**3.2.1 C-reactive protein**

332 Myocarditis

Fig. 4. Poor heart function in TRIF deficient mice is associated with elevated sST2 during acute CVB3 myocarditis. Male C57BL/6 (BL/6) or TRIF deficient (TRIF-/-) mice were infected intraperitoneally with heart-passaged coxsackievirus B3 (CVB3) containing infectious CVB3 (103 plaque forming units) and heart proteins on day 0 and myocarditis examined at day 10 post infection (pi) using end systolic pressure-volume relationships (ESPVR). Ees, a measure of LV end systolic stiffness/elastance, was 7.1 in BL/6 and 5.1 in TRIF-/- mice (*P* = 0.04) while V0, the X-intercept of the ESPVR, was -5.4 in BL/6 and 26.8 in TRIF-/- mice (*P* = 0.0001). End diastolic volume (EDV) was 24±1.2 in BL/6 and 33±3.3 in TRIF-/- mice (*P* < 0.01). Thus, elevated sST2 in the sera of TRIF-/- mice (not shown) was associated with dilation and heart failure in TRIF deficient mice in an autoimmune model of

IL-6 and serum amyloid A protein (SAA), are part of the acute phase response arising in the liver and although they are strongly associated with disease they may simply infer the presence of an inflammatory state. In clinical studies inflammatory mediators have been found to predict progression to HF similar to injury biomarkers and/or neurohormones (Table 2) (Mann, 2005). Inflammatory biomarkers have been shown in animal studies and the clinical setting to increase LV dysfunction, increase edema, and induce endothelial dysfunction and cardiomyocyte apoptosis, as well as other deleterious effects (Table 5). A recent long-term study of myocarditis patients revealed that inflammation was the best predictor for the progression to HF following acute myocarditis (Kindermann et al., 2008). Viruses like CVB3, adenovirus, parvovirus B19 and hepatitis C virus are often detected in patient myocardial biopsies (Cooper, 2009; Gupta et al., 2008). Antiviral treatments such as interferon- reduce inflammation and HF in animal models and patients, implying that viral infections are an important cause of myocarditis cases that lead to HF (Kuhl et al., 2003; Wang et al., 2007). Inflammation appears to be etiologically linked with the development of HF, not only because heart failure is a consequence of inflammatory CVDs but because patients with chronic HF that have elevated levels of inflammatory mediators have a worse prognosis (Robinson et al., 2011). Evidence exists that both cellular and auto/antibodymediated damage contribute to the progression to DCM and HF following myocarditis (Cooper, 2009; Fairweather et al., 2008; Kallwellis-Opara et al., 2007). Similar to atherosclerosis, acute myocardial inflammation is associated with an elevated Th1 response in males (Daniels et al., 2008; Frisancho-Kiss et al., 2007; Huber and Pfaeffle, 1994; Nishikubo et al., 2007). A Th17 response has been shown to increase fibrosis leading to DCM in the experimental autoimmune myocarditis (EAM) model in mice (Baldeviano et al., 2010).

CVB3 myocarditis.

Interest in the study of inflammatory mediators in patients with HF began in 1954 when an assay for CRP was first developed (Braunwald, 2008). CRP is an acute phase protein synthesized in the liver in response to IL-6 and released to the circulation during inflammation (Pepys & Hirschfield, 2003). Its levels are synergistically increased by IL-1. In phagocytes CRP has been shown to bind Fc receptor I and II and to function in the clearance of apoptotic and necrotic cells (Devaraj et al., 2009; Rhodes et al., 2011). In 1956 a study was published showing that CRP was detectible in the sera of 30 out of 40 patients with chronic HF, and that elevated CRP levels were associated with more severe disease (Braunwald, 2008; Elster et al., 1956). Since then many studies have shown that CRP independently predicts adverse outcomes in patients with acute or chronic HF (Braunwald, 2008; Osman et al., 2006). Higher levels of CRP are associated with more severe HF and independently associated with morbidity and mortality (Anand et al., 2005). Additionally, elevated CRP levels identified asymptomatic elderly individuals who were at a high risk of developing HF in the future (Vasan et al., 2003). The main problem with CRP as a biomarker is that it lacks specificity for CVD. That is, CRP levels are elevated in the sera during most conditions that increase inflammation such as acute or chronic infection, cigarette smoking, ACS and some autoimmune diseases (Pepys & Hirschfield, 2003; Perez-De-Lis et al., 2010; Rhodes et al., 2011). There is increasing evidence that CRP may be able to exert direct proinflammatory effects on the heart by increasing matrix metalloproteinase-1 (MMP)-1 and IL-8 in endothelial cells and by increasing CD11b and CC-chemokine receptor 2 (CRR2) in monocytes, for example (Table 6) (Devaraj et al., 2009; Osman et al., 2006; Venugopal et al., 2005).

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 335

humans. Thus, CRP deficient or transgenic mice may provide only limited information on

The inability of hemodynamic factors to fully explain HF cases led to the hypothesis that cytokines released from cardiac tissue and/or inflammatory cells also contribute to disease progression. According to the "cytokine hypothesis", HF develops because cytokine cascades that are activated following myocardial injury or stress exert deleterious effects on heart function (Table 5) (Anker & von Haehling, 2004; Braunwald, 2008; Seta et al., 1996). Cytokines can induce hemodynamic abnormalities and/or direct toxic effects on the heart. Since the original report in 1990 by Levine et al. there have been many studies showing that an increase in circulating TNF levels directly relates to a patient's NYHA classification and predicts patient mortality (Anker & von Haehling, 2004; Mann, 2005; Seta et al., 1996; Vasan et al., 2003). Similar relationships between IL-1, IL-6 or IL-18 and HF have been found (Anker & von Haehling, 2004; Hedayat et al., 2010; Jefferis et al., 2011; Vasan et al., 2003). Cardiac myocyte hypertrophy, contractile dysfunction, cardiac myocyte apoptosis and extracellular matrix (ECM) remodeling contribute critically to the progression from cardiac

More than any other category of biomarker (Table 2), the role of cytokines in the pathogenesis of myocarditis and DCM has been studied by researchers (Fairweather & Rose, 2005; Hedayat et al., 2010). TNF, IL-1 and IL-18 have all been shown to play a role in myocarditis by inducing myocyte hypertrophy, contractile dysfunction, myocyte apoptosis and contributing to ECM remodeling, a step critical in the progression from myocarditis to DCM (Cain et al., 1999; Fairweather et al., 2004a; 2004b; Hedayat et al., 2010). In one study, TNF mRNA expression was found to be elevated more often in myocarditis patients when viral genomes were also detected, and greater mRNA levels of TNF and its receptor TNFRI correlated with impaired cardiac function (Calabrese et al., 2004). In a mouse model of CVB3 myocarditis, TNF was found to increase CD1d expression on lymphocytes resulting in increased inflammation in males (Huber, 2010). However, viral replication and acute CVB3 myocarditis was not altered in TNFRI deficient mice (Fairweather et al., 2005). Out of the 13 or so TLRs that have been described so far in humans and mice, TLR4 is unique in its ability to work with the inflammasome to produce bioactive IL-1 and IL-18 in the heart (Fairweather et al., 2003; Vallejo, 2011). TLR2 and TLR4 signaling increase TNF levels, and TLR2 can act with TLR4 to increase IL-1 levels (Vallejo, 2011). TLR4 mRNA expression has been found to be higher in patients with myocarditis than controls, and to correlate with viral RNA levels in the heart (Satoh et al., 2003). Myocarditis patients with active viral replication had higher levels of TLR4 that was associated with lower systolic function. In a mouse model of CVB3 myocarditis, our laboratory found that TLR4 deficient mice develop reduced acute inflammation and lower IL-1 and IL-18 levels in the heart (Fairweather et al., 2003). The importance of TLR4 signaling in a strictly autoimmune model of myocarditis was demonstrated by Nishikubo et al. where TLR4 signaling was found to be necessary to mount a Th1-type immune response (Nishikubo et al., 2007). We have shown that TLR4 is upregulated on macrophages and mast cells during the innate immune response to CVB3 and during acute CVB3 myocarditis and this response results in increased inflammation and progression to DCM and HF in males compared to females (Frisancho-Kiss et al., 2007; 2009; Onyimba et al., 2011). A Th1 response in male mice was found to be due to TLR4-derived IL-

the role of CRP in HF promotion.

injury to HF (Hedayat et al., 2010).

**3.2.2 Cytokines: TNF, IL-1 and IL-18** 


Table 6. Inflammatory effects of C-reactive protein. (Adapted from Devaraj et al., 2009) Abbreviations: AT, angiotensin receptor; CCR2, CC-chemokine receptor-2; CD40L, CD40 ligand; EPC, endothelial progenitor cell; ET, endothelin; HMGB1, high-mobility group protein B1; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP, monocyte chemotactic protein; M-CSF, macrophage colonystimulating factor; MMP, matrix metalloproteinase; oxLDL, oxidative low-density lipoprotein; PAI, plasminogen activator inhibitor; ROS, reactive oxygen species; TNF, tumor necrosis factor; tPA, tissue plasminogen activator; VCAM, vascular cell adhesion molecule; VSMC, vascular smooth muscle cell.

Although many studies have examined the relationship between serum CRP levels and HF, few studies have examined CRP levels in myocarditis patients. In one study, 31 patients with clinical and histological evidence of lymphocytic myocarditis were found to have elevated plasma CRP levels that correlated positively with the NYHA functional class (Kaneko et al., 2000; Osman et al., 2006). Five of these patients who died of HF during the study had significantly higher levels of CRP, suggesting that CRP measurement may be a useful tool for determining prognosis in myocarditis patients. A separate study found that 80% of patients with clozapine-induced myocarditis (an antipsychotic drug used to treat schizophrenic symptoms) had elevated levels of CRP compared to a control group (Ronaldson et al., 2010). Several studies have examined CRP levels in idiopathic/ nonischemic DCM patients where CRP levels have been found to independently predict disease outcome (Ishikawa et al., 2006; Kaneko et al., 1999; Senes et al., 2008). CRP levels increased with the severity of symptoms and the level of systolic impairment in DCM patients, while ongoing statin treatment was found to decrease CRP levels (De Gennaro et al., 2008). Interestingly, CRP has been found to co-express with TNF, macrophages and complement in the myocardium of DCM patients suggesting that CRP may play a role in the pathogenesis of disease (Satoh et al., 2005; Zimmermann et al., 2009). One of the obstacles to understanding the role of CRP in myocarditis and DCM is that mouse CRP appears only in trace amounts during the acute phase response (Pepys & Hirschfield, 2003). Instead of CRP mice upregulate serum amyloid P component (SAP), which is a non-acute phase protein in humans. Thus, CRP deficient or transgenic mice may provide only limited information on the role of CRP in HF promotion.

### **3.2.2 Cytokines: TNF, IL-1 and IL-18**

334 Myocarditis

Increased AT-1 and VSMC migration and

Increased neointimal formation *in vivo*

proliferation

Increased iNOS

**Endothelial cells Monocyte-macrophages Smooth muscle cells** 

Increased tissue factor

myeloperoxidase

decreased IL-10

Decreased prostacyclin Increased CD11b and CCR2 Increased ROS

Increased M-CSF and

Table 6. Inflammatory effects of C-reactive protein. (Adapted from Devaraj et al., 2009) Abbreviations: AT, angiotensin receptor; CCR2, CC-chemokine receptor-2; CD40L, CD40 ligand; EPC, endothelial progenitor cell; ET, endothelin; HMGB1, high-mobility group protein B1; ICAM, intercellular adhesion molecule; IL, interleukin; iNOS, inducible nitric oxide synthase; MCP, monocyte chemotactic protein; M-CSF, macrophage colonystimulating factor; MMP, matrix metalloproteinase; oxLDL, oxidative low-density lipoprotein; PAI, plasminogen activator inhibitor; ROS, reactive oxygen species; TNF, tumor necrosis factor; tPA, tissue plasminogen activator; VCAM, vascular cell adhesion molecule;

Although many studies have examined the relationship between serum CRP levels and HF, few studies have examined CRP levels in myocarditis patients. In one study, 31 patients with clinical and histological evidence of lymphocytic myocarditis were found to have elevated plasma CRP levels that correlated positively with the NYHA functional class (Kaneko et al., 2000; Osman et al., 2006). Five of these patients who died of HF during the study had significantly higher levels of CRP, suggesting that CRP measurement may be a useful tool for determining prognosis in myocarditis patients. A separate study found that 80% of patients with clozapine-induced myocarditis (an antipsychotic drug used to treat schizophrenic symptoms) had elevated levels of CRP compared to a control group (Ronaldson et al., 2010). Several studies have examined CRP levels in idiopathic/ nonischemic DCM patients where CRP levels have been found to independently predict disease outcome (Ishikawa et al., 2006; Kaneko et al., 1999; Senes et al., 2008). CRP levels increased with the severity of symptoms and the level of systolic impairment in DCM patients, while ongoing statin treatment was found to decrease CRP levels (De Gennaro et al., 2008). Interestingly, CRP has been found to co-express with TNF, macrophages and complement in the myocardium of DCM patients suggesting that CRP may play a role in the pathogenesis of disease (Satoh et al., 2005; Zimmermann et al., 2009). One of the obstacles to understanding the role of CRP in myocarditis and DCM is that mouse CRP appears only in trace amounts during the acute phase response (Pepys & Hirschfield, 2003). Instead of CRP mice upregulate serum amyloid P component (SAP), which is a non-acute phase protein in

proliferation

dysfunction *in vivo* Increased MMPs and HMGB1

Increased superoxide and

Increased proinflammatory cytokines (e.g. TNF) and

Promoted oxLDL uptake and

decreases cholesterol efflux Increased tissue factor

Increased VCAM, ICAM-1, E-selectin, MCP-1 and monocyte adhesion

Increased PAI-1, IL-8, CD40/CD40L, MMP-1, ET-1

Increased superoxide and

Impaired EPC number and

VSMC, vascular smooth muscle cell.

Promoted endothelial

function *in vitro*

and M-CSF

iNOS

Decreased tPA

The inability of hemodynamic factors to fully explain HF cases led to the hypothesis that cytokines released from cardiac tissue and/or inflammatory cells also contribute to disease progression. According to the "cytokine hypothesis", HF develops because cytokine cascades that are activated following myocardial injury or stress exert deleterious effects on heart function (Table 5) (Anker & von Haehling, 2004; Braunwald, 2008; Seta et al., 1996). Cytokines can induce hemodynamic abnormalities and/or direct toxic effects on the heart. Since the original report in 1990 by Levine et al. there have been many studies showing that an increase in circulating TNF levels directly relates to a patient's NYHA classification and predicts patient mortality (Anker & von Haehling, 2004; Mann, 2005; Seta et al., 1996; Vasan et al., 2003). Similar relationships between IL-1, IL-6 or IL-18 and HF have been found (Anker & von Haehling, 2004; Hedayat et al., 2010; Jefferis et al., 2011; Vasan et al., 2003). Cardiac myocyte hypertrophy, contractile dysfunction, cardiac myocyte apoptosis and extracellular matrix (ECM) remodeling contribute critically to the progression from cardiac injury to HF (Hedayat et al., 2010).

More than any other category of biomarker (Table 2), the role of cytokines in the pathogenesis of myocarditis and DCM has been studied by researchers (Fairweather & Rose, 2005; Hedayat et al., 2010). TNF, IL-1 and IL-18 have all been shown to play a role in myocarditis by inducing myocyte hypertrophy, contractile dysfunction, myocyte apoptosis and contributing to ECM remodeling, a step critical in the progression from myocarditis to DCM (Cain et al., 1999; Fairweather et al., 2004a; 2004b; Hedayat et al., 2010). In one study, TNF mRNA expression was found to be elevated more often in myocarditis patients when viral genomes were also detected, and greater mRNA levels of TNF and its receptor TNFRI correlated with impaired cardiac function (Calabrese et al., 2004). In a mouse model of CVB3 myocarditis, TNF was found to increase CD1d expression on lymphocytes resulting in increased inflammation in males (Huber, 2010). However, viral replication and acute CVB3 myocarditis was not altered in TNFRI deficient mice (Fairweather et al., 2005). Out of the 13 or so TLRs that have been described so far in humans and mice, TLR4 is unique in its ability to work with the inflammasome to produce bioactive IL-1 and IL-18 in the heart (Fairweather et al., 2003; Vallejo, 2011). TLR2 and TLR4 signaling increase TNF levels, and TLR2 can act with TLR4 to increase IL-1 levels (Vallejo, 2011). TLR4 mRNA expression has been found to be higher in patients with myocarditis than controls, and to correlate with viral RNA levels in the heart (Satoh et al., 2003). Myocarditis patients with active viral replication had higher levels of TLR4 that was associated with lower systolic function. In a mouse model of CVB3 myocarditis, our laboratory found that TLR4 deficient mice develop reduced acute inflammation and lower IL-1 and IL-18 levels in the heart (Fairweather et al., 2003). The importance of TLR4 signaling in a strictly autoimmune model of myocarditis was demonstrated by Nishikubo et al. where TLR4 signaling was found to be necessary to mount a Th1-type immune response (Nishikubo et al., 2007). We have shown that TLR4 is upregulated on macrophages and mast cells during the innate immune response to CVB3 and during acute CVB3 myocarditis and this response results in increased inflammation and progression to DCM and HF in males compared to females (Frisancho-Kiss et al., 2007; 2009; Onyimba et al., 2011). A Th1 response in male mice was found to be due to TLR4-derived IL-

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 337

myocarditis in rodents (Liu et al., 2006; Marchant & McManus, 2009; Westermann et al., 2010). For example, MMP9 deficient mice had increased CVB3 replication and inflammation, and worse heart function than wild type controls, indicating that MMP9 protects the heart from CVB3 myocarditis (Cheung et al., 2008). In contrast, TIMP-1 deficient mice were protected from CVB3 myocarditis indicating that TIMP-1 increases disease (Crocker et al., 2007). Although a number of studies have examined the role of MMPs and TIMPs in myocarditis, the relationship between serum levels of these factors and the progression to

Fig. 5. Development of fibrosis and DCM following acute myocarditis. Macrophages and lymphocytes present within the myocardium during acute myocarditis release profibrotic cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-4 and IL-13 that activate fibroblasts to release collagen. Inflammation additionally stimulates the release of growth factors like platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor (TGF)-1, which act with cytokines to increase collagen production. Cytokines and growth factors released from inflammatory cells and cardiac

tissues contribute to pathology. Normally a balance exists between matrix

resulting in dilated cardiomyopathy (DCM) and heart failure.

metalloproteinases (MMPs) that proteolytically degrade fibrillar collagen and tissue inhibitors of MMPs (TIMPs). However, during acute myocarditis an imbalance in MMPs and TIMPs contributes to collagen deposition, ventricular dilatation and remodeling

DCM and HF is not yet clear from animal models.

18, which was originally named IFN--inducing factor, rather than to a classical IL-12/STAT4-induced Th1 response (Frisancho-Kiss et al., 2006). We were surprised to find that TLR4 was expressed on alternatively activated M2 macrophages (induced by Th2 cytokines) rather than classically activated M1 macrophages (induced by Th1 cytokines) within the heart during acute CVB3 myocarditis (Fairweather & Cihakova 2009; Frisancho-Kiss et al., 2009). These CD11b+GR1+F4/80+ M2 macrophages expressed TLR4 and IL-1 (Frisancho-Kiss et al., 2009). We, and others, have shown that IL-1 is particularly important in the cardiac remodeling that leads to fibrosis, DCM and HF following acute myocarditis (Blyszczuk et al., 2009; Cihakova et al., 2008 Fairweather et al., 2004a; 2006). Further work is needed to better understand the role of innate TLRs and cytokine production/regulation in the heart in order to determine whether anti-cytokine therapies will be effective once disease has progressed to the point of being clinically apparent (Mann, 2005). Another area that needs to be addressed in animal models is the relationship between sera levels of proinflammatory cytokines and the stage of disease (i.e. acute myocarditis vs. DCM) and whether increases in sera levels of cytokines predict HF.

#### **3.3 Biomarkers of extracellular matrix remodeling**

Remodeling of the ventricles plays an important role in the progression to HF (Braunwald, 2008). The extracellular matrix provides a framework for cardiac myocytes, mediates cell adhesion and cell-to-cell communication, mediates diastolic stiffness, promotes cell survival or apoptosis, and is a reservoir for growth factors and cytokines (Liu et al., 2006). Release of cytokines and growth factors at the site of tissue injury and by inflammatory cells induces fibroblast proliferation and deposition of collagen, which is the primary component of the ECM resulting in scar tissue (Figure 5). Profibrotic cytokines, such as TNF and IL-1 and growth factors, like transforming growth factor (TGF)1 and fibroblast growth factor (FGF), induce collagen production from fibroblasts. Normally a balance exists between matrix metalloproteinases (MMPs) that proteolytically degrade fibrillar collagen and tissue inhibitors of MMPs (TIMPs). However, during inflammatory CVDs an imbalance in MMPs and TIMPs contributes to collagen deposition, ventricular dilatation and remodeling resulting in DCM and HF. The activity of MMPs has been shown to be increased in the progression to HF (Bradham et al., 2002; Tyagi et al., 1996). Serum MMP9, for example, has been found to predict CVD mortality better than other traditional prognostic markers such as cholesterol, CRP or IL-6 (Blankenberg et al., 2003; Liu et al., 2006).

The progression from myocarditis to fibrosis, DCM and HF has been well documented in clinical studies and animal models (Fairweather et al., 2004a; Kania et al., 2009; Looi et al., 2010). Studies in our laboratory have revealed a two-stage process where increases in profibrotic mediators during acute CVB3 myocarditis, which occurs from day 8 to 12 post infection, result in a gradual remodeling that progresses to fibrosis and DCM by day 35 post infection (Figure 5) (Fairweather et al., 2004a; 2006; Fairweather & Rose, 2007). In both animal and human studies of DCM, genes associated with extracellular matrix remodeling and fibrosis are upregulated with disease (Piro et al., 2010; Yung et al., 2004). TNF, IL-1, IL-4, IL-6, IL-17 and TGF- have all been found to initiate remodeling (Baldeviano et al., 2010; Blyszczuk et al., 2009; Fairweather et al., 2004a; Heymans, 2006; Kania et al., 2009). There are many MMPs, but only 4 known TIMPS. MMPs are upregulated in the heart during EAM and during viral myocarditis (Marchant & McManus, 2009; Tang et al., 2007; Westermann et al., 2010). Individual MMPs and TIMPs have been found to differ in their effects on

18, which was originally named IFN--inducing factor, rather than to a classical IL-12/STAT4-induced Th1 response (Frisancho-Kiss et al., 2006). We were surprised to find that TLR4 was expressed on alternatively activated M2 macrophages (induced by Th2 cytokines) rather than classically activated M1 macrophages (induced by Th1 cytokines) within the heart during acute CVB3 myocarditis (Fairweather & Cihakova 2009; Frisancho-Kiss et al., 2009). These CD11b+GR1+F4/80+ M2 macrophages expressed TLR4 and IL-1 (Frisancho-Kiss et al., 2009). We, and others, have shown that IL-1 is particularly important in the cardiac remodeling that leads to fibrosis, DCM and HF following acute myocarditis (Blyszczuk et al., 2009; Cihakova et al., 2008 Fairweather et al., 2004a; 2006). Further work is needed to better understand the role of innate TLRs and cytokine production/regulation in the heart in order to determine whether anti-cytokine therapies will be effective once disease has progressed to the point of being clinically apparent (Mann, 2005). Another area that needs to be addressed in animal models is the relationship between sera levels of proinflammatory cytokines and the stage of disease (i.e. acute myocarditis vs. DCM) and

Remodeling of the ventricles plays an important role in the progression to HF (Braunwald, 2008). The extracellular matrix provides a framework for cardiac myocytes, mediates cell adhesion and cell-to-cell communication, mediates diastolic stiffness, promotes cell survival or apoptosis, and is a reservoir for growth factors and cytokines (Liu et al., 2006). Release of cytokines and growth factors at the site of tissue injury and by inflammatory cells induces fibroblast proliferation and deposition of collagen, which is the primary component of the ECM resulting in scar tissue (Figure 5). Profibrotic cytokines, such as TNF and IL-1 and growth factors, like transforming growth factor (TGF)1 and fibroblast growth factor (FGF), induce collagen production from fibroblasts. Normally a balance exists between matrix metalloproteinases (MMPs) that proteolytically degrade fibrillar collagen and tissue inhibitors of MMPs (TIMPs). However, during inflammatory CVDs an imbalance in MMPs and TIMPs contributes to collagen deposition, ventricular dilatation and remodeling resulting in DCM and HF. The activity of MMPs has been shown to be increased in the progression to HF (Bradham et al., 2002; Tyagi et al., 1996). Serum MMP9, for example, has been found to predict CVD mortality better than other traditional prognostic markers such

The progression from myocarditis to fibrosis, DCM and HF has been well documented in clinical studies and animal models (Fairweather et al., 2004a; Kania et al., 2009; Looi et al., 2010). Studies in our laboratory have revealed a two-stage process where increases in profibrotic mediators during acute CVB3 myocarditis, which occurs from day 8 to 12 post infection, result in a gradual remodeling that progresses to fibrosis and DCM by day 35 post infection (Figure 5) (Fairweather et al., 2004a; 2006; Fairweather & Rose, 2007). In both animal and human studies of DCM, genes associated with extracellular matrix remodeling and fibrosis are upregulated with disease (Piro et al., 2010; Yung et al., 2004). TNF, IL-1, IL-4, IL-6, IL-17 and TGF- have all been found to initiate remodeling (Baldeviano et al., 2010; Blyszczuk et al., 2009; Fairweather et al., 2004a; Heymans, 2006; Kania et al., 2009). There are many MMPs, but only 4 known TIMPS. MMPs are upregulated in the heart during EAM and during viral myocarditis (Marchant & McManus, 2009; Tang et al., 2007; Westermann et al., 2010). Individual MMPs and TIMPs have been found to differ in their effects on

whether increases in sera levels of cytokines predict HF.

as cholesterol, CRP or IL-6 (Blankenberg et al., 2003; Liu et al., 2006).

**3.3 Biomarkers of extracellular matrix remodeling** 

myocarditis in rodents (Liu et al., 2006; Marchant & McManus, 2009; Westermann et al., 2010). For example, MMP9 deficient mice had increased CVB3 replication and inflammation, and worse heart function than wild type controls, indicating that MMP9 protects the heart from CVB3 myocarditis (Cheung et al., 2008). In contrast, TIMP-1 deficient mice were protected from CVB3 myocarditis indicating that TIMP-1 increases disease (Crocker et al., 2007). Although a number of studies have examined the role of MMPs and TIMPs in myocarditis, the relationship between serum levels of these factors and the progression to DCM and HF is not yet clear from animal models.

Fig. 5. Development of fibrosis and DCM following acute myocarditis. Macrophages and lymphocytes present within the myocardium during acute myocarditis release profibrotic cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, IL-4 and IL-13 that activate fibroblasts to release collagen. Inflammation additionally stimulates the release of growth factors like platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor (TGF)-1, which act with cytokines to increase collagen production. Cytokines and growth factors released from inflammatory cells and cardiac tissues contribute to pathology. Normally a balance exists between matrix metalloproteinases (MMPs) that proteolytically degrade fibrillar collagen and tissue inhibitors of MMPs (TIMPs). However, during acute myocarditis an imbalance in MMPs and TIMPs contributes to collagen deposition, ventricular dilatation and remodeling resulting in dilated cardiomyopathy (DCM) and heart failure.

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 339

2010). Eosinophils are potent inducers of ECM remodeling, releasing many profibrotic factors including IL-1, IL-6, TGF-, MMPs, and TIMPs (Shamri et al., 2011). Additionally, eosinophils release potent prothrombotic agents such as major basic protein, eosinophilic cationic protein, and eosinophil peroxidase, as well as directly and/or indirectly activating TF (Ames et al., 2010). Eosinophilia, fibrosis and thrombosis are characteristics of eosinophilic cardiovascular diseases like Churg Strauss syndrome, a form of vasculitis, hypereosinophilic syndrome, eosinophilic myocarditis and giant cell myocarditis (Ames et al., 2010; Cooper, 2000; 2009; Kleinfeldt et al., 2010; Rezaizadeh et al., 2010). Circulating biomarkers that may indicate a hypercoagulable state include complement C3, C4, IL-6, fibrinogen or antithrombin III (Abdo et al., 2010). Complement components were recently found to be elevated in the sera of mycarditis and DCM patients (Cooper et al., 2010). We have found that complement receptor 1 deficient mice, a receptor that regulates C3 levels, develop severe CVB3 myocarditis, dilation and HF with elevated levels of IL-1 in the heart and fibrosis (Fairweather et al., 2006). Overactivation of the terminal complement complex (C5b-9) has been shown to contribute to the progression of myocarditis to DCM in mice, indicating the importance of complement in regulating disease (Zwaka et al., 2002). Overall, these findings suggest that biomarkers of coagulation and/or thrombosis are likely to be

important indicators of progression to DCM and HF following myocarditis.

Biomarkers are an important clinical tool for assessing progression to heart failure. DCM often leads to HF, and myocarditis is an important cause of acute (sudden death) and chronic (arising from DCM) forms of HF. Even though myocarditis is known to be an important cause of HF, few clinical studies have been conducted to determine the presence or usefulness of HF biomarkers in predicting adverse outcomes in myocarditis patients. Studies that have been conducted in myocarditis/DCM patients or animal models suggest that many of the biomarkers used to assess the likelihood of progression to heart failure in other CVDs will also provide useful information in myocarditis/DCM patients. More studies examining the ability of circulating HF biomarkers to predict poor outcome and HF in animal models of myocarditis/DCM are needed. Animal models will also provide valuable information on the potential role of HF biomarkers in the pathogenesis of disease. This knowledge is critical in determining the ability of therapies targeted to these

The authors thank Adriana Bucek for technical assistance and Norman Barker for photography. The research discussed by the authors in this review was funded by a

Abdo, A.S., Kemp, R., Barham, J. & Geraci, S.A. (2010) Dilated cardiomyopathy and role of antithrombotic therapy. *American Journal of Medical Science* 339: 557-560. Afanasyeva, M., Wang, Y., Kaya, Z., Park, S., Zilliox, M.J., Schofield, B.H., Hill, S.L. & Rose,

N.R. (2001) Experimental autoimmune myocarditis in A/J mice is an interleukin-4 dependent disease with a Th2 phenotype. *American Journal of Pathology* 159: 193-203.

**4. Conclusions** 

biomarkers to prevent disease progression.

National Institutes of Health Grant HL087033 to Dr. Fairweather.

**5. Acknowledgements** 

**6. References** 

#### **3.4 Thrombosis biomarkers**

Atherosclerosis is a major initiator of thrombi formation that can restrict blood flow and lead to a heart attack (Carter, 2005). Cardiac mural thrombi can arise from a myocardial infarction, infection, inflammation (e.g. myocarditis) or rheumatic heart disease, for example. Although research has led to a clear understanding of conditions that induce thrombosis, the precise pathology leading to disease remains unclear. Thrombi can develop anywhere in the cardiovascular system like in the ventricular or atrial chambers, arteries, veins or capillaries. The size and shape of individual thrombi vary depending on the circumstances leading to their development. They often are found at sites of endothelial injury. Once thrombi have formed (acute) they may accumulate more platelets and fibrin and grow larger, or dislodge and travel to other sites, or be removed by fibrinolytic activity, and finally they can attract inflammation, undergo remodeling with deposition of collagen and be reincorporated into the vessel or myocardium (Kumar et al., 2005) (Figure 6).

Fig. 6. Mural thrombi develop during acute CVB3 autoimmune myocarditis in mice and reincorporate into the myocardium. Male BALB/c mice were infected intraperitoneally with heart-passaged coxsackievirus B3 (CVB3) containing infectious CVB3 (103 plaque forming units) and heart proteins on day 0 and thrombus formation examined at day 10 post infection (pi) during acute myocarditis (**A**) or during chronic myocarditis/DCM at day 35 pi (**B**). H&E staining shows ventricular mural thrombus at day 10 pi (magnification, x100) (**A**). Masson's trichrome stains bright blue revealing collagen composition of mural integrated ventricular thrombus at day 35 pi (magnification, x100) (**B**).

Damage of cardiac tissue by viruses and inflammation is known to release tissue factor (TF), the main initiator of the coagulation cascade that results in the formation of fibrin and thrombotic clots (Mackman, 2009). Mural thrombi are known to occur in viral models of myocarditis in mice as well as in myocarditis patients (Antoniak et al., 2008; Kojima et al., 1988; Kuh & Seo, 2005). Furthermore, DCM patients demonstrate a high frequency of LV thrombi and prothrombotic characteristics like high levels of circulating fibrinogen and antithrombin III (Abdo et al., 2010). Many studies have shown that TF can increase inflammation by stimulating release of IL-6, a cytokine that along with TNF and IL-1 has been strongly associated with poor CVD outcome (Braunwald, 2008; Carter, 2005; Mackman, 2009; Rao et al., 2006). Complement components also contribute to thrombosis by depositing at sites of tissue damage and by activating platelets (Fairweather et al., 2006; Peerschke et al., 2010). Eosinophils are potent inducers of ECM remodeling, releasing many profibrotic factors including IL-1, IL-6, TGF-, MMPs, and TIMPs (Shamri et al., 2011). Additionally, eosinophils release potent prothrombotic agents such as major basic protein, eosinophilic cationic protein, and eosinophil peroxidase, as well as directly and/or indirectly activating TF (Ames et al., 2010). Eosinophilia, fibrosis and thrombosis are characteristics of eosinophilic cardiovascular diseases like Churg Strauss syndrome, a form of vasculitis, hypereosinophilic syndrome, eosinophilic myocarditis and giant cell myocarditis (Ames et al., 2010; Cooper, 2000; 2009; Kleinfeldt et al., 2010; Rezaizadeh et al., 2010). Circulating biomarkers that may indicate a hypercoagulable state include complement C3, C4, IL-6, fibrinogen or antithrombin III (Abdo et al., 2010). Complement components were recently found to be elevated in the sera of mycarditis and DCM patients (Cooper et al., 2010). We have found that complement receptor 1 deficient mice, a receptor that regulates C3 levels, develop severe CVB3 myocarditis, dilation and HF with elevated levels of IL-1 in the heart and fibrosis (Fairweather et al., 2006). Overactivation of the terminal complement complex (C5b-9) has been shown to contribute to the progression of myocarditis to DCM in mice, indicating the importance of complement in regulating disease (Zwaka et al., 2002). Overall, these findings suggest that biomarkers of coagulation and/or thrombosis are likely to be important indicators of progression to DCM and HF following myocarditis.
