Unbalance between sarcoplasmic reticulum Ca2 + uptake and release: A first step toward Ca2 + triggered arrhythmias and cardiac damage

The present review focusses on the regulation and interplay of cardiac SR Ca2+ handling proteins involved in SR Ca2+ uptake and release, i.e., SERCa2/PLN and RyR2. Both RyR2 and SERCA2a/PLN are highly regulated by post-translational modifications and/or different partners’ proteins. These control mechanisms guarantee a precise equilibrium between SR Ca2+ reuptake and release. The review then discusses how disruption of this balance alters SR Ca2+ handling and may constitute a first step toward cardiac damage and malignant arrhythmias. In the last part of the review, this concept is exemplified in different cardiac diseases, like prediabetic and diabetic cardiomyopathy, digitalis intoxication and ischemia-reperfusion injury.Fil: Federico, Marilén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Valverde, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Mattiazzi, Ramona Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Palomeque, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. Universidad Abierta Interamericana. Secretaría de Investigación. Centro de Altos Estudios En Ciencias Humanas y de la Salud - Sede Buenos Aires; Argentin

Empujando los límites de la medicina

La identificación de las causas moleculares de las enfermedades junto con el desarrollo de la industria biotecnológica en general y de la farmacéutica en particular permiten el desarrollo de mejores métodos de diagnóstico, la identificación de blancos terapéuticos para el desarrollo de fármacos personalizados, y una mejor medicina preventiva. Se han logrado avances en un gran número de aspectos que en otros tiempos hubiesen sido impensables; tales son los casos de las técnicas de resonancia magnética, cirugías a distancia (telemedicina) gracias a Internet2, utilización de microprocesadores con diferentes usos médicos, decodificadores genéticos, utilización de tecnologías inalámbricas, entre otros. Los avances formidables en el campo de la biología molecular de las últimas décadas generan, además una gran cantidad de información que podría resultar inmanejable. Este inmenso caudal de nueva información requiere para su análisis el uso de herramientas de cálculo altamente especializadas.Facultad de Informátic

A low-cost Portable Device to Deliver Smoke, Volatile or Vaporized Substances to Drosophila melanogaster, Useful for Research and/or Educational Assays

Drosophila melanogaster has been used to test drugs of abuse, substances with potential benefits for medical purposes, as well as contaminants and hazardous volatile compounds. This model has also been used for the characterization of behavioral changes, physiopathological consequences, and subcellular mechanisms of the use of cocaine, methamphetamines, ethanol, nicotine, cannabinoids, toluene, and other airborne volatile organic compounds. When testing these substances, routes of administration are important to define. Admixing the test compounds with water or food is one suitable option in many cases, but the inhalation route is especially suitable when the administration of one or more volatile compounds is desired. One advantage of the administration of substances via the inhalation route is its rapid exchange and distribution throughout the cuticle and the tracheal system. In addition, this route allows treating a large group of individuals simultaneously. Moreover, the inhalation route is frequently used to administer different drugs to humans. A good model system shares physiology and molecular pathways with humans, and D. melanogaster possesses almost 75% homologous genes associated with human diseases. Methodologies to deliver the abovementioned substances usually include customized devices. Herein, we focus on the development of a low-cost customized device useful to deliver smoke or vaporizable compounds to D. melanogaster. This approach might be applied for acute or chronic exposure to vaporized substances. In particular, our device was utilized for testing cigarette smoke and vaporized cannabis extract on cardiac performance of adult individuals during chronic treatment. We are describing how to set up this low-cost portable device, useful for research and/or educational assays, taking advantage of the amenability of D. melanogaster to test different compounds in relatively short periods, and especially including a large number of individuals at the same time.Fil: Santalla, Manuela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Gómez, Ivana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Valverde, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Ferrero, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentin

Phospholamban phosphorylation sites enhance the recovery of intracellular Ca2+ after perfusion arrest in isolated, perfused mouse heart

Objective: To investigate the importance of the phosphorylation of Ser16 and Thr17 sites of phospholamban (PLN) on intracellular Ca2+ (Cai2+) handling and contractile recovery of the stunned myocardium. Methods: Cai2+ (Rhod-2, pulsed local-field fluorescence microscopy) and contractility (isovolumic left ventricular developed pressure, LVDP) were simultaneously measured in Langendorff perfused hearts from transgenic mice expressing either intact PLN (PLN-WT) or PLN with both phosphorylation sites mutated to Ala (PLN-DM), subjected to 12 min of global ischemia followed by a reperfusion period of 30 min. Results: Pre-ischemic values of Cai2+ and LVDP were similar in both groups. In PLN-WT, a transient increase in Thr17 phosphorylation at early reperfusion preceded a recovery of Ca2+ transient amplitude, virtually completed by the end of reperfusion. LVDP at 30 min reperfusion was 67.9 ± 7.6% of pre-ischemic values, n = 14. In contrast, in PLN-DM, there was a poor recovery of Cai2+ transient amplitude and LVDP was significantly lower (28.3 ± 6.7%, n = 11, 30 min reperfusion) than in PLN-WT hearts. Although myofilament Ca2+ responsiveness and troponin I (TnI) degradation did not differ between groups, the episodes of mechanical alternans, typical of Cai2+ overload, were significantly prolonged in PLN-DM vs. PLN-WT hearts. Conclusions: PLN phosphorylation appears to be crucial for the mechanical and Cai2+ recovery during stunning and protective against the mechanical abnormalities typical of Cai2+ overload. The importance of PLN phosphorylation would primarily reside in the Thr17 residue, which is phosphorylated during the critical early phase of reperfusion. Our results emphasize that, although ablation of PLN phosphorylation does not affect basal contractility, it does alter Ca2+ handling and mechanical performance under stress situations.Facultad de Ciencias Médica

Pharmacological inhibition of translocon is sufficient to alleviate endoplasmic reticulum stress and improve Ca2+ handling and contractile recovery of stunned myocardium

Introduction: The function of endoplasmic reticulum (ER), a Ca2+ storage compartment and site of protein folding, is altered by disruption of intracellular homeostasis. Misfolded proteins accumulated in the ER lead to ER stress (ERS), unfolded protein response (UPR) activation and ER Ca2+ loss. Myocardial stunning is a temporary contractile dysfunction, which occurs after brief ischemic periods with minimal or no cell death, being oxidative stress and Ca2+ overload potential underlying mechanisms. Myocardial stunning induces ERS response with negatively impact on the post-ischemic mechanical performance through an unknown mechanism. Aims: In this study, we explored whether ER Ca2+ efflux through the translocon, a major Ca2+ leak channel, contributes to Ca2+ mishandling and the consequent contractile abnormalities of the stunned myocardium. Methods: Mechanical performance, cytosolic Ca2+, UPR markers and oxidative state were evaluated in perfused rat/mouse hearts subjected to a brief ischemia followed by reperfusion (I/R) in absence or presence of the translocon inhibitor, emetine (1 μM), comparing its effects with those of the chaperones TUDCA (30 μM) and 4-PBA (3 mM). Results: Emetine treatment precluded the I/R-induced increase in UPR signaling markers and improved the contractile recovery together with a remarkable attenuation in myocardial stiffness when compared to I/R hearts with no drug. This alleviation of I/R-induced mechanical abnormalities was more effective than that obtained with the chemical chaperones, TUDCA and 4-PBA. Moreover, emetine treatment produced a striking improvement in diastolic Ca2+ handling with a partial recovery of the I/R-induced oxidative stress. Conclusion: Blocking ER Ca2+ store depletion via translocon suppressed ER stress and improved mechanical performance and diastolic Ca2+ handling of stunned myocardium. Modulation of translocon permeability emerges as a therapeutic approach to face dysfunctional consequences of the I/R injury.Fil: Mariángelo, Juan Ignacio Elio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Valverde, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Vittone, Leticia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Said, Maria Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Mundiña, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentin

Producción de forraje hidropónico de maíz (Zea mays L.) utilizando fertilizantes químicos y orgánicos

The province of Manabí occupies the first place in the meat production in Ecuador, the deficiency of food during the dryseason is one of its main problems. The production of green hydroponic fodder (GHF) is proposed as an alternativesolution, for which the following objectives were: i) determine the appropriate fertilizer for the hydroponic production ofmaize, ii) to establish the maize variety with the highest yield as GHF and iii) to define the appropriate harvest time inthe exploitation of GHF. In order to develop this research, 2 mL/L of water of an inorganic fertilizer (IF) based on phosphorus was applied and 2 g/L of powdered mycorrhizal water were applied, which were tested in the varieties of whiteand yellow maize, With airing of 48 and 72 h respectively. The experimental design was completely randomized withfactorial arrangement of 2x2x2 with eight treatments, generating 64 experimental units, considering as variables ofresponse, weight, root size and leaf height of the GHF. The results determined higher weight of white maize with FI with1.69 kg in trays of 40 x 50 cm and with 0.45 kg of maize per tray, at height level no statistical difference was determinedand for root size, the best treatment was white corn with FI with 12.49 cm in length. It is concluded that white maize responds better to IF under hydroponic conditions, with an airing time of 48 h.La provincia de Manabí ocupa el primer lugar en la producción cárnica en Ecuador, la deficiencia de alimento durante laépoca seca es uno de sus principales problemas, se plantea la producción de forraje verde hidropónico (FVH) comoalternativa de solución, para lo que se plantearon los siguientes objetivos: i) determinar el fertilizante apropiado para laproducción hidropónica de maíz, ii) establecer la variedad de maíz con mayor rendimiento como FVH y iii) definir eltiempo de oreo ideal en la explotación de FVH. Para desarrollar la investigación se aplicó 2 mL/L de agua de un fertilizante inorgánico (FI) a base de fosforo y se aplicó 2 g/L de agua de micorriza en polvo, se contrastaron en las variedadesde maíz blanco y amarillo, con oreos de 48 y 72 h respectivamente. El diseño experimental aplicado fue completamenteal azar con arreglo factorial de 2x2x2 con ocho tratamientos, generando 64 unidades experimentales, considerando comovariables de respuesta, peso, tamaño de raíz y altura de hojas del FVH. Los resultados determinaron mayor peso del maízblanco con FI con 1.60 kg en bandejas de 40 x 50 cm y con 0.45 kg de maíz por bandeja, a nivel de altura no se determinó diferencia estadística y en lo que respecta al tamaño de raíz, el mejor tratamiento fue el maíz blanco con FI con 12.49cm de longitud. Se concluye que el maíz blanco responde de mejor manera a la FI en condiciones hidropónicas, con untiempo de oreo de 48 h

Smoking flies: Testing the effect of tobacco cigarettes on heart function of Drosophila melanogaster

Studies about the relationship between substances consumed by humans and their impact on health, in animal models, have been a challenge due to differences between species in the animal kingdom. However, the homology of certain genes has allowed extrapolation of certain knowledge obtained in animals. Drosophila melanogaster, studied for decades, has been widely used as model for human diseases as well as to study responses associated with the consumption of several substances. In the present work we explore the impact of tobacco consumption on a model of 'smoking flies'. Throughout these experiments, we aim to provide information about the effects of tobacco consumption on cardiac physiology. We assessed intracellular calcium handling, a phenomenon underlying cardiac contraction and relaxation. Flies chronically exposed to tobacco smoke exhibited an increased heart rate and alterations in the dynamics of the transient increase of intracellular calcium in myocardial cells. These effects were also evident under acute exposure to nicotine of the heart, in a semi-intact preparation. Moreover, the alpha 1 and 7 subunits of the nicotinic receptors are involved in the heart response to tobacco and nicotine under chronic (in the intact fly) as well as acute exposure (in the semi-intact preparation). The present data elucidate the implication of the intracellular cardiac pathways affected by nicotine on the heart tissue. Based on the probed genetic and physiological similarity between the fly and human heart, cardiac effects exerted by tobacco smoke in Drosophila advances our understanding of the impact of it in the human heart. Additionally, it may also provide information on how nicotine-like substances, e.g. neonicotinoids used as insecticides, affect cardiac function.Fil: Santalla, Manuela. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Pagola, Lucia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Gómez, Ivana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Balcazar, Dario Emmanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; ArgentinaFil: Valverde, Carlos Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Ferrero, Paola Viviana. Universidad Nacional del Noroeste de la Provincia de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentin

Evaluación de tres métodos de injertación en cacao en el Cantón Jipijapa Provincia de Manabí. (Original)

The research was carried out at the State University of the South of Manabí in the career of Agricultural Engineering in the Canton Jipijapa, 216 plants were grafted with the objective of evaluating three grafting methods in cocoa (Theobroma cacao) in time of stay in the nursery and quality of the seedlings. The research was carried out in six months, developing three months of field work, which consisted in the preparation of the nursery, obtaining the patterns, preparing the rods, grafting and maintenance of the nursery. The completely randomized statistical design with three treatments and three repetitions was used, T1 terminal spike, T2 lateral spike and T3 patch, the statistical analysis was performed using the free software infostat. The results in percentage of seizure showed that the best treatment was T1 with 88%, followed by T2 with 0.71%; while T3 showed the lowest uptake with 0.13%. Regarding the diameter of the stem and the height of the pattern, the largest diameter used was the terminal spike graft, the intermediate diameter was the lateral spike and the smallest diameter was the patch graft, therefore, three methods were applied, so that the whole plant is grafted at once. Recommending Manabí farmers to use the terminal barb, which was shown with the analysis of variance to be highly significant with p <0.01.La investigación se llevó a cabo en la Universidad Estatal del Sur de Manabí en la carrera de Ingeniería Agropecuaria, en el Cantón Jipijapa. Se injertaron 216 plantas con el objetivo de evaluar tres métodos de injertación en cacao (Theobroma cacao) en tiempo de estancia en vivero y calidad de las plántulas. La investigación se realizó en seis meses, desarrollando tres meses de trabajo de campo que consistió en la preparación del vivero, obtención de los patrones, preparación de las varetas, injertación y mantenimiento del vivero. Se utilizó el diseño estadístico completamente al azar con tres tratamientos y tres repeticiones. T1 púa terminal, T2 púa lateral y T3 parche; el análisis estadístico se realizó aplicando el software libre infostat. Los resultados en porcentaje de prendimiento mostraron que el mejor tratamiento fue el T1 con 88%, seguido del T2 con un 0,71%; mientras que el T3 mostró el más bajo prendimiento con un 0,13%. En cuanto al diámetro de tallo y altura del patrón, el mayor diámetro que se utilizó fue el injerto de púa terminal, el diámetro intermedio fue de púa lateral y el de menor diámetro fue el injerto en parche, por lo tanto, se aplicaron tres métodos para que quede toda la planta injertada de una vez. Recomendando a los agricultores manabitas utilizar el de púa terminal que fue el que se demostró con el análisis de varianza que es altamente significativo con el valor p< 0,01

Aging and CaMKII alter intracellular Ca2+ transients and heart rhythm in Drosophila melanogaster

Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations. Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca 2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.Centro de Investigaciones CardiovascularesCentro de Investigaciones Inmunológicas Básicas y Aplicada

Reperfusion after Ichemia Causes Cytosolic Calcium Overload Due to Rapid Calcium Release from the Sarcoplasmic Reticulum

After a brief ischemic insult, a sustained contractile dysfunction occurs manifested as a sluggish recovery of pump function (myocardial stunning). Substantial evidence supports that myocardial dysfunction is triggered by Ca2+ overload during reperfusion (R). Previous results from different laboratories including our own, describe a cascade of events triggered by R that involves the activation of Na+/H+ and Na+/Ca2+ (NCX) exchangers, with enhanced Ca2+ influx. Whether this Ca2+ influx directly produces the increase in cytosolic Ca2+ or this increase occurs as a consequence of sarcoplasmic reticulum (SR) Ca2+ release triggered in turn by the Ca2+ influx, is not known. To address this issue, we performed 12 min of global no-flow ischemia followed by R in the isovolumic Langendorff perfused mouse heart positioned on a Pulsed Local Field Fluorescence microscope and loaded with fluorescent dyes (Rhod-2 or Mag-Fluo-4 to assess cytosolic or SR Ca2+, respectively). The results indicated an initial increase in diastolic Ca2+ during early R that gradually returned to pre-ischemic levels. This increase was associated with a decrease in SR Ca2+ content that recovered within 10 min, as a mirror image of the diastolic Ca2+ profile. Additional experiments in which caffeine pulses (20 mM) were applied, confirmed that SR Ca2+ content was greatly diminished at the onset of R and gradually recovered within 10 min of R. The present findings indicate that the increase in diastolic Ca2+ that occurs upon R is due to a SR Ca2+ release and not just because of the Ca2+ entry through the reverse NCX mode, as has been previously thought.Facultad de Ciencias Médica