Study Of Effects Of Rosuvastatin, A HGM-CoA Reductase Inhibitor, On Experimental Autoimmune Myocarditis

BackgroundMyocarditis (see Glossary) is broadly defined as an inflammatory condition of the heart. It represents one of the most challenging clinical problems in cardiology, associated with a broad spectrum of pathological triggers and a wide range of clinical presentations that vary from mild dyspnea to acute heart failure and sudden death. It frequently affects young, previously healthy individuals and has been estimated to account for up to12%of sudden deaths in patients under40years of age. Although most individuals recover from acute myocarditis, genetically susceptible individuals may go on to develop chronic myocarditis and dilated cardiomyopathy (DCM) resulting in congestive heart failure. Despite this simplistic definition, the diagnosis and treatment of myocarditis continue to present clinical problems.Developing good animal models for myocarditis is crucial to advance understanding of molecular and immunological disease mechanisms. Experimental autoimmune myocarditis (EAM) in genetically susceptible mice may be elicited by immunization of cardiac myosin or a myocardiotogenic peptide derived from the cardiac a-myosin heavy chain or cardiac troponin I emulsified in complete Freund’s adjuvant, and EAM in mice mimics human fulminant myocarditis in the acute phase and human DCM in the chronic phase. The animal models of experimental autoimmune myocarditis offer an excellent tool to study heart-specific autoimmune responses and cardiac inflammation. These models hopefully will aid in the development of new diagnostic and therapeutic strategies.AimsIn our study, we will construct an EAM derived from BALB/c mice.MethodsFemale BALB/c mice (wild-type;13~18g body weight, purchased from the animal experiment center of Shandong University, China) were housed under specific pathogen-free conditions at the Laboratory Animal Center. Six-week-old mice were used in all experiments. All of the animal procedures were approved by the Institutional Authority for Laboratory Animal Care and were performed in accordance with the Guidelines for Animal Experiments of Medical College of Shandong University.All BALB/c mice were divided into four groups randomly, including normal control group, low-dose peptide group, middle-dose peptide group and high-dose peptide group. Forty-five mice were immunized twice to establish LAM. A specific peptide derived from murine cardiac u-myosin heavy chain (MyIIc-u614-629[Ac-SLKLMATLFSTYASAD-OH|) was used as an antigen. The peptide (purity≥98.75%; GL Biochem (Shanghai) Ltd, China) was dissolved in physiological saline and emulsified in an equal volume of Freund’s complete adjuvant (Sigma-Aldrich. St Louis. MO, USA). Lach mouse was injected subcutaneously in the back with peptide on days0and7respectively. On days16,21.26and63after immunization, echocardiography was carried out. the severities of myocarditis and interstitial fibrosis were detected by histopathological evaluation.ResultsIn the LAM model, immunization of susceptible mice with a myocardiotogenic peptide derived from the α-cardiac heavy chain emulsified in complete Fround’s adjuvant (FCA) induces myocarditis in mice with a peak of inflammation in the heart around day21. On day63, inflammation largely resolve, but the process of pathological remodelling continues and many animals develop ventricular dilation and heart failure on follow-up.Conclusion Despite the fact that the EAM models are rather artificial, they offer the great advantage to study disease pathogenesis and autoimmune mechanisms in vivo in the absence of an infective agent. From the EAM model we can not only learn about autoimmune mechanisms contributing to disease development, but the model also allows us to study the pathophysiology of inflammatory heart disease and helps us in the design of novel, immunomodulating treatment strategies. In addition, the EAM model might offer a potential tool to refine the diagnostic accuracy of currently available and future imaging modalities in inflammatory cardiomyopathy. BackgroundMyocarditis is defined as inflammation of the myocardium with consequent myocardial injury. It can lead to sudden death, and about10-20%of patients with histological evidence of myocardial inflammation, even when asymptomatic, will develop a chronic disease eventually leading to dilated cardiomyopathy (DCM). Many cases are associated with enteroviruses infections such as cardiotropic coxsackievirus B3, adenoviruses, or parvovirus B19. EAM induced in susceptible rodent animals by injection of cardiac myosin is an animal model of human myocarditis and post-myocarditis DCM. Cardiac myosin is one of the dominant autoantigens in virus-induced myocarditis in mice. The later phase of enteroviruses-induced heart disease can be mimicked by immunization of mice with purified murine cardiac myosin in the absence of viral infection. An EAM derived from BALB/c mice was constructed to understand the mechanisms of myocardial injury and to develop an effective therapeutic strategy for myocarditis. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatroy effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Published studies suggest they may be beneficial for T cell-mediated diseases by suppressing inducible class II MHC expression and costimulator on APCs, favoring Th2versus Thl differentiation of helper T cell. Statins can also protect endothelial function and the integrity of the microvasculature, increase nitric oxide (NO) bioavailability and exert antioxidant effects. Based on these beneficial effects of statins, this study tested whether rosuvastatin in an RAM BALB/c mouse model can affect the progression of myocarditis and apoptosis of the myocardium cell in vivo, and further enhance cardiac function.Aims1.To investigate the effect of rosuvastatin on the acute phase of myocarditis.2.To test the effect of rosuvastatin on the progression of apoptosis of the myocardium cell in vivo.3.To investigate the effect of rosuvastatin on cardiac function.Methods1.ModeI of EAM and medicationFemale BALB/c mice were housed under specific pathogen-free conditions at the Laboratory Animal Center, six-week-old mice were used in all experiments. All BALB/c mice were divided into four groups randomly, including normal control group (group C). low-dose rosuvastatin group (group L), high-dose rosuvastatin group (group H) and non-treated LAM group (group N). Group L. group H and group N mice were immunized twice to establish LAM. Lach mouse was injected subcutaneously in the back with200ug/200μ1of peptide on days0and7respectively. Rosuvastatin therapy started at the same time of immunization. Group N received physiological saline instead of drugs. The acute phase of LAM was analyzed on day21after the first immunization.2.EchocardiographyLchocardiographie studies were performed in40mice. After determination of body weight, transthoracie echocardiography was recorded under anaesthesia. Left ventricular internal dimensions at end-systole and end-diastole (LVLDs and LVLDd) were measured digitally on the M-mode tracings. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVF’S) were then both calculated.3.Histologic examination and assessment of severity of myocarditis All mice were sacrificed under pentobarbital sodium anesthesia on day21. Heart and body weights were measured, and the ratio of heart weight to body weight (HW/BW) was calculated. The heart was removed, fixed in formalin for24h, embedded in paraffin, and stained with hematoxylin and eosin. Myocarditis was determined by identifying both infiltrating mononuclear cells and myocyte necrosis. The percentage of myocardial inflammation was determined by semi-quantitative image analysis.4. Enzyme-linked immunosorbent assay of inflammatory cytokinesELISA assay was performed to determine serum levels of IL-6and TNF-a in mice. Serum IL-6and TNF-α levels were quantified with the use of IL-6and TNF-α kits.5. Serum lipids levelsOn day21, the levels of serum total cholesterol (TC) and triglycerides (TGs) were determined with an automated enzymatic technique, and low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol levels were detected with an automated chemically modified technique.6. TUNEL assayApoptotic cells in myocardium were detected by the terminal transferase-mediated DNA nick end labeling (TUNEL) assay following the manufacturer’s protocol (Roche Diagnostics GmbH, Germany).7. Immunohistochemisty stainingImmunohistochemistry were performed for detecting the expressions of active caspase3, according to the instructions of the manufacturer.8. Western blotting analysisWestern blotting was performed to detect activation of caspase3,8, and9.Results1. Effects of rosuvastatin on heart weight/body weight ratio and serum lipids levelsThe heart weight/body weight ratio was significantly increased in group N, compared with that in group C. The heart weight/body weight ratio of group H and group L was lower than that of group N. Rosuvastatin treatment suppressed the increase in that value in a dose-dependent manner. On day21, there was no significant difference in the serum lipids level among the four groups.2.Improvement of cardiac function by rosuvastatinAfter immunization on day21, the echocardiographic analyses exhibited the left ventricular dysfunction following myocarditis, and that the rosuvastatin treatment significantly prevented the progression of heart failure.3.Effects of rosuvastatin on myocarditis-affected areas and histoscoreOn day21after immunization, Histopathological scores of the myocarditis revealed that treatment with rosuvastatin suppressed the prevalence and severity on inilammation and that the effect of rosuvastatin tended to be dose-dependent.4.Suppression of expression of inflammatory cytokinesKLISA was performed for serum cytokines of the four groups. Rosuvastatin suppressed the increased inflammatory cytokine production on myocarditis mice in a dose-dependent manner. These results suggest that treatment with rosuvastatin on myocarditis mice inhibits pro-inflammatory cytokine production.5. Apoptosis of cardiomyocyte and active caspase3in myocardiumThe number of apoptotic cells was determined by TUNKL immunolluorescence assay, and the apoptotic cells were stained green. The positive staining cells with active Caspase-3protein were mostly distributed in the myocardium around the areas of inilammation. In addition, some infiltration lymphocytes were stained positively. Similarly, large amounts of apoptotic cardiomyocytes and the positive staining cells with active Caspase-3protein were detected in group N, compared with that of other groups. A few apoptotic cardiomyocytes and the positive staining cells with active Caspase-3protein were seen in the mice receiving low-dose rosuvastatin. Only very few apoptotic cardiomyocytes and the positive staining cells with active C’aspase-3protein were found in group H. No TUNEL-positive cardiomyocytes and no activation of caspase-3were found in group C. These results showed that rosuvastatin treatment suppressed cardiomyocyte apoptosis6.Effects of rosuvastatin on Intracellular Molecules Activated caspase8and activated caspase9were analyzed at the peak of disease on day21, and theirvlevels were significantly up-regulated in group N compared with group C, group L and group H. Treatment with rosuvastatin dose-dependently decreased the myocardial levels of activated caspase8and activated caspase9significantly.ConclusionThe present study demonstrated for the first time that rosuvastatin administration markedly interfered with the progression of experimental autoimmune myocarditis through inhibiting cardiac inflammatory infiltration, suppressing release of proinflammatory cytokines, and resisting apoptosis of cardiomyocyte, all of which can contribute to the improvement of LV function and the attenuation of progressive LV remodeling in HAM. BackgroundMyocarditis is defined as inflammation of the myocardium with consequent myocardial injury. Myocarditis often progresses to dilated cardiomyopathy (DCM), a major cause of heart failure. This condition is characterized hy infiltration of inflammatory cells into the myocardium with consequent loss of myocytes and development of fibrosis and necrosis. Clinical observations and animal experiments suggest that autoimmunity plays an important role in myocarditis.Experimental autoimmune myocarditis is a mouse model of postinfectious myocarditis that can be induced in susceptible mouse strains by immunization with cardiac myosin or a myocardiotogenic peptide derived from the cardiac α-myosin heavy chain or cardiac troponin I. It has been demonstrated to progress into the clinieopathological state similar to DCM in the chronic phase, and has been found to be characterized by the enlargement of the heart, dilatation of ventricles, diffuse and extensive myocardial fibrosis, and hypertrophie and atrophic changes of myocardial fibers, resembling human cardiomyopathy. We constructed an HAM derived from BALB/c mice to investigate the pathogenesis of myocarditis induced by autoimmune mechanism.The3-hydroxy-3-methylgutaryl coenzyme A (HMG-CoA) reduetase inhibitors, commonly referred to as "statins", are well-known potent lipid lowering agents. In addition to their primary effects, the statins have been shown to have pleiotropic effects on the cardiovascular system, including immunomodulation, antiinflammatory, antioxidative, endothelial protective effects, cellular senescence and cardiac remodeling. Based on these beneficial effects of statins, this study was designed to examine whether rosuvastatin in an EAM BALB/c mouse model can affect the myocarditis progression and cardiac remodeling in vivo, and to elucidate the probable mechanisms.Aims1. To assess the effect of rosuvastatin on the chronic phase of EAM.2. To investigate the effect of rosuvastatin on cardiac function.3. To elucidate the molecular mechanisms of rosuvastatin therapentic effects on cardiac remodeling, especially cardiac myocyte hypertrophy, and interstitial fibrosis in the EAM.Methods1. Model of EAMBALB/c mice were immunized at6weeks of age with a peptide derived from murine cardiac α-myosin heavy chain (MyHc-α614-629[Ac-SLKLMATLFSTYASAD-OH]), as described previously. Mice with EAM were divided into three groups:non-treated EAM group (group N); low-dose rosuvastatin group (1mg/kg/day, group L), and high-dose rosuvastatin group (10mg/kg/day, group H). The other BALB/c mice, received neither immunization nor statins therapy, were used as normal controls (group C).2. Rosuvastatin treatmentRosuvastatin therapy started at the same time of immunization. They were administered orally by gastric gavage for9weeks from day0to day63after immunization. Group N and group C received physiological saline instead of drugs.3. EchocardiographyEchocardiography was performed on days21and63after the first immunization. Echocardiographic images were taken from2D M-mode, parasternal long axis views, and short axis views at the mitral valve and mid-papillary muscle levels, the left ventricular (LV) end diastolic dimension (LVEDD), LV end systolic dimension (LVESD), LV septal wall thickness at end diastole (IVSED) and LV posterior wall thickness at end diastole (PWTED) were measured digitally on the M-mode tracings and averaged from at least three cardiac cycles. FS and EF were then calculated.4. Histological assessment of severity of myocarditisMice were evaluated for the development of EAM at the peak of disease on day 21. The heart sections were stained by haematoxylin and eosin. The percentage of myocardial inflammation was determined by semi-quantitative image analysis.5. Enzyme-linked immunosorbent assay of inflammatory cytokinesSerum concentrations of IL-6and TNF-α were quantified with the use of IL-6and TNF-α ELISA kits on days21and63, according to the manufacturer’s instructions.6. Histological assessment of cardiac myocyte hypertrophy and fibrosisMice were euthanized in chronic stage of KAM on day63after immunization. Histological analysis was performed on deparaffinized5-μm-thick tissue sections, which were stained with H&E to assess cardiomyocyte hypertrophy, with Masson trichrome for evaluation of fibrosis and with picrosirius red for evaluation of collagen deposition and orientation.7. Western blotting analysisThe protein levels of p-ERK1/2, t-ERK1/2, p-JNK, t-JNK, p-p38and t-p38were analyzed by Western blot measurement at the peak of disease on day21. TGF-β and p-Smad2/3protein were analyzed by Western blot measurement In chronic stage of KAM on day63.Results1.Effects of rosuvastatin on cardiac structure and functionThe echocardiographic analyses exhibited the left ventricular dysfunction and remodeling following myocarditis. Treatment with high-dose rosuvastatin which of effectiveness was better than low-dose rosuvastatin prevented the left ventricular remodeling, dysfunction and the progression of heart failure. And rosuvastatin administration from day0to day63further improved cardiac function and suppressed cardiac remodeling compared with that administration from day0to day21in group H and group L2.Rosuvastatin ameliorated myocarditis progression in EAM miceSevere inflammatory lesions were observed in the hearts of group N mice. In contrast, area of cellular infiltration into the myocardium was significantly decreased in both group L and group H, compared with group N. Histopathological scores of the myocarditis also revealed that treatment with rosuvastatin suppressed the prevalence and severity on inflammation and that the effect of rosuvastatin tended to be dose-dependent.3.Effects of rosuvastatin on serum inflammatory cytokines ELISA was performed for serum cytokines of the four groups on day21and63. The serum levels of IL-6and TNF-a were significantly lower in the two rosuvastatin-treated groups than that in group N, and rosuvastatin also suppressed the increased inflammatory cytokine production on myocarditis mice in a dose-dependent manner.4. Effect of rosuvastatin on myocardial Fibrosis and remodelingMarked interstitial fibrosis and collagen deposition were detected in the hearts of mice in each group on day63. Rosuvastatin treatment significantly reduced the areas of fibrosis and collagen deposition in a dose-dependent manner compared with those in group N. The myocyte size was significantly increased in group N. and dose-dependently reduced in the treatment with rosuvastatin groups compared with those in group N.5. Effects of rosuvastatin on Intracellular Signaling Kinases and MoleculesTreatment with rosuvastatin dose-dependently decreased the myocardial levels of pERK1/2, pJNK, p-p38, TGF-β1and pSmad2/3protein significantly. These results showed that rosuvastatin treatment down-regulated Mitogen-activated protein kinase, and TGF-β1-Smad2/3signaling pathways in the hearts of EAM mice.ConclusionThis investigation demonstrates for the first time that rosuvastatin attenuates cardiac remodeling, especially cardiac myocyte hypertrophy, and interstitial fibrosis in the EAM mice, all of which can contribute to the improvement of LV function. These beneficial effects of rosuvastatin treatment are related partially to down-regulated Mitogen-activated protein kinase, TGF-β1-Smad2/3and caspase-mediated signaling pathways. Further clinical studies are needed to clarify whether this beneficial effect can be translated into clinical practice.