Francisco Moraga

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains.

Relation between oxidative stress, catecholamines, and impaired chronotropic response to exercise in patients with chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy

O stress has been implicated in the pathogenesis of chronic heart failure (HF). Different studies have shown that reactive oxygen species are produced in the failing myocardium, causing injury in cardiac myocytes.1–3 Different factors classically associated with cardiomyocyte dysfunction and death in chronic HF, such as increased plasma catecholamine levels, are well-known stimuli for the generation of reactive oxygen species.4 In patients with advanced chronic HF at rest, the circulating norepinephrine concentrations are much higher, generally 2 to 3 times the level found in normal subjects.5,6 During comparable levels of exercise, much greater elevations in circulating norepinephrine occur in patients with chronic HF than in normal subjects. However, despite the increase in norepinephrine with exercise, patients with chronic HF had an attenuated heart rate response to exercise; this finding has been attributed to postsynaptic desensitization of the -adrenergic receptor pathway.7 A relation between cardiac exercise capacity and oxidative stress determined by malondialdehyde (MDA) plasma levels, a marker of lipid peroxidation, has been proposed.8 Experimental data also suggest that hydrogen peroxide may attenuate the -adrenoceptor– linked signal transduction in the heart by changing the functions of Gs proteins and the catalytic subunit of the adenylyl cyclase.9 In the present study we investigated the association between MDA plasma levels, catecholamines at peak exercise, and impaired heart rate response to exercise in patients with chronic HF. • • • We enrolled 27 patients with chronic HF secondary to coronary heart disease (n 15) or idiopathic dilated cardiomyopathy (n 12). They fulfilled the following criteria: (1) chronic stable HF in New York Heart Association functional classes II to IV; (2) ability to complete a symptom-limited treadmill exercise test; (3) evidence of left ventricular (LV) dilation and LV ejection fraction 40% as determined by radionuclide-gated pool scan; and (4) treatment with diuretics, digitalis, and vasodilators. We excluded patients with (1) coronary artery bypass surgery, angioplasty, or myocardial infarction in the last 6 months; (2) chronic angina; (3) uncontrolled hypertension (systolic blood pressure 160 mm Hg or diastolic blood pressure 90 mm Hg); (4) hypertensive myocardiopathy; (5) change in maintenance therapy or use of blockers in the last 2 months; (6) implanted pacemaker; (7) significant valvular disease; and (8) presence of other conditions that affect determination of oxidative stress status, such as renal insufficiency (plasma creatinine 2 mg/dl), autoimmune diseases, neoplasia, advanced liver or pulmonary disease, and acute or chronic inflammation. All patients signed an informed consent approved by our institutional review board and ethics committee. For clinical assessment we used New York Heart Association functional class and the Mahler et al10 clinical score (range 0 to 12 points), which evaluates the severity of dyspnea. The score depends on ratings for 3 different categories: functional impairment, magnitude of task, and magnitude of effort. Dyspnea is rated in 5 degrees from 0 (severe) to 4 (unimpaired) for each category. The ratings for each of the 3 categories are added to form the score. LV end-diastolic and LV end-systolic diameters were determined by Doppler 2-dimensional echocardiography, and LV ejection fraction was determined by radionuclide ventriculography. Each patient performed a 6-minute corridor walk test and a maximal exercise test with gas exchange. Plasma norepinephrine and epinephrine specimens were collected from an indwelling venous line after patients had been in the supine position in a quiet room for 30 minutes. Measurements were repeated at maximal exercise. Determination of catecholamines was performed by high-performance liquid chromatography using a commercial kit (Chromsystems Instruments & Chemicals GmbH, Munchen, Germany). The interand intra-assay coefficients were 6% and 5%, respectively. The ratio of the increment in heart rate divided by the increment in norepinephrine from From the Department of Cardiovascular Diseases, Faculty of Medicine, P. Catholic University of Chile; and the Departments of Biochemistry and Molecular Biology and Chemical Pharmacology and Toxicology, Faculty of Chemical and Pharmaceutical Sciences, Faculty of Medicine and the FONDAP Center for Molecular Studies of the Cell, University of Chile, Santiago, Chile. Dr. Castro was supported in part by Grant FONDECYT 1010992, Santiago; and Dr. Lavandero was supported in part by Grant FONDAP 15010006, Santiago, Chile. Dr. Castro’s address is: Department of Cardiovascular Diseases, Faculty of Medicine, P. Catholic University of Chile, Marcoleta 367, Santiago, Chile. E-mail: pcastro@med.puc.cl. Manuscript received January 23, 2003; revised manuscript received and accepted April 7, 2003.

Matrix metalloproteinase-9 activity is associated to oxidative stress in patients with acute coronary syndrome

Abstract In the present work we evaluate the relationship between oxidative stress and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activities in 44 patients with non ST-elevation acute coronary syndrome. We found an early increase in malondialdehyde (MDA) levels (oxidative stress marker) and MMP-9, with decrease of both at day five. A positive correlation was found between fractional changes of MDA and MMP-9, suggesting a common role in the pathophysiology of the acute coronary syndrome.

Osmotically-induced genes are controlled by the transcription factor TonEBP in cultured cardiomyocytes.

Changes in cardiac osmolarity occur in myocardial infarction. Osmoregulatory mechanisms may, therefore, play a crucial role in cardiomyocyte survival. Tonicity-responsive enhancer binding protein (TonEBP) is a key transcription factor participating in the adaptation of cells to increases in tonicity. However, it is unknown whether cardiac TonEBP is activated by tonicity. Hypertonicity activated transcriptional activity of TonEBP, increased the amounts of both TonEBP mRNA and protein, and induced both the mRNA and protein of TonEBP target genes (aldose reductase and heat shock protein-70). Hypotonicity decreased the amount of TonEBP protein indicating bidirectional osmoregulation of this transcription factor. Adenoviral expression of a dominant negative TonEBP suppressed the hypertonicity-dependent increase of aldose reductase protein. These results indicated that TonEBP controls osmoregulatory mechanisms in cardiomyocytes.

Adiposidad visceral y su asociación con lípidos séricos e insulinemis en adolescentes obesas

BACKGROUND Increased visceral or abdominal adipose tissue in children and adults is strongly associated with metabolic and a variety of chronic diseases. AIM To study the association between visceral or external body measurements of adiposity with blood lipids, glucose and insulin levels, in obese female adolescents. MATERIAL AND METHODS In a cross-sectional study, 47 obese female adolescents (body mass index (BMI) >95th percentile) aged 10 to 15 years, were analyzed. Weight, height, BMI, Tanner pubertal stages, skinfold thickness, waist circumference, waist-to-hip ratio, fasting and 120 min post prandial blood glucose, serum insulin, and lipid profile were studied. Visceral fat was assessed by computed tomography at the L4-L5 level, measuring the fat area or the length of a straight drawn line between the spine and the internal border of the rectus abdominus muscle. RESULTS No association between lipid profile and BMI or external body measurements (skinfold thickness, waist circumference, waist-to-hip ratio) was observed. Total serum cholesterol >170 mg/dL was positively associated with the straight line over 63 mm (a cut-off obtained by ROC analysis (RR 2.64; 1.15-6.08). This association was statistically significant in girls in Tanner I + II (n =21; Fisher, p <0.023), but not with Tanner III + IV (n=26) stages. Increased cholesterol (>170 mg/dL) was also positively associated with a serum insulin >17 uU/mL in the Tanner I + II group (Fisher p<0.05), but not with the homeostasis model assessment of insulin resistance (HOMA). CONCLUSIONS No external body measurement of adiposity was associated to increased serum cholesterol in these obese female adolescents. Increased total cholesterol (>170 mg/dL) was associated with visceral fat (evaluated through the straight line spina-rectus abdominus muscle), and also with a serum insulin >17 uU/ml in those teenagers with Tanner I or II pubertal stages.

Persistencia del estrés oxidativo postrasplante cardíaco: estudio comparativo entre pacientes con trasplante cardíaco y con insuficiencia cardíaca crónica estable

Introduccion y objetivo Existe estres oxidativo en pacientes con insuficiencia cardiaca cronica (ICC). El trasplante cardiaco, alternativa terapeutica importante en estos pacientes, podria disminuir el estres oxidativo al mejorar la funcion cardiaca. Nuestro objetivo fue evaluar el estres oxidativo postrasplante cardiaco. Pacientes y metodo Fueron estudiados 3 grupos experimentales: a) trasplantados cardiacos, sin evidencia de rechazo (n = 11); b) pacientes con ICC capacidad funcional III de la NYHA (n = 19), y c) sujetos controles sanos (n = 14). El estres oxidativo se evaluo determinando valores plasmaticos de malondialdehido (MDA), y actividades de glutation peroxidasa (GSH-Px), catalasa (CAT) y superoxido dismutasa (SOD). Resultados Las caracteristicas demograficas fueron similares entre los grupos. El tiempo postrasplante fue 20,0 ± 4,8 meses. Los valores de MDA en trasplantados y con ICC fueron significativamente mayores que en sujetos normales (3,35 ± 0,8; 3,27 ± 1,7, y 0,90 ± 0,3 µM, respectivamente). La actividad de GSH-Px aumento en trasplantados respecto al grupo control (0,40 ± 0,07 y 0,33 ± 0,05 U/g Hb, respectivamente). La actividad de SOD fue menor en trasplantados respecto al grupo control ICC (0,44 ± 0,1 frente a 0,87 ± 0,6 U/mg Hb). No hubo diferencias en las actividades de CAT entre trasplantados y pacientes con ICC. Conclusion Los pacientes sometidos a trasplante cardiaco tienen un aumento del estres oxidativo, evidenciado por una elevacion del MDA y por una disminucion de la actividad de SOD, a pesar de una mayor actividad de GSH-Px. Este aumento del estres oxidativo fue similar al encontrado en pacientes con ICC estable CF III de la NYHA, y se observo en ausencia de episodios reconocidos de infeccion o rechazo.

Mediciones de adiposidad intraabdominal por ultrasonido y factores asociados con riesgo cardiovascular en niños obesos

BACKGROUND Cardiovascular risk factors are commonly present in obese children. AIM To evaluate the association among radiological measurements of intra-abdominal adipose tissue, and cardiovascular risk factors, in prepuberal obese children. PATIENTS AND METHODS We evaluated 30 obese (body mass index > p95) children aged 6 to 12 years (15 males). Anthropometry and blood pressure were measured. Subcutaneous and intra-abdominal fat thickness and fat area were measured by ultrasound (US) and computed tomography. Serum insulin, glucose and lipid profile were measured in a fasting blood sample. Homeostasis model assessment (HOMA) was calculated as an index of insulin resistance. RESULTS There was a significant correlation between US intra-abdominal fat thickness and HOMA (r = 0.47, p < 0.01), serum triglycerides (r = 0.46, p < 0.05) and with positive criteria for metabolic syndrome (r = 0.66, p < 0.01). A receiver operating curve (ROC) analysis showed that, above a cut-off of 45 mm for intra-abdominal fat thickness, US was able to identify insulin resistance with a sensibility and specificity of 79 and 69% respectively and metabolic syndrome with sensibility and specificity of 100 and 67% respectively. US and computed tomography measurements for intra-abdominal fat thickness were significantly correlated (r= 0.62, p < 0.01). CONCLUSIONS US measurements of intra-abdominal fat thickness identify obesity-associated damage in childhood. Age-specific measurements of intra-abdominal adipose tissue may improve the detection power of this approach.

Effects of Carvedilol on Functional Capacity, Left Ventricular Function, Catecholamines, and Oxidative Stress in Patients With Chronic Heart Failure

INTRODUCTION AND OBJECTIVE Carvedilol is an antioxidant and adrenergic antagonist with demonstrated benefits in terms of mortality from heart failure (HF). The aim of the present study was to evaluate clinical parameters, left ventricular function, oxidative stress levels and neurohumoral status at baseline and after 6 months of treatment with carvedilol in patients with chronic HF. PATIENTS AND METHOD Thirty patients with chronic HF that was stable without beta blocker treatment were included. Functional class was II or III, and left ventricular ejection fraction (LVEF) was < 40%. Mahler score, distance walked in 6 min, peak oxygen consumption, LVEF, plasma catecholamine (norepinephrine) concentration and oxidative stress parameters were evaluated at baseline and after 6 months of treatment with carvedilol. RESULTS Mean age was 59 (2) years, and 23 patients were men. After 6 months of treatment there were clinical improvements as measured by the Mahler score (from 6.8 to 11.0 points; P=.001) and the 6-min walk distance (from 499 [18] to 534 [17] m; P =.032), but no changes in peak oxygen consumption. The LVEF increased from 24 (1) to 31 (2)% (P=.003). In patients with chronic HF, plasma malondialdehyde concentration was significantly lower after 6 months (decrease from 2.4 [0.2] to 1.1 [0.2] micromol/l; P<.001). No significant changes were observed in plasma catecholamine levels or antioxidant enzyme activities. CONCLUSIONS In patients with chronic HF, carvedilol treatment for 6 months was associated with clinical improvements, increased left ventricular function and decreased plasma concentrations of malondialdehyde, with no changes in plasma catecholamine levels.

Interacción entre los polimorfismos del receptor ß1 y ß2 adrenérgico como predictor de riesgo de insuficiencia cardiaca crónica

e ha propuesto que los polimorfismos geneti-cos se asocian a la genesis o desarrollo dediversas enfermedades cardiovasculares y a lavariabilidad individual a la respuesta farmacologi-ca. Los receptores s-adrenergicos (s-RA) sonreguladores importantes de la homeostasis cardio-vascular, siendo ademas un atractivo modelo parainvestigar las interacciones entre sus polimorfis-mos con la respuesta a farmacos o su relacion conla susceptibilidad o progresion de las enfermeda-des cardiovasculares