Research On The Function And Mechanism Of MORN4 And NR4A2 In The Apoptosis Of Ischemic Cardiomyocytes

Background and objective Ischemic heart disease is one of the leading causes of world mortality.Thedecreased blood flux is not sufficient for cardiomyocyte survival.Cardiomyocyte apoptosis stimulated by deficiency of oxygen and nutrient is the main mechanism leading to heart failure.Loss of cardiomyocytes would cause irreversible damage to ventricle because heart possesses limited regeneration capacity.Thus how to inhibit cardiomyocyte apoptosis and decrease cell loss is significant for therapy.Autophagy is another important process of ischemic cardiomyocytes.By clearing disordered organelles or longevity protein,cells provide essential energy to survive.A moderate amount of autophagy is beneficial to cell survival while excessive autophagy may lead to cell death.There is a complex crosstalk between apoptosis and autophagy,key factors work in apoptosis and autophagy still need more research for potent drug targets.Sphingosylphosphorylcholine(SPC)is a naturally occurred bioactive sphingolipid,which could regulate cardiomyocyte apoptosis and autophagy in our previous reports.We intended to use SPC as a useful tool to find new proteins participated in cardiomyocyte apoptosis.The microarray assay was used to detecte genes regulated by SPC in ischemic cardiomyocytes.We found that MORN4 and NR4A2 were regulated by SPC and may be key factors involved in ischemic cardiomyocyte autophagy and apoptosis.MORN4(the membrane occupation and recognition nexus repeats 4 times)is a protein with 4 MORN repeat motifs.Although proteins containing morn motif exist widely in protozoan,plant and animal and the motif number varies from 2 to 17,very little is known about the function of MORN motif in animals,and much little about MORN4.The MORN motif was known to mediate PIPK localization to membrane in plants.It is reported that morn repeat motif could affecting chloroplast cleavage by the reason that ARC3 could localize to thylakoid membrane through MORN.Function of MORN4 was repoted less except research on photosensitive reaction of Drosophila.Till now,MORN4 roles in hypoxia-caused cardiomyocyte apoptosis and the underlying mechanisms are not known.NR4A2(Nuclear receptor subfamily 4,group A,member 2),also named as Nurr1,consist NR4A orphan nuclear receptor family with NR4A1,and NR4A3.NR4A family belong to the immediately response transcription factors,which is able to response to changes of environment quickly.By regulating differentiation,apoptosis,migration and proliferation,NR4A2 was involved in cancer,obesity,diabetes and neurological disease in particular.But whether NR4A2 associated with ischemic heart disease is not clear.Now there have been some reports of NR4A2 in apoptosis,such as NR4A2 combining with p53 in lung cancer cells to inhibit the expression of Bax and apoptosis.But whether NR4A2 participated in the apoptosis of myocardial cells is not clear.To answer the above scientific questions,this study investigated function of MORN4 and NR4A2 in ischemic cardiomyocytes and their involvement in autophagy and apoptosis.Mechanism of the two molecules was clarified further and our study provide novel drug target for ischemic heart disease therapy.Results1.SPC protects the heart against ischemic damage throughJNK/MORN4/MFN2-regulated autophagic flux1.1 SPC up-regulated expression of MORN4H9c2 cells were deprived of FBS to mimic ischemic condition and changes of MORN4 were detected.Western blot results showed that SPC upregulated MORN4 levels at 5 and 10 ?M.The increased expression of MORN4 by SPC at 5 ?M was also confirmed by QPCR and immunofluescence assay respectively.1.2 Expression of MORN4 in cardiomyocytes exposed to hypoxia increased both in vitro and in vivo.To analyze the possible involvement of MORN4 in hearts exposed to ischemic condition,we detected its changes and functions in hypoxia stress firstly.As was shown,when hypoxia for more than 4 h,apoptosis markers including cleaved PARP and caspase 3 increased as reported.MORN4 increased accompanied with the changes of PARP and caspase 3 in cardiomyocytes exposed to ischemia situation.We confirmed changes of MORN4 in ischemic hearts using the myocardial infarction(MI)models.MI caused apoptosis of cardiac cells as evidenced by the cleavage of PARP and caspase 3.Furthermore,MORN4 in MI mouse hearts was higher at protein and mRNA levels than sham controls.The result is in accordance with in vitro results,which suggested that MORN4 might be important in hypoxia induced cardiomyocyte apoptosis.1.3 MORN4 knockdown promoted apoptosis of H9c2 cardiomyocytes and NRCMs.To evaluate MORN4 function in hypoxia-caused cardiomyocyte apoptosis,we used MORN4 siRNA to knockdown its expression.RT-PCR results showed that siRNA at 80 nM could down regulate MORN4 expression significantly.QPCR and immunofluorescence staining assays were both used to verify the knockdown efficiency.The levels of cleaved PARP and caspase3 in cardiomyocytes with MORN4 knockdown were higher than controls and more cells labeled by FITC,which suggested that MORN4 knockdown promoted apoptosis in H9c2 cells and NRCMs in hypoxia condition.So MORN4 performed a protection role in hypoxia stress.1.4 MORN4 knockdown inhibited autophagic flux in H9c2 and NRCMs.The increased LC3-? level and LC3 patches in cardiomyocytes with MORN4 knockdown revealed that MORN4 changed cardiomyocyte autophagy.The accumulation of LC3-? could be caused by autophagic initiation promotion or autophagic flux blocking.3-methyladenine(3MA)inhibits autophagic initiation by blocking formation of autophagosome.Bafilomycin A1 could inhibit fusion of lysosomes and autophagosomes,and used as a blocker for autophagic flux.Western blot results show that MORN4 knockdown block the autophagic flux.The mTOR inhibitor rapamycin increased LC3-? levels in cardiomyocytes with MORN4 knockdown confirmed that MORN4 affectedautophagic flux,with the addictive effects ofrapamycin in promotion autophagy initiation,so LC3-? levels increased further.1.5 MORN4 knockdown promoted cardiomyocyte apoptosis through impeding autophagic flux.To know the effect of autophagy in MORN4 regulated apoptosis,we used the autophagy blocker and promoter.The autophagy blocker,bafilomycin A1,did not affect MORN4 knockdown-promoted apoptosis as evidenced by no changes of cleaved PARP levels and caspase3.But rapamycin,which promotes mTOR-dependent autophagy,inhibited apoptosis of cardiomyocytes in control and MORN4 knockdown group.The results suggested that MORN4 knockdown promoted apoptosis via impeding autophagic flux,and promotion of autophagy will alleviate apoptosis.1.6 SPC performs cardiomyocyte protection under hypoxia condition through MORN4The involvement of MORN4 in protection of SPC was tested.SPC inhibited the increasing of cleaved PARP and caspase3 in cells with MORN4 normal expression in hypoxia.When MORN4 was knockdown,SPC could not inhibit the cleavage of PARP and caspase 3,which suggested that SPC protected cardiomyocytes through MORN4.1.7 MORN4 affects autophagic flux through interaction with mitofusion 2(MFN2)MFN2 might be the downstream factor of MORN4.Western blot results showed that MFN2 was increased with hypoxia.Furthermore,knockdown of MORN4 decreased and overexpression of MORN4 increased its levels.CoIP results showed that MORN4 did associate with MFN2,which further confirmed that MFN2 is the downstream effector of MORN4.Furthermore,SPC increased MFN2 in cells with MORN4 knockdown,which supported the evidence that SPC might regulate autophagy through MORN4.1.8 SPC promotes MORN4 expression through JNK phosphorylation and MORN4 feedback negatively to JNK.SPC promoted cardiomyocyte autophagy through JNK phosphorylation in our previous study.The relationship between MORN4 and JNK was tested.The results showed JNK phosphorylation was upregulated in cells with MORN4 knockdown,and JNK inhibitor decreased MORN4 expression stimulated by SPC.So it was JNK that regulated MORN4 expression and MORN4 performed a negative feedback to JNK.2.NR4A2 protects cardiomyocyte from apoptosis through enhance autophagic flux 2.1 Expression of NR4A2 in ischemic cardiomyocytes increasedThe changes of NR4A2 in ischemia conditions in H9c2 cardiomyocyteswere analyzed.As was shown,when hypoxia for more than 4 h,NR4A2 increased as shown by RT-PCR,western blot and immunofluorescence,which suggested that NR4A2 might be important in ischemia-induced cardiomyocyte apoptosis.2.2 NR4A2 protected cardiomyocytes from apoptosis.To evaluate the function of NR4A2 in ischemia-caused cardiomyocyte apoptosis,we used NR4A2 siRNA to knockdown the expression of NR4A2.RT-PCR and western results showed that siRNA could down regulate NR4A2 expression significantly.The levels of cleaved PARP and cleaved caspase3 in cardiomyocytes with NR4A2 knockdown were higher than controls,and TUNEL staining showed more FTTC positive cells,which suggested that NR4A2 knockdown promoted apoptosis in cardiomyocytes.In addition,overexpressing of NR4A2 decreased level of cleaved PARP and caspase3.So NR4A2 performs a protection role in ischemia stress.2.3 NR4A2 enhanced autophagic flux in cardiomyocytes.NR4A2 knockdown increased LC3-? level and LC3 patches in cardiomyocytes,and NR4A2 overexpression decreased LC3-?,revealing that NR4A2 changed cardiomyocyte autophagy.To further confirm the change of LC3-? caused by autophagic initiation promotion or autophagic flux blocking,Baf A1,NH4Cl and 3MA were used and the western blot results showed that NR4A2 knockdown blocked autophagic flux.2.4 NR4A2 knockdown promoted cardiomyocyte apoptosis by inhibiting autophagic flux.To know the relationship of autophagy mediated by NR4A2 and apoptosis,the autophagy inhibitor baf Aland promotor rapamycin were used.After baf Alwas added,cleaved PARP and caspase3 did not increase furtherwhile rapamycin decreased apoptosis of NR4A2 knockdown.The results suggested that NR4A2 knockdown promoted apoptosis via impeding autophagic flux,and promotion of autophagy will alleviate apoptosis.2.5 NR4A2 inhibits apoptosis through interaction with p53P53 might be the downstream factor of NR4A2 through which inhibiting expression of Bax and apoptosis.NR4A2 was knockdown or overexpressed to test the hypothesis.Western blot results showed that NR4A2 knockdown increased p53 and Bax level while NR4A2 overexpression decreased p53 and Bax.Co-IP results revealed that p53 could bind with NR4A2 as reported.Immunofluorescence showed that NR4A2 affected location of p53.This data revealed that p53 was the downstream of NR4A2.2.6 NR4A2 was the target of miR-212-3pExpression of miR-212-3p decreased when cardiomyocytes were exposed to ischemia in our microarray data while website prediction showed that miR-212-3p targeted NR4A2.The RT-PCR test confirmed decreased expression of miR-212-3p in ischemia.Inhibitor of miR-212-3p inhibited cardiomyocyte apoptosis while miR-212 mimics did not affect apoptosis.Double luciferase reporter gene assay,RT-PCR and western blot all revealed that NR4A2 was the target of miR-212-3p.MiR-212-3p could also regulate p53 and Bax,which confirmed miR-212 was the upstream of NR4A2.In conclusion,SPC could protect cardiomyocytes against apoptosis through autophgic flux regulated by interaction of MORN4 and MFN2.NR4A2 was targeted by miR-212-3p and protect against apoptosis by p53/Bax.