Effect Of Trimetazidine On Autophagy-related Gene Beclin1 Induced By Ischemia Reperfusion Injury In Cardiomyocytes In Vitro

Objective: Coronary heart disease is the leading cause of death in cardiovascular diseases.With the improvement of people’s living standard,the number of patients with coronary heart disease rises year by year and the acute myocardial infarction patiens increase significantly at the same time.Now the therapeutic strategies of acute myocardial infarction are used to clear the blocked blood vessel for saving dying myocardium including thrombolysis,percutaneous coronary intervention(PCI)and coronary artery bypass grafting(CABG),however,the subsequent myocardial ischemia reperfusion(IR)injury which seriously affecting treatment effect and prognosis has drawn a height attention among medical scientists.Myocardial IR injury involves a variety of molecular and cellular mechanisms,such as increased production of reactive oxygen species(ROS),calcium overload,inflammation,mitochondria damage,apoptosis and so on,while recent papers suggest that another form of programmed cell death,autophagy,regarded as a new research hotspot,may play an important role in myocardial IR injury.Autophagy is an evolutionarily conserved process responsible for the degradation of damaged and dysfunctional protein and cellular organelles.Autophagy plays a “double-edge sword” role in myocardial IR injury,including protective effect in ischemia stage and harmful effect in reperfusion period when excessive activation of autophagy may lead to cell death.The process of autophagy is very complicated,in which the formation of autophagosome is the critical step.Majority of autophagy-related genes(Atg)take part in the regulation of autophagy,one of which is Beclin1 as homolog of heast Atg6.Beclin1 is the essential element in the process of autophagosome formation for the reasons that it can mediate theautophagy-related genes to locate in the autophagic membrane and regulate the formation and maturation of autophagosome with a variety of protein.Therefore,inhibiting the expression of Beclin1 can effectively block autophagy.Drugs in myocardial energy metabolism have attracted much attention in treatment of myocardial ischemia combining with conventional drugs to improve blood flow(for example,nitrates,beta blockers,calcium antagonist)in recent years.With the increasing research on myocardial energy metabolism,it has become a new spotlight that trimetazidine can optimize myocardial metabolism.Trimetazidine,as a myocardial metabolism regulating agent,through partly inhibiting the oxidation of free fatty acids,decreasing the generation of oxygen free radical(OFR)and reducing the calcium overload,plays the pharmacological effects of mitochondria protection and anti-oxigative stress to achieve the purpose of protecting the myocardium,to maintain the ATP generation and the energy metabolism function of ischemic cardiomyocytes.But it is still unknown whether trimetazidine protect myocardium via inhibiting autophagy of cardiomyocytes.Therefore,cultured neonatal rat cardiomyocytes were prepared for ischemia reperfusion injury model in vitro by hypoxia reoxygenation(HR),and were interfered with different concentrations trimetazidine.It was used to evaluate myocardium injury and the protective effect of trimetazidine on myocardial IR injury by comparing cardiomyocyte vitality and LDH detection in different groups,and RT-PCR was performed to detect the m RNA level of autophagy-related gene Beclin1 to identify the expression of Atg in myocardial ischemia reperfusion injury and explore the effect of trimetazidine during IR,and provide new method and basis for clinical medication.Methods: Cardiomyocytes were dissociated and cultured from the 2-3days old healthy Sprague Dawley(SD)rats of Hebei medical university animal center,and were randomly divided into the follwing five groups:1)control group(A group): The cardiomyocytes were cultured in DMEM culture medium containg 10% fetal bovine serum with 5%CO2 and 95% airfor 26.5h.2)HR group(B group): The cardiomyocytes were cultured in serum-free low sugar DMEM medium for 30 minutes,and then cultured with 24 h hypoxia(92%N2,5%CO2,3%O2)and reinstated in normal culture medium with 2h reoxygentation(5%CO2,95%air)to mimic ischemia reperfusion injury.3)0.2 μ mol/L TMZ pre-treatment +HR group(C group): The cardiomyocytes were treated with TMZ of 0.2μmol/L 30 min before HR.4)1.0 μ mol/L TMZ pre-treatment +HR group(D group): The cardiomyocytes were treated with TMZ of 1.0μmol/L 30 min before HR.5)5.0 μ mol/L TMZ pre-treatment +HR group(E group): The cardiomyocytes were treated with TMZ of 5.0μmol/L 30 min before HR.After treatment in each group,the MTT method was used to compare the cardiomyocytes viability,LDH was measured by automatic biochemical analyzer and RT-PCR was performed to detect the m RNA expression level of Beclin1.Results:1 The cardiomyocytes viability results: It was found that the cell viability of group B was decreased to 61.26±6.30% compared with group A(P<0.05),while that in group C、D and E was 70.34±7.17%,79.33±6.90%,89.43±6.02% compared with group A,respectively,which was higher than group B(P<0.05),especially in group E.2 The LDH detection results: It was found that LDH in group B、C、D and E was significantly higher than group A(176.96±12.70 U/L,157.87±13.38U/L,126.44±12.41U/L,97.13±12.14U/L vs 65.18±16.99U/L)(P<0.05),while that in group C、D and E was lower than group B(P<0.05),and group E was lower than group C and D(P<0.05).The results suggested that the IR model in vitro was effective by HR,in which cardiomyocytes was injuried,and was protected by TMZ and it was concentration-dependented with the results of cardiomyocytes viability.3 The RT-PCR results: The RT-PCR results showed that the Beclin1 m RNA of group B was 2.99±0.19 times than group A(P<0.05),and that ofgroup C、 D、E was 2.65±0.24,2.19±0.17,1.64± 0.20 than group A,respectively,which was lower than group B(P<0.05),especially in group E.These suggested that TMZ could attenuate Beclin1 m RNA expression induced by HR,and it had certain concentration dependence.Conclusions:1 The method of hypoxia-reoxygenation culture was effective for ischemia reperfusion model because it could significantly decrease the cardiomyocytes viability and increase LDH content.2 Trimetazidine lightened the cardiomyocyte damage by ischemia reperfusion and it had certain concentration dependence.3 The expression level of autophagy-related gene,Beclin1,increased in ischemia reperfusion injury.4 Trimetazidine attenuated the cardiomyocyte autophagy induced by ischemia reperfusion by reducing Beclin1 m RNA expression,and it had certain concentration dependence.