Effect Of EPHA4 Knockout On Cardiac Morphology And Function In Rat And The Preliminary Underlying Mechanisms

Aims:EPHA4 is one of the most special members in EPH receptors which belong to receptor tyrosine kinase family.It has been reported that EPHA4 can interact with almost all of the ephrin ligands,including type A and type B.The interaction subsequently activated various downstream signaling pathways depending on tissue and cell types,and played a variety of biological functions.EPHA4 has been well documented in central nervous system(CNS)containing both embryo and adult stages,and was involved in development of CNS,as well as pathophysiological process of multiple neurological disorders.Recent years,EPHA4 has also been found in other tissues besides nervous system with important roles as well.EPHA4 regulated the development of vasculature and organs such as kidney.thymus and thyroid.Besides.it also took part in other biological processes such as hormone secretion and monocyte adhesion,and occurrence,metastasis and prognosis of a variety of cancers.To examine whether there ’s some special expression and effect of EPHA4 which had not been found in peripheral tissue,our study mainly focused on three goals:(1)building a gene-editing rat line that could accurately report EPHA4 expression pattern,and realized EPHA4 knockout(named SD.EPHA4(TM-mCherry)-GC/ILAS,referred to as EphA4-/-)at the same time;(2)using the gene reporting system of EphA4--rat model to investigate whether there’s any special expression of EphA4 in tissues without recognition before;(3)investigating the EPHA4 function and underlying mechanisms in tissues with special EPHA4 expression by EphA4-/-rat which systematically deleted EPHA4 expression.Methods:The mCherry cassette was inserted downstream of the EphA4 promoter,replacing the exon 1 of EphA4 and resulted in its knockout in rat.Meanwhile,this system allowed to detect the EPHA4 expression pattern through the reporter gene,mCherry.By living observation.the phenotypes of EphA 4-/-rat were evaluated,including survival rate,body weight and locomotive gait.The EPHA4 expression pattern was detected using the fluorescence of mCherry in frozen sections from different tissues of EphA4-/-rat.The morphology and function of heart,where it had a special mCherry expression,were analyzed with the echocardiography,magnetic resonance imaging and catherization in EphA4-/-rat.The electrocardiography was used to examine cardiac conduction in EphA4-/-rat.The histological staining was used to analyze cardiac remodeling on the tissue level.Besides,PCR,immunoblot and RNA sequencing were used to detect the molecular changes in EphA 4-/-rat.Results:We successfully established a EPHA4 knockout rat line,EphA4-/-which expressed mCherry and replaced the endogenous EPHA4 expression.The EphA4-/-rats exhibited obvious retardation in growth,typical hopping gait and short lifespan.The fluorescence observation showed that mCherry unevenly expressed in different tissues and specific cells were reported by mCherry in EphA4-1-rats.The atria were found to highly express EPHA4,which had not gained recognition before.The atria specific expression of EPHA4 was also confirmed in adult human heart tissues,EPHA4 knockout induced atrial enlargement and gradual hypertrophy in rat since 2 months old,with decreased atrial ejection fraction and increased central venous pressure.While,left ventricular ejection fraction slightly decreased but ventricular morphology had little changed.EPHA4 deletion also induced abnormal electrocardiography in EphA4-/-rats.which occurred before the atrial enlargement,showing disappeared p waves,abnormal QRS complexes and prolonged QT intervals.Histological staining showed that EphA4-/-exhibited increased atrial cardiomyocyte size and extracellular collagen expression.Moreover,the sinoatrial node tissue in EphA4-/-was obviously different compared with that of EphA4-/-.RNA sequencing revealed that EPHA4 knockout altered the transcription of multiple genes involved in transcription and translation,ion binding,metabolism and cell adhesion,qPCR showed that ion channel related genes in EphA4-/-expressed different from the EphA4-/-rats.Deletion of EPHA4 reduced IGF1 mRNA and protein expression,suggesting that IGF1 could be involved in cardiac remodeling.Conclusions:We successfully established the EPHA4 knockout rat model with the mCherry reporter gene replacing the endogenous EPHA4 expression.The mCherry reported that EPHA4 unevenly expressed in specific cells and different tissues.Our data first demonstrated that EPHA4 was highly expressed in rat atria.EPHA4 knockout caused atrial dysfunction and abnormal cardiac conduction.Our results showed that EPHA4 knockout changed IGF1 and a certain number of genes in rat atria,which were involved in atrial remodeling and arrhythmia.Our results suggested that EPHA4 had critical role in maintaining normal cardiac conduction,and might play biological roles in diseases related to atrial remodeling,such as cardiomyopathy,atrial fibrillation and hypertension.