NADPH Oxidase 2 Mediates Cardiac Sympathetic Nerve Terminal Dysfunction In Heart Failure

Cardiovascular disease(CVD)is the leading cause of death globally.Heart failure,a complex clinical syndrome,is a serious stage of various heart disease.Myocardial infarction(MI)is a major cause of heart failure.Cardiac sympathetic nerve system is activated in heart failure after myocardial infarction(MI),but cardiac sympathetic nerve terminal density and function are reduced.Reactive oxygen species(ROS)and oxidative stress are increased in the remote non-infarcted myocardium in heart failure after MI.NADPH oxidase is a main sources of ROS in myocardium.NADPH oxidase 2(Nox2)is the main subunit of NADPH oxidase.In heart failure,myocardial NADPH oxidase activity and Nox2 expression are increased,and cardiac sympathetic nerve endings density and function are decreased.However,the relationship between NADPH oxidase activity and cardiac sympathetic terminal dysfunction in heart failure has not been elucidated.It is unclear whether Nox2 mediates cardiac sympathetic terminal dysfunction in heart failure.In this study,we tested the hypothesis that Nox2 mediates cardiac sympathetic nerve terminal dysfunctional in heart failure.First,we determined the relationship between NADPH oxidase activity and the changes in cardiac sympathetic terminal function in a rabbit model of MI-induced heart failure.Second,we determined whether NADPH oxidase mediates cardiac sympathetic nerve terminal abnormalities in the rabbit model of MI-induced heart failure using an NADPH oxidase inhibitor.Finally,we further investigated whether Nox2 mediated cardiac sympathetic nerve terminal dysfunction in heart failure using Nox2 deficient mice.The thesis includes three parts as followings:Part1.The relationship between NADPH oxidase and cardiac sympathetic nerve terminal dysfunction in heart failure after myocardial infarctionObjective: To determine whether NADPH oxidase activation is associated with cardiac sympathetic nerve terminal abnormalities in heart failure after MI.Methods: The New Zealand white rabbits(2-2.5kg)were used in this study.MI was induced by the ligation of left anterior descending coronary artery.The animals were divided into two groups: Sham-operated group(Sham)and post-MI heart failure group(MI-HF).All rabbits underwent echocardiographic and hemodynamic examinations 4 weeks after surgery.At the end of study,the animals were sacrificed,the heart,the left and right atria and ventricles,the lungs and the liver were weighed.Left ventricular myocardial tissue was collected for assays.We measured myocardial NADPH oxidase activity,myocardial tissue fluorescence for catecholamine levels,oxidative stress by immunofluorescence,the expression levels of tyrosine hydroxylase(TH)and PGP9.5 by immunohistochemistry and the m RNA expression levels of PGP9.5,norepinephrine transporter(NET)and GAP43 by RT-PCR.Results: Compared with the Sham group,the left ventricular(LV)end-diastolic volume(EDV)and LV end-systolic volume(ESV)were increased in the MI-HF group.LV ejection fraction(EF)and LV d P/dt were decreased.In the MI-HF group,NADPH oxidase activity and the expression of 4-hydroxynonenal(4-HNE,an indicator of oxidative stress)in the remote non-infarcted myocardium increased.The fluorescence of myocardial catecholamine was decreased in the MI-HF group.The expressions of TH and PGP9.5 proteins was decreased in the MI-HF group.The expressions of PGP9.5,NET and GAP43 m RNA was decreased in the MI-HF group.Part 2.Activation of NADPH oxidase mediates cardiac sympathetic nerve terminal abnormalities in heart failure after myocardial infarctionObjective: To investigate whether NADPH oxidase activation mediates abnormalities in cardiac sympathetic nerve endings in heart failure after myocardial infarction.Methods: New Zealand white rabbits(2-2.5kg)were used in this study.MI was produced by the ligation of the anterior descending branch of the left coronary artery.The animals were divided into three groups: Sham operation + normal saline(Sham placebo),MI + normal saline group(MI placebo),and MI + Diphenyleneiodonium group(MI DPI).The animals were received an NADPH oxidase inhibitor DPI(0.8mg/kg/day,i.p.)or saline.All rabbits underwent cardiac hemodynamic examination.At the end of study,the animals were sacrificed,the heart,the left and right atria and ventricles,the lungs and the liver were weighed.Left ventricular myocardial tissue was collected for assays.We measured myocardial NADPH oxidase activity,myocardial tissue fluorescence for catecholamine levels,the expression levels of tyrosine hydroxylase(TH)and PGP9.5 by immunohistochemistry and the expression level of TH,PGP9.5 and GAP43 proteins by Western Blot.Results: Compared with Sham Placebo(PLA)group,LV d P/dt was decreased in the MI PLA group,DPI treatment alleviated the reduction of LV d P/dt in the MI rabbits.Compared with the Sham PLA group,the tissue fluorescence for catecholamine and TH expression were decreased in the MI PLA group,and these changes were attenuated by the DPI treatment.Compared with the MI PLA group,the alteration in the expressions of TH,PGP9.5 and GAP43 proteins were improved in the MI DPI group.Part 3 NADPH oxidase 2 mediates cardiac sympathetic nerve terminal abnormalities in heart failureObjective: Nox2 is a major subunit of NADPH oxidase.NADPH oxidase is activated,and Nox2 expression is increased in heart failure.In the third part of this study,Nox2 knockout mice were used to further explore whether Nox2 mediates abnormalities in cardiac sympathetic nerve endings in heart failure.Methods: Wild type(WT)and NOX2 knockout(KO)mice were used in this study.The animals were divided into 3 groups: WT + saline group,WT + Doxorubicin group(WT Dox),NOX2 KO + Doxorubicin group(NOX2 KO Dox).The animals were administered Dox(a total dose of 15 mg/kg)or saline for 1 to 2 weeks.All animals underwent cardiac echocardiographic examination.At the end of study,the animals were sacrificed,the heart,the left and right atria and ventricles,the lungs and the liver were weighed.Left ventricular myocardial tissue was collected for assays.The level of myocardial catecholamine was detected by tissue fluorescence.The expression levels of 8-hydroxy-2-deoxyguanosine(8-OHd G,an indicator of oxidative stress)and tyrosine hydroxylase(TH)were detected by immunohistochemistry,and the expression levels of GAP43 and PGP9.5 proteins were detected by Western Blot.Results:Compared with the WT saline group,LV FS was decreased in the WT Dox group.Nox2 KO mice improved LV FS.The expression of 8-OHd G in the WT Dox group was increased,Nox2 KO reduced the increase in 8-OHd G in the Dox group.In the WT Dox group,the catecholamine fluorescence and TH expression were decreased,these changes were attenuated in the Nox2 KO Dox group.The expression of PGP9.5 and GAP43 proteins were decreased in WT Dox group,Nox2 KO improved the expression of PGP9.5 and GAP43 proteins.Conclusion: NADPH oxidase activation mediates cardiac sympathetic nerve terminal abnormalities in heart failure.NADPH oxidase 2 plays a crucial role in the process.The present study suggests that NADPH oxidase inhibitors or specific Nox2 inhibitors may be of value in the improvement of cardiac sympathetic nerve terminal function in heart failure,and implicated in the treatment of heart failure.