Jolanta Sawicka

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.

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.

Effect of duration of ischemia on myocardial proteome in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a serious problem resulting from clinical setting of coronary revascularization. Despite extensive studies on I/R injury, the molecular bases of cardiac dysfunction caused by I/R are still unknown, but are likely to result from alterations in protein expression. Isolated rat hearts were subjected to 15–30 min of no‐flow ischemia without (Ischemia protocol) or with 30 min of reperfusion (I/R protocol). 2‐DE analysis of heart proteins from both experimental protocols showed wide‐ranging changes in protein levels. In the Ischemia protocol, 39 protein spots were changed in ischemic groups and those changes correlated with duration of ischemia. Ninety percent of the affected proteins were increased. In contrast to increased protein levels, the total messenger RNA (mRNA) level decreased approximately two fold. Compared to the Ischemia protocol, changes in protein levels in the I/R protocol did not correlate with the duration of ischemia and the degree of recovery of mechanical function. The decrease of affected protein from I/R protocol was associated with the increase in total protein level in reperfusate. Our studies show that the protein increase is correlated with the mechanical function of the I/R hearts and the increase is not likely associated with an increase in protein synthesis.

Ischemia induced peroxynitrite dependent modifications of cardiomyocyte MLC1 increases its degradation by MMP-2 leading to contractile dysfunction

Damage to cardiac contractile proteins during ischemia followed by reperfusion is mediated by reactive oxygen species such as peroxynitrite (ONOO−), resulting in impairment of cardiac systolic function. However, the pathophysiology of systolic dysfunction during ischemia only, before reperfusion, remains unclear. We suggest that increased ONOO− generation during ischemia leads to nitration/nitrosylation of myosin light chain 1 (MLC1) and its increased degradation by matrix metalloproteinase‐2 (MMP‐2), which leads to impairment of cardiomyocyte contractility. We also postulate that inhibition of ONOO− action by use of a ONOO− scavenger results in improved recovery from ischemic injury. Isolated rat cardiomyocytes were subjected to 15 and 60 min. of simulated ischemia. Intact MLC1 levels, measured by 2D gel electrophoresis and immunoblot, were shown to decrease with increasing duration of ischemia, which correlated with increasing levels of nitrotyrosine and nitrite/nitrate. In vitro degradation of human recombinant MLC1 by MMP‐2 increased after ONOO− exposure of MLC1 in a concentration‐dependent manner. Mass spectrometry analysis of ischemic rat cardiomyocyte MLC1 showed nitration of tyrosines 78 and 190, as well as of corresponding tyrosines 73 and 185 within recombinant human cardiac MLC1 treated with ONOO−. Recombinant human cardiac MLC1 was additionally nitrosylated at cysteine 67 and 76 corresponding to cysteine 81 of rat MLC1. Here we show that increased ONOO− production during ischemia induces MLC1 nitration/nitrosylation leading to its increased degradation by MMP‐2. Inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO− scavenger FeTPPS (5,10,15,20‐tetrakis‐[4‐sulfonatophenyl]‐porphyrinato‐iron[III]), or inhition of MMP‐2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility.

Neonatal asphyxia induces the nitration of cardiac myosin light chain 2 that is associated with cardiac systolic dysfunction.

Hypoxia followed by reoxygenation (H-R) observed during perinatal asphyxia is a serious complication with high mortality and morbidity rates that may cause adverse cardiovascular effects in neonates. Our aim was to determine if oxidative stress related to H-R induces peroxynitrite-dependent modifications of the cardiac contractile protein, myosin regulatory light chain 2 (MLC2), and whether this is associated with development of cardiac systolic dysfunction. Twelve newborn piglets were acutely instrumented for hemodynamic monitoring and randomized to a control group ventilated with only atmospheric air or to the H-R study group exposed to alveolar normocapnic hypoxia followed by reoxygenation. Afterward, animals were euthanized, and the hearts were harvested for biochemical analyses. Systolic function as well as cardiac MLC2 levels decreased in H-R animals, whereas nitrates and nitrotyrosine levels increased. Negative correlations between nitrates, nitrotyrosine, and MLC2 levels were observed. Moreover, H-R induced nitration of two tyrosine residues within the MLC2 protein. Similarly, in vitro exposure of MLC2 to peroxynitrite resulted in the nitration of tyrosine, which increased the susceptibility of MLC2 to subsequent degradation by matrix metalloproteinase 2. Substitution of this tyrosine with phenylalanine prevented the matrix metalloproteinase 2-dependent degradation of MLC2. In addition, a large decrease in MLC2 phosphorylation caused by H-R was observed. Oxidative stress related to asphyxia induces nitration of cardiac MLC2 protein and thus increases its degradation. This and a large decrease in MLC2 phosphorylation contribute to the development of systolic dysfunction. Inhibition of MLC2 nitration and/or direct inhibition of its degradation by MMP-2 could be potential therapeutic targets aiming at reduction of myocardial damage during resuscitation of asphyxiated newborns.

Inhibition of endogenous nitric oxide in the heart enhances matrix metalloproteinase‐2 release

1 Matrix metalloproteinase (MMP) activity is upregulated in pathologies such as atherosclerosis during which endogenous nitric oxide (NO) biosynthesis is reduced. Diminished levels of NO, an antioxidant species, may result in higher oxidative stress. Oxidants are capable of activating MMPs from their zymogen forms. We examined whether basal biosynthesis of NO in the coronary circulation regulates MMP‐2 activity. 2 In isolated rat hearts perfused with Krebs–Henseleit buffer at a constant flow of 10 ml min−1, we measured the release of MMP‐2 into the coronary effluent by gelatin zymography. The main gelatinolytic activity of 72‐kDa corresponds to MMP‐2. Infusion of the NO synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) concentration dependently increased coronary perfusion pressure (CPP) (by 48±11 mmHg with 100 μM) and enhanced the release of the 72‐kDa MMP‐2 in the effluent. Coinfusion of the NO donor S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP, 1 μM) with L‐NAME abolished both the increase in CPP and the enhanced MMP‐2 release. 3 The thromboxane A2 mimetic U46619 increased CPP to the same extent as L‐NAME without increasing 72‐kDa activity in the effluent, suggesting that MMP‐2 release is not caused simply by enhanced perfusion pressure. 4 Infusion of either L‐NAME or U46619 did not significantly enhance LDH release. 5 L‐NAME infusion concentration dependently increased the level of lipid hydroperoxides in homogenates prepared from the perfused hearts. Coinfusion of SNAP prevented this increase. 6 These data reveal another cytoprotective mechanism of endogenous NO biosynthesis in the heart, the inhibition of MMP‐2 release.

Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase 2.

Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase 2 (MMP‐2) during myocardial ischemia/reperfusion (I/R) has been demonstrated. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP‐2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). When hearts were subjected to I/R in the presence of ML‐7 (a myosin light‐chain kinase inhibitor) or doxycycline (an MMP inhibitor), improved recovery of contractile function was observed compared to aerobic controls, and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP‐2 for the phosphorylated form of MLC1 compared to non‐phosphorylated MLC1. We conclude that MLC1 phosphorylation is an important mechanism controlling the intracellular action of MMP‐2 and promoting degradation of MLC1. These results further support previous findings implicating post‐translational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.

Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion.

Ischemia/reperfusion (I/R) injury is a major consequence of a cardiovascular intervention. The study of changes of the left and right ventricle proteomes from hearts subjected to I/R may be a key to revealing the pathological mechanisms underlying I/R‐induced heart contractile dysfunction. Isolated rat hearts were perfused under aerobic conditions or subjected to 25 min global ischemia and 30 min reperfusion. At the end of perfusion, right and left ventricular homogenates were analyzed by 2DE. Contractile function and coronary flow were significantly reduced by I/R. 2DE followed by mass spectrometry identified ten protein spots whose levels were significantly different between aerobic left and right ventricles, eight protein spots whose levels were different between aerobic and I/R left ventricle, ten protein spots whose levels were different between aerobic and I/R right ventricle ten protein spots whose levels were different between the I/R groups. Among these protein spots were ATP synthase beta subunit, myosin light chain 2, myosin heavy chain fragments, peroxiredoxin‐2, and heat shock proteins, previously associated with cardiovascular disease. These results reveal differences between proteomes of left and right ventricle both under aerobic conditions and in response to I/R that contribute to a better understanding of I/R injury.