Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.The authors thank the Centro Nacional de Investigaciones Cardiovasculares (CNIC) Viral Vectors Unit for producing the adeno-associated virus serotype 9. Confocal experiments were conducted at the CNIC Microscopy and Dynamic Imaging Unit. The authors thank the CNIC Bioinformatics Unit for generating the in silico homology modeling simulations, F-function analysis, and helpful discussions. The authors also thank the Centro de Supercomputación de Galicia for the use of the Finis Terrae III supercomputer to perform molecular dynamics studies. The CNIC was supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia, Innovación y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033). This work was supported by the National heart, Lung and Blood Institute under National Institutes of Health (NIH) grant R01HL163943; the La Caixa Banking Foundation project code HR18-00304 (grant LCF/PR/HR19/52160013); grants PI-FIS-2020, PI20/01220, PI-FIS-2023, and PI23/01039 from the Instituto de Salud Carlos III and cofunded by the Fondo Europeo de Desarrollo Regional (FEDER) and the European Union, respectively; grants PID2020-116935RB-I00 and BFU2016-75144-R funded by MICIU/AEI/10.13039/501100011033; the Fundación La Marató de TV3 (736/C/2020) amb el suport de la Fundació La Marató de TV3; the CIBER (Centro de Investigación Biomédica en Red) de Enfermedades Cardiovasculares (grant CB16/11/00458); the European Union’s Horizon 2020 grant agreement GA-965286; and the Program S2022/BMD7229-CM ARCADIACM funded by the Comunidad de Madrid to J. Jalife; grant PID2021-126423OB-C22 (to M. Martín-Martínez) funded by MICIU/AEI/10.13039/501100011033; and European Regional Development Fund (ERDF) grant PID2022-137214OB-C22 (to M. Gutierrez-Rodríguez) funded by MICIU/AEI/10.13039/501100011033. The imaging studies were performed in the TRIMA@CNIC (Infraestructura de Imagen Traslacional Avanzada del CNIC) node of the ICTS ReDIB (Infraestructuras Científicas y Técnicas Singulares: Red Distribuida de Imagen Biomédica) grant ICTS-2018- 04-CNIC-16 funded by MICIU/AEI/10.13039/501100011033 and ERDF, and project EQC2018-005070-P funded by MICIU/AEI/10.13039/501100011033 and FEDER. A.I. Moreno-Manuel holds an formación profesional universitaria (FPU) contract (FPU20/01569) from the Ministerio de Universidades. J.M. Ruiz Robles holds an FPU contract (FPU22/03253) from the Ministerio de Universidades. L.K. Gutiérrez holds an FPI contract (PRE2018-083530) from the Ministerio de Economía y Competitividad de España cofunded by the Fondo Social Europeo, attached to project SEV-2015-0505-18-2. I. Martínez-Carrascoso holds a PFIS (Contratos predoctorales de formación en investigación en salud) contract (FI21/00243) funded by Instituto de Salud Carlos III and the Fondo Social Europeo Plus cofunded by the European Union. M.L. Vera-Pedrosa held contract PEJD-2019-PRE/BMD15982 funded by the Consejería de Educación e Investigación de la Comunidad de Madrid y Fondo Social Europeo.S

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome

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Acute Molecular Changes in Synovial Fluid Following Human Knee Injury:Association With Early Clinical Outcomes.

We investigated whether molecules found to be up-regulated within hours of surgical joint destabilisation in the mouse were also elevated in the analogous human setting of acute knee injury, how this molecular response varied between individuals, and whether it related to patient-reported outcomes in the 3 months after injury.7 candidate molecules were analysed in blood and synovial fluid (SF) of 150 participants with recent structural knee injury at baseline (<8 weeks from injury) and in blood at 14 days and 3 months following baseline. KOOS4 was collected at baseline and 3 months. Assays were by MesoScale Discovery™platform or ELISA, and compared with controls.6/7 molecules were significantly elevated in human synovial fluid immediately after injury: IL-6, MCP-1, MMP-3, TIMP-1, activin-A and TSG-6. There was low-moderate correlation with blood measurements. 3/6 molecules were significantly associated with baseline KOOS4 (those with higher SFIL-6, TIMP-1 or TSG-6 had lower KOOS4 ). These 3, MMP-3 and activin-A were all significantly associated with greater improvement in KOOS over 3 months, adjusting for other relevant factors. Of these, IL-6 alone significantly accounted for the molecular contribution to baseline KOOS4 , and its difference over 3 months.Our findings validate relevant human biomarkers of tissue injury identified in a mouse model. Analysis of SF rather than blood more accurately reflects this response. The response is associated with patient-relevant outcomes over this early period, SF IL-6 acting as a single representative marker. Longitudinal outcomes will determine if these molecules are biomarkers of subsequent disease risk. This article is protected by copyright. All rights reserved

Protein traffic is an intracellular target in alcohol toxicity

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains

Crocetin isolated from the natural food colorant saffron reduces intracellular fat in 3T3-L1 adipocytes

14 pags., 5 figs., 1 tab. -- This article belongs to the Special Issue Food Innovation as a Means of Developing Healthier and More Sustainable FoodsSaffron, as a food colorant, has been displaced by low-cost synthetic dyes. These have unhealthy properties; thus, their replacement with natural food colorants is an emerging trend. Obesity is a worldwide health problem due to its associated comorbidities. Crocetin esters (crocins) are responsible for the red saffron color. Crocetin (CCT) exhibits healthful properties. We aimed to broaden the existing knowledge on the health properties of CCT isolated from saffron, to facilitate its consideration as a healthy natural food colorant in the future. We evaluated the ability of CCT (1 and 5 μM) to reduce lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Intracellular fat was quantified by Oil Red O staining. CTT cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The number and size of lipid droplets were analyzed using WimLipid software. The expression of adipogenic genes (CCAAT/enhancer-binding protein (C/EBPβ, C/EBPδ, C/EBPα), and peroxisome proliferator-activated receptor γ (PPARγ)) was analyzed using quantitative real-time PCR (qRT-PCR). CCT 5 μM decreased intracellular fat by 22.6%, without affecting viability or lipid droplet generation, via a decrease in C/EBPα expression, implicated in lipid accumulation. Thus, CCT is a potential candidate to be included in dietary therapies aimed at reversing adipose tissue accumulation in obesity.Peer reviewe

Micro-ribonucleic acids and extracellular vesicles repertoire in the spent culture media is altered in women undergoing In Vitro Fertilization

Abstract MicroRNAs (miRNAs) are class of small RNA molecules with major impact on gene regulation. We analyzed the potential of miRNAs secreted from pre-implantation embryos into the embryonic culture media as biomarkers to predict successful pregnancy. Using microarray analysis, we profiled the miRNome of the 56 spent culture media (SCM) after embryos transfer and found a total of 621 miRNAs in the SCM. On average, we detected 163 miRNAs in SCM of samples with failed pregnancies, but only 149 SCM miRNAs of embryos leading to pregnancies. MiR-634 predicted an embryo transfer leading to a positive pregnancy with an accuracy of 71% and a sensitivity of 85%. Among the 621 miRNAs, 102 (16.4%) showed a differential expression between positive and negative outcome of pregnancy with miR-29c-3p as the most significantly differentially expressed miRNA. The number of extracellular vehicles was lower in SCM with positive outcomes (3.8 × 109/mL EVs), as compared to a negative outcome (7.35 × 109/mL EVs) possibly explaining the reduced number of miRNAs in the SCM associated with failed pregnancies. The analysis of the miRNome in the SCM of couples undergoing fertility treatment lays the ground towards development of biomarkers to predict successful pregnancy and towards understanding the role of embryonic miRNAs found in the SCM