Functional Analysis Of Pdgfr? In Mouse Cadiac Development

PDGF?platelet-derived growth factor?family are major mitogens for a number of cell types.During the embryonic development,PDGFs are important for the development of mesenchymal cells of different organs,such as mesangial cells of the kidney,alveolar smooth muscle cells of the lung,smooth muscle cells and pericytes of blood vessels,and glial cells of the central nervous system.There are four isoforms of PDGFs?PDGFA,PDGFB,PDGFC and PDGFD?.Biologically active PDGF is a dimer of two PDGF protein chains.There are a total of 5 biologically active PDGF proteins because in addition to the four homodimers?PDGF-AA,PDGF-BB,PDGF-CC and PDGF-DD?of PDGF,there is one heterodimer,PDGF-AB.PDGFs exert their biological effects through the activation of two tyrosine kinase receptors,?and?-receptors?designated as PDGFR?and PDGFR?,respectively?.Binding of ligand to PDGFR dimerizes PDFGRs for triggering the receptor's kinase activity.Thus,transduction of PDGF signaling is via three forms of PDGFR?PDGFR?and PDGFR?homodimers,PDGFR??heterodimer?.PDGF signaling has been demonstrated to be extensively involved in cardiovascular development,especially in the formation and maturation of blood vessels.Despite there are some studies that reveal the expression of Pdgfr in the developing murine heart,the knowledge of PDGF signaling in heart development is incomplete.In this study,we comprehensively analyzed the function of Pdgfr?in the developing mouse heart using gain-of-function and lost-of function mouse genetic model,providing ample evidences for understanding of the function and underlying mechanism of PDGF signaling in hear development.We firstly thoroughly examined the expression pattern of Pdgfr?in wildtype mouse embryonic hearts using immunofluorescent staining with an anti-Pdgfr?antibody that has been demonstrated to specifically recognize Pdgfr?in literatures.Our results reveal that a high expression levels of Pdgfr?were detected in cardiac cells of the ventricle,but not in those of the atrial at E10.5-11.5.The expression of Pdgfr?in cardiac cells of ventricle was apparently decreased at E12.5.The presentation of Pdgfr?proteins were not observed even in the ventricle of E13.5 heart.The expression of Pdgfr?restored in the ventricle at E14.5-16.5,however these Pdgfr?proteins were not present in working myocardium,but other types of cardiac cells,such as myofibroblasts,which was evidenced by co-staining of Nkx2.5,a marker of working myocardium.We also analyzed the expression pattern of Pdgfr?in the SAN?Sino-atrial node?of developing hearts.We observed that the strong expression of Pdgfr?in the SAN from E12.5-15.5,but dramatically decreased expression of Pdgfr?at E16.5,when the development of SAN is complete.Our expression data implies the involvement of Pdgfr?in heart development and prompts us to analyze the function of Pdgfr?in cardiogenesis.PDGF signaling has been reported to play important roles in the cardiac development.A previous study utilizing a Pdgfr?^Egfp/Egfpknock-in allele reported hypoplasia of the sinus venosus myocardium including the sinoatrial node?SAN?accompanied by increased expression of Nkx2.5.In this study,we revisited this observation by generation of specific ablation of Pdgfr?in the SAN using Shox2-Cre and Pdgfr?^flox/floxmouse lines.Resulted homozygous mutant mice did not exhibit malformation of SAN as compared to their wild-type littermates.Further analysis of cardiac function revealed no differences between wild-type and mutant mice regarding heart rate and electrocardiogram,indicating dispensability of Pdfgr?in the development of SAN.We then further re-examined the heart histology and Nkx2.5expression in conventional Pdgfr?null mouse embryos.We found that mice lacking Pdgfra exhibited normal SAN morphology without alerted Nkx2.5 expression as compared with wildtype embryos.Our data unambiguously demonstrate that Pdgfr?is dispensable for SAN development in mice.We observed sponged morphology of ventricular wall in the heart of Pdgfr?^Egfp/Egfpmice,resembling the symptom of noncompaction of the ventricular myocardium in humans.Since embryos of Pdgfr?^Egfp/Egfpmice exhibit sever abnormalities and die around E8.5 due to the conventional knockout of Pdgfr?in the genome.A very few embryos survive to E13.5 after adiminstration with isoprenaline.To further investigate the function of Pdgfr?in the heart development,we generated a mouse line with conditional knockout of Pdgfr?in the heart by crossing c Tn T-Cre mice with Pdgfr?^flox/floxmice to avoid early death.We examined the rates of proliferation and apoptosis in E13.5 ventricles.Our results reveal that deletion of Pdgfr?in myocardium dramatically attenuated cell proliferation,but do not affected cell apoptosis,in comparison with their wildtype littermate contral.We further performed transcriptome analysis to uncover the molecular events involved.We found 133 obvious upregulated genes?log₂Flodchange>2,p.value<0.05?and 52 obvious downregulated genes?log₂Flodchange>2,p.value<0.05?.Gene ontology enrichment analysis demonstrated these up-and downregulated genes in myocardium of Pdgfr?knockout mice were functionally enriched in terms associated with heart tissue development and heart growth.Among them,we further identified a three-fold upregulation of Nkx2.5 expression.This upregulation was further verified by real time PCR and immunostaining.Nkx2.5 has been reported to be a negative factor regulating myocardium proliferation,decreased Nkx2.5 promotes myocardium proliferation and represses its differentiation and vice versa.We conclude that Pdgfr?signaling plays an important role in heart development.Pdgfr?is upstream of Nkx2.5 and regulates the proliferation of cardiac cells via Nkx2.5 signaling during heart development.To investigate if gain of Pdgfr function would affect heart development,we generated the mouse lines with conditional activation of Pdgfr?signaling in cardiac cells by crossing c Tn T-Cre mice with Pdgfr?^J/+or Pdgfr?^K/+mice.Pdgfr?^J/+or Pdgfr?^K/+alells autonmously activate Pdgfr?signaling in the cells where CRE enzyme is present.c Tn T-Cre;Pdgfr?^K/+allel is five fold active than c Tn T-Cre;Pdgfr?^J/+one.c Tn T-Cre;Pdgfr?^J/+and c Tn T-Cre;Pdgfr?^K/+mice survived normally.Analysis of electrocardiodiagram indicated,as compared to wildtype litter mate,cardiac electric axis was abnormally right slant and ST segment was abviouly depressed in c Tn T-Cre;Pdgfr?^+/Kmice,but almost nornal in c Tn T-Cre;Pdgfr?^+/Jmice?We examined morphology of c Tn T-Cre;Pdgfr?^K/+and c Tn T-Cre;Pdgfr?^J/+adult hearts.Results revealed that c Tn T-Cre;Pdgfr?^K/+adult mice exhibit cardiomegely,but not c Tn T-Cre;Pdgfr?^J/+mice.Toluidinge blue staining demonstrated higher containment of collagenous fibers in the mid myocardium of c Tn T-Cre;Pdgfr?^K/+adult mice,but not in c Tn T-Cre;Pdgfr?^J/+adult mice.Higher proportion of collagene III can be detected by Sirius red staining in c Tn T-Cre;Pdgfr?^K/+mice.Also higher expression level of Collagen can be observed in c Tn T-Cre;Pdgfr?^K/+mice.These results support the idea that enhanced Pdgfr?signaling promotes cardiac fibrosis.In addition,we found an enhanced rate of cell proliferation,but not apoptosis,in the hearts of c Tn T-Cre;Pdgfr?^K/+mice in comparison with wildtype ones.To uncover the underlying mechanism,we performed RNA-Seq analysis.Transcriptome data exhibit that 132 genes are obviously upregulated?log2Flodchange>2,p.value<0.05?and 35 genes are abviously downregulated?log2Flodchange<-2,p.value<0.05?in the heart tissues of c Tn T-Cre;Pdgfr?^K/+mice compared to wildtype mice.Gene ontology enrichment analysis demonstrated these upregulated genes in c Tn T-Cre;Pdgfr?^K/+mice were functionally enriched in terms associated with fibrosis,but no functional enrichment could be identified in downregulated genes.However,we identified downregulation of several genes associated with repression of cardiac cell proliferation,such as Cdkn2a and Meis1.We verified the expression levels of these with realtime PCR.In conclusion,we provide evidences that highly excessive activation of Pdgfr?signaling would promote proliferation of cardiac cells and progress of heart fibrosis.