The Effect Of TRPC6-NFATc4 On Myocardial Cells Injured By Daunorubicin

Anthracyclines,represented by Daunorubicin(DNR),are widely used anti-tumor drugs in clinical practice.Its accompanying cardiotoxicity is the most common and serious adverse reaction,which greatly restricts its clinical application.Anthracyclinebased drugs to induce cardiac toxicity(Anthracycline induced cardiotoxicity,AIC)in the pathogenesis of complex and diverse,the latest reviews related to myocardial apoptosis and mitochondrial damage to each of the points,close ties.Many studies have shown that TRPC6(Transient receptor potential cation channel,member 6)plays an important role in the regulation of cardiovascular functions and diseases,and the potential target genes of TRPC6 are involved in many ways,and their expression and regulatory functions are very complex.Our previous study found that the Ca N-NFAT signaling pathway was significantly activated,especially NFATc4 nuclear translocation was significant,and ERK1/2 was also involved in the cardiotoxicity of anthracyclines.Whether the activation of TRPC6 Ca N cause the activation of Ca NNFAT signaling pathway to form a positive feedback pathway,and how it regulates mitochondrial function remains to be studied.This study aims to clarify the relationship between TRPC6 and NFATc4,so as to understand whether TRPC6-NFATc4 can regulate mitochondria in vivo and its effect on mitochondrial function,its effect on apoptosis of AIC cardiomyocytes,and how ERK1/2 signaling pathway changes,and what pathophysiological role it plays is still unclear and needs to be studied and discussed.In this study,H9c2 rat cardiomyocytes were treated with 1μMDNR to establish AIC model.The changes of TRPC6 gene expression and NFATC4 in H9c2 cardiomyocytes after DNR injury were detected.The interaction between TRPc6 and NFATc4 in DNR injured H9c2 cardiomyocytes: the effects of knocking down TRPC6 and NFTAc4 on each other’s expression,the effects on the morphology and function of mitochondria,and the effects on the proliferation and apoptosis of DNR injured H9c2 cardiomyocytes were observed.The effect of TRPC6 knockdown and NFATc4 knockdown on mitochondrial dynamic regulation signal ERK1/2-DRP1 and mitochondrial apoptosis-related Bax,Bcl-2 and Caspase-3 in DNR-injured H9c2 cardiomyocytes.The above experiments are helpful to understand TRPC6 in AIC and further enrich the understanding of the pathogenesis of AIC.To provide the necessary theoretical basis for the prevention and treatment of AIC targeting TRPC6 and NFATc4.Objective: The interaction between TRPC6 and NFATc4 in H9c2 cardiomyocytes treated with DNR was observed,and the effects of TRPC6-NFATc4 on mitochondrial dynamics and apoptosis of cardiomyocytes were analyzed.To explore the possible mechanism of its influence on mitochondrial dynamics.Methods:(1)1 μM DNR treated H9c2 rat cardiomyocytes for 24 hours to establish an AIC model;(2)Real-time PCR to detect the changes of TRPC6 gene expression in H9c2 cardiomyocytes of DNR damage and the knockdown effect of TRPC6;(3)Western blot and immunofluorescence techniques to detect TRPC6 knockdown in DNR damage Changes in protein content of H9c2 cardiomyocytes and changes in immune fluorescence intensity;(4)Real-time PCR dynamic monitoring of TRPC6 knockdown in DNR damage H9c2 cardiomyocytes TRPC6,NFATc4 gene expression changes;(5)Real-time PCR dynamic monitoring of NFATc4 knockdown in DNR damage H9c2 cardiomyocytes Changes in gene expression of TRPC6 and NFATc4;(6)Detection of NFATc4 content and nuclear translocation after Western detection of TRPC6 knockdown;(7)Mito-Tracker(Green)detects the effects of TRPC6 knockdown and NFATc4 knockdown on mitochondrial morphology and membrane potential;(8)Western Blot detection of ERK1/2-DRP1 protein expression changes in H9c2 cardiomyocytes damaged by TRPC6 knockdown and NFATc4 knockdown in DNR;(9)Ed U cell proliferation test,Annexin V-FITC test to detect cell survival rate and apoptosis rate;(10)TRPC6 knockdown and NFATc4 knockdown damage DNR Changes of apoptosis proteins such as Bcl-2,Bax,Caspase-3 in cardiomyocytes.Results:(1)compared with the normal group,the mrna content of TRPC6 was significantly increased after DNR injured myocardial cells for 24 hours,and the gene content of trpc6 decreased by 84.00% after transfection of TRPC6 m RNA;(2)the expression of TRPC6 protein in H9c2 rat cardiomyocytes treated with DNR was obviously up-regulated,and TRPC6 si RNA reduced the protein content of dnr group by74.87%.The above results indicated that TRPC6 of H9c2 cardiomyocytes damaged by DNR was successfully knocked down by si RNA.(3)in dnr group,the expression of NFATc4 increased,and nuclear translocation was significantly activated.Transfection of NFATc4 si RNA reduced NFATc4 m RNA content by 70.00%,whole cell protein immunofluorescence intensity by 68.62%,and nuclear plasma ratio by 75.75%.NFATc4 knockdown was successful in H9c2 cardiomyocytes injured by DNR.(4)knocking down TRPC6,the fluorescence intensity of NFATc4 whole cell protein decreased by 44.50%,the content of NFATc4 in nucleus decreased by about 50.98%,and the activation of NFATc4 nuclear translocation was obviously inhibited.(5)When NFATc4 was knocked down,the m RNA and protein content of TRPC 6 in myocardial cells damaged by DNR decreased by 62.50% and 70.90%,respectively.(6)both trpc6knock-down and NFATc4 knock-down can inhibit the excessive mitochondrion division of H9c2 cardiomyocytes injured by DNR,and the mitochondrial morphological factors increased to 70.00% and 53.97%.The diameter increased to 63.23% and 65.29%respectively,the network morphology of mitochondria recovered obviously,the fluorescence intensity of rhodamine 123 decreased by 29.87% and 58.30%,and the collapse of mitochondrial membrane potential improved obviously(7)Low knockdown of TRPC 6 and NFATc4 increased the survival rate of myocardial cells injured by DNR by 16.88% and 19.66% respectively,and decreased the apoptosis rate by 44.02% and 47.67%.(8)knockdown of trpc6 and NFATc4 can reduce the protein content of P-ERK1/2 and DRP1-S616 in H9c2 cardiomyocytes injured by DNR,and inhibit the activation of ERK1/2-DRP1 pathway.(9)knockdown of TRPC6 and NFATc4 can increase Bc1-2 protein content,up-regulate Bcl-2/Bax ratio and inhibit Caspase-3 activity in myocardial cells injured by DNR.Conclusion: The expression of TRPC6 and NFATc4 in DNR injured H9c2 rat cardiomyocytes was significantly up-regulated and activated,and the expression of TRPC6 and NFATc4 could regulate each other.TRPC6 and NFATc4 have certain functional consistency,and may act as a functional complex to participate in mitochondrial overdivision induced by DNR through ERK1/2-DRP1,and further affect mitochondrial membrane potential and cardiac cell apoptosis and death.