MicroRNA Biophysically Modulates Cardiac Action Potential by Direct Binding to Ion Channel
Authors: Mauricio G S Costa
Thomas J Hund
Ji-Dong Fu
Peter J Mohler
Catherine Vénien-Bryan
Daniel H Gratz
Zhihua Wang
Charline Fagnen
Kenneth R Laurita
Xiaoping Wan
Dandan Yang
Xianyao Xu
Isabelle Deschênes
Adrienne T Dennis
Emre Bektik
Journal: Circulation (2021)
Discipline: CLINICAL MEDICINE
Keywords: arrhythmias, cardiac; cardiac electrophysiology; ion channels; microRNAs.
Affiliations: 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.)., Institute of Mineralogy, Materials Physics and Cosmochemistry, UMR 7590, Sorbonne Université, CNRS, MNHN, Paris, France (M.G.S.C., C.F., C.V.-B.)., Oswaldo Cruz Foundation, Scientific Computing Program, Vice Presidency of Education, Information and Communication, Rio de Janeiro, Brazil (M.G.S.C.)., Biomedical Engineering and Internal Medicine (X.X., D.H.G., T.J.H.), The Dorothy M. Davis Heart and Lung Research Institute, Frick Center for Heart Failure and Arrhythmia, The Ohio State University, Columbus.

Abstract:  Background: 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. Methods: 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. 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 IK1 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. 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. 

Retracted: No

PubPeer comments:
 The authors described the out-of-the-box role of a particular miRNA in regulating membrane ion channels by direct binding to specific regions of the protein. Looking to the results by the point of view of the structural biology, there are many open questions not answered within the manuscript or the supplementary material, and not discussed by the authors. It is really surprising that such a high level impact journal did not take into consideration these facts. Just as a summary of my personal concerns: 1.- The authors claimed that mature mirR-1 forms a short hairpin-like structure which appears to be required for the binding to the ion channel. Other miRNAs also could potentially form these structures as described by Belter and coworkers in a very classical paper not cited by the authors (Belter et al, 2014, PLoS ONE 9(11): e113848). The question is how the authors could explain the unique features of miR-1 in terms of its ability to interact with the ion channel? Are these features also present in other miRNAs? 2.- Another striking observation is that the predicted miR-1 interaction patch in the surface of the ion channel is very close to the membrane region of the protein. Since a miRNA is an intrinsically negative charged molecule, what is the influence of the membrane over the interaction? Electrostatic repulsion forces should be expected between the phospholipid heads and the negatively charged RNA. How the authors can conclude that this is the putative interaction patch over the surface of the protein? What kind of chemical interactions should be involved? The interaction patch appears to be hydrophobic by looking at the electrostatic potential surface representations. It would be more predictable to observe this interaction in a positively-charged region of the protein. Moreover, the information about the specific protocols used for the docking of the miRNA onto the surface of the protein are missing from the material and methods. Do the authors used flexible, rigid or blind docking and what was the exact software and parameters used? 3.- The paper lacks a deep molecular analysis of the interaction between miR-1 and the channel. Since this is a very unusual phenomenon for miRNAs, it should deserve a deeper look. Molecular dynamics simulations of the miRNA-protein complex within a membrane environment should be performed to determine if the miRNA-channel complex is stable enough during time to justify a molecular interaction. Molecular dynamics could also say if the miR-1 hairpin structure is stable in solution and in the RNA-protein complex. I am not arguing against the presented results, but rather trying to pinpoint the factors that I consider as weak in the paper. The direct interaction between the miRNA and the protein was demonstrated by using pull-down experiments with peptides comprising the putative miRNA-binding site. These experiments would also need to be taken with a lot of care, due to their intrinsic limitations that take out of context a small piece of the protein. In consequence, a very good paper in a very reputated journal, showing interesting results but limited molecular evidence of the proposed mechanism. Such a relevant paper in a very good journal, that even proposes a new mechanism for miRNAs out of the canonical one described more than 20 years ago should deserve a careful look from the scientific community, and a more careful peer-review process involving specialists from several areas and not only from the field of cardiology. ,