Long QT Syndrome KCNH2 Variant Induces hERG1a/1b Subunit Imbalance in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

[Figure: see text].

Keywords: electrophysiology; ion channel; long QT syndrome; phenotype; stem cell.

Figure 1.

hERG1-H70R patient-specific iPSCs. A. Schematic representation of hERG1a and hERG1b isoforms with the H70R variant located in the N-terminal PAS domain of hERG1a. B. Schematic of genomic structure of KCNH2 encoding hERG1a and hERG1b with sequence analysis of PCR-amplified genomic DNA from iPSCs (hERG1-H70R, hERG1-H70R^corr, and DF 19–9-11 T). The heterozygous missense variant c.209 A>G is detected in exon 2 of KCNH2 gene (c.209A>G, NM_000238.3), resulting in an H70R substitution only in the hERG1-H70R iPSCs line. C. Electrocardiogram of the hERG1-H70R LQT2 patient showing prolonged QTc interval (507 ms). D. Ventricular arrhythmia torsades de pointes recorded by ICD from hERG1-H70R patient. E. Immunofluorescence analysis of pluripotency markers SSEA4 (red), OCT4 (green) and NANOG (magenta) in a representative hERG1-H70R clone and hERG1-H70R^corr, with nuclear staining (DNA, blue). Scale bars: 100 μm.

Figure 2.

Characterization of iPSC-CMs from hERG1-H70R, hERG1-H70R^corr and DF19–9-11T. A. Immunofluorescence images of hERG1-H70R, hERG1-H70R^corr, DF19–9-11T iPSC-CMs immunolabeled with antibodies to α-actinin (red) and cardiac troponin (cTnT, green). Scale bars: 100 μm. B. Representative action potentials from single iPSC-CM paced at 1Hz (Temp 36 ± 1°C). C. D. Action potential parameters for action potential amplitude (APA), maximum diastolic potential (MDP), action potential duration at 30, 50, 70, 80, and 90% of repolarization (APD30, APD50, APD70, APD80 and APD90) for individual cells from the three groups as indicated; mean (thick bar) ± S.D. (thin bars); * p<0.05 for indicated comparisons.

Figure 3.

Reduced I_Kr with accelerated deactivation in hERG1-H70R iPSC-CMs. A. Representative whole-cell voltage clamp current traces from hERG1-H70R, hERG1-H70R^corr and DF19–9-11T iPSC-CMs elicited upon 4 s depolarizing voltage steps to −40mV, −20mV, 0mV, +10mV and +20mV before and after the application of 1 μM E-4031. The E-4031-sensitive current is defined as I_Kr. Inset: voltage protocol. B. C. D. Average current–voltage relationships for I_Kr measured at the end of the test pulses (B), peak tail I_Kr resulting from repolarization to −40 mV (C), and peak tail I_Kr normalized to the maximal current following repolarization to −40mV from iPSC-CMs. Values are represented as mean ± S.D, hERG1-H70R(n=14), hERG1-H70R^corr (n=9), DF19–9-11T (n=9); Temp 36 ± 1°C, * p<0.05 for hERG1-H70R relative to hERG1-H70R^corr or DF19–9-11T. E. Representative tail I_Kr at −40 mV measured after a depolarizing step to +20mV from iPSC-CMs. F. G. H. Fit I_Kr tail current deactivation at −40 mV following depolarizing step to +20 mV to a bi-exponential decay function for fast time constant, τ_fast (F); slow time constant, τ_slow (G); and fraction of fast deactivating I_Kr (H) from hERG1-H70R (n=9), hERG1-H70R^corr (n=8), and DF19–9-11T (n=7) iPSC-CMs. Values are for single cells with mean (thick bar) ± S.D. (thin bars); Temp 36 ± 1°C; ‡ p<0.005.

Figure 4.

Heterologous expression of hERG1-H70R shows reduced I_hERG without changing deactivation kinetics. A. Representative current traces for I_hERG from HEK293 transfected with equal total plasmid amounts encoding hERG1a (WT), hERG1a(H70R), and hERG1a (WT+H70R) homomeric channels as well as hERG1a (WT)/1b and hERG1a (H70R)/1b heteromeric channels. B. C. D. Average current–voltage relationships for I_hERG, measured at the end of the test pulses (B), peak tail I_hERG resulting from repolarization to −50 mV (C), and peak tail current normalized to the maximal current following repolarization to −50mV. Values are represented as mean ± S.D. E. Representative deactivation current traces I_hERG from hERG1a (WT), hERG1a (H70R), hERG1a (WT+H70R) homomeric channels and hERG1a (WT) /1b, hERG1a (H70R)/1b heteromeric channels. F. G. H. Fit I_hERG tail current deactivation at −40 mV following depolarizing step to +40 mV to a bi-exponential decay function for fast time constant, τ_fast (F); slow time constant, τ_slow (G); and fraction of fast deactivating I_hERG (H) from hERG1a (WT), hERG1a (H70R), hERG1a (WT+H70R) homomeric channels and hERG1a (WT)/1b, hERG1a (H70R)/1b heteromeric channels. Individual cell values are plotted with mean (thick bar) ± S.D. (thin bars). hERG1a (WT): n=5, hERG1a(H70R): n=7, hERG1a(WT+H70R): n=7, hERG1a(WT)/1b: n=6; hERG1a(H70R)/1b: n=8, * p<0.05.

Figure 5.

hERG1a and hERG1b expression in hERG1-H70R, hERG1-H70R^corr and DF19–9-11TiPSC-CMs. A. qRT-PCR analysis of hERG1a, hERG1b and KCNQ1 mRNA from hERG1-H70R, hERG1-H70R^corr, and DF19–9-11T iPSC-CMs. Expression values are relative to those of hERG1-H70R^corr, normalized to GAPDH, mean (thick bar) ± S.D. (thin bars); n = 4, * p<0.05. B. Representative immunoblots of hERG1subunit proteins from hERG1-H70R, hERG1-H70R^corr and DF19–9-11T iPSC-CMs with 3 independent experiments (Set1, Set2, Set3). Complex-glycosylated and core glycosylated hERG1 (hERG1a:155 /135 kDa, hERG1b: 90/80 kDa respectively) are indicated. β-actin is shown as a loading control. C. D. Immunoblot densitometry determined relative expression levels of complex glycosylated 155KDa hERG1a normalized to β-actin (C) and of complex glycosylated 90KDa hERG1b (D) normalized to β-actin mean (thick bar) ± S.D. (thin bars), n = 5, * p<0.05. E. F. Trafficking efficiency of hERG1a (E) and hERG1b (F) calculated from ratio of complex glycosylated immunoblot band intensity to total intensity of complex and core glycosylated proteoforms, mean (thick bar) ± S.D. (thin bars), n = 5, *p<0.05, n.s.= not significant.

Figure 6.

Reduced hERG1a immunofluorescence in hERG1-H70R relative to hERG1-H70R^corr and DF19–9-11T iPSC-CMs in contrast to unchanged hERG1b immunofluorescence. A. B. Immunofluorescence images for (A) hERG1a- and (B) hERG1b-specific N-terminal antibodies (green) in representative iPSC-CMs derived from hERG1-H70R, hERG1-H70R^corr and DF19–9-11T iPSC and co-labeled with antibodies for endoplasmic reticulum marker protein disulfide isomerase (PDI, red) and for the myocyte marker sarcomeric myosin (MF20, cyan). Nuclei are labeled with 4’,6-diamidino-2-phenylindole for DNA (blue). Scale bars: 25 μm. C. Primary antibodies were omitted for background fluorescence signal. D. E. Quantification of hERG1a (D) and hERG1b (E) immunofluorescence signal intensity in iPSC-CM groups. Data points are for single cells with mean (thick bar) ± SD (thin bars), * p<0.05.

Figure 7.

smFISH analysis reveals increase in hERG1b/1a ratio in mRNA complexes without change in nuclear nascent transcript abundance in hERG1-H70R iPSC-CMs. A. Diagram of hERG1 smFISH probe design. The gene structure of KCNH2 is shown with exons (blue boxes) and introns (black solid lines). To visualize hERG1a and hERG1b and distinguish between them, two smFISH probes sets were used, with spectrally distinct fluorophores. The Probe Set a (PSa) targets exons only in hERG1a (black dashed lines) and thus visualizes only hERG1a. The Probe Set ab (PSab) targets 3’ region that hERG1a and hERG1b share (magenta dashed lines) and thus visualizes both isoforms. Each probe set comprises 48 of 20-nucleotide-long probes. Diagram is not to scale. B. hERG1 smFISH with hERG1-H70R (top), hERG1-H70R^corr (middle) and DF 19–9-11 T (bottom).smFISH were conducted with PSa (left, yellow) and PSab (middle, magenta). DNA is stained with Hoechst (right, cyan). Square boxes (right) are 9X magnification of white boxes. Arrow: hERG1a mRNA (seen both with PSa and PSab); arrowhead: hERG1b mRNA (seen only with PSab); asterisk: hERG1a&1b mRNA complex. Scale bar: 10 μm. C. Left: hERG1a mRNAs can be seen in both PSa and PSab channels whereas hERG1b mRNAs can be seen only in PSab channel. Right: the equations for counting the number of each isoform in the RNA complex. D. hERG1 isoform ratio (1b/1a) per mRNA complex is shown for hERG1-H70R (red, n=324), hERG1-H70R^corr (blue, n=117) or DF 19–9-11 T (black, n=160)., §: p< 0.001. E. hERG1 isoform ratio (1b/1a) of total hERG1 mRNAs in the cytoplasm. (n= 28, 23, and 28 cells from left). F. G. Total number of nascent hERG1a (F) or hERG1b (G) transcripts in each nucleus is estimated based on its intensity relative to the average single mRNA intensity. n= 59, 30, and 39 nuclei from left). D-G. mean (thick middle bar) ± S.D. (thin bars). n.s., not significant.

Figure 8.

Model of hERG1-H70R variant pathogenesis of LQT2. Cotranslational assembly of hERG1 complexes occurs at the ER where hERG1a is required for dimer formation initially leading to hERG1a-1a and hERG1a-1b complexes that ultimately form tetrameric hERG1 channels. In this simplified model, the hERG1a-H70R encoded subunit misfolds and undergoes ER-associated degradation (ERAD) along with associated subunits. This disproportionately impacts dimeric hERG1a-1a complexes (75% contain mutant allele) relative to hERG1a-1b complexes (50% contain mutant allele). Post-transcriptional changes in mRNA levels result in increased hERG1b mRNA abundance with an increase in the ratio of hERG1b:1a in mRNA complexes. The net result is to increase the relative ratio of hERG1b:1a in tetrameric channels at the membrane with an overall reduction in the number of total channels relative to control. (RNA-binding proteins, RBP)