The Molecular Basis Of Hcn Ion Channels In Pacemaker Function

In the rescent years, the development of pacemarker proposes a novel strategy to treat arrhythmia in order to save the lives of those who suffered.However, the implantation of pacemaker can only help relieve symptom but can not remove the disease from the root .Further more ,it is risky to implant electric pacemaker devices in the patients.Especially for the youth, it is quite difficult to do this because their hearts is developing.As a result ,a novel stratery present itself accordingly.The heartbeat originates from a so-called If ion channel expressed in sinoatrial node (SAN),activated by membrane hyperpolarozation,and is an important comtributer to pace making.HCN is termed as hyperpolarization-activated,cyclic-nucl etide-modulated(HCN) cation channel.This gene family encoded protein can produce If current ,and this family has been cloned. It turns out to be a hot research topic in gene therapy to cure arrhythmia.The research used lentivirus expression system to carry mHCN1 and HCN2 genes respectively. We use molecular biology technology to modify the vector pLL3.7:delete the original mU6 promoter ,insert EF-1αand HCN genes between XbaⅠand NotⅠrestriction sites.Then we tranfected 293FT with packing plasmids and constructed expressing plasmids,collected the lentivirus to infect tool cells and target cells ,and compared current among different clonies.Finally we use the function-enhancing virus to do in vivo expriments ,rendering potential pacemaker cells to functional pacemaker cells, knocked out the native artrial node to examine pace making restoration.It is proved that the lentivirus expression system could long-termly and stably express target genes, the differenct among the clonies are respectively: HCN1-ΔEVY>HCN1E272A>HCN1E272A-ΔEVY>HCN1WT;HCN2E324A>HCN2WT>HCN2-ΔEVY>HCN E324A-ΔEVY. But the expression efficiency and virus'MOI is too low to do in vivo expriments even the virus is 100 times concentrated , so the vector need to be further modified or replaced.Since HCN's role in pacemaking has been tested ,my experiment had done further study on the properties of them through several mutations and we have got pacemaking-enhancing clonies. We adopt lentivector as the vehicle which can integrate the gene of interest into host's genome so as to express it in a long-term and more steady way ,bue in my case it needs further reconstruction.