J. of Cardiovasc. Trans. Res. (2014) 7:212–225 DOI 10.1007/s12265-013-9528-2

Targeting Matrix Metalloproteinase Activity and Expression for the Treatment of Viral Myocarditis Reid G. Hendry & Leanne M. Bilawchuk & David J. Marchant

Received: 9 August 2013 / Accepted: 29 November 2013 / Published online: 1 January 2014 # Springer Science+Business Media New York 2014

Abstract Infectious agents including viruses can infect the heart muscle, resulting in the development of heart inflammation called myocarditis. Chronic myocarditis can lead to dilated cardiomyopathy (DCM). DCM develops from the extensive extracellular matrix (ECM) remodeling caused by myocarditis and may result in heart failure. Epidemiological data for viral myocarditis has long suggested a worse pathology in males, with more recent data demonstrating sex-dependent pathogenesis in DCM as well. Matrix metalloproteinases (MMPs), long known modulators of the extracellular matrix, have important roles in mediating heart inflammation and remodeling during disease and in convalescence. This ability of MMPs to control both the inflammatory response and ECM remodeling during myocarditis makes them potential drug targets. In this review, we analyze the role of MMPs in mediating myocarditis/DCM disease progression, their sexdependent expression, and their potential as drug targets during viral myocarditis and DCM. Keywords Matrix metalloproteinases . Viral myocarditis . Gender . Inhibitor

Introduction Myocarditis Heart disease is the number one killer in the USA and is responsible for one in every three deaths with cardiomyopathies being responsible for 24,000 of these deaths annually [1]. One type of cardiomyopathy, myocarditis, is inflammation of R. G. Hendry : L. M. Bilawchuk : D. J. Marchant (*) Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada e-mail: [email protected]

the heart muscle and it is caused by infection or an autoimmune response [2]. The infectious agents that trigger myocarditis range from viruses, bacteria, and parasites to toxins or allergens [3]. Some of the most common causes of viral myocarditis are the enteroviruses, such as coxsackievirus type B3 (CVB3). CVB3 is one of the most common causes of viral myocarditis, and thus, it is the most commonly used pathogen to induce viral myocarditis experimentally. In the mouse model, inflammation can persist past the point of viral clearance, particularly in male mice [4]. This is a crucial feature of the disease as it suggests an importance of other factors in mediating continued inflammation after viral clearance. After long-term inflammation, cardiac remodeling, and matrix turnover, the disease can progress to dilated cardiomyopathy (DCM). Dilated Cardiomyopathy DCM is a sequela of myocarditis that is associated with decreased left ventricular function and heart failure; the only cure for which is heart transplantation. It has been well established that enhanced extracellular matrix turnover in the heart, particularly collagen remodeling, is correlated with decreased ejection fraction and progression to heart failure during DCM [5–7]. The levels of collagens I and III detected in the heart [5, 7, 8] and serum [6] are associated with the degree severity of DCM. This elevation in free collagens is accompanied by matrix metalloproteinases (MMPs) 2 and 9, which can also be detected in the heart and serum. Given the spatial, temporal coincidence and activity of MMPs for collagens, it is logical to connect MMP action to the genesis of DCM, particularly since high MMP levels can be detected in the heart before the onset of DCM. However, matrix remodeling must take place to a degree in order to repair and regenerate damaged tissue as a part of normal wound repair and tissue reclamation. The questions that remain involve the triggers and whether infectious or immune etiologies result in

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prolonged MMP expression. MMP activity that lingers for too long results in a cycle of extracellular matrix breakdown and deposition that ultimately weakens the connections between the cardiomyocytes. The result is a spongy myocardium with focal regions of hypertrophied cardiomyocytes. Therefore, a better understanding of extracellular matrix remodeling and the persistent queues that insist on prolonged remodeling will provide an understanding of how sequelae like DCM can arise from myocarditis. The transition from myocarditis to DCM is not understood, but significant clues have been provided by the matrix metalloproteinases (MMPs). An Introduction to MMPs MMPs are extracellular matrix proteins that were initially discovered by Gross and Lapiere in 1962 [9]. There are over 23 MMP genes in vertebrates with broad overlapping substrate specificities, and so, the differences in MMP functions lie primarily in their cell-specific expression. All MMPs are synthesized as inactive zymogens and share two conserved pro-peptide and catalytic domains. Several of the MMPs contain additional linker-hinge and hemopexin-binding domains [10]. Activation of MMPs is regulated by a “cysteine switch” mechanism that relies on a conserved cysteine residue within the pro-domain of all MMPs that is dysfunctional only in MMP26 [11, 12]. When this switch is in the “on” position, it acts to sterically block interaction between substrates and the Zn2+ active site within the protease; flipping this switch into the “off” position or removing it entirely results in proteolytic activity of the MMP. The control or removal of this integral cysteine switch is mediated by many chemicals and proteins including plasminogen activators, membrane-type MMPs, serine proteases, and oxidation [11]. In addition, all MMPs have the ability to activate themselves through autocatalytic cleavage; however, cleavage is relatively slow in all MMPs except MMP12 [13]. MMP Substrates Many of the MMPs digest collagen, which is the most abundant extracellular protein in the myocardium. DCM, a sequela of myocarditis, is a result of prolonged extracellular matrix remodeling. The MMPs are key mediators of extracellular matrix (ECM) turnover so they have been studied in the context of myocarditis in the CVB3 myocarditis mouse model [14]. Recent studies have elucidated the existence of many more MMP substrate targets other than just collagens. Most notably, these new targets include cytokines and chemokines [15–18]. As far as cytokine and chemokine digestion by MMPs is concerned, the purpose is not to merely degrade the cytokine or chemokine and to remove it from the extracellular matrix but to modulate cytokine and chemokine activity. MMP digestion results in the formation of cleavage

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products with activities that have agonist [19] and even antagonistic properties [16] with respect to the parent molecule. MMPs also have very similar and overlapping substrate specificities due to a high degree of homology in the catalytic domain. For example, a broad range of MMPs (MMP1, 2, 3, 7, 8, 9, 12, 13, and 14) all cleave chemokine C-C motif ligands (CCL)-15, 16, and 23 into their active forms increasing monocyte recruitment in response to injury [20]. The specificity of MMP roles is provided by differing cell and tissue-dependent expression profiles that are a result of diverse MMP gene promoter arrangements [11, 21]. In summary, MMPs are involved in the response, resolution, and even the genesis of many diseases including cancer, emphysema, arthritis, heart disease, and Alzheimer's disease by extracellular matrix breakdown and alteration of cytokine and chemokine activities [21–26], and in the treatment of some of these diseases, it may be appropriate to inhibit MMP action. In this review, we explore the involvement of MMPs in the response and genesis of myocarditis and DCM from MMP activity before and during disease. We are learning lessons of MMP action during heart disease in particular to help us understand whether MMP digestion of various substrates is deleterious or reparative in the heart. If the MMPs are truly destructive and their inhibition is indicated, we will discuss later how the overlapping substrate specificity of MMPs makes them difficult molecules to target pharmacologically. The TIMPs and Other Endogenous Inhibitors of MMPs To control the activity of MMPs and maintain homeostasis, the body expresses specific inhibitors of MMP activity. There are four endogenous cognate inhibitors of MMPs termed tissue inhibitors of matrix metalloproteases (TIMPs) 1–4 [27]. The TIMPs inhibit MMPs by binding to the MMP active site in a 1:1 stoichiometric ratio. Each of the four TIMPs can inhibit the action of most of the 23 different MMPs with a few exceptions such as the inability of TIMP1 to inhibit the activity of MT1-MMP (MMP14) [28]. The ability of TIMPs to inhibit MMPs varies, with TIMP2 being the most potent MMP inhibitor [29]. However, it is TIMP1 that is expressed during CVB3-induced viral myocarditis and the other TIMPs 2, 3, and 4 are expressed only marginally during virus infection. This suggests that TIMP1 is the predominant TIMP active during CVB3 myocarditis [29]. While the main role of TIMPs is to inhibit MMP action, they are also involved in the activation of MMPs. TIMP2 for instance plays a crucial role in the MT1-MMP dependent activation of MMP2, and upon ablation of the Timp2 gene in mice, a dysfunction in MMP2 activation is seen [30, 31]. In addition, it is important to note that although TIMP proteins are classically thought of as inhibitors of MMPs, MMPs also inhibit the action of TIMPs. This change in thinking regarding MMP/TIMP interdependence becomes critical as we consider the MMP-

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independent action of TIMPs during viral myocarditis. Any imbalance in TIMP1 expression could lead to pathological enhancement or inhibition that exacerbates myocarditis, potentially leading to its sequelae, DCM. In summary, when considering MMP activity during pathological states, one must also take into account the TIMPs because an imbalance in TIMP levels will affect the activity of MMPs and vice versa. α-2-Macroglobulin Systemic dissemination of MMPs is prevented through the action of potent inhibitors of MMP action that are expressed constitutively into the circulation. The α-macroglobulins are general endopeptidase inhibitors that inhibit MMP action in the blood by presenting a decoy substrate to the MMP and absorbing it into the macroglobulin [32]. The macroglobulins are present in high concentrations in the blood and act to temper global protease activity. MMPs During Heart Disease When MMP activity is beyond inhibitors in the circulation, it is subject to less control and can thus exacerbate illness if activity remains unchecked. MMP expression during different types of heart disease has been well defined and is typically associated with a worsening disease outcome. For instance, MMP activity in atherosclerotic plaques leads to plaque instability and dislodging of the lesion creating an embolus [33, 34], particularly in the case of emboli caused by infective endocarditis [35]. However, the assumption that MMPs are only exacerbating factors during heart disease is not only broad and overreaching but it is wrong in the case of viral myocarditis. In this review, we explore the importance of MMP action during viral myocarditis, while investigating their gender-related expression to highlight valid treatment targets.

The Roles of MMPs During Viral Myocarditis Cells of the Immune Response Mediate Inflammation by Secreting MMPs MMPs are enzymes that are secreted by cells in response to stimuli to carry out effector functions. Therefore, it is the secreting immune cells that are the origin and central culprits of MMP action and any consequences thereof. Cells of the immune system mediate inflammation and pathogenesis of viral myocarditis by secreting MMPs [36]. However, the involvement of MMPs varies significantly depending on the stage of infection since different immune cell subsets are involved in the immune response at different points after the onset of inflammation. MMP2 and MMP9 expression is

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increased at 3 days post-infection in the mouse model [37]. This is most likely due to resident fibroblasts, endothelial cells, and cardiomyocytes responding to virus-induced damage as significant inflammation has yet to occur. Once immune infiltration of neutrophils and macrophages into the heart has occurred, these cells begin to secrete MMP8/9 and MMP12, respectively [38–40]. This results in a marked increase in MMP8, MMP9, and MMP12 levels during acute viral myocarditis [37, 41]. As we discuss MMPs in this review, it is important to remember that the significant changes in MMP secretion seen are dependent upon the recruitment of various leukocytes and the activation states of the pertinent MMP producing cell types in the myocardium. The Acute Mechanisms of MMP Action on Heart Inflammation and Viral Replication During an immune response, MMPs are secreted at the immune cell migration front to degrade the extracellular matrix. This allows diapedesis of immune cells from the circulation into the infected heart tissue [42]. Endothelial cell secreted MMPs 2 and 9, which are activated immediately upon VCAM-1 binding by lymphocytes during an immune response, are required for lymphocyte extravasation [43]. These MMPs digest and open up endothelial junctions in the vasculature, which allows lymphocytes to move between and through endothelial layers and into the underlying tissue [44, 45]. For example, addition of exogenous TIMP2 blocks T cell migration through venules suggesting an essential role of MMPs during an acute immune response [46]. Lymphocyteassociated MMPs on the other hand are activated several hours after diapedesis suggesting that lymphocyte-associated MMPs are important in extravascular tissue migration rather than venule migration [42, 43]. In summary, MMPs are pivotal in digesting extracellular matrix for immune cell migration out of vascular beds into and through tissue to the site of insult. In support of the importance of MMPs during acute viral myocarditis, mice infected with CVB3 show an increase in MMP2, MMP3, MMP8, MMP9, MMP12, and TIMP1 7 days post-infection (Table 1, [37, 41, 47]). This MMP increase coincides with a 13-fold increase in CD3+ T cells in heart tissue as well as a 29-fold increase in myocytolysis [41]. In these same studies, TIMP3 and TIMP4 levels were decreased during the acute phase of CVB3 myocarditis, suggesting changes in the MMP/TIMP ratio could be to blame for deleterious immune cell invasion [37]. It should be noted, however, that the action of MMPs during acute viral myocarditis is not always deleterious. Mmp 2−/− mice for example are unable to inactivate CCL7 (a normal cleavage product of MMP2), and as a result, there is greater macrophage recruitment into the myocardium [48]. These results suggest that MMP2 normally acts to moderate inflammation through control of

J. of Cardiovasc. Trans. Res. (2014) 7:212–225 Table 1 Expression levels of selected MMPs and TIMPs during viral myocarditis

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Viral myocarditis Protein

Acute

Chronic

Sex-related expression

Role of MMP

References

Protective Protective No role Protective Unknown

[4, 37, 41, 48] [4, 41, 114] [4, 41, 49] [4, 37, 49] [37]

Deleterious Unknown Unknown Unknown

[4, 41] [37] [37, 41] [37, 41]

Matrix metalloproteinases

↑ increase, ↓ decrease, — return to control levels

MMP2 ↑ ↑ Unknown MMP3 ↑ — ↓ With testosterone treatment MMP8 ↑ — ↑ With testosterone treatment MMP9 ↑ — — With testosterone treatment MMP12 ↑ ↑ Unknown Tissue inhibitors of matrix metalloproteinases TIMP1 ↑ ↓ ↑ With testosterone treatment TIMP2 — — Unknown TIMP3 ↓ — Unknown TIMP4 ↓ — Unknown

macrophage invasion. Mmp9 −/− mice also have increased pathology during acute viral myocarditis due to enhanced viral load in the heart [49]. In this study, enhanced viral load in the absence of MMP9 was attributed to immune dysregulation caused by increased IFN-β concentration, which is normally inactivated by MMP9 cleavage [50]. In summary, acute heart dysfunction wrought by myocarditis may be due to the increase in certain MMPs, allowing for sustained immune cell invasion that can lead to DCM. Other MMPs, like MMP2 and MMP9, may be playing a beneficial role by regulating antiviral arms of the immune response. The loss of function of beneficial MMPs during acute viral myocarditis demonstrably causes enhanced viral replication and dysregulation of immunity—the use of inhibitors of MMPs as drugs to treat myocarditis, therefore, must be approached with caution. The Consequences of Chronic MMP Action Chronic inflammation that can accompany viral myocarditis is signified by sustained extracellular matrix remodeling and catastrophic fibrosis and scarring (Fig. 1a), ultimately leading to ventricular dysfunction and DCM. Chronic inflammation in the heart is accompanied by the sustained increase of certain MMPs as well, which is signified by a change in the MMP/ TIMP ratio beyond 28 days post-infection in the mouse model [36, 41]. It was posited in a paper by Heymans et al. that longterm MMP activity leads to exacerbated disease in the mouse model of coxsackievirus-induced myocarditis [51]. Treatment of mice with an inhibitor of urokinase-type plasminogen activator (uPA, activates MMPs) led to decreased severity and incidence of chronic myocarditis in the mice [51]. This chronic model of MMP expression and pathogenesis is consistent with other models of CVB3-infected mice where MMP2 and MMP12 are significantly enhanced 30 days post-infection and beyond (Table 1, [37, 41, 47]). TIMP1 is

detectable in significantly lower quantities 28 days postinfection while TIMP2, TIMP3, and TIMP4 return to control levels at this late time point [41]. Decreased TIMP1 levels result in a change in the MMP/TIMP ratio in the chronic phase of myocarditis. An increased ratio of MMP2, MMP9, and MMP12 to TIMP1 could lead to greater activity of these MMPs. This is just one example of where a change in the MMP/TIMP ratio could result in immune dysregulation, leading to chronic remodeling and DCM [36]. Interestingly, tissue plasminogen activator and uPA are both increased in mice at 7 days post-infection and are still up beyond 28 days postinfection [41]. The high levels of both activators seen at late time points post-infection fosters the continued infiltration of immune cells and the active state of MMPs, resulting in a hypothetical positive feedback loop where inflammatory stimuli beget inflammation. This continued immune infiltration likely contributes to the pathology seen during viral myocarditis and DCM.

Reconciling the Benefits and Detriments of MMP Action From the evidence cited already, using −/− MMP models of viral myocarditis and looking at long-term time points of infection, it is quite clear that the MMPs are mediating, at least in part, the transition from acute myocarditis to DCM (Fig. 1b). However, acutely, as described in the previous section, MMPs are regulating the immune response in a positive way. The MMPs are secreted to regulate cytokine and chemokine action to clear the heart of virus infection. What is clear is that lingering inflammation and MMP action is deleterious in the long term. Therefore, therapeutic strategies need to be developed whereby the beneficial roles of MMPs are preserved and the detrimental action of MMPs is inhibited.

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A

Healthy heart muscle H and E

Heart failure due to myocarditis Chronic infection and persistent inflammation and MMP activation promotes chronic remodeling = fibrosis and cardiomyocyte dropout

H and E

B Viral infection

0-4d

4-14d Cytokines

Immune infiltration Resident macrophages MMP9, MMP12

Virus infection

MMP

Cardiomyocyte

>15d

Reclamation of the heart, remodeling and fibrosis

Monthsyears

Dilation and prolonged inflammation TIMP1 MMP

Continued cytokine cleavage by MMP2 and 12 secreted by lingering inflammatory cells encourages chronic immune infiltration

Prolonged decrease in TIMP1 levels allows continued activation of MMPs secreted by inflammatory cells

Fig. 1 a Hematoxylin and eosin stained healthy and enterovirus-infected patient heart samples shows the drastic change in heart morphology due to viral myocarditis that leads to dilated cardiomyopathy. b The progres-

sion of viral myocarditis to DCM. Numbers represent days after viral infection of the mouse model

MMP Inhibition as a Therapeutic Strategy

treat a wide array of illnesses. MMPs were initially identified as possible drug targets to treat various cancers over 30 years ago. Since then, several MMP inhibitors have gone to clinical trials, which have been “drastically underwhelming” in the treatment of cancer [52]. A majority of these failings were a direct result of the inhibitors lacking direct MMP specificity. Generally, these first generation inhibitors (Table 2, [53–63])

First Generation MMP Inhibitors as Drugs There are apparent benefits observed by inhibiting MMP activity in laboratory experiments that study viral myocarditis [51]. Drugs are being developed to target MMPs in order to

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Table 2 The differences in selectivity between first generation and third generation matrix metalloproteinases inhibitors Drug

Specificity

Target

Targeted applications

First use

First generation inhibitors Batimastat Ilomastat Marimastat Prinomastat Metastat

Broad Broad Broad Broad Broad

None None None None Higher affinity to MMP2/9

Cancer, tumor cell proliferation, and angiogenesis Encephalomyelitis and cancer Cancer, tumor cell proliferation, and angiogenesis Cancer, tumor cell proliferation, and angiogenesis Cancer, tumor cell proliferation, and angiogenesis

199363 199257 199658 199653 199959

Selective inhibitors RXP470.1 DX-2400 MMP408

Selective Selective Selective

MMP12 MMP14 MMP12

Atherosclerosis Cancer and proMMP2 processing Chronic obstructive pulmonary disease

200655 200961 200962

Selective Selective Selective

MMP3 MMP9 MMP13

Chronic dermal conditions Hypertension and myocardial infarction Osteoarthritis

200156 201360 201254

UK-356618 Salvianolic Acid A 14D10

are strong Zn2+ chelators; therefore, they lack specific MMP targeting. These types of drugs cause negative side effects such as musculoskeletal pain, general inflammation, stiffness, joint swelling, and arthralgia [64, 65]. There are, however, several more important reasons for the failure of MMP inhibitors in the clinic other then drug side effects. In mouse models, pretreatment with MMP inhibitors before the induction of a disease event (e.g., virus inoculation) is the ideal drug regimen. This method of treatment is not only an unrealistic proposal in humans but is also directly opposed to how most clinical trials operate. Patients are typically directed to drug trials after failing the accepted standard of treatment for their respective disease. For example, in cancer, MMPs are believed to aid in tumor detachment and angiogenesis. Logic would then assume that MMP inhibitors should be applied before tumor migration can occur; however, this is rarely the case as a majority of the clinical trials looking into MMPs have been in advanced stage already metastasized cancers. Another problem is that upon obtaining activity-based protein profiles for various MMP inhibitors, it was determined that these inhibitors often bound other classes of MMPs [66, 67]. Off-target effects cause a decrease in the therapeutic index, meaning that the effective dose of the drug becomes closer to the toxic dose of the drug. This is likely the reason why MMP inhibitor use often causes adverse effects and why these same adverse effects were associated with higher patient survival [52, 68]. Lastly, broad MMP inhibition is inefficient as it can inhibit MMPs that are irrelevant during the disease [69]. All of these lessons were instrumental in shaping how MMP inhibitors are now being developed. New Candidate Drugs The need for ultra-selective MMP inhibitors has not only been driven in part by clinical trial failings but also by the continued

elucidation of MMP action during injury. A recent review by Dufour and Overall identified 16 MMPs that have beneficial roles during various infections/disease processes with the remaining MMPs (seven in humans) likely having yet unknown beneficial properties [70]. MMP inhibition therefore must be targeted and efficient to protect the positive effects of MMPs. In acute viral myocarditis, for example, the beneficial effects of both MMP2 and MMP9 should be protected, while the negative effects of TIMP1 should likely be targeted [48, 49, 71]. Newly developed MMP inhibitors can now target the zinc-binding SAXZ group of MMPs while simultaneously targeting specific conformational differences between MMPs, predominately at the S1′ loop. This loop, appropriately termed the “selectivity pocket”, differs in size, shape, and sometimes contains an attached S1′* loop which allows for ultra-selective MMP inhibitors [72]. RXP470.1, MMP408, and UK-356618 (Table 2) all take advantage of MMP S1′ loop differences to ensure nanomolar specificity to their respective MMP [56, 62, 73]. In addition to chemical compounds, development of monoclonal antibodies (mAb) like DX-2400 and 14D10 as well as natural compounds like salvianolic acid A provide other avenues for investigation [54, 60, 61]. Each potential drug provides a unique opportunity in disease treatment. For example, a mAb to MMP14 has been shown to block MMP14-dependent cleavage of pro-MMP2, while preserving other functions of MMP14 [74]. This provides the possibility that only negative roles of a specific MMP could be targeted while positive roles remain preserved. Important differences among these new drugs also exist in the form of varying biological availability, solubility, and toxicity. It is crucial that each potential drug class undergoes continued development ensuring the wide variety of MMP-related illnesses can be treated effectively with appropriate and relevant drugs.

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Viral Myocarditis and MMP Inhibition Trials? Clinical trials have not yet investigated MMP inhibition during viral myocarditis. It is apparent that MMP activity is beneficial in controlling acute infection in the heart. However, the long-term roles that MMPs play may exacerbate chronic myocarditis through prolonged collagen matrix turnover, leading to weakening of the heart muscle and DCM. Treatment of chronic myocarditis patients with MMP inhibitors may alleviate their fibrosis and extracellular matrix turnover, which could promote resolution of inflammation, thus preventing the progression of the disease to DCM. However, before such an endeavor could take place, lessons gleaned from the failure of cancer MMP trials should be remembered and considered in the context of viral myocarditis/DCM. In vivo models of myocarditis should now investigate the effects of MMP inhibition starting later in infection as pretreatment or treatment during acute myocarditis with MMP inhibitors is not realistic in human therapy. Such experimental work could aid in identifying which MMPs are key to the continuing inflammation and remodeling seen in chronic myocarditis and DCM. Off-target regulators of MMP activation are target therapeutic candidates. A study looking at the effects of cyclophilin A (CyPA), a ligand of extracellular MMP-inducer, and its inhibition during CVB3-induced murine myocarditis demonstrated that inhibition of CyPA early in infection enhanced virus replication by decreasing MMP9 activation, while CyPA inhibition after viral clearance reduced myocardial fibrosis [75]. This study supports the model whereby drug administration at different stages of viral myocarditis could be responsible for drastically different outcomes. Targets that are not MMPs, but control or regulate MMP activation, like CyPA could be considered. Additional support for this strategy comes from the study of inhibition of uPA during viral myocarditis. It was shown that the inhibition or absence of uPA during CVB3 murine myocarditis resulted in a decrease in myocardial inflammation and overall cardiac injury [51]. This presents another “off-target” target for MMP reduction. It is hard to know what exactly will happen once next generation drugs begin testing in clinical trials. With the vast amount of substrate overlap between the MMPs (Fig. 1 in [70]), it is impossible to predict all possible complications. The use of ultra-selective inhibitors allows targeted therapy with the benefit of identifying physiological side effects that are directly associated with one MMP. In summary, the MMP inhibitor field has come a long way since clinical trial failings and appears to be ready for another round.

Gender-Dependent Differences in MMP Expression An understanding of MMP expression between genders may lend insight to the increased prevalence of myocarditis in

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males [76, 77]. Discrepancies in MMP expression will certainly have to be elucidated prior to any clinical trial that targets MMP activity. Like myocarditis, gender-based disparities in disease are seen in cancer, heart disease, diabetes mellitus, and chronic obstructive pulmonary diseases, which are all top 10 killers in the USA [1, 78–81]. Androgens and estrogens themselves have been directly implicated in cancers of the prostate, breast, ovary, colon, and endometrial layer as well heart diseases such as atherosclerosis, hypertension, and congestive heart failure (reviewed in [82–84]). In addition, neurodegenerative diseases such as Alzheimer's disease and Kennedy's disease as well diabetes mellitus and liver disease have been directly linked to sex hormone or sex hormone receptor disparities [85–88]. Since androgens and estrogens clearly regulate disease processes, it begs the question what is the effect of these major hormone classes in the context of myocarditis and DCM? Gender-Specific Immune Responses During Myocarditis and Dilated Cardiomyopathy Acute viral myocarditis in children and adolescents can present with an array of symptoms ranging from fever, poor feeding, flu-like illness, and gastroenteritis all the way to more severe symptoms such as apnea, diaphoresis, or complete cardiovascular collapse [89, 90]. The insidious nature of myocarditis requires careful examination in all possible cases especially in young males as they have been shown to have a worse disease outcome in both DCM and myocarditis when compared to females [76, 77]. The incidence of myocarditis is estimated to be anywhere from 0.12 to 12 % with a majority of the patients being young adult males [91–93]. Sex-related variance at the genetic level during idiopathic DCM (IDCM) was recently confirmed by Haddad et al. [94]. By comparing 11 patients (five men) with 11 age-matched controls, they found that 55 genes were differentially expressed (37 upregulated and 18 downregulated) in female patients while 30 genes were differentially expressed (17 upregulated and 13 downregulated) in male IDCM patients. Male and female IDCM samples shared a total of 13 upregulated genes and 6 downregulated genes with none of the genes differentially expressed. This study could not identify gene targets that were drastically different between males and females but confirmed the gender-specific differences in the context of human IDCM. Additionally, the CVB3 murine model used to study viral myocarditis, which has long been known to accurately mimic the human disease state, shows sex-related differences. Although male and female mice have the same viral loads in the heart male mice progress to DCM more often than their female counterparts [4]. Using this murine model, it has also been demonstrated that testosterone is deleterious in viral myocarditis while estrogen confers protective properties [95]. Sex hormones in CVB3-infected mice have been

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implicated in various physiological processes including Th1/ Th2 cell balance, macrophage polarization, cardiomyocyte death, cardiomyocyte survival, and ultimately myocarditis disease progression [4, 96–100]. The consensus of these findings is that estrogen acts as a cardioprotective molecule while testosterone acts in a cardiopathic manner during myocarditis and eventually DCM. Gender-Specific Expression of MMPs MMPs are important mediators of cardiac fibrosis development and can aid in predicting the outcome of cardiac injury. In healthy humans, MMP3 and TIMP-1 levels have been correlated with age in men and women. However, generally speaking, men have significantly higher levels of MMP3 compared to women [101, 102]. During ECM remodeling, estrogen alters the TIMP/MMP balance protecting the heart from adverse outcomes such as DCM [103]. Voloshenyuk and Gardner have shown that MMP2, estrogen receptor-α, and estrogen receptor-β were downregulated in ovariectomized rats [103]. This is in contrast to the inhibitory effect that β-estradiol has on the MMP2 promoter but active repression of MMP2 promoter requires the estrogen receptor [104], which is also downregulated after ovariectomy [103]. MMP9 and MT1-MMP (MMP14) were upregulated after ovariectomy and were associated with loss of interstitial collagen in the heart during volume overload [103]. Estrogen treatment after ovariectomy improved TIMP2/MMP2 and TIMP1/MMP9 protein balance, thereby preventing MMP9 activation, perivascular collagen accumulation, and development of heart failure. Therefore, these results suggest that estrogen has a protective effect by preventing undesirable ECM remodeling and DCM through modulation of TIMP and MMP expression [103]. MMP sex-determined expression has also been characterized in several diseases such as abdominal aortic aneurysms (AAAs), myocardial infarction, coronary artery disease, and myocarditis. Treatment of rat aortic smooth muscle cells (RASMCs) with concentrations of IL-1β that are known to induce AAAs in vivo demonstrated that male derived cells have 10× more MMP9 expression and significantly higher TIMP1 and pro-MMP2 levels [105, 106]. However, treatment of these same male RASMCs with 17β-estradiol did not reduce MMP9 levels in vitro, but rats pretreated with 17βestradiol for 3 weeks before artery explantation and IL-1β stimulation showed significantly reduced MMP9 levels. This suggests a role of 17β-estradiol in MMP9 regulation in vivo that cannot be replicated in vitro. The in vivo rat model for AAAs further confirmed an increase of MMP9 in males and also demonstrated an increase in MMP13 when compared to females [107, 108]. This MMP increase coincides with increased macrophage and neutrophil infiltration as well as a reduction in type I and type III collagens. This points to MMP

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expression as a predictor for pathology in AAAs. Interestingly, in humans with AAAs, the differences in MMP9 levels are reversed, but still correlated to estrogen levels [109]. In a study of 54 AAA patients (18 men), MMP9 levels were reduced while estradiol levels were increased in men. This MMP9/estradiol reversal makes sense in the context of humans where elderly men have been found to have higher levels of estradiol then elderly women [110, 111]. In myocardial infarction, murine model male mice have increased MMP9 expression that is associated with increased rupture when compared to females, further confirming a role of MMPs in gender-related differences in disease pathogenesis [112]. Human coronary artery disease patients show the opposite with female patients having increased levels of MMP9 [113]. Studies of differences in MMP expression between the sexes in myocarditis and DCM are only preliminary with very little information available in the current literature. A study by Coronado et al. demonstrated sex-related expression of MMP3, MMP8, and TIMP1 in CVB3-infected mice. Testosterone increased the levels of MMP8 and TIMP1 in the hearts of male gonadectomized mice while decreasing MMP3 and having no effect on levels of MMP9 (Table 1, [4]). It was also found that male mice suffer more inflammation and LV remodeling without any differences in viral load between male and female mice. It has been previously demonstrated that TIMP1 is deleterious in CVB3 myocarditis while decreases in MMP3 have been associated with increased fibrosis, suggesting a protective role of MMP3 during myocarditis [4, 71, 114]. So, it would be no surprise that an increase in TIMP1 and a decrease in MMP3, as observed in male mice, could be responsible for the enhanced pathology observed in male mice. With the known importance of MMPs in the pathology of myocarditis, it is imperative that sex-related differences of their expression are defined. Sex hormone dependent MMP expression could illuminate opportunities for treatment, hopefully resulting in better MMP inhibitor targeting and treatment.

Intracellular Roles of MMPs Complicate Simple MMP Therapeutic Strategies MMPs have Evolved Intracellular Roles Although all MMPs were initially identified by their ability to proteolytically cleave extracellular matrix proteins, we now know that several MMPs can localize inside cells and cleave intracellular proteins. MMP2, 3, 9, 12, 13, 14, and 26 have all been shown to be present in the cytoplasm or nucleus of various cell types [115–123]. The exact function and overall knowledge of these roles vary greatly from one MMP to

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another. For example, MMP2 is involved in the processing of troponin I, myosin light chain 1, α-actin, and poly(ADPribose) polymerase (PARP) in myocytes, while MMP12 possesses direct antibacterial properties in murine macrophages [115, 118, 119]. This variance in intracellular properties is another possible reason as to why broad-spectrum MMP inhibitors had unexpected side effects in clinical trials. It is a key that we uncover the intracellular roles (if any) of all MMPs for without doing so, we will continue to see unexpected side effects with MMP inhibitor use. With that said, could there be intracellular roles for MMPs during viral myocarditis? MMP Intracellular Action in Viral Myocarditis No published study to our knowledge looks directly at the intracellular action of MMPs in the context of viral myocarditis. However, given what now appears to be an evolving level of substrate promiscuity by MMPs, circumstantial observations can be made. For example, the inducible nitric oxide synthase (iNOS) pathway that results in nitric oxide (NO) and peroxynitrite (ONOO−) production has long been known to be involved in the pathology of many heart diseases, including viral myocarditis (reviewed in [124–126]). In addition, several studies have reported both cytopathic and cytoprotective effects by the iNOS pathway during viral myocarditis [127–133]. Further investigation into the peroxynitrite pathway has revealed one of its downstream effecters, PARP, as a potential target in cardiovascular disease [134]. PARP is a nuclear localized substrate of MMP2 [119] and is activated during heart injury causing apoptosis and necrotic death of cardiomyocytes [135–139]. PARP inhibition is protective during heart insult likely through the avoidance of PARP overactivation that causes an increase in pro-inflammatory cytokines and ATP/NAD+ depletion resulting in necrotic death followed by improper immune invasion [136, 140–143]. This suggests a potential intracellular role of MMP2 in the avoidance of necrotic death of cardiomyocytes during heart diseases like myocarditis. Intracellular MMP2 may shift the form of cardiomyocyte death to apoptotic pathways that are known to be anti-inflammatory and crucial for the repair of the endothelium [144].

Conclusion Help and Hurt Prolonged extracellular matrix remodeling by MMPs expressed during viral myocarditis may shift disease states from acute viral myocarditis to DCM. The ability of MMPs to act as immune modulators is crucial during the initial stages of infection but may be devastating during later phases of

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disease. Invaluable lessons are being learned about beneficial and deleterious MMP expression, particularly MMP9 activity, from the models that study gender differences in the genesis of heart disease. The prospect that disease progression may be caused principally by MMPs allows a unique opportunity for drug treatment. The newest generation of ultra-selective inhibitors could possibly be used to prevent the transition of myocarditis to DCM and heart failure. This does not rule out the targeting of MMPs during acute myocarditis, however, as only MMP2 and MMP9 have been demonstrated as beneficial during the acute phase of infection. Further research should examine the temporal expression of MMPs during viral myocarditis to better define the best time for drug targeting. Mouse MMP knockout models should also be challenged with virus infection to examine the respective importance of each MMP during infection as thus far only MMP2, MMP8, and MMP9 null mice have been challenged with viral myocarditis causing agents. Lastly, it is our belief that the intracellular roles of MMPs must be thoroughly examined as they are likely, not simple bystanders during infection, but rather essential to the function of viral immunity. Acknowledgments DM is a Canada Research Chair in Viral Pathogenesis, The Department of Medical Microbiology and Immunology, The Li Ka Shing Institute of Virology at the University of Alberta.

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