© 1996 American Heart Association, Inc.
Articles |
Differential Response to Na+ Channel Blockade, ß-Adrenergic Stimulation, and Rapid Pacing in a Cellular Model Mimicking the SCN5A and HERG Defects Present in the Long-QT Syndrome
From the Centro di Fisiologia Clinica e Ipertensione, Ospedale Maggiore di Milano IRCCS (S.G.P., C.N., F.C., P.J.S.), Università di Milano (Italy); Case Western Reserve University Rammelkamp Center (A.M.B.), MetroHealth Campus, Cleveland, Ohio; and Dipartimento di Cardiologia (P.J.S.), Università di Pavia, and IRCCS Ospedale S. Matteo, Pavia, Italy.
Correspondence to Peter J. Schwartz, MD, Professor of Cardiology, Dipartimento di Cardiologia, Università di Pavia, Policlinico S. Matteo, Piazzale Golgi 2, 27100 Pavia, Italy.
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Abstract |
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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 ß-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.
Key Words: long-QT syndrome • Na+ channel blockade • sudden death • genetic defects • sympathetic stimulation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, ß-adrenergic stimulation, and Na+ channel blockade.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Myocyte Isolation
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.
Experimental Protocol
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 M
) 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.
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Results |
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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).
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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
).
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, n=5) and dofetilide (
, n=5) and during
combined exposure to both drugs plus mexiletine (mex) (n=5 for both
groups). There was a significant difference (P<.01) between
the two groups during combined exposure with mexiletine.
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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.
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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).
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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.
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, n=6) and
anthopleurin-treated (
, n=5) cells during pacing at 0.5, 1,
1.5, 2, and 2.5 Hz. *P<.05 vs control cells
(Scheffé's post hoc test); **P<.05 vs control and
dofetilide-treated cells (Scheffé's post hoc test).
Data for all groups are presented in Tables 1 and 2.
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Discussion |
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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 ß-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.
Genetic Background
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 (
KPQ) 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
KPQ 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 ß-Adrenergic Stimulation
ß-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 ß-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.
Conclusions
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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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.
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