Protective Effects Of Brain-derived Neurotrophic Factor On Cardiac Apoptosis And Underlying Mechanisms

Background and PurposeCardiomyocyte apoptosis plays critical roles in many cardiac diseases such as myocardial infarction?MI?,ischemia-reperfusion injury,chemotherapeutic agents-induced cardiotoxicity and congestive heart failure.Brain-derived neurotrophic factor?BDNF?is one of the most abundant neurotrophins in the mammalian central nervous system that promotes neuronal survival and differentiation.Previous studies suggested that BDNF is expressed in the myocardium.Growing studies have documented that circulating BDNF is closely related to the development and prognosis of several heart diseases such as coronary artery diseases and heart failure.Moreover,recent studies proved that BDNF inhibited apoptosis of various cancer cells,neurons and pancreatic?cells by binding to its specific receptor tropomyosin receptor kinase B?Trk B?.However,the roles of BDNF in cardiac apoptosis and dysfunction remain unclear.Therefore,the present studies were performed to investigate the impact of BDNF on acute ischemia and chronic doxorubicin-induced cardiomyocyte apoptosis and underlying mechanisms.Our study firstly evaluated the anti-apoptotic effect of BDNF and the relationship between BDNF and miRNA or Akt signaling pathway.MethodsHealthy male Wistar rats were employed and acute MI model?ischemia time 1 h,6 h or 24 h?was induced by the ligation of left coronary artery.Rat ventricles and/or blood samples were collected from MI rats and patients.Plasma BDNF level,protein expression of BDNF and Trk B were examined by enzyme-linked immunosorbent assay and Western blot.Cardiomyocyte apoptosis,infarct size,lactate dehydrogenase?LDH?activity and cardiac function were measured after intra-myocardium injection with recombinant human BDNF in male Kunming mice.Meanwhile,expression and/or activity of apoptotic related protein Bcl-2 and caspase-3 were examined.miR-195 expression in different regions?infarct zone,border zone,and remote zone?after ischemia for different times was examined by Real-time PCR.And primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide?H₂O₂,100?M?.miR-195 mimic,inhibitor or negative control was transfected into the cardiomyocytes.Cell viability and apoptosis were detected by MTT assay and Td T-mediated d UTP nick end labeling?TUNEL?staining,respectively to assess the effect of miR-195 on cardiomyocyte apoptosis.Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195.Luciferase assay,Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF.Sprague-Dawley?SD?rats were injected with Dox?2.5 mg/kg,3 times/week,i.p.?,in the presence or absence of recombinant BDNF?0.4?g/kg,i.v.?for two weeks.Transmission electron microscopy,HE staining,TUNEL assay and echocardiography were used to validate the role of BDNF in Dox-induced cardiac injury and apoptosis.H9c2 cells were treated with Dox?1?M?and/or BDNF?400 ng/m L?for 24 h.Functional roles of BDNF against Dox-induced cardiac injury and regulatory effects on Akt/mammalian target of rapamycin?m TOR?-Bad,p38 MAPK and ERK signals were examined both in vivo and in vitro by TUNEL staining,flow cytometery and Western blot.ResultsCirculating BDNF was significantly enhanced in MI rats and patients.Protein expression of BDNF and Trk B were upregulated in MI.3 days post-MI,BDNF treatment markedly reduced the infarct size and serum LDH activity.Meanwhile,echocardiography indicated that BDNF significantly improved ejection fraction?EF?and fractional shortening?FS?of MI mice.Furthermore,BDNF markedly inhibited cardiomyocyte apoptosis by upregulating Bcl-2 expression and downregulating caspase-3 expression and activity in ischemic myocardium.In neonatal rat ventricular myocytes,cell viability was dramatically increased by BDNF in hypoxia,which was restored by BDNF scavenger Trk B-Fc.miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H₂O₂-treated cardiomyocytes.Meanwhile,BDNF and Trk B protein levels were rapidly increased in H₂O₂-treated cardiomyocytes.Apoptosis in both hypoxic and H₂O₂-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor.Moreover,inhibition of miR-195 significantly improved cardiac function of MI rats.Bcl-2 was validated as the direct target of miR-195.Furthermore,BDNF abolished the pro-apoptotic role of miR-195,which was reversed by its scavenger Trk B-Fc.Protein level of BDNF was reduced in Dox-treated rat ventricles,whereas BDNF and Trk B were markedly upregulated after BDNF administration.BDNF significantly inhibited Dox-induced cardiomyocyte apoptosis,oxidative stress and cardiac dysfunction in rats.Meanwhile,BDNF increased cell viability,inhibited apoptosis and DNA damage of Dox-treated H9c2 cells.Investigations of the underlying mechanisms revealed that BDNF activated Akt and preserved phosphorylation of m TOR and Bad without affecting p38 mitogen-activated protein kinase?MAPK?and extracellular regulated protein kinase?ERK?pathways.Furthermore,the beneficial effect of BDNF was abolished by BDNF scavenger Trk B-Fc or Akt inhibitor.ConclusionsmiR-195 promoted ischemic cardiomyocyte apoptosis,and inhibition of miR-195improved cardiac function of ischemic rats.BDNF/Trk B alleviated cardiac ischemic injury and inhibited cardiomyocytes apoptosis by regulating miR-195/Bcl-2 pathway.BDNF attenuated Dox-induced cardiotoxicity by activating Akt/m TOR-Bad signaling.The present study firstly uncovered the anti-apoptotic effect and mechanisms of BDNF in cardiomyocytes,which provides a novel potential therapeutic strategy for cardiac injury.