Identification And Functional Study Of A Novel Connexin45 Mutation Associated With Idiopathic Atrial Fibrillation

Background: Atrial fibrillation(AF)is the most common sustained cardiac arrhythmia in clinical practice.The mechanisms of AF are still unknown.Gap junctions,consist of connexins(Cx),are the key channels to coordinate the mechanical and electrophysiological activities in cardiac cells.There are three kinds of connexin in human myocardium:Cx40,Cx43 and Cx45.Recent studies have shown that germline mutants in Cx40 may predispose individuals to AF,indicating that genetic variation in Cx45 may be responsible for AF.Objective: The aim of the study was to identify a novel Cx45 mutation associated with AF,and light the cellular electrophysiological mechanisms of the mutation.Methods: A cohort of 102 unrelated patients with idiopathic AF and 300 healthy individuals used as the controls were recruited.Clinical data and blood samples were collected,and genomic DNA was extracted from blood leucocytes.The entire coding region and flanking splice junction sites of the Cx45 gene was amplified by polymerase chain reaction(PCR).The amplified products were purified and PCR-sequenced.A Cx45 sequence variation may be detected by comparing the acquired Cx45 sequences with those released from GenBank.In order to identify a novel Cx45 mutation associated with AF,the frequency distributions of Cx45 variation in patients with idiopathic AF and healthy control individuals were analyzed by sequencing,and alignment of multiple Cx45 proteins across different species was performed.The wild-type Cx45 gene was cloned by molecular cloning technique,and the mutant-type Cx45 was obtained by site-directed mutagenesis.The recombinant eukaryotic expression plasmids for both wild-type Cx45 and mutant-type Cx45 were constructed.Then the plasmids were transfected into tool cells.Electrophysiological recordings by patch clamp were carried out to analyze the effect of a Cx45 mutation on the electrophysiological characteristics of the gap junction.And the permeability of the gap junction was evaluated by laser scanning confocal microscopy to unveil the cellular electrophysiological mechanism by which the mutation predisposes to atrial fibrillation.Results: A novel heterozygous missense mutation in the Cx45 gene was identified in a patient with idiopathic AF.The mutation resulted in a substitution of leucine for methionine at amino acid position 235 of the Cx45 protein(p.M235L),which was found in 300 healthy control individuals.Alignment of multiple Cx45 protein sequences among various species showed that the methionine at amino acid 235 of Cx45 was highly conserved evolutionarily.The identified Cx45 mutation was predicted to be pathogenic by several softwares.Studies by patch clamp showed that this mutation led to impaired electrical current conduction of gap junctions.But the permeability of gap junction was normal.Triggered activities and reentry arised from the atrium may lead to the occurrence of AF.Conclusions: A novel heterozygous missense mutations(p.M235L)in the Cx45 gene is associated with AF.The p.M235 L mutation exerts impaired electrical current conduction of gap junctions,which may contribute to atrial fibrillation.