| BackgroundIrisin is mainly secreted by muscle tissue.It is a peptide fragment of fibronectin typeⅢ domain-containing protein 5(FNDC5),which can be released into blood after being hydrolyzed by proteolytic enzyme.Irisin plays an important role in metabolism and cardiovascular diseases and promotes the browning of white adipose tissue.Irisin promotes energy consumption and heat production,improving glucose and lipid metabolism and insulin resistance in diabetic mice.Numerous studies have reported that the serum irisin level in patients with type 2 diabetes is downregulated,and the level of irisin is negatively correlated with the symptoms of diabetes.It has been reported that irisin inhibits atherosclerosis in mice.Besides,irisin can improve cardiac function by promoting angiogenesis after MI.However,the effect of irisin on heart failure remains to be fully elucidated.Heart failure can be divided into two broad categories,heart failure with preserved ejection fraction(HFpEF)and heart failure with reduced ejection fraction(HFrEF).Overall,the incidence rate and mortality rate of HFpEF and HFrEF are similar.It is of great significance to explore new therapeutic strategies for treatment of HFpEF and HFrEF.HFpEF accounts for approximately 50%of HF cases.Diabetes is a major cause of HFpEF.Diabetic cardiomyopathy is one of the cardiovascular complications caused by diabetes.The initial clinical manifestation of diabetic cardiomyopathy is HFpEF,which is characterized by diastolic dysfunction and normal or near-normal systolic function.Timely detection and intervention of diastolic dysfunction is essential to delay the development of heart failure.A large number of studies have shown that superoxide can damage tissue cells.When superoxide combines with nitric oxide,it will produce a highly toxic substance,peroxynitrite anion(ONOO-),which causes extensive damage to lipids,proteins and nucleic acids.Oxidative/nitrosative stress plays an important role in the occurrence and development of diabetic cardiomyopathy,and is also considered to be the common pathway and central link of diabetes and its complications.It is unknown whether irisin can ameliorate diabetic cardiomyopathy by inhibiting oxidative/nitrosative stress.Heart failure with ejection fraction less than 40%is known as heart failure with reduced ejection fraction(HFrEF).Ischemic heart disease is the most common cause of HFrEF.How to effectively control the occurrence and development of heart failure after myocardial infarction is still an urgent issue.Pathological remodeling is an important cause of heart failure after myocardial infarction.Macrophage polarization plays an important role in regulating pathological remodeling.Promoting macrophage polarization to M2 phenotype is beneficial to myocardial tissue repair after myocardial infarction.There is no report concerning whether irisin affects the polarization of myocardial macrophages after myocardial infarction.On these grounds,we seek to investigate whether irisin exerts effects on the development of diabetic cardiomyopathy induced HFpEF and MI induced HFrEF and whether the enhancement of FNDC5 expression or the exogenous supplement of irisin attenuates diabetic cardiomyopathy induced HFpEF and MI induced HFrEF.What are the mechanisms underlying the effects of FNDC5/irisin?The aim of this study is to investigate the role of irisin in diabetic cardiomyopathy induced by type 2 diabetes mellitus in vivo and in vitro.Simultaneously,we aim to study the effect of irisin on macrophage numbers and macrophage polarization after myocardial infarction,and to explore the possible molecular mechanisms.The effects of irisin on pathological cardiac remodeling and cardiac function after MI will also be evaluated.ObjectivesFNDC5/Irisin and HFpEF1.To verify whether myocardial overexpression of FNDC5 or supplement of exogenous irisin exerts therapeutic effect on diabetic cardiomyopathy caused by type 2 diabetes mellitus.To verify whether FNDC5/irisin plays a direct protective role on cardiomyocytes at the cellular level.2.To verify the molecular mechanism of irisin’s cytoprotective effect in HG/HF treated cardiomyocytes.FNDC5/Irisin and HFrEF1.To verify whether myocardial overexpression of FNDC5 reduces the pathological remodeling and regulates the number and polarization of cardiac macrophages after myocardial infarction.2.To investigate the molecular mechanisms involved in the irisin-regulated macrophage polarization.MethodsFNDC5/Irisin and HFpEFPart one:the therapeutic effect of myocardial FNDC5 overexpression or exogenous supplementation of irisin on diabetic cardiomyopathy.The direct protective effect of FNDC5/irisin on cardiomyocytes.1.In order to investigate the expression of FNDC5/irisin in diabetic cardiomyopathy,we detected the protein expression of FNDC5/irisin in the left ventricles of db/db mice and db/+mice fed for 16 weeks and determined the plasma level of irisin.2.To verify whether FNDC5/irisin can ameliorate diabetic cardiomyopathy,db/db mice were fed for 16 weeks to establish diabetic cardiomyopathy model.Myocardial injection of Ad-FNDC5 or abdominal implantable osmotic pump supplemented with exogenous irisin(0.25 μg/g body weight/day)were used for 4 weeks.The diastolic and systolic functions were evaluated by echocardiography.Cardiomyocyte apoptosis was detected by Western blot and TUNEL assay.Myocardial fibrosis was detected by Sirius red staining,and myocardial hypertrophy was measured by WGA staining.Simultaneously,the effect of FNDC5/irisin on the ultrastructure of myocardial mitochondria was observed.3.In order to determine whether FNDC5/irisin affects the oxidative stress level of diabetic myocardium,the protein expression of NOX2/iNOS of myocardial tissue was detected.4.H9C2 cells and neonatal rat ventricular myocytes(NRVMs)were treated with HG/HF(25 mM glucose,300 μM sodium palmitate)to simulate type 2 diabetes in vitro.H9C2 cells were treated with irisin(1 μg/mL)and NRVMs were treated with Ad-FNDC5.Western blot and TUNEL assay were used to detect apoptosis.JC-1 staining was used to detect mitochondrial membrane potential,and Western blot was used to detect the release of mitochondrial cytochrome c.The effect of FNDC5/irisin on mitochondria dependent apoptosis of cardiomyocytes was observed.In order to determine whether FNDC5/irisin affects the oxidative/nitrosative stress level of cardiomyocytes,Western blot was used to detect the expression of NOX2,iNOS,and 3-NT.Part two:the molecular mechanism involved in the role of irisin on alleviating cardiomyocyte apoptosis and oxidative/nitrosative stress induced by HG/HF1.In order to determine whether integrin αV/β5 receptor is involved in the cardioprotective effect of irisin,small interference RNA was used to inhibit the expression of integrin αV/β5 in H9C2 cells,and then cells were co-incubated with irisin and HG/HF.Western blot and flow cytometry were used to determine whether irisin alleviated oxidative/nitrosative stress and apoptosis.2.In order to determine whether AKT is a signal molecule of irisin’s cytoprotective effect,H9C2 cells were treated with AKT inhibitor,and then they were incubated with irisin and HG/HF.Western blot and flow cytometry were used to detect whether irisin alleviated oxidative/nitrosative stress and apoptosis.FNDC5/Irisin and HFrEFPart three:myocardial overexpression of FNDC5 attenuates myocardial pathological remodeling after MI,improving cardiac function and promoting macrophage M2 polarization1.In order to determine the expression of FNDC5/irisin in myocardial tissue after MI,Western blot was used to measure the protein expression of FNDC5/irisin in left ventricle on the day 1,3 and 7 post-MI.2.In order to determine whether FNDC5 can reduce cardiac pathological remodeling after MI,Ad-FNDC5 was used for intromyocardial injection,and then MI model was established to observe the effect of FNDC5 overexpression on cardiac fibrosis,cardiac hypertrophy and microvascular angiogenesis after MI.Cardiac function was detected by echocardiography.3.In order to determine whether myocardial overexpression of FNDC5 regulates the number and polarization of macrophages after MI,C57BL/6J mice were first irradiated with 60Co-γ rays.Then they received bone marrow cell transplantation(BMT)from GFP mice and underwent MI surgery.They were divided into two groups:(1)BMT Ad-con MI 7d group(2)BMT Ad-FNDC5 MI 7d group.Immunofluorescence staining and flow cytometry were used to detect the number of GFP positive cells and the number of GFP and F4/80 double positive cells(macrophages)in myocardial tissue to observe the effect of FNDC5 on cell recruitment after MI,and the expression levels of M1 marker(iNOS)or M2 marker(Arg-1)in GFP and F4/80 double positive cells were measured to determine the effect of FNDC5 on macrophage polarization after MI.Part four:the mechanism involved in the role of irisin on regulating macrophage polarization1.To determine whether irisin can regulate the polarization of macrophage,RAW264.7 cells were treated with LPS for M1 polarization,and then they were incubated with irisin to observe the changes of mRNA and protein expression levels of M1 macrophage markers.RAW264.7 cells were incubated with irisin to observe the mRNA and protein expression changes of M2 macrophage markers.2.In order to clarify the mechanisms involved in the role of irisin on regulating macrophage polarization,we screened the possible mechanism of irisin regulating macrophage polarization from the RNA sequencing results of our previous work.Small interference RNAs were used to inhibit IRAK-3 and SOCS3 respectively,and the effect of irisin on M2 polarization was examined.Small interference RNA of NFKBIA was used to observe whether the inhibitory effect of irisin on M1 polarization was alleviated.The inhibition of integrin αV/β5 by small interference RNA was used to observe whether the inhibition of M1 polarization and the promotion of M2 polarization were alleviated.ResultsFNDC5/Irisin and HFpEF1.Compared with db/+mice,the protein expression levels of FNDC5 and irisin of the left ventricles of 16-week-old db/db mice were significantly decreased,and the plasma irisin level was significantly decreased.Compared with db/+mice,cardiac fibrosis and hypertrophy were significantly increased while diastolic dysfunction was impaired in 16-week-old db/db mice,but there was no difference in systolic function between the groups.After 4 weeks of treatment with myocardial overexpression of FNDC5 or supplement of exogenous irisin,diastolic dysfunction,myocardial interstitial fibrosis and myocardial hypertrophy were ameliorated in db/db mice.2.Abnormalities in mitochondria number and size were ameliorated and the reduced electron density of mitochondria and numbers of ruptured cristae in db/db hearts were ameliorated.Overexpression of FNDC5 in myocardium or supplement of exogenous irisin increased the plasma irisin level in db/db mice.Myocardial overexpression of FNDC5 or exogenous irisin supplement reduced the protein expression levels of iNOS and NOX2 in db/db hearts.HG/HF induced H9C2 cell apoptosis,which was alleviated by irisin.HG/HF induced apoptosis of NRVMs,which was alleviated by FNDC5 overexpression.HG/HF induced the collapse of mitochondrial membrane potential and release of mitochondrial cytochrome c into the cytoplasm and apoptosis of H9C2 cells,which was attenuated by irisin.HG/HF induced the oxidative/nitrosative stress in H9C2 cells and NRVMs(as evidenced by increased protein expression levels of iNOS,NOS2 and 3-NT),were inhibited by irisin or FNDC5 overexpression.HG/HF led to oxidative/nitrosative stress and apoptosis in H9C2 cells,which was alleviated by irisin.Inhibition of integrin αV/β5 expression by siRNA partly abolished the above-mentioned effect of irisin.Irisin alleviated the oxidative/nitrosative stress and apoptosis induced by HG/HF,which was compromised by an AKT inhibitor,MK2206.These results suggested that integrinαV/β5-AKT pathway is the possible signaling pathway.FNDC5/Irisin and HFrEF1.Compared with Sham group,the protein expression levels of FNDC5 and irisin in left ventricles were decreased at dayl,3 and 7 post-MI.Myocardial FNDC5 overexpression reduced myocardial fibrosis and cardiac hypertrophy,improving microvascular angiogenesis and systolic function after MI.Compared with BMT Ad-con MI 7d group,the numbers of GFP positive cells and GFP/F4/80 double positive cells were increased in BMT Ad-FNDC5 MI 7d group,suggesting that myocardial FNDC5 overexpression promoted the recruitment of cells and increased the proportion of macrophages after MI.Compared with BMT Ad-con MI 7d group,the number of GFP,F4/80 and iNOS positive cells in BMT Ad-FNDC5 MI 7d group decreased,and the number of GFP,F4/80 and Arg-1 positive cells increased,indicating that myocardial FNDC5 overexpression promoted the polarization of macrophages to M2 like phenotype after MI.Irisin increased CSR2RB protein expression in BMDMs,indicating that CSF2RB is the potential mechanism involved in the effect of FNDC5 on recruiting cells.2.Compared with CON group,the mRNA levels of M1 markers IL-6,CCL2,CXCL10,IL-1β and TNF-α in LPS group were significantly increased.Compared with LPS group,the expression levels of these genes were significantly decreased in LPS+irisin group.Compared with CON group,the expression levels of CD86 and iNOS in LPS group were significantly increased,while those in LPS+irisin group were significantly decreased.Compared with CON group,the mRNA and protein expression levels of Arg-1 and Fizz-1 in irisin group were significantly increased.These results suggested that irisin inhibited M1 polarization and promoted M2 polarization.3.Irisin increased SOCS3 expression and M2 polarization,which was attenuated by i nhibition of IRAK-3 expression by small interference RNA.Irisin inhibited M1 polarization,which was attenuated by the inhibition of NFKBIA(IκB)by small interference RNA.Irisin upregulated the expressions of IκB,IRAK-3 and SOCS3,which was compromised by inhibition of integrin αV/β5 by small interference RNA.Irisin inhibited M1 polarization and promoted M2 polarization,which was also alleviated by inhibition of integrin αV/β5 by small interference RNA.These results suggested that irisin inhibited M1 polarization of macrophages through integrin αV/β5-IκB pathway and promoted M2 polarization of macrophages through integrin αV/β5-IRAK-3-SOCS3 pathway.Conclusion1.Protein expression levels of cardiac FNDC5 and irisin are significantly decreased in diabetic cardiomyopathy caused by type 2 diabetes.Myocardial FNDC5 overexpression or supplement of exogenous irisin increases the level of circulating irisin.FNDC5/Irisin alleviates myocardial apoptosis.Irisin attenuates pathological cardiac remodeling,including myocardial hypertrophy and fibrosis in diabetic heart.The diastolic function is ameliorated.FNDC5/irisin attenuates oxidative/nitrosative stress and apoptosis induced by HG/HF.2.Irisin alleviates the oxidative/nitrosative stress and mitochondrial dependent apoptosis induced by HG/HF through integrin αV/β5 receptor-AKT signaling pathway.3.The cardiac expression of FNDC5/irisin is significantly decreased after myocardial infarction.FNDC5 overexpression reduces pathological cardiac remodeling and ameliorates systolic function after myocardial infarction.FNDC5 overexpression increases the recruitment of macrophages derived from blood monocytes into myocardium after myocardial infarction.CSF2RB is the potential molecular mechanism.4.FNDC5 overexpression promotes macrophage polarization to anti-inflammatory type(M2 type)after MI.Irisin inhibits macrophage M1 polarization through integrin αV/β5 receptor-IκB pathway,and promotes macrophage M2 polarization through integrin αV/β5 receptor-IRAK3-SOCS3 pathway. |
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