Effects Of HUCMSCs And Their Exosomes On Cardiac Function And The Expression Of SCN5A MRNA And Its Regulated Sodium Channel Nav1.5 Protein In Myocardial Tissue Of DCM Rats

Objective:To investigate the effects of human umbilical cord mesenchymal stem cells(hUCMSCs)and hUCMSCs-derived exosome(hUCMSCs-Ex)administration via tail vein injection on the cardiac function and the changes of SCN5A,and its regulated sodium channel Nav1.5 protein in the myocardial tissue of rats with dilated cardiomyopathy(DCM)induced by doxorubicin hydrochloride.Methods:1.Establishment of DCM rat model:100 healthy SD rats weighing approximately200g were randomly divided into five groups:normal group(n=20),DCM group(n=20),DCM+phosphate buffer saline group(DCM+PBS group)(n=20),DCM+hUCMSCs group(n=20),and DCM+hUCMSCs-Ex group(DCM+EXO group)(n=20).The normal group was treated as the control,while the other four groups were given intraperitoneal injections of doxorubicin hydrochloride solution,1mg/kg/time,twice a week,with a total injection cycle of 8 weeks.The rats in the normal group were injected with normal saline solution for 8 weeks.The cardiac function of the rats in each group was evaluated by ultrasound cardiogram(UCG).2.Isolation and identification of primary hUCMSCs and hUCMSCs-Ex:Primary hUCMSCs were cultured using the adherent tissue culture.Flow cytometry was used to identify the specific markers of hUCMSCs:CD44,CD45,and CD146;the hUCMSCs-Ex was extracted by ultracentrifugation.The specific surface proteins CD81 and CD9 of hUCMSCs-Ex were determined using the western blotting.The morphology of exosomes was observed by transmission electron microscopy(TEM),and the particle size of the exosomes was analyzed by nanoparticle tracking analysis(NTA).3.Effects of hUCMSCs and hUCMSCs-Ex injected into tail vein on cardiac function and expression of SCN5A m RNA,and its regulated sodium channel Nav1.5 protein in myocardium of DCM rats:The DCM+hUCMSCs group and DCM+EXO group were injected with 1×10~7 hUCMSCs and hUCMSCs-Ex 100μg/kg through tail vein,respectively,and DCM+PBS rats were injected with 1m L/kg of PBS once a week for 4weeks.One week after administration of the drug,the rats’cardiac function were evaluated by UCG.The differential expression genes were screened using Illumina sequencing.The level of SCN5A m RNA in myocardium was determined via real-time PCR(RT-PCR),and the expression of the Nav1.5 protein in myocardium was detected using the western blotting.Results:1.Establishment of the DCM rat model:The eating and activity of rats in the normal group were normal,but the rats injected with doxorubicin hydrochloride exhibited heart failure,gradually decreasing food intake and even weight loss,abdominal distension,vomiting,hematochezia,and so on.Later accompanied by heart failure,the amount of activity decreased,and presented progressive aggravation.After the injection cycle of doxorubicin,UCG showed that the extensive decline in ventricular wall mobility,and the left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)in the doxorubicin hydrochloride group were significantly larger than those in the normal group(P<0.01).The left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS),which reflect the cardiac function,were significantly decreased than those in the normal group(P<0.01).2.Isolation and identification of hUCMSCs and its hUCMSCs-Ex:Third generation hUCMSCs with stable growth were selected to evaluate whether the hUCMSCs grow in a long fusiform or polygonal adherent with clear and bright edges.Flow cytometry showed that the specific surface protein molecule of hUCMSCs,CD44,was highly expressed,while CD45 and CD146 were poorly expressed.The exocrine was successfully extracted via ultracentrifugation,and the regular elliptical bubble structure of hUCMSCs-Ex was observed via TEM.The western blot showed that the exosome expressed specific CD81and CD9 protein molecules.NTA showed that the diameter of the exocrine particles was concentrated between 60-130nm.3.The effects of hUCMSCs and hUCMSCs-Ex injection via tail vein on cardiac functionand the changes of SCN5A m RNA,and its regulated sodium channel Nav1.5protein expression in the myocardium of DCM rats:After injecting hUCMSCs and hUCMSCs-Ex,the survival rate of the rats in the DCM+hUCMSCs group and DCM+EXO group significantly increased,and the symptoms of heart failure was significantly lower than those of the DCM group and DCM+PBS group.The LVEDD and LVESD of the two groups were significantly lower than those of the DCM group and DCM+PBS group before the drug use(P<0.05),and the LVFS and LVEF of the two groups were significantly higher than those of the pre-injection group,DCM group,and DCM+PBS group(P<0.05).The results of Illumina sequencing showed that 31 genes changed in the all group,DCM group,and DCM+EXO group.And the expression of the SCN5A gene in the normal group and DCM+EXO group was higher than that in DCM group.RT-PCR showed that the m RNA level of SCN5A in the rats’myocardial tissue in the DCM+hUCMSCs group and DCM+EXO group was significantly higher than that in DCM group and DCM+PBS group(P<0.05).Western blotting showed that the expression of sodium channel Nav1.5 protein regulated by SCN5A gene in myocardium of rats in DCM+hUCMSCs group and DCM+EXO group was significantly higher than that in DCM group and DCM+PBS group.Conclusion:1.The hUCMSCs were successfully isolated from human umbilical cord and cultured in accordance with the typical characteristics of stem cells.2.Exosomes with high concentration and purity were successfully extracted from the supernatant of hUCMSCs culture,and verified by TEM,NTA and Western blot to meet the typical characteristics of exosomes.3.The caudal intravenous injection of hUCMSCs and hUCMSCs-Ex in DCM rats can improve the cardiac function of rats,and it was proposed for the first time that hUCMSCs and hUCMSCs-Ex may play a role by up-regulating the expression level of SCN5A gene and its regulated Nav1.5 protein in myocardial tissue.