Functional Characterization Of Nup155 And Gle1 In The Heart Development Of Zebrafish

Cardiac arrhythmia is one of the most serious cardiovascular diseases,and is a cardiac rhythm or rate disorder caused by abnormal heart impulse or impulse conduction.Atrial fibrillation is the most comMon arrythmia in the clinic,and confers a high risk of morbidity and mortality.Studies with atrial fibrillation pedigrees and GWAS have linked many genes to atrial fibrillation.These findings provide important insights into the development of atrial fibrillation.Our team previously reported that NUP155 mutation R391H causes atrial fibrillation and sudden cardiac death in 2008.This is the first time that NUP155,a non-ion channel gene,is linked to atrial fibrillation.This finding provides a broader perspective on the pathogenesis and treatment of atrial fibrillation.GLE1 is first identified as an essential mRNA export factor.In addition to mRNA export,a novel role for GLE1 in mediating efficient translation initiation and termination weare also identified.Interestingly,NUP155 and GLE1 interact with each other at the nuclear pore complex to regulate the nuclear export of mRNA.All these work revealed that GLE1 is a key regulator of multiple post-transcriptional gene expression.However,to date,the physiological role of GLE1 in vertebrate development is largely unknown.In this thesis project,we used zebrafish as a model system to study the in vivo physiological roles of nup155 and gle1.We knocked down and overexpressed nup155 and gle1 in zebrafish by injecting Morpholino oligos and mRNA,respectively,to explore their effects on the structure and function of the heart during early development of zebrafish.We then used the system to probe the molecular regulation of heart rhythm and cardiac action potentials involving nup155 and gle1,which provides important insights into mechanism of atrial fibrillation.First,the expression profile of nup155 during zebrafish embryogenesis was analyzed using whole-mount in situ hybridization.Based on the result of whole-mount in situ hybridization,the nup155 expression was first detected at one-cell-stage embryos,suggesting that nup155 was maternally expressed.During two-cell-stage to 48 hpf stage,expression of nup155 was ubiquitous in the embryo.By comparing with the expression profile of gle1 in zebrafish,we found that the expression of nup155 and gle1 was highly consistent to each other.They both were highly expressed at the places where the cell division was strong.All these results showed that the expression of nup155 and gle1 was accompanied with the embryonic development,which may be because of the important role of nup155 and gle1 in the mRNA export at NPC.It also implied an important role of nup155 and gle1 during the embryogenesis.The hearts were excised and total RNA was isolated for real-time RT-PCR analysis.The result showed that nup155 and gle1 were expressed in 24 hpf,48 hpf and adult fish hearts.Second,we analyzed the morphological changes of the heart and focused on myocardium differentiation.We examined whether the expression of genes involved in the myocardium differentiation were influenced after nup155 and gle1 knockdown by their respective morpholinos.We specifically analyzed the expression of gata4,nkx2.5and hand2 by whole mount in situ hybridization.We found that knockdown of either nup155 or gle1 decreased the expression level of nkx2.5 mRNA in zebrafish embryos and knockdown of gle1 also decreased the expression level of gata4 mRNA,but not that of hand2.Interestingly,knockdown of gle1 affected the normal looping of the heart at 48 hpf.Further experiments showed that the expression of the left-right asymmetry regulatory factors spaw and pitx2 and the pancreas specific expression maker ins was altered.The data suggested that gle1 may play an important role in left-right asymmetry and cardiac lopping.Our data thus identified a novel role of gle1 in left and right asymmetry in vertebrates.Third,because NUP155 mutation causes AF in humans,we analyzed the effect of knockdown of nup155 and gle1 on the heart rate and cardiac action potential duration in zebrafish,which were intricately connected with development of AF.By recording and analyzing the heart rate changes at 48 hpf,we found that knockdown of nup155 and gle1caused a significant up-regulation of the zebrafish heart rate.We then examined the expression levels of heart rate-related genes hcn4,cav1.3,kcnj2 and scn5a at the mRNA level by using real-time RT-PCR analysis.We found that knockdown of nup155 and gle1selectively decreased the expression level of hcn4 mRNA in zebrafish embryos,but not that of cav1.3,kcnj2 and scn5a.These data suggested that nup155 and gle1 affected the heart rate of zebrafish possibly through affecting hcn4 expression.An important factor in the development of atrial fibrillation is the change of action potential during of cardiomyocytes.Therefore,we recorded and analyzed the action potential duration of the zebrafish heart at 24 hpf.We found that the action potential duration(APD₉₀)of zebrafish hearts was significantly shortened after nup155 knockdown.RT-PCR analysis showed that the expression of kcnq1 was affected by nup155 knockdown.However,knockdown of the gle1 did not affect the action potential duration in zebrafish.Finally,we detected the markers of embryonic arterial vessels and venous vessels after knockdown of nup155 and gle1.The result showed that the expression of dab2,flt4and notch did not change,indicating that nup155 and gle1 might only affect cardiac development,but not that of vascular development.We found that injection of nup155 and gle1 mRNA into embryos to overexpress either of the two genes rescued the respective phenotypes of knockdown of nup155 and gle1 by morpholionos.The data suggest that the effects of knockdown by nup155 and gle1morpholinos are specific to nup155 and gle1,respectively.In summary,our studies found that nup155 regulated the morphogenesis of the embryonic heart through regulating expression of nkx2.5,and affected the heart rate and action potential duration of embryonic hearts by regulating hcn4 and kcnq1.These genes are closely related to the development of atrial fibrillation.Meanwhile,we also found that gle1,which interacts with nup155,also regulated the morphogenesis of the heart during embryonic development through regulating nkx2.5 as well as gata4,and modulated the heart rate of the embryo by regulating hcn4.Altogether,these results uncovered novel roles of nup155 and gle1 in the morphogenesis and development of zebrafish hearts for the first time,and demonstrated that both nup155 and gle1 affected the heart rate.Moreover,nup155 also regulates the action potential duration of zebrafish hearts.These interesting results provided important insights into the pathogenic mechanisms for the development of atrial fibrillation.