Multichannel Dysfunction Associated With a Dominant-Negative KCNH2 Missense Variant, p.G604S, Results in Action Potential Duration Prolongation and Early Afterdepolarizations in a Patient-Specific, Reengineered Cardiomyocyte Model of Type 2 Long-QT Syndrome.
TL;DR
Fux dynamics analysis indicated that this persistent Ca2+ entry suppresses the forward-mode sodium-calcium exchanger, forcing a compensatory reliance on sarcoplasmic-endoplasmic reticulum Ca-ATPase that primes for calcium instability.
Abstract
Background
Type 2 long-QT syndrome results from KCNH2-encoded Kv11.1 loss-of-function, but whether secondary inward-current dysfunction amplifies dysfunction remains unclear.
Methods
We characterized sodium (SCN5A, INa) and L-type calcium channel (CACNA1C, ICa) function in KCNH2-G604S and variant-corrected isogenic control induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Whole-cell patch-clamp measured action potential duration at 90% repolarization (APD90), and potassium (IKr), sodium (INa), and calcium (ICa, L) currents. Fluo-4 imaging quantified calcium transients. A modified Kernik-Clancy iPSC-CM model incorporated experimentally measured persistent INa,L and ICa,L fractions and simulated isolated and combined current defects.
Results
KCNH2-G604S caused severe IKr loss-of-function (72.5% reduction). APD90 was prolonged in G604S-iPSC-CMs (765±58 ms) compared with isogenic controls (368±37 ms; P<0.005), with early afterdepolarizations in 50% of G604S cells. Nifedipine, verapamil, and mexiletine successfully shortened APD90 and abolished early afterdepolarizations. Compared with isogenic control, INa,L (0.60±0.1% versus 0.26±0.06%; P<0.01), persistent ICa,L (2.88±0.55% versus 1.26±0.35%; P<0.05), and calcium transient decay times (P<0.0001) were greater in G604S iPSC-CMs. Increased persistent ICa,L was phenocopied by E-4031 in isogenic controls and reduced by nifedipine in G604S iPSC-CMs. In silico modeling revealed that the IKr defect alone prolonged APD90 to only 620 ms. The addition of the observed INa,L defect had a small additive effect (+20 ms); however, incorporating persistent ICa,L synergistically extended APD90 to 758 ms, replicating the experimental phenotype. Flux dynamics analysis indicated that this persistent Ca2+ entry suppresses the forward-mode sodium-calcium exchanger, forcing a compensatory reliance on sarcoplasmic-endoplasmic reticulum Ca-ATPase that primes for calcium instability.
Conclusions
The KCNH2-G604S variant drives a severe multihit phenotype via primary IKr loss-of-function and secondary state-dependent gain-of-function in persistent ICa,L. These comprehensive validations demonstrate that the remodeled persistent ICa,L acts as the essential amplifier driving extreme action potential duration prolongation and Ca2+ mishandling.