**3. Toll-like receptors in the inflamed heart**

Despite a wealth of information regarding the symptomatology and clinical course of the disease, the complex pathophysiological mechanisms underlying inflammatory heart disease are only partially understood. Lessons learned from transgenic mouse models have shed some light on the essential role of endogenous receptors and transcriptional regulators engaged in early anti-viral response. From clinical and animal studies we know that the host´s innate immune system acts as the first line of defense against viral replication in a wide array of pathogenic viruses. The innate immune system, which senses pathogen invasion and primes antigen-specific adaptive immunity, has long been considered to be only non-specific and somewhat simpler than that of the adaptive system. However, recent findings on pattern-recognition receptors and their downstream signalling pathways have

Pattern-Recognition Receptors

Sensing Viral Infection in Myocarditis and Inflammatory Heart Disease 205

Fig. 1. Signalling pathways engaged in the detection of highly conserved, relatively

invariant structural motifs of pathogens. Depicted are different pathways for the recognition

led to a reconsideration of the role of innate immunity now seen as a highly potent defense apparatus against microbial pathogens which works in close cooperation with adaptive immunity.

Initial sensing of invading microorganisms by the innate immune system is mediated by pattern-recognition receptors (PRRs), which include toll-like receptors (TLRs), RIG-I-likereceptors, NOD-like receptors, and C-type lectin receptors (Akira et al., 2006; Bauer et al., 2009; Yajima and Knowlton, 2009; Kawai and Akira, 2010; Takeuchi and Akira, 2010). These four classes of germline-encoded PRR families are responsible for recognizing exogenous structures conserved among microbial species, which are called pathogen-associated molecular patterns (PAMPs). Currently, the paradigm of PRRs has changed, since it has been shown that PRRs also recognize endogenous molecules released from damaged cells, collectively termed damage-associated molecular patterns (DAMPs) (Piccinini and Midwood, 2010; Lamkanfi, 2011).

Among the PRRs, toll-like receptors and C-type lectin receptors are transmembrane glycoproteins, whereas retinoic acid-inducible gene (RIG)-I-like receptors and NOD-like receptors function as cytosolic PRRs (Figure 1). Toll-like receptors were first discovered in Drosophila as evolutionary ancient molecules that function as receptors for endogenous ligands such as proteolytically cleaved Spätzle protein, but later were found to be present also in the mammalian system where they respond to microbial components as well as endogenous ligands including heat shock proteins HSP60, HSP70, and gp96 (Medzhitov et al., 1997; Ohashi et al., 2000; Vabulas et al., 2002; Vabulas et al., 2002b; Kim et al., 2009; Arnot et al., 2010). Toll-like receptors are expressed on macrophages, dendritic cells, endothelial cells, and interestingly also on cardiac myocytes (Boyd et al., 2006). Structurally, they are characterized by a highly variable amino-terminal region containing a leucine-rich repeat (LRR) ectodomain, followed by a hydrophobic transmembrane region and a cytoplasmic toll/interleukin 1 receptor (TIR) homology domain, which mediates interaction between TLRs and downstream signalling molecules (Choe et al., 2005; Bell et al., 2006; Jin et al., 2007; Kang et al., 2009; Park et al., 2009). Ligand binding is mediated by the extracellular LRR ectodomain, which is composed of 19-25 tandem copies of the "xLxxLxLxx" motif (Jin and Lee, 2008). In humans, 10 members of the germline-encoded TLR family have been identified so far, TLR1-TLR9 being conserved in humans and mice. Due to a retroviral insertion, TLR10 is not functional in mice and the murine TLRs 11-13 are not present in humans (O'Neill, 2008; Kawai and Akira, 2010).

Ligand binding of PAMPs by TLRs occurs at the plasma membrane (TLR1, TLR2, TLR4-6) as well as in endolysosomal compartments and the endoplasmic reticulum (TLR3, TLR7-9) (Frantz et al., 2007. Kawai and Akira, 2010; Takeuchi and Akira, 2010; Yamamoto and Takeda, 2010). The difference in the downstream signalling cascades activated can be partly explained by the individual TLR molecule, which recognizes a specific subset of PAMPs and recruits different TIR domain-containing adaptors to the receptor. Ligands for TLRs include a broad range of various microbial components, such as bacterial lipoprotein moieties (TLR1-2, TLR6), lipopolysaccharide (TLR4), and flagellin protein (TLR5). Toll-like receptor 3, the first TLR family member to be implicated in the recognition of viral nucleic acids, binds to double-stranded RNA (dsRNA) molecules, which are produced as intermediates during the replication cycle of many viruses (Alexopoulou et al., 2001). TLR7 and TLR8 receptors recognize single-stranded RNA (ssRNA) and are expressed in a variety of immune cells, including dendritic cells, lymphocytes, monocytes, and NK cells (Bauer et al., 2008). Triantafilou and co-workers reported that inflammatory responses in human myocarditis

led to a reconsideration of the role of innate immunity now seen as a highly potent defense apparatus against microbial pathogens which works in close cooperation with adaptive

Initial sensing of invading microorganisms by the innate immune system is mediated by pattern-recognition receptors (PRRs), which include toll-like receptors (TLRs), RIG-I-likereceptors, NOD-like receptors, and C-type lectin receptors (Akira et al., 2006; Bauer et al., 2009; Yajima and Knowlton, 2009; Kawai and Akira, 2010; Takeuchi and Akira, 2010). These four classes of germline-encoded PRR families are responsible for recognizing exogenous structures conserved among microbial species, which are called pathogen-associated molecular patterns (PAMPs). Currently, the paradigm of PRRs has changed, since it has been shown that PRRs also recognize endogenous molecules released from damaged cells, collectively termed damage-associated molecular patterns (DAMPs) (Piccinini and

Among the PRRs, toll-like receptors and C-type lectin receptors are transmembrane glycoproteins, whereas retinoic acid-inducible gene (RIG)-I-like receptors and NOD-like receptors function as cytosolic PRRs (Figure 1). Toll-like receptors were first discovered in Drosophila as evolutionary ancient molecules that function as receptors for endogenous ligands such as proteolytically cleaved Spätzle protein, but later were found to be present also in the mammalian system where they respond to microbial components as well as endogenous ligands including heat shock proteins HSP60, HSP70, and gp96 (Medzhitov et al., 1997; Ohashi et al., 2000; Vabulas et al., 2002; Vabulas et al., 2002b; Kim et al., 2009; Arnot et al., 2010). Toll-like receptors are expressed on macrophages, dendritic cells, endothelial cells, and interestingly also on cardiac myocytes (Boyd et al., 2006). Structurally, they are characterized by a highly variable amino-terminal region containing a leucine-rich repeat (LRR) ectodomain, followed by a hydrophobic transmembrane region and a cytoplasmic toll/interleukin 1 receptor (TIR) homology domain, which mediates interaction between TLRs and downstream signalling molecules (Choe et al., 2005; Bell et al., 2006; Jin et al., 2007; Kang et al., 2009; Park et al., 2009). Ligand binding is mediated by the extracellular LRR ectodomain, which is composed of 19-25 tandem copies of the "xLxxLxLxx" motif (Jin and Lee, 2008). In humans, 10 members of the germline-encoded TLR family have been identified so far, TLR1-TLR9 being conserved in humans and mice. Due to a retroviral insertion, TLR10 is not functional in mice and the murine TLRs 11-13 are not present in

Ligand binding of PAMPs by TLRs occurs at the plasma membrane (TLR1, TLR2, TLR4-6) as well as in endolysosomal compartments and the endoplasmic reticulum (TLR3, TLR7-9) (Frantz et al., 2007. Kawai and Akira, 2010; Takeuchi and Akira, 2010; Yamamoto and Takeda, 2010). The difference in the downstream signalling cascades activated can be partly explained by the individual TLR molecule, which recognizes a specific subset of PAMPs and recruits different TIR domain-containing adaptors to the receptor. Ligands for TLRs include a broad range of various microbial components, such as bacterial lipoprotein moieties (TLR1-2, TLR6), lipopolysaccharide (TLR4), and flagellin protein (TLR5). Toll-like receptor 3, the first TLR family member to be implicated in the recognition of viral nucleic acids, binds to double-stranded RNA (dsRNA) molecules, which are produced as intermediates during the replication cycle of many viruses (Alexopoulou et al., 2001). TLR7 and TLR8 receptors recognize single-stranded RNA (ssRNA) and are expressed in a variety of immune cells, including dendritic cells, lymphocytes, monocytes, and NK cells (Bauer et al., 2008). Triantafilou and co-workers reported that inflammatory responses in human myocarditis

immunity.

Midwood, 2010; Lamkanfi, 2011).

humans (O'Neill, 2008; Kawai and Akira, 2010).

Fig. 1. Signalling pathways engaged in the detection of highly conserved, relatively invariant structural motifs of pathogens. Depicted are different pathways for the recognition

Pattern-Recognition Receptors

(Hardarson et al., 2007).

infection.

degradation of the bacteria was critically impaired.

Sensing Viral Infection in Myocarditis and Inflammatory Heart Disease 207

activation of kinases of the IB kinase complex (Häcker et al., 2000). When infected with the spirochete *Borrelia burgdorferi*, mice deficient in MyD88 expression develop myocarditis and arthritis similar to the disease in wild-type mice (Liu et al., 2004). However, the pathogen burden was much higher in MyD88-/- mice than in wild-type mice, probably because

In response to stimulation with dsRNA, TLR3 recruits another adaptor molecule referred to as TRIF (TIR domain-containing adaptor protein inducing interferon-β, also known as TICAM), which associates with TRAF6 and RIP1 (receptor interacting protein 1) (Yamamoto et al., 2003). TRIF plays a critical role in MyD88-independent TLR3 signalling via TRAF6 and TANK-binding kinase (TBK)-1, leading to the activation of two distinct transcription factors, NF-B and interferon-regulatory factor 3 (Sato et al., 2003). Hardarson and colleagues reported that TLR3 is an essential component of the innate stress response in encephalomyocarditis virus (EMCV)-induced cardiac injury (Hardarson et al., 2007). Mice lacking TLR3 expression were more susceptible to EMCV infection and had a significantly higher viral load in the heart, but lesser inflammatory changes of the myocardium as compared to control mice. TLR3-deficient mice had impaired proinflammatory cytokine and chemokine production in the heart, while expression of interferon- was not impaired

Satoh and colleagues reported that in 44 patients with myocarditis increased expression of TLR4 was associated with replication of enteroviral RNA and that these RNA levels were related to cardiac dysfunction (Satoh et al., 2003). In TLR4 signalling, the ligand-bound receptor utilizes MyD88 and TIRAP (toll-interleukin 1 receptor (TIR) domain-containing adaptor protein) for MyD88-dependent as well as TRIF and TRAM (TRIF-related adaptor molecule) for MyD88-independent pathways (Yamamoto et al., 2003b; Fitzgerald et al., 2003). The adaptor protein TRAM is required for activation of TRIF and recently a splice variant of TRAM called TAG (TRAM adaptor with GOLD domain) has been identified that acts as a negative regulator of TRIF-dependent signalling (Palsson-McDermott et al., 2009). TLR1, TLR2, TLR4, and TLR6 signalling requires, in addition, TIRAP which is important for bridging between the cytoplasmic TLR tail and MyD88 (Fitzgerald et al., 2001). Recently, it was shown that infection with Coxsackie virus group B serotype 3 (CVB3) resulted in cardiac remodelling, severe heart failure, and high mortality in TRIF-deficient mice, while wild-type mice showed only mild myocarditis and normal survival postinfection (Riad et al., 2011). Furthermore, virus control was markedly reduced in mice lacking TRIF expression and, interestingly, TRIF-deficient myocytes displayed a TLR4-dependent suppression of interferon-β. These findings suggest that TRIF confers cardioprotection against CVB3

The recruitment of these adaptors triggers a cascade of signalling events, which leads to the activation of the transcription factors NF-B and interferon-regulatory factors (IRFs). These transcription factors ultimately induce the expression of various inflammatory cytokines, which execute important functions in anti-viral defence. The first step in the synthesis of cytokines leads to the activation of interleukin 1 receptor-associated kinase 4 (IRAK4), which functions as a serine/threonine kinase with an aminoterminal death domain (DD) (Suzuki et al., 2002; Suzuki et al., 2002b). Subsequently, IRAK1 and IRAK2 are phosphorylated by IRAK4 and, after dissociation of MyD88, a complex with TRAF3 and TRAF6 is formed (Kawagoe et al., 2007; Kawagoe et al., 2008; Lin et al., 2010). TRAF6 acts as an E3 ligase in conjugation with the E2 ubiquitin-conjugating enzymes Ubc13 and Uev1A and catalyzes the formation of a lysine63-linked polyubiquitin chain on target proteins, including TRAF6

of microbial infection including toll-like receptor (TLR)-mediated MyD88-dependent and TRIF-dependent pathways as well as cytosolic sensors for foreign nucleic acid sequences (STING, RIG-I, and MDA5). For details on key receptors, their signalling adaptors and downstream mediators see text. **Abbreviations:** IKK; IB kinase, IRAK; IL1-receptorassociated kinase, IRF; interferon-regulatory factor, MDA5; melanoma differentiationassociated gene 5, MyD88; myeloid differentiation primary response gene 88, NEMO; NF-B essential modulator, NF-B; nuclear factor-B, RIG-I; retinoic acid-inducible gene-I, STING; stimulator of interferon genes, TAK1; transforming growth factor--activated kinase 1, TBK1; TANK-binding kinase 1, TIRAP; toll-interleukin 1 receptor (TIR) domain-containing adaptor protein, TLR; toll-like receptor, TRAF; tumour necrosis factor receptor-associated factor, TRIF; TIR-domain-containing adaptor protein inducing interferon-β (also known as TICAM), TRAM; TRIF-related adaptor molecule.

induced by Coxsackie virus B3 (CVB3) are mediated through TLR8 and to a lesser extent through TLR7 (Triantafilou et al., 2005). Elevated expression levels of TLR8 have been associated with heart failure and adverse clinical outcome in patients with enterovirusassociated dilated cardiomyopathy (Satoh et al., 2007). Toll-like receptor 9 functions as a sensor for unmethylated cytosine-phosphate-guanine (CpG) sequences in bacterial and viral DNA, which are rarely found in vertebrates (Barton et al., 2006; Guggemoos et al., 2008). Riad and colleagues demonstrated that, in the acute phase of CVB3-induced myocarditis, TLR9 knockout mice displayed improved LV function associated with reduced cardiac inflammation as compared to CVB3-infected wild-type mice. The cardioprotective effects due to TLR9 deficiency were associated with suppression of the TLR9 downstream pathway as indexed by reduced cardiac levels of the adapter protein MyD88 and the proinflammatory cytokine TNF-α (Riad et al., 2010).

In non-infected cells, TLR3 and TLR7-9 reside in the endoplasmic reticulum, whereas after uncoating and exposure to viral nucleic acids they traffic to the endosomal compartments, where they finally trigger a signal cascade resulting in the activation of the transcription factor NF-B. The subcellular localization of the various TLR family members appears to be tightly regulated, probably because this avoids unbalanced activation to self-DNA in the absence of viral encounter. The diverse distributions of individual TLRs allow for the surveillance of different intracellular compartments, as viral entry usually occurs by receptor-mediated endocytosis and endosomal fusion or by direct fusion with the plasma membrane (Barton et al., 2006; Barton and Kagan, 2009).

Structural studies have revealed that the hydrophobic ligands of TLR1, TLR2, and TLR4 interact with internal protein pockets on the ectodomain, while hydrophilic dsRNA binds to the solvent-exposed surface of TLR3. Binding to cognate ligands induces homodimerization of the TLR ectodomains, whereas TLR2 forms heterodimers with TLR1 or TLR6 which interact with triacyl- and diacyl lipoproteins, respectively (Jin et al., 2007; Kang et al., 2009; Kawai and Akira, 2010). The membrane-adjacent carboxy-termini of the extracellular domains then converge and probably facilitate dimer formation of the cytoplasmic TIR domains to activate intracellular signalling.

Upon stimulation, the dimeric TLR molecules, except for TLR3, recruit a cytoplasmic adaptor called MyD88 (myeloid differentiation primary response gene 88), which is composed of a death domain (DD) in addition to a TIR domain (Muzio et al., 1997; Frantz et al., 2007; Kawai and Akira, 2010). CpG-DNA activates the TLR signaling pathway via MyD88 and TRAF6 (tumour necrosis factor receptor-associated factor 6), leading to

induced by Coxsackie virus B3 (CVB3) are mediated through TLR8 and to a lesser extent through TLR7 (Triantafilou et al., 2005). Elevated expression levels of TLR8 have been associated with heart failure and adverse clinical outcome in patients with enterovirusassociated dilated cardiomyopathy (Satoh et al., 2007). Toll-like receptor 9 functions as a sensor for unmethylated cytosine-phosphate-guanine (CpG) sequences in bacterial and viral DNA, which are rarely found in vertebrates (Barton et al., 2006; Guggemoos et al., 2008). Riad and colleagues demonstrated that, in the acute phase of CVB3-induced myocarditis, TLR9 knockout mice displayed improved LV function associated with reduced cardiac inflammation as compared to CVB3-infected wild-type mice. The cardioprotective effects due to TLR9 deficiency were associated with suppression of the TLR9 downstream pathway as indexed by reduced cardiac levels of the adapter protein MyD88 and the

In non-infected cells, TLR3 and TLR7-9 reside in the endoplasmic reticulum, whereas after uncoating and exposure to viral nucleic acids they traffic to the endosomal compartments, where they finally trigger a signal cascade resulting in the activation of the transcription factor NF-B. The subcellular localization of the various TLR family members appears to be tightly regulated, probably because this avoids unbalanced activation to self-DNA in the absence of viral encounter. The diverse distributions of individual TLRs allow for the surveillance of different intracellular compartments, as viral entry usually occurs by receptor-mediated endocytosis and endosomal fusion or by direct fusion with the plasma

Structural studies have revealed that the hydrophobic ligands of TLR1, TLR2, and TLR4 interact with internal protein pockets on the ectodomain, while hydrophilic dsRNA binds to the solvent-exposed surface of TLR3. Binding to cognate ligands induces homodimerization of the TLR ectodomains, whereas TLR2 forms heterodimers with TLR1 or TLR6 which interact with triacyl- and diacyl lipoproteins, respectively (Jin et al., 2007; Kang et al., 2009; Kawai and Akira, 2010). The membrane-adjacent carboxy-termini of the extracellular domains then converge and probably facilitate dimer formation of the cytoplasmic TIR

Upon stimulation, the dimeric TLR molecules, except for TLR3, recruit a cytoplasmic adaptor called MyD88 (myeloid differentiation primary response gene 88), which is composed of a death domain (DD) in addition to a TIR domain (Muzio et al., 1997; Frantz et al., 2007; Kawai and Akira, 2010). CpG-DNA activates the TLR signaling pathway via MyD88 and TRAF6 (tumour necrosis factor receptor-associated factor 6), leading to

of microbial infection including toll-like receptor (TLR)-mediated MyD88-dependent and TRIF-dependent pathways as well as cytosolic sensors for foreign nucleic acid sequences (STING, RIG-I, and MDA5). For details on key receptors, their signalling adaptors and downstream mediators see text. **Abbreviations:** IKK; IB kinase, IRAK; IL1-receptorassociated kinase, IRF; interferon-regulatory factor, MDA5; melanoma differentiationassociated gene 5, MyD88; myeloid differentiation primary response gene 88, NEMO; NF-B essential modulator, NF-B; nuclear factor-B, RIG-I; retinoic acid-inducible gene-I, STING; stimulator of interferon genes, TAK1; transforming growth factor--activated kinase 1, TBK1; TANK-binding kinase 1, TIRAP; toll-interleukin 1 receptor (TIR) domain-containing adaptor protein, TLR; toll-like receptor, TRAF; tumour necrosis factor receptor-associated factor, TRIF; TIR-domain-containing adaptor protein inducing interferon-β (also known as

TICAM), TRAM; TRIF-related adaptor molecule.

proinflammatory cytokine TNF-α (Riad et al., 2010).

membrane (Barton et al., 2006; Barton and Kagan, 2009).

domains to activate intracellular signalling.

activation of kinases of the IB kinase complex (Häcker et al., 2000). When infected with the spirochete *Borrelia burgdorferi*, mice deficient in MyD88 expression develop myocarditis and arthritis similar to the disease in wild-type mice (Liu et al., 2004). However, the pathogen burden was much higher in MyD88-/- mice than in wild-type mice, probably because degradation of the bacteria was critically impaired.

In response to stimulation with dsRNA, TLR3 recruits another adaptor molecule referred to as TRIF (TIR domain-containing adaptor protein inducing interferon-β, also known as TICAM), which associates with TRAF6 and RIP1 (receptor interacting protein 1) (Yamamoto et al., 2003). TRIF plays a critical role in MyD88-independent TLR3 signalling via TRAF6 and TANK-binding kinase (TBK)-1, leading to the activation of two distinct transcription factors, NF-B and interferon-regulatory factor 3 (Sato et al., 2003). Hardarson and colleagues reported that TLR3 is an essential component of the innate stress response in encephalomyocarditis virus (EMCV)-induced cardiac injury (Hardarson et al., 2007). Mice lacking TLR3 expression were more susceptible to EMCV infection and had a significantly higher viral load in the heart, but lesser inflammatory changes of the myocardium as compared to control mice. TLR3-deficient mice had impaired proinflammatory cytokine and chemokine production in the heart, while expression of interferon- was not impaired (Hardarson et al., 2007).

Satoh and colleagues reported that in 44 patients with myocarditis increased expression of TLR4 was associated with replication of enteroviral RNA and that these RNA levels were related to cardiac dysfunction (Satoh et al., 2003). In TLR4 signalling, the ligand-bound receptor utilizes MyD88 and TIRAP (toll-interleukin 1 receptor (TIR) domain-containing adaptor protein) for MyD88-dependent as well as TRIF and TRAM (TRIF-related adaptor molecule) for MyD88-independent pathways (Yamamoto et al., 2003b; Fitzgerald et al., 2003). The adaptor protein TRAM is required for activation of TRIF and recently a splice variant of TRAM called TAG (TRAM adaptor with GOLD domain) has been identified that acts as a negative regulator of TRIF-dependent signalling (Palsson-McDermott et al., 2009). TLR1, TLR2, TLR4, and TLR6 signalling requires, in addition, TIRAP which is important for bridging between the cytoplasmic TLR tail and MyD88 (Fitzgerald et al., 2001). Recently, it was shown that infection with Coxsackie virus group B serotype 3 (CVB3) resulted in cardiac remodelling, severe heart failure, and high mortality in TRIF-deficient mice, while wild-type mice showed only mild myocarditis and normal survival postinfection (Riad et al., 2011). Furthermore, virus control was markedly reduced in mice lacking TRIF expression and, interestingly, TRIF-deficient myocytes displayed a TLR4-dependent suppression of interferon-β. These findings suggest that TRIF confers cardioprotection against CVB3 infection.

The recruitment of these adaptors triggers a cascade of signalling events, which leads to the activation of the transcription factors NF-B and interferon-regulatory factors (IRFs). These transcription factors ultimately induce the expression of various inflammatory cytokines, which execute important functions in anti-viral defence. The first step in the synthesis of cytokines leads to the activation of interleukin 1 receptor-associated kinase 4 (IRAK4), which functions as a serine/threonine kinase with an aminoterminal death domain (DD) (Suzuki et al., 2002; Suzuki et al., 2002b). Subsequently, IRAK1 and IRAK2 are phosphorylated by IRAK4 and, after dissociation of MyD88, a complex with TRAF3 and TRAF6 is formed (Kawagoe et al., 2007; Kawagoe et al., 2008; Lin et al., 2010). TRAF6 acts as an E3 ligase in conjugation with the E2 ubiquitin-conjugating enzymes Ubc13 and Uev1A and catalyzes the formation of a lysine63-linked polyubiquitin chain on target proteins, including TRAF6

Pattern-Recognition Receptors

TLR2 Lipoprotein Plasma

Lipopolysaccharide (LPS)

TLR5 Flagellin Plasma

lipoprotein

TLR6 Diacyl

RIG-I Short

inflammatory heart disease.

TLR4

TLR7

**PRRs Ligand Localization Origin of** 

membrane

TLR3 dsRNA Endolysosome Viruses

Plasma membrane

Plasma membrane

(TLR8) ssRNA Endolysosome Bacteria,

TLR9 CpG-DNA Endolysosome Bacteria,

dsRNA Cytoplasm Viruses

Sensing Viral Infection in Myocarditis and Inflammatory Heart Disease 209

Bacteria, viruses

Bacteria, viruses

Bacteria, viruses

viruses

viruses

membrane Bacteria

**the ligand Selected references** 

(Nozaki et al., 2004).

type mice (Ha et al., 2005).

et al., 2007).

2008).

al., 2007)

Table 1. Pattern recognition receptors (PRR) and their ligands engaged in myocarditis and

2007).

Following coronary artery ligation, *tl*r2-/- mice show reduced mortality and preserved left ventricular function as compared to wild-type mice (Shishido et al., 2003; Riad et al., 2008). Ischemia-reperfusion results in smaller infarct size (Favre et al., 2007). *Tl*r2-/- mice are protected from doxorubicin-induced cardiomyopathy

*Tl*r3-/- mice are more susceptible to EMCV infection and have a higher viral load, but lesser myocardial inflammation (Hardarson et al.,

*Tl*r4-/- mice are protected from ischemiareperfusion injury (Chong et al, 2004; Oyama et al., 2004; Kim et al., 2007) and LPS-induced mortality and cardiac dysfunction (Tavener et al., 2004; Nemoto et al, 2009). *Tl*r4-/- mice develop less-severe cardiac hypertrophy following pressure overload by aortic banding than wild-

Exposure to the TLR5 ligand flagellin triggers cardiac innate immune responses that result in acute contractile dysfunction (Rolli et al., 2010).

*Tl*r7-/- mice show markedly reduced myocardial cellular infiltration in experimental autoimmune myocarditis (Pagni et al., 2010). TLR8 expression is higher in DCM patients than in controls (Satoh

TLR6 Ser249Pro polymorphism has been associated with lower left ventricular thickness in hypertensive women (Sales et al., 2010).

Upon murine cytomegalovirus-induced myocarditis, *tl*r9-/- mice show higher severity of myocardial infiltration compared to wild-type (Pagni et al., 2010). Myocardial TLR9 expression is reduced in DCM patients (Ruppert et al.,

RIG-I mRNA is expressed at high levels in normal heart tissue (Ellis et al., 2002). Interferon upregulates RIG-I in pericardial mesothelial cells, suggesting that RIG-I may be involved in the pathogenesis of pericarditis (Hatakeyama et

itself, IRAK, and the NF-B essential modulator (NEMO) (Deng et al., 2000). Transforming growth factor--activated kinase 1 (TAK1) is recruited and ubiquitinylated by TRAF6 (Wang et al., 2001). Subsequently, the IKK complex composed of IKK, IKK and NEMO is formed which phosphorylates inhibitor of NF-B (IB) kinase-ß (IKK). The activated IKK complex then induces phosphorylation and subsequent degradation of IB by the proteasome. Upon degradation of IB, the freed NF-B is no longer sequestered in the cytosol, but translocates into the nucleus, where it drives the expression of cytokine genes. Simultaneously, TAK1 activates the mitogen-activated protein kinase (MAPK) cascade leading to the activation of the transcription factor AP-1, which also targets gene expression of cytokine genes (Wang et al., 2001).
