The novel p.V210G gain-of-function mutation in the SCN5A gene
of MEPPC patients causes a negative shift in voltage
dependence of activation
Sterre van Piggelen (11073152)
Bachelor thesis Biomedical Science – Universiteit van Amsterdam Date of submission: 16 juli 2020
Supervised by Arie Verkerk & Ronald Wilders
Abstract
The SCN5A gene encodes Nav1.5, a Voltage gated sodium channels crucial for the electrical impulse conduction of the heart. Mutations in SCN5A have been correlated with several arrhythmic disorders such as long QT, Brugada, and sick sinus syndrome and, more recently, multifocal ectopic Purkinje-related premature contractions (MEPPCs). A novel mutation, p.V210G, was identified in a Dutch family in which multiple members suffer from MEPPCs. To get insight in the underlying electrophysiological mechanism of how this mutation influences the function of the Nav1.5 channel, patch clamp measurements in the voltage clamp mode of the whole-cell configuration were performed on human embryonic kidney cells expressing mutant or wild type Nav1.5 channels, to determine current density, voltage dependence of (in)activation, inactivation kinetics and recovery from inactivation. Current density was increased in mutant channels compared to wild type. In addition, voltage dependence of activation showed a 11 mV shift towards more negative potentials. This leads to a lower threshold needed for channel activation, explaining the hyperexcitability of the Purkinje cells and therefore the premature contractions seen in patients suffering from MEPPCs.
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Introduction
Heart and cardiovascular diseases are the leading cause of death among humans worldwide. Sudden cardiac death (SCD) is often a consequence of structural heart disorders (cardiomyopathies), but in active and previously healthy individuals between the ages of 16-64 years, 5% of sudden deaths are inexplicable and not identified in autopsy (Priori et al., 2014). These cases are labeled as sudden arrhythmic death syndrome (SADS) and occur in 25-35% of sudden deaths of individuals <40 years. Post-mortem genetic testing showed that primary electrical disorders are responsible for 20-35% of these cases (Ackerman et al., 2011; Tester and Ackerman, 2017). Primary electrical disorders are caused by cardiac ion channel dysfunction and are often not recognized as a consequence of the absence of structural abnormalities (Offerhaus et al., 2020).
Cardiac sodium channels are crucial for the electrical impulse conduction of the heart (Buchanan et al., 1985). These voltage gated ion channels are responsible for the fast inward sodium current (INa), which induces the action potential upstroke of working myocardial cells and Purkinje cells by influx of Na+ ions, causing fast depolarization of their cell membrane (Weidmann, 1955). This fast depolarization is essential for the fast propagation of impulses through the heart. Sodium channels consist of an α-subunit forming the pore and accessory β-subunits (Gellens et al., 1992).The nine members of the SCNxA gene family encode the α-subunits of the different sodium channels (Nav1.1 – Nav1.9) (Meisler, O’Brien and Sharkey, 2010). SCN5A encodes the α-subunit Nav1.5, which is the pore-forming subunit of the sodium channel that is responsible for generating the INa in cardiomyocytes, and is primarily expressed in atrial and ventricular myocytes, and cardiac Purkinje cells (Remme et al., 2009).
The Nav1.5 α-subunit consists of four conserved, homologous transmembrane domains (DI-DIV), connected by three cytoplasmic, intracellular loops (DI–DII, DII–DIII, and DIII–DIV) (Figure 1). Each of the domains consists of six transmembrane segments of which five segments (S1, S2, S3, S5 and S6) are hydrophobic, and one segment (S4) is shown to have a strong positive charge. This is caused by several arginine or lysine residues, interspersed with nonpolar amino-acids (Noda et al., 1984). Stühmer et al. (1989) presented the function of S4 as voltage sensor involved in activation of the channel by gating. They showed that a decrease of the positive charge in S4 caused a change in the voltage dependence of activation. Additionally, a strong reduction of the inactivation rate was found when domains III and IV were disconnected, emphasizing the importance of the connecting loop in inactivation of the channel (Vassilev et al., 1988). Indeed, the DIII-DIV loop has been demonstrated to be critical for fast inactivation of the channel, as it forms an inactivation gate which can occlude the open pore (Patton et al., 1992).
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Mutations in SCN5A have been correlated with several arrhythmic disorders such as long QT syndrome (Wang et al., 2004; Olesen et al., 2012), Brugada syndrome (Frustaci et
al., 2005; Wilde and Amin, 2018), sick sinus syndrome (Smits et al., 2005; Abe et al., 2014)
and, more recently, multifocal ectopic Purkinje-related premature contractions (MEPPCs) (Laurent et al., 2012; Mann et al., 2012). MEPPCs are associated with dilated cardiomyopathy (DCM), frequent premature ventricular contractions (PVCs), and atrial arrhythmias due to hyperexcitability of Purkinje cells. Currently, only a very limited amount of mutations correlated with MEPPCs have been identified (see Figure 1 and Table 1) and pre-clinical and clinical findings differ substantially between studies. A manifestation common in all known mutations associated with MEPPCs is an increased and/or hyperpolarized INa window current (see Table 1). Window current describes channel availability, and is determined by the range of membrane potentials in which steady-state activation and inactivation curves overlap (Han et
al., 2018). An increased window current indicates a higher channel availability which leads to
a gain of function and is often associated with cardiac arrhythmias.
Figure 1. Schematic representation of the Nav1.5 sodium channel α-subunit, together with the
interacting β subunits. The four domains (DI-DIV) of the α-subunit, consisting of six identical segments S1–
S6, are linked by intracellular loops. The red dots indicate the known locations of the mutations that have been linked to MEPPC. The β-subunits one, two, three, and four are thought to influence cell surface expression, but are still largely unknown. The accentuated p.V210G mutation is subject of the present study.
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Although the increase and/or hyperpolarized window current is common to all mutations in SCN5A associated with MEPPCs, the underlying mechanism varies. For example, one of the mutations found in the SCN5A gene of patients suffering from MEPCC, p.R222Q, causes a negative shift in voltage dependence of both activation and inactivation (Mann et al., 2012). This causes a slightly enlarged, significantly hyperpolarized window current, resulting in an increased availability of the channels at the resting membrane potential of Purkinje cells. In contrast, the positive shift in voltage dependence of inactivation causes the gain of function seen in patients harboring the p.M1851V mutation (Lieve et al., 2017). Also, gain-of-function mutations in SCN5A associated with MEPCCs have been found to be a result of an increased window current due to a negative shift in voltage dependence of activation, e.g. the p.A204E, p.G213D, and p.L828F mutations (Calloe et al., 2018; Doisne et
al., 2018; Ter Bekke et al., 2018). Apart from an increased window current, the negative shift
in voltage dependence of activation will also lead to increased excitability of the Purkinje cells due to a lowered threshold for Nav1.5 channel activation.
Recently, a novel heterozygous mutation, c.629T>G, was discovered in the SCN5A gene of a Dutch family in which members from multiple generations suffer from MEPPCs (Holl
et al., 2018). This mutation results in substitution of a valine for a glycine at position 210
(p.V210G), and causes a phenotype consisting of sinus, atrial and junctional beats competing with multifocal PVCs and in some cases sudden death. Due to the absence of cellular electrophysiological data, it remains unknown how this mutation in SCN5A affects the function of the NaV1.5 channel, and thus INa. Therefore, the aim in this present study is to elucidate the relation between the p.V210G mutation in the SCN5A gene and MEPPCs by determining the electrophysiological differences between wild-type (WT) and heterozygous mutant NaV1.5 channels expressed in HEK 293T cells. From the MEPCC in patients and the previous findings
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in MEPCC-related SCN5A mutations, a gain of function of p.V210G INa channels is expected due to a negative shift in activation, a positive shift in inactivation, and/or an increased current density. When focusing on the location of the mutation it can be noticed that it is, similar to the known p.A204E mutation, located in segment 3 of domain 1 of the Nav1.5 channel. This suggests similarities in the electrophysiological mechanism underlying the MEPPCs. To determine the electrophysiological characteristics of the novel p.V210G mutation, patch clamp measurements were performed on HEK 293T cells expressing wild type (WT) and p.V210G mutant Nav1.5 channels.
Methods
Cell culture and transfection
HEK 293T cells were cultured in t25 flasks using Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 5% glutamax, supplemented with 1% penicillin and streptomycin, and 10% fetal bovine serum (FBS; Biowest). Cells were incubated at 37ºC in 5% CO2 and split every 3-4 days. The c.629T>G point mutation (p.V210G) was introduced into the WT SCN5A (in a bicistronic GFP vector) by site directed mutagenesis using Quick Change XL kit (Agilent Technologies, Santa Clara, USA) using standard procedures. To express WT or mutant
SCN5A, cells were transfected at a confluency of approximately 70%, with WT or mutant
α-subunit construct (2.5 μg) together with β-α-subunit construct (2.5 μg) using polyethylenimine (PEI: construct 1:3; Sigma-Aldrich) following standard protocols. A gene construct containing green fluorescent protein (GFP) was used to enable identification of transfected cells with an epifluorescent microscope. To obtain cells in suspension, cells were dissociated for 2 minutes in stove (37ºC, 5% CO2) using trypsin (0.05% in DMEM; Gibco), one or two days after transfection. Electrophysiological experiments were performed on single, GFP-positive cells.
Patch clamp experiments
INa in HEK 293T cells was measured in the voltage clamp (VC) mode of the whole-cell configuration of the patch clamp technique, using an Axopatch 200B patch clamp amplifier (Molecular Devices Corporation, Sunnyvale, CA, USA). Micropipettes with a resistance of 2.5-3 MΩ were pulled from borosilicate glass (Harvard Apparatus) and filled with pipette solution consisting of (in mM): 110 CsF, 10 CsCl, 10 NaF, 1 CaCl2, 2 Na2ATP, 11 EGTA, 10 HEPES, pH 7.2 (CsOH). Before making the seal between the pipette and the cell membrane, any pipette offset potential was set to zero. During measurements, cells were perfused constantly, in a custom-made perfusion chamber using bath solution. The bath solution was kept at room temperature (≈21°C) throughout the measurements and consisted of (in mM): 140 NaCl, 10 CsCl, 2 CaCl2, 1 MgCl2, 5 glucose, 10 HEPES, pH 7.4 (NaOH). Custom VC protocols (see
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insets Figures 2A and 3A) were run by accomplishing voltage control, data acquisition, and analysis by means of custom-written software running in the OS9.X operating system on an Apple Macintosh G4 computer. Signals were low-pass filtered with a cutoff frequency of 5 kHz, and recorded at a sampling rate of 20 kHz. Series resistance was electronically compensated by ≥80%. Membrane capacitance of the cell (Cm, in pF) was used to determine cell size, and was obtained by dividing the time constant of the decay of the capacitive transient after a -5 mV step from -40 mV by the series resistance.
A holding potential of -120 mV was used during the VC protocols to determine peak INa current density, voltage dependence of (in)activation, and recovery from inactivation. Average current density of peak INa (in pA/pF), as displayed in the current-voltage (I-V) curve, was determined by dividing the INa amplitude (in pA), defined by the difference between peak and steady-state current, by Cm. Voltage dependence of INa (in)activation was measured using custom VC protocols with a VC cycle length of 5 sec (Figures 2 & 3, insets) and obtained data were corrected for INa driving force and normalized to maximum peak current. To determine the membrane potential of half-maximal (in)activation (V1/2) and the slope factor (k), the steady-state activation and inactivation curves were fit using the Boltzmann equation (I/Imax = A / {1.0 + exp[(V1/2 − V) / k]}), in which I represents the peak current at the membrane potential V, Imax represents the maximum peak current, and A is a dimensionless fitting parameter. A double exponential curve was fitted through the decay phase of the INa current to determine the fast and slow time constants of inactivation (eq: I/Imax=Afast * exp(-t/fast) + Aslow * exp(-t/fast) ), where t denotes time, fast and slow are the time constants of average INa inactivation components, and Afast and Aslow express the mutual distribution of those components in fractional amplitudes. INa recovery from inactivation was measured using a custom VC two-pulse protocol (Figure 5, inset). Peak INa amplitudes in response to the test pulse (P2) were normalized to the associated peak amplitudes at the inactivating pre-pulse (P1) and plotted versus the interpulse interval. The average data were fitted with a double exponential function (I/Imax =Afast * [1.0 − exp(−t/fast] + Aslow × [1.0 − exp(−t/slow)]), where t represents the recovery time interval, fast and slow the time constants of the fast and slow components of recovery, and Afast and Aslow the fractions of the fast and slow components of recovery, respectively.
Statistical analysis
All data are expressed as mean ± SEM. Differences between mutant and WT INa were analyzed with unpaired, two-tailed t-tests or two-way repeated measures ANOVA tests where appropriate. Statistical analyses were carried out with Graphpad Prism 8.2.1. The results were considered statistically significant when p < 0.05.
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Results
p.V210G mutant INa peak current density is increased
Amplitudes of peak INa were measured using a custom VC protocol with a testpulse of 50 ms at membrane potentials ranging from -80 to 65 mV with increments of 5 mV (see Figure 2A, inset). Figure 2A, top, shows typical examples of peak INa measurements, which appear to be larger in mutant channels compared to WT. The speed of activation also appears to be increased in mutant channels. To determine the peak current density, INa amplitudes were divided by cell size (in pF). As shown in Figure 2A, bottom, the mean current density of mutant INa is significantly greater at membrane potentials of 35 mV (-401 ± 44 pA/pF) compared to WT (-123 ± 22 pA/pF) (two-way repeated measures ANOVA, p = 0.011). At 40 mV, density of mutant INa was also significantly greater (-347 ± 36 pA/pF) compared to WT (-110 ± 27 pA/pF) (two-way repeated measures ANOVA, p = 0.011). Lastly, at a membrane potential of 45 mV, mutant INa density was again increased significantly (-300 ± 32 pA/pF) compared to WT (-89 ± 25 pA/pF) (two-way repeated measures ANOVA, p = 0.011). Figure 2A also suggests a larger mutant INa density at other test potentials between −60 and +50 mV, but statistical significance was not obtained at these other potentials. Of note, cell size (Figure 2B) did not
Figure 2. Current-voltage (I-V) relationship of WT and mutant INa. A: Top: Typical examples of INa peak current.
Bottom: INa peak current density in HEK 293T cells at potentials ranging from -80 to 60 mV (n = 5 and n = 7,
respectively). Inset shows custom VC protocol for current density measurements, cycle length was 5 seconds. B: Cell size of WT and P.V210G mutant expressing HEK 293T cells (n = 5 and n = 7, respectively).
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differ significantly between WT and mutant cells (two-tailed t-test, p = 0.56), with values of 7.1 ± 0.8 and 7.9 ± 1.1 pF, respectively. Overall, these results indicate that the p.V210G mutation causes an increased peak INa current density.
The p.V210G mutation causes an increased window current due to a negative shift in voltage dependence of activation
Characteristically, activation of INa starts near -60 mV and peaks near -30 mV, whereafter the reduction in Na+ driving force dominates, causing decline in current amplitude (Figure 2A, open circles). As indicated by filled circles in Figure 2A, mutant INa starts to activate at ~ -70 mV and reaches its peak at -40 mV, i.e. 10 mV more negative than the WT values of −60 and −30 mV, respectively. To verify whether the hyperpolarized INa peak current through p.V210G channels can be attributed to changes in inactivation and/or activation characteristics of the channel, inactivation and activation curves were determined.
Voltage dependence of channel inactivation of WT and mutant was determined using a two-pulse protocol, with a prepulse at potentials ranging from -120 to 0 mV to induce steady-state inactivation, followed by a test pulse of 50 ms at -20 mV (see left inset to Figure 3A). Figure 3A shows the Boltzmann fits to the average data of these measurements. As shown by open squares, WT INa started inactivating at -120 mV and complete inactivation was reached at -60 mV. Inactivation of mutant channels was shown to be unaffected, as inactivation curves were almost completely overlapping with WT (Figure 3A, open and closed squares, respectively). Voltage dependence of activation of INa was determined by recording INa for 50 ms at test potentials ranging from -80 mV to 65 mV with 5 mV increments (see right inset to Figure 3A). Figure 3A shows the Boltzmann fits to the average data of these measurements. As shown in Figure 3A by open and closed circles (WT and mutant, respectively) and listed in Table 2, mutant INa activated at more negative membrane potentials with a WT value of −44.3 ± 1.3 mV versus a mutant V1/2 value of −55.1 ± 2.6 mV (two-tailed t-test, p = 0.008). Based on the negative shift of voltage dependence of activation and unaffected inactivation of mutant INa channels, a change in INa window current is suggested. From the INa (in)activation curves of Figure 3A, the window current was derived to give an overview of the active channels at different test potentials (Figure 3B). As shown in Figure 3B, and in line with the values of V1/2 and k listed in Table 2, the activation curve of the mutant channel displays a hyperpolarized shift compared to WT, while the inactivation curves are overlapping, thus remaining unaffected. As a result, the mutant window current is larger and hyperpolarized compared to WT (light and dark gray area, respectively).
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Speed of inactivation is unaffected by the p.V210G mutation
To characterize the speed of inactivation slow and fast time constants (slow and
fast, respectively) of p.V210G mutant INa decay were determined and compared to WT. Figure 4A shows that both slow and fast tend to decrease with increasing membrane potentials. The fast in mutant INa (Figure 4A, open circles) is increased compared to WT at all test potentials, but this was not found to be significant (two-way repeated measures ANOVA, p > 0.99). Mutant
slow (Figure 4A, open squares) was increased at test potentials ranging from -30 mV to 15Wild type Mutant P-value
Activation V1/2 -44.3 ± 1.3 -55.1 ± 2.6 0.008 * k 4.5 ± 0.3 3.5 ± 0.7 0.3 n 5 7 Inactivation V1/2 -83.3 ± 3.2 -85.9 ± 2.8 0.6 k 4.5 ± 0.1 4.6 ± 0.3 0.7 n 5 7
Table 2. V1/2 and k values of activation and inactivation of WT and mutant INa.
Figure 3. The gating properties of WT and p.V210G mutant channels. A: Steady-state (in)activation curves
of WT and mutant INa (n = 5 and n = 7, respectively). Inset shows custom VC protocol of inactivation (left) and
activation (right), a cycle length of 5 seconds was used. B: Window current of WT and mutant INa (n = 5 and n =
10
mV, which was also found to be nonsignificant (two-way repeated measures ANOVA, p > 0.99). To determine the distribution, amplitudes of fast and slow time constants of p.V210G mutant INa were determined and compared to WT and displayed in Figures 4B (filled circles) and C (filled squares), respectively. The amplitudes of the fast and slow components (Figures 4B and C) did not differ significantly (two-way repeated measures ANOVA, p ≥ 0.31).
Figure 4. The time constants and relative amplitudes of INa inactivation. A: The fast and slow time constant
of inactivation of WT and mutant INa (n = 4 and n = 7, respectively) on a logarithmic timescale (τfast and τslow,
respectively). B: The average relative amplitudes of the fast constant of inactivation (Afast). C: The average relative
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Recovery from inactivation is unaffected by the p.V210G mutation
To complete the characterization of the electrophysiological behavior of the mutant Nav1.5 channel, it was tested whether the p.V210G mutant channel is associated with changes in recovery from inactivation. A two-pulse protocol was used, with a 1000 ms prepulse (P1, -20 mV) to inactivate the channels followed by a 50 ms test pulse (P2, -20 mV) after a variable interval ranging from 1 to 1000 ms after P1, at a recovery potential of -120 mV (see inset to Figure 5). As shown in Figure 5, the degree of recovery from inactivation in WT and mutant (open and filled circles, respectively), as determined by normalizing peak INa during P2 to peak INa during P1, is increasing with increasing time intervals. Both WT and mutant channels show a full recovery from inactivation within 1000 ms. Recovery from inacivation did not differ significantly (two-tailed t-test, p = 0.63)
Discussion
In this study, we provided insights into the underlying mechanism in which MEPPCs arise in patients suffering from Purkinje-related premature contractions of the heart, caused by the novel p.V210G mutation in SCN5A as identified in a Dutch family suffering from MEPPC. The electrophysiological characteristics of the mutant channel, as measured in HEK 293T cells, showed a significant increase of current density and an 11 mV shift towards more negative potentials of voltage dependence of activation, leading to an increased sodium-influx during upstroke and phase 1 of the action potential (AP) as well as activation of channels near the Purkinje cell resting membrane potential. This gain of function is likely to cause
Figure 5. Recovery from WT and mutant INa inactivation. Relative amplitude of WT and mutant peak INa (n =
3 and n = 6, respectively) assessed using interpulse interval ranging from 1 to 1000 ms on a logarithmic time scale. Inset shows custom VC protocol using interpulse intervals ranging from 1 to 1000 ms.
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hyperexcitability of the cardiac Purkinje cells, explaining the premature contractions seen in the phenotype of MEPPC patients.
When comparing the results to earlier research, INa peak current density was, in contrast to other known MEPPC related mutations (Table 1), significantly greater at membrane potentials of 35, 40 and 45 mV (Figure 2). However, it has to be mentioned that both WT (-610 ± 90 pA/pF) and mutant (-1862 ± 469 pA/pF) maximum peak current density was relatively high, as sodium current amplitude ranged from 0 to 21.5 nA, much larger than the range of 0.1 to 2.5 nA required for high quality voltage clamp experiments in the whole cell configuration (Trapani & Korn, 2003). This indicates that transfection of SCN5A in HEK 293T cells was highly efficient, affecting the reliability of the current measurements. In further experiments, a less efficient transfection should be performed by, for example, reducing the transfection time, or changing the ratio of PEI versus DNA construct. Nevertheless, the difference between WT and mutant INa peak current density was significant at membrane potentials of 35, 40 and 45 mV, at which sodium current amplitude is relatively small, allowing reliable measurements. Based on this, the results can be considered conclusive.
In addition to the increase in peak current density, mutant channels showed an 11 mV negative shift in the V1/2 of voltage dependence of activation (Figure 3A, Table 2). This affects window current (Figure 3B) as well as availability of the channels. The negative shift in activation brings the voltage threshold of channel opening to a more negative potential, declining the voltage stimulus essential for depolarization of the cells and thus increasing sodium-influx closer to the resting membrane potential of Purkinje cells. In addition, the increased window current widens the voltage spectrum of channel activation probability. Taken together, this causes a gain of function phenotype, leading to hyperexcitability of the cardiac Purkinje cells, explaining the premature contractions seen in patients suffering from MEPPC. Although window current is visually increased and hyperpolarized, no separate experiments were carried out to directly measure and quantify window current, which made statistical analysis impossible. In future experiments, a custom protocol for window current measurements should be performed to be able to test significance of differences between WT and mutant window current. Though, as window current is a direct derivative of steady-state (in)activation, and steady-state activation shifted significantly towards more negative potentials, whereas no change was observed in steady-state inactivation, it is plausible to state that window current is increased and hyperpolarized. The increase in window current is a common biophysical effect of all known mutations correlated with MEPPC (Table 1), but aforementioned characteristics of the p.V210G mutation show specifically great similarity to the known electrophysiological characteristics of the p.A204E mutation. Earlier research of this mutation showed an 8 mV negative shift in voltage dependence of activation while inactivation remains unchanged, resulting in an increased window current (Doisne et al.,
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2018). In addition, treatment of one patient was started using hydroquinidine, which caused complete disappearance of PVCs. When considering that both p.V210G and p.A204E mutations are located in the same segment of the Nav1.5 channel and show similar electrophysiological characteristics underlying the MEPPCs, these results indicate potential therapeutic applications of hydroquinidine in patients with the V210G (Doisne et al., 2018). Hydroquinidine is a class-1A drug known for its PVC reducing capacities, as well as lessening DCM in patients displaying these symptoms (Laurent et al., 2012). Other options to reduce hyperexcitability of the Purkinje cells in patients with MEPPC-like phenotypes are Class-1C antiarrhythmic drugs. These drugs are also known to decrease PVCs in patients (Hyman et
al., 2018) through blockage of the NaV1.5 channel (Roden, 2014). For example, flecainide is a Class-1C drug found to be highly effective in decreasing the PVC burden in patients suffering from MEPPC (Laurent et al., 2012; Mann et al., 2012; Remme and Wilde, 2014). In addition, amiodarone is also known to reduce PVCs by blocking NaV1.5 channels (Beckermann et al., 2014), and was found to be effective in the p.G213D mutation associated with MEPPC (Calloe
et al., 2018). Research regarding the application of these drugs in treatment of MEPPC
patients harboring the p.V210G mutation remains to be determined.
Fast and slow inactivation of the mutant channel did not show statistically significant differences with WT (Figure 4), but additional experiments should be carried out to allow a firmer conclusion. The absence of a change in kinetics of inactivation is in line with the location of the p.V210G mutation, which is not in a segment that has been associated with inactivation (Figure 1). Recovery from inactivation was not affected in mutant SCN5A channels, indicating no influence from recovery of inactivation on the phenotype of patients suffering from MEPPC carrying the p.V210G mutation. A slowed recovery from inactivation would reduce the sodium current density, while in this study, an increase of current density was found in mutant INa. A faster recovery from inactivation could underly the increase in current density, but due to the cycle length of 5 seconds, while recovery is completed in 1 second, this is unlikely to affect our voltage clamp measurements.
In this study we provided the electrophysiological characterization of the novel mutation in SCN5A, p.V210G, found in a Dutch family in which multiple members have been diagnosed with MEPPC. Treatment of patients with a high PVC burden is necessary to reduce the risk of DCM and sudden death and increase the quality of life, and these results contribute to potentially treating these patients.
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Appendix
Supplementary data
