An Investigation Into Mechanisms Responsible For Cardiac Fibrosis In CVB₃-induced Murine Viral Heart Diseases And Its Pharmaceutic Treatment

Myocardial fibrosis is characteristic by excessive fibrous tissue accumulation, collagen contents or ratio alteration in heart tissue. The abnormal collagen accrementition is the characteristic pathological change in viral heart disease including acute and chronic myocarditis and partial dilated cardiomyopathy (DCM). Excessive cardiac fibrosis causes abnormal ventricular construction and dysfunction and is known as a key factor in the pathogenesis from myocarditis to cardiomyopathy, however, mechanisms of which are not well understood. The present study was to investigate the mechanisms of myocardial fibrosis in viral heart disease and search for effective medical resolutions. The whole study included four parts as follow:Part I: Acute, Chronic Myocarditis and Dilated Cardiomyopathy Animal Model Setup[Objective] To replicate animal models of acute, chronic myocarditis and dilated cardiomyopathy. [Methods] One hundred Balb/c mice were randomized into 3 groups they were groups of day7 (n=30), month 3 (n=30) and month 9 (n=40). Ten mice selected randomly from each group were injected ip with Eagle's minimal essential medium (EMEM) solution, and served as controls; the remaining of them were inoculated ip with diluted coxsackievirus B₃(CVB₃) EMEM solution to establish animal model of acute and chronic myocarditis and DCM. All mice were examined by echocardiography at the end of day7, mon3 and mon9 postinoculation, respectively and then were sacrificed. Samples of heart tissue were processed for histological and morphological examination. [Results] Degeneration and necrosis of cardiomyocyte, inflammatory infiltration, collapse of cardiac muscle fibers and little fibrosis located around necrosis were observed in hearts of CVB3-infected mice on day 7; less inflammatory infiltration and manifest interstitial fibrosis were seen in infected mice at the end of the third month, left ventricular (LV) systolic function was found decreased in comparison with controls; no inflammatory infiltration but massive diffused fibrosis was shown at the end of mon9, LV dilation and dysfunction were verified by echocardiography. [Conclusion] Acute, chronic and DCM animal models were successfully established with single or repeated CVB3 infection, which closely resemble human myocarditis and DCM.Part II : Study the Role and Mechanism of ADAMTS-1 in Myocardial Fibrosis of Viral Heart Disease[Objective] To investigate the role and mechanism of ADAMTS-1 in the development of myocardial fibrosis of viral heart disease. [Methods] Both in vivo and in vitro experiments were performed. In vivo study, the collagen specific picrosirius red staining was applied on slides of heart samples from controls and mice of each phase of viral heart disease, and then the collagen volume fraction (CVF) was calculated. Collagen I and III mRNA and TGF-beta1 expression were detected with RT-PCR. ADAMTS-1 mRNA was determined with Real-time PCR and the relationship with myocardial fibrosis was further studied. In in vitro study, Balb/c neonatal cardiac fibroblasts were isolated and cultured and treated with ADAMTS-1, TGF-beta, individually and in combination. Variations of Collagen I mRNA were identified with RT-PCR. [Results] In vivo experiments showed that collagen I was generated in chronic myocarditis and DCM and it was most significant in DCM. Collagen III mRNA increased in DCM. TGF-beta1 expression increased in viral heart diseased mice, especially higer in chronic myocarditis and DCM. Comparing to controls, ADAMTS-1 mRNA significantly increased in three stages of viral heart diseases, and especial higher in group mon3. In vitro experiments showed that collagen I expression increased after TGF-beta treatment, while in cells pre-treated with ADAMTS-1, collagen I mRNA were decreased. [Concusion] ADAMTS-1mRNA increased in three stages of viral heart disease, most significantly in samples from group of mon3. Further In vitro experiment proved that ADAMTS-1 can down-regulate the mRNA expression of collagen I via suppressing TGF-beta activity. Part III: Effect of Spironolactone on Remodeling Caused by Myocardial Fibrosis in Murine CVB₃-induced DCM[Objective] To study the mechanisms of spironlactone on remodeling resulted from myocardial fibrosis in murine DCM. [Methods] Sixty Balb/c were randomized into 3 groups and served as groups of control(n= 10), DCM (n=30) and DCM+Spi (n=20) , respectively. Mice in DCM and DCM+Spi were inoculated ip with CVB3 while controls with EMEM. Mice in group control and DCM were fed with drinking water and group DCM+Spi with Spiro-added drinking water at a concentration of 10mg/L. All survived mice were killed at the end of mon 9. Remodeling caused by cardiac fibrosis in each group of mice was determined by echocardiography and histopathological methods. Expression of cell signaling proteins, TGF-β1 and its downstream Smads in hearts of each of mice was confirmed by Western Blot. PCPE-1 mRNA and ADAMTS-1 mRNA in hearts tissue were analysised with RT-PCR. [Results] Spironolactone reduced CVF and alleviated myocardial fibrosis, which contributed to improving remodeling of heart in DCM. Western blot revealed that spironolactone downregulated TGF-β1, p-Smad2/3 and Smad4 expression protein, while significantly increased level of Smad7 in hearts tissue compared to group DCM. Similarly, Spironalactone significantly attenuated PCPE-1 mRNA expression up-regulated in DCM. Spironolactone has no effect on ADAMTS-1 mRNA in hearts. [Conclusions] Spironolactone ameliorated remodeling through antifibrosis. The possible mechanism is that spironolactone upregulated Smad7 and downregulated the expression of TGF-beta1, p-Smad2/3, Smad4 protein and PCPE-1 mRNA.Part IV: Effect of Astragaloside on Myocardial Fibrosis in Murine DCM[Objective] To test the effect and its mechanisms of astragaloside(Astr), one of the active components of astragulus membranaceus, on myocardial fibrosis in murine DCM and its mechanisms. [Methods] Sixty Balb/c mice were randomized into group of control (n=10), DCM (n=30) and DCM+Astr (n=20) , respectively. Mice in group DCM and Astr were inoculated ip with CVB₃, monthly, for 9 times, while the controls with EMEM. Mice in group control and DCM were fed with drinking water and group Astra with Astr-added drinking water at a concentration of 300mg/L. All survived mice were killed at the end of mon 9. Serum concentrations of procollagen peptides (PICP and PINP) were measured by ELISA and the PICP/PINP ratio was further caculated. Histopathological examination was performed as mentioned above and collagen I was detected with polarimicroscope. CVF and collagen I was caculated assisted with image analysis software. The expressions of TGF-β1, p-Smad2/3, Smad4, Smad7 and phosphoralated activity p-P38MAPK in hearts of each group of mice were detected by Western blot analysis. Collagen I mRNA and ADAMTS-1 mRNA were assessed by RT-PCR. [Results] Astragaloside significantly lowered the mortality of DCM (35% vs 56%, P<0.05) ; PICP and PICP/PINP ratio were decreased in mice treated with astragaloside. Increase of CVF and Collagen I deposition in DCM was degraded in group Astr. Western blot showed that increased TGF-β1, p-Smad2/3 and phosphorylation of P-38 MAPK proteines in DCM were repressed with treatment of astragaloside, while Smad7 showed no evident variations. Collagen I, the target gene of TGF-β1/Smad and TGF-β1/p38MAPK signal transduction pathway, was cut down in group Astr. Astragaloside reduced ADAMTS-1 mRNA expression in hearts. [Conclusions] Astragaloside raised survival rate of mice with DCM and alleviated myocardial fibrosis. The antifibrotic effect of Astr resulted from inhibiting expression of TGFβ1, p-Smad2/3, Smad4 and p-P38 MAPK in heart tissues.