The Study On The Mechanism Of NRSF Inhibiting Cardiac Hypertrophy Induced By Myocardin

Cardiac hypertrophy is an adaptive response of the heart to external stimuli.In previous work,it has been reported that myocardin can induce cardiac hypertrophy by activating the expression of fetal genes,such as ANF and BNP.Neuron-restrictive silencer factor(NRSF),also known as RE-1 Silencing Transcription Factor(REST),can inhibit the expression of neuronal genes and non-neuronal genes.Study confirmed that NRSF could inhibit the expression of multiple fetal genes,but the interaction between NRSF and myocardin in cardiac hypertrophy has been unknown.This study attempted to clarify the relationship between NRSF and myocardin on cardiac hypertrophy.Neonatal rat cardiomyocytes(NRCMs)and H9c2 cells were treated with Ang ? and ET-1 for 48h,and then ANF,BNP and Sk-?-actin were detected by real-time PCR,western blotting and immunofluorescence.These results showed that Ang? and ET-1 could induce cardiac hypertrophy.The expression of NRSF was detected in hypertrophic cardiomyocytes by real-time PCR and western blotting.The results showed that the expression of NRSF was decreased in hypertrophic cardiomyocytes.The knockdown of NRSF using siRNA targeting.NRSF in neonatal rat cardiomyocytes(NRCMs)and H9c2 cells up-regulated the expression of hypertrophy genes,such as ANF,BNP,Sk-?-actin and myocardin.To investigate the mechanism underlying NRSF regulating cardiac hypertrophy,we detected the role of NRSF on the transcription activity of myocardin by luciferase reporter gene assay.The results showed that NRSF could decrease the myocardin-induced transcription activity of ANF,BNP and SK-?-actin.The H9c2 cells with stably overexpressed NRSF were constructed by lentiviral infection technique.The expression of myocardin was down-regulated and the inhibition of myocardin transcription was observed in NRSF-transfected cardiac cells by real-time PCR and western blotting.Furthermore,the result of Co-IP showed the protein-protein interaction between NRSF and myocardin.ChIP experiments showed that NRSF inhibited the binding of myocardin to CArG box.Gel mobility shift showed that NRSF can inhibit the formation of nyocardin to SRF/CArG.This study shows that NRSF can not only inhibit the expression of myocardin,but also inhibit the binding of myocardin to SRF/CArG box.In conclusion,this study mainly demonstrates that NRSF inhibit the expression of fetal gene and cardiomyocyte hypertrophy by inhibiting the transcription and expression of myocardin and the binding of myocardin to CArG box.