School of Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
School of Physiology, Pharmacology & Neuroscience, Faculty of Biomedical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.
J Mol Cell Cardiol. 2022 Mar;164:29-41. doi: 10.1016/j.yjmcc.2021.11.004. Epub 2021 Nov 22.
The fast transient outward potassium current (I) 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 I 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 I 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 I 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 I agonist). Variation in the density of the expressed I, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca transient. I expression also reduced AP triangulation but did not affect I and I magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.
快速瞬态外向钾电流（I）在人心律失常动作电位（AP）的 1 期复极化中起着关键作用，其在心力衰竭（HF）中的减少导致收缩力丧失。因此，恢复 I 可能有益于治疗 HF。在 Kv4.3 和 KChIP2.1 基因之间，克隆了编码 P2A 肽的序列，通过 Western blot 证实核糖体跳跃。在转染的 HEK293 细胞中，由于 KChIP2.1 与 Kv4.3 的结合，观察到具有缓慢失活和从失活中加速恢复的典型 I 特性。两个双顺反子成分都转运到质膜上，在腺病毒转导的兔心肌细胞中，t 小管和肌膜结构的标记都出现。所得电流与人心室肌细胞中的 I 相似，在 ~0.8 mmol/l 4-氨基吡啶时约有 50%被阻断，5 μmol/l NS5806（I 激动剂）时增加约 30%。在兔心肌细胞中表达的 I 密度的变化，重现了 AP 形态的典型种间变化。同时进行电压记录和细胞内 Ca 成像显示，将 1 期修饰为非衰竭人类表型可改善 Ca 瞬变的上升速度和幅度。I 表达还减少了 AP 三角化，但不影响 I 和 I 的幅度。这为基于选择性 1 期修饰的 HF 的新基因治疗方法提供了可能性。
