Molecular Mechanism Of Naoxintong In The Treatment Of Cardiomyopathy In Zebrafish

Cardiomyopathy is a group of non-homogeneous cardiomyopathies caused by various etiologies.It is a progressive impairment of cardiac function due to structural changes in the heart chambers and impaired myocardial function,which is clinically manifested by abnormal enlargement of the heart,arrhythmias,and eventually heart failure.Cardiomyopathies can be classified as hypertrophic cardiomyopathy,dilated cardiomyopathy,restrictive cardiomyopathy,arrhythmogenic right ventricular cardiomyopathy and unclassified cardiomyopathy depending on the phenotype.NXT,as a compound Chinese medicinal preparation,has the pharmacological effects of benefiting Qi,activating blood circulation,resolving blood stasis and opening blood channels,which can be widely used in the treatment of cardiac panic,palpitation and blood vessel disorders caused by Qi stagnation and blood stasis.Therefore,we constructed a zebrafish cardiomyopathy model to investigate the molecular mechanisms and drug targets of NXT in the treatment of cardiomyopathy.In this study,we successfully constructed a zebrafish dilated cardiomyopathy model using the cardiovascular toxic drug terfenadine（TFD）as a model and treated it with NXT.By measuring the embryonic heart rate,venous sinus-to-arterial bulb（SV-BA）spacing,heart capsule area and blood flow velocity in zebrafish,we found that NXT significantly restored the TFD-induced cardiomyopathy such as slowed heart rate,abnormal heart enlargement and blood flow arrest in zebrafish,indicating that NXT could treat TFD-induced zebrafish cardiomyopathy.Using Tg（cmlc2:e GFP）heart transgenic zebrafish,embryonic hearts were isolated from zebrafish embryos by manual fragmentation dissection and subjected to RNA-seq analysis to investigate the molecular mechanism and drug action targets of NXT for cardiomyopathy.Transcriptome analysis of the TFD and TFD+NXT groups identified a total of 961 differentially expressed genes（DGES）,of which 441 were up-regulated and 520 were down-regulated.By GO analysis,we found that differential genes were mainly enriched in the development of cardiovascular system,including the important processes of cardiac development,blood circulation,cardiomyocyte and tissue development,etc.By KEGG analysis,we found that differential genes were enriched in signaling pathways mainly in myocardial contraction pathway,cardiomyocyte adrenergic signaling pathway and ECM-receptor interaction pathway,RNA-seq data analysis and q RT-PCR showed that the therapeutic effect of NXT was closely related to myocardial contraction pathway and cardiovascular development direction.Through an in-depth analysis of the zebrafish embryonic heart transcriptome,among them,HEG1,a gene associated with cardiomyopathy and involved in the biological process of heart development,and its mediated HEG1-CCM pathway attracted our attention.We verified the HEG1-CCM pathway by q RT-PCR and found that the abnormal expression of HEG1-CCM pathway（heg1,ccm1,ccm2,ccm2l,klf2a）genes induced by TFD was restored after NXT treatment,which led to the speculation that HEG1-CCM pathway might be one of the targets of NXT for cardiomyopathy treatment.To test this conjecture,the present study was conducted to investigate the mechanism of action of NXT in the treatment of cardiomyopathy using the heg1^?25 mutant,a zebrafish model of congenital cardiomyopathy,treated with NXT for further validation.Measurements of heart rate,SV-BA spacing,heart capsule area and blood flow velocity in zebrafish embryos showed that NXT significantly restored the abnormally enlarged heart,reduced heart rate and blood flow stagnation caused by cardiac dysfunction in heg1^?25mutant zebrafish.The restorative effect of NXT on the cardiac vasculature of heg1^?25 mutant zebrafish was further verified by Tg(heg1^?25;cmlc2:e GFP)cardiac transgenic zebrafish and Tg(heg1^△25;fik1:e GFP)vascular transgenic zebrafish,and it was found that after NXT treatment of heg1^?25mutant zebrafish,the heg1^?25 mutant zebrafish The abnormal enlargement,asymmetry of atrial position and abnormal increase of vascular endothelial cells in the embryonic heart were significantly improved by NXT treatment.In addition,the abnormal expression of heg1^?25 mutant zebrafish myocardial tissue-specific markers cmlc2,myh6,myh7,and vascular markers vegfc,scl,and flt4 were restored to normal to near-normal levels after NXT treatment,as verified by q RT-PCR.The experimental results showed that NXT has a therapeutic effect on heg1^?25 mutant zebrafish congenital cardiomyopathy,and HEG1-CCM signaling may be one of the molecular targets of NXT for the treatment of cardiomyopathy.Finally,the analysis of the main active components of NXT revealed that Paeoniflorin,Salvianolic acid B,Hydroxysafflor yellow A,Ferulic acid and Ligustilide were the main components of NXT.Among them,paeoniflorin and tannic acid B,the main components with cardioprotective,vasodilating and thrombogenic inhibitory effects,were selected for further validation by treating cardiomyopathic zebrafish with either individual or combined administration.Measurements of embryonic heart rate,SV-BA spacing,pericardial area and blood flow velocity in zebrafish revealed that both Paeoniflorin and Salvianolic acid B administered alone or in combination could improve the symptoms of abnormally enlarged heart,reduced heart rate and slowed blood flow in zebrafish with cardiomyopathy.q RT-PCR validation revealed that paeoniflorin and salvianolic acid B administered alone or in combination could repair the expression of cardiovascular molecular markers in zebrafish with cardiomyopathy levels.The results showed that Paeoniflorin and Salvianolic acid B,the main active components of NXT,had significant therapeutic effects on zebrafish cardiomyopathy.In this study,TFD induced dilated cardiomyopathy model and heg1 mutant zebrafish congenital dilated cardiomyopathy model were used to find that NXT has a good therapeutic effect on zebrafish cardiomyopathy.Its therapeutic effect is to regulate the cardiac contractile pathway and the heart development process to protect the heart and cardiovascular.The mechanism of the effect is related to HEG1-CCM pathway.This study provides a scientific and theoretical basis for the study of NXT drug action mechanism.