The Protective Effects Of Ivabradine In Preventing Progression From Viral Myocarditis To Dilated Cardiomyopathy

Background:Elevated heart rate is associated with increased cardiovascular morbidity.Heart rate-controlling treatments exert beneficial effects on coronary artery disease and heart failure.Sinus tachycardia is almost invariably observed in myocarditis.Both rest and heart rate-lowering drugs could help to control heart rate.The selective If current inhibitor ivabradine reduces heart rate without affecting cardiac contractility,and has been shown to be cardioprotective in the failing heart.Ivabradine also exerts some of its beneficial effects by decreasing cardiac proinflammatory cytokines and inhibiting collagen accumulation in congestive heart failure.Recently,we demonstrated that ivabradine decreased the expression of pro-inflammatory cytokines and provided protection in a murine model of acute viral myocarditis.However,the protective role of ivabradine in dilated cardiomyopathy caused by chronic viral myocarditis remains unclear.Therefore,in the present study,we investigated whether ivabradine attenuates inflammation and prevents progression from viral myocarditis to dilated cardiomyopathy in mice with chronic viral myocarditis induced by coxsackievirus B3.Heart rate,cardiac size and function,myocardial fibrosis,cardiomyocyte apoptosis,inflammatory cytokines,MAPK protein expression within the myocardium will be determined.Part I Effects of ivabradine in preventing progression from chronic viral myocarditis to dilated cardiomyopathyObjectives:To study the beneficial effects of ivabradine in dilated cardiomyopathy mice,which evolved from coxsackievirus B3?CVB3?-induced chronic viral myocarditis.Methods:One hundred and fifty 4-6 week-old,male Balb/c mice were randomly assigned to three groups:normal control group 1?CON-35,n=10?,normal control group 2?CON-65,n=10?and chronic viral myocarditis group?CVMC,n=130?.All mice in the CON-35 and CON-65 groups were killed on days 35 and 65,respectively.The CVMC group mice were inoculated intraperitoneally with 100 TCID₅₀ CVB3?strain Nancy?0.2ml,0.25 ml and 0.3 ml on day 1,day 14 and day 28,respectively.We defined the day of the first virus inoculation as day 1.The normal control mice were inoculated intraperitoneally with an equivalent dose of Eagle's MEM.The development of chronic viral myocarditis and dilated cardiomyopathy was assessed,and 7 mice from the chronic viral myocarditis group were killed on day 35?CVMC-35,n=7?.Then,the remaining surviving chronic myocarditis mice in the CVMC group were divided into two groups randomly on day 35:an untreated chronic viral myocarditis group?CVMC-65,n=35?and an ivabradine treatment group?IVA,n=20?.The treatment group was treated by intragastric administration with ivabradine?10 mg/kg per day?for 30 days consecutively,and the untreated group mice were given a gavage of normal saline of the same volume.Heart rate,survival rate,myocardial histopathological changes,myocardial fibrosis,cardiac function,inflammatory cytokines,collagen content,p38-MAPK protein expression within the myocardium were studied.Results:1)Mice inoculated with CVB3 manifested the virus infection syndrome,such as loose hair,idle,bad appetite and reduced body weight.2)The survival rate was100%in normal control group.The survival rate of chronic viral myocarditis mice was about 47.69%?62/130?.Sixty-five days later,the survival rate of Ivabradine treatment group was 80%?16/20?,while untreated group was 48.57%?17/35?.3)Masson stain indicated that the degree of myocardial fibrosis was significantly decreased in Ivabradine treatment group compared with the untreated group?P?27?0.05?.Western blot showed that the expression level of collagen was significantly upregulated in chronic viral myocarditis mice,and Ivabradine decreased the production of collagen,reversed the fibrosis and ventricular remodeling?P?27?0.05?.4)The chronic viral myocarditis mice with a lower left ventricular ejection fraction?LVEF?compared with normal control group?P?27?0.001?.Ivabradine significantly improved the heart function,increased LVEF and fractional shortening?LVFS?,and decreased the size of left ventricle?P?27?0.05?.5)Western blot revealed that Ivabradine attenuated the production of TNF-?,IL-1?and IL-6 in treatment group compared with the untreated group significantly?P?27?0.001?.6)The ratio of phospho-p38 MAPK to total-p38 was calculated.Compared with the untreated CVMC mice,ivabradine significantly lowered the production of phospho-p38MAPK?P?27?0.05?.Conclusions:The findings indicate the therapeutic effect of ivabradine in preventing the progression from viral myocarditis to dilated cardiomyopathy in mice with chronic viral myocarditis induced by coxsackievirus B3,is associated with inhibition of the p38MAPK pathway,downregulated inflammatory responses and decreased collagen expression.Ivabradine appears a promising approach for the treatment of patients with viral myocarditis.Part II Effects of ivabradine on cardiomyocyte apoptosis in a murine model of chronic viral myocarditisObjectives:To explore the effects of ivabradine?IVA?on cardiomyocyte apoptosis in a murine model of chronic viral myocarditis?CVMC?.Methods:Ninety male Balb/c mice were divided into the following 6 groups:normal control group?group 1?,CVMC1 group?group 2,n=20?,normal control group?group 3,n=10?,Normal+IVA group?group 4,n=10?,CVMC+IVA group?group 5,n=20?,and CVMC2 group?group 6,n=20?.Mice in groups 2,5,and 6 were inoculated intraperitoneally with Coxsackievirus B3?CVB3?at days 1,14,and 28 using 0.20 ml,0.25ml,and 0.30 ml of CVB3,respectively.On day 42,the mice in groups 1 and 2 were checked by echocardiography,and the hearts were fixed for hematoxylin-eosin?HE?staining after sacrifice.On day 42,the mice from groups 4 and 5 were gavaged with IVA?10 mg/kg-1·d-1?for 30 days until the 72^nd day.Concurrently,mice from groups 3 and 6mice received normal saline?10 mg/kg-1·d-1?.Mice in groups 3,4,5 and 6 were checked with electrocardiogram and echocardiography at day 72 days post-infection.Then,the hearts were fixed for HE staining,and the ends of DNA nucleotide transferase-mediated Nick end labeling?TUNEL?and stored at-80°C for western blot?WB?analysis to detect the expression of Bax,Bcl-2,and Caspase-3.Results:On day 72,echocardiography showed that the left ventricular diameter of group 6 was significantly dilated?P<0.05?,and LVEF and LVFS was significantly decreased?P<0.05?versus normal control group 3.Compared with the CVMC mice in group 6,IVA significantly reduced the heart rate and left ventricular diameter and improved LVEF and LVFS in group 5.The heart chambers were significantly shrunken,and less interstitial fibroblasts infiltrated after treatment with IVA in group 5 when compared to group 6,but they still showed more dilation and fibroblasts than that of group3.The positive apoptosis of myocardial cells in CVMC group 6 were significantly higher than that of normal control group 3 and obviously reduced in group 5 after treatment with IVA.The expression levels of Bax and Caspase-3 in group 6 were significantly higher than those of group 3 and obviously reduced in group 5 versus group 6 after treatment with IVA.Bcl-2 was overexpressed in groups 3 and 4 and under-expressed in group 6,but its levels in group 5 were higher than that of group 6.None of those proteins showed any difference between groups 3 and 4.Conclusions:IVA significantly reduced the heart rate of both normal and CVMC mice,improved the impairment of left ventricular function,and reversed heart remodeling.IVA can upregulate the Bax level and downregulate the Bcl-2 level in CVMC mice and then reduce the Caspase-3 level to inhibit cardiomyocyte apoptosis.