Divergent effects of adrenaline in human induced pluripotent stem cell-derived cardiomyocytes obtained from hypertrophic cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiac disease that affects the heart muscle with diverse clinical outcomes. HCM can cause sudden cardiac death (SCD) during or immediately after mild to rigorous physical activity in young patients. However, the mechanism causing SCD as a result of exercise remains unknown, but exercise-induced ventricular arrhythmias are thought to be responsible for this fatal consequence. To understand the disease mechanism behind HCM in a better way, we generated patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We extensively investigated the effects of low to high concentrations of adrenaline on action potential characteristics, and the occurrence of arrhythmias in the presence of various concentrations of adrenaline and in wash-out condition. We classified and quantified different types of arrhythmias observed in hiPSC-CMs, and found that the occurrence of arrhythmias was dependent on concentrations of adrenaline and positions of mutations in genes causing HCM. In addition, we observed ventricular tachycardia types of arrhythmias in hiPSC-CMs carrying the TPM1-Asp175Asn mutation. We additionally examined the antiarrhythmic potency of bisoprolol in HCM-specific hiPSC-CMs. However, bisoprolol could not reduce the occurrence of arrhythmias during administration or during the wash-out condition of adrenaline in HCM-specific hiPSC-CMs. Our study demonstrates hiPSC-CMs as a promising tool for studying HCM. The experimental design used in this study could be suitable and beneficial for studying other components and drugs related to cardiac disease in general.

Keywords: Adrenaline; Arrhythmia; Bisoprolol; HCM; hiPSC-CMs.

Fig. 1.

Voltage-gated ionic currents in ventricle-like WT-CMs, HCMT-CMs and HCMM-CMs. (A) Current-voltage (I-V) relationship of L-type calcium current (I_Ca) (above) and voltage clamp protocol used (below). P<0.05 from −10 mV to 60 mV (WT-CMs versus HCMT-CMs, one-way ANOVA, post hoc Tukey test). P<0.05 from 10 mV to 60 mV (WT-CMs versus HCMM-CMs, one-way ANOVA, post hoc Tukey test). (B) I-V relationship of transient outward potassium current (I_to) (above) and voltage clamp protocol used (below). P<0.05 from 30 mV to 70 mV (WT-CMs versus HCMT-CMs and WT-CMs versus HCMM-CMs; one-way ANOVA, post hoc Tukey test). (C) I-V relationship of inward rectifier outward current (I_K1) (above) and voltage clamp protocol used (below) (ns at all test potentials, WT-CMs versus HCMT-CMs and WT-CMs versus HCMM-CMs; one-way ANOVA, post hoc Tukey test). Data are mean±s.e.m. Values inside parentheses represent the number of CMs used.

Fig. 2.

Classification of arrhythmias recorded in ventricle-like hiPSC-CMs. (A) Representative AP recording with DADs (Aa), its first derivative (Ab), its phase plot (Ac) and percentage of cells exhibiting DADs (Ad). (B) Representative AP recording with phase-3 EAD (Ba), its first derivative (Bb), its phase plot (Bc) and percentage of cells exhibiting phase-3 EAD (Bd). (C) Representative AP recording with burst EAD (Ca), its first derivative (Cb), its phase plot (Cc) and its occurrence, calculated as number of bursts/total number of cells (Cd). (D) Representative AP recording with QES-EAD (Da), its first derivative (Db), its phase plot (Dc) and its occurrence, calculated as number of QES-EADs/total number of cells (Dd). (E) Representative AP recording with VT-EAD (Ea), its first derivative (Eb), its phase plot (Ec) and percentage of cells exhibiting VT (Ed). Arrowheads indicate the corresponding arrhythmias in APs. Dashed line represents 0 mV. (For more detail, see Fig. S3.)

Fig. 3.

Representative AP profile of VT arrhythmias recorded in HCMT-CMs. (A) AP with non-sustained ventricular tachycardia (NSVT, duration <30 s) (B) non-recovered ventricular tachycardia (NRVT) and (C) sustained ventricular tachycardia (SVT, >30 s). Dashed line represents 0 mV.

Fig. 4.

Summary of variabilities in hiPSC-CMs. (A-F) Comparison of SD1, SD2, SDRR, SDSD, STV-APD50 and STV-APD90 in WT-CMs (n=63), HCMT-CMs (n=154) and HCMM-CMs (n=79). Data are mean±s.e.m. *P<0.05, **P<0.005 and ***P<0.0001; one-way ANOVA, post hoc Tukey test). (G-L) Representative AP traces (G,J), with superimposed 30 APs (H,K) and Poincaré plots of these APs from hiPSCs exhibiting low (black circles) and high (maroon circles) variabilities (I,L). Dashed line represents 0 mV. (For more detail, see Figs S6-S9.)

Fig. 5.

Superimposed traces representing the effects of different concentrations of adrenaline on AP characteristics. (A-F) Percentage change in BPM (A), APD50 (B), APD90, (C) MDP, (D) APA (E) and dV/dt (F), with respect to pre-adrenaline administration condition in WT-CMs, HCMT-CMs and HCMM-CMs. One concentration of adrenaline was tested at a time. Data are mean±s.e.m. *P<0.05, WT versus HCMT or HCMM; ^#P<0.05, HCMT versus HCMM; one-way ANOVA, post hoc Tukey test. Table shows the number of cells used in each group at different adrenaline concentrations. (For more detail, see Tables S4-S7.)

Fig. 6.

Superimposed traces representing the effects of different concentrations of adrenaline on beat rate and repolarization variabilities. (A-F) Percentage change in SD1 (A), SD2 (B), SDRR (C), SDSD (D), STV-APD50 (E) and STV-APD90 (F) with respect to pre-adrenaline administration condition in WT-CMs, HCMT-CMs and HCMM-CMs. Data are mean±s.e.m. *P<0.05, WT-CMs versus HCMT-CMs or HCMM-CMs; ^#P<0.05, HCMT-CMs versus HCMM-CMs; one-way ANOVA, post hoc Tukey test. Table shows the number of cells used in each group at different adrenaline concentrations. (For more detail, see Tables S13-15.)

Fig. 7.

Treatment of HCMT-CMs with bisoprolol. (A-E) Quantification of the occurrence of arrhythmia in the presence of 0.5 nM adrenaline (0.5 nM A), with addition of 1 μM (0.5 nM A+1 μM B) and 10 μM (0.5 nM A+10 μM B) bisoprolol. (F-I) Summary of SD1, SD2, STV-APD50 and STV-APD90 in the presence of 0.5 nM adrenaline with addition of 1 μM and 10 μM bisoprolol (n=14 for 0.5 nM A, n=4 for 0.5 nM A+1 μM B and n=7 for 0.5 nM A+10 μM B). (J-N) Quantification of the occurrence of arrhythmia during the wash-out condition of 0.5 nM adrenaline with 0 μM (no B), 1 μM (1 μM B) and 10 μM (10 μM B) bisoprolol (n=24 for 0.5 nM A/no B, n=9 for 0.5 nM A+1 μM B/1 μM B and n=15 for 0.5 nMA+10 μM B/10 μM B).

Fig. 8.

Treatment of HCMM-CMs with bisoprolol. (A-D) Quantification of the occurrence of arrhythmia in the presence of 10 nM adrenaline (10 nM A), with addition of 1 μM (10 nM A+1 μM B) and 10 μM (10 nM A+10 μM B) bisoprolol. (E-H) Summary of SD1, SD2, STV-APD50 and STV-APD90 following the administration of 10 nM A, 10 nM A+1 μM B and 10 nM A+10 μM B (n=12 for 10 nM A; n=17 for 10 nM A+1 μM B; and n=12 for 10 μM B). (I-L) Quantification of the occurrence of arrhythmias during wash-out condition of 10 nM A with 0 μM (no B), 1 μM (1 μM B) and 10 μM (10 μM B) bisoprolol (n=18 for 10 nM A/no B, n=25 for 10 nM A+1 μM B/1 μM B and n=15 for 10 nM A+10 μM B/10 μM B).