Grzegorz Sawicki

Intracellular Action of Matrix Metalloproteinase-2 Accounts for Acute Myocardial Ischemia and Reperfusion Injury

Background—Matrix metalloproteinases are best recognized for their ability to degrade the extracellular matrix in both physiological and pathological conditions. However, recent findings indicate that some of them are also involved in mediating acute processes such as platelet aggregation and vascular tone. The acute contractile defect of the heart after ischemia-reperfusion may involve the proteolytic degradation of the thin filament protein troponin I; however, the protease responsible for this remains obscure. Methods and Results—Here we report that matrix metalloproteinase-2 is colocalized with troponin I within the thin myofilaments of cardiomyocytes in ischemic-reperfused hearts and that troponin I is a novel intracellular target for proteolytic cleavage by matrix metalloproteinase-2. Inhibition of matrix metalloproteinase-2 activity prevented ischemia-reperfusion-induced troponin I degradation and improved the recovery of mechanical function of the heart. Conclusions—These data reveal for the first time a novel molecular mechanism by which matrix metalloproteinase-2 causes acute myocardial dysfunction after ischemia-reperfusion-injury and that matrix metalloproteinase-2 has a biological action within the cell.

Matrix Metalloproteinase-2 Contributes to Ischemia-Reperfusion Injury in the Heart

BACKGROUND Matrix metalloproteinases (MMPs) contribute to collagen degradation and remodeling of the extracellular matrix after myocardial infarction; however, their role in myocardial dysfunction immediately after ischemia and reperfusion is unknown. METHODS AND RESULTS We measured the release of MMPs into the coronary effluent of isolated, perfused rat hearts during aerobic perfusion and reperfusion after ischemia. Aerobically perfused control hearts expressed pro-MMP-2 and MMP-2, as well as an unidentified 75-kDa gelatinase. These enzymes were also detected in the coronary effluent. After 20 minutes of global no-flow ischemia, there was a marked increase in pro-MMP-2 in the coronary effluent that peaked within the first minute of reperfusion. The release of pro-MMP-2 into the coronary effluent during reperfusion was enhanced with increasing duration of ischemia and correlated negatively with the recovery of mechanical function during reperfusion (r(2)=0.99). MMP-2 antibody (1.5 to 15 microg/mL) and the inhibitors of MMPs doxycycline (10 to 100 micromol/L) and o-phenanthroline (3 to 100 micromol/L) improved whereas MMP-2 worsened the recovery of mechanical function during reperfusion. CONCLUSIONS These results show that acute release of MMP-2 during reperfusion after ischemia contributes to cardiac mechanical dysfunction. The inhibition of MMPs may be a novel pharmacological strategy for the treatment of ischemia-reperfusion injury.

Degradation of Myosin Light Chain in Isolated Rat Hearts Subjected to Ischemia-Reperfusion Injury A New Intracellular Target for Matrix Metalloproteinase-2

Background—Matrix metalloproteinase-2 (MMP-2) contributes to cardiac dysfunction resulting from ischemia-reperfusion (I/R) injury. MMP-2 not only remodels the extracellular matrix but also acts intracellularly in I/R by degrading troponin I. Whether other intracellular targets exist for MMP-2 during I/R is unknown. Methods and Results—Isolated rat hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The impaired recovery of mechanical function of the heart was attenuated by the MMP inhibitors o-phenanthroline or doxycycline. Quantitative 2D electrophoresis of homogenates of aerobically perfused hearts (control) or those subjected to I/R injury (in the presence or absence of MMP inhibitors) showed 3 low-molecular-weight proteins with levels that were significantly increased upon I/R injury and normalized to control levels by MMP inhibitors. Mass spectrometry analysis identified all 3 proteins as fragments of myosin light chain 1, which possesses theoretical cleavage recognition sequences for MMP-2 and is rapidly degraded by it in vitro. The association of MMP-2 with the thick myofilament in fractions prepared from I/R hearts was observed with immunogold electron microscopy, gelatin zymography for MMP-2 activity, and immunoprecipitation. MMP-2 was found to cleave myosin light chain 1 between tyrosine 189 and glutamine 190 at the C terminus. Conclusions—Our results demonstrate that myosin light chain 1 is another novel substrate for MMP-2 in the cardiomyocyte and that its degradation may contribute to contractile dysfunction resulting from I/R injury to the heart.

Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro

Matrix metalloproteinases (MMPs) are traditionally known for their role in extracellular matrix remodeling. Increasing evidence reveals several alternative substrates and novel biological roles for these proteases. Recent evidence showed the intracellular localization of MMP‐2 within cardiac myocytes, colocalized with troponin I within myofilaments. Here we investigated the presence of MMP‐2 in the nucleus of cardiac myocytes using both immunogold electron microscopy and biochemical assays with nuclear extracts. The gelatinase activity found in both human heart and rat liver nuclear extracts was blocked with MMP inhibitors. In addition, the ability of MMP‐2 to cleave poly (ADP‐ribose) polymerase (PARP) as a substrate was examined as a possible role for MMP‐2 in the nucleus. PARP is a nuclear matrix enzyme involved in the repair of DNA strand breaks, which is known to be inactivated by proteolytic cleavage. PARP was susceptible to cleavage by MMP‐2 in vitro in a concentration‐dependent manner, yielding novel degradation products of ~66 and <45 kDa. The cleavage of PARP by MMP‐2 was also blocked by MMP inhibitors. This is the first characterization of MMP‐2 within the nucleus and we hereby suggest its possible role in PARP degradation.

Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2

OBJECTIVES Peroxynitrite (ONOO(-)) mediates in part both ischemia-reperfusion and pro-inflammatory cytokine-induced injury to the heart. As oxidants like ONOO(-) are known to activate matrix metalloproteinases (MMPs), we examined whether they play a role in the detrimental action of ONOO(-) in isolated perfused rat hearts. METHODS Hearts were isolated from Sprague-Dawley rats and perfused retrogradely with Krebs-Henseleit buffer under constant flow. Peroxynitrite (30 and 80 microM) was infused into the hearts for 15 min. The release of MMPs into the coronary effluent and level of MMPs in the myocardium were measured by gelatin zymography. RESULTS The main gelatinolytic activity in control effluent was 72-kDa corresponding to pro-MMP-2. Infusion of ONOO(-) (80 microM) for 15 min caused a vasodilatation which peaked at 5 min and then converted into vasoconstriction by 15 min. It also caused a rapid increase in the release of 72-kDa activity within 10 min and a progressive decline in cardiac mechanical function. In contrast, decomposed ONOO(-) caused no change in vascular tone, the release of 72-kDa activity or mechanical function. The MMPs inhibitor PD-166793 prevented the ONOO(-)-induced loss in myocardial mechanical function. Detoxification of ONOO(-) with glutathione prevented both the enhancement in coronary effluent 72-kDa activity and the decline in mechanical function. CONCLUSIONS Acute cardiac toxicity induced by ONOO(-) is mediated by MMP-2.

Activation and modulation of 72 kDa matrix metalloproteinase-2 by peroxynitrite and glutathione

Matrix metalloproteinase-2 (MMP-2) has emerged as a key protease in various pathologies associated with oxidative stress, including myocardial ischemia-reperfusion, heart failure or inflammation. Peroxynitrite (ONOO(-)), an important effector of oxidative stress, was reported to activate some full length MMP zymogens, particularly in the presence of glutathione (GSH), but whether this occurs for MMP-2 is unknown. Treating MMP-2 zymogen with ONOO(-) resulted in a concentration-dependent regulation of MMP-2, with 0.3-1 microM ONOO(-) increasing and 30-100 microM ONOO(-) attenuating enzyme activity. The enzymes V(max) was also significantly increased by 1 microM ONOO(-). Comparable responses to ONOO(-) treatment were observed using the intracellular target of MMP-2, troponin I (TnI). GSH at 100 microM attenuated the effects of ONOO(-) on MMP-2. Mass spectrometry revealed that ONOO(-) can oxidize and, in the presence of GSH, S-glutathiolate the MMP-2 zymogen or a synthetic peptide containing the cysteine-switch motif in the enzymes autoinhibitory domain. These results suggest that ONOO(-) and GSH can modulate the activity of 72 kDa MMP-2 by modifying the cysteine residue in the autoinhibitory domain of the zymogen, a process that may be relevant to pathophysiological conditions associated with increased oxidative stress.

Matrix metalloproteinase-2 mediates cytokine-induced myocardial contractile dysfunction.

OBJECTIVE Pro-inflammatory cytokines depress myocardial contractile function by enhancing peroxynitrite production, yet the mechanism by which peroxynitrite does this is unknown. As matrix metalloproteinases (MMPs) can be activated by peroxynitrite and can proteolytically cleave troponin I in hearts, we determined whether this occurs in cytokine-induced myocardial dysfunction. METHODS Isolated working rat hearts were perfused with buffer containing interleukin-1 beta, interferon-gamma, and tumor necrosis factor-alpha. RESULTS Cytokines induced a marked decline in mechanical function during 60-120 min of perfusion. This decline was accompanied by increased myocardial inducible NO synthase activity and perfusate dityrosine (a marker of peroxynitrite), compared to control hearts. Before the decline in mechanical function there was enhanced MMP-2 activity in the perfusate. This was accompanied by decreased tissue levels of MMP-2, tissue inhibitor of matrix metalloproteinases-4 and troponin I in cytokine-treated hearts. The collagen content of the heart was not affected by cytokine treatment. A neutralizing anti-MMP-2 antibody or the MMP inhibitors Ro31-9790 or PD166793 attenuated the decline in myocardial function. Moreover, the MMP-2 antibody prevented the decline in myocardial MMP-2 and troponin I levels. CONCLUSIONS Myocardial contractile dysfunction caused by pro-inflammatory cytokines results in MMP-2 activation and a decline in tissue inhibitor of matrix metalloproteinases-4 in the heart. Troponin I is also a target for the proteolytic action of MMP-2 during acute heart failure triggered by pro-inflammatory cytokines. Inhibition of MMPs may be a novel pharmacological strategy for the treatment of acute inflammatory heart disease.

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloprotein‐ases (MMP) has been studied extensively due to the fundamental roles these zinc‐endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP‐2 contains 29 potential phosphorylation sites. Mass spectrometryreveals that at least five of these sites are phosphorylated in hrMMP‐2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP‐2 immunoreactivity on 2D immuno‐blots consistent with phosphorylation, and purified MMP‐2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP‐2 from HT1080 cell‐conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP‐2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP‐2 significantly affects its enzymatic properties. Consistent with this, dephos‐phorylation of MMP‐2 immunoprecipitated from HT1080 conditioned medium with alkaline phospha‐tase significantly increases its activity. We conclude that MMP‐2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.—Sariahmetoglu, M., Crawford, B. D., Leon, H., Sawicka, J., Li, L., Ballermann, B. J., Holmes, C., Berthiaume, L. G., Holt, A., Sawicki, G., Schulz, R. Regulation of matrix metalloproteinase‐2 activity by phosphorylation. FASEB J. 21, 2486–2495 (2007)

Imbalance Between Tissue Inhibitor of Metalloproteinase-4 and Matrix Metalloproteinases During Acute Myoctardial Ischemia-Reperfusion Injury

Background—We have previously reported that matrix metalloproteinase-2 (MMP-2) contributes to myocardial ischemia-reperfusion injury by degradation of troponin I, a regulatory element of the contractile proteins. MMP activities are also tightly regulated by tissue inhibitors of metalloproteinase (TIMPs). The change in TIMPs during acute myocardial ischemia-reperfusion injury is not clear. Methods and Results—Isolated rat hearts were perfused either aerobically for 75 minutes or subjected to 15, 20, or 25 minutes of global, no-flow ischemia followed by 30 minutes of aerobic reperfusion. During reperfusion after ischemia, there was a rapid, enhanced release of TIMP-4, the most abundant TIMP in the heart, into the coronary effluent, as shown both by reverse zymography and Western blot. There was a negative correlation between the recovery of cardiac mechanical function and the release of TIMP-4 during reperfusion in hearts subjected to different durations of ischemia. Immunogold electron microscopy revealed a close association of TIMP-4 with the sarcomeres in aerobically perfused hearts. Moreover, TIMP-4 was present only in thin myofilaments prepared from aerobically perfused hearts but not in ischemic-reperfused hearts. An enhanced MMP activity was shown in ischemic-reperfused hearts by in situ zymography. Conclusions—Loss of TIMP-4 from the cardiac myocyte leads to an increase in net myocardial MMP activity that contributes to acute myocardial stunning injury.

The role of nitric oxide and metalloproteinases in the pathogenesis of hyperoxia-induced lung injury in newborn rats

1 The effects of nitric oxide (NO) and metalloproteinases (MMP‐2 and MMP‐9) in the pathogenesis of hyperoxia‐induced lung damage in newborn rats were examined. 2 Three‐day‐old rat pups were subjected to hyperoxia (95% O2) or room air for 7 and 14 days. 3 Some animals were treated with NG‐L‐nitro‐L‐arginine methyl ester (L‐NAME, 10 mg kg−1, s.c., daily). 4 Histology, morphometry, oedema, Ca2+‐dependent and ‐independent NO synthase (NOS) activities, expression of NOS isoforms and the activities of MMP‐2 and MMP‐9 were measured in lungs of hyperoxic and control animals. 5 Exposure of rats to hyperoxia for 7 days resulted in alveolar sac injury characterized by the presence of cellular debris, red cell extravasation and inflammatory infiltration with mononuclear cells. Lung water content, epithelial, smooth muscle layers and total airway thickness was similar to controls. 6 In contrast, exposure of rats to hyperoxia for 14 days resulted in lung oedema, inflammation and epithelial proliferation. 7 Hyperoxia caused a decrease in Ca2+‐dependent NOS activity, an effect that was associated with increased expression of eNOS protein. 8 In control rats, Ca2+‐dependent NOS activity and expression of eNOS were reduced at 14 days. 9 Hyperoxia caused 10 fold increase in the activity of Ca2+‐independent NOS that remained significantly elevated after 14 days of exposure to hyperoxia. The activity of this enzyme was unchanged in control rats. 10 In lungs of hyperoxic rats, the immunoblot showed time‐dependent, biphasic expression (peak at 7 days) of iNOS. The profile of expression of iNOS in control rats was similar. 11 The activities of MMPs were increased in lungs of hyperoxic animals. 12 The L‐NAME treatment of hyperoxic animals reduced lung oedema and epithelial proliferation, but enhanced the activities of MMPs. L‐NAME exerted no significant effects in control rats. 13 It is concluded that increased generation of NO contributes to the pathogenesis of hyperoxia‐induced lung damage in newborn rats.