Inhibition of Urokinase-Type Plasminogen Activator or Matrix Metalloproteinases Prevents Cardiac Injury and Dysfunction During Viral Myocarditis
Background— Acute viral myocarditis is an important cause of cardiac failure in young adults for which there is no effective treatment apart from general heart failure therapy. The present study tested the hypothesis that increased expression of the proteinases urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) is implicated in cardiac inflammation, injury, and subsequent failure during Coxsackievirus-B3 (CVB3)–induced myocarditis.
Methods and Results— First, we showed increased expression and activity of uPA and MMP-9 in wild-type mice at 7 days of CVB3-induced myocarditis. Targeted deletion of uPA, which resulted in reduced MMP activity and cytokine expression or inhibition of MMPs by adenoviral gene overexpression of tissue inhibitor of metalloproteinases-1, decreased cardiac inflammation and reduced myocardial necrosis at 7 days and decreased cardiac fibrosis at 35 days after CVB3 infection. Importantly, loss of uPA or MMP activity prevented CVB3-induced cardiac dilatation and dysfunction, as determined by serial echocardiography.
Conclusions— Loss of uPA or MMP activity reduces the cardiac inflammatory response after CVB3 infection, thereby protecting against cardiac injury, dilatation, and failure during CVB3-induced myocarditis.
Received September 26, 2005; revision received February 24, 2006; accepted May 26, 2006.
Acute viral myocarditis is an important cause of cardiac failure in young, otherwise healthy patients and may result in “idiopathic” dilated cardiomyopathy. The clinical course of acute viral myocarditis is mostly insidious, with limited cardiac inflammation and dysfunction. However, overwhelming inflammation may occur in a subset of patients, leading to fulminant cardiac injury and severe cardiac failure. To date, there is no effective treatment apart from general supportive therapy and standard anti-heart failure regimens.1,2
Clinical Perspective p 573
Acute viral myocarditis caused by Coxsackievirus-B3 (CVB3) is characterized by viral infection of cardiomyocytes followed by a pronounced infiltration of inflammatory cells. Both direct infection of myocytes by the coxsackievirus and the immune reaction after viral infection may cause myocyte necrosis and cardiac dysfunction.2 Importantly, a subset of patients with fulminant viral myocarditis and cardiac dilatation completely recovers, with normalization of cardiac dimensions and function.1,3 The pathogenesis of fulminant viral myocarditis remains unclear. Earlier reports have suggested that disruption of coupling between cardiomyocytes and the cardiac matrix may play a role.4,5
The proteolytic enzymes urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) have been implicated in cardiac repair and remodeling after cardiac injury or stress. Inactivation of the uPA gene impaired wound healing after acute myocardial infarction6 and prevented pressure overload-induced cardiac failure,7 whereas absence of MMP-2 or MMP-9 prevented cardiac rupture6,8 and dilatation9 after myocardial infarction. A single study showed increased transcript levels of MMP-9 together with decreased transcript levels of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-4 during CVB3-induced myocarditis10 (reviewed in Pauschinger et al11). Given the possible importance of the cardiac matrix in myocarditis, the present study aimed to investigate whether increased expression of the proteolytic enzymes uPA and MMPs may mediate acute inflammation and cardiac dilatation during CVB3-induced viral myocarditis. Therefore, uPA gene inactivation and adenoviral overexpression of TIMP-1 or plasminogen activator inhibitor (PAI)-1 were evaluated for their ability to halt the cardiac consequences of viral myocarditis in a mouse model of human pathogenic CVB3-induced myocarditis.
In the present study, we demonstrate increased expression and activity of uPA and MMPs during acute viral myocarditis. Loss of uPA activity or reduction in MMP activity in the heart offered protection against cardiac inflammation and necrosis, preventing cardiac dilatation and dysfunction during viral myocarditis.
Mouse Model of Viral Myocarditis
All mice were bred and maintained in an open animal facility (Leuven University, Leuven, Belgium), and experiments were performed according to the guidelines for care and use of laboratory animals approved by the institution animal care committee of Leuven University. Six- to 8-week-old inbred C57BL/6 uPA knockout mice and wild-type (WT) littermates of either sex weighing 15 to 18 g were used. On day 0, mice were inoculated intraperitoneally with 1×107 cell culture 50% infective dose (CCID50) of CVB3 diluted in 0.2 mL of saline.12 Because C57BL/6 mice demonstrated minimal myocarditis after CVB3 infection alone, 25 μg of lipopolysaccharide (LPS; Salmonella minnesota, Sigma Re 595, St. Louis, Mo) was injected at day 0 and day 4 after CVB3 infection to promote CVB3-induced autoimmune myocarditis in genetically resistant mice. LPS alone did not induce cardiac inflammation, concordant with previous studies.13 Sham infection (n=7 per group at both 7 and 35 days) consisted of injection of 0.2 mL of saline intraperitoneally together with LPS at day 0 and day 4. Mice were euthanized at 7 days (n=9 in uPA WT and n=8 in uPA knockout mice) and 35 days (n=8 per group) and prepared for further molecular and histological analysis.
Virus Titration of Tissue Homogenate and Viral Load in Blood
In a separate group (uPA knockout [n=6] and WT littermates [n=6]), CVB3 load (RNA) was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in serum 2 days after intraperitoneal injection of CVB3. Briefly, CVB3 RNA was extracted with the viral RNA Mini Kit (Qiagen, Valencia, Calif), and RNA was quantified with the TaqMan fluorogenic detections system on an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, Calif).12 In addition, heart, pancreas, and spleen were dissected aseptically at 48 hours after infection. Tissue homogenates 20% (wt/vol) were prepared in Eagle’s minimal essential medium (Gibco Laboratories, Grand Island, NY). Vero cells were grown to confluence in microtiter trays and infected with serial dilutions of the homogenates and incubated for 2 hours at 37°C, after which the virus was removed. Virus-induced cytopathic affect was recorded microscopically at 3 days and expressed as CCID50.12
Histology, Immunostaining, and Morphometry
At 7 and 35 days, hearts were taken out and prepared for microscopic analysis.6,7 The extension of myocarditis at 7 days after CVB3 injection was determined with an ocular grid that contained 121 equally spaced points, and a myocardial score was defined as described previously.12
Immunostaining was performed with rat anti-mouse CD4, CD8 (Pharmingen, San Jose, Calif), and myeloperoxidase (Dako, Glostrup, Denmark) antibodies. Myocardial necrosis was determined by percentage of necrotic cardiomyocytes per total cardiac area. Collagen was stained with Sirius red, and the amount of collagen was quantified as percentage of Sirius red staining area per total cardiac area, excluding fibrosis around large cardiac vessels.6,7
Expression of mRNA by RT-PCR
Total RNA was extracted from the myocardial samples by the Trizol method (GIBCO BRL, Carlsbad, Calif). RT-PCR was used to detect and semiquantify the mRNA abundance of uPA, tissue plasminogen activator (tPA), MMP-2, MMP-3, MMP-8, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3, TIMP-4, interleukin (IL)-1β, IL-6, and transforming growth factor (TGF)-β1 based on methods described previously.10,14
Adenoviral Gene Transfer of TIMP-1 and PAI-1
One hundred microliters of 1.3×109 plaque-forming units of replication-deficient adenovirus carrying the TIMP-1 gene (AdTIMP-1) or PAI-1 gene (AdPAI-1) or replication-deficient adenovirus carrying a control RR5 gene (AdRR5) diluted in saline was injected in the tail vein,6,7 1 day before (AdTIMP-1) or 4 days after (AdTIMP-1 and AdPAI-1) inoculation with 107 CCID50 of CVB3 in 4-week-old male CVB3-susceptible C3H/HeNHsd mice (Harlan Laboratories, Horst, the Netherlands) weighing 15 g. In human patients, as described in mice,13 the susceptibility to develop acute myocarditis after CVB3 infection depends on a certain immune background.15 Therefore, to address the hypothesis that uPA/MMP inhibition may serve as a therapeutic tool to reduce inflammation, injury, and dysfunction due to acute myocarditis, we preferred to use CVB3-susceptible mice, which makes a more compelling case for treatment in human subjects.
TIMP-1 and PAI-1 plasma levels were measured in 100 μL of blood, sampled from the retro-orbital plexus with a commercially available ELISA for TIMP-1 (Amersham, Life Science, Inc, Leiden, the Netherlands) or PAI-16 at 4 days after gene transfer. CVB3-injected mice were analyzed at 7 and 35 days (n=8 to 11 per group) after CVB3 or sham injection for cardiac function, followed by histology and molecular analysis.
Proteinase Activity and Molecular Analysis
Zymography of tPA, uPA, MMP-2, and MMP-9 activities in extracts of hearts at 7 days after CVB3 or sham injection was determined as described previously.6 The amount of lysis (area×intensity) was analyzed quantitatively with Quantimed 600 image analysis software (Leica Instruments, Hamburg, Germany) and expressed in arbitrary units of lysis. Immunoblotting was performed as described previously.16
In addition, MMP activity at 7 days after CVB3 or sham injection was investigated with a biochemical substrate for combined MMP-2/9, MMP-3, MMP-8, and MMP-13 activity (Calbiochem, EMD Biosciences, Inc, Darmstadt, Germany). A total of 3.7 μg of protein of cardiac extracts was assayed in a 96-well polystyrene plate (Corning, Inc, Corning, NY) containing 5 μmol/L of the fluorogenic MMP substrate. The fluorescence was followed at excitation 320 nm and emission 405 nm with a Shimadzu RF-5301 PC spectrofluorometer (Shimadzu Scientific, Columbia, Md). Relative fluorescent units are compared between different experimental groups within 240 minutes.
M-mode echocardiography at 7 and 35 days after CVB3 injection was performed as described previously.7 Views in the parasternal short axis were obtained for guided M-mode measurements (at the midpapillary level) of the intraventricular septum, left ventricular posterior wall, left ventricular end-diastolic diameter (LVEDD), and left ventricular end-systolic diameter (LVESD), with the leading-edge-to-leading-edge convention. Percent fractional shortening was calculated by the formula: [(LVEDD−LVESD)/LVEDD]×100. For each measurement, 3 consecutive cycles were measured and averaged.
All data are expressed as mean±SEM. The Mann-Whitney U test or 2-sided ANOVA test assessed statistical significant difference between groups, with 2-sided probability values (P<0.05).
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
Increased Activity and Expression of uPA and MMPs During Acute Viral Myocarditis
In the present study, we first investigated whether acute CVB3-induced myocarditis increased uPA/MMP expression. Transcript levels of uPA and MMP-2, -3, -8, -9, and -13 but not tPA were significantly increased in hearts of CVB3-infected WT compared with sham-treated WT mice at 7 days (Table 1). Zymographic activity of uPA and MMP-9 was significantly increased in CVB3-infected hearts compared with sham-treated hearts at 7 days (Figure 1A and 1B), accompanied by a significant upregulation of protein expression of MMP-9 (2.5-fold increase, P<0.05; Figure 1D). tPA activity remained unaffected (Figure 1A). MMP activity assays with fluorogenic substrates revealed significantly increased combined activity of MMP-2/MMP-9 (Figure 1C; Table 2), MMP-8, and MMP-13 but not MMP-3 (Table 2) in CVB3-infected hearts compared with control WT hearts at 7 days. Together, these data reveal increased expression and activity of uPA and MMPs during acute CVB3-induced viral myocarditis.
Reduced Inflammation and Cardiac Fibrosis in Absence of uPA
To provide genetic evidence for a role of uPA in cardiac inflammation and injury during CVB3-induced myocarditis, we infected uPA knockout and WT littermates with CVB3 intraperitoneally. Myocarditis score in the left ventricle was significantly reduced in uPA knockout compared with WT mice at 7 days (Table 3; Figure 2A through 2D). In concordance, the number of CD4- and CD8-immunoreactive T lymphocytes and myeloperoxidase-immunoreactive neutrophils was significantly decreased in uPA knockout compared with WT hearts (Table 3). Focal myocarditis resulted in pronounced myocardial necrosis in WT mice but significantly less in uPA knockout mice (percent area of necrosis per total myocardial area: 15±1.2% in WT mice versus 7.4±0.4% in uPA knockout mice, P<0.05).
Collagen content did not differ significantly between CVB3- and sham-infected mice at 7 days after CVB3 infection, either in uPA knockout or in WT mice (Table 3; Figure 2E and 2F). Hence, viral infection of the heart and associated myocardial inflammation and necrosis resulted in increased collagen deposition at 35 days after CVB3 infection in WT mice (Table 3; Figure 2G). Decreased inflammation and necrosis in uPA knockout mice was associated with significantly reduced deposition of collagen in uPA knockout hearts compared with WT hearts at 35 days (Table 3; Figure 2G and 2H).
To investigate whether loss of uPA might directly affect viral load and infectivity shortly after CVB3 injection, viral RNA load and infectivity were quantified in blood and infected tissues, respectively, at 2 days after intraperitoneal CVB3 injection. Viral load in serum did not differ significantly between uPA knockout and WT mice at 2 days (log viral copies of RNA per microliter of serum: 1.6±0.1 in uPA WT mice compared with 1.26±0.23 in uPA knockout mice, P=NS); viral infectivity in pancreas, spleen, and heart also did not differ significantly in CVB3-infected uPA knockout and WT mice at 2 days after infection (Figure 1E).
Reduced Plasmin and MMP Activity in Absence of uPA
Active plasmin was significantly reduced in hearts of uPA knockout mice at 7 days of CVB3 injection (Figure 1F; ratio of active 68-kDa plasmin to inactive 93-kDa plasminogen 0.60±0.05 in WT compared with 0.38±0.04 in uPA knockout mice; n=6; P=0.02). Induction of MMP-9 activity, which occurs in part through plasmin,6 was significantly attenuated in CVB3-infected uPA knockout mice, as revealed by a significant reduction of MMP-9 zymographic activity in hearts of CVB3-infected uPA knockout compared with WT mice (P<0.01; Figure 3A and 3B). Associated with the slight but nonsignificant increase of MMP-2 activity in WT hearts after CVB3 infection (Figure 1A and 1B), MMP-2 activity showed only a moderate but nonsignificant decrease (P=0.08) in CVB3-infected hearts of uPA knockout mice compared with WT mice (Figure 3A and 3B). Decreased gelatinase activity in the absence of uPA was confirmed with a fluorogenic substrate for combined MMP-2/MMP-9 activity, showing significantly decreased activity in CVB3-infected uPA knockout hearts compared with WT hearts at 7 days (Figure 3C), whereas MMP-2/MMP-9 activity did not significantly differ between sham-treated uPA knockout and WT mice (Table 2). Concordantly, the increase in expression of pro-MMP-2 and –MMP-9 protein was significantly reduced by 24% and 50%, respectively, in uPA knockout mice compared with WT mice at 7 days after CVB3 infection (P<0.05 for both; Figure 3D). In addition, MMP-8 and MMP-13 activity was significantly decreased in hearts of uPA KO compared with WT at 7 days, whereas sham levels did not differ (Table 2). Decreased MMP activity or protein expression in uPA knockout compared with WT hearts was not due to differences in MMP or TIMP transcript levels (Table 1).
Reduced Cytokine Expression in Absence of uPA
To investigate whether absence of uPA and associated reduction in inflammation and MMP activity may alter cytokine expression,11 transcript levels of IL-6, Il-1β, and TGF-β were determined in cardiac extracts of CVB3- or sham-infected hearts at 7 days. Absence of uPA decreased the transcript levels of proinflammatory cytokines, including IL-6 by 47%, IL-1β by 55% (P<0.05 for both), and TGF-β by 13% (P=0.08) compared with WT levels at 7 days after CVB3 infection, whereas transcript levels were very low and did not differ significantly in sham-treated uPA knockout hearts compared with WT hearts (data not shown).
MMP Inhibition by TIMP-1 Gene Overexpression Reduces Focal Myocarditis
To investigate whether reduction of cardiac MMP activity as observed in uPA knockout mice may reduce myocardial inflammation and injury during viral myocarditis, a replication-deficient adenovirus overexpressing the human AdTIMP-1 or a control AdRR5 adenovirus was injected intravenously in C3H mice, followed by intraperitoneal injection of CVB3 1 day later. AdTIMP-1 injection increased plasma protein levels of TIMP-1 (33±4 μg/mL) within 5 days in the AdTIMP-1–treated group. TIMP-1 gene overexpression resulted in a significant reduction of cardiac MMP-2 and MMP-9 zymographic activity by 61% and 38%, respectively, compared with control RR5 gene overexpression at 7 days after CVB3 infection (arbitrary units lysis, MMP-2 2.4±0.3 with AdTIMP-1 versus 5.9±0.5 with AdRR5; MMP-9 3.1±0.3 with AdTIMP-1 versus 5.2±0.1 with AdRR5; P<0.05 for both). TIMP-1 gene overexpression significantly reduced the myocarditis score and the influx of CD4- and CD8-immunoreactive T lymphocytes and myeloperoxidase-immunoreactive neutrophils compared with control RR5-gene-treated mice at 7 days (Table 3; Figure 4A through 4D). This resulted in decreased interstitial fibrosis in TIMP-1–treated compared with RR5-treated mice at 35 days after CVB3 infection (Table 1; Figure 4G and 4H).
To investigate whether TIMP-1 or PAI-1 may reduce cardiac inflammation and injury well after induction of myocarditis by CVB3 injection, thereby mimicking a clinically more relevant situation, AdTIMP-1 or AdPAI was injected 4 days after CVB3 injection, which resulted in elevated TIMP-1 (21±4 μg/mL) and PAI-1 (23±4 μg/mL) levels at 7 days after CVB3 injection. Both TIMP-1 and PAI-1 gene overexpression after CVB3 injection still significantly reduced cardiac injury and interstitial fibrosis compared with control RR5 gene overexpression (Table 3; Figure 5A through 5F). These results indicate that uPA/MMP inhibitors, even given well after the initial infection, may serve as a novel therapeutic tool to reduce harmful cardiac inflammation and the resulting fibrosis caused by CVB3-induced myocarditis.
Loss of uPA or MMP Protects Against Cardiac Dilatation and Systolic Dysfunction
To investigate whether reduction in focal myocarditis and cardiac fibrosis by inhibition of uPA/MMPs may influence overall cardiac function and dimensions, echocardiographic recordings were performed. Fractional shortening and heart rate were similar in sham WT and uPA knockout mice (Table 4). Cardiac function or dimensions did not change statistically within 7 days after CVB3 infection. LVEDD increased significantly, and left ventricular fractional shortening decreased significantly at 35 days after CVB3 infection in WT- and AdRR5-treated mice (Table 4). However, absence of uPA or adenoviral TIMP-1 or PAI-1 gene overexpression, induced either 1 day before or 4 days after CVB3 injection, protected against LV dilatation and systolic dysfunction. Heart rates did not differ between groups (Table 4).
The present study unveils a novel and important role for the cardiac proteinases uPA and MMPs in cardiac inflammation, injury, and dilatation during CVB3-induced myocarditis. First, we show increased expression and activity of uPA and MMPs after CVB3 infection. Loss of uPA by gene inactivation, or inhibition of plasminogen activator/MMPs by adenoviral PAI-1/TIMP-1 overexpression, reduced cardiac inflammation and injury and prevented left ventricular dilatation and dysfunction during CVB3-induced myocarditis.
Infection of the heart with CVB3 results in a pronounced infiltration of inflammatory cells, including neutrophils, CD4 lymphocytes, and cytotoxic CD8 lymphocytes. The present study demonstrates that absence of uPA or inhibition of MMPs significantly decreased infiltration of inflammatory cells. Reduced inflammation prevented cardiac injury and resulting fibrosis, in concordance with previous studies demonstrating a pivotal role of inflammation in cardiac injury during CVB3-induced myocarditis.17,18 This suggests that uPA and MMPs may regulate cardiac inflammation during viral myocarditis in different ways.
First, uPA-mediated plasmin promotes degradation of the interstitial matrix of the heart, which serves as a barrier for invading inflammatory cells. However, uPA and plasmin alone are not able to degrade all matrix elements within the heart. uPA-mediated plasmin activates MMP-9 and MMP-13, as indicated by their decreased MMP activity in the absence of uPA. Increased MMP activity, however, may also result from increased activation by reactive oxygen metabolites, elastases, or cathepsins released by neutrophils.19 Thus, the combined upregulation of uPA and MMP activity provides complementary proteolytic degradation of the extracellular matrix. This in turn allows infiltration of different inflammatory cells in the heart during viral myocarditis, as indicated by the present findings that both uPA and MMP inhibition similarly decrease neutrophil and T-cell infiltration. Importantly, uPA, MMP-2, and MMP-9 are the only matrix-cleaving enzymes that have been described in T cells, facilitating the passage of T cells into the extracellular matrix.19 The present study demonstrates that the absence of uPA or a reduction of MMP-9 activity during CVB3-induced myocarditis decreases infiltration of inflammatory cells, including T cells. In accordance with these results, Lee et al5 also showed reduced cardiac inflammation, necrosis, and inflammation during viral myocarditis by inhibiting serine elastase, another serine proteinase.
Second, uPA is an important direct modulator of T-cell and neutrophil activation and modulates cytokine responses.20,21 The present finding of decreased T-cell infiltration after cardiac CVB3 infection in the absence of uPA is in line with earlier findings showing a decreased T-cell response to pulmonary Cryptococcus infection in the absence of uPA.20 uPA may also promote myocardial inflammation, dysfunction, and adverse matrix remodeling during CVB3 infection by stimulating the expression of proinflammatory cytokines, including IL-1β, IL-6, and TGF-β, that stimulate myocardial injury during CVB3-induced myocarditis.22 In addition to their role in modulating lymphocyte function, these proinflammatory cytokines also disturb the balance between MMPs and TIMPs, which results in overall increased MMP activity.11 Reduced cytokine expression in the absence of uPA may also have resulted from a reduced number of inflammatory cells.
Finally, uPA-mediated degradation of matrix elements promotes activation of T cells and macrophages23,24 and may thereby further facilitate myocarditis. In particular, uPA-mediated degradation of tenascin-C, a marker of viral myocarditis,25 promotes T-lymphocyte migration in vitro without affecting their function.23 The in vivo relevance of the latter and whether other matricellular proteins, including thrombospondins, osteopontin, and secreted protein acidic and rich in cysteine (SPARC)/osteonectin, which all affect T-cell function (reviewed in Kuznetsova and Roberts26), may be involved during viral myocarditis requires further investigation.
The absence of uPA and overexpression of PAI-1 or TIMP-1 protected against myocarditis-induced loss of cardiac function. Reduced inflammation, cytokine expression, and MMP activity in the absence of uPA protected against cardiac dilatation and dysfunction during viral myocarditis. A central role for uPA in cardiac inflammation and dysfunction during CVB3-induced myocarditis is further suggested by reduced cardiac inflammation and related dysfunction by PAI-1 gene transfer. Reduced MMP activity in the absence of uPA protects against proteolytic degradation of the extracellular matrix, thereby preventing myocardial slippage27 and the resulting cardiac dilatation and dysfunction during viral myocarditis. Reduced cytokine expression in the absence of uPA further protects against cardiac dilatation and dysfunction, in agreement with previous observations that show a deleterious effect of exaggerated inflammation and cytokine expression during CVB3-induced viral myocarditis.13
In conclusion, the present study clearly reveals a crucial role for uPA and MMPs in mediating cardiac inflammation and necrosis during CVB3-induced viral myocarditis in mice. The absence of uPA or overexpression of the TIMP-1 or PAI-1 gene both before or after CVB3 infection in mice reduced inflammation and protected against cardiac injury and dysfunction during CVB3-induced viral myocarditis. These data encourage the use of proteinase inhibition as a novel therapeutic approach to prevent deleterious cardiac inflammation and cardiac dysfunction during CVB3-induced myocarditis.
Sources of Funding
This study was supported by a Dr Dekker grant from the Netherlands Heart Foundation (NHS, 2003T036) to Dr Heymans, a research grant from the Leuven University, Belgium (OT-0346) to Dr Heymans and Dr Van de Werf, a VIDI grant (016.036.346) from the Netherlands Organization for Scientific Research (NWO) to Dr Pinto, and a grant of the Deutsche Forschungsgemeinschaft through SFB Transregio 19 (grant SFB Transregio 19 A2) to Dr Pauschinger.
Kearney MT, Cotton JM, Richardson PJ, Shah AM. Viral myocarditis and dilated cardiomyopathy: mechanisms, manifestations, and management. Postgrad Med J. 2001; 77: 4–10.
Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nube O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. In: Nat Med. 1999; 1135–1142.
Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest. 2000; 106: 55–62.
Li J, Schwimmbeck PL, Tschope C, Leschka S, Husmann L, Rutschow S, Reichenbach F, Noutsias M, Kobalz U, Poller W, Spillmann F, Zeichhardt H, Schultheiss HP, Pauschinger M. Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc Res. 2002; 56: 235–247.
Pauschinger M, Rutschow S, Chandrasekharan K, Westermann D, Weitz A, Peter Schwimmbeck L, Zeichhardt H, Poller W, Noutsias M, Li J, Schultheiss HP, Tschope C. Carvedilol improves left ventricular function in murine coxsackievirus-induced acute myocarditis association with reduced myocardial interleukin-1beta and MMP-8 expression and a modulated immune response. Eur J Heart Fail. 2005; 7: 444–452.
Schroen B, Heymans S, Sharma U, Blankesteijn WM, Pokharel S, Cleutjens JPM, Porter JG, Evelo CTA, Duisters R, van Leeuwen REW, Janssen BJA, Debets JJM, Smits JFM, Daemen MJAP, Crijns HJGM, Bornstein P, Pinto YM. Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy. Circ Res. 2004; 95: 515–522.
Gyetko MR, Aizenberg D, Mayo-Bond L. Urokinase-deficient and urokinase receptor-deficient mice have impaired neutrophil antimicrobial activation in vitro. J Leukoc Biol. 2004; 76: 648–656.
Gundersen D, Tran-Thang C, Sordat B, Mourali F, Ruegg C. Plasmin-induced proteolysis of tenascin-C: modulation by T lymphocyte-derived urokinase-type plasminogen activator and effect on T lymphocyte adhesion, activation, and cell clustering. J Immunol. 1997; 158: 1051–1060.
Vaday GG, Lider O. Extracellular matrix moieties, cytokines, and enzymes: dynamic effects on immune cell behavior and inflammation. J Leukoc Biol. 2000; 67: 149–159.
Sato M, Toyozaki T, Odaka K, Uehara T, Arano Y, Hasegawa H, Yoshida K, Imanaka-Yoshida K, Yoshida T, Hiroe M, Tadokoro H, Irie T, Tanada S, Komuro I. Detection of experimental autoimmune myocarditis in rats by 111In monoclonal antibody specific for tenascin-C. Circulation. 2002; 106: 1397–1402.
Spinale FG, Coker ML, Bond BR, Zellner JL. Myocardial matrix degradation and metalloproteinase activation in the failing heart: a potential therapeutic target. Cardiovasc Res. 2000; 46: 225–238.
Viral myocarditis is an important cause of acute heart failure in young, otherwise healthy adults but also is thought to account for 60% of so-called chronic idiopathic cardiomyopathies. Acute viral myocarditis is characterized by viral infection of cardiomyocytes, followed by infiltration of inflammatory cells. Whereas viral infection of myocytes only results in limited injury of cardiac cells, it is the exaggerated inflammatory reaction against the virus itself that causes extensive injury of the heart, with resulting dilatation and failure. The present study investigated whether degradation of the interstitial matrix is essential for these severe consequences of viral myocarditis. Our results reveal that inhibition of the 2 main proteolytic systems that degrade the cardiac matrix, the plasminogen activator and the matrix metalloproteinase (MMP) system, prevented exaggerated cardiac inflammation and dilatation during coxsackievirus-B3–induced viral myocarditis. Overexpression of the tissue inhibitor of metalloproteinases-1 or plasminogen activator inhibitor-1 significantly reduced the invasion of inflammatory cells and prevented cardiac dilatation and failure, without affecting the viral load. Inhibition of MMP is therefore a promising novel approach to treat the damaging cardiac inflammatory reaction. Development of specific MMP inhibitors targeting MMP-2 and -9, but without cytokine side effects, may therefore provide a novel form of treatment of viral myocarditis and related “inflammatory cardiomyopathies” such as those seen in acute cardiac ischemia, cardiac sarcoidosis, and doxorubicin (Adriamycin, Pharmacia & Upjohn, Piscataway, NJ)-induced cardiomyopathy.
↵*The first 2 authors contributed equally to this article.