Investigation Of The Role Of The Chromogranin A-derived Peptide Vasostatin-2 In Post-MI Heart Failure

Objective: The present study was designed to investigate the role of the chromogranin a-derived peptide vasostatin-2 in post-MI heart failure.1.To analyze the serum levels of vasostatin-2 in CHF patients with previous myocardial infarction(MI).To analyze the relation of vasostatin-2 levels with major adverse cardiac events(MACE).2.To test the impact of vasostatin-2 on post-MI heart failure,recombinant vasostatin-2 was injected intra-peritoneally in rat models with myocardial infarction every other day afterwards.The biological effects and mechanisms of vasostatin-2 in vitro and in vivo were evaluated.Methods: 1.This study started with 515 patients of post-MI CHF.These patients were consecutively admitted in Department of Cardiology,Rui Jin Hospital for the diagnosis and treatments of ST-segment elevation MI from September 2009 to May 2011.After further exclusion of patients with cancer,AIDS and loss to contact during 3-year follow-up,450 CHF patients were finally included in the present study.Control group consisted of 149 healthy subjects,who were from a district near our hospital.All patients received regular medications recommended for CHF and MI.For approximately 3 years,the patients were followed up in a special outpatient clinic for MI and CHF or by telephone contact(every 3 months after discharge).During follow-up,blood samples were drawn from patients and control subjects after an overnight fast,or 24 hours after hospitalization.Serum was isolated and stored until use.A complete clinical laboratory measurement and echocardiography were performed in all subjects.Serum concentrations of high-sensitivity C-reactive protein(hs CRP)and tumor necrosis factor-α(TNF-α)and N-terminal pro-brain natriuretic peptide(NT-pro BNP)were measured using ELISA.Serum vasostatin-2 levels were determined with a commercially available human vasostatin-2 ELISA kit.CHF patients were followed up for three years and composite major adverse cardiac events(MACE)were recorded,which was defined as heart transplantation,death due to cardiovascular causes and refractory heart failure requiring hospitalizations.Besides,events regarding reinfarction,target-vessel revascularization,stroke and all-cause death were also documented.2.To test the impact of vasostatin-2 on post-MI heart failure.The biological effects and mechanisms of vasostatin-2 in vitro and in vivo were evaluated.2.1.Cloning and preparation of human recombinant vasostatin-2 protein.Vasostatin-2 c DNA was amplified using RT-PCR from human total RNA,confirmed by sequencing,and cloned into pet15 b vector.Expression of His-tagged vasostatin-2 was induced in BL21 by addition of 1m M IPTG for 8 h,and the protein was isolated by binding to Ni-agarose beads and eluted with imidazole.Next,vasostatin-2 protein was purified by reverse-phase HPLC using a SOURCE 15 RPC column(GE Healthcare).Purified fractions were analyzed by SDS/PAGE and western blotting,and then lyophilized and stored at-80 oC.2.2.Sprague-Dawley rat models(250 to 300g)with myocardial infarction(n=60)were established as described previously,with sham control group set(n=30).Briefly,anesthesia was induced by inhalation of isofluorane mix and maintained with continuous flow of 1.5% isofluorane.The animals were subjected to orotracheal intubation,and ventilated on a Harvard ventilator.Ligation of left anterior descending artery was made at middle part.Those MI rats died within 48 hours were excluded from experiments.To evaluate the influence of vasostatin-2 on the ischemic heart failure,vasostatin-2(100mg)or same amount of saline was injected intra-peritoneally every other day after 48 hours of MI,which resulted in the formation of two experiment groups(saline and vasostatin-2 groups;for each group,n=30).The rat models were raised for 8 weeks.Echocardiography and hemodynamic detections were performed in these rats at 48 hours and 8 weeks of MI.Animal models were euthanized after completion of experiments,and cardiac tissues of infarct area and non-infarct area were isolated and prepared for molecular biology experiments and immunohistochemistry.2.3.To decipher the mechanisms of vasostatin-2 on ischemic HF and also to bypass the masking effects of structural proteins in myocardial tissue,we performed m RNA microarray analysis of vasostatin-2-treated H9C2 cells and rat peritoneal macrophages,followed by bioinformatics analysis.Results: 1.As expectation,post-MI patients with CHF had greatly elevated levels of NT-pro BNP,and significantly lowered LVEF,as compared with controls(for all these comparison,P<0.001).Serum levels of vasostatin-2 were significantly decreased,and in contrast,TNF-a and hs CRP levels were increased in patients with CHF than in controls(for all comparison,P<0.001).2.During a mean follow-up period of approximately 3 years,272 patients(60.4%)experienced MACE,including reinfarction in 54 patients(12%),stroke in 27 patients(6%),target-vessel revascularization in 26 patients(5.8%),heart failure requiring hospitalization in 261 patients(58%),cardiovascular death in 46 patients(10.2%).Notably,serum levels of vasostatin-2 were lower but levels of NT-pro BNP,TNF-α and hs CRP were higher in patients with MACE than those without MACE significantly(for these comparison,P<0.001).Vasostatin-2 level was correlated with HF stages(Spearman’s r=-0.288,P<0.05),LVEF(r= 0.377,P<0.05)and pro-BNP level(r=-0.294,P<0.05).Multivariable logistic regression analysis suggested that decreased vasostatin-2 level and other conventional risk factors(older age,hypertension,diabetes,renal function impairment,low GFR,severe heart failure stages,and lower left ventricular EF)and serum inflammatory cytokines(pro-BNP,hs CRP and TNF-a)were independently associated with MACE in post-MI CHF patients.(all P<0.05).3.Echocardiography at 48 hours showed no statistical difference of parameters in these rats before saline or vasostatin-2 administration.During follow-up,5 rats in saline group and 2 rats in vasostatin-2 group died of apparent cardiac causes.Notably,echocardiography and hemodynamic examinations by a Millar pressure recording catheter at 8 weeks demonstrated that vasostatin-2 provided a significant preservation of post-MI cardiac function.An improvement of left ventricular ejection fraction by 6.7%,and a reduction of left ventricular end-diastolic diameter by 0.54 mm and left ventricular end-diastolic pressure by 3.4 mm Hg were observed in vasostatin-2-treated rats as compared with saline-treated rats(for these comparisons,P<0.05).Vasostatin-2 treatment also resulted in a higher positive dp/dt and a lower negative dp/dt value(both P<0.01),indicating a protective effect on cardiac function.4.Vasostatin-2 reduces myocardial remodeling,interstitial fibrosis and inflammation in rats with myocardial infarction.In histological study of rats with post-MI heart failure,the cross-sectional area of cardiomyocytes,an index reflecting cardiac hypertrophy was significantly attenuated in vasostatin-2-treated rats,when compared with PBS treatment.Increased interstitial fibrosis of post-MI heart failure rats was significantly decreased by vasostatin-2 treatment.Consistent with this,MI-induced expression of several key extracellular matrix protein,collagen I,collagen III,fibronectin,MMP2 and MMP9 was reduced by vasostatin-2 treatment.The expression of interleukin-6 and TNF-α was significantly lower in vasostatin-2-treated rats than in PBS-treated rats using Western blot.Moreover,immunohistochemistry of myocardial tissue was performed in sham and post-MI infarction rats.The levels of TNF-a,interleukin-6,TLR-4,MMP-2,MMP-9 were greatly up-regulated and infiltration of macrophages CD68 and cytotoxic T lymphocytes CD8 was enhanced in heart failure rats versus sham rats,as expectation.Such pathology,however,was significantly suppressed by vasostatin-2 treatment(for all these comparisons,P<0.05),suggesting that vasostatin-2 attenuates post-MI heart failure mainly through modulation of Rho,Wnt and TLR-4 pathway,and RAAS.5.Vasostatin-2 down-regulated the expression of genes with regard to promotion of inflammatory reaction,macrophage activation,positive cytoskeletal organization,promotion of remodeling and cancer growth,and ACE level of renin-angiotensin system.This peptide,on the other hand,up-regulated the expression of ACE2 level of RAAS,and genes negatively modulating cytoskeletal organization,or genes suppressing inflammatory reactions.Conclusions: 1.Decreased serum vasostatin-2 level is associated with heart failure and MACE in post-MI CHF patients,providing a certain guidance for CHF about the prognostic evaluation,diagnosis and treatment.2.Vasostatin-2 exhibiting heart function improvement and reduction of infarct size,remodeling and fibrosis in vasostatin-2-treated rats with post-MI heart failure,as compared with those of PBS treatment.Vasostatin-2 upregulates ACE2 and downregulates ACE,inhibits Rho A and Rac1-PAK1 pathway,de-activates Wnt-β-catenin pathway.Moreover,vasostatin-2 decreases the expression of TLR-4 and its partners,and suppresses inflammatory reaction by downregulating inflammatory cytokine levels.Collectively,vasostatin-2 prevents post-MI heart failure through multiple mechanisms.