**3.1.2 BNP and NT-pro-BNP**

328 Myocarditis

higher in-hospital mortality, independent from other predictive values, than those who were negative for troponin (Peacock et al., 2008). Troponin T was found to be an important independent variable that predicted increased risk of death in patients with chronic HF (Latini et al., 2007). These findings demonstrate that troponin measurement is an important

tool in early risk assessment of myocarditis/ DCM patients.

Acute coronary syndrome

Dilated cardiomyopathy Acute heart failure Pulmonary embolism

Acute aortic dissection Tachyarrhythmias Cardiac contusion

Tako-tsubo cardiomyopathy

Sympathomimetic drugs

Chemotherapy

Agewall et al., 2011)

Strenuous exercise e.g. marathon runners

Table 3. Cardiac conditions that can result in acutely elevated troponins. (Adapted from

Interestingly, autoantibodies against circulating troponin I have been found in patients with ACS and acute myocardial infarction (Eriksson et al., 2005; Leuschner et al., 2008; Shmilovich et al., 2007). These autoantibodies were discovered because they interfered with troponin detection assays (Eriksson et al., 2005). This discovery led to the realization that autoantibodies against troponin I were also present in the sera of patients with DCM and heart failure (Miettinen et al., 2008; Shmilovich et al., 2007). One study of DCM patients found that troponin I, but not troponin I autoantibodies, were associated with dilation and poor outcome (Miettinen et al., 2008). In another study a significant number of DCM patients had autoantibodies against troponin I, but the autoantibodies were not found to bind to cardiac myocytes or activate Ca2+ currents (Shmilovich et al., 2007). Myocardial infarction patients with elevated troponin I autoantibodies had poor recovery of LV EF suggesting that troponin autoantibodies affect heart function (Leuschner et al., 2008). Further evidence that troponin autoantibodies may directly affect heart function comes from animal studies. PD-1 receptor deficient mice were found to develop severe DCM with high levels of autoantibodies against tropoinin I (Kaya et al., 2010; Nishimura et al., 2001). These troponin I autoantibodies were found to bind to heart tissue and to induce Ca2+ influx in cardiac myocytes. Inoculation of mice with recombinant troponin I with complete Freund's adjuvant was found to induce severe myocarditis and increased proinflammatory cytokines that progressed to fibrosis, DCM and heart failure (Goser et al., 2006; Kaya et al., 2008; 2010). However, this inflammatory response was only observed for troponin I but not troponin T inoculation. More research is needed to better understand the relationship of circulating

**Condition** 

Myocarditis Pericarditis Endocarditis

Stroke Sepsis

> In contrast to cardiac myosin or troponins that are released due to cell wall compromise, BNP is synthesized in healthy cardiac myocytes from its precursor NT-pro-BNP (Braunwald, 2008; Chen et al., 2010). The prohormone BNP is only released to the circulation when the ventricles become dilated, hypertrophic or during other conditions that induce wall distension and stretching, and by neurohormonal activation (Table 4). Prohormone BNP is cleaved by an endoprotease, corin, in the circulation into two polypeptides: the inactive NT-pro-BNP and the bioactive BNP. BNP causes arterial vasodilation, natriuresis and diureses while reducing the renin angiotensin system and adrenergic response (Braunwald, 2008; Palazzuoli et al., 2011). Elevated plasma BNP levels occur during hypertrophic cardiomyopathy, diastolic dysfunction and LV hypertrophy, and have been shown to be directly proportional to NYHA class and inversely related to cardiac output (Silver et al., 2004). Although few studies specifically address the topic, BNP has been found to be elevated in the serum of patients with myocarditis or DCM and in animal models of myocarditis (Grabowski et al., 2004; Miller et al., 2007; Ogawa et al., 2008; Talvani et al., 2004; Tanaka et al., 2011). Plasma BNP levels are also elevated in patients with acute myocardial infarction and this relationship persists into the late phases of cardiac remodeling (Hirayama et al., 2005). Many studies have linked higher levels of circulating BNP with heart failure diagnosis and worse outcome (Miller et al., 2007; Palazzuoli et al., 2011). BNP levels are a better predictor of death than norepinephrine or endothelin-1 (Braunwald, 2008). Several studies have found that NT-pro-BNP was better than BNP for predicting death or re-hospitalization for heart failure, probably due to the longer half-life of NT-pro-BNP in sera (Masson et al., 2006; Omland et al., 2007). However, BNP is a better

al., 2008).

myocarditis and DCM patients.

10 mice per group. \**P* < 0.05; \*\*\**P* < 0.001.

**3.2 Biomarkers of inflammation** 

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 331

cardioprotective using animal models where recombinant (r)IL-33 treatment reduced hypertrophy and fibrosis following pressure overload induced by transverse aortic constriction (TAC) (Sanada et al., 2007). This effect was reversed by treating mice with sST2 prior to TAC, providing evidence that sST2 functions as a decoy receptor for IL-33. Additionally, rIL-33 treatment was found to decrease atherosclerosis in ApoE deficient mice fed a high fat diet by skewing the immune response from a Th1 to a Th2 response (Miller et

Currently there are no reports on the role of sST2 or IL-33 in the development of myocarditis or DCM even though sST2 is known to be a good biomarker predicting heart failure. Our laboratory is investigating the role of sST2/IL-33 signaling in an autoimmune model of coxsackievirus B3 (CVB3) myocarditis and DCM in mice. We found that IL-33 mRNA was upregulated in the heart during acute CVB3 myocarditis and chronic DCM (Figure 3A,B). Additionally, sST2 levels were elevated in the sera during acute CVB3 myocarditis in mice (Figure 3C) and correlated with poor heart function as assessed by echocardiography (not shown) or pressure-volume relationships (Figure 4). Serum sST2 is a good marker of disease because it could not be detected in the sera of undiseased mice (Figure 3C). Our findings of elevated sST2 levels in the sera of mice with CVB3 myocarditis and its relation to poor heart function suggest that sST2 may serve as a useful biomarker to predict progression to HF in

Fig. 3. IL-33 and sST2 are increased during autoimmune CVB3 myocarditis in mice. 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 myocarditis examined at day 10 post infection (pi) and dilated cardiomyopathy (DCM) at day 90 pi. Saline inoculated age-matched mice were used as controls. Interleukin (IL)-33 mRNA was assessed by quantitative RT-PCR in the heart at day 10 (**A**) and 90 (**B**) pi and normalized to hypoxanthine phosphoribosyltransferase (HPRT) levels. sST2 levels were assessed in the sera of mice during acute myocarditis at day 10 pi by ELISA (**C**). Data are expressed as mean relative gene expression (RGE) ±standard error of the mean (SEM) in 7 to

Inflammation is important in the pathogenesis of many of the conditions that lead to HF. Traditionally, inflammatory biomarkers have been considered to be risk markers rather than risk factors because their role in disease pathogenesis is not always clear (Rao et al., 2006). Many inflammatory biomarkers found in the circulation, such as C-reactive protein (CRP),

predictor than NT-pro-BNP of worse outcome in ACS (Palazzuoli et al., 2011). Natriuretic peptides were also found to be useful in screening asymptomatic subjects at risk of developing HF such as those with hypertension, diabetes and coronary artery disease (Braunwald, 2008). Noncardiac conditions that increase plasma BNP levels such as age, race, obesity and renal dysfunction should be taken into consideration when using this biomarker (Table 4). Overall, BNP and NT-pro-BNP are biomarkers with a high sensitivity and specificity in predicting HF in a number of conditions including myocarditis and DCM.


Table 4. Conditions that increase plasma natriuretic peptide levels*.* (Adapted from Braunwald, 2008 and Palazzuoli et al., 2011)

#### **3.1.3 Soluble ST2**

Soluble ST2 (sST2), an interleukin (IL)-1 receptor (R) (IL-1R) family member, is basally expressed by cardiomyocytes and upregulated in the heart by mechanical strain and IL-1 (Weinberg et al., 2002). The ST2 gene is known to encode at least 3 isoforms of ST2 by alternative splicing: ST2L, a transmembrane receptor; sST2, a secreted soluble form of ST2 that can serve as a decoy receptor for the ST2 ligand, IL-33; and ST2V, a variant of ST2 found in the gut of humans (Miller & Liew, 2011). ST2L is a member of the Toll-like receptor (TLR)/IL-1R superfamily that share a common structure with an extracellular domain of three linked immunglobulin-like motifs, a transmembrane segment and a cytoplasmic Tollinterleukin-1 receptor (TIR) domain. ST2L forms a complex with IL-1R accessory protein (IL-1RAcP) that is required for IL-33 signaling (Ali et al., 2007). IL-33 signaling recruits the adaptor protein MyD88 to the TIR domain leading to activation of the transcription factors NF-B and AP-1 and production of inflammatory mediators including proinflammatory tumor necrosis factor (TNF), IL-1, IL-6, and the T helper (Th)2-associated cytokines IL-5, IL-13 and IL-10 (Liew et al., 2010; Miller & Liew, 2011). Both ST2L and sST2 are induced in cardiomyocytes by biomechanical strain (Weinberg et al., 2002). Elevated levels of sST2 in the sera are associated with poor prognosis in patients with acute myocardial infarction or chronic heart failure where sST2 levels correlate positively with creatine kinase and negatively with EF (Weinberg et al., 2002; 2003). In patients with severe chronic NYHA class III or IV heart failure, the change in sST2 levels was an independent predictor of subsequent mortality or transplantation (Weinberg et al., 2003). IL-33 is expressed largely within fibroblasts in the heart and is thought to be released from necrotic cells due to tissue damage caused by direct damage or infection (Liew et al., 2010). IL-33 has been shown to be

predictor than NT-pro-BNP of worse outcome in ACS (Palazzuoli et al., 2011). Natriuretic peptides were also found to be useful in screening asymptomatic subjects at risk of developing HF such as those with hypertension, diabetes and coronary artery disease (Braunwald, 2008). Noncardiac conditions that increase plasma BNP levels such as age, race, obesity and renal dysfunction should be taken into consideration when using this biomarker (Table 4). Overall, BNP and NT-pro-BNP are biomarkers with a high sensitivity and specificity in predicting HF in a number of conditions including myocarditis and DCM.

> **Cardiac condition Noncardiac condition**  Systolic dysfunction Acute pulmonary embolism Diastolic dysfunction Pulmonary hypertension

Table 4. Conditions that increase plasma natriuretic peptide levels*.* (Adapted from

Soluble ST2 (sST2), an interleukin (IL)-1 receptor (R) (IL-1R) family member, is basally expressed by cardiomyocytes and upregulated in the heart by mechanical strain and IL-1 (Weinberg et al., 2002). The ST2 gene is known to encode at least 3 isoforms of ST2 by alternative splicing: ST2L, a transmembrane receptor; sST2, a secreted soluble form of ST2 that can serve as a decoy receptor for the ST2 ligand, IL-33; and ST2V, a variant of ST2 found in the gut of humans (Miller & Liew, 2011). ST2L is a member of the Toll-like receptor (TLR)/IL-1R superfamily that share a common structure with an extracellular domain of three linked immunglobulin-like motifs, a transmembrane segment and a cytoplasmic Tollinterleukin-1 receptor (TIR) domain. ST2L forms a complex with IL-1R accessory protein (IL-1RAcP) that is required for IL-33 signaling (Ali et al., 2007). IL-33 signaling recruits the adaptor protein MyD88 to the TIR domain leading to activation of the transcription factors NF-B and AP-1 and production of inflammatory mediators including proinflammatory tumor necrosis factor (TNF), IL-1, IL-6, and the T helper (Th)2-associated cytokines IL-5, IL-13 and IL-10 (Liew et al., 2010; Miller & Liew, 2011). Both ST2L and sST2 are induced in cardiomyocytes by biomechanical strain (Weinberg et al., 2002). Elevated levels of sST2 in the sera are associated with poor prognosis in patients with acute myocardial infarction or chronic heart failure where sST2 levels correlate positively with creatine kinase and negatively with EF (Weinberg et al., 2002; 2003). In patients with severe chronic NYHA class III or IV heart failure, the change in sST2 levels was an independent predictor of subsequent mortality or transplantation (Weinberg et al., 2003). IL-33 is expressed largely within fibroblasts in the heart and is thought to be released from necrotic cells due to tissue damage caused by direct damage or infection (Liew et al., 2010). IL-33 has been shown to be

Myocarditis Anemia Pericarditis Septic shock Myocardial fibrosis Hyperthyroidism Dilated cardiomyopathy *Cor pulmonale*  Heart failure Renal failure

Valvular heart disease Coronary artery disease

Hypertension Atrial fibrillation

Braunwald, 2008 and Palazzuoli et al., 2011)

**3.1.3 Soluble ST2** 

cardioprotective using animal models where recombinant (r)IL-33 treatment reduced hypertrophy and fibrosis following pressure overload induced by transverse aortic constriction (TAC) (Sanada et al., 2007). This effect was reversed by treating mice with sST2 prior to TAC, providing evidence that sST2 functions as a decoy receptor for IL-33. Additionally, rIL-33 treatment was found to decrease atherosclerosis in ApoE deficient mice fed a high fat diet by skewing the immune response from a Th1 to a Th2 response (Miller et al., 2008).

Currently there are no reports on the role of sST2 or IL-33 in the development of myocarditis or DCM even though sST2 is known to be a good biomarker predicting heart failure. Our laboratory is investigating the role of sST2/IL-33 signaling in an autoimmune model of coxsackievirus B3 (CVB3) myocarditis and DCM in mice. We found that IL-33 mRNA was upregulated in the heart during acute CVB3 myocarditis and chronic DCM (Figure 3A,B). Additionally, sST2 levels were elevated in the sera during acute CVB3 myocarditis in mice (Figure 3C) and correlated with poor heart function as assessed by echocardiography (not shown) or pressure-volume relationships (Figure 4). Serum sST2 is a good marker of disease because it could not be detected in the sera of undiseased mice (Figure 3C). Our findings of elevated sST2 levels in the sera of mice with CVB3 myocarditis and its relation to poor heart function suggest that sST2 may serve as a useful biomarker to predict progression to HF in myocarditis and DCM patients.

Fig. 3. IL-33 and sST2 are increased during autoimmune CVB3 myocarditis in mice. 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 myocarditis examined at day 10 post infection (pi) and dilated cardiomyopathy (DCM) at day 90 pi. Saline inoculated age-matched mice were used as controls. Interleukin (IL)-33 mRNA was assessed by quantitative RT-PCR in the heart at day 10 (**A**) and 90 (**B**) pi and normalized to hypoxanthine phosphoribosyltransferase (HPRT) levels. sST2 levels were assessed in the sera of mice during acute myocarditis at day 10 pi by ELISA (**C**). Data are expressed as mean relative gene expression (RGE) ±standard error of the mean (SEM) in 7 to 10 mice per group. \**P* < 0.05; \*\*\**P* < 0.001.

#### **3.2 Biomarkers of inflammation**

Inflammation is important in the pathogenesis of many of the conditions that lead to HF. Traditionally, inflammatory biomarkers have been considered to be risk markers rather than risk factors because their role in disease pathogenesis is not always clear (Rao et al., 2006). Many inflammatory biomarkers found in the circulation, such as C-reactive protein (CRP),

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 333

Interestingly, Th2 responses have also been implicated in the pathogenesis of myocarditis leading to HF (Afanasyeva et al., 2001; Fairweather et al., 2004a; 2004b). IFN- deficient mice, which have elevated IL-4 levels and a Th2 response, progress to severe DCM and HF

Table 5. Deleterious effects of inflammatory biomarkers on acute and chronic heart failure.

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.,

(Adapted from Anker & von Haehling, 2004; Braunwald, 2008; Mann, 2005)

following CVB3 myocarditis (Fairweather et al., 2004a).

**Deleterious effects** 

Pulmonary edema Cardiomyopathy Inflammation

Left ventricular dysfunction

Left ventricular remodeling Endothelial dysfunction

Anorexia and cachexia

**3.2.1 C-reactive protein** 

Inducible nitric oxide synthase activation Decreased skeletal-muscle blood flow

Receptor uncoupling from adenylate cyclase

Activation of the fetal-gene program Apoptosis of cardiac myocytes

**Known** 

**Potential** 

2005).

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 CVB3 myocarditis.

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). Interestingly, Th2 responses have also been implicated in the pathogenesis of myocarditis leading to HF (Afanasyeva et al., 2001; Fairweather et al., 2004a; 2004b). IFN- deficient mice, which have elevated IL-4 levels and a Th2 response, progress to severe DCM and HF following CVB3 myocarditis (Fairweather et al., 2004a).


Table 5. Deleterious effects of inflammatory biomarkers on acute and chronic heart failure. (Adapted from Anker & von Haehling, 2004; Braunwald, 2008; Mann, 2005)
