Hernando Leon

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.

Effect of fish oil on arrhythmias and mortality: systematic review.

Objective To synthesise the literature on the effects of fish oil—docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)—on mortality and arrhythmias and to explore dose response and formulation effects. Design Systematic review and meta-analysis. Data sources Medline, Embase, the Cochrane Library, PubMed, CINAHL, IPA, Web of Science, Scopus, Pascal, Allied and Complementary Medicine, Academic OneFile, ProQuest Dissertations and Theses, Evidence-Based Complementary Medicine, and LILACS. Studies reviewed Randomised controlled trials of fish oil as dietary supplements in humans. Data extraction The primary outcomes of interest were the arrhythmic end points of appropriate implantable cardiac defibrillator intervention and sudden cardiac death. The secondary outcomes were all cause mortality and death from cardiac causes. Subgroup analyses included the effect of formulations of EPA and DHA on death from cardiac causes and effects of fish oil in patients with coronary artery disease or myocardial infarction. Data synthesis 12 studies totalling 32 779 patients met the inclusion criteria. A neutral effect was reported in three studies (n=1148) for appropriate implantable cardiac defibrillator intervention (odds ratio 0.90, 95% confidence interval 0.55 to 1.46) and in six studies (n=31 111) for sudden cardiac death (0.81, 0.52 to 1.25). 11 studies (n=32 439 and n=32 519) provided data on the effects of fish oil on all cause mortality (0.92, 0.82 to 1.03) and a reduction in deaths from cardiac causes (0.80, 0.69 to 0.92). The dose-response relation for DHA and EPA on reduction in deaths from cardiac causes was not significant. Conclusions Fish oil supplementation was associated with a significant reduction in deaths from cardiac causes but had no effect on arrhythmias or all cause mortality. Evidence to recommend an optimal formulation of EPA or DHA to reduce these outcomes is insufficient. Fish oils are a heterogeneous product, and the optimal formulations for DHA and EPA remain unclear.

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.

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.

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)

Inhibition of matrix metalloproteinases prevents peroxynitrite-induced contractile dysfunction in the isolated cardiac myocyte.

The potent oxidant peroxynitrite (ONOO−) induces mechanical dysfunction in the intact heart in part through activation of matrix metalloproteinase‐2 (MMP‐2). This effect may be independent of the proteolytic actions of MMPs on extracellular matrix proteins. The purpose of this study was to examine the effects of ONOO− on contractile function at the level of the single cardiac myocyte and whether this includes the action of MMPs.

Do Calcium Channel Blockers Increase the Diagnosis of Heart Failure in Patients With Hypertension

Calcium channel blockers (CCBs) are widely used to control hypertension. Previous work suggested that their use could increase heart failure (HF), which is 1 of the consequences of uncontrolled hypertension. Information about the effect of CCBs on incident HF in patients with hypertension is scarce. A systematic review was conducted to evaluate patients with hypertension treated with CCBs and incident HF. An electronic search of publications was conducted using 8 major databases. Studies were eligible if they (1) were randomized clinical trials, (2) performed comparisons of CCBs versus active control, (3) randomized >200 patients, (4) had follow-up periods >6 months, and (5) provided data regarding incident HF. Trials of renal transplantation patients, placebo-controlled trials, and HF trials were excluded. A total of 156,766 patients were randomized to CCBs or control, with a total of 5,049 events. The analysis indicated a significant increase in the diagnosis of HF in patients allocated to CCBs (odds ratio 1.18, 95% confidence interval 1.07 to 1.31). The effect observed was independent of incident myocardial infarction. Subgroup analyses indicated that patients with diabetes were at higher risk for developing HF (odds ratio 1.71, 95% confidence interval 1.21 to 2.41). In conclusion, the results suggest that patients with hypertension treated with CCBs have increased incident HF.

Hydrogen peroxide causes cardiac dysfunction independent from its effects on matrix metalloproteinase-2 activation.

Hydrogen peroxide (H2O2) causes cardiac dysfunction through multiple mechanisms. As oxidative stress can activate matrix metalloproteinases (MMPs) and, in particular, MMP-2 activity is associated with oxidative stress injury in the heart, we hypothesized that MMP-2 activation by H2O2 in isolated rat hearts contributes to cardiac dysfunction in this model. Isolated working rat hearts were perfused at 37 degrees C with a recirculating Krebs-Henseleit buffer+/-5 mmol/L pyruvate, known to protect hearts from oxidative stress. H2O2 (300 micromol/L) was added as a single bolus after 20 min of equilibration, and cardiac function was monitored for 60 min. MMPs activities in both the heart and perfusate samples were assessed by gelatin zymography. Tissue high energy phosphates were analysed by HPLC. The actions of 2 MMP inhibitors, doxycycline (75 micromol/L) or Ro 31-9790 (3 micromol/L), were also assessed. H2O2 at 300 micromol/L produced a rapid decline in cardiac mechanical function, which was maximal at 5 min. A peak in perfusate MMP-2 activity was also observed at 5 min. The deleterious effect of H2O2 on cardiac function was abolished by pyruvate but not by the MMPs inhibitors. This study suggests that in intact hearts, H2O2 induces contractile dysfunction independent of MMPs activation.

Turmoil in the Cardiac Myocyte: Acute Intracellular Activation of Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. From their first description in the process of tadpole tail resorption [1] it was recognized that they play an active role in regulating the extracellular matrix. Thus‚ over the course of the last forty years most research has focused on the extracellular role of MMPs in long term (days to months) physiological and pathological processes such as wound healing‚ embryogenesis‚ metastasis‚ atherosclerosis‚ and heart failure. Recently‚ however‚ it has been recognized that MMPs may also act: a) intracellularly‚ b) on non-extracellular matrix substrates‚ and c) over an acute time frame (i.e.‚ seconds-minutes). This chapter will review these recent developments with a special focus on the intracellular role MMPs play in acute ischemia-reperfusion injury in the heart.