Inducing Ito,f and phase 1 repolarization of the cardiac action potential with a Kv4.3/KChIP2.1 bicistronic transgene

Nan Wang,Eef Dries,Ewan D Fowler,Stephen C Harmer,Jules C Hancox,Mark B Cannell

doi:10.1016/j.yjmcc.2021.11.004

Abstract

The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 μmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.

Highlights

Differences in outward K+ currents due to variations in K+ channel and/or accessory subunit(s) expression are primary determinants of species- and region-dependent variations in cardiac action potential (AP) morphology [1,2,3]
We recently showed that electrophysio logical restoration of phase 1 of the AP can improve sarco plasmic reticulum (SR) release syn chrony, suppress arrhythmogenic late Ca2+ spark production and increase the amplitude of the Ca2+ transient in a heart failure model [16] in agreement with the earlier demonstration that the loss of human phase 1 repolarization impairs excitation-contraction coupling (ECC) [14]
We have shown that a robust Ito,f, which requires both Kv4.3 and KChIP2.1, can be produced in cardiomyocytes by using a bicistronic gene construct and viral transduction

Summary

Introduction

Differences in outward K+ currents due to variations in K+ channel and/or accessory subunit(s) expression are primary determinants of species- and region-dependent variations in cardiac action potential (AP) morphology [1,2,3]. As the depth/rate of phase 1 repolarization decreases, dyssynchronous and reduced Ca2+ release from the sarco plasmic reticulum (SR) develops [12,13] due to the reduction in trig gering L-type Ca2+ current (ICa,L) [12,14] and this contributes to the reduction in contractility other cellular changes play a role (for review see [15]). Such results support the idea that modulation of Ito,f could be beneficial in heart failure [4]. The effects of NS5806 on the heart could be complicated by off-target effects such as Na+ current inhibition [20,21]

Methods

Results

Discussion

Conclusion