The spatio-temporal properties of calcium transients in hippocampal pyramidal neurons in vitro

The spatio-temporal properties of calcium signals were studied in cultured pyramidal neurons of the hippocampus using two-dimensional fluorescence microscopy and ratiometric dye Fura-2. Depolarization-induced Ca2+ transients revealed an asynchronous delayed increase in free Ca2+ concentration. We found that the level of free resting calcium in the cell nucleus is significantly lower compared to the soma, sub-membrane, and dendritic tree regions. Calcium release from the endoplasmic reticulum under the action of several stimuli (field stimulation, high K+ levels, and caffeine) occurs in all areas studied. Under depolarization, calcium signals developed faster in the dendrites than in other areas, while their amplitude was significantly lower since larger and slower responses inside the soma. The peak value of the calcium response to the application of 10 mM caffeine, ryanodine receptors (RyRs) agonist, does not differ in the sub-membrane zone, central region, and nucleus but significantly decreases in the dendrites. In the presence of caffeine, the delay of Ca2+ signals between various areas under depolarization significantly declined. Thirty percentage of the peak amplitude of Ca2+ transients at prolonged electric field stimulation corresponded to calcium release from the ER store by RyRs, while short-term stimulation did not depend on them. 20 μM dantrolene, RyRs inhibitor, significantly reduces Ca2+ transient under high K+ levels depolarization of the neuron. RyRs-mediated enhancement of the Ca2+ signal is more pronounced in the central part and nucleus compared to the sub-membrane or dendrites regions of the neuron. In summary, using the ratiometric imaging allowed us to obtain additional information about the involvement of RyRs in the intracellular dynamics of Ca2+ signals induced by depolarization or electrical stimulation train, with an underlying change in Ca2+ concentration in various regions of interest in hippocampal pyramidal neurons

Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants

Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b6/f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (Fv/Fm, rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis

Ryanodine receptor dispersion disrupts Ca2+ release in failing cardiac myocytes

Reduced cardiac contractility during heart failure (HF) is linked to impaired Ca2+ release from Ryanodine Receptors (RyRs). We investigated whether this deficit can be traced to nanoscale RyR reorganization. Using super-resolution imaging, we observed dispersion of RyR clusters in cardiomyocytes from post-infarction HF rats, resulting in more numerous, smaller clusters. Functional groupings of RyR clusters which produce Ca2+ sparks (Ca2+ release units, CRUs) also became less solid. An increased fraction of small CRUs in HF was linked to augmented ‘silent’ Ca2+ leak, not visible as sparks. Larger multi-cluster CRUs common in HF also exhibited low fidelity spark generation. When successfully triggered, sparks in failing cells displayed slow kinetics as Ca2+ spread across dispersed CRUs. During the action potential, these slow sparks protracted and desynchronized the overall Ca2+ transient. Thus, nanoscale RyR reorganization during HF augments Ca2+ leak and slows Ca2+ release kinetics, leading to weakened contraction in this disease

Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse

Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91phox, p67phox and rac 1 were increased 2–3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91phox and p67phox were increased 3–4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca2+ entry, a key mechanism for muscle damage and functional impairment

Determinants of Ca2+ release restitution: Insights from genetically altered animals and mathematical modeling

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.Fil: Cely Ortiz, Diana Cataloina Alejandra. 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: Felice, Juan Ignacio. 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: Diaz Zegarra, Leandro Agustin. 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: 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: 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"; ArgentinaFil: Wehrens, Xander H.T.. Cardiovascular Research Institute. Baylor College of Medicine. Center for Space Medicine. Departments of Molecular Physiology and Biophysics, Medicine (in Cardiology), Neuroscience, Pediatrics; Estados UnidosFil: Kranias, Evangelina G.. University of Cincinnati; Estados UnidosFil: Aiello, Ernesto Alejandro. 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: Lascano, Elena Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Negroni, Jorge Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; 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"; Argentin

Quantitative evaluation of the beneficial effects in the mdx mouse of epigallocatechin gallate, an antioxidant polyphenol from green tea

In two separate previous studies, we reported that subcutaneous (sc) or oral administration of (−)-epigallocatechin-3-gallate (EGCG) limited the development of muscle degeneration of mdx mice, a mild phenotype model for Duchenne muscular dystrophy (DMD). However, it was not possible to conclude which was the more efficient route of EGCG administration because different strains of mdx mice, periods of treatment and methods of assessment were used. In this study, we investigated which administration routes and dosages of EGCG are the most effective for limiting the onset of dystrophic lesions in the same strain of mdx mice and applying the same methods of assessment. Three-week-old mdx mice were injected sc for 5 weeks with either saline or a daily average of 3 or 6 mg/kg EGCG. For comparison, age-matched mdx mice were fed for 5 weeks with either a diet containing 0.1% EGCG or a control diet. The effects of EGCG were assessed quantitatively by determining the activities of serum muscle-derived creatine kinase, isometric contractions of triceps surae muscles, integrated spontaneous locomotor activities, and oxidative stress and fibrosis in selected muscles. Oral administration of 180 mg/kg/day EGCG in the diet was found the most effective for significantly improving several parameters associated with muscular dystrophy. However, the improvements were slightly less than those observed previously for sc injection started immediately after birth. The efficacy of EGCG for limiting the development of dystrophic muscle lesions in mice suggests that EGCG may be of benefit for DMD patients