 10, glucose 10, and CaCl2 1.2), at 37°C and pH 7.38, was performed to wash out the blood from the coronary bed. Only hearts in which the onset of this procedure restored spontaneous, rhythmic, and vigorous contractions were used for cell dissociation. Subsequently, on a separate apparatus the heart was perfused with Ca2+-free oxygenated HEPES-buffered solution (37°C, pH 7.35) for 10 minutes after cessation of the spontaneous contractions. In this step, as well as in the subsequent collagenase perfusion, a constant perfusion flow was maintained by means of a peristaltic pump at 6 to 7 mL/min. After 10 minutes of nominally 0 Ca2+ perfusion, collagenase (Worthington type II) was added at a concentration of 0.28 to 0.30 mg/mL for another 15 minutes.
Only rod-shaped myocytes (with clear cross striations) showing no spontaneous contractions were considered for the study protocol. Transmembrane action potentials were recorded with glass microelectrodes (with an input resistance of 30 to 50 MΩ) filled with 3 mol/L KCl. Action potentials were recorded through an electrometer (Axoclamp A2, Axon Instruments), analog-to-digital conversion was performed using a Labmaster TL-1 125 (Axon Instruments), and data were subsequently stored on an optical disk (Laser Bank 1000, Micro Design International) at an acquisition frequency of 1.5 kHz.
After cell impalement, a 10-minute equilibration period was allowed in order to reach a stable action potential morphology, duration, and resting membrane potential. Only cells with a normal resting membrane potential (−90 mV), with no spontaneous activity or afterdepolarizations during pacing, were used for the subsequent study protocol. Cells were studied in an experimental bath (volume, 5 mL), with temperature and pH kept in a physiological range (37.5°C, pH 7.38), and continuously superfused (constant flow, 5 mL/min) with oxygenated HEPES-buffered solution containing 1.3 mmol/L Ca2+. Cells were paced at a cycle length of 1000 ms (stimulator model S8800, Grass Instruments); in 20 cells exposed to anthopleurin (n=10) and dofetilide (n=10) and in 10 cells in control conditions, pacing protocols were performed at 2.5 Hz for 40 consecutive beats in order to assess APD adaptation to changes in cycle length. In 16 cells previously studied with pacing at 1 and 2.5 Hz, pacing was performed at 0.5, 1, 1.5, 2, and 2.5 Hz in order to evaluate APD90 adaptation at intermediate rates (control, n=5; anthopleurin, n=5; and dofetilide, n=6).
Block of the inactivation of INa was obtained by exposure of cells to 10 nmol/L anthopleurin (Sigma Chemical Co) for 15 minutes, and the effect of INa blockade (n=5) was assessed by adding 10 μmol/L mexiletine to the solution containing anthopleurin. Washout was then performed by washing the solution with the active substances and replacing it with HEPES-buffered solution. K+ channel blockade was performed (n=5) by exposure to 10 μmol/L dofetilide, a selective IKr blocker, for 15 minutes; 10 μmol/L mexiletine was then added to the dofetilide solution, and combined exposure was maintained for an additional 15 minutes before washout. Thirteen cells pretreated with dofetilide were exposed to mexiletine at higher dosages (50 μmol/L [n=6] and 100 μmol/L [n=7]) in order to evaluate whether the response to Na+ channel blockade was shifted to the right compared with anthopleurin-treated cells.
Six cells treated with dofetilide and six cells treated with anthopleurin were exposed to 10 nmol/L isoproterenol. Combined exposure to dofetilide and isoproterenol or to anthopleurin and isoproterenol was maintained for 15 minutes until steady state APD values were obtained.
Throughout the study, cells were kept at a temperature of 37.5°C, and APD was measured at APD90 in at least five consecutive beats at 5-minute intervals by Axotape software (Axon Instruments).
Differences between the means in the groups of cells studied were analyzed by paired and unpaired t tests, and one-way ANOVA was used for multiple comparisons. Post hoc analysis was performed using Scheffé’s method. A value of P<.05 was accepted as statistically significant.
The study was performed in 54 isolated guinea pig myocytes after obtaining a stable resting membrane potential and APD. Eight of these cells were studied in control conditions.
Effect of Anthopleurin and of Dofetilide
The effect of anthopleurin or dofetilide was studied in 46 cells; 35 of them were studied also with either mexiletine or isoproterenol (Table 1⇓), and 11 cells were used for the pacing protocol from 0.5 to 2.5 Hz. In the 46 cells, the mean duration of APD90 in control conditions at a basic drive of 1 Hz was 213±33 ms. Sixteen cells were exposed to anthopleurin (10 nmol/L), and 30 cells were exposed to dofetilide (10 μmol/L). Both substances prolonged APD. The mean steady state prolongation was 54±13 ms in cells treated with anthopleurin (n=16, from 220±29 to 274±25 ms; ANOVA, P<.001) and 62±16 ms in cells treated with dofetilide (n=30, from 209±35 to 272±36 ms; ANOVA, P<.001).
Effect of Mexiletine
The effect of the Na+ channel blocker mexiletine (10 μmol/L)17 was studied in 23 cells. Mexiletine was added to the perfusion solution, and it reduced the APD prolongation induced by anthopleurin (n=5, from 264±38 to 226±32 ms; ANOVA, P<.001), whereas it did not affect the prolongation induced by dofetilide (n=5, from 314±61 to 314±64 ms; P=NS) (Figs 1⇓ and 2⇓). The effect of mexiletine in the two groups of cells was significantly different, as the mean APD difference after mexiletine in the anthopleurin group was −37±8 versus +1±12 ms in the dofetilide group (P<.001). Thirteen additional cells pretreated with dofetilide were also exposed to higher dosages of mexiletine. In the six cells exposed to 50 μmol/L mexiletine and in the seven cells exposed to 100 μmol/L mexiletine, APD90 remained unchanged (Table 1⇑).
Effect of Isoproterenol
The effect of isoproterenol was studied in 15 cells. Three cells received isoproterenol (10 nmol/L) in control conditions, and in none of them was APD modified (from 208±34 to 205±47 ms, P=NS),18 nor did EADs develop.19 In the cells (n=6) that were exposed first to anthopleurin and subsequently to anthopleurin plus isoproterenol, a prompt shortening of APD was obtained after 1 minute (from 277±19 to 240±22 ms; ANOVA, P<.001). By contrast, in the cells (n=6) exposed to dofetilide, the addition of isoproterenol initially further prolonged APD from 280±25 to 313±20 ms (ANOVA, P<.001) but after 15 minutes reduced it to control values (229±22 ms; ANOVA, P<.001) (Figs 3⇓ and 4⇓). During the initial prolongation induced by isoproterenol added to dofetilide, EADs always developed; in contrast, EADs did not develop in any of the cells exposed to anthopleurin and isoproterenol.
Effect of Pacing
The effect of pacing was studied in 30 cells. Pacing at 1 Hz and at 2.5 Hz was performed in all of them. Sixteen cells were subsequently studied with incremental pacing from 0.5 to 2.5 Hz by steps of 0.5 Hz, whereas the remaining 14 cells after the pacing protocol were entered in either the mexiletine or the isoproterenol protocols (Table 2⇓, Fig 5⇓).
The 30 cells (10 in control conditions, 10 exposed to anthopleurin, and 10 exposed to dofetilide) were studied with pacing protocols to evaluate APD shortening at faster rates. At a 1-Hz driving frequency, the mean steady state APD was 202±15 ms in control cells, and it was longer (P<.005) in cells exposed to anthopleurin (277±19 ms) and in cells exposed to dofetilide (278±31 ms). When the pacing rate was increased to 2.5 Hz, mean APD decreased by 10% to 182±11 ms in control cells, by 13% to 240±26 ms in the dofetilide group, and by 22% to 217±18 ms in the anthopleurin group (ANOVA, P<.001) (Fig 5⇑).
Pacing from 0.5 to 2.5 Hz by steps of 0.5 Hz was performed in 16 cells: five control cells, six cells treated with dofetilide, and five cells treated with anthopleurin. APD90 shortening was higher in dofetilide- and anthopleurin-treated cells than in control cells, and anthopleurin-treated cells had a more pronounced adaptation than did dofetilide-treated cells. Data are shown in Fig 6⇓.
The present study describes differences in the behavior of the APD in myocytes after pharmacological modification of INa and IKr. Our goal was to provide a means of distinguishing between phenotypes of prolonged ventricular repolarization potentially relevant to the hereditary LQTS.1 2 3 The findings demonstrate the existence of different responses to Na+ channel blockade, to β-adrenergic stimulation, and to rapid pacing in cells pretreated with anthopleurin and with dofetilide. These different responses in myocytes have striking similarities with the differential responses to analogous interventions observed in LQTS patients with mutations on the SCN5A and HERG genes.20 The present data support the developing concept that the different and recently identified forms of LQTS may respond differently to therapeutic interventions. Furthermore, they suggest that in the context of recent advances in molecular genetics, cellular models could be used to identify novel approaches to optimize therapy in relationship to a specific genotype.
LQTS is the first recognized inherited myocardial ion channel disease.21 Recently, three defective ion channel genes have been identified in LQTS patients: the gene for the cardiac Na+ channel (SCN5A),9 10 the gene for the HERG K+ channel,11 and the gene for a yet undefined K+ channel.8 Prolongation of the QT interval is the common phenotypic manifestation.
Because the LQTS deletion appears to be located on the III-IV linker of the Na+ channel putative topology, it was initially predicted that the mutation would affect the inactivation of INa.9 However, Bennett et al21 have expressed the first identified mutation (ΔKPQ) and have found that mutant channels fluctuate between normal and noninactivating gating modes. These channels burst for prolonged periods of time, producing persistent inward current, which explains the prolongation of cardiac action potentials. Moreover, Dumaine et al22 have recently expressed not only the ΔKPQ mutation but also two more recently described10 single residue substitutions (R1644H and N1325S) and found that the late INa produced by all three mutations is blocked by mexiletine.
The mutations identified in LQT2 affect HERG (human Ether a-go-go–related gene), a newly defined human K+ channel described in 1994.23 Sanguinetti et al12 expressed HERG in Xenopus oocytes and recorded a delayed rectifier K+ current that was interpreted as encoding the human protein for the IKr channel. They also showed that the different mutations observed in HERG lead to a reduced function of the channel by diminishing K+ current.24
The Present Study
On the basis of evidence that the mutant SCN5A Na+ channels produce an increased amount of slowly inactivating inward INa,21 22 anthopleurin was selected as the pharmacological intervention that might mimic LQT3.
Since LQT2 consists of an alteration of the IKr channel encoded by the HERG gene,12 it is expected that this would reduce the expression of functional channels. Accordingly, in an attempt to mimic the condition of LQT2, we used dofetilide, a selective blocker of IKr.25
In these two models, we tested the response of APD to (1) exposure to the Na+ channel blocker mexiletine, (2) exposure to isoproterenol, and (3) its adaptation to increases in stimulation frequency.
Exposure to a Na+ Channel Blocking Agent
Both anthopleurin and dofetilide induced a prolongation of APD, confirming that either a reduced IKr or a persistent inward INa can cause the classic phenotypic alteration of LQTS, ie, prolongation of the QT interval. Exposure to the Na+ channel blocker mexiletine significantly reduced APD in cells treated with anthopleurin, whereas it did not modify the prolongation induced by dofetilide. Furthermore, we exposed dofetilide-treated cells to higher dosages of mexiletine in order to evaluate whether the dose-response curve to the Na+ channel blockers was shifted to the right compared with the response in anthopleurin-treated cells. The lack of shortening induced by mexiletine at the higher dosages supports a lack of effect on APD90 prolongation induced by K+ channel blockade. The latter result may appear in contrast with the reports of shortening of APD induced by mexiletine26 27 and lidocaine28 in Purkinje fibers pretreated with the K+ channel blocker sotalol. However, the use of Purkinje fibers whose plateau currents differ from those of myocytes29 30 and of sotalol (which, at variance with dofetilide,31 32 also exerts an inhibitory effect on INa33 ) makes comparisons hazardous.
Our observations suggested that a Na+ channel blocker might reduce APD, and therefore the QT interval, in LQT3 patients. In striking confirmation of this hypothesis, when we administered mexiletine to 13 LQTS patients,20 we found that the QT interval shortened significantly, by an average value of 90 ms, in the LQT3 patients (n=6), whereas it remained almost unchanged in the LQT2 patients (n=7).
Effect of β-Adrenergic Stimulation
β-Adrenergic stimulation performed in control cells did not modify APD, as previously described.34 However, when isoproterenol was added to anthopleurin, a prompt shortening of APD toward control values was observed. Also, the cells treated with dofetilide shortened their APD to control values when exposed to isoproterenol; however, during the first 3 minutes of exposure to isoproterenol, APD was further prolonged and EADs developed. Only after 15 minutes of exposure did APD shorten to control values.
This differential effect of β-adrenergic stimulation according to pretreatment with anthopleurin or dofetilide may suggest that vulnerability to catecholamines may also differ in LQTS patients according to the gene involved. In agreement with this, we found that the majority of cardiac arrests occur during stress among LQT2 patients and during sleep or at rest among LQT3 patients.20
Adaptation of APD to Changes in Rate
As expected,35 36 cells exposed to dofetilide shortened APD during rapid pacing more than the control cells did. However, anthopleurin-treated cells had an even greater shortening of APD to rapid pacing than did the control and dofetilide-treated cells. A pacing protocol at frequencies ranging from 0.5 to 2.5 Hz was performed in 16 cells and confirmed the more pronounced adaptation to the fast rate of anthopleurin-treated cells. Consistent with these findings are data showing that tetrodotoxin prolongs the restitution of cardiac APD, thus suggesting that blockade of Na+ channels alters adaptation.37 This suggests that increased shortening in response to fast rate is probably not a specific consequence of anthopleurin but rather an effect of increased INa. Once more, our observations in myocytes fit with the finding that chromosome 3–linked patients shortened their QT intervals more during physiologically induced increases in heart rate than do chromosome 7–linked patients and healthy control subjects.20
Limitations of the Study
The model used in the present study represents the first attempt to replicate in vitro the electrophysiological consequences of the genetic abnormalities identified in patients affected by two variants (LQT2 and LQT3) of LQTS. As with many experimental preparations, data should be interpreted with caution, especially when attempting clinical extrapolation. Four potential limitations apply to our preparation: (1) The model is based on guinea pig ventricular cells, which have several important differences compared with myocytes of other species,38 including humans.39 The transient outward current is absent in guinea pigs, and the slow component of the delayed rectifier is more prominent than in most species.38 (2) Differences have been described in myocytes originating from epicardium, endocardium, and midmyocardium,40 which may influence the results; we have not attempted any characterization of the site of origin of ventricular cells used in the present study. (3) The use of dofetilide to block IKr is made on the assumption24 that genetic defects in HERG result in a reduction of the native current. (4) Experiments performed with mexiletine are based on the assumption that the binding affinity to Na+ channels is not modified by anthopleurin and dofetilide. We have addressed this issue by performing dose-response curves showing that mexiletine, even at higher dosages (50 and 100 μmol/L), does not affect APD prolongation induced by dofetilide; however, since pharmacological data on the effects of anthopleurin and dofetilide on mexiletine binding are not available, we cannot rule this out as a potential confounding factor in our model.
Pharmacological tools in isolated myocytes have allowed an attempt to develop a cellular model designed to mimic, within the constraints of experimental electrophysiology, the two forms of hereditary LQTS dependent on mutations on the SCN5A (LQT3) and HERG (LQT2) genes. The two preparations have shown differential responses to the interventions tested. When, on this experimental basis, similar interventions have been assessed in LQT3 and LQT2 patients, we have observed responses very similar to those present in the cells pretreated with anthopleurin and with dofetilide, respectively. These findings are encouraging for the exciting possibility of using cellular models, which have originated from the progress in molecular genetics, to guide novel therapeutic approaches for a life-threatening disease such as LQTS.
Selected Abbreviations and Acronyms
|anthopleurin||=||anthopleura toxin A|
|APD||=||action potential duration|
|APD90||=||APD at 90% repolarization|
|IKr||=||rapid component of the delayed rectifier K+ current|
The financial support of Telethon (grant 748) is gratefully acknowledged. This study was also supported by a BIOMED research grant (PL-950028). We wish to express our gratitude to Dr Thomas J. Colatsky, Princeton, NJ, for reading the manuscript and providing useful and constructive criticisms. We are also grateful to Pinuccia De Tomasi for editorial support.
- Received September 25, 1995.
- Accepted February 29, 1996.
- © 1996 American Heart Association, Inc.
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