MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel

Abstract

Background: MicroRNAs (miRs) play critical roles in regulation of numerous biological events, including cardiac electrophysiology and arrhythmia, through canonical RNA interference (RNAi) mechanism. However, it remains unknown if endogenous miRs modulate the physiological homeostasis of the heart through noncanonical mechanisms. Methods: We focused on the predominant miR of the heart--miR1 and investigated if miR1 could physically bind with ion channels in cardiomyocytes by electrophoretic mobility shift assay (EMSA), in situ proximity ligation assay (PLA), RNA pull down and RNA Immunoprecipitation (RIP) 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 utilized to understand the underlying mechanism. The effect on the ex vivo heart was demonstrated through investigating arrhythmia-associated human single nucleotide-polymorphisms (hSNPs) with miR1-deficient mice. Results: 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 K1 at sub-pmol/L concentration, which is close to endogenous miR-expression level. Acute presence of miR1 depolarized resting membrane potential (RMP) and prolonged final repolarization of the action potential in cardiomyocytes. We identified three 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 RNAi seed region. This biophysical modulation is involved in the dysregulation of gain-of-function Kir2.1-M301K mutation in short-QT/AF patients. We found that an arrhythmia-associated hSNP of miR1--hSNP14A/G specifically disrupts the biophysical modulation while retaining the RNAi function. Remarkably, miR1 but not hSNP14A/G relieved the hyperpolarized RMP in miR1-deficient cardiomyocytes, improved the conduction velocity, and eliminated the high inducibility of arrhythmia in miR1-deficient hearts ex vivo . Conclusions: 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.

Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1

“…The location of Kir2.1 at this microdomain corresponded to the M line where Kir2.1 colocalized with Ankyrin-B (Figure 4E). Furthermore, while a double Kir2.1 band pattern has not been specifically reported previously, it is clearly visible in several published illustrations from different laboratories 10,12,17,18 (Online Figure IV). To test whether the double Kir2.1 band pattern was not mouse-specific we analyzed Kir2.1 immunolocalization in rat cardiomyocytes.…”

Ventricular arrhythmias in mouse models of diabetic kidney disease

“…with a ketamine (100 mg/Kg)/xylazine (10 mg/kg) mixture. Hearts were excised and cannulated for Langendorff perfusion with 34°–36° O 2 -bubbled Tyrode solution containing (mM) NaCl 140, KCl 4.5, CaCl 2 1.25, MgCl 2 0.7, HEPES 10, and dextrose 5.5 (pH 7.4) and placed in a heated custom-built Plexiglas chamber, as described previously 74 . Electrocardiograms, perfusion pressure (40–60 mmHg), and bath temperature were measured continuously during the entire experiment.…”

Genomic Variants and Multilevel Regulation of ABCA1, ABCG1, and SCARB1 Expression in Atherogenesis

“…In addition to interaction with mRNA and post-transcriptional regulation of gene expression, miRNA can exert post-translational functions. The direct binding of miRNA to proteins that modulate protein function has been observed recently [95,96]. The mechanisms that modulate miRNA activity, stability, and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, transport from the cytoplasm, and regulation of miRNA-target interactions were reviewed elsewhere [97].…”