Disease Phenotypes and Mechanisms of iPSC-Derived Cardiomyocytes From Brugada Syndrome Patients With a Loss-of-Function SCN5A Mutation

Abstract

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of I _Na density, a 69.5% reduction of Na_V1.5 expression, and the impaired localization of Na_V1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The I _to in BrS-CMs was significantly augmented, and the I _CaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of I _to in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Keywords: Brugada syndrome; SCN5A mutation; depolarization; disease modeling; induced pluripotent stem cells; repolarization.

FIGURE 1

The expression of cardiac-specific proteins in Ctrl- and BrS-CMs. (A) Immunostaining of Ctrl- and BrS-CMs for cardiac markers cTnT. Scale bar, 20 μm. (B) Double-immunostaining of α-actinin and Na_V1.5. Scale bar, 20 μm. (C) Double-immunostaining of Cx43 and α-actinin. Scale bar, 20 μm. (D) Double-immunostaining of Ctrl- and BrS-CMs with antibodies against Cx43 and Na_V1.5. Scale bar, 20 μm. (E) Shown are original western blots for the decoration of Na_V1.5 α subunit, Cx43, Ca_V1.2, K_V4.3, and cTnT. The arrows represent the target bands used for quantification. Quantitative analysis of Na_V1.5 α subunit (F), Cx43 (G), Ca_V1.2 (H), and K_V4.3 (I) expression (normalized to cTnT). Data are presented as mean ± SEM. Two-tailed unpaired Student’s t-test was used for statistical analysis: #P < 0.01.

FIGURE 2

Field potential and conduction velocity of Ctrl- and BrS-CMs. (A) Represented original field potential traces of Ctrl2- and BrS1-CMs were enlarged to show PPA, PPD, and PPS. (B) Represented beats of Ctrl2- and BrS1-CMs were dissected to 10 frames (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 ms), respectively. The width and length of the electrodes distributed area were 1.43 mm. The metrics of MEA measurements including beating frequency (C), PPA (D), PPD (E), PPS (F), CV (G), and normalized SD of inter-beat interval (H) in Ctrl-CMs, BrS1-CMs, and BrS2-CMs. Gray triangle represents Ctrl1 and gray rectangle represents Ctrl2. Data are mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis: ^∗P < 0.05, §P < 0.001. PPA, peak-to-peak amplitude; PPD, peak-to-peak duration; PPS, peak-to-peak slope; CV, conduction velocity; SD, standard deviation.

FIGURE 3

I_Na characterization in Ctrl- and BrS-CMs. (A) Examples of original I_Na traces elicited by 5 mV-step depolarization from −95 to 5 mV at a holding potential of −100 mV. Left, Na⁺ current in one Ctrl1-CM. Right, Na⁺ current in one BrS1-CM. (B) Average I–V relationships of the I_Na in Ctrl- and BrS-CMs. The protocol is shown as inset. (C) Current–voltage relationships normalized to the maximum peak I_Na show a positive shift of the peak I_Na in BrS1- and BrS2-CMs compared to Ctrl1- and Ctrl2-CMs. No differences were observed between two controls or two BrS. (D) Average of the steady-state voltage dependence of activation of the I_Na in Ctrl- and BrS-CMs. The protocol is shown as inset. (E) Average of the steady-state voltage dependence of inactivation of I_Na in Ctrl- and BrS-CMs. The protocol is shown as inset. (F) Average of the recovery from inactivation of I_Na in Ctrl- and BrS-CMs. The protocol is shown as inset. (G) Average current–voltage relationships of the I_Na in Ctrl-CMs with and without 2.5 or 5 μM TTX treatment. Protocol is shown as inset. (H) Average steady-state voltage dependence of activation in Ctrl-CMs treated with and without 2.5 μM TTX. Protocol is shown as inset. (I) V_max in Ctrl-CMs with and without 2.5 or 5 μM TTX treatment. Data are presented as mean ± SEM. Two-way repeated measures ANOVA (B–H) and one-way ANOVA followed by Tukey’s post hoc test were used for statistical analysis: ^∗P < 0.05, #P < 0.01, §P < 0.001.

FIGURE 4

I_CaL and I_to characterization of Ctrl- and BrS-CMs. (A) Examples of original I_to traces elicited by 10 mV-step depolarization from −40 to 60 mV at a holding potential of −90 mV. Top, I_to in one Ctrl1-CM. Bottom, I_to in one BrS1-CM. (B) Average I–V relationships of the I_to current in Ctrl- and BrS-CMs. Protocol is shown as inset. (C) Average of the steady-state voltage dependence of inactivation I_to current in Ctrl- and BrS-CMs. The pulse protocol is shown as inset. No significant differences were observed. The I_to current density in Ctrl-CMs (D) and BrS-CMs (E) with and without 4-AP (1 mM) treatment for 1 min. (F) Average of recovery from inactivation of I_to current in Ctrl- and BrS-CMs. The pulse protocol is shown as inset. No significant differences were observed. Expression profile of I_to-related genes: KCND3 (G), KCND2 (H), KCNA4 (I), and TNNT2 (J). Data were presented relative to RPL32 expression (Ctrl: n = 6, BrS1: n = 6, BrS2: n = 6 independent differentiation experiments). (K) Examples of original I_CaL traces of a Ctrl2-CM and a BrS1-CM. (L) Average I–V relationships of the I_CaL in Ctrl-, BrS1-, and BrS2-CMs. The protocol is shown as inset. (M) The steady-state of activation (G/G_max) and steady-state of inactivation (I/I_max) are overlaid to show the window current of I_CaL. Window currents are the areas under the intersecting current–voltage curves. The impulse for inactivation is shown as inset. (N) The probability being within I_CaL window current is plotted. (O) Average of recovery from inactivation of I_CaL in Ctrl-, BrS1-, and BrS2-CMs. The pulse protocol is shown as inset. Data are presented as mean ± SEM. Two-way repeated measures ANOVA was used for I_to and I_CaL statistical analysis. One-way ANOVA was used for gene expression analyses. ^∗P < 0.05, #P < 0.01, §P < 0.001.

FIGURE 5

Action potential characterization of Ctrl- and BrS-CMs. (A) Representative traces of spontaneous action potentials (APs) measured in ventricular-, atrial-, and nodal-like CMs. (B) V_max in ventricular- and atrial-like BrS- and Ctrl-CMs. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis: #P < 0.01, §P < 0.001. (C) Representative traces of spontaneous AP recordings from Ctrl- and BrS-CMs. (D) Percentage of Ctrl- and BrS-CMs with spontaneously (ar)rhythmic beating. Two-tailed Fisher’s exact test was used for statistical analysis.

FIGURE 6

Effects of PDE inhibitor cilostazol or milrinone on electrophysiological properties of Ctrl- and BrS-CMs. Average current–voltage relationships of I_to in Ctrl-CMs (A), BrS1-CMs (B), and BrS2-CMs (C) with and without cilostazol (10 μM) or milrinone (2.5 μM) treatment for half an hour. Data are presented mean ± SEM. (D) Representative original action potential (AP) traces of Ctrl- and BrS-CMs after 10 μM cilostazol treatment. (E) Percentages of Ctrl- and BrS-CMs with rhythmic and arrhythmic APs after 10 μM cilostazol treatment. (F) Representative original AP traces of Ctrl- and BrS-CMs after 2.5 μM milrinone treatment. (G) Percentages of Ctrl- and BrS-CMs with rhythmic and arrhythmic APs after 2.5 μM milrinone treatment. Average I–V curves of I_Na (automated patch-clamp) in Ctrl-CMs (H), BrS1-CMs (I), and BrS2-CMs (J) with and without 2.5 μM milrinone treatment for half an hour. No statistically significant differences were observed. (K) Quantitative analysis of the conduction velocity in Ctrl-, BrS1-, and BrS2-CMs with and without cilostazol (10 μM) treatment. No statistically significant differences were observed. (L) Quantitative analysis of the conduction velocity in Ctrl-, BrS1, and BrS2-CMs with and without 2.5 μM milrinone treatment. No statistically significant differences were observed. Two-way repeated measures ANOVA (A–C,H–J) and two-tailed paired Student’s t-test (K,L) were used for statistical analysis: ^∗P < 0.05, #P < 0.01, §P < 0.001.