Study On Calcium Regulatory Proteins And NF-?B In MDMA Induced Neonatal Rat Cardiacmyocytes

[Objective]Ecstasy has become an unavoidable social and public health issue worldwide.The toxic effects of 3,4-methamphetamine(MDMA),a major component of ecstasy,has aroused widespread concern and become one of the hot topics in forensic toxicology.In addition to the more extensive and in-depth study of neurotoxicity,the cardiovascular toxicity of MDMA has been recognized.MDMA can rise changes in the function and morphology of the cardiovascular system,which is characterized by elevated blood pressure,arrhythmia,cardiomyopathy and even sudden death from heart failure.The researches showed that the direct myocardial toxicity of MDMA could lead to the disequilibrium of calcium ion concentration and the change of calcium oscillation mode,however the specific mechanism was still not elucidated.In this reseahch,the primary neonatal rat cardiomyocytes was cultured in vitro,to observe the cardiac toxicity as well as effect on the morphology induced by MDMA in different concentrations and different intoxicate time.The expression of target calcium regulatory proteins,activation and translocation of NF-?b,and activity of PKA and PKC were detected.With the application of inhibitors of target proteins,in order to define the most appropriate concentration and intoxicate time of MDMA to establish the research subject,to explore the effect of MDMA on calcium regulatory protein and NF-?b which are involved in cardiac myocyte calcium cycling and its potential mechanism.[Methods]1.Neonatal rat cardiomyocyte was isolated and cultured in vitro and inoculated at Day6 to establish MDMA induced myocyte.2.Set up groups by concentration in 10?1000?M and intoxicate time in 1?24h of rat cardiac myocyte cultured and exposed to MDMA.The morphological changes of myocardial cells were observed by microscope,cardiac toxicity of MDMA was detected by LDH method and CCK-8 method.3.Rat cardiac myocyte was cultured and induced by inhibitors of Ry R2 and Cav1.2 as well as 10?1000?M MDMA for 24 h.Expressions of m RNA transcription,total protein and phosphorylation of target calcium regulatory proteins were detected.4.Rat cardiac myocyte was cultured and induced by inhibitors of NF-?B,PKA,PKC,Ry R2 and Cav1.2 as well as 10?1000?M MDMA for 24 h.Immunofluorescence staining was applied to observe the translocation of NF-?B,activites of PKA and PKC,and expressions of target calcium regulatory proteins were also detected.[Results]1.It is demenstrated that MDMA can cause the morphological changes of primary cultured neonatal rat cardiomyocytes,which were vacuoles,cell shrinkage,spontaneous beating rhythm of cardiac myocytes,the manifestations were correlated with the concentration and intoxicate time of MDMA.2.10?1000?M MDMA has toxic damage to cardiomyocytes,and toxicity is proportional to the time of MDMA exposure,and 1000?M MDMA reduces the viability of cardiac myocytes.3.MDMA increased the expression of Cav1.2,which may lead to increase extracellular calcium ion influx.MDMA raising the expression of Ry R2 and its phosphorylated protein,may resulte in increasing intracellular calcium overload.NCX1 protein expression was also up regulated,promoting the protective effect by evacuate intracellular calcium.MDMA also improved the expression of PLN,which affected the recovery of calcium ions.4.MDMA enhanced the activites of PKA and PKC.5.MDMA amplified the activated and translocation of NF-?B,which was correlated to the concentration MDMA exposed.6.When PKA or NF-?B was inhibited,compared to Group 1000?M MDMA,expression alternations of target calcium regulatory proteins induced by MDMA have varied.[Conclusion]1.Cardiac myocyte intoxication of MDMA has positive correlation to ite exposure time and concentration,the most appropriate condition for intoxicated myocyte model is incubated in 1000?M MDMA for 24 h.2.MDMA has corresponding effect on expressions of calcium regulatory proteins,which was related to the MDMA induced concentration.3.MDMA amplified the activated and translocation of NF-?B,which was correlated to the concentration MDMA exposed.4.PKA can produce positive regulation to Cav1.2 and Ry R2 to maintain the balance of calcium ions after the infection of MDMA.After the infection of MDMA,Cav1.2 and Ry R2 could be mediated by NF-?B pathway,which may be resulted from the alternation of calcium concentration regulated by Cav1.2 or RyR2.