The Role And Mechanism Of Alternative Splicing Factor PTBP1 In Heart Development

Background:The heart is one of the earliest organ in the developing embryo to form and function with a delicately regulated process of various cells.The formation and compaction of trabecular myocardium is an important process of ventricular morphogenesis.At the early stage,the trabecular myocardium facilitates oxygen uptake.While along with the progresses,this function of the trabecular myocardium gradually degenerates and is replaced by coronary system.Majority of trabecular myocardium compacts against the ventricular wall,eventually forming a mature ventricular chamber.Trabecular myocardium formation and compaction defects are often manifested as noncompaction of ventricular myocardium（NVM）,also known as left ventricular noncompaction（LVNC）since it mainly affects the left ventricle.LVNC is characterized by abnormally thick trabeculae in the ventricle and deep crypts interlaced between trabeculae,and is considered to be a class of inherited cardiomyopathy associated with genetic mutations.Being the third most frequent cardiomyopathy after dilated and hypertrophic cardiomyopathy,LVNC can lead to systemic embolism,malignant arrhythmia,heart failure and sudden death.However,the pathogenesis of LVNC remains unclear,which limits the research of interventions.In mammals,the alternative splicing of pre-m RNA（Precursor messenger RNA）participates in and controls many key biological processes to maintain genetic diversity.Regulation of alternative splicing factors mainly include hn RNPs（Heterogeneous nuclear ribonucleoproteins）and SR（serine/arginine-rich）protein families.PTBP1（Polypyrimidine Tract Binding Protein 1）,which belongs to the subfamily of hn RNPs,is one of the core alternative splicing factors and also considered as an RNA binding protein.PTBP1 has been found to play important roles in the development of the nervous system and tumorigenesis,while its role in cardiovascular diseases is poorly understood.Our research revealed the role of PTBP1in heart development and explored its regulatory mechanism.Methods:We examined the localization of PTBP1 at different embryonic stages utilizing the genetic lineage tracing system in combination with immunofluorescence staining;constructed endocardial(Nfatc1-Cre;Ptbp1^fl/fl)and two pan-endothelial(Cdh5-Cre ERT2;Ptbp1^fl/fl and Tie2-Cre;Ptbp1^fl/fl)specific Ptbp1 knockout mouse lines to explore the effect of PTBP1 deficiency on heart development;via Hematoxylin and Eosin（H&E）staining to detect the ventricular structure of Ptbp1-deficient mice;confirmed the thickness of compact myocardium and trabecular myocardium and expression of trabecular marker（CX40）through immunofluorescence staining of endomucin（EMCN）and trabecular marker CX40.To clarify the cellular processes underlying noncompaction of ventricular myocardium in Ptbp1-deficient mice,we examined cardiomyocyte proliferation in the trabecular and compact myocardium through immunofluorescence staining of cell proliferation marker p H3（Phospho S10+T11）;detected the migration of endothelial cells in Ptbp1-deficient mice thorough the genetic lineage tracing system in combination with whole-mount immunofluorescence staining.In addition,embryonic ventricular explant culture and HUVECs（human umbilical endothelial cells）migration assay were carried out to further investigate the regulation of PTBP1 on the endothelial migration and the effect on the proliferation of cardiomyocytes.Finally,we explored the mechanism of PTBP1mediated alternative splicing in regulating heart development by RNA-seq and alternative splicing analysis;confirmed that ARRB1 was the key downstream target of PTBP1 regulating heart development.Results:Results showed that PTBP1 was rarely localized within the myocardium,while enriched expression was observed in endothelial cells.Then H&E and EMCN immunofluorescence staining of embryonic hearts of Ptbp1-deficient mice were performed and abnormal ventricular structure,decreased thickness of the compact myocardium and the expanded trabecular myocardium toward the ventricle were observed in E13.5 Nfatc1-Cre;Ptbp1^fl/fl,Tie2-Cre;Ptbp1^fl/fl and E15.5 Cdh5-Cre ERT2;Ptbp1^fl/fl embryonic hearts.Trabecular marker CX40 immunofluorescence staining showed that E13.5 Nfatc1-Cre;Ptbp1^fl/fl,Tie2-Cre;Ptbp1^fl/fl and E15.5 Cdh5-Cre ERT2;Ptbp1^fl/fl hearts all had an abnormal expression when compared to Ptbp1^fl/flhearts.These results suggested that Ptbp1 deletion affects embryonic ventricular morphology and leads to LVNC phenotype.To clarify the cellular process of ventricular noncompaction in Ptbp1-deficient mice,we first examined the proliferation of cardiomyocytes in the trabecular and the compact layer.Increased proliferation of trabecular cardiomyocytes was observed in E13.5 Nfatc1-Cre;Ptbp1^fl/fl,Tie2-Cre;Ptbp1^fl/fl and E15.5 Cdh5-Cre ERT2;Ptbp1^fl/flhearts,while the proliferation of cardiomyocytes was decreased in compact layer.Further immunofluorescence staining analysis showed that the migration of endothelial cells in Ptbp1-deficient mice was inhibited.Results of embryonic ventricular explant culture and HUVECs migration assay further confirmed the role of PTBP1 in endothelial cells and the effect on the proliferation of cardiomyocytes.To identify the mechanisms underlying ventricular noncompaction in Ptbp1-deficient mice,we performed alternative splicing analysis of the RNA sequencing data and validated these results in vitro.Results showed that Ptbp1 deletion altered the expression ratio of the“short”ARRB1 isoform（ARRB1-S,lacking exon 17）and the“long”ARRB1 isoform（ARRB1-L,including exon 17）in the E13.5 endothelial cells of Nfatc1-Cre;Ptbp1^fl/fl,Tie2-Cre;Ptbp1^fl/fland Cdh5-Cre ERT2;Ptbp1^fl/fl hearts.HUVECs scratch assay showed that ARRB1-S overexpression reversed the reduced cell migration induced by Ptbp1 knockdown,indicated that ARRB1-S is the key target of PTBP1.These data suggested that PTBP1-mediated alternative splicing of Arrb1regulated endothelial cell migration.Conclusion:PTBP1 was shown to colocalize with endothelial cells during heart development.Ptbp1 deletion inhibited the migration of endothelial cells,thereby disrupting the proliferation of cardiomyocytes,and led to the occurrence of LVNC.Mechanistic studies revealed that Ptbp1 deletion led to a series of cardiac development-related alternative splicing events,in which the alternative splicing of Arrb1 was identified as the core event that mediates the regulation of endothelial cell migration by PTBP1.