PMC3536519_01
Clinical and genetic profile of the LQTS family. (A) Genotype of SCN5A and KCNH2 of each family member. SCN5A_1473F/F is referred to as WT SCN5A in the text. See text (Results section hERG channel activity in iPSC-CMs) for definitions of KCNH2 variants. (B, top) Surface electrocardiography lead II recorded from the LQT-3 proband before this study. QTc, QT interval corrected for heart rate. (bottom) Example of ICD detection and termination of ventricular tachyarrhythmia in proband.
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Although this regimen improved the patient's condition, it provided an incomplete control of his arrhythmia, as multiple episodes of arrhythmia were still detected daily by his ICD, and numerous shocks by the ICD were required to terminate sustained arrhythmic events (Fig. 1 B). Steady-state availability (inactivation) of Na+ channels in the proband's iPSC-CMs was right shifted compared with channels recorded in the parents' iPSC-CMs (Fig. 5 C and Table 1), whereas activation curves were not changed (Fig. 5 E and Table 1). In addition, Na+ channel recovery from inactivation in the proband's iPSC-CMs was significantly faster than in WT cells (Fig. 5 D and Table 1).
Characterization of iPSC clones from each family member. (A) Karyotype in one clone from each family individual (father, HR-I-15; mother, HR-II-9; proband, OA6 17Cr8). (B) Expression of pluripotency markers in one clone from each family individual (father, HR-I-15; mother, HR-II-9; proband, OA6 17Cr8). (C) Teratoma assay indicated trilineage in vivo differentiation of the proband iPSC clone (OA6 17Cr8). Bars, 200 mum.
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The resulting iPSC clones exhibited a normal karyotype (Fig. 2 A), expressed stem cell markers (Fig. 2 B), and displayed functional pluripotency in teratoma assays (Fig. 2 C).
Action potential phenotypes in iPSC-CMs. (A) In a total of 22 cells, 2 different types of action potentials were recorded: atrial-like (left; mean of two cells) and ventricular-like (right; mean of 20 cells). Action potentials were elicited with 3-ms suprathreshold stimuli at 0.2 Hz. (B) Distribution of CMs according to their action potential phenotype: out of 22 cells, 2 cells were atrial-like and 20 cells were ventricular-like.
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Current clamp showed that iPSC-CMs from all clones were predominantly characterized by what has been referred to as ventricular-like action potentials when paced with suprathreshold current pulses, and we detected the same distribution of action potential waveforms in the iPSC-CMs as we reported previously in human embryonic stem cells (hESCs) derived using the same procedures (Fig. 3).
Na+ channel currents in response to adult ventricular action potential waveforms. (A and B) Adult ventricular action potential waveforms were computed and applied as command waveforms in whole-cell voltage-clamp mode on iPSC-CMs from father (A) and proband (B) using recording solutions to reveal Na+ channel currents. Mexiletine-dissected Na+ channel currents are shown, averaged for four (A) and five (B) cells.
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Imposing voltage-clamp waveforms of adult ventricular muscle action potentials clearly revealed the pathophysiological contribution of mutation increase noninactivating or INaL current apparent in cells from the proband (Fig. 4 B), but not from either parent (Fig. 4 A).
Biophysical properties of Na+ channels in iPSC-CMs. (A) TTX (50 microM)-sensitive averaged Na+ current traces recorded from father (WT, n = 27, three clones; left) and proband (LQT-3, n = 41, clone OA6 9Cr8; right) iPSC-CMs at -10 mV (100-ms pulses at 0.2 Hz from a -90-mV holding potential). Dashed lines, zero current. (insets) INa normalized to peak current reveal the presence of INa late current (INaL; arrow) in LQT-3 but not in WT iPSC-CMs. (B) Percentage of INaL with respect to peak current measured in three clones from the father, two clones from the mother, and three clones from the proband. (All three LQT-3 clones are P < 0.01 vs. each WT clone). The number of cells tested is indicated above each bar. (C) Steady-state availability in LQT-3 (proband, n = 27, averaged from two clones) and WT iPSC-CMs (father, n = 23, averaged from three clones; mother, n = 3, one clone; both are P < 0.001 vs. LQT-3). For each individual, data obtained from each clone are detailed in Table 1. (D) Recovery from inactivation in LQT-3 (proband, n = 6, clone OA6 9Cr8) and WT iPSC-CMs (father, n = 4, clone HR-I-2R 2Cr). Half-time in WT iPSC-CMs is P < 0.01 versus LQT-3. (E) Activation curve in LQT-3 (proband, n = 3, clone OA6 9Cr8) and WT iPSC-CMs (mother, n = 3, clone HR-II-9). V1/2 in WT iPSC-CMs is not significantly different from LQT-3. Data are shown as means +- SEM.
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We then used square wave voltage-clamp protocols designed to reveal critical biophysical properties of these channels in multiple iPSC clones of both parents and the proband (Fig. 5). A mutation-dependent increase in INaL was detected in all three clones from the proband and none of the parents' clones (Fig. 5, A and B; and Table 1). Steady-state availability (inactivation) of Na+ channels in the proband's iPSC-CMs was right shifted compared with channels recorded in the parents' iPSC-CMs (Fig. 5 C and Table 1), whereas activation curves were not changed (Fig. 5 E and Table 1). In addition, Na+ channel recovery from inactivation in the proband's iPSC-CMs was significantly faster than in WT cells (Fig. 5 D and Table 1).
hERG K897T polymorphism does not alter biophysical properties of hERG channels in iPSC-CMs. (A) IKr currents recorded from father (897K/K), mother (897T/T), and proband (897K/T) iPSC-CMs during 2-s pulses (0.1 Hz) to -40, -10, and 20 mV and for 2 s after return to a -40-mV holding potential. Dashed lines, zero current. (B and C) Current versus test pulse voltage. (B) Current measured at 2 s during test pulse (n = 4-15). (C) Normalized tail current measured at -40 mV (n = 12-22). (D) Bar graph summary showing IKr midpoint of activation (V1/2) for each clone tested and calculated from a Boltzmann fit of normalized tail current. Data are shown as means +- SEM.
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We also compared these results with previously measured hERG activity in hESC-CMs. hERG channel activity (IKr) was readily detected as E4031-sensitive current (Fig. 6 A) with hallmark rectification in all clones from each family member, as can be seen in the current traces as well as the I-V curves illustrated in the figure (Fig. 6 B). IKr properties, neither rectification that is seen in the I-V curves (Fig. 6 B) nor in the voltage dependence of activation (Fig. 6, C and D), was observed among all family members and all clones. Importantly, the properties of hERG channels recorded in the iPSC-CMs were remarkably similar to those measured in hESC-CMs that had been differentiated using the same protocols with no significant differences between IKr properties in hESC-CMs and IKr in any of the iPSC-CM lines (Fig. 6).
Mexiletine corrects F1473C-altered Na+ channel inactivation. (A and B, top) Averaged INa current traces recorded at -10 mV (100-ms pulses applied at 0.2 Hz from a -90-mV holding potential) in father (A; WT, n = 5, clone HR-I-2R 2Cr) and proband (B; LQT-3, n = 18, clone OA6 9Cr8) iPSC-CMs in control conditions (gray traces and arrows) and in the presence of 50 microM mexiletine (black traces and arrows) at low and high (insets) gain. (insets) INa at high gain shows the absence of mexiletine-sensitive INaL in father's iPSC-CMs (A) but reveals the presence of INaL in LQT-3 iPSC-CMs, which was significantly blocked by 50 microM mexiletine (B, arrow). (A and B, bottom) Steady-state availability in the absence (open symbols) and presence (closed symbols) of 50 microM mexiletine in WT (A; n = 5, clone HR-I-2R 2Cr) and LQT-3 (B; n = 8, clone OA6 9Cr8) cells. Data are shown as means +- SEM.
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Fig. 7 compares the effects of mexiletine on Na+ channel currents from one of the parent's (Fig. 7 A, WT channels) and the proband's (Fig. 7 B) iPSC-CMs. Recordings are illustrated at low and high (Fig. 7, A and B, insets) gains. INaL for WT channels (Fig. 7 A, top). In addition, we also found that there was no difference in mexiletine block of INa peak in F1473C (see aforementioned data) and WT (14.5 +- 1.9%; n = 5) iPSC-CMs. V1/2 = -71.5 +- 0.8 mV with mexiletine; n = 5, P < 0.05; Fig. 7 A, bottom) and iPSC-CMs from the proband (V1/2 = -58.5 +- 1.6 mV in control vs. V1/2 = -64.1 +- 1.9 mV with mexiletine; n = 8, P < 0.05; Fig. 7 B, bottom). In the case of proband iPSC-CMs, the drug-induced shift rescues inactivation voltage dependence of drug-free iPSC-CM Na+ channels from the parents (compare closed symbols in Fig. 7 B with open symbols in Fig. 7 A).
Mexiletine blocks IKr channels. (A, top) IKr current traces recorded in father and proband iPSC-CMs during a 2-s depolarizing test pulse to 30 mV followed by a 2-s repolarizing pulse to -40 mV (holding potential was -40 mV) at 0.1 Hz in control conditions (black traces and arrows) and in the presence of 50 microM mexiletine (gray traces and arrows). 30 microM chromanol 293B was added to block IKs. (bottom) Mexiletine (50 microM)-sensitive potassium currents in iPSC-CMs subtracted from the top recordings. Dashed lines, zero current. (B) 50 microM mexiletine significantly blocked IKr tail current at -40 mV in iPSC-CMs from the father (WT = 897K/K, P < 0.05 vs. control) and proband (897K/T, P < 0.05 vs. control). Data are shown as means +- SEM.
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We found mexiletine inhibits IKr in cells from both the father (897K/K) and proband (897K/T; Fig. 8 A) with no significant impact of the K897T polymorphism on mexiletine block (Fig. 8 B).
Impact of stimulation frequency on INaL and IKr in the absence and presence of drugs. (A) Averaged sodium current traces recorded at -10 mV (100-ms pulses applied from a -90-mV holding potential) in LQT-3 iPSC-CMs in control conditions (left; n = 5) and in the presence of 50 microM mexiletine (right; n = 5) at 0.2 Hz (gray traces and arrows) and at 1.6 Hz (black traces and arrows) at low and high (insets) gain. Dashed lines, zero current. (B) Percent block of INa peak current (INa peak) and INa late current (INaL) at 0.2 and 1.6 Hz by 50 microM mexiletine alone (n = 6) and 50 microM mexiletine plus 5 microM flecainide (n = 3). One-way ANOVA followed by Tukey's test: *, P < 0.01 versus paired INa peak; #, P < 0.01 versus INa peak at 0.2 Hz in the presence of mexiletine; delta, P < 0.01 versus INaL at 0.2 Hz in the presence of mexiletine. (C) Block of IKr channels by 50 microM mexiletine (Mex) and 5 microM flecainide (Flec) at 1.6 Hz in WT iPSC-CMs depolarized to 30 mV for 200 ms, from a holding potential of -40 mV, and repolarized at -40 mV for 200 ms. (D) Change in IKr tail current density measured at -40 mV after 200-ms test pulses at 30 mV in control condition (at 0.2 and 1.6 Hz), in the presence of 50 microM mexiletine and after the addition of 5 microM flecainide. *, P < 0.05 versus paired control; #, P < 0.05 versus mexiletine at 1.6 Hz. Data are shown as means +- SEM.
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Even in the absence of drug, increasing the pulse rate from 0.2 to 1.6 Hz resulted in a 50% decrease in INaL, and when mexiletine was added, INaL was reduced further to <15% of drug-free values recorded at the slower pulse rate (Fig. 9, A and B). We found that addition of the second drug, flecainide, had no significant further increase in INaL block at the higher pulse frequency but further reduced INa peak (Fig. 9 B), predicting little clinical benefit by the addition of the second Na+ channel blocker. The rate-dependent increase in mexiletine reduction of INaL was not accompanied by changes in mexiletine IKr block, but further addition of flecainide more than doubled pro-arrhythmic IKr inhibition (Fig. 9, C and D).