Susceptibility to Ventricular Arrhythmias Resulting from Mutations in FKBP1B, PXDNL, and SCN9A Evaluated in hiPSC Cardiomyocytes

Figures

• 

  Figure 1. (a) ECG analysis of patient MMRL 1126 showing early repolarization phenotype. A prominent ST segment elevation and slurring of the QRS complex is apparent in several leads. (b) Infusion of dobutamine caused the appearance of monomorphic VT in MMRL1126 which persisted after dobutamine was discontinued. (c) The arrhythmogenic focus was ablated and patient subsequently exhibited RBBB apparent in lead V1. (d) Patient was challenged with ajmaline resulting in unmasking of BrS.

• 

  Figure 2. DNA sequencing analysis of SCN9A and FKBP1b. The FKBP1b gene was mutated to a C deletion at location 137 in exon 3 resulting in complexed amino acid changes. The amino acid change was proline to leucine at position 46; the next 22 amino acids were framed shifted culminating in a stop codon 22 amino acides downstream (P46L fsx22). The SCN9A gene was mutated from a T to C substitution at position 3253 in exon 17, resulting in an amino acid change from serine to proline at position 1085 (S1085P). The PXDNL gene was mutated from a G to A at position 1172 in exon 14, resulting in an amino acid change from arginine to glutamine at position 391 (R391Q). Mutations in SCN9A and FKBP1b are highly conserved across the various species. PXDNL is only expressed in certain mammals including primates.

• 

  Figure 3. Pedigree of the proband (denoted with arrow) and family. Genetic analysis revealed that each parent carried different heterozygous mutation(s). The father (1239) carried the FKBP1b mutation, whereas the mother (1240) carried the PXDNL and SCN9A mutations. The sister of the proband (1241) only inherited the mutation in SCN9A. Both parents and the sister were asymptomatic.

• 

  Figure 4. Line scans recorded from WT, MMRL 1239, and MMRL1126 hiPSC-CMs showing spontaneous Ca2+ transients in all hiPSC-CMs. However, fluorescence intensity was much lower in MMRL 1126, and spontaneous activity was episodic in nature.

• 

  Figure 5. Action potential (AP) recordings obtained from WT and MMRL1126 monolayers. WT hiPSC-CMs were more hyperpolarized and had a larger Vmax. In addition, hiPSC-CMs from MMRL1127 exhibited slower spontaneous activity, and some showed episodic activity.

• 

  Figure 6. Representative INa recordings from a WT (a) and MMRL1126 hiPSC-CM (b). Current recordings were obtained at test potentials between −80 and 25 mV in 5 mV increments. The holding potential was −120 mV. (c) I–V relation for WT (n = 13) and MMRL1126 (n = 14) showing no difference in INa magnitude. (d) Steady-state activation relation for WT and MMRL1126 hiPSC-CM cells. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in (a) and (b). Data were normalized and plotted against their test potential. Representative steady-state inactivation recordings from a WT (e) and MMRL1126 hiPSC-CM (f). The voltage clamp protocol is shown at the top of the figure. Peak current was normalized to their respective maximum values and plotted against the conditioning potential. The mean data for the steady-state inactivation relation is shown (g).

• 

  Figure 7. RT-qPCR analysis of SCN9A and SCN5A in hiPSC-CMs (normalized to GAPDH). SCN5A message was highly expressed, whereas levels of SCN9A were negligible in the hiPSC-CMs (a). RT-qPCR analysis of SCN9A and SCN5A in TSA201 cells transfected with SCN5A and SCN9A plasmid at a 1 : 1 molar ratio. SCN9A message could readily be detected in transfected TSA201 cells (b). RT-qPCR analysis of PXDN and PXDNL in hiPSC-CMs (c). PXDNL was expressed at low levels in both WT and MMRL1126 hiPSC-CMs.

• 

  Figure 8. (a) Representative INa recordings from a WT and R391Q mutant in PXDNL cotransfected with SCN5A. I–V relation for WT and R391Q mutant in PXDNL cotransfected with SCN5A. The I-V relation was generated using same voltage clamp protocol described in Figure 5. A small reduction in INa density was noted with R391Q mutation. Representative Ito recordings from a WT and R391Q (a) mutant in PXDNL cotransfected with KCND3. Current recordings were obtained at test potentials between −60 and 50 mV from a holding potential was −80 mV. (c) I–V relation for WT and R391Q showing a significant difference in Ito magnitude at potentials above -20 mV.

• 

  Figure 9. Representative traces showing ICa recorded from (a) WT and (b) MMRL1126. Ca2+ currents were recorded during a 300 ms step depolarization from -40 to +50 mV in 10 mV increments. (c) Current-voltage relationship for ICa. (d) RT-PCR showing a reduction of Cav1.2 and Cav1.3 message in MMRL1126.