Associaton Study Of AKAP10with Sinus Node Dysfunction In Han Chinese Population And The Mechanism Of Novel Mutations Of AKAP10Associated With Sinus Node Dysfuntion

Bradyarrhythmia is one of the serious threat to people’ s health clinic lethal diseases, including damage to the sinus node automaticity and atrial-ventricular block, both clinically often in combination. Among them, especially sick sinus syndrome is most common. Sick sinus syndrome is a cardiac arrhythmia caused by sinus rhythm dysfunction and accounts for more than a half of patients undergo pacemaker transplantation. The major clinical manifestations of sick sinus syndrome is sinus arrhythmia （including bradycardia, tachycardia, or both in combination）, and its etiology is various, also pathogenesis is not clear at present. The understanding of its pathogenesis mainly focused on the following three aspects:（1） abnormal gene expression of ion channels;（2） reduction of the sinus node cells;（3） cumulative component of connective tissue. Among them, the changes in ion channel gene expression caused by genetic factors are common, which is mainly reported in foreign documents. For example, the mutations of HCN4, SCN5A, etc. However, the progress in biological pacemaker is not ideal as expected, which suggests that there are a lot of new sick sinus syndrome-related genes remain to be discovered. Therefore, the genetic research in sick sinus syndrome will contribute to reveal the pathogenesis of sinus node dysfunction and provide the basis for effective treatment of and biological pacemaker study. Our study systematically explore the role of A-kinase anchoring protein AKAP10in the pathogenesis of sick sinus syndrome, and also analyze its molecular mechanism, with the methods of molecular biology, cell biology, and genetics. Our main findings are:1. Genetic associaton study of AKAP10with sinus node dysfunction in Han Chinese population Sick sinus syndrome accounts for more than a half of the implantable cardiac pacemaker surgery, however, the mechanisms of sick sinus syndrome is still unclear. We determined based on a large number of documents retrieved arrhythmia with four closely related genes （AKAP10, Kcnj8, Cacnald, MIR-1） as candidate genes using the PCR-SSCP and direct sequencing methods and finaly determined several SNPs of AKAP10are associated with sinus arrest Firsly we collected456cases with sick sinus syndrome （which including227patients with sinus arrest） and467control. By PCR-SSCP and direct sequencing, we two novel AKAP10mutations:c.682₆87delAGAACT （p. Arg228_Thr229de1） in exon4and c.1062-2A> G in intron6. And we also found two SNPs （rs203462and rs4925060） are both significantly correlated with sinus node dysfunction. In summary, we for the first time found two novel AKAP10mutations and two SNPs in sinus node dysfunction patients, which indicates that the AKAP10gene may be involved in the mechanism of sinus node dysfunction, especially in the occurrence of sinus arrest.The first study found that in patients with sinus arrest AKAP10multiple sites （which contains two new mutations and two SNP loci） change, suggesting that this gene may be involved in AKAP10sick sinus syndrome, especially occurrence of sinus arrest development.2. The Mechanism of novel mutations of AKAP10associated with sinus node dysfuntionWe constructed the micro gene of AKAP10to prove that the novel mutation c.1062-2A> G in intron6of AKAP10can lead to abnormal RNA splicing, resulting in missing exon7, which may be the possible mechanism of sinus node dysfunction. We detected the transcrips of AKAP10in different human cell lines, including human umbilical vein endothelial cells （HUVEC）, human aortic smooth muscle cells （HASMC）, human monocytic cell line （THP-1） and myocardial samples from several patients. We found different transcripts of AKAP10in different cell lines （containing exon7or missing exon7）,but in the myocardium we found only one transcript of AKAP10（containing exon7）, which indicate that the lack of exon7in AKAP10may lead to sick sinus syndrome, and different transcripts of AKAP10may vary in function. To prove the novel mutation c.682₆87delAGAACT （p. Arg228_Thr229de1） in exon4of AKAP10can lead to sinus node dysfunction, we established the earlier （3dpf） zebrafish electrocardiogram detection platform, by injected the RNA to overpressed p. Arg228_Thr229del and then successfully simulated the sinus arrest-like phenotype. We further overexpressed p. Arg228_Thr229del and HCN4/HCN2in HEK293A, and then using the confocol microscopy scaning. Finally we found that compared to the wild type group, the mutant group （p. Arg228_Thr229del） can reduced the expression of HCN4in membrane, which may be the mechanism of occurrence of sinus node dysfunction.Takern together, by genetic and functional study on AKAP10, we found its essenpial role in regulating the expression of HCN4in membrane, suggesting that AKAP10is possibly involved in the pathogenesis of sinus node dysfunction. The new discoveries would refresh our knowledge in the occurrence and development of sinus node dysfunction and provided new theoreticalComprehensive two-part research, we adopted the A-type kinase anchoring protein AKAP10investigate genetic and functional analysis, first found its role in regulating the expression of HCN4membrane, suggesting that AKAP10possibly through a mechanism involved in the sinoatrial node cells HCN4trafficking process. The results deepen our understanding of the pathogenesis of diseases associated with sick sinus syndrome, and for the clinical diagnosis and treatment of sick sinus syndrome provides a new theoretical basis for clinical diagnosis and treatment.