Study Of Effects Of Valsartan On Experimental Autoimmune Myocarditis And The Mechanisms Involved

Backgroud:Myocarditis, a heterogeneous myocardial inflammatory disease, is characterized by myocyte necrosis and degeneration with mononuclear cell infiltration in the presence or absence of fibrosis. The clinical manifestation ranges from the asymptomatic state due to the focal inflammation to the fatal congestive heart failure due to diffuse myocarditis. Most patients with myocarditis can completely recover, while some may develop dilated cardiomyopathy (DCM) and manifest cardiac dilation and heart failure. Myocarditis is the major cause of sudden unexpected death in patients less than 40 years of age.To date, the pathogenesis of myocarditis and the pathogenesis of myocarditis developing into DCM remains unclear. The etiology of myocarditis appears to be rather complicated involving idiopathic, invasion of virus (the major cause), invasion of bacterium and parasite, infectious exposure to drugs and toxins and autoimmune factors. Autoimmunity and immune mediators are known to play a critical role in the pathogenesis of an array of cardiovascular diseases including myocarditis. So the autoimmunity factor becomes a worldwide focus. There is substantial evidence suggesting that autoimmune responses to heart antigens, particularly cardiac myosin, following viral infection may contribute to the disease process. An experimental model of autoimmune myocarditis (EAM) has been induced in susceptible strains of rats produced by immunization with cardiac myosin, which resembles myocarditis in humans and can develop into DCM. So EAM is an excellent experimental model for studying the pathogenesis of myocarditis and the effective therapy. Domestic reports in this field are still relatively rare.Because the pathogenesis of myocarditis and DCM remains unclear, treatments for myocarditis are directed toward reducing or eliminating the inciting agent when possible and tailoring therapy toward the associated the symptoms and complications, such as congestive heart failure, cardiogenic shock, conduction abnormalities and arrhythmia. Recent focus has been on the point that autoimmune response plays an important role in the development of myocarditis. It has been reported that EAM in rats is a CD4+T cell-mediated disease and is thought to be related to Thl responses in the acute phase. Proinflammatory cytokines and Thl/Th2 imbalance play crucial roles in the induction of EAM and in the progression of myocardial injury in this disease.Recently, because evidences suggest that autoimmune response plays important role in the development of myocarditis, immune therapies are being taken more and more attention for the treatment of myocarditis. However, their effects are still controversial. The renin-angiotensin system (RAS) is primarily responsible for regulating vascular tone, which is a key target of vasodilator therapy. The effects of angiotensin II type 1 receptor blockers (ARBs) on the treatment of hypertension, heart failure, and other cardiovascular diseases have been confirmed extensively. However, recent studies have emphasized the nonhemodynamic effects of these drugs. Recently, interest has focused on the nonhemodynamic effects of these drugs. Some Ang-Ⅱreceptor type 1 (AT1) antagonists have been reported to be effective agents in modulating inflammation, adhesion molecule expression, and fibrosis. The cardioprotection of these drugs may be partly due to their regulation of inflammation as a result of removal of overproduced cytokines. Nevertheless, the effects of the ARBs on autoimmune diseases such as autoimmune myocarditis have not been well investigated.Objective:In the present study, the EAM rat model was established by injecting cardiac myosin twice and valsartan, a novel ARB, has been tested in the experimental model of autoimmune myocarditis. It was aimed at investigating the effects of valsartan on the development of EAM and the immune mechanisms involved, In addition, the present study might provide a promising new strategy for myocarditis and DCM. It represents a paradigm of immunosuppressive therapy on immune-mediated diseases.Methods:Thirty-two male Lewis rats (6 weeks old) were maintained in the study. Twenty-four susceptible rats were immunized with porcine cardiac myosin to establish EAM. All Lewis rats were randomly divided into four groups:1) Normal control group (Group C, n=8):Rats were immunized with Freund's complete adjuvant alone, and they received physiological saline instead of durgs administration for three weeks; 2) Non-treated group (Group N, n=8):Purified porcine cardiac myosin (1 mg) was injected subcutaneously in the rear foot pads mixed with an equal volume of Freund's complete adjuvant supplemented with Mycobacterium tuberculosis on days 0 and 7 respectively to establish EAM, and they also received physiological saline instead of durgs administration; 3) Low-dose valsartan (3 mg/kg/day, Group L, n=8): Rats were immunized twice with porcine cardiac myosin to establish EAM and they were administrated orally by valsartan (3mg/kg/day) at the same time of immunization from day 0 to day 21; 4) High-dose valsartan (10 mg/kg/day, Group H, n=8):Rats were immunized twice with porcine cardiac myosin to establish EAM and they received valsartan therapy (10 mg/kg/day) for three weeks.At the time of immunization, we noted the changes of body weight. Heart rate, systolic and diastolic blood pressure were measured by tail-cuff method using a photoelectric tail-cuff detection system and recorded regularly. All rats were killed 21 days after the first immunization.21 days after the after the first immunization, Lewis rats were anesthetized and secured to a warming table. To evaluate the cardiac function and heart structure of the rats, transthoracic echocardiographic analysis was performed by the accurate method using Agilent sonos 5500 echocardiograph with 11-13 L transducer. We obtained the M-mode echocardiogram in the two-dimensional short-axis view of the left ventricular at the chordae tendineae level. After the echocardiography examination, all the rats were sacrificed and their hearts were immediately removed and weighted, then the ratio of heart weight/body weight (g/kg) was calculated. The heart specimens were fixed in 4.0%formaldehyde. Heart sections were stained were stained by the hematoxylin-eosin or Masson's trichrome. Inflammation and fibrosis was examined by light microscopy. Pathologic score was calculated. In order to observe lymphocyte proliferation to cardiac myosin, spleens were collected and cell suspensions were prepared on day 21 after the first immunization and used to preform lymphocyte proliferation assay by MTT colorimetric technique immediately. In addition, serum samples were obtained and the serum concentrations of IFN-y, interleukin (IL)-2 as representative Thl cytokines and IL-4, IL-10 as representative Th2 cytokines were analyzed by ELISA.Results:1. There was no statistically significant difference in blood pressure or heart rate values between the groups. Systolic blood pressure and diastolic blood pressure in Group N were slightly lower than Group C. However, the valsartan dosages did not affect blood pressure.2. Body weights were significantly lower in Group N compared with the groups, the effect of which was ablated by valsartan treatment. Following 3 weeks of autoimmune disease, hearts were significantly enlarged in rats from Group N compared with Group C or Group H. With regard to the heart weight/body weight ratio, it was increased in Group N compared with that in Group C. Valsartan treatment suppressed the increase in that value in a dose-dependent manner.3. Transthoracic echocardiography was performed to assess heart function in all three rat groups. Compared with Group C, LVEDs and LVEDd were significantly enhanced, left ventricular fractional shortening (LVFS) was greatly impaired in Group N, and treatment with high-dose valsartan prevented the left ventricular dysfunction. Moreover, the experimental autoimmune myocarditis significantly enhanced IVS and LVPW, while treatments with both low and high-dose valsartan inhibited the increase. Echocardiography analysis demonstrated that heart structure and cardiac function were greatly impaired in EAM rats, and the treatment with valsartan significantly prevented the progression of autoimmune myocarditis-induced left ventricular dysfunction and ventricular remodeling.4. On day 21 after the first immunization, hearts from Group N were markedly enlarged and contained large grayish areas. Autoimmune myocarditis elicited typical cardiac histopathological changes in myocardium which were manifested as presence of fragments of necrotic myocardial fibers, mononuclear cells, polymorphonuclear neutrophils, eosinophils and giant cells. Masson's trichrome staining showed extensive myocardial fibrosis under autoimmune myocarditis. Treatment with 3 and 10 mg·kg-1·day-1 valsartan significantly reduced the severity of the disease as assessed by detecting the typical signs of myocarditis. Our further quantitative evaluation revealed that the severity of EAM and affected area was significantly suppressed by valsartan in a dose-dependent manner.5. Our data from lymphocyte proliferation assay suggested that lymphocytes obtained from rats in Group N had remarkable proliferative response to porcine cardiac myosin. And this antigen-specific proliferative response to myosin stimulation was significantly suppressed by valsartan treatment in a dose-dependent manner. However, the difference did not reach statistical significance in lymphocytes proliferative reaction to the non-specific mitogen ConA among the four groups.6. Serum analysis revealed significantly elevated serum levels of the Thl cytokines IFN-y and IL-2 in rats from Group N compared with groups, the effect of which was abrogated by valsartan treatment in a dose-dependent manner. In contrast, production of Th2-type cytokines, including IL-4 and IL-10 was significantly higher in the two valsartan-treated groups compared with Group N. These results suggested that valsartan treatment may shift the balance of helper T cells from Thl to Th2.Conclusions: The collective results suggest that valsartan inhibites the progression of myocardial function, remodeling and morphological alteration in a rat model of experimental autoimmune myocarditis in a dose-dependent manner. These data favor the notion that the mechanism of amelioration of the disease by this inhibitory agent can be at least partly explained by the removal of proinflammatory cytokines and the Th1/Th2 imbalance independent of BP-lowering effects. Backgroud:Myocarditis is one of the important causes of dilated cardiomyopathy and severe congestive heart failure. Although myocarditis is habitually viewed as a chronological sequence of several pathologically distinct phases, its precise pathogenesis remains unclear. To date, there is no universally accepted effective therapy for myocarditis. Treatments for myocarditis are not directed toward the disease itself but instead of managing the symptoms and complications. It is a heterogeneous myocardial inflammatory disease, the etiology of myocarditis appears to be rather complicated involving idiopathic, infectious or autoimmune factors. Myocarditis is a disease associated with immune dysfunction and inflammatory reaction that frequently precedes the development of dilated cardiomyopathy. It is now known that proinflammatory cytokines are important for the activation of the detrimental autoimmune responses, such as myocarditis. Cardiac myosin-induced experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by immune response. The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway was initially discovered as a major intracellular signal transduction pathway of the cytokine superfamilies. It is activated by many cytokines and growth factors. Binding of ligands to receptors leads to the activation of the JAK tyrosine kinase family, and activated JAK kinases phosphorylate various combinations of STAT transcription factors, which form dimers, translocate into the nucleus, and bind response elements in the promoters of target genes to stimulate transcription. Different cytokines and growth factors activate different combinations of JAK kinases and STAT transcription factors. JAK-STAT plays a crucial role in various inflammatory reaction. The JAK/STAT pathway has recently been shown to be an integral part of the response of the myocardium to various cardiac insults, including myocardial infarction, oxidative damage, myocarditis, hypertrophy and remodeling. The majority of data involving STAT activity in the heart is confined to STAT1 and STAT3. However, these two STAT proteins have not been well have not been well investigated.The renin-angiotensin system (RAS) is a key target of vasodilator therapy. AngiotensinⅡ(AngⅡ) is a multifunctional hormone that influences the function of cardiovascular cells through a complex series of intracellular signaling events initiated by the interaction of AngⅡwith AT1 and AT2 receptors. Previous studies have reported that Ang II, acting through the AT1 receptor, played an important role in the immunopathology involved in renal injury, chronic allograft rejection and graft coronary artery disease, as well as acute inflammation, such as viral myocarditis. Evidence suggests that the renin-angiotensin system(RAS)Contributes to several steps of the inflammatory process. Studies in inflammatory diseases suggested that Ang II was involved in immune and inflammatory responses and RAS antagonists produced effects by blocking the action markedly. The effects of RAS antagonists on EAM have only recently been addressed. In addition, recent studies have revealed that Ang II activates the JAK-STAT pathway via the AT1 receptor. It has been reported that the JAK/STAT pathway is activated in the rat heart or cardiomyocytes and angiotensinⅡis involved in. However, the effects of angiotensinⅡreceptor blocker valsartan on JAK-STAT signal transduction pathway of experimental models of antigen-induced autoimmune myocarditis have not been addressed.Objective:In our study, Lewis rats were immunized with porcine cardiac myosin to induce experimental autoimmune myocarditis, which is an ideal animal model of human myocarditis. Valsartan was administered orally for 3 weeks to rats with EAM at the same time of immunization. We investigated whether activation of the JAK/STAT pathway was involved in the process of EAM and whether blockade of the renin-angiotensin system by an AT1 receptor antagonist (valsartan) inhibited the JAK/STAT pathway in the rat heart of EAM.Methods:Inbred 6-week-old male Lewis rats (SPF) were immunized with purified porcine cardiac myosin to induce experimental autoimmune myocarditis (EAM). All Lewis rats (n=24) were divided into three groups at random, namely non-treated group (Group N, n=8), normal control group (Group C, n=8), and valsartan-treated group (Group H, n=8). The purified porcine cardiac myosin (10.0 mg/ml) was mixed with an equal volume of Freund's complete adjuvant supplemented with 10.0 mg/ml of Mycobacterium tuberculosis.1) Group N:A total volume of 0.2 ml of the emulsion (lmg porcine cardiac myosin) was injected subcutaneously in each rear footpad of rats on days 0 and 7 respectively; 2) Group C:Rats in control group were given an equal volume of Freund's complete adjuvant alone; 3) Group H:Rats were immunized twice on days 0 and 7 with 0.2 ml of the emulsion.At the same time of immunization. Rats in Group H were received valsartan (10 mg/kg/day) orally by gastric gavage for 3 weeks from day 0 to day 21 prior to sacrifice. Rats from Group C and Group N received physiological saline instead of valsartan.During the study, the changes of body weight were noted. We measured heart rate, systolic and diastolic blood pressure regularly by tail-cuff method using a photoelectric tail-cuff detection system. Twenty-one days after the first immunization, transthoracic echocardiography was performed on animals using Agilent sonos 5500 echocardiograph with 11-13 L transducer. The examination of transthoracic echocardiography was the same as described in part 1. After the transthoracic echocardiographic analysis, the rats were sacrificed and their hearts were immediately removed. Histologic evaluation and lymphocyte proliferation assay were performed also the same as described in part 1.In addition, rat serum samples were obtained 3 weeks postimmunization before sacrificed. Cardiac myosin (10μg/mL) was coated in flat-bottom 96-well ELISA plates, and anti-cardiac myosin autoantibody assay were performed. Absorbance was evaluated as indicator for the levels of anti-cardiac myosin autoantibody using a test wavelength of 405 nm. Furthermore, after the animals were sacrificed, the rat hearts samples were immediately harvested, frozen in liquid nitrogen and stored at-80℃until analysis. We detected tyrosine-phosphorylated protein members of the JAK-STAT family (p-JAK2, p-STAT1 and p-STAT3) in rat myocardial tissue by western blot. To normalize protein loading, the membranes were cut and the lower molecular weight portion was analyzed with the primary antibody forβ-actin.Results:1. There were no statistically significant differences between the three groups with regard to systolic blood pressure, diastolic blood pressure and heart rate values between the groups.2. The results of transthoracic echocardiography, histologic evaluation and lymphocyte proliferation assay showed that valsartan improved EAM significantly. In summary, valsartan treatment significantly prevented the left ventricular dysfunction and severely altered heart structure following myocarditis, reduced the myocarditis-affected histopathologic areas, and suppressed antigen-specific proliferative response to cardiac myosin stimulation.3. Effect of valsartan treatment on serum anti-cardiac myosin autoantibody was examined. Rats in Group N produced large numbers of anti-cardiac myosin autoantibody and the titer of the autoantibody was significantly higher in Group N than in Group C and Group H. These data indicated that autoimmunity triggered by porcine cardiac myosin significantly facilitated large numbers of anti-cardiac myosin autoantibody, the effect of which was nullified by valsartan treatment.4. Western blot showed that expression of phosphorylation JAK2 (p-JAK2) in myocardium of EAM rats were much higher than Group C and Group H. The date suggested that JAK2 was activated in myosin-induced cardiac injury, and the activation of p-JAK2 was abrogated by valsartan treatment.5. Tyrosine-phosphorylated members of the STAT family (p-STAT1 and p-STAT3) were also detected by western blot. The expression levels of p-STAT1 and p-STAT3 were significantly increased in Group N compared with Group C. Interestingly, this expression was attenuated by valsartan administration. This suggests that the activation of STATs is induced by autoimmune myocarditis and valsartan might block the induction.Conclusions:As is refered in our article, the JAK-STAT signal transduction pathway was significantly activated in cardiac myosin-induced autoimmune myocarditis, and valsartan treatment suppressed the over-expression of phosphorylated JAK2, STAT1 and STAT3. Valsartan could ameliorate EAM independent of BP-lowering effects. The mechanism involved might be the effects of valsartan by which blocking the activity of JAK-STAT signal transduction pathway.Key words:Valsartan; experimental autoimmune myocarditis; JAK-STAT signal transduction pathway.