**2. Experimental autoimmune myocarditis (EAM)**

The EAM model has been extensively used as a disease model of human myocarditis (Kodama et al., 1990). Experimental data revealed several similarities between this model

Experimental Autoimmune Myocarditis: Role of Renin Angiotensin System 311

the EAM hearts at the onset of the disease (Okura et al., 1998). Subsequently, mRNA of IL-1β, interferon γ, and tumor necrosis factor (TNF) α increases (Maeda et al., 2005). In the recovery phase IL-10 appears in the heart. IL1, IL2, IL6 and TNFα are involved in the impairment of cardiac contractility, IL1 and IL6 may induce hypertrophy of myocytes whereas IL1 and TNFα may play a role in the development of myocardial fibrosis. Th2 cytokine IL-10 plays a protective role in the development of EAM (Watanabe et al., 2001). IL10 possesses immunomodulatory properties involving the inhibition of macrophage

In rat EAM, infiltration of the inflammatory cells into the myocardium may be mediated by monocyte chemoattractant protein-1 (MCP-1) or other chemokines as MCP-1 mRNA is strongly expressed in the heart coincident with the onset of the disease and persists until the recovery phase (Goser et al., 2005). Nitric oxide may also play a role in the development of autoimmune myocarditis, by exacerbating inflammatory responses to cardiac infections

Angiotensin (Ang) II, the principal effector peptide of the renin–angiotensin system (RAS), has been reported to induce immune and inflammatory response in various cardiac disease conditions, including atherosclerosis, hypertension, left ventricular hypertrophy, myocardial infarction, heart failure and myocarditis (Ferrario & Strawn, 2006; Schmieder et

The RAS is a central element of the physiological and pathological responses of the cardiovascular system. Its primary effector hormone, Ang II, not only intercedes immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, hypertension and heart failure (Opie & Sack, 2001). Many of the cellular effects of Ang II appear to be mediated by ROS generated by NAD(P)H oxidase (Koumallos et al., 2011). Two subtypes of Ang II receptors have been defined on the basis of their differential pharmacological and biochemical properties: Ang II type 1 receptors (AT1), which are involved in most of the well-known physiological effects of Ang II, and Ang II type 2 receptors (AT2), which have a less well-defined role but appear capable of counterbalancing some of the effects of AT1 stimulation. AT1 transactivates growth pathways and mediates major Ang II effects such as vasoconstriction, increased cardiac contractility, renal tubular sodium reabsorption, cell proliferation, vascular and cardiac hypertrophy, inflammatory responses, and oxidative stress. AT2 is believed to induce essentially opposite effects, including vasodilation, antigrowth and antihypertrophic effects, and to play a significant role in blood pressure (BP) regulation (Oudit & Penninger,

2011; Horiuchi et al., 1999; Matsubara, 1998; Siragy, 2000; Carey et al., 2001).

The discovery of Ang (1–7), an endogenous peptide which opposes the pressor, proliferative, profibrotic, and prothrombotic actions mediated by Ang II has contributed to the realization that the RAS is composed of two opposing arms: the pressor arm constituted by the enzyme angiotensin-converting enzyme (ACE), Ang II as the product, and the AT1 receptor as the main protein mediating the biological actions of Ang II; the second arm is composed of the monocarboxypeptidase ACE2, Ang (1–7) produced through hydrolysis of Ang II, and the Mas receptor as the protein conveying the vasodilator, antiproliferative,

antifibrotic, and antithrombotic effects of Ang (1–7) (Petty et al., 2009; Ferrario, 2011).

function and the production of proinflammatory cytokines (Nishio et al., 1999).

(Kittleson et al., 2005).

**3. Renin angiotensin system (RAS)** 

al., 2007).

and the original disease in human. The current EAM model will provide the opportunity for further fundamental research into myocarditis.

EAM is similar to the giant cell myocardits and is more likely to progress into dilated cardiomayopathy (DCM) (Kodama et al., 1990). EAM can be induced by injection of cardiac myosin with Freund's adjuvant in to the footpads of Lewis rats. The immunized rats become ill and immobile at day 14 and then their activity gradually recovers beginning in the fourth week. The diseased rats show severe myocardial damage with inflammatory cell infiltration. Rats with EAM that survive the acute phase develop postmyocarditis DCM after 4 months or more (Watanabe et al., 2001).

Rat EAM is characterized by its high morbidity and mortality (Kamal et al., 2010). Pericardial effusions, cardiac enlargement and discoloration of the cardiac surface are the macroscopic findings of EAM (Kodama et al., 1992). These findings are common in the autopsy of the patients with myocarditis and EAM Lewis rats but rarely reported in other experimental animal models of myocarditis.

#### **2.1 Pathogenesis of EAM**

In the rat model of EAM, cardiac myosin is one of the major inflammation inducing agents used commonly. It is composed of two heavy chains of about 2000 amino acids and four light chains. This protein acts as antigen and stimulates the inflammatory reactions in the rat especially targeting the myocardium. In the rat immune system, T cells recognize 10-20 amino acid residues, and B cells recognize 5-10 amino acid peptides as antigens. Amino acid residues 1539-1555 of the rat cardiac myosin α-chain would be the myocarditogenic epitope of EAM (Pummerer et al., 1996). Direct sub fragment analysis revealed that actually several myocarditogenic epitopes existed on cardiac myosin. The most effective epitope is present on the residues 1070-1165 of the porcine cardiac myosin β-chain (Inomata et al., 1995). Antigen-specific breakdown of self-tolerance due to the molecular mimicry of myocarditogenic epitopes initiates the autoimmune reaction (Jones et al., 1997). This is followed by the stimulation and proliferation of myocarditogenic T cells. Activated T cells secrete many cytokines, chemokines and other mediators, which recruit and activate other inflammatory cells. The inflammatory mediators damage the myocardium and interfere with the cardiac function (Smith & Allen, 1992; Goren et al., 1998; Ishiyama et al., 1998).

Administration of porcine cardiac myosin with complete Freund's adjuvant, which comprises of inactivated and dried *Mycobacterium tuberculosis* leads to antigen presentation of cardiac myosin-specific T cells in the peripheral lymphatic organs. Freund's adjuvant, an immunopotentiator, plays an important role in the cell-mediated immunity leads to the breakdown of self-tolerance by activation of antigen presenting cells, enhancement of the expression of major histocompatibility molecules as well as increases in vascular permeability. T cells produced de novo in the bone marrow play a major role in the pathogenesis of the autoimmune myocarditis (Bergelson et al., 1997). Release of cardiac myosin from the damaged heart leads to further activation of specific T cells. Autoantibodies developed against the myosin bind to the injured heart and destroy them (Fedoseyeva et al., 2002).

#### **2.2 Role of inflammation in myocarditis**

Cytokines play important roles in the pathogenesis of myocarditis (Ding et al., 2010; Yuan et al., 2010; Huang et al., 2009; Ingkanisorn et al., 2006). Interleukin (IL) -2 mRNA appears in

and the original disease in human. The current EAM model will provide the opportunity for

EAM is similar to the giant cell myocardits and is more likely to progress into dilated cardiomayopathy (DCM) (Kodama et al., 1990). EAM can be induced by injection of cardiac myosin with Freund's adjuvant in to the footpads of Lewis rats. The immunized rats become ill and immobile at day 14 and then their activity gradually recovers beginning in the fourth week. The diseased rats show severe myocardial damage with inflammatory cell infiltration. Rats with EAM that survive the acute phase develop postmyocarditis DCM after 4 months

Rat EAM is characterized by its high morbidity and mortality (Kamal et al., 2010). Pericardial effusions, cardiac enlargement and discoloration of the cardiac surface are the macroscopic findings of EAM (Kodama et al., 1992). These findings are common in the autopsy of the patients with myocarditis and EAM Lewis rats but rarely reported in other

In the rat model of EAM, cardiac myosin is one of the major inflammation inducing agents used commonly. It is composed of two heavy chains of about 2000 amino acids and four light chains. This protein acts as antigen and stimulates the inflammatory reactions in the rat especially targeting the myocardium. In the rat immune system, T cells recognize 10-20 amino acid residues, and B cells recognize 5-10 amino acid peptides as antigens. Amino acid residues 1539-1555 of the rat cardiac myosin α-chain would be the myocarditogenic epitope of EAM (Pummerer et al., 1996). Direct sub fragment analysis revealed that actually several myocarditogenic epitopes existed on cardiac myosin. The most effective epitope is present on the residues 1070-1165 of the porcine cardiac myosin β-chain (Inomata et al., 1995). Antigen-specific breakdown of self-tolerance due to the molecular mimicry of myocarditogenic epitopes initiates the autoimmune reaction (Jones et al., 1997). This is followed by the stimulation and proliferation of myocarditogenic T cells. Activated T cells secrete many cytokines, chemokines and other mediators, which recruit and activate other inflammatory cells. The inflammatory mediators damage the myocardium and interfere with the cardiac function (Smith & Allen, 1992; Goren et al., 1998; Ishiyama et al., 1998). Administration of porcine cardiac myosin with complete Freund's adjuvant, which comprises of inactivated and dried *Mycobacterium tuberculosis* leads to antigen presentation of cardiac myosin-specific T cells in the peripheral lymphatic organs. Freund's adjuvant, an immunopotentiator, plays an important role in the cell-mediated immunity leads to the breakdown of self-tolerance by activation of antigen presenting cells, enhancement of the expression of major histocompatibility molecules as well as increases in vascular permeability. T cells produced de novo in the bone marrow play a major role in the pathogenesis of the autoimmune myocarditis (Bergelson et al., 1997). Release of cardiac myosin from the damaged heart leads to further activation of specific T cells. Autoantibodies developed against the myosin bind to the injured heart and destroy them

Cytokines play important roles in the pathogenesis of myocarditis (Ding et al., 2010; Yuan et al., 2010; Huang et al., 2009; Ingkanisorn et al., 2006). Interleukin (IL) -2 mRNA appears in

further fundamental research into myocarditis.

experimental animal models of myocarditis.

or more (Watanabe et al., 2001).

**2.1 Pathogenesis of EAM** 

(Fedoseyeva et al., 2002).

**2.2 Role of inflammation in myocarditis** 

the EAM hearts at the onset of the disease (Okura et al., 1998). Subsequently, mRNA of IL-1β, interferon γ, and tumor necrosis factor (TNF) α increases (Maeda et al., 2005). In the recovery phase IL-10 appears in the heart. IL1, IL2, IL6 and TNFα are involved in the impairment of cardiac contractility, IL1 and IL6 may induce hypertrophy of myocytes whereas IL1 and TNFα may play a role in the development of myocardial fibrosis. Th2 cytokine IL-10 plays a protective role in the development of EAM (Watanabe et al., 2001). IL10 possesses immunomodulatory properties involving the inhibition of macrophage function and the production of proinflammatory cytokines (Nishio et al., 1999).

In rat EAM, infiltration of the inflammatory cells into the myocardium may be mediated by monocyte chemoattractant protein-1 (MCP-1) or other chemokines as MCP-1 mRNA is strongly expressed in the heart coincident with the onset of the disease and persists until the recovery phase (Goser et al., 2005). Nitric oxide may also play a role in the development of autoimmune myocarditis, by exacerbating inflammatory responses to cardiac infections (Kittleson et al., 2005).

Angiotensin (Ang) II, the principal effector peptide of the renin–angiotensin system (RAS), has been reported to induce immune and inflammatory response in various cardiac disease conditions, including atherosclerosis, hypertension, left ventricular hypertrophy, myocardial infarction, heart failure and myocarditis (Ferrario & Strawn, 2006; Schmieder et al., 2007).
