Evaluating the role of herg potassium-channel protein in the development of heart and vasculature

The role of human ether a-go-go related gene(hERG) in electrically excitable cells has long been known.hERG currents IKr contribute to the re-polarisation phase 3 of the cardiac action potential. Mutations of this channel causes long QT syndrome. N629D hERG mutation(GFGN to GFGD) alters the pore selectivity signature sequence.N629D was over-expressed, via adenoviral gene transfer,in car-diomyocytes derived from mouse embryonic stem cells,the "IKr" showed outward rectification and an inward tail current,while WT IKr showed inward rectification and a positive tail current.N629D "IKr" phenotype also altered resting membrane potential and caused arrhythmia.Since hERG was reported to express in early stage of cardiogenesis,the role of the ERG potassium channel in cardiac development was elaborated in an in vivo model of a homozygous. The hERG N629D mutation was introduced into the orthologous mouse gene,mERG,by homologous recombination in mouse embryonic stem cells. N629D/N629D homozygous mutation results in embryonic lethality(died by E11.5).The mutation displayed defect cardiac morphogenesis including altered looping architecture,poorly developed bulbus cordis,and distorted aortic sac and branchial arches. N629D/N629D myocytes from embryonic day 9.5 embryos manifested complete loss of IKr function, depolarized resting potential,prolonged action potential duration(LQT),failure to repolarize,and propensity to oscillatory arrhythmias.Because deletion of transcription factor Hand2 produces apoptosis in similar regions and with a similar final developmental phenotype,Hand2 expression was evaluated. Robust decrease in Hand2 expression was observed in the secondary heart field in N629D/N629D embryos. mERG protein expression in the developing embryonic heart is not homogeneous.The protein expression is exaggerated in the right ventricle and in the outflow tract.N629D/N629D embryos manifest extensive apoptosis particularly in the first branchial arch and the facial region.Given that cells from the branchial arch populate the outflow tract,the early apoptosis,in the branchial arch and facial region would prevent those cells from contributing to the development of the outflow tract in N629D/N629D hearts.The working model is that the Hand2 expres- sion is down regulated in N629D/N629D embryonic right ventricle and outflow tract because progenitor cells that populate the outflow tract undergo apoptosis while in the facial region and branchial arch. Thus tissues that would be expected to express Hand2 are absent,simply because those structures fail to develop.N629D/N629D embryos also displayed defects in both extraembryonic and intraem-bryonic vasculature.Yolk sac from N629D/N629D homozygous embryos revealed primary vascular networks formed,while they failed to remodel into more complex vascular structures,unlike wild-type yolk sacs at E9.5 N629D/N629D embryo yolk sacs at E9.5 display absence of visible vessels.Intraembry-onic vessels in the mutant showed less complex branching in comparison with the normal vessels structure in WT embryo,dorsal aorta exhibited abnormally formation and small lumens.Whole mount in situ hybridization displayed hERG was also expressed in E9.5 yolk sac and dorsal aorta.Immunofluorescence showed the co-localization of hERG and Cd31 and smooth muscle actin in E10.5. The role of hERG protein in the development of vasculature is further evaluated by using Cre-loxP-based mouse model for tissue specific hERG mutation expression.