**1. Introduction**

292 Myocarditis

Zhang, H.M., Yuan, J., Cheung, P., Luo, H., Yanagawa, B., Chau, D., Stephan-Tozy, N.,

Zhang, Y., Li, Z., Xinna, G., Xin, G., and Yang, H. (2011). Autophagy promotes the replication of encephalomyocarditis virus in host cells. Autophagy *7*.

viral replication. J Biol Chem *278*, 33011-33019.

Wong, B.W., Zhang, J., Wilson, J.E.*, et al.* (2003). Overexpression of interferongamma-inducible GTPase inhibits coxsackievirus B3-induced apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway and inhibition of

> Viral myocarditis, the inflammation of the myocardium caused by viral infection, is an important cause of dilated cardiomyopathy (DCM) – a major cause of morbidity and mortality worldwide (Mason, 2003; Esfandiarei & McManus, 2008; Cooper, 2009). In North America, viral myocarditis and DCM together account for 20% of the sudden deaths and heart failure in children and adolescents (Okuni *et al.*, 1975; Drory *et al.*, 1991). To date, there is no effective therapeutic against these diseases. Patients diagnosed with late stage DCM are limited to supportive treatments such as ventricular assist device implantation and heart transplantation.

> The clinical presentation of viral myocarditis comes in various severities. Most people have contracted and subsequently recovered from multiple viral infections of the heart without overt symptoms. Yet, retrospective studies revealed that ~20% of subclinical cases later develop congestive heart failure. In addition, some may experience acute fulminant viral myocarditis or persistent chronic myocarditis symptoms. About one-third of these patients with viral myocarditis subsequently develop DCM (Esfandiarei & McManus, 2008). A combination of new diagnostic technologies for viral myocarditis such as cardiovascular magnetic resonance techniques with conventional diagnostics including clinical presentation, histopathological examination, cardiac antibody assessment, and viral polymerase chain reaction (PCR), now helps better define disease stage and its respective management protocol (Baughman, 2006).

> The presence of viral genome in the myocardium is associated with significantly worse outcome over two years (Why *et al.*, 1994). Analysis of human failing hearts by PCR unveiled trails of previous viral infection. The identified viruses include enterovirus, adenovirus, parvovirus B19, herpes simplex virus 6, cytomegalovirus, hepatitis C virus, and human immunodeficiency virus, which are clinically associated with viral myocarditis (Grist & Reid, 1997; Calabrese *et al.*, 2010). Among them, coxsackievirus B3 (CVB3), an enterovirus in the picornavirus family, is highly implicated in clinical cases of viral myocarditis, particularly in neonates and young children, and is the most thoroughly studied causative agent in experimental viral myocarditis models (Froeschle *et al.*, 1966; Abelmann, 1971; Reyes & Lerner, 1985; McManus *et al.*, 1988). CVB3 replicates rapidly in short infection cycles that begin with viral receptor engagement and subsequent internalization, followed by translation of viral RNA, amplification of viral genome, viral assembly, and complete with viral progeny release.

Impaired Cardiac Function in Viral Myocarditis 295

the extracellular matrix (ECM) by forming the dystrophin glycoprotein complex (DGC), is cleaved during CVB3 infection by protease 2A (Badorff *et al.*, 1999; Badorff & Knowlton, 2004) (Fig. 1A). Dystrophin has three domains that serve different purposes. Its N-terminal domain anchors to the actin cytoskeleton and its rod domain provides the linkage to the Cterminal domain, which binds to -dystroglycan that in turn connects to the sarcolemma and the extracellular matrix (Fig. 1A). Furthermore, dystrophin-deficient mice have been shown to have an increased susceptibility to viral myocarditis and develop severe cardiomyopathy (Xiong *et al.*, 2002). Human genetic mutations of the dystrophin gene cause Duchenne Muscular Dystrophy (DMD) (Nigro *et al.*, 1990). Approximately 20% of DMD patients suffer and die from a resultant cardiomyopathy. Other mutations of the dystrophin gene also cause X-linked DCM (Ferlini *et al.*, 1999). CVB3-induced dystrophin cleavage occurs at its 3' hinge and therefore breaks its connection to the ECM. As a result, the sarcolemmal integrity is compromised and force transmission is reduced. This can further lead to cardiomyocyte necrosis due to the increased sarcolemmal permeability (Fig. 1A). Thereafter, dystrophin cleavage has been viewed as a major mechanism in enteroviral cardiomyopathy. However, dystrophin knockout mice (Mdx) display only mild cardiomyopathy phenotype, due to the compensatory upregulation of utrophin, a dystrophin homologue (Deconinck *et al.*, 1997; Grady *et al.*, 1997). Thus, other mechanisms may also contribute to the severe cardiomyopathy phenotype in viral protease 2A

**2.3 Cleavage of serum response factor by viral protease 2A is associated with** 

Recent efforts have demonstrated for the first time that serum response factor (SRF) is cleaved in CVB3-infected mouse hearts and cultured murine cardiomyocytes (unpublished). SRF, which belongs to the MADS-box (MCM1, Agamous, Deficiens, and SRF) protein superfamily, is a muscle-enriched transcription factor that regulates the expression of contractile and regulatory genes, as well as microRNAs (miRNAs) (Miano, 2003; Niu *et al.*, 2007; Oka *et al.*, 2007) (Fig. 1B). SRF interacts with tissue specific cofactors such as myocardin, Nkx2.5, c-Fos, and binds to the serum response element (SRE) of its target genes. It contains two major domains: the N-terminal DNA binding and dimerization domain and the C-terminal transactivation domain (Fig. 1B). Genomic studies have identified over 1200 SRE containing genes and more than 250 of these have been verified (Sun *et al.*, 2006). Cardiac contractile genes under SRF regulation include cardiac -actin, -myosin heavy chain, myosin light chain, cardiac troponin I, etc. SRF is indispensable for mesoderm formation and plays a central role in cardiac development and function (Niu *et al.*, 2007). Therefore, SRF knockout results in embryonic lethality (Parlakian *et al.*, 2004). The construction of cardiac-specific inducible SRF knockout transgenic mice overcomes this problem and helps illustrate the importance of SRF in cardiac function (Parlakian *et al.*, 2005). It was shown that SRF knockout in the adult mouse heart results in damaged cardiac function, and subsequent progression to DCM. Genetic mutations of SRF in humans have not been described likely due to the associated lethality. However, the expression of alternatively spliced SRF isoforms, which was shown to have inhibitory effects on wild-type SRF, is increased in failing human and animal hearts (Davis *et al.*, 2002). Furthermore, a cleaved form of SRF lacking the transactivation domain was also found in human failing hearts as a result of caspase-3 activation during cardiomyocyte apoptosis (Chang *et al.*,

expressing mice.

**impaired cardiac function** 

CVB3 infection of myocarditis susceptible mice results in severe heart failure. The disease progression of viral myocarditis in the experimental infection model can be classified into three phases: acute (viremia), subacute (inflammatory), and chronic (remodeling) phases. The acute (viremia) phase is signified by active viral replication and direct virus-induced cardiomyocyte damage. The subacute (inflammatory) phase is characterized by the infiltration of immune cells that helps viral clearance but nonetheless adds to myocardial damage. The chronic (remodeling) phase is featured by the continual efforts of the impaired heart to meet the hemodynamic demand by remodeling the myocardium. Cardiac hypertrophy is triggered during remodeling to compensate for reduced contractile function due to myocyte loss and interstitial fibrosis in the earlier phases. However, such an adaption is unsustainable in the longer term in face of increasingly hostile environments, i.e. reduced blood supply and increased reactive oxidative stress, thus leading to cardiomyocyte death and triggering further inflammation and fibrosis. The pathological remodeling process eventually leads to DCM and heart failure.

This book chapter focuses on the virus-host protein interactions in cardiomyocytes during viral myocarditis. We discuss the role of virus-induced protein cleavage and dysregulation of the host protein degradation systems in the pathogenesis of viral myocarditis and its subsequent progression to DCM.
