**1. Introduction**

268 Myocarditis

Sukumaran, V., K. Watanabe, et al. (2011). "Telmisartan ameliorates experimental

Szalay, G., M. Sauter, et al. (2006). "Sustained nitric oxide synthesis contributes to

Tanaka, K., M. Ito, et al. (2011). "Sulfated polysaccharide fucoidan ameliorates experimental autoimmune myocarditis in rats." J Cardiovasc Pharmacol Ther 16(1): 79-86. Triantafilou, K., G. Orthopoulos, et al. (2005). "Human cardiac inflammatory responses

van Kuppeveld, F. J., J. G. Hoenderop, et al. (1997). "Coxsackievirus protein 2B modifies

Vaughan, M. B., E. W. Howard, et al. (2000). "Transforming growth factor-beta1 promotes

Wang, Y., M. Afanasyeva, et al. (1999). "Characterization of murine autoimmune

Why, H. J., B. T. Meany, et al. (1994). "Clinical and prognostic significance of detection of

Wing, K. and S. Sakaguchi (2010). "Regulatory T cells exert checks and balances on self

Wittner, B., B. Maisch, et al. (1983). "Quantification of antimyosin antibodies in experimental

Yajima, T. and K. U. Knowlton (2009). "Viral Myocarditis: From the Perspective of the

Yuan, J., Z. Liu, et al. (2009). "CXCL10 inhibits viral replication through recruitment of

Yuan, J., M. Yu, et al. (2010). "Th17 cells contribute to viral replication in coxsackievirus B3-

Yue, Y., J. Gui, et al. (2011). "Direct Gene Transfer with IP-10 Mutant Ameliorates Mouse

Yue, Y., J. Gui, et al. (2011). "Gene therapy with CCL2 (MCP-1) mutant protects CVB3-

Zhang, P., C. J. Cox, et al. (2009). "Cutting edge: cardiac myosin activates innate immune

Zou, W. and N. P. Restifo (2010). "T(H)17 cells in tumour immunity and immunotherapy."

stress." Eur J Pharmacol 652(1-3): 126-135.

facilitates virus release." EMBO J 16(12): 3519-3532.

cardiomyopathy." Circulation 89(6): 2582-2589.

Virus." Circulation 119(19): 2615-2624.

tolerance and autoimmunity." Nat Immunol 11(1): 7-13.

Woodruff, J. F. (1980). "Viral myocarditis. A review." Am J Pathol 101(2): 425-484.

induced acute viral myocarditis." J Immunol 185(7): 4004-4010.

responses through TLRs." J Immunol 183(1): 27-31.

Nat Rev Immunol 10(4): 248-256.

Am J Pathol 169(6): 2085-2093.

Cell Microbiol 7(8): 1117-1126.

Res 257(1): 180-189.

162.

239-247.

638.

e18186.

713.

autoimmune myocarditis associated with inhibition of inflammation and oxidative

immunopathology in ongoing myocarditis attributable to interleukin-10 disorders."

triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent."

endoplasmic reticulum membrane and plasma membrane permeability and

the morphological and functional differentiation of the myofibroblast." Exp Cell

myocarditis induced by self and foreign cardiac myosin." Autoimmunity 31(3): 151-

enteroviral RNA in the myocardium of patients with myocarditis or dilated

myocarditis by a new solid phase fluorometric assay." J Immunol Methods 64(1-2):

natural killer cells in coxsackievirus B3-induced myocarditis." Circ Res 104(5): 628-

CVB3-Induced Myocarditis by Blunting Th1 Immune Responses." PLoS ONE 6(3):

induced myocarditis by compromising Th1 polarization." Mol Immunol 48(4): 706-

Since the discovery of coxsackievirus type B3 (CVB3) more than 50 years ago there has been considerable progress in the understanding of viral heart disease. Coxsackievirus was first discovered as a filterable agent associated with a paralytic syndrome, so named for its identification in Coxsackie, New York (coxsackievirus type A) (Dalldorf and Sickles, 1948). Coxsackievirus type B (CVB) was isolated the following year from patients with aseptic meningitis and by the mid-1950s an association with acute myocarditis in humans was apparent (Melnick et al., 1949). Many other viruses have since been shown to cause myocarditis and its long term sequelae, arrhythmias, dilated cardiomyopathy (DCM) and heart failure. By example, adenovirus, herpes viruses, and influenza can cause myocarditis in humans and models of heart failure. The viral agent most often reported as being the cause of viral myocarditis is CVB3. For example, CVB3 RNA can be detected in the heart muscle of 10 - 35 % of DCM patients (Feldman and McNamara, 2000; Hosenpud et al., 2001), depending on the study cohort.

#### **1.1 Myocarditis etiologies**

Inflammation of the heart muscle can have many causes, not least of which is viral infection. Cocaine [(Jentzen, 1989) reviewed in (Maraj et al., 2010)], virus induced, autoimmune (possibly due to resolved microbial infection), and even vaccine induced myocarditis (Cassimatis et al., 2004) have all been reported with a wide degree of occurrence. Though very rare, the vaccine induced aetiology is classified as idiopathic due to the poorly understood mechanism of inflammation. This manifestation of myocarditis is so rare it is probably very complex in cause and mechanism, requiring both environmental and genetic susceptibility factors of the host to trigger myocardial immune invasion. The subject of this chapter is one of the most common forms of myocarditis, and perhaps the most studied: myocarditis due to viral infection. In such cases, myocyte dropout and provisional matrix fibrosis, focal lesions of immune cell invasion are marked and key observations in the diagnosis of these cases.

#### **1.2 Clinical presentation**

With an experimental understanding of CVB3 induced myocarditis comes a better understanding of the breadth of symptoms presented by the admitted patient, before,

Cellular and Immunological Regulation of Viral Myocarditis 271

The advent of virus replication in the heart of an individual is thought to occur secondarily to a more general and systemic virus infection (Carthy et al., 1997; Mena et al., 2000; Vuorinen et al., 1994), the spleen, gut, pancreas and lymph nodes are often infected; patients often report malaise, fatigue and more general 'flu-like' symptoms about 1- 2 weeks prior to chest pains and cardiac rhythm disturbances. Immune infiltration in the heart has taken hold by the time the patient has been seen by a physician and any evidence of direct virusinduced damage has been superseded by T cell invasion at the foci of infection. It is not surprising that there have been many reports that claim the predominant source of damage is immune infiltration of the myocardium, as inflammation is what defines this disease. Autoimmune etiologies due to an antiviral response gone awry have been proposed as one of the major causes of pathology (Lv et al., 2011). Regardless, there are numerous publications that suggest a large amount of damage is inflicted by the virus itself, in the murine model, early post-infection, that is correlated with the number of myocarditic lesions spatially coincident with positivity by *in situ* hybridization for CVB3 genome (Cheung et al., 2008; Chow et al., 1992; Marchant et al., 2009a; McManus et al., 1993). These observations are supported by findings in autopsy cases, particularly in infants (Iwasaki et al., 1985a; Iwasaki

Coxsackievirus B3 is a member of the *Enteroviridae* of the *Picornaviridae* family, which are fast replicating and exquisitely cytotoxic viruses. Another member of this family is poliovirus, which is also highly cytotoxic, producing a paralytic syndrome reminiscent of the aseptic meningitis and encephalitis sometimes caused by CVB3. The very nature of enterovirus replication makes these viruses very cytotoxic because the infected cell must burst to permit the release of progeny virus virions for infection of bystander cells and further replication cycles. This aspect of CVB3 replication means that every virus infected cell will die within 8 – 24 hrs of infection, releasing numerous rounds of progeny virus into the heart before the innate immune system has had a chance to control infection. The degree of immune infiltration and damage inflicted on the heart muscle is therefore a function of the amount of virus inoculum that takes hold in the heart, dictating the degree of damage

The virus polarises the protein expression machinery entirely to the benefit of the virus by a number of mechanisms and so in combination with the inability of the cell to perform its natural housekeeping functions inevitably leads to cell destruction. The viral proteases act to cleave capped cellular mRNAs thereby skewing protein translation in the cell from capdependent translation initiation to internal ribosome entry site (IRES) translation. Coxsackievirus B3 translates it's RNA genome via IRES translation initiation so destruction of the cell's own capped mRNAs removes any competition of the virus' own IRES RNAs for the cell's ribosomes. This usurping of control of the cell's protein expression machinery

The massive amount of protein produced by the virus, devolution of the endoplasmic reticulum (ER) and other membranous structures combined with the oxidative stress placed upon the cell by virus replication, all destabilise the homeostasis of the infected cell. The large protein-nucleic acid aggregates that make up progeny virion are not normal to the cell and induce numerous UPR responses; due to their toxic nature, protein aggregates are

effectively brings the normal housekeeping function of the cell to a halt.

**2. Viral replication as the central regulator of viral myocarditis** 

**2.1 The cytotoxic nature of enterovirus replication** 

et al., 1985b).

done to the myocardium.

during and after a hospital visit. The patient is most often admitted to hospital with general chest pain or discomfort, combined with other symptoms of an infection, which are often suspected as other ailments (Brady et al., 2004). These symptoms of infection can be so apparent that they divert the attention of the clinician away from cardiac dysfunction. Further work-up of the case reveals elevated creatinine phosphokinase and troponin with rhythmic disturbances and reduced cardiac output, often presented by shortness of breath with minimal exertion (Brady et al., 2004). Most cases resolve with little or no supportive therapy, but a minor percentage of cases progress to dilated cardiomyopathy and congestive heart failure, for which there is no cure other than heart transplantation.

#### **1.3 Diagnosis: the Dallas criteria**

The Dallas criteria for myocarditis are a set of histology based criteria proposed by a publication in 1987 by Aretz et al. (Aretz et al., 1987). These criteria rely upon histological observations made of left ventricular biopsies, obtained by either the Cordis or Scholten bioptomes. Several biopsies of the ventricle are required for a definitive diagnosis and there is a high possibility of false negatives using this method. Though not necessarily a problem inherent in the method itself but the need for a large number of samples due to the often localised and focal nature of the disease. The chances of false negatives combined with procedural discomfort are driving forward a need to develop new and less invasive procedures like blood-based biomarker-detection.

The Dallas criteria for diagnosis of myocarditis require an inflammatory infiltrate with associated myocyte necrosis, or otherwise damage that is not characteristic of ischemia. Less intense immune infiltration with little or no evidence of myocyte destruction is classified as borderline myocarditis (Aretz et al., 1987).

Understanding the etiology of myocarditis will improve the success rate of diagnosis, which in turn, improves management of the disease and prognosis (Baughman, 2006; Felker et al., 2000). The Dallas criteria were a good stepping stone toward a standardised diagnostic method, however there remain significant drawbacks to this biopsy based histological criteria. It has been demonstrated that myocarditis could only be accurately diagnosed in 25 % of single biopsies obtained from patients who had died of myocarditis (Chow et al., 1989; Hauck et al., 1989), and more than 5 biopsies are required to diagnose myocarditis in two thirds of patients (Hauck et al., 1989), and 17 biopsies are required for an 80 % level of confidence in diagnosis (Hauck et al., 1989; Schultz et al., 2009). Therefore only positive samples obtained by endomyocardial biopsy should be considered diagnostic. These poor rates of definitive diagnosis are a product of the small size of the bioptome sample from a relatively large area afflicted with a disease that is often focal and of relatively narrow localisation.

The Dallas criteria were a good start for a disease that had no diagnostic criteria at the time of publication (1987). To this day, the Dallas criteria are used to diagnose myocarditis from endomyocardial biopsies, though the existence of sensitive methods for disease detection, particularly in the blood, biomarker discovery should make this methodology supplementary or even obsolete. Therefore, there is a need for the development of sensitive methods for the differential and definitive diagnosis of non-ischemic cardiomyopathies like myocarditis. This chapter will not cover diagnosis or new detection strategies any further as these topics are covered in greater detail, elsewhere in this book.

during and after a hospital visit. The patient is most often admitted to hospital with general chest pain or discomfort, combined with other symptoms of an infection, which are often suspected as other ailments (Brady et al., 2004). These symptoms of infection can be so apparent that they divert the attention of the clinician away from cardiac dysfunction. Further work-up of the case reveals elevated creatinine phosphokinase and troponin with rhythmic disturbances and reduced cardiac output, often presented by shortness of breath with minimal exertion (Brady et al., 2004). Most cases resolve with little or no supportive therapy, but a minor percentage of cases progress to dilated cardiomyopathy and congestive

The Dallas criteria for myocarditis are a set of histology based criteria proposed by a publication in 1987 by Aretz et al. (Aretz et al., 1987). These criteria rely upon histological observations made of left ventricular biopsies, obtained by either the Cordis or Scholten bioptomes. Several biopsies of the ventricle are required for a definitive diagnosis and there is a high possibility of false negatives using this method. Though not necessarily a problem inherent in the method itself but the need for a large number of samples due to the often localised and focal nature of the disease. The chances of false negatives combined with procedural discomfort are driving forward a need to develop new and less invasive

The Dallas criteria for diagnosis of myocarditis require an inflammatory infiltrate with associated myocyte necrosis, or otherwise damage that is not characteristic of ischemia. Less intense immune infiltration with little or no evidence of myocyte destruction is classified as

Understanding the etiology of myocarditis will improve the success rate of diagnosis, which in turn, improves management of the disease and prognosis (Baughman, 2006; Felker et al., 2000). The Dallas criteria were a good stepping stone toward a standardised diagnostic method, however there remain significant drawbacks to this biopsy based histological criteria. It has been demonstrated that myocarditis could only be accurately diagnosed in 25 % of single biopsies obtained from patients who had died of myocarditis (Chow et al., 1989; Hauck et al., 1989), and more than 5 biopsies are required to diagnose myocarditis in two thirds of patients (Hauck et al., 1989), and 17 biopsies are required for an 80 % level of confidence in diagnosis (Hauck et al., 1989; Schultz et al., 2009). Therefore only positive samples obtained by endomyocardial biopsy should be considered diagnostic. These poor rates of definitive diagnosis are a product of the small size of the bioptome sample from a relatively large area afflicted with a disease that is often focal and of relatively narrow

The Dallas criteria were a good start for a disease that had no diagnostic criteria at the time of publication (1987). To this day, the Dallas criteria are used to diagnose myocarditis from endomyocardial biopsies, though the existence of sensitive methods for disease detection, particularly in the blood, biomarker discovery should make this methodology supplementary or even obsolete. Therefore, there is a need for the development of sensitive methods for the differential and definitive diagnosis of non-ischemic cardiomyopathies like myocarditis. This chapter will not cover diagnosis or new detection strategies any further as

these topics are covered in greater detail, elsewhere in this book.

heart failure, for which there is no cure other than heart transplantation.

**1.3 Diagnosis: the Dallas criteria** 

procedures like blood-based biomarker-detection.

borderline myocarditis (Aretz et al., 1987).

localisation.
