**3. Biomarkers in heart failure**

Biomarkers are frequently used in cardiovascular medicine where they provide valuable information regarding diagnosis, treatment, identification of individuals at risk for HF, and potentially the pathogenesis of disease. For a biomarker to be clinically useful it should fulfill several criteria: 1) biomarker levels should be able to be accurately assessed using widely available and cost-efficient methods, 2) biomarkers should provide additional information from the tests already conducted such as MRI, and 3) biomarker information should aid in medical decision making (Braunwald, 2008; Morrow & de Lemos, 2007). A growing list of enzymes, hormones, markers of cardiac stress or necrosis, cytokines and other biological agents have been examined as possible biomarkers for HF (Table 2). Although biomarkers are discussed in this review by category (e.g. those associated with cardiac damage or inflammation), in reality many of these biomarkers interact or are associated with one another suggesting that combinations of biomarkers are likely to provide the best assessment of HF risk. This review will focus on HF biomarkers from Table 2 that have been studied in myocarditis/ DCM patients or experimental models.

#### **3.1 Biomarkers of cardiovascular injury or stress**

Myocyte injury can occur from many causes including infections, oxidative stress, inflammation or severe ischemia (Brauanwald, 2008). Cardiac myosin is typically not used as a biomarker for HF because it is rapidly cleared from the circulation. Cardiac troponin I and T are current standard biomarkers used to diagnose acute myocardial infarction and to stratify patient risk in acute coronary syndromes (ACS) because of their long half-life in the circulation (Hochholzer et al., 2010). B-type natriuretic peptide (BNP) and N-terminal probrain natriuretic peptide (NT-pro-BNP) are important indicators of cardiovascular stress having the advantage of being able to distinguish acute from chronic HF. Recently ST2 has emerged as another biomarker of cardiac damage that has prognostic ability in patients suspected of HF.

#### **3.1.1 Troponins I and T**

The cardiac troponins are proteins located in myocytes that are responsible for regulating cardiac muscle contraction. Cardiac troponin is composed of three subunits that are products of different genes- troponin C, troponin I and troponin T. Compared to myosin and actin, troponins are present at low levels in the heart. However, both troponin I and T are ideally suited to detect myocardial damage because they are expressed as cardiacspecific isoforms (Agewall et al., 2011). Elevated sera troponins are associated with

myocarditis. Unique features of myocarditis including myocardial edema, hyperemia, increased capillary permeability due to inflammation, and fibrosis can be identified using a combination of T1 and T2-weighted images (Blauwet & Cooper, 2010; Karamitsos & Neubauer, 2011). Endomyocardial biopsy is used to verify inflammation in the heart and to assess whether certain cell populations such as eosinophils or giant cells are present in the myocardium for diagnostic purposes (i.e. eosinophilic or giant cell myocarditis). Due to the focal nature of myocarditis and the fact that foci are frequently located in the peri/myocardium, endomyocardial biopsies often miss inflammation and so often do not aid in diagnosis (Maisch, 1994; Olimulder et al., 2009). Although the complication rate is

Biomarkers are frequently used in cardiovascular medicine where they provide valuable information regarding diagnosis, treatment, identification of individuals at risk for HF, and potentially the pathogenesis of disease. For a biomarker to be clinically useful it should fulfill several criteria: 1) biomarker levels should be able to be accurately assessed using widely available and cost-efficient methods, 2) biomarkers should provide additional information from the tests already conducted such as MRI, and 3) biomarker information should aid in medical decision making (Braunwald, 2008; Morrow & de Lemos, 2007). A growing list of enzymes, hormones, markers of cardiac stress or necrosis, cytokines and other biological agents have been examined as possible biomarkers for HF (Table 2). Although biomarkers are discussed in this review by category (e.g. those associated with cardiac damage or inflammation), in reality many of these biomarkers interact or are associated with one another suggesting that combinations of biomarkers are likely to provide the best assessment of HF risk. This review will focus on HF biomarkers from Table

2 that have been studied in myocarditis/ DCM patients or experimental models.

Myocyte injury can occur from many causes including infections, oxidative stress, inflammation or severe ischemia (Brauanwald, 2008). Cardiac myosin is typically not used as a biomarker for HF because it is rapidly cleared from the circulation. Cardiac troponin I and T are current standard biomarkers used to diagnose acute myocardial infarction and to stratify patient risk in acute coronary syndromes (ACS) because of their long half-life in the circulation (Hochholzer et al., 2010). B-type natriuretic peptide (BNP) and N-terminal probrain natriuretic peptide (NT-pro-BNP) are important indicators of cardiovascular stress having the advantage of being able to distinguish acute from chronic HF. Recently ST2 has emerged as another biomarker of cardiac damage that has prognostic ability in patients

The cardiac troponins are proteins located in myocytes that are responsible for regulating cardiac muscle contraction. Cardiac troponin is composed of three subunits that are products of different genes- troponin C, troponin I and troponin T. Compared to myosin and actin, troponins are present at low levels in the heart. However, both troponin I and T are ideally suited to detect myocardial damage because they are expressed as cardiacspecific isoforms (Agewall et al., 2011). Elevated sera troponins are associated with

low, patients are at a risk of death from the procedure.

**3.1 Biomarkers of cardiovascular injury or stress** 

suspected of HF.

**3.1.1 Troponins I and T** 

**3. Biomarkers in heart failure** 

**Biomarker Myocyte inury**  Cardiac-specific troponins I and T Myosin light-chain kinase I Creatine kinase MB fraction **Myocyte stress**  Brain natiuretic peptide N-terminal pro-brain natriuretic peptide ST2/ interleukin-33 **Inflammation**  C-reactive protein Tumor necrosis factor Fas (APO-1) Interleukins 1, 6, 18 Galectin-3 **Oxidative stress**  Oxidized low-density lipoproteins Myeloperoxidase Urinary and plasma isoprostanes **Neurohormones**  Norepinephrine Renin Angiotensin II Aldosterone Endothelin **Extracellular-matrix remodeling**  Matrix metalloproteinases Tissue inhibitors of metalloproteinases Collagen propeptides

Table 2. Biomarkers in heart failure. (Adapted from Braunwald, 2008)

myocardial ischemia and necrosis and have been found to be excellent diagnostic and prognostic biomarkers for thrombotic ACS. But many cardiac conditions can lead to elevated troponins in addition to ACS including myocarditis, DCM and HF (Table 3). With new high-sensitivity detection methods for troponins, very minor changes in cardiac damage can be detected. Although troponins released to the circulation do not identify the type of heart damage, their levels may indicate the severity of damage (Figure 2) (Miller et al., 2007). A number of studies have been conducted examining troponin release during myocarditis, DCM or heart failure in patients (Smith et al., 1997; Brandt et al., 2001; Imazio et al., 2008; Miller et al., 2007; Peacock et al., 2008). Troponin I and T have also been found to predict the severity of myocarditis and the short-term prognosis in children with acute and fulminant myocarditis and DCM (Soongswang et al., 2002; Al-Biltagi et al., 2010). Overall, troponin levels were found to increase in relation to the severity of myocardial inflammation or ventricular wall stress caused by remodeling (Agewall et al., 2011; Miller et al., 2007). DCM patients with elevated serum troponin I levels were more dilated and had a worse outcome that troponin I-negative patients (Miettinen et al., 2008). Additionally, acute decompensated heart failure patients who were positive for troponin had a lower EF and

failure.

2011.

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

Biomarkers of Heart Failure in Myocarditis and Dilated Cardiomyopathy 329

troponin I and its autoantibodies in the progression of myocarditis to DCM and heart

Fig. 2. Relationship between serum troponin levels and the severity of cardiac damage caused by myocarditis, heart failure or myocardial infarction. Adapted from Agewall et al.,

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

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.


Table 3. Cardiac conditions that can result in acutely elevated troponins. (Adapted from Agewall et al., 2011)

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 troponin I and its autoantibodies in the progression of myocarditis to DCM and heart failure.


Fig. 2. Relationship between serum troponin levels and the severity of cardiac damage caused by myocarditis, heart failure or myocardial infarction. Adapted from Agewall et al., 2011.
