Overlap Arrhythmia Syndromes Resulting from Multiple Genetic Variations Studied in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for genetic models of cardiac diseases. We report an arrhythmia syndrome consisting of Early Repolarization Syndrome (ERS) and Short QT Syndrome (SQTS). The index patient (MMRL1215) developed arrhythmia-mediated syncope after electrocution and was found to carry six mutations. Functional alterations resulting from these mutations were examined in patient-derived hiPSC-CMs. Electrophysiological recordings were made in hiPSC-CMs from MMRL1215 and healthy controls. ECG analysis of the index patient showed slurring of the QRS complex and QTc = 326 ms. Action potential (AP) recordings from MMRL1215 myocytes showed slower spontaneous activity and AP duration was shorter. Field potential recordings from MMRL1215 hiPSC-CMs lack a "pseudo" QRS complex suggesting reduced inward current(s). Voltage clamp analysis of I_Ca showed no difference in the magnitude of current. Measurements of I_Na reveal a 60% reduction in I_Na density in MMRL1215 hiPSC-CMs. Steady inactivation and recovery of I_Na was unaffected. mRNA analysis revealed ANK2 and SCN5A are significantly reduced in hiPSC-CM derived from MMRL1215, consistent with electrophysiological recordings. The polygenic cause of ERS/SQTS phenotype is likely due to a loss of I_Na due to a mutation in PKP2 coupled with and a gain of function in I_K,ATP due to a mutation in ABCC9.

Keywords: action potentials; depolarization; electrophysiology; sodium current; stem cells.

Figure 1

Representative ECG of MMRL1215 illustrating Early Repolarization Syndrome phenotype. A slurring of the QRS complex is apparent in multiple leads (denoted by the arrows). In addition, a QTc = 326 ms was noted.

Figure 2

(A): Extracellular field potential (EFP) signals recorded in WT (left side) and MMRL1215 hiPSC-CMs (right side). Compared to WT hiPSC, myocytes derived from MMRL1215 showed a complete lack of a pseudo QRS complex, suggesting a major reduction in inward current(s). Panel (B): Action potential (AP) recordings obtained from WT and MMRL1215 monolayers. MMRL1215 hiPSC-CMs had a shorter action potential duration compared to WT.

Figure 3

Line scans recorded from WT and MMRL1215 hiPSC-CMs showing spontaneous Ca²⁺ transients. Fluorescence intensity was much lower in MMRL 1215, and in some myocytes the spontaneous activity was episodic in nature.

Figure 4

Representative traces showing I_Ca recorded from WT (n = 11) and MMRL1215 (n = 9) (A,B). Ca²⁺ currents were recorded during a 300 ms step depolarization from −40 to +50mV. (C) Current-voltage relationship for I_Ca.

Figure 5

Representative whole cell I_Na recordings from WT and MMRL1215 hiPSC-CMs (A,B). Current recordings were obtained at test potentials between −80 and 30 mV (protocol at top of figure). (C): I–V relation for WT (n = 18) and MMRL1215 (n = 25) myocytes. (D): Steady-state activation curves for WT and MMRL1215 myocytes. Data were normalized and plotted against their test potential. * Statistically significant from WT, p < 0.05.

Figure 6

Representative steady-state inactivation recordings from WT and MMRL1215 myocytes (A,B) in response to the voltage clamp protocol (top of the figure). The steady state-inactivation relation for hiPSC-CMs (C) yielded similar mid-inactivation potential for WT (n = 25) and MMRL1215 (n = 29) cells.

Figure 7

Representative recovery from inactivation traces recorded from WT and MMRL1215 myocytes (A,B). Recovery was measured using two identical voltage clamp steps to −20 mV from a holding potential of either −120 mV or −100 mV separated by selected time intervals. (C): Mean data showing recovery time-course of I_Na recorded at −120 mV for WT (n = 24) and MMRL1215 (n = 26). (D): Recovery time-course of I_Na recorded at −100 mV for WT (n = 16) and MMRL1215 (n = 19).

Figure 8

Expression profile of genes PKP2, ANK2 and SCN5A obtained using real-time PCR.