Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes

Aim: In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodeling in cardiac diseases is associated with down-regulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signaling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes. Methods and results: Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodeling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with Methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions: JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signaling complexes to regulate LTCC activity

Ursodeoxycholic acid prevents ventricular conduction slowing and arrhythmia by restoring T-type calcium current in fetuses during cholestasis

Background Increased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated. Methods High-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes. Results TC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (−10.2±1.5 versus −5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (−22.3±1.1 versus −9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L. Conclusion Our findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA

The Protective Effect of Ursodeoxycholic Acid in an in vitro model of the Human Fetal Heart occurs via Targeting Cardiac Fibroblasts

Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts.Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential.We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells.UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts in human fetal hearts. Hypoxia further increased the number of human fetal and rat neonatal myofibroblasts. However, chronically administered UDCA reduced the number of myofibroblasts and prevented hypoxia-induced depolarisation.In conclusion, our results show that the protective effect of UDCA involves both the reduction of fibroblast differentiation into myofibroblasts, and hyperpolarisation of myofibroblasts, most likely through the stimulation of potassium channels, i.e. KATP channels. This could be important in validating UDCA as an antifibrotic and antiarrhythmic drug for treatment of failing hearts and fetal arrhythmia

Ball Grid Array Module with Integrated Shaped Lens for 5G Backhaul/Fronthaul Communications in F-Band

In this paper, we propose a ball grid array (BGA) module with an integrated 3-D-printed plastic lens antenna for application in a dedicated 130 GHz OOK transceiver that targets the area of 5G backhaul/fronthaul systems. The main design goal was the full integration of a small footprint antenna with an energy-efficient transceiver. The antenna system must be compact and cost effective while delivering an approximately 30 dBi gain in the working band, defined as 120 to 140 GHz. Accordingly, a 2×2 array of aperture-coupled patch antennas was designed in the 7×7×0.362 mm3 BGA module as the feed antenna of the lens. This achieved a 7.8 dBi realized gain, broadside polarization purity above 20 dB, and over 55% total efficiency from 110 to 140 GHz (20% bandwidth). A plastic elliptical lens 40 mm in diameter and 42.3 mm in height was placed on top of the BGA module. The antenna achieved a return loss better than ?10 dB and a 28 dBi realized gain from 114 to 140 GHz. Finally, active measurements demonstrated a >12 Gbps Tx/Rx link at 5 m with bit error rate (BER) < 10?6 at 1.6 pJ/b/m. These results pave the way for future cost-effective, energy-efficient, high-data rate backhaul/fronthaul systems for 5G communications.info:eu-repo/semantics/acceptedVersio

Towards precision medicine: defining and characterizing adipose tissue dysfunction to identify early immunometabolic risk in symptom-free adults from the GEMM family study

Interactions between macrophages and adipocytes are early molecular factors influencing adipose tissue (AT) dysfunction, resulting in high leptin, low adiponectin circulating levels and low-grade metaflammation, leading to insulin resistance (IR) with increased cardiovascular risk. We report the characterization of AT dysfunction through measurements of the adiponectin/leptin ratio (ALR), the adipo-insulin resistance index (Adipo-IRi), fasting/postprandial (F/P) immunometabolic phenotyping and direct F/P differential gene expression in AT biopsies obtained from symptom-free adults from the GEMM family study. AT dysfunction was evaluated through associations of the ALR with F/P insulin-glucose axis, lipid-lipoprotein metabolism, and inflammatory markers. A relevant pattern of negative associations between decreased ALR and markers of systemic lowgrade metaflammation, HOMA, and postprandial cardiovascular risk hyperinsulinemic, triglyceride and GLP-1 curves was found. We also analysed their plasma non-coding microRNAs and shotgun lipidomics profiles finding trends that may reflect a pattern of adipose tissue dysfunction in the fed and fasted state. Direct gene differential expression data showed initial patterns of AT molecular signatures of key immunometabolic genes involved in AT expansion, angiogenic remodelling and immune cell migration. These data reinforce the central, early role of AT dysfunction at the molecular and systemic level in the pathogenesis of IR and immunometabolic disorders

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor

Background and Purpose Flecainide is a use-dependent blocker of cardiac Na+ channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na+ channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca2+-release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na+ and/or RyR2 channels. Experimental Approach We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca2+ sparks in intact adult rat cardiac myocytes. Key Results Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca2+ spark frequency. Conclusions and Implications Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from Na+ channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin-proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA 2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression. © 2003 Cancer Research UK

Ibero-American Consensus on Low- and No-Calorie Sweeteners: Safety, Nutritional Aspects and Benefits in Food and Beverages

International scientific experts in food, nutrition, dietetics, endocrinology, physical activity, paediatrics, nursing, toxicology and public health met in Lisbon on 2-4 July 2017 to develop a Consensus on the use of low- and no-calorie sweeteners (LNCS) as substitutes for sugars and other caloric sweeteners. LNCS are food additives that are broadly used as sugar substitutes to sweeten foods and beverages with the addition of fewer or no calories. They are also used in medicines, health-care products, such as toothpaste, and food supplements. The goal of this Consensus was to provide a useful, evidence-based, point of reference to assist in efforts to reduce free sugars consumption in line with current international public health recommendations. Participating experts in the Lisbon Consensus analysed and evaluated the evidence in relation to the role of LNCS in food safety, their regulation and the nutritional and dietary aspects of their use in foods and beverages. The conclusions of this Consensus were: (1) LNCS are some of the most extensively evaluated dietary constituents, and their safety has been reviewed and confirmed by regulatory bodies globally including the World Health Organisation, the US Food and Drug Administration and the European Food Safety Authority; (2) Consumer education, which is based on the most robust scientific evidence and regulatory processes, on the use of products containing LNCS should be strengthened in a comprehensive and objective way; (3) The use of LNCS in weight reduction programmes that involve replacing caloric sweeteners with LNCS in the context of structured diet plans may favour sustainable weight reduction. Furthermore, their use in diabetes management programmes may contribute to a better glycaemic control in patients, albeit with modest results. LNCS also provide dental health benefits when used in place of free sugars; (4) It is proposed that foods and beverages with LNCS could be included in dietary guidelines as alternative options to products sweetened with free sugars; (5) Continued education of health professionals is required, since they are a key source of information on issues related to food and health for both the general population and patients. With this in mind, the publication of position statements and consensus documents in the academic literature are extremely desirable