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

The regulation of matrix metalloproteinases (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 spectrometry reveals 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 immunoblots 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, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase 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.

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.

Cardiovascular autonomic function testing in asymptomatic T. cruzi carriers: a sensitive method to identify subclinical Chagas' disease.

BACKGROUND: Although impaired cardio-vagal response characterizes full-blown Chagas' disease, this feature among otherwise healthy T. cruzi serology carriers (SERO[+]) requires confirmation. The purpose of this study was to determine whether abnormal cardio-vagal responses were different among SERO[+] subjects with varying ECG alterations. METHODS: We assessed cardio-vagal reflex response in 57 randomly selected healthy blood donors (36 SERO[+], 15 with ECG rhythm/conduction abnormalities). The following cardiac autonomic tests were performed: (1) short-term heart rate variability (HRV), (2) Deep breathing test (DBT), (3) cold face test, (4) cold pressor test (CPT), (5) Valsalva maneuver, and (6) baroreflex sensitivity after administration of nitroprusside (BRS-NTP) and phenylephrine (BRS-PNP). RESULTS: Overall, SERO[+] subjects had 161/324 (49.7%) abnormal responses, compared to 41/189 (21.7%) in SERO[-] (p<0.001). Similar rates were found in SERO[+] according to ECG status (68/135, 50.4% in ECG[+] and 93/189, 49.2% in ECG[-], p=0.836). Covariate-adjusted pooled odd ratios (95%CI) for abnormal responses compared to SERO[-] were: 2.73 (1.71-4.35) for SERO[+], and 2.63 (1.63-4.34) for SERO[+]/ECG[-] (p<0.001). BRS-NTP, CPT and DBT individually showed significant differences between SERO[-] and SERO[+] groups. Conversely, ECG changes among SERO[+] were not associated with a significant excess of autonomic abnormality either overall (OR=1.09, 95%CI: 0.67-1.78, p=0.719) or by any individual test. CONCLUSIONS: Early cardio-vagal dysfunction was documented in SERO[+] subjects regardless of ECG status. Cardiac autonomic evaluation may be useful for identification of subclinical disease in SERO[+] subjects.

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.

Activation and modulation of 72kDa 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 enzyme's 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 enzyme's 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.

Inhibiting matrix metalloproteinase-2 reduces protein release into coronary effluent from isolated rat hearts during ischemia-reperfusion.

BACKGROUND: Previous studies have shown that the disruption of the coronary endothelium and the increase in its permeability during ischemia-reperfusion (I/R), are linked to matrix metalloproteinase-2 (MMP-2) activity. Studies from our group have shown that during I/R, activity of MMP-2 in the coronary effluent increases and this increase is associated with cardiac dysfunction, which in turn, can be prevented by MMP inhibitors. Therefore, we hypothesize that inhibiting MMPs reduces the MMP-2 dependent disruption of the coronary endothelium and subsequent protein release during I/R. METHODS: Isolated rat hearts were perfused in the Langendorff mode at a constant pressure and subjected to 15, 20 or 30 min no-flow ischemia followed by 30 min of reperfusion. The MMP inhibitors, o-phenanthroline (Phen, 100 microM) or doxycycline (Doxy, 30 microM) an inhibitors of MMPs, were added to the perfusion solution 10 min before ischemia and for the first 10 min of reperfusion. The coronary effluents were collected during perfusion for protein analysis. Creatine kinase was measured as an index of cellular damage. Endothelial integrity was assessed by measuring coronary flow and by measuring the levels of serotransferrin and interstitial albumin in the coronary effluent. Additionally, damage to the endothelium was assessed histologically by light microscopy analysis of the cellular structure of the myocardium. MMP-2 activity was measured by zymography in hearts subjected to 15, 20 and 30 min of ischemia without reperfusion. RESULTS: MMP-2 activity was increased in heart tissue at the end of ischemia and was correlated with duration of ischemia. The post-ischemia decrease in coronary flow, and the increase in the release of serotransferrin and albumin were attenuated by Phen. Edema (another indirect marker of endothelial damage) was observed in I/R heart and the edema was abolished in I/R heart treated with MMP inhibitors. CONCLUSION: MMP inhibition not only reduces cardiac mechanical dysfunction but also reduces endothelial damage resulting from cardiac I/R injury.

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.

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.

Impaired baroreflex gain in patients with inappropriate sinus tachycardia.

INTRODUCTION: The aim of this study was to determine the characteristics of heart rate variability (HRV), blood pressure variability (BPV), and baroreflex gain (BRG) at rest and during orthostatic stress in patients with clinical criteria of inappropriate sinus tachycardia (IST). METHODS AND RESULTS: Beat-to-beat HRV and BPV, measured by time- and frequency-domain methods, and noninvasive BRG, calculated by cross-spectral analysis, were obtained during 10 minutes both at rest and during the stabilization phase (5-15 min) of orthostatic stress at 60 degrees in 8 patients with clinical criteria of IST and 9 healthy volunteers (CON). IST patients had a higher resting mean heart rate (78.8 +/- 5.3 vs 58.5 +/- 4.2 beats/min, P=0.01) and mean blood pressure (90.4 +/- 2.4 vs 72.0 +/- 4.2 mmHg; P=0.002). RMSSD, pNN50m, and BRG were significantly reduced in IST patients at rest. BRG during orthostatic stress (7.2 +/- 0.8 vs 20.3 (2.4 ms/mmHg, P <0.01) was significantly reduced in IST patients. Delta BRG (-16.9%+/- 11 vs -50.1%+/- 5, P=0.02) was markedly blunted during orthostatic stress in IST patients. CONCLUSION: BRG was markedly impaired both at rest and during orthostatic stress in IST patients. This alteration may be responsible for the higher resting heart rate and mean blood pressures seen at rest and may facilitate tachycardia during orthostatic stress. A primary alteration in sinus node automaticity coupled with impaired BRG determines heart rate response to orthostatic stress in patients with IST.

Pyruvate prevents cardiac dysfunction and AMP-activated protein kinase activation by hydrogen peroxide in isolated rat hearts.

Ischemia-reperfusion injury in the heart results in enhanced production of H2O2 and activation of AMP-activated protein kinase (AMPK). Since mutations in AMPK result in cardiovascular dysfunction, we investigated whether the activation of AMPK mediates the H2O2-induced reduction in cardiac mechanical function. Isolated working rat hearts were perfused at 37 degrees C with Krebs-Henseleit solution. Following a 20-minute equilibration period, a single bolus of H2O2 (300 micromol/L) was added and the hearts were perfused for an additional 5 min. H2O2 induced a dramatic and progressive reduction in cardiac function. This was accompanied by rapid and significant activation of AMPK, an increase in Thr-172 phosphorylation of AMPK, and an increase in the creatine to phosphocreatine (Cr/PCr) ratio. Addition of pyruvate (5 mmol/L) to the perfusate prevented the H2O2-mediated reduction in cardiac mechanical dysfunction, activation of myocardial AMPK activity, increase in AMPK phosphorylation and the increase in the Cr/PCr ratio. Hearts challenged with H2O2 (300 micromol/L) in presence of either AMPK inhibitor Compound C (10 micromol/L) or its vehicle (dimethyl sulfoxide (DMSO), 0.1%) showed reduced impairment in cardiac mechanical function. Compound C but not its vehicle significantly inhibited myocardial AMPK activity. Thus, H2O2 induces cardiac dysfunction via both AMPK-dependent and independent mechanisms.

Una válvula de de exclusión de gases eficiente y de bajo costo

Es ampliamente conocido de todos los anestesiólogos, la patología ocupacional producida por la inhalación crónica de agentes anestésicos volátiles cuando se trabaja con máquinas de anestesia que no tienen incorporada válvulas de exclusión de gases fuera de los quirófanos. El excedente de los gases que no es utilizado por el paciente va a parar a la atmósfera ambiental del anestesiólogo. En el mercado existe una gran variedad de formas, tipos y usos de válvulas; las más comunes son la de globo pistón, aguja, orificios y la de bola. Nosotros nos proponemos presentar la válvula de bola o de media vuelta como se la conoce comunmente. Se consigue en PVC, latón, bronce, acero cromado y acero inoxidable, recomendamos esta última. El actor principal de la válvula es una esfera que tiene una gran perforación de lado a lado. Esta esfera se gira para darle graduación, con la ayuda de una palanca, determinando la posición deseada, lo que implica el grado de restricción al flujo circulante. Este control nos lleva a variaciones de presión en el circuito respiratorio. A la salida de la válvula se le conecta una manguera transparente plástica tipo jardín y se saca fuera del quirófano o se coloca en la rejilla de aspiración del aire acondicionado central. Dados los resultados excelentes obtenidos con esta válvula nos permitimos recomendar su uso en todo tipo de aparato de anestesia, incluyendo los pediátricos

Dolor ovulatorio / Ovulatory pain

I. La disimilitud de las estadísticas sobre el dolor ovulatorio se deriva de la dificultad en la apreciación del síntoma por parte de la paciente y el médico. II. El autor entiende el dolor ovulatorio como entidad patológica únicamente cuando el síntoma es de por si suficiente para llamar seriamente la atención de la enferma. III. Presenta 2 casos sobre 897 historias clíncas revisadas, o sea el 0.22 por ciento. IV. Buenos resultados terapéuticos con 50 miligramos de testosterona inyectados en dos dosis el día anterior y 3 días antes de la fecha de ovulación

Epítope reumatoide y alelo HLA-DRB1*0404 se asocian con susceptibilidad para artritis reumatoide en mestizos peruanos alelo HLA-DRB1*1402 en duda y la combinación maligna *0401/0404 ausente / Rheumatoide Epitope and I stupefy Hla-drb1*0404 are associated with susceptibility for rheumatoid arthritis in Peruvian mestizos I stupefy Hla-drb1*1402 in 0401/0404 doubt and combination vitiates* absentee

Objetivo: Evaluar en mestizos peruanos con artritis reumatoide (AR) la asociación existente entre la enfermedad (características diagnósticas y clínicas) y los alelos portadores del epítope reumatoide (ER). Pacientes y métodos: Estudio de corte transversal en 52 pacientes con AR (criterios ACR) y 81 controles sanos - no consanguíneos-apareados por edad y sexo. Tipificación de baja resolución para HLA-DRB1 (DR1, DR",DR3/5/5/8, DR4, DR7, DR9, DR10) así como para DQA1 y DQB1. Tipificación de alta resolución para alelos específicos. Los alelos HLA_DR4 fueron confirmados por secuencia y directa del exón 2. Resultados: El ER estuvo presente en 32 (61.5 por ciento de los pacientes y 25 (31 por ciento) de los controles (OR:= 3.58; 95 por ciento IC = 1.73, 7.44; p =0.0004). Sólo el alelo HLA-DRB1* 0404 (en 11 pacientes y en 4 controles se asoció con AR: (OR = 5.16;95 por ciento IC=1.55,17.24;p = 0.003). No encontramos asociación entre AR y el alelo HLA-DRB1*1402, tampoco entre características de la AR y algún alelo específico DRB1. Ningún paciente fue DRB1*0401/10404. Conclusiones: La asociación entre AR y el ER en mestizos peruanos es más débil que la informada en caucásicos, pero mayor que la informada en afro-americanos. El alelo DBR1*0404 es el demayor prevalencia y el único con asociación estadísticamente significativa. La asociación con el DRB1*1402 no fue significativa. El DBR1*0401 tiene escasa prevalencia y no se combinó con el DBR1*0404 en ningún caso. La mayor discrepancia con un estudio previo obedece a diferencias observadas entre los grupos control.

Cambios en la función automática cardíaca en sujetos seropositivos a T. cruzi asintomáticos

La secuencia fisiopatológica que conduce a la enfermedad de Chagas crónica a partir de un sujeto con infección asintomática no ha sido aclarada aun. Con el fin de evaluar su función autonómica cardíaca, se estudiaron donantes de sangre clínicamente sanos sero (+)(n=34; 32,6ñ1.9 años) o sero (-) (n=22; 38,7ñ1.8 años) a T. cruzi. El grupo sero (+) se subdividió en Chagas 1 (n=22) con electrocardiograma normal y Chagas 2 (n=12), con bradicardia sinusal, bloqueos de conducción o extrasístoles ventriculares. Usando el software CAFTS (Medykro) se analizaron la frecuencia cardíaca (FC) y la presión arterial media (PAM) basales, el poder espectral (TPS) y la relación entre los componentes de baja y alta frecuencia (LF/HF), la varianza de los intervalos R-R normales (RMSSD); la disminución porcentual de la FC a la prueba de frio en cara (CFT), porcentaje de aumento de la PAM en la prueba presora al frio (CPT), y los índices FC náxima/mínima en respiración profunda controlada (DBT), de Valsava (VM) y de sensibilidad barorrefleja (BRS). Los anteriores hallazgos demuestran la existencia de trastornos de la función autonómica cardíaca previos al desarrollo de la enfermedad clínica. Una disminución de los reflejos cardiovagales asociada a un tono simpático aumentado carcterizan al seropositivo asintomático. Estos hallazgos pueden estar relacionados con el desarrollo de la cardiomiopatía chagásica

Disfunción autonómica en sujetos seropositivos para T. cruzi asintomáticos

La enfermedad de Chagas crónica se caracteriza por daño cardiaco microvascular, contráctil y autonómico, sin que se comprenda completamente cuál de ellos determina el inicio de la enfermedad. Con el fin de edentificar alteraciones autonómicas en sujetos asintomáticos seropositivos para Tripanosoma cruzi, se evaluó la función autonómica cardíaca en 18 donantes de banco seropositivos (11 "Chagas 1") con electrocardiograma normal y siete "Chagas 2" con alteraciones del ritmo o de conducción) y 24 individuos seronegativos. Se realizaron tres pruebas de eferencia parasimpática (frecuencia cardíaca máxima/mínima en respiración profunda, frío en cara y respuesta presora al frío) y dos de eferencia simpática (respuesta cronotrópica y presora en la mesa inclinada). Durante el procedimiento se registró también el número variaciones de la frecuencia cardíaca (VFC) utilizando un oxímetro de pulso. Se encontraron diferencias significativas (p<0.033) en la frecuencia cardíaca basal y en las pruebas de respiración profunda, frío en cara y la respuesta presora a la mesa inclinada. La VFC fue menor en el grupo Chagas 2 en la mayoría de las pruebas. La respuesta parasimpática fue menor para el grupo Chagas 2. La función simpática se caracterizó por aumento del tono basal en el grupo Chagas 1. Estos hallazgos sugieren disautonomía en sujetos seropositivos asintomáticos. Se requieren estudios con mejores instrumentos y mayores poblaciones, para consolidar los conocimientos en este campo