Oxidative stress-induced, peroxynitrite-dependent, modifications of myosin light chain 1 lead to its increased degradation by matrix metalloproteinase-2

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function. In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at 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. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction

Effects of MMP-9 inhibition by doxycycline on proteome of lungs in high tidal volume mechanical ventilation-induced acute lung injury

Abstract Background Although mechanical ventilation (MV) is a major supportive therapy for patients with acute respiratory distress syndrome, it may result in side effects including lung injury. In this study we hypothesize that MMP-9 inhibition by doxycycline might reduce MV-related lung damage. Using a proteomic approach we identified the pulmonary proteins altered in high volume ventilation-induced lung injury (VILI). Forty Wistar rats were randomized to an orally pretreated with doxycycline group (n = 20) or to a placebo group (n = 20) each of which was followed by instrumentation prior to either low or high tidal volume mechanical ventilation. Afterwards, animals were euthanized and lungs were harvested for subsequent analyses. Results Mechanical function and gas exchange parameters improved following treatment with doxycycline in the high volume ventilated group as compared to the placebo group. Nine pulmonary proteins have shown significant changes between the two biochemically analysed (high volume ventilated) groups. Treatment with doxycycline resulted in a decrease of pulmonary MMP-9 activity as well as in an increase in the levels of soluble receptor for advanced glycation endproduct, apoliporotein A-I, peroxiredoxin II, four molecular forms of albumin and two unnamed proteins. Using the pharmacoproteomic approach we have shown that treatment with doxycycline leads to an increase in levels of several proteins, which could potentially be part of a defense mechanism. Conclusion Administration of doxycycline might be a significant supportive therapeutic strategy in prevention of VILI.</p

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

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