The Remodeling And Transcriptional Regulation Factor Of The L-type Ca²⁺ Channel α1c Subunit In Chronic Fibrillated Atrium In Patients With Rheumatic Heart Disease

Atrial fibrillation(AF) is the most common cardiac arrhythmia in humans.It is characterized by heterogeneity in the electrical activation pattern and loss of contractility. AF has the tendency to become more persistent over time.This is illustrated by the fact that a large percentage of patients with paroxysmal AF eventually will develop persistent or even permanent AF.The presence of AF is associated with a considerable increase in morbidity and in mortality.Despite the clinical importance of AF,the molecular mechanisms underlying this arrhythmia are poorly understood,and treatment options are limited.Experimental and clinical studies have pointed at two major mechanisms involved in the intrinsically progressive nature of AF.The first consists of a change in the electrical properties of the atrium,notably a shortening of the atrial effective refractory period(AERP) and a loss of rate adaptation,and hence was named electrical remodeling.Furthermore,it has been considered that AF is also associated with elaborate cell morphological changes, which was denominated structural remodeling.Several groups have investigated the mechanisms underlying electrical remodeling in various animal models of long-lasting rapid atrial pacing and in humans with chronic AF. All studies have pointed to a marked reduction in the density of currents through L-type Ca²⁺ channels(Ica,L) as the major factor.The fewer expression of L-type Ca²⁺ channel in AF myocytes was presumedly responsible for the reduction of Ica,L.However,some results that have been published conflict each other.The Kamp's comment suggested that the patient populations,technical differences,regional heterogeneity,and remodeling extent in atrial etc.might have contributed to the disparate results.Severe cellular structural alterations are observed in the atrial myocardium of patients with chronic AF,including fibrosis and abundant large dystrophic myocytes showing extensive myolysis.The nature of this cellular remodeling is still unknown.A degenerative process is ruled out by the lack of lysosomal degradation.Several lines of evidence indicate that these atrial myocytes after sustained AF resemble immature muscle cells.The phenotypic resemblance with foetal cardiomyocytes point towards the nature of dedifferentiation of fibrillating atria that render the cardiomyocytes able to survive stress conditions.AF is the only clinical manifestation of a process of profound tissular and cellular remodeling of the atrial myocardium.The similar response between fibrillating atria and embryonic myocardium has led to the hypothesis that there are common pathways involved in the regulation of gene expression.The shared pathways would result in part in a common molecular phenotype among these tissue states.Transcriptional activator ELK-1,one of members of ternary complex factor(TCF) family,has been shown to play an important role in regulating cell differentiation and proliferation.It's reasonable to presume that ELK-1 participated in the molecular and structural remodeling by which AF is sustained.In order to further investigate the molecular mechanisms of AF electrical remodeling and ascertain the correlation between transcriptional activator Elk-1 and the pathogenesis of chronic AF,we carried out this study.The specific aims and results of our research are summarized below:1.The gene expression changes of L-type ca2+ channelα1c subunit in chronic fibrillated atrium1) The gene expression of L-type Ca²⁺-channelα1c subunits in atrial myocardium of AF patients was compared with that in controls.In a semi-quantitative polymerase chain reaction(PCR) the cDNA of interest were amplified and separated by ethidium bromide-stained gel electrophoresis.It was found that the mRNA expression corresponding to theâ… -â…¡link of a l c subunits was not altered in the patients with chronic AF compared to the controls,while that corresponding to the IV trans-membrane ofα1c subunits was reduced.The result suggests that reduced Ica,L of fibrillating atria might be due to downregulated expression of the isoform ofα1c subunits.2) To elucidate the different changes of mRNA expression detected by PCR on theâ… -â…¡link and theâ…£trans-membrane ofα1c subunits gene in chronic AF atria,the DNA segments after PCR were elaborately separated to identify the alternative splicing in the amplified domains.The results showed that:1)the PCR segment ofâ… -â…¡link was encoded by exon(12+15+17+20) rather than by exon(12+15+16+17+20),and the splicing of exon14 insertion was skipped over;2) Three types of alternative splicing were observed in theâ…£trans-membrane ofα1c subunits gene in chronic AF atria.Thus,the alteration of splicing isoforms of L-type ca²⁺ channel underlies the molecular mechanisms of atrial electrical remodeling in chronic AF.3) To further investigate the transcriptional regulation mechanism of theα1c subunits gene in atria during chronic AF,the isoforms ofα1c subunits gene with different transcription start sites were identified in fibrillating atria.The results showed that two alternatively spliced first exons ofα1c subunits gene were observed in cDNA of atria tissue of chronic AF.Thus,two distinct promoters regulate the gene expression of isoforms of L-type ca²⁺ channelα1c subunits,which might have different activity during chronic AF.2 The role of transcriptional activator Elk-1 in the pathogenesis of chronic AF1) To prove the hypothesis that transcriptional activator Elk-1 participate the pathogenesis of chronic AF,the expression of ELK-1 protein in chronically fibrillating atria compared to that in normal rhythmic atria was detected.It was found that the protein expression of ELK-1 was significantly reduced in atrial tissue of chronic AF patients compared to that in the controls.Our results suggest that the downregulated expression of transcriptional activator ELK-1 may play an important role in the pathogenesis of AF.2) To investigate the role of the MEK/ERK pathway in signal transduction of cardiomyocytes remodeling during chronic AF,the expression of signal proteins in chronic fibrillating atria were compared with that in controls.The protein expression of MEK,ERK, and p-ERK was not altered in atrial myocardium of AF patients.The results show that the capability of signal conduction along MEK/ERK pathway that targets ELK-1 was not altered in atrial cells of chronic AF.3) To further discuss the role of Elk-1 in electrical remodeling of chronic AF,the regulation of Elk-1 on the transcription ofα1c subunits gene was predicted by the technique of bioinformatics.The 5'flanking region of human CACNA1C exon1b contains ELK-1 and SRF binding sites,whereas the 5'flanking region of human CACNA1C exon1a only contains ELK-1 binding sites.Thus,the ELK-1 might activate the promoter before exon1b but suppress the promoter before exon1a of human CACNA1C. In summary,this study investigated the molecular mechanisms of atrial electrical remodeling of chronic AF and the role of transcriptional activator Elk-1 in the pathogenesis of chronic AF.Firstly,our results suggest that reduced Ica,L of fibrillating atria might be due to downregulated expression of the isoform of a lc subunits,and the alteration of splicing isoforms of L-type ca²⁺ channel underlies the molecular mechanisms of atrial electrical remodeling in chronic AF.Secondly,we firstly observed that the expression of transcriptional activator ELK-1 was significantly reduced in atrial tissue of chronic AF patients,which suggests that the ELK-1 might play an important role in cardiomytes remodeling of chronic AF.