| The heart is the earliest functional organ formed during vertebrate development and is the power pump that drives the flow of blood through the blood vessels.The development of the vertebrate heart is a dynamic process,controlled at different levels,with many transcription factors,signaling pathways and epigenetic factors forming a complex network to regulate the generation,migration and remodeling of cardiomyocytes.Aberrant heart development can lead to diseases including atrial/ventricular septal defect,Fallot’s tetralogy,and arterial stenosis.Therefore,the understanding of heart developmental mechanisms is increasingly important for the purpose to help establish novel and efficient strategies for congenital heart diseases.The systolic-diastolic coupling of the heart requires the synergistic action of various Ca2+channels,Ca2+ion pumps and transporters.A previous study found that the Na+-Ca2+exchanger(NCX1)in the cell membrane plays an important role in zebrafish heart development,suggesting that the maintenance of calcium homeostasis can indeed influence early embryonic heart development.To further explore the relationship between calcium homeostasis and the embryonic heart development,we have systematically explored the effects of disturbances of calcium homeostasis on the developmental processes of the embryonic heart,with a focus on Atp2a2.This study first made RNA probes and detected the spatial-temporal expression map of zebrafish atp2a2(atp2a2a,atp2a2b)at the embryonic developmental stages by whole mount in situ hybridization.The result shows that atp2a2a gene is specifically expressed in cardiomyocytes,while atp2a2b gene is not.This is very similar to the expression pattern in mammals,suggesting that the function of Atp2a2a is highly conserved across species and that Atp2a2a may be involved in the regulation of embryonic heart development.To test whether Atp2a2a regulates embryonic heart development,we treated zebrafish embryos with thapsigargin,a specific inhibitor of the calcium-transporting protein Atp2a2,and found that inhibition of Atp2a2 leads to abnormalities in zebrafish heart looping.Knockdown of Atp2a2a by morpholino antisense oligonucleotides(Atp2a2a MO)reveals similar result.The data demonstrates that the calcium transporter Atp2a2a is indeed involved in the regulation of embryonic heart development.To investigate the relationship between Atp2a2a and calcium homeostasis,we constructed a transgenic zebrafish line Tg(myl7:GCaMP6s)that specifically senses Ca2+signals in cardiomyocytes,and then monitored the dynamics of Ca2+fluctuationin cardiomyocytes in real time using spinning disk confocal microscopy.The result reveals that the Ca2+in different cardiomyocytes showed similar fluctuation patterns.However,the fluctuation pattern of Ca2+is disturbed after inhibition of Atp2a2a activity by thapsigargin.We then tested the relative concentration of Ca2+ by flow cytometry,and found the accumulation of Ca2+in the cardiomyocytes in Atp2a2a inhibited embryos.These results suggest that the calcium transporter protein Atp2a2a does regulate calcium homeostasis in zebrafish embryonic cardiomyocytes,and also implies a negative effect of disturbed calcium homeostasis in embryonic heart development.The accumulation of Ca2+in the cytoplasm activates downstream calcium-regulated neurophosphatase(CaN)activity,leading to dephosphorylation of Nfat,which permits its translocation into the nucleus to regulate downstream genes expression.To verify whether the Nfat signaling pathway is involved in Atp2a2a-regulated cardiac development,we used Cyclosporine A(CsA),an inhibitor that specifically targets this pathway,to inhibit Nfat dephosphorylation,and found that the embryonic heart looping defect due to the inhibition of Atp2a2 or knockdown of Atp2a2a by atp2a2a MO,can be rescued by addition of CsA.This suggests that the calcium transporter Atp2a2a regulates zebrafish heart development through Ca2+downstream-Nfat signaling pathway.In summary,using zebrafish as a model animal,this study demonstrates the calcium transporter protein Atp2a2a,modulates the calcium homeostasis and the downstream Nfat signaling pathway to regulate the embryonic cardiac development.This provides new theoretical support for understanding the relationship between calcium homeostasis and the vertebrate heart development.The study also helps to facilitate the development of new therapeutic targets and related drugs for cardiac development-related diseases with the regulation of calcium homeostasis as the core.Meanwhile,we have also constructed a transgenic zebrafish model that specifically senses Ca2+ in cardiac myocytes,which provides a powerful tool to support subsequent in-depth studies on calcium homeostasis and cardiac developmental processes. |
PDF Full Text Request