**2.** *In vivo* **experimental systems**

Mice provide a model system to distinguish characteristics of disease between autoimmune myocarditis and viral myocarditis. Mice are advantageous models because they share similar genetics to humans, they are cost-effective in handling and breeding, many transgenic strains are available and they are responsive to cardiotropic viruses (Cunningham 2001; Fairweather et al. 2001; Esfandiarei et al. 2008). Coxsackievirus B3 has been detected in 30-50% of dilated cardiomyopathy patients, providing support for a coxsackievirus B3-induced myocarditis mouse model (Escher et al. 2011). Following a single dose of coxsackievirus B3, acute myocarditis, encephalomeningitis, hepatitis, and even pancreatitis can ensue. Severe systemic pathogenicity observed with coxsackievirus B3 infection has also been related to the presence of sarcoma (Src) family kinase Lck (p56lck) (Liu et al. 2000). With coxsackievirus B3 infection, mice tend to either develop chronic dilated cardiomyopathy mirroring clinical disease after recovering from viral infection or mice die from severe cytopathic effects, thus making coxsackievirus B3-induced myocarditis an excellent yet challenging model to study (Liu et al. 2000).

Studies with mice have demonstrated the significant contribution of the Th1 immune response to disease severity even though coxsackievirus B3 directly targets and destroys the myocardium. The contribution of a Th1 response to viral myocarditis pathogenesis is supported by studies modulating and inhibiting immune components and improving cardiac damage and function (Jiang et al. 2008).

Interestingly and in parallel with human myocarditis, male mice infected with coxsackievirus B3 experience acute myocarditis with greater severity compared to females. Greater disease severity in males has been linked to enhanced cardiac and splenic mast cell and macrophage TLR-4 expression at 12 hours post-infection (Frisancho-Kiss et al. 2006; Frisancho-Kiss et al. 2007). In males virally infected, signalling through cardiac TLR-4 enhances the production of cardiac IL-1β and IL-18 and promotes a Th1 skewed immune response (Fairweather et al. 2003). Also, male mice infected with coxsackievirus B3 harbour macrophages of a different phenotype and have more severe disease compared to infected female mice though viral replication is at similar levels in the heart (Frisancho-Kiss et al. 2006; Frisancho-Kiss et al. 2007). Moreover, genes relating to cholesterol metabolism in macrophages and to androgen receptor, which are known predictors for myocarditis, dilated cardiomyopathy and heart failure in male mice and humans, are upregulated considerably in spleens of male mice (Onyimba et al. 2011). This not only affords support for a gender difference in the susceptibility to viral-myocarditis, but critically implicates the innate response in disease severity. Furthermore, to bring male mice to the same immunological plane as female mice during myocarditis, Frisancho-Kiss et al removed the gonads from BALB/c mice and observed an increase in IL-4 production, the activation of macrophages and induction of regulatory T cells in the heart (Frisancho-Kiss et al. 2009).

Myocarditis can be induced in female mice with TNF-α treatment on days 1 and 3 post coxsackievirus B3 infection (Huber 2010). The lack of disease susceptibility in females is

protein 2B (van Kuppeveld et al. 1997; Esfandiarei et al. 2008). Though many groups have identified key virus and host interactions throughout the coxsackievirus B life cycle, there still remains many stages unsolved. Nevertheless, the role of coxsackievirus B in the onset of myocarditis has been extensively studied thus far in animal models and cell culture systems. Here, we will discuss clinical and mouse studies that have investigated coxsackievirus-

Mice provide a model system to distinguish characteristics of disease between autoimmune myocarditis and viral myocarditis. Mice are advantageous models because they share similar genetics to humans, they are cost-effective in handling and breeding, many transgenic strains are available and they are responsive to cardiotropic viruses (Cunningham 2001; Fairweather et al. 2001; Esfandiarei et al. 2008). Coxsackievirus B3 has been detected in 30-50% of dilated cardiomyopathy patients, providing support for a coxsackievirus B3-induced myocarditis mouse model (Escher et al. 2011). Following a single dose of coxsackievirus B3, acute myocarditis, encephalomeningitis, hepatitis, and even pancreatitis can ensue. Severe systemic pathogenicity observed with coxsackievirus B3 infection has also been related to the presence of sarcoma (Src) family kinase Lck (p56lck) (Liu et al. 2000). With coxsackievirus B3 infection, mice tend to either develop chronic dilated cardiomyopathy mirroring clinical disease after recovering from viral infection or mice die from severe cytopathic effects, thus making coxsackievirus B3-induced myocarditis

Studies with mice have demonstrated the significant contribution of the Th1 immune response to disease severity even though coxsackievirus B3 directly targets and destroys the myocardium. The contribution of a Th1 response to viral myocarditis pathogenesis is supported by studies modulating and inhibiting immune components and improving

Interestingly and in parallel with human myocarditis, male mice infected with coxsackievirus B3 experience acute myocarditis with greater severity compared to females. Greater disease severity in males has been linked to enhanced cardiac and splenic mast cell and macrophage TLR-4 expression at 12 hours post-infection (Frisancho-Kiss et al. 2006; Frisancho-Kiss et al. 2007). In males virally infected, signalling through cardiac TLR-4 enhances the production of cardiac IL-1β and IL-18 and promotes a Th1 skewed immune response (Fairweather et al. 2003). Also, male mice infected with coxsackievirus B3 harbour macrophages of a different phenotype and have more severe disease compared to infected female mice though viral replication is at similar levels in the heart (Frisancho-Kiss et al. 2006; Frisancho-Kiss et al. 2007). Moreover, genes relating to cholesterol metabolism in macrophages and to androgen receptor, which are known predictors for myocarditis, dilated cardiomyopathy and heart failure in male mice and humans, are upregulated considerably in spleens of male mice (Onyimba et al. 2011). This not only affords support for a gender difference in the susceptibility to viral-myocarditis, but critically implicates the innate response in disease severity. Furthermore, to bring male mice to the same immunological plane as female mice during myocarditis, Frisancho-Kiss et al removed the gonads from BALB/c mice and observed an increase in IL-4 production, the activation of macrophages and induction of regulatory T cells in the heart (Frisancho-Kiss et al. 2009). Myocarditis can be induced in female mice with TNF-α treatment on days 1 and 3 post coxsackievirus B3 infection (Huber 2010). The lack of disease susceptibility in females is

induced myocarditis and the role of key immune players in disease pathogenesis.

**2.** *In vivo* **experimental systems** 

an excellent yet challenging model to study (Liu et al. 2000).

cardiac damage and function (Jiang et al. 2008).

attributed to low mRNA and protein levels of TNF-α and IL-Iβ as well as reduced CD1d expression on splenic lymphocytes. CD1d is an important non-classical major histocompatibility complex antigen that can be regulated by TNF-α to induce myocarditis susceptibility in female mice (Huber 2010).

Not only is susceptibility to developing myocarditis dictating by sex, but development of chronic disease depends on the mouse strain. A.BY/SnJ & SWR/J are susceptible mouse strains that can develop ongoing myocarditis, where viral RNA is detected within the myocardium. C57BL/6J & DBA/1J mice are resistant strains capable of eliminating virus just after the early acute phase of disease. Tomioka and colleagues investigated neutralizing antibodies and their role in virus-induced myocarditis and B-cell-mediated immunity using BALB/c mice (Esfandiarei et al. 2008). NK-deficient mice have been used to look at the role of natural killer (NK) cells in killing virus-infected cardiomyocytes (Godeny et al. 1986). Perforin knockout mice inoculated with coxsackievirus B3 have also been used to describe the interplay of virus infection and lymphocyte infiltration in the myocytes and their effect on disease outcome (Godeny et al. 1987). Interestingly, with coxsackievirus B3 infection in suckling, weaning and adolescent mice, coxsackievirus B3 replicates in the heart, pancreas, spleen, and brain and causes human disease-like symptoms. In fact, following IP injection, three distinct immunovirological phases of disease have been observed.

In mice, coxsackievirus B3 can induce two forms of inflammatory heart disease, acute only or acute and chronic (biphasic) autoimmune disease (Horwitz et al. 2000; Cunningham 2001; Fairweather et al. 2001). Interestingly, coxsackievirus B3 replication is mainly observed in the pancreas and to a lesser extent in the heart. In genetically susceptible mice, such as A/J2, Balb/c and NOD mice, chronic autoimmune myocarditis after coxsackievirus B3 infection is observed. Autoimmune myocarditis in mice presents as early as day 7 pi with inflammatory cell infiltration in the heart and the formation of multifocal inflammatory lesions. At this stage, autoantibodies (autoAbs) against heart antigens, like cardiac myosin (cardiac myosin), are seen. In NOD mice, isotype switching ensues after 2 to 3 weeks, with the autoAbs switching from IgM to IgG subclasses (Kaya et al. 2001; Kaya et al. 2002). It is particularly remarkable to note that the chronic autoimmune heart disease induced by coxsackievirus B3 in mice resembles inflammatory heart disease seen with myocarditis and dilated cardiomyopathy in humans. There are still many complications with the full characterization and study of chronic virus and/or autoimmune induced myocarditis in mice and humans. One such complication is the acute infection that precedes chronic disease.
