**3. Dysregulation of host protein degradation systems in viral cardiomyopathy**

#### **3.1 Protein degradation systems**

Protein degradation is an integral part in the maintenance of cellular homeostasis. It allows for the selective removal of host proteins that are misfolded, damaged, and unnecessary, and balances the ongoing protein synthesis that is driven to provide the desirable cellular function in response to environmental changes. The dynamic interaction between protein synthesis and degradation is of particular importance to cardiomyocytes, the basic contractile units of the heart, due to the plasticity of the heart. It drives the hypertrophy or atrophy of individual cardiomyocytes, which contributes to sufficient contractile force generation that meets the hemodynamic demands. Consequently, the dysregulation of protein degradation jeopardizes cardiomyocyte vitality and function and plays an important role in the development of various cardiac diseases (Zheng & Wang, 2010).

Enteroviral infection leads to the dysregulation of host protein degradation pathways, which include the ubiquitin/proteasome system (UPS) and autophagy. Such viral-induced manipulation plays key roles not only in viral propagation, but also in the pathogenesis of viral myocarditis and the subsequent development of DCM. The following sections discuss the contributions of UPS and autophagy in viral propagation and the development of cardiomyopathy.

#### **3.2 The ubiquitin/proteasome system**

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotic cells that accounts for ~80% of host protein recycling (Zheng & Wang, 2010). By controlling the longevity/half-life of most proteins (predominantly short-lived but also some long-lived proteins), the UPS extends its role beyond protein recycling and regulates most aspects of cellular functions. UPS substrate selectivity is achieved by protein polyubiquitination, i.e. the attachment of ubiquitin (Ub) molecules onto the target protein. Ubiquitin is a small 76 amino acid protein modifier molecule that conjugates to target protein or to another Ub through one of its seven lysine residues. Ubiquitin linkage at different lysine residues serves different functions. For example, polyubiquitination via lysine 48 targets protein for UPS degradation, polyubiquitination via lysine 63 promotes signal transduction or targets degradation through the autophagy pathway, whereas monoubiquitination modulates protein intracellular localization and protein function.

Protein ubiquitination is regulated in a multi-step manner (Hershko & Ciechanover, 1998) (Fig. 2A). First, Ub is activated by the ubiquitin-activating enzyme (E1) using ATP. Then, it is transferred to ubiquitin-conjugating enzyme (E2). Finally, it is conjugated onto the target protein selectively brought in by the ubiquitin ligase (E3). A polyUb chain is formed by repeating the ubiquitination process. The expression of these important enzymes that regulate protein ubiquitination may change according to physiological stimuli. For instance, in cardiac atrophy mouse model, the E2 enzyme UbcH2 expression is upregulated in atrophic hearts to increase the capacity of protein degradation (Razeghi *et al.*, 2006).

The 26S proteasome is composed of the 20S proteolytic core and the 19S proteasome activator lid(s) (Luo *et al.*, 2010). The 20S proteolytic core is made up of two outer (αsubunits) and two inner (β-subunits) rings. It contains caspase-like, trypsin-like, and chymotrypsin-like protease activities conveyed by subunits β1, β2, and β5, respectively. The 19S lid(s), also known as proteasome activator 700 (PA700), helps the recognition and

**3. Dysregulation of host protein degradation systems in viral cardiomyopathy** 

Protein degradation is an integral part in the maintenance of cellular homeostasis. It allows for the selective removal of host proteins that are misfolded, damaged, and unnecessary, and balances the ongoing protein synthesis that is driven to provide the desirable cellular function in response to environmental changes. The dynamic interaction between protein synthesis and degradation is of particular importance to cardiomyocytes, the basic contractile units of the heart, due to the plasticity of the heart. It drives the hypertrophy or atrophy of individual cardiomyocytes, which contributes to sufficient contractile force generation that meets the hemodynamic demands. Consequently, the dysregulation of protein degradation jeopardizes cardiomyocyte vitality and function and plays an important

Enteroviral infection leads to the dysregulation of host protein degradation pathways, which include the ubiquitin/proteasome system (UPS) and autophagy. Such viral-induced manipulation plays key roles not only in viral propagation, but also in the pathogenesis of viral myocarditis and the subsequent development of DCM. The following sections discuss the contributions of UPS and autophagy in viral propagation and the development of

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotic cells that accounts for ~80% of host protein recycling (Zheng & Wang, 2010). By controlling the longevity/half-life of most proteins (predominantly short-lived but also some long-lived proteins), the UPS extends its role beyond protein recycling and regulates most aspects of cellular functions. UPS substrate selectivity is achieved by protein polyubiquitination, i.e. the attachment of ubiquitin (Ub) molecules onto the target protein. Ubiquitin is a small 76 amino acid protein modifier molecule that conjugates to target protein or to another Ub through one of its seven lysine residues. Ubiquitin linkage at different lysine residues serves different functions. For example, polyubiquitination via lysine 48 targets protein for UPS degradation, polyubiquitination via lysine 63 promotes signal transduction or targets degradation through the autophagy pathway, whereas monoubiquitination modulates

Protein ubiquitination is regulated in a multi-step manner (Hershko & Ciechanover, 1998) (Fig. 2A). First, Ub is activated by the ubiquitin-activating enzyme (E1) using ATP. Then, it is transferred to ubiquitin-conjugating enzyme (E2). Finally, it is conjugated onto the target protein selectively brought in by the ubiquitin ligase (E3). A polyUb chain is formed by repeating the ubiquitination process. The expression of these important enzymes that regulate protein ubiquitination may change according to physiological stimuli. For instance, in cardiac atrophy mouse model, the E2 enzyme UbcH2 expression is upregulated in

The 26S proteasome is composed of the 20S proteolytic core and the 19S proteasome activator lid(s) (Luo *et al.*, 2010). The 20S proteolytic core is made up of two outer (αsubunits) and two inner (β-subunits) rings. It contains caspase-like, trypsin-like, and chymotrypsin-like protease activities conveyed by subunits β1, β2, and β5, respectively. The 19S lid(s), also known as proteasome activator 700 (PA700), helps the recognition and

atrophic hearts to increase the capacity of protein degradation (Razeghi *et al.*, 2006).

role in the development of various cardiac diseases (Zheng & Wang, 2010).

**3.1 Protein degradation systems** 

cardiomyopathy.

**3.2 The ubiquitin/proteasome system** 

protein intracellular localization and protein function.

docking of polyubiquitinated target protein. 19S also serves to detach and hence recycle the Ub by its deubiquitinating enzyme (DUB) activity. Furthermore, 19S unfolds the target protein and feeds it to the 20S core for degradation.

The immunoproteasome is an alternative version of the proteasome expressed to accommodate inflammatory responses upon stimulation with interferon- (Rivett & Hearn, 2004). The immunoproteasome has a 20S core that substitutes the constitutive catalytic βsubunits with inducible β-counterparts (β1i, β2i, and β5i), which offer different proteolytic function and activity to generate small peptides suitable for antigen presentation by major histocompatibility complex (MHC) class I (Griffin *et al.*, 1998) (Fig. 2A). In addition to the 19S proteasome, the immunoproteasome can also have a different lid(s) – the 11S proteasome, also known as PA28 (proteasome activator 28). Different compositions of 11S exist: heteroheptamer of PA28 and PA28 that are induced by interferon- under intensified immune response (Murray *et al.*, 2000) and homoheptamer of PA28 that resides in the nucleus and assists ATP- and ubiquitin-independent proteasomal activity (Mao *et al.*, 2008). Sometimes, hybrid proteasomes with both 11S and 19S lids are also observed. However, their functions remain to be explored.

#### **3.2.1 The UPS and heart diseases**

UPS dysregulation is a common phenomenon in heart diseases. It is accentuated with the accumulation of Ub-protein conjugates and is associated with markedly reduced proteasome proteolytic activity in failing human hearts as compared to non-failing hearts (Predmore *et al.*, 2010). This suggests that ubiquitinated proteins in hearts are not degraded due to impaired proteasomal function. While no changes were noted in protein expression of proteasome subunits (i.e. 20S, 19S, 11S), elevated levels of protein carbonyls and 4-hydroxynonenylated proteins were observed in failing hearts. Also, oxidative modification to the 19S ATPase subunit Rpt5 was found in these failing hearts. Together, these oxidative modifications to proteasome subunits and substrate proteins may lead to impaired proteasomal function. On the other hand, microarray studies demonstrate reduced transcript levels of some 20s α- and β-subunits in the failing hearts as compared to controls (Hwang *et al.*, 2002; Kaab *et al.*, 2004). The incongruence between protein and mRNA expression of proteasome subunits may be attributed to myocyte loss and fibrosis in the failing hearts.

Animal models of cardiac diseases also have an increased ubiquitinated protein expression, but are acommpanied with changes in their proteasome expression profile. Upregulation in protein expression of proteasome subunits was observed in a left ventricular hypertrophy mouse model (Depre *et al.*, 2006). Post-translational modifications of the proteasome subunits were also reported in these hypertrophic hearts (Depre *et al.*, 2006). Treatment with proteasome inhibitor effectively prevents cardiac hypertrophy development, suggesting that the upregulation of proteasome expression is central to this physiological adaptation. Similar beneficial effects of proteasome inhibition in the regression of cardiac hypertrophy were observed in other studies (Meiners *et al.*, 2008; Stansfield *et al.*, 2008). Besides hypertrophic cardiomyopathy, the accumulation of Ub-conjugated proteins was observed in hyperglycemia-induced cardiomyopathy mouse model (Powell *et al.*, 2008). A parallel drop in the basal ATP-dependent proteasomal activity was observed in these mice. However, an increased ATP-independent chymotryptic proteasomal activity was observed, which is accompanied by an increased expression of 11S lid subunits PA28 and PA28, as well as

Impaired Cardiac Function in Viral Myocarditis 301

The other host protein degradation system is autophagy, i.e. "self-eating". It proceeds by the engulfing of a portion of the cytoplasm including long-lived and misfolded proteins and organelles by the autophagic membranes to form double-membraned vesicles called autophagosomes, followed by their delivery to lysosomes for degradation (Levine & Kroemer, 2008) (Fig. 2B). Autophagy is activated in two parallel cascades of enzymatic actions that are similar to the process of protein ubiquitination (Ravikumar *et al.*, 2010). First, Atg12 (autophagy-related gene 12) and Atg8 (also known as LC3, microtubule-associated protein light chain 3, in mammalian cells) are activated by the E1-like activating enzyme Atg7 using ATP. Then, Atg12 is conjugated to its E2 Atg10, while Atg8 is attached to another E2 Atg3. Atg12 is then transferred to its designated partner Atg5 forming the Atg12- Atg5 complex and further matures by the conjugation to Atg16. Finally, the Atg12-Ag5- Atg16 complex acts as an E3 to help Atg8 lipidation, forming the Atg8-PE (phosphatidylethanolamine) complex. Lipidation of Atg8 helps its incorporation onto the autophagic membrane. Atg8-PE then takes part in the elongation of the autophagic

Under baseline conditions, autophagy represents an important homeostatic mechanism. However, excessive activation of the autophagy machinery has been suggested to be involved in the pathogenesis of various disease conditions, including cardiac diseases. LC3 activation was observed early and was well-sustained in pressure-overload cardiomyopathy mouse model (Zhu *et al.*, 2007). Autophagy activation in this model promotes cardiac remodeling. Overexpression of Beclin-1, also known as Atg6, accentuates pathological remodeling and interstitial fibrosis, whereas heterozygous knockout of Beclin-1 improves systolic function and delays cardiac remodeling. In desmin-mediated cardiomyopathy mouse model, early activation of autophagy was observed well before any measurable decline in cardiac function (Tannous *et al.*, 2008). Autophagy pathway impairment by heterozygous inactivation of Beclin-1 leads to accumulation of polyubiquitinated protein aggregates, as well as acceleration to heart failure and early mortality (Tannous *et al.*, 2008). Similarly, autophagy is activated in both ischemia and subsequent reperfusion, but via two different initiation pathways (Takagi *et al.*, 2007). The AMPK pathway drives autophagy during ischemia, while Beclin-1 initiates autophagy upon reperfusion. Autophagy during ischemia is considered a cell survival response as it helps to sustain the starved cardiomyocytes during ischemia; however, autophagy during reperfusion is viewed as a

Autophagy also plays an important part in the host innate defense system by direct sequestration of invading pathogens (bacteria, fungi, and virus) for clearance through lysosomal degradation (Jackson *et al.*, 2005). In addition, autophagy helps antigen presentation to class II MHC in order to mount an adaptive immune response (Dengjel *et al.*, 2005). However, this innate defense machinery is subverted by certain viruses to facilitate their replication (Jackson *et al.*, 2005; Wong *et al.*, 2008). It was shown that LC3-PE expression, a hallmark of autophagosome formation, is increased after CVB3 infection with dramatic reorganization of intracellular membranes (Wong *et al.*, 2008) (Fig. 2B). Inhibition

membrane and its enclosure to form the autophagosome.

pathological response as it promotes autophagic cell death.

**3.3.2 Autophagy and viral myocarditis** 

**3.3.1 Autophagy and cardiac diseases** 

**3.3 Autophagy** 

the 20S subunits 3 and 5. Together, these data suggest a shift to immunoproteasomal activity is induced under hyperglycemic stress conditions to help the degradation of accumulated proteins.

The difference in cardiac proteasome expression profiles between human heart failure and various cardiomyopathy mouse models can be attributed to the limited time frame of animal studies. It is likely that stressed human hearts also induce the compensatory upregulation of proteasomal expression and activity at the earlier disease stages, but fail to maintain these changes over time.

#### **3.2.2 The UPS and viral myocarditis**

The UPS is also dysregulated in viral myocarditis (Fig. 2A). Accumulation of Ub-protein conjugates, as in other cardiomyopathies, was observed in CVB3-infected mouse hearts and cultured cells (Luo *et al.*, 2003; Gao *et al.*, 2008; Si *et al.*, 2008). The expression of several enzymes in the UPS pathway such as E1 enzyme E1A/E1B, E2 enzyme UbcH7, and DUB UCHL1 (ubiquitin carboxyl-terminal hydrolase L1) is upregulated in CVB3-infected mouse hearts, while ATP-dependent proteasomal activity is unaltered (Gao *et al.*, 2008). *In vitro* application of proteasome inhibitors such as MG132 (Luo *et al.*, 2003), lactacystin (Luo *et al.*, 2003), pyrrolidine dithiocarbamate (PDTC) (Si *et al.*, 2005), and curcumin (Si *et al.*, 2007) effectively attenuates viral RNA replication and protein synthesis. In addition, depletion of Ub by RNA interference also inhibits viral replication (Si *et al.*, 2008). Furthermore, it was shown that viral RNA-dependent polymerase 3D is ubiquitinated during viral replication, which may help its anchorage to intracellular membrane platforms required for the assembly of viral RNA replication machinery (Si *et al.*, 2008). *In vivo* administration of proteasome inhibitor to CVB3-infected mice also improves the outcome of viral myocarditis with reduced myocardial damage and inflammatory infiltration (Gao *et al.*, 2008). The viral titer, however, is not significantly reduced in the treated mice. This suggests that proteasome inhibitor treatment ameliorates viral myocarditis via multiple mechanisms: direct viral inhibition and immunomodulation. It was further demonstrated that the expression of 11S subunit PA28 plays a role in CVB3 replication (Gao *et al.*, 2010). CVB3 infection leads to the redistribution of PA28 from the nucleus to the cytosol, where it interacts with host proteins, such as tumor suppressor protein p53, and promotes their degradation via UPS. Overexpression of PA28 enhances viral replication while its knockdown does the opposite.

Szalay et al. explored the involvement of the immunoproteasome in viral myocarditis. They found that the catalytic subunits of the immunoproteasome, LMP2 (β1i), LMP7 (β5i), and MECL-1 (β2i), are upregulated in CVB3-infected myocarditis-susceptible mouse hearts as compared to infected hearts from resistant mouse strains (Szalay *et al.*, 2006). Increased activity of the immunoproteasome in the susceptible myocardium helps generate the MHC class I-restricted peptide, boost antigen presentation and mount the subsequent adaptive immune response. A recent study demonstrates a differential immunoproteasome expression pattern between myocarditis-susceptible and -resistant mouse strains (Jakel *et al.*, 2009). In this study, immunoproteasome formation peaks early after CVB3 challenge in resistant mice, while it is postponed and expressed in greater extent in susceptible mice. The timing and magnitude of immunoproteasome activation determine in part the effectiveness of early viral clearance and the extent of direct viral-mediated damages, as well as the injury incurred during adaptive immune responses.

#### **3.3 Autophagy**

300 Myocarditis

the 20S subunits 3 and 5. Together, these data suggest a shift to immunoproteasomal activity is induced under hyperglycemic stress conditions to help the degradation of

The difference in cardiac proteasome expression profiles between human heart failure and various cardiomyopathy mouse models can be attributed to the limited time frame of animal studies. It is likely that stressed human hearts also induce the compensatory upregulation of proteasomal expression and activity at the earlier disease stages, but fail to

The UPS is also dysregulated in viral myocarditis (Fig. 2A). Accumulation of Ub-protein conjugates, as in other cardiomyopathies, was observed in CVB3-infected mouse hearts and cultured cells (Luo *et al.*, 2003; Gao *et al.*, 2008; Si *et al.*, 2008). The expression of several enzymes in the UPS pathway such as E1 enzyme E1A/E1B, E2 enzyme UbcH7, and DUB UCHL1 (ubiquitin carboxyl-terminal hydrolase L1) is upregulated in CVB3-infected mouse hearts, while ATP-dependent proteasomal activity is unaltered (Gao *et al.*, 2008). *In vitro* application of proteasome inhibitors such as MG132 (Luo *et al.*, 2003), lactacystin (Luo *et al.*, 2003), pyrrolidine dithiocarbamate (PDTC) (Si *et al.*, 2005), and curcumin (Si *et al.*, 2007) effectively attenuates viral RNA replication and protein synthesis. In addition, depletion of Ub by RNA interference also inhibits viral replication (Si *et al.*, 2008). Furthermore, it was shown that viral RNA-dependent polymerase 3D is ubiquitinated during viral replication, which may help its anchorage to intracellular membrane platforms required for the assembly of viral RNA replication machinery (Si *et al.*, 2008). *In vivo* administration of proteasome inhibitor to CVB3-infected mice also improves the outcome of viral myocarditis with reduced myocardial damage and inflammatory infiltration (Gao *et al.*, 2008). The viral titer, however, is not significantly reduced in the treated mice. This suggests that proteasome inhibitor treatment ameliorates viral myocarditis via multiple mechanisms: direct viral inhibition and immunomodulation. It was further demonstrated that the expression of 11S subunit PA28 plays a role in CVB3 replication (Gao *et al.*, 2010). CVB3 infection leads to the redistribution of PA28 from the nucleus to the cytosol, where it interacts with host proteins, such as tumor suppressor protein p53, and promotes their degradation via UPS. Overexpression of PA28 enhances viral replication while its

Szalay et al. explored the involvement of the immunoproteasome in viral myocarditis. They found that the catalytic subunits of the immunoproteasome, LMP2 (β1i), LMP7 (β5i), and MECL-1 (β2i), are upregulated in CVB3-infected myocarditis-susceptible mouse hearts as compared to infected hearts from resistant mouse strains (Szalay *et al.*, 2006). Increased activity of the immunoproteasome in the susceptible myocardium helps generate the MHC class I-restricted peptide, boost antigen presentation and mount the subsequent adaptive immune response. A recent study demonstrates a differential immunoproteasome expression pattern between myocarditis-susceptible and -resistant mouse strains (Jakel *et al.*, 2009). In this study, immunoproteasome formation peaks early after CVB3 challenge in resistant mice, while it is postponed and expressed in greater extent in susceptible mice. The timing and magnitude of immunoproteasome activation determine in part the effectiveness of early viral clearance and the extent of direct viral-mediated damages, as well as the injury

accumulated proteins.

maintain these changes over time.

knockdown does the opposite.

incurred during adaptive immune responses.

**3.2.2 The UPS and viral myocarditis** 

The other host protein degradation system is autophagy, i.e. "self-eating". It proceeds by the engulfing of a portion of the cytoplasm including long-lived and misfolded proteins and organelles by the autophagic membranes to form double-membraned vesicles called autophagosomes, followed by their delivery to lysosomes for degradation (Levine & Kroemer, 2008) (Fig. 2B). Autophagy is activated in two parallel cascades of enzymatic actions that are similar to the process of protein ubiquitination (Ravikumar *et al.*, 2010). First, Atg12 (autophagy-related gene 12) and Atg8 (also known as LC3, microtubule-associated protein light chain 3, in mammalian cells) are activated by the E1-like activating enzyme Atg7 using ATP. Then, Atg12 is conjugated to its E2 Atg10, while Atg8 is attached to another E2 Atg3. Atg12 is then transferred to its designated partner Atg5 forming the Atg12- Atg5 complex and further matures by the conjugation to Atg16. Finally, the Atg12-Ag5- Atg16 complex acts as an E3 to help Atg8 lipidation, forming the Atg8-PE (phosphatidylethanolamine) complex. Lipidation of Atg8 helps its incorporation onto the autophagic membrane. Atg8-PE then takes part in the elongation of the autophagic membrane and its enclosure to form the autophagosome.

#### **3.3.1 Autophagy and cardiac diseases**

Under baseline conditions, autophagy represents an important homeostatic mechanism. However, excessive activation of the autophagy machinery has been suggested to be involved in the pathogenesis of various disease conditions, including cardiac diseases. LC3 activation was observed early and was well-sustained in pressure-overload cardiomyopathy mouse model (Zhu *et al.*, 2007). Autophagy activation in this model promotes cardiac remodeling. Overexpression of Beclin-1, also known as Atg6, accentuates pathological remodeling and interstitial fibrosis, whereas heterozygous knockout of Beclin-1 improves systolic function and delays cardiac remodeling. In desmin-mediated cardiomyopathy mouse model, early activation of autophagy was observed well before any measurable decline in cardiac function (Tannous *et al.*, 2008). Autophagy pathway impairment by heterozygous inactivation of Beclin-1 leads to accumulation of polyubiquitinated protein aggregates, as well as acceleration to heart failure and early mortality (Tannous *et al.*, 2008). Similarly, autophagy is activated in both ischemia and subsequent reperfusion, but via two different initiation pathways (Takagi *et al.*, 2007). The AMPK pathway drives autophagy during ischemia, while Beclin-1 initiates autophagy upon reperfusion. Autophagy during ischemia is considered a cell survival response as it helps to sustain the starved cardiomyocytes during ischemia; however, autophagy during reperfusion is viewed as a pathological response as it promotes autophagic cell death.

#### **3.3.2 Autophagy and viral myocarditis**

Autophagy also plays an important part in the host innate defense system by direct sequestration of invading pathogens (bacteria, fungi, and virus) for clearance through lysosomal degradation (Jackson *et al.*, 2005). In addition, autophagy helps antigen presentation to class II MHC in order to mount an adaptive immune response (Dengjel *et al.*, 2005). However, this innate defense machinery is subverted by certain viruses to facilitate their replication (Jackson *et al.*, 2005; Wong *et al.*, 2008). It was shown that LC3-PE expression, a hallmark of autophagosome formation, is increased after CVB3 infection with dramatic reorganization of intracellular membranes (Wong *et al.*, 2008) (Fig. 2B). Inhibition

A.

E1 E2 E3

E1A/E1B (E1) UbcH7 (E2) UCHL1 (DUB)

ATP

Ubiquitin

Isolation membrane

been shown to block viral replication *in vitro*.

B.

Impaired Cardiac Function in Viral Myocarditis 303

Monoubiquitination: Functional modulation

Polyubiquitination

26S Proteasome

β1 β2 β5 β1i β2i β5i

Autolysosome

Fig. 2. Dysregulation of the host protein degradation systems in viral cardiomyopathy. The ubiquitin/proteasome system (UPS) and autophagy are the two major protein

reactions (El - ubiquitin activating enzyme, E2 - ubiquitin conjugating enzyme, E3 -

degradation mechanisms in eukaryotic cells. A. The UPS function by a cascade of enzymatic

ubiquitin ligase) that conjugate ubiquitin, a small protein modifier, onto the target proteins. The type of ubiquitin conjugation linkage determines the target protein's fate: functional modulation or degradation. Polyubiquitinated target proteins are recognized by the 26S proteasome for degradation, whereas monoubiquitination serves to help endocytosis, endosomal sorting, DNA repair, histone regulation, and nuclear export. Ubiquitins are recycled by the deubiquitinating enzymes (DUBs). CVB3 infection causes the dysregulation of the UPS. An increased expression of ubiquitin-protein conjugates, E1A/E1B (El), UbcH7 (E2), UCH-L1 (DUB) was observed in CVB3-infected mouse hearts. Proteasome inhibitor application attenuates viral replication *in vitro* and reduces myocardial lesion and fibrosis *in vivo*. B. Autophagy begins with the enwrapping of organelles and cytoplasmic proteins by the isolation membrane which elongates and encloses to form a double-membraned vesicle called the autophagosome. The autophagosome fuses with lysosomes to degrade the sequestrated materials. Autophagy plays an important role in host defense by trapping and degrading invading pathogens. However, certain viruses including CVB3 evolve strategies to subvert autophagic mechanism to facilitate their own replication. Autophagosome formation is upregulated during CVB3 infection. Inhibition of the autophagy pathway has

Degradation

DUB

Lysosome

Autophagosome

Ub-proteins

3D ubiquitination

Interferon Immunoproteasome

LC3-PE Autophagosomes

Degradation

β1i β2i β5i

of autophagy by 3-methyladenine which targets the upstream signaling class III PI3-kinase, and by siRNA knockdown of Atg7 expression effectively block viral replication (Wong *et al.*, 2008). Recent work in mouse models also suggests that autophagy is activated *in vivo* after CVB3 infection (unpublished data). LC3-PE expression is elevated in CVB3-infected organs such as the heart, liver, and pancreas. Kemball *et al.* also reported the induction of autophagosome formation in pancreatic acinar cells in CVB3-infected mice (Kemball *et al.*, 2010). This theme of virus-induced autophagy activation is further extended to coxsackievirus B4-infected rat primary neurons (Yoon *et al.*, 2008). Nonetheless, virusinduced autophagy only serves to help viral replication without increasing protein degradation as suggested by the unchanged expression level of p62, a selective autophagy substrate, after virus infection (Wong *et al.*, 2008).

Subversion of the autophagy machinery by enteroviruses may contribute to the pathogenesis of viral myocarditis beyond impacting cardiomyocyte viability. Recent research demonstrates that cellular autophagy plays a role in nucleic acid-sensing toll-like receptor 3 (TLR3) signaling, which is necessary for the antiviral interferon pathway (Gorbea *et al.*, 2010). TLR3-deficient mice show vulnerability to CVB3 infection and develop acute myocarditis (Negishi *et al.*, 2008). Dysregulation of the autophagy pathway in CVB3-infected cardiomyocytes may interfere with TLR3-mediated antiviral response, resulting in compromised viral clearance and increased myocardial damage during viremia. In addition, autophagy is known to be a pro-survival response against apoptosis. The dysregulation of autophagy may decrease the viability of virus-infected cardiomyocytes because it cannot protect the host from virus-induced apoptosis. Furthermore, angiotensin II receptors type I & II (AT1 & AT2) regulate cardiomyocyte autophagy activity (Porrello *et al.*, 2009). AT1 expression triggers autophagy in neonatal cardiomyocytes as well as subsequent autophagic cell death, while AT2 expression counteracts AT1-induced autophagic activity. Further modulation by angiotensins may have an adverse effect on virus-infected cardiomyocytes as it may further activate autophagy, thus triggering autophagic cell death.

### **4. Potential therapeutics targeting viral proteases, UPS, and autophagy**

The current knowledge of the roles of viral proteases and the host protein degradation systems in viral myocarditis may lead to new diagnostic and therapeutic approaches for the disease. Virus-induced SRF cleavage fragments may be utilized as a biomarker to detect acute phase myocarditis. Early diagnosis of viral myocarditis opens up the optimal timeframe for treatment. Successful medical interventions during acute infection can limit viral replication and its associated damage, limit viral spreading, as well as minimize the damage caused by immune activation. Since the viral proteases and the protein degradation systems all play important roles in viral propagation, a combinatorial therapy of highly specific viral protease inhibitors, proteasome inhibitors, and autophagy inhibitors during viremia would limit viral infection effectively. Furthermore, application of proteasome inhibitors and autophagy inhibitors provides additional benefits in immunomodulation to control the inflammatory response. On the other hand, viral myocarditis patients in the chronic phases may be managed differently. Since DCM patients have depressed proteasomal function, proteasome inhibitor treatment may further exacerbate myocardial damage. Moreover, long-term application of inhibitors against UPS will have adverse effects as demonstrated in the increased incidence of heart failure in cancer patients undergoing proteasome inhibitor treatment (Enrico *et al.*, 2007; Hacihanefioglu *et al.*, 2008).

of autophagy by 3-methyladenine which targets the upstream signaling class III PI3-kinase, and by siRNA knockdown of Atg7 expression effectively block viral replication (Wong *et al.*, 2008). Recent work in mouse models also suggests that autophagy is activated *in vivo* after CVB3 infection (unpublished data). LC3-PE expression is elevated in CVB3-infected organs such as the heart, liver, and pancreas. Kemball *et al.* also reported the induction of autophagosome formation in pancreatic acinar cells in CVB3-infected mice (Kemball *et al.*, 2010). This theme of virus-induced autophagy activation is further extended to coxsackievirus B4-infected rat primary neurons (Yoon *et al.*, 2008). Nonetheless, virusinduced autophagy only serves to help viral replication without increasing protein degradation as suggested by the unchanged expression level of p62, a selective autophagy

Subversion of the autophagy machinery by enteroviruses may contribute to the pathogenesis of viral myocarditis beyond impacting cardiomyocyte viability. Recent research demonstrates that cellular autophagy plays a role in nucleic acid-sensing toll-like receptor 3 (TLR3) signaling, which is necessary for the antiviral interferon pathway (Gorbea *et al.*, 2010). TLR3-deficient mice show vulnerability to CVB3 infection and develop acute myocarditis (Negishi *et al.*, 2008). Dysregulation of the autophagy pathway in CVB3-infected cardiomyocytes may interfere with TLR3-mediated antiviral response, resulting in compromised viral clearance and increased myocardial damage during viremia. In addition, autophagy is known to be a pro-survival response against apoptosis. The dysregulation of autophagy may decrease the viability of virus-infected cardiomyocytes because it cannot protect the host from virus-induced apoptosis. Furthermore, angiotensin II receptors type I & II (AT1 & AT2) regulate cardiomyocyte autophagy activity (Porrello *et al.*, 2009). AT1 expression triggers autophagy in neonatal cardiomyocytes as well as subsequent autophagic cell death, while AT2 expression counteracts AT1-induced autophagic activity. Further modulation by angiotensins may have an adverse effect on virus-infected cardiomyocytes as

it may further activate autophagy, thus triggering autophagic cell death.

**4. Potential therapeutics targeting viral proteases, UPS, and autophagy** 

proteasome inhibitor treatment (Enrico *et al.*, 2007; Hacihanefioglu *et al.*, 2008).

The current knowledge of the roles of viral proteases and the host protein degradation systems in viral myocarditis may lead to new diagnostic and therapeutic approaches for the disease. Virus-induced SRF cleavage fragments may be utilized as a biomarker to detect acute phase myocarditis. Early diagnosis of viral myocarditis opens up the optimal timeframe for treatment. Successful medical interventions during acute infection can limit viral replication and its associated damage, limit viral spreading, as well as minimize the damage caused by immune activation. Since the viral proteases and the protein degradation systems all play important roles in viral propagation, a combinatorial therapy of highly specific viral protease inhibitors, proteasome inhibitors, and autophagy inhibitors during viremia would limit viral infection effectively. Furthermore, application of proteasome inhibitors and autophagy inhibitors provides additional benefits in immunomodulation to control the inflammatory response. On the other hand, viral myocarditis patients in the chronic phases may be managed differently. Since DCM patients have depressed proteasomal function, proteasome inhibitor treatment may further exacerbate myocardial damage. Moreover, long-term application of inhibitors against UPS will have adverse effects as demonstrated in the increased incidence of heart failure in cancer patients undergoing

substrate, after virus infection (Wong *et al.*, 2008).

Fig. 2. Dysregulation of the host protein degradation systems in viral cardiomyopathy. The ubiquitin/proteasome system (UPS) and autophagy are the two major protein degradation mechanisms in eukaryotic cells. A. The UPS function by a cascade of enzymatic reactions (El - ubiquitin activating enzyme, E2 - ubiquitin conjugating enzyme, E3 ubiquitin ligase) that conjugate ubiquitin, a small protein modifier, onto the target proteins. The type of ubiquitin conjugation linkage determines the target protein's fate: functional modulation or degradation. Polyubiquitinated target proteins are recognized by the 26S proteasome for degradation, whereas monoubiquitination serves to help endocytosis, endosomal sorting, DNA repair, histone regulation, and nuclear export. Ubiquitins are recycled by the deubiquitinating enzymes (DUBs). CVB3 infection causes the dysregulation of the UPS. An increased expression of ubiquitin-protein conjugates, E1A/E1B (El), UbcH7 (E2), UCH-L1 (DUB) was observed in CVB3-infected mouse hearts. Proteasome inhibitor application attenuates viral replication *in vitro* and reduces myocardial lesion and fibrosis *in vivo*. B. Autophagy begins with the enwrapping of organelles and cytoplasmic proteins by the isolation membrane which elongates and encloses to form a double-membraned vesicle called the autophagosome. The autophagosome fuses with lysosomes to degrade the sequestrated materials. Autophagy plays an important role in host defense by trapping and degrading invading pathogens. However, certain viruses including CVB3 evolve strategies to subvert autophagic mechanism to facilitate their own replication. Autophagosome formation is upregulated during CVB3 infection. Inhibition of the autophagy pathway has been shown to block viral replication *in vitro*.

Impaired Cardiac Function in Viral Myocarditis 305

Drory Y, Turetz Y, Hiss Y, Lev B, Fisman EZ, Pines A & Kramer MR. (1991). Sudden unexpected death in persons less than 40 years of age. *Am J Cardiol* 68, 1388-1392. Enrico O, Gabriele B, Nadia C, Sara G, Daniele V, Giulia C, Antonio S & Mario P. (2007).

Esfandiarei M & McManus BM. (2008). Molecular biology and pathogenesis of viral

Ferlini A, Sewry C, Melis MA, Mateddu A & Muntoni F. (1999). X-linked dilated cardiomyopathy and the dystrophin gene. *Neuromuscul Disord* 9, 339-346. Froeschle JE, Feorino PM & Gelfand HM. (1966). A continuing surveillance of enterovirus

Gao G, Wong J, Zhang J, Mao I, Shravah J, Wu Y, Xiao A, Li X & Luo H. (2010). Proteasome

Gao G, Zhang J, Si X, Wong J, Cheung C, McManus B & Luo H. (2008). Proteasome

Gorbea C, Makar KA, Pauschinger M, Pratt G, Bersola JL, Varela J, David RM, Banks L,

Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS & Sanes JR. (1997). Skeletal

Griffin TA, Nandi D, Cruz M, Fehling HJ, Kaer LV, Monaco JJ & Colbert RA. (1998).

Grist NR & Reid D. (1997). Organisms in myocarditis/endocarditis viruses. *J Infect* 34, 155. Hacihanefioglu A, Tarkun P & Gonullu E. (2008). Acute severe cardiac failure in a myeloma patient due to proteasome inhibitor bortezomib. *Int J Hematol* 88, 219-222. Hershko A & Ciechanover A. (1998). The ubiquitin system. *Annu Rev Biochem* 67, 425-479. Huber M, Watson KA, Selinka HC, Carthy CM, Klingel K, McManus BM & Kandolf R.

kinase in the course of coxsackievirus B3 replication. *J Virol* 73, 3587-3594. Hwang JJ, Allen PD, Tseng GC, Lam CW, Fananapazir L, Dzau VJ & Liew CC. (2002).

central nervous system disease. *Am J Epidemiol* 83, 455-469.

and dilated cardiomyopathy. *J Biol Chem* 285, 23208-23223.

(IFN-gamma)-inducible subunits. *J Exp Med* 187, 97-104.

Duchenne muscular dystrophy. *Cell* 90, 729-738.

end-stage heart failure. *Physiol Genomics* 10, 31-44.

intracellular source proteins. *Proc Natl Acad Sci U S A* 102, 7922-7927. Depre C, Wang Q, Yan L, Hedhli N, Peter P, Chen L, Hong C, Hittinger L, Ghaleh B,

*Circulation* 114, 1821-1828.

*Haematol* 138, 396-397.

myocarditis. *Annu Rev Pathol* 3, 127-155.

degradation. *J Virol* 84, 11056-11066.

*Physiol Heart Circ Physiol* 295, H401-408.

(2005). Autophagy promotes MHC class II presentation of peptides from

Sadoshima J, Vatner DE, Vatner SF & Madura K. (2006). Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy.

Unexpected cardiotoxicity in haematological bortezomib treated patients. *Br J* 

infection in healthy children in six United States cities. II. Surveillance enterovirus isolates 1960-1963 and comparison with enterovirus isolates from cases of acute

activator REGgamma enhances coxsackieviral infection by facilitating p53

inhibition attenuates coxsackievirus-induced myocardial damage in mice. *Am J* 

Huang CH, Li H, Schultheiss HP, Towbin JA, Vallejo JG & Bowles NE. (2010). A role for Toll-like receptor 3 variants in host susceptibility to enteroviral myocarditis

and cardiac myopathies in mice lacking utrophin and dystrophin: a model for

Immunoproteasome assembly: cooperative incorporation of interferon gamma

(1999). Cleavage of RasGAP and phosphorylation of mitogen-activated protein

Microarray gene expression profiles in dilated and hypertrophic cardiomyopathic
