The Structural–Functional Crosstalk of the Calsequestrin System: Insights and Pathological Implications

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Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1 dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

In cardiac myocytes, β‐adrenergic stimulation enhances Ca2+ cycling through an integrated signalling cascade modulating L‐type Ca2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca2+/calmodulin‐dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole‐cell voltage‐clamp and confocal line‐scan imaging with Fluo‐4 as a [Ca2+]i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca2+ sparks, which are assigned to coupled and non‐coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces spark frequency in coupled RyRs only; NOS1 inhibition mimics the effect of CaMKII inhibition. Moreover, ISO induces the repetitive activation of coupled RyR clusters through CaMKII activation. Immunostaining shows high levels of CaMKII phosphorylation at the dyadic cleft. CaMKII inhibition reduces ICaL and local Ca2+ transients during depolarizing steps but has only modest effects on amplitude or relaxation of the global Ca2+ transient. In contrast, protein kinase A (PKA) inhibition reduces spark frequency in all RyRs concurrently with a reduction of sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude and relaxation. In conclusion, CaMKII activation during β‐adrenergic stimulation is restricted to the dyadic cleft microdomain, enhancing LTCC‐triggered local Ca2+ release as well as spontaneous diastolic Ca2+ release whilst PKA is the major pathway increasing global Ca2+ cycling. Selective CaMKII inhibition may reduce potentially arrhythmogenic release without negative inotropy

Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation.

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels' pore cytosolic exit affects the channel's gating behavior thereby explaining the enhanced ligand-channel's sensitivity.The authors received the support of research grants from the National Institutes of Health grant/award numbers R01GM111397 to S. R. W. Chen, M. Fill, and J. Ramos‐Franco, R01HL057832 to M. Fill and by the Canadian Institutes of Health Research, grant/award number PJT‐173352. S. R. W. Chen holds the Heart and Stroke Foundation Chair in Cardiovascular Research (END611955). A. Tambeaux was supported by the Graduate College of Rush University Medical Center.S

Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Aims In ventricular myocytes from humans and large mammals, the transverse and axial tubular system (TATS) network is less extensive than in rodents with consequently a greater proportion of ryanodine receptors (RyRs) not coupled to this membrane system. TATS remodelling in heart failure (HF) and after myocardial infarction (MI) increases the fraction of non-coupled RyRs. Here we investigate whether this remodelling alters the activity of coupled and non-coupled RyR sub-populations through changes in local signalling. We study myocytes from patients with end-stage HF, compared with non-failing (non-HF), and myocytes from pigs with MI and reduced left ventricular (LV) function, compared with sham intervention (SHAM).Methods and resultsSingle LV myocytes for functional studies were isolated according to standard protocols. Immunofluorescent staining visualized organization of TATS and RyRs. Ca2+ was measured by confocal imaging (fluo-4 as indicator) and using whole-cell patch-clamp (37°C). Spontaneous Ca2+ release events, Ca2+ sparks, as a readout for RyR activity were recorded during a 15 s period following conditioning stimulation at 2 Hz. Sparks were assigned to cell regions categorized as coupled or non-coupled sites according to a previously developed method. Human HF myocytes had more non-coupled sites and these had more spontaneous activity than in non-HF. Hyperactivity of these non-coupled RyRs was reduced by Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition. Myocytes from MI pigs had similar changes compared with SHAM controls as seen in human HF myocytes. As well as by CaMKII inhibition, in MI, the increased activity of non-coupled sites was inhibited by mitochondrial reactive oxygen species (mito-ROS) scavenging. Under adrenergic stimulation, Ca2+ waves were more frequent and originated at non-coupled sites, generating larger Na+/Ca2+ exchange currents in MI than in SHAM. Inhibition of CaMKII or mito-ROS scavenging reduced spontaneous Ca2+ waves, and improved excitation–contraction coupling.ConclusionsIn HF and after MI, RyR microdomain re-organization enhances spontaneous Ca2+ release at non-coupled sites in a manner dependent on CaMKII activation and mito-ROS production. This specific modulation generates a substrate for arrhythmia that appears to be responsive to selective pharmacologic modulation

Altered adrenergic response in myocytes bordering a chronic myocardial infarction underlies <i>in vivo</i> triggered activity and repolarization instability

Ventricular arrhythmias are a major complication early after myocardial infarction (MI). The heterogeneous peri‐infarct zone forms a substrate for re‐entry while arrhythmia initiation is often associated with sympathetic activation. We studied the mechanisms triggering these post‐MI arrhythmias in vivo and their relation to regional myocyte remodelling. In pigs with chronic MI (6 weeks), in vivo monophasic action potentials were simultaneously recorded in the peri‐infarct and remote regions during adrenergic stimulation with isoproterenol (ISO). Sham animals served as controls. During infusion of ISO in vivo, the incidence of delayed afterdepolarizations (DADs) and beat‐to‐beat variability of repolarization (BVR) was higher in the peri‐infarct than in the remote region. Myocytes isolated from the peri‐infarct region, in comparison to myocytes from the remote region, had more DADs, associated with spontaneous Ca2+ release, and a higher incidence of spontaneous action potentials when exposed to ISO (9.99 ± 4.2 vs. 0.16 ± 0.05 APs/min, p = 0.004); these were suppressed by CaMKII inhibition. Peri‐infarct myocytes also had reduced repolarization reserve and increased BVR (26 ± 10 ms vs. 9 ± 7 ms, p 2+ handling at baseline and myocyte hypertrophy were present throughout the LV. Expression of some of the related genes was however different between the regions. In conclusion, altered myocyte adrenergic responses in the peri‐infarct, but not in the remote region, provide a source of triggered activity in vivo and of repolarization instability amplifying the substrate for re‐entry. These findings stimulate further exploration of region‐specific therapies targeting myocytes and autonomic modulation

100 años investigando el mar. El IEO en su centenario (1914-2014).

Se trata de un libro que pretende divulgar a la sociedad las principales investigaciones multidisciplinares llevadas a cabo por el Instituto Español de Oceanografía durante su primer siglo de vida, y dar a conocer la historia del organismo, de su Sede Central y de los nueve centros oceanográficos repartidos por los litorales mediterráneo y atlántico, en la península y archipiélagos.Kongsberg 20

Evidence for soil pesticide contamination of an agroecological farm from a neighboring chemical-based production system

The global chemical-based agriculture (CBA) production system brought social and environmental consequences such as the contamination of soils, waters, bottom sediments and food, as well as negative effects on non-target species. As an alternative, a new paradigm emerged: agroecology-based agriculture (ABA), based on ecosystem services and the reduction of chemical inputs. More and more establishments have adopted this form of production; however, they are located next to crops to which pesticides are applied. The objective of this work was to study, through the spatio-temporal characterizations of pesticides in soils, how an ABA production system can be affected by the CBA fields around it. Two sampling campaigns were conducted and soil samples were obtained from "La Aurora", an agricultural establishment located in the Argentine pampa and recognized by the FAO for its agroecological practices, and from neighboring fields with CBA productions. The samples were tested for 19 herbicides (including 3 metabolites) and 3 fungicides by UPLC-MS/MS, of which we detected glyphosate and its metabolite AMPA, 2,4-D, atrazine, acetochlor, metsulfuron-methyl, desethyl-atrazine, epoxiconazole, and tebuconazole. Three or more pesticides co-occurred in 93% and 32% of the CBA and ABA samples, respectively. Glyphosate and AMPA, with the highest detection frequency, also accounted for 90% of the total pesticide load in both systems. The maximum concentrations (μg kg−1 dry weight) in the CBA/ABA fields, respectively, were glyphosate (1268.92/98.93), AMPA (2919.17/114.01), followed by 2,4-D (38.52/31.12), and epoxiconazole (13.35/18.41). No significant temporal differences were found in glyphosate concentration within each establishment, corroborating its pseudo-persistence in CBA establishments, and establishing it in ABA field. Moreover, glyphosate was found in the ABA field more than 300 m from the limit with the CBA fields. Glyphosate and AMPA concentrations are in the order of those reported to cause sublethal and lethal effects in soil organisms. These results highlight the mobility of pesticides, as the ABA establishment is affected by its surroundings where pesticides are used, even at sites far from the interface between them. Given their higher detection frequencies and environmental concentrations in comparison to the other pesticides, glyphosate and AMPA are proposed as environmental tracers of conventional agroproductive activities.Fil: Demetrio, Pablo Martin. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones del Medio Ambiente; ArgentinaFil: Demetrio, Pablo Martin. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones del Medio Ambiente; ArgentinaFil: Alonso, Lucas Leonel. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones del Medio Ambiente; ArgentinaFil: Mac Loughlin, Tomas Mariano. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones del Medio Ambiente; ArgentinaFil: Cerdá, Eduardo. Red Nacional De Municipios y Comunidades que Fomentan La Agroecología; ArgentinaFil: Sarandón, Santiago J.. Red Nacional De Municipios y Comunidades que Fomentan La Agroecología; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Marino, Damian Jose Gabriel. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigaciones del Medio Ambiente - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones del Medio Ambiente; Argentin