Function and mechanism analysis of short QT syndrome related KCNH2 gene variants

2020 Fiscal Year Research-status Report

• Project/Area Number: 20K17103
• Research Institution: National Cardiovascular Center Research Institute
• Principal Investigator: Wang Qi 国立研究開発法人国立循環器病研究センター, 研究所, リサーチフェロー (70756767)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: patch-clamp / SLC4A3 / AE3

Outline of Annual Research Achievements

In our short QT syndrome (SQTS) family, we previously found a novel KCNH2-H70Y mutation, which was thought as the pathogenic mutation. We have performed patch-clamp analysis of IKr currents in KCNH2-WT or H70Y transfected HEK293 cells. However, the gating kinetics of hERG channel was not significantly changed in the H70Y mutant cells. Compared to the serious phenotype of the short QT syndrome (SQTS) proband, the changes in the H70Y cells were mild. Therefore, we suspected that KCNH2-H70Y might not play a key role in abbreviating the QTc interval.
To figure out our suspicion, we performed whole exome sequencing for our SQTS family and identified a novel SLC4A3 variant. The SLC4A3 was reported as a responsible gene for SQTS in 2017. It encodes AE3 channel, which works as a Cl-/HCO3- exchanger and modulates intracellular pH (pHi) level. The SLC4A3-deficient zebrafish showed a shorter QTc interval and higher pHi level. Unlike other SQTS related genes directly involved in cardiac action potential, it is still unclear how the SLC4A3 mediates QTc interval.
Therefore, the newly found SLC4A3 mutation might correspond to our SQTS family. We established SLC4A3 WT and mutant stable cell lines for further explore the molecular relationship between SLC4A3 and SQTS.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We have been working on this SQTS project. For the further exploration of the genetic background in this SQTS family, we performed whole exome sequencing and found a novel SLC4A3 mutation. We obtained a SLC4A3 plasmid and constructed a mutant SLC4A3 plasmid and successfully established SLC4A3 WT and mutant HEK293 stable cell lines.
To explore the molecular mechanism of this novel mutation causing QTc shortening, we recorded IKr, IKs and IK1 currents, which are involved in the phase 3 and 4 of cardiac action potential. However, the electrophysiological study in the SLC4A3 mutant stable cells did not show any significant difference from that in the WT cells. We are now using pHi indicator, BCECF-AM, to measure pHi by live cell imaging technique.

Strategy for Future Research Activity

For understanding the pathogenesis of the short QT syndrome, we established SLC4A3 WT and mutant stable cell lines for studying AE3 function, including AE3 protein expression, basic and dynamic intracellular pH (pHi). For understanding the relationship between pHi and QTc interval, we also record IKr, IKs, IK1 currents, which is active in directly controlling the cardiac action potential.
Next, for in vivo study, we are constructing a SLC4A3 knock-in zebrafish to mimic the SQTS phenotype. We can monitor the QTc interval on ECGs, occurrence of ventricular arrhythmias and prognosis. Isolated cardiomyocytes of knock-in zebrafish are used for the evaluation of pHi, protein expression and electrophysiology study.

Causes of Carryover

To elucidate the pathophysiology of SQTS in our family, we had to change our target from a variant in KCNH2 to SLC4A3. Therefore, we could not use the research fee as we planned. This year, we are going to use the research fee for establishing the knock-in zebrafish and analyzing it.