The Role And Mechanism Of BMP9 In Doxorubicin-Induced Cardiotoxicity

BackgroundDoxorubicin(DOX)is an anthracycline broad-spectrum anti-tumour drug and has a good effect on breast cancer,osteosarcoma,leukaemia and other malignant tumours.However,DOX causes severe and fatal cardiotoxicity in a dose-dependent manner,resulting in arrhythmia,cardiomyopathy,and left ventricular dysfunction,and eventually developing into congestive heart failure.Therefore,there is an urgent need to alleviate DOX-induced cardiotoxicity.DOX increases oxygen absorption and produces reactive oxygen species(ROS).The excess formation of ROS induces oxidative stress injury.Most importantly,DOX induces mitochondrial oxidative stress,affects cell ultrastructure and energy metabolism,leads to cardiomyocyte apoptosis,and finally leads to cardiac functional abnormalities.Bone morphogenetic protein 9(BMP9)is related to bone differentiation,metabolism,and malignancy.New evidence suggests that BMP9 plays a vital role in vascular development and cardiovascular disease.Exogenous BMP9 supplementation promotes Smad1 activation and inhibits TGF-β in cardiac fibroblasts,reducing pressure load-induced heart failure.Therefore,BMP9 has an important protective effect on the pathological process of the cardiovascular system.However,the role of BMP9 in DOX-induced cardiotoxicity has not been studied.Peroxisome proliferator-activated receptor coactivator-1α(PGC1α)plays a protective role in stabilizing mitochondria.PGC1α activation reduces cardiac injury and pathological remodelling after myocardial infarction.PGC1α reduces mitochondrial oxidative stress and alleviates myocardial ischemia/ reperfusion injury.Notably,DOX treatment significantly reduced PGC1α level in the heart,aggravating its cardiotoxicity.Therefore,PGC1α activation may be a possible way to reduce DOX-induced cardiotoxicity.Methods Part one:The DOX-induced cardiotoxicity model was established in murine.Male C57 mice were intraperitoneally injected with 5mg/kg DOX on day 0,day 7,and day 14 to establish DOX-induced cardiotoxicity model.The mice were euthanised on the28 th day,and their hearts were collected.The expression of BMP2,BMP4,BMP6,and BMP9 m RNA levels were detected by RT-PCR.The expression of BMP9 in mouse hearts was detected by Western blot,ELISA,and immunohistochemistry(IHC).Neonatal rat cardiac fibroblasts(NRCFs)and neonatal rat cardiomyocytes(NRCMs)were extracted.Twelve hours after DOX stimulation,cellular proteins were extracted to detect the expression of BMP9.Part two:Healthy male C57 mice were randomly divided into four groups: Saline+Ig G group,Saline+rh BMP9 group,DOX+Ig G group,and DOX+rh BMP9 group.Recombinant human BMP9(rh BMP9)was administered from one week before DOX injection to the end of the experiment,and the dose was 72 ng/d.The blood of mice was collected on the third day after the first injection of DOX.On the 28 th day,echocardiography and hemodynamic parameters were used to evaluate cardiac function.At the same time,the heart tissue of mice was collected.A ten-week survival experiment evaluated the survival rate.CK-MB and LDH in mouse serum were detected to evaluate the degree of heart injury.The oxidative stress and apoptosis levels in mouse hearts were evaluated by DHE and TUNEL staining.The levels of 4-HNE,3-NT,MDA,and the ratio of GSH/GSSG in mouse hearts were detected by immunohistochemical staining and ELISA.The in-vitro experiment is divided into two parts.Firstly,NRCMs were used for the experiment and divided into four groups: PBS+Ig G,PBS+BMP9 NAb,DOX+Ig G,and DOX+BMP9 NAb.Next,NRCFs supernatant was used as the culture medium of NRCMs.DHE and TUNEL staining detected the apoptosis of NRCMs.ELISA detected the level of oxidative stress-related molecules,and RT-PCR detected the expression of apoptosis-related gene m RNA.Part three:C57 mice were randomly divided into “Saline+Ig G”,“Saline+rh BMP9”,“DOX+Ig G”,and “DOX+rh BMP9”.The administration methods and doses of saline,DOX,and rh BMP9 were the same as before.Western blot detected oxidative stress and apoptosis proteins in mouse hearts.In vitro,NRCMs were divided into four groups: "PBS+Ig G","PBS+rh BMP9","DOX+Ig G",and "DOX+rh BMP9".The protein expression levels of PGC1α,Nrf1,UCP2,and TFAM in NRCMs were detected by Western blot.Then,we performed experiments using PGC1α-c KO mice.PGC1α-c KO mice were produced by PGC1α-flox mice mating with α-MHC-cre mcie.Tamoxifen induces a specific knockout of cardiac PGC1α.Mice were divided into WT+Ig G,WT+rh BMP9,PGC1α-c KO+Ig G,and PGC1α-c KO+rh BMP9.All mice received DOX stimulation,and the dose and time were the same as before.DHE and TUNEL staining were used to detect cardiac oxidative stress and apoptosis.ELISA detected3-NT and MDA to evaluate the level of oxidative stress in mouse hearts.Serum myocardial injury markers were used to evaluate the degree of cardiac injury in mice;Echocardiography and hemodynamics were used to evaluate cardiac function.In vivo,sh PGC1α was transfected to silence PGC1α in NRCMs.The cardiomyocyte experiment was divided into eight groups: sh RNA+PBS+Ig G,sh RNA+PBS+rh BMP9,sh RNA+DOX+Ig G,sh RNA+DOX+rh BMP9,sh PGC1α+PBS+Ig G,sh PGC1α+PBS+rh BMP9 group,sh PGC1α+DOX+Ig G,and sh PGC1α+DOX+rh BMP9.The expression of Nrf1 in NRCMs was detected by immunofluorescence staining.The protein expressions of Nrf1,UCP2 and TFAM were detected by Western blot.The levels of ROS,mt DNA,MDA and ATP,as well as the activities of Caspase-3 and Caspase-3/7,were measured to evaluate the oxidative stress and apoptosis of NRCMs.Furthermore,C57 were divided into four groups: Saline,DOX,DOX+rh BMP9+sh RNA,and DOX+rh BMP9+sh FGF21.AAV-sh FGF21 was injected into the NRCMs to silence FGF21.DHE and TUNEL staining were used to detect cardiac oxidative stress and apoptosis.Oxidative stress injury was evaluated by4-HNE,3-NT,and MDA levels.CK-MB,LDH,ANP,and BNP levels were detected to evaluate mice’s cardiac injury and heart failure.Echocardiography and hemodynamics were used to evaluate cardiac function.In vitro,NRCMs were divided into four groups,namely DOX+Ig G+sh RNA,DOX+rh BMP9+sh RNA,DOX+Ig G+sh FGF21,DOX+rh BMP9+sh FGF21.The protein expression levels of PGC1α,Nrf1,UCP2 and TFAM in NRCMs were detected by Western blot.Results Part one:On day 28 of DOX intraperitoneal injection,cardiac BMP2,BMP4,and BMP6 m RNA levels were unchanged,while BMP9 m RNA expression was significantly decreased.Western blot,ELISA,and IHC also confirmed the decrease of BMP9.In vitro immunofluorescence staining showed that BMP9 was highly expressed in NRCFs but not in NRCMs.BMP9 expression in NRCFs was significantly decreased after DOX stimulation.Part two:The survival rate of mice in the “DOX+rh BMP9” group was higher than that in the “DOX+Ig G” group.On the third day after DOX injection,rh BMP9 treatment reduced the increase of CK-MB and LDH induced by DOX.On the 28 th day after DOX treatment,rh BMP9 treatment significantly reduced the reduction of HW/TL.In addition,exogenous BMP9 treatment weakened the pathological changes of EF,FS,LVEDd,and ±dp/dt caused by DOX.ELISA showed that ANP and BNP levels were significantly decreased after rh BMP9 treatment.Further,rh BMP9 treatment reduced ROS production.Exogenous BMP9 treatment partially corrected abnormal3-NT,MDA,4-HNE,and GSH/GSSG levels.In vitro,NRCFs supernatant pretreatment reduced DOX-induced ROS production and apoptosis in NRCMs.The therapeutic effect of NRCFs supernatant disappears after using the BMP9 neutralizing antibody.In addition,NRCFs supernatant pretreatment increased DOX-stimulated mt DNA levels in NRCMs,whereas mt DNA levels decreased after using BMP9 neutralizing antibodies.C-Caspase3 activity,C-Caspase3/7 activity,and BAX/BCL2 ratio in NRCMs co-incubated with NRCFs supernatant decreased.This beneficial effect of NRCFs supernatant was eliminated after adding BMP9 NAb.Part three:BMP9 synergistic treatment upregulated PGC1α and its downstream Nrf1,UCP2,and TFAM expression in vivo and in vitro.In NRCMs,DOX stimulation significantly reduced the expression level of Nrf1,and rh BMP9 attenuated the decrease of Nrf1.However,the rescue of Nrf1 by rh BMP9 disappeared after PGC1αsilencing.Western blot showed that rh BMP9 failed to increase the levels of Nrf1,UCP2 and TFAM after PGC1α silencing.In addition,in PGC1α-silenced NRCMs,rh BMP9 failed to reduce ROS,MDA level,and Caspase-3 activity or increase mt DNA and ATP levels.After being treated with DOX and rh BMP9,the cardiac DHE densities and TUNEL positivity in PGC1α-c KO mice were higher compared with WT mice.In PGC1α-c KO mice,BMP9 could not increase mt DNA,ATP,GSH/GSSG or decrease4-HNE,3-NT,MDA,and Caspase3 levels.Treatment with rh BMP9 increased EF and FS,and PGC1α-c KO abolished the treatment effect.ELISA confirmed that rh BMP9 failed to reduce CK-MB,LDH,ANP,and BNP levels in PGC1α deficiency mice.BMP9 treatment increased FGF21 m RNA expression in mouse heart tissue.FGF21 silencing prevents BMP9 from activating the protein expression of PGC1α,Nrf1,UCP2 and TFAM in vitro.Meanwhile,BMP9 failed to reduce DOX-induced cardiac ROS production and apoptotic cell density in FGF21-silenced mice.FGF21 deficiency markedly increased 4-HNE,3-NT and MDA levels and decreased mt DNA and ATP levels,whereas rh BMP9 treatment failed to alleviate this change.The activities of Caspase3 and Caspase3/7 in the "sh FGF21+DOX+rh BMP9" group were also significantly higher than in other groups.Meanwhile,FGF21 silencing aggravated the elevation of CK-MB,LDH,ANP,and BNP caused by DOX.FGF21 deficiency exacerbated DOX-induced reductions in EF and FS,whereas rh BMP9 treatment failed to reverse the decline in cardiac function.Conclusions1.The expression level of BMP9 decreased significantly in DOX-induced cardiotoxicity in mice;2.BMP9 protected the cardiac function of mice and alleviated oxidative stress and apoptosis induced by DOX;3.BMP9 was mainly expressed in cardiac fibroblasts and acts on cardiomyocytes through paracrine pathway;4.BMP9 activated Nrf1,UCP2 and TFAM signals by PGC1α-dependent pathway to alleviate DOX-induced cardiotoxicity;5.Activation of the PGC1α signaling pathway by BMP9 was dependent on FGF21.