Departments of Physiology and Cell Biology (D.Y., X.W., P.J.M., I.D., J.-D.F.), The Dorothy M. Davis Heart and Lung Research Institute, Frick Center for Heart Failure and Arrhythmia, The Ohio State University, Columbus.
Department of Medicine, Heart and Vascular Research Center, The MetroHealth System, Case Western Reserve University, Cleveland, OH (A.T.D., E.B., K.R.L., J.-D.F.).
Circulation. 2021 Apr 20;143(16):1597-1613. doi: 10.1161/CIRCULATIONAHA.120.050098. Epub 2021 Feb 16.
MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through a canonical RNA interference mechanism. It remains unknown whether endogenous miRs modulate physiologic homeostasis of the heart through noncanonical mechanisms.
We focused on the predominant miR of the heart (miR1) and investigated whether miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay, in situ proximity ligation assay, RNA pull down, and RNA immunoprecipitation assays. The functional modulations of cellular electrophysiology were evaluated by inside-out and whole-cell patch clamp. Mutagenesis of miR1 and the ion channel was used to understand the underlying mechanism. The effect on the heart ex vivo was demonstrated through investigating arrhythmia-associated human single nucleotide polymorphisms with miR1-deficient mice.
We found that endogenous miR1 could physically bind with cardiac membrane proteins, including an inward-rectifier potassium channel Kir2.1. The miR1-Kir2.1 physical interaction was observed in mouse, guinea pig, canine, and human cardiomyocytes. miR1 quickly and significantly suppressed I at sub-pmol/L concentration, which is close to endogenous miR expression level. Acute presence of miR1 depolarized resting membrane potential and prolonged final repolarization of the action potential in cardiomyocytes. We identified 3 miR1-binding residues on the C-terminus of Kir2.1. Mechanistically, miR1 binds to the pore-facing G-loop of Kir2.1 through the core sequence AAGAAG, which is outside its RNA interference seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short QT or atrial fibrillation. We found that an arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts the biophysical modulation while retaining the RNA interference function. It is remarkable that miR1 but not hSNP14A/G relieved the hyperpolarized resting membrane potential in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo.
Our study reveals a novel evolutionarily conserved biophysical action of endogenous miRs in modulating cardiac electrophysiology. Our discovery of miRs' biophysical modulation provides a more comprehensive understanding of ion channel dysregulation and may provide new insights into the pathogenesis of cardiac arrhythmias.
microRNAs（miRs）通过经典的 RNA 干扰机制在调节许多生物事件中发挥关键作用，包括心脏电生理学和心律失常。目前尚不清楚内源性 miRs 是否通过非经典机制调节心脏的生理稳态。
我们专注于心脏中的主要 miR（miR1），并通过电泳迁移率变动分析、原位邻近连接分析、RNA 下拉和 RNA 免疫沉淀测定来研究 miR1 是否可以与心肌细胞中的离子通道发生物理结合。通过内向外和全细胞膜片钳来评估细胞电生理学的功能调节。通过突变 miR1 和离子通道来理解潜在的机制。通过 miR1 缺陷型小鼠研究与心律失常相关的人类单核苷酸多态性，证明了对心脏的体外作用。
我们发现内源性 miR1 可以与心脏膜蛋白，包括内向整流钾通道 Kir2.1 发生物理结合。在小鼠、豚鼠、犬和人源心肌细胞中均观察到 miR1-Kir2.1 物理相互作用。miR1 以亚皮摩尔浓度快速且显著地抑制 I，该浓度接近内源性 miR 的表达水平。miR1 急性存在可使心肌细胞的静息膜电位去极化，并延长动作电位的终末复极化。我们在 Kir2.1 的 C 末端识别出 3 个 miR1 结合残基。在机制上，miR1 通过核心序列 AAGAAG 与 Kir2.1 的孔面向 G 环结合，该序列位于其 RNA 干扰种子区域之外。这种生物物理调节参与了短 QT 或心房颤动中功能获得性 Kir2.1-M301K 突变的失调。我们发现 miR1 的一个与心律失常相关的人类单核苷酸多态性（hSNP14A/G）特异性地破坏了生物物理调节，同时保留了 RNA 干扰功能。值得注意的是，miR1 而不是 hSNP14A/G 可以减轻 miR1 缺陷型心肌细胞的超极化静息膜电位，改善传导速度，并消除 miR1 缺陷型心脏的体外高诱导性心律失常。
本研究揭示了内源性 miRs 在调节心脏电生理学方面的一种新的进化保守的生物物理作用。我们对 miR 生物物理调节的发现提供了对离子通道失调的更全面理解，并可能为心律失常的发病机制提供新的见解。
