Heart Membranes Research Articles

Coronavirus disease 2019 and cardiovascular disease: what we have learnt during the last 2 years.

Coronavirus disease 2019 and cardiovascular disease: what we have learnt during the last 2 years.

Analysis of Mitochondrial Function in Cell Membranes as Indicator of Tissue Vulnerability to Drugs in Humans.

Drug side effects are one of the main reasons for treatment withdrawal during clinical trials. Reactive oxygen species formation is involved in many of the drug side effects, mainly by interacting with the components of the cellular respiration. Thus, the early detection of these effects in the drug discovery process is a key aspect for the optimization of pharmacological research. To this end, the superoxide formation of a series of drugs and compounds with antidepressant, antipsychotic, anticholinergic, narcotic, and analgesic properties was evaluated in isolated bovine heart membranes and on cell membrane microarrays from a collection of human tissues, together with specific inhibitors of the mitochondrial electron transport chain. Fluphenazine and PB28 promoted similar effects to those of rotenone, but with lower potency, indicating a direct action on mitochondrial complex I. Moreover, nefazodone, a drug withdrawn from the market due to its mitochondrial hepatotoxic effects, evoked the highest superoxide formation in human liver cell membranes, suggesting the potential of this technology to anticipate adverse effects in preclinical phases.

Ultrastructural and proteomic profiling of mitochondria-associated endoplasmic reticulum membranes reveal aging signatures in striated muscle

Aging is a major risk for developing cardiac and skeletal muscle dysfunction, yet the underlying mechanism remains elusive. Here we demonstrated that the mitochondria-associated endoplasmic reticulum membranes (MAMs) in the rat heart and skeletal muscle were disrupted during aging. Using quantitative morphological analysis, we showed that the mitochondria-endoplasmic reticulum contacts (MERCs) were reduced by half over the lifespan with an early onset of accelerated thickening in the clefts. The ultrastructural changes were further validated by proteomic profiling of the MAM fractions. A combination of subcellular fractionation and quantitative mass spectrometry identified 1306 MAM-enriched proteins in both heart and skeletal muscle, with a catalog of proteins dysregulated with aging. Functional mapping of the MAM proteome suggested several aging signatures to be closely associated with the ER-mitochondria crosstalk, including local metabolic rewiring, calcium homeostasis imbalance, and impaired organelle dynamics and autophagy. Moreover, we identified a subset of highly interconnected proteins in an ER-mitochondria organization network, which were consistently down-regulated with aging. These decreased proteins, including VDAC1, SAMM50, MTX1 and MIC60, were considered as potential contributors to the age-related MAM dysfunction. This study highlights the perturbation in MAM integrity during the striated muscle aging process, and provides a framework for understanding aging biology from the perspective of organelle interactions.

Guanylyl cyclase-A phosphorylation decreases cardiac hypertrophy and improves systolic function in male, but not female, mice.

Atrial natriuretic peptide (NP) and BNP increase cGMP, which reduces blood pressure and cardiac hypertrophy by activating guanylyl cyclase (GC)-A, also known as NPR-A or Npr1. Although GC-A is highly phosphorylated, and dephosphorylation inactivates the enzyme, the significance of GC-A phosphorylation to heart structure and function remains unknown. To identify in vivo processes that are regulated by GC-A phosphorylation, we substituted glutamates for known phosphorylation sites to make GC-A8E/8E mice that express an enzyme that cannot be inactivated by dephosphorylation. GC-A activity, but not protein, was increased in heart and kidney membranes from GC-A8E/8E mice. Activities were threefold higher in female compared to male cardiac ventricles. Plasma cGMP and testosterone were elevated in male and female GC-A8E/8E mice, but aldosterone was only increased in mutant male mice. Plasma and urinary creatinine concentrations were decreased and increased, respectively, but blood pressure and heart rate were unchanged in male GC-A8E/8E mice. Heart weight to body weight ratios for GC-A8E/8E male, but not female, mice were 12% lower with a 14% reduction in cardiomyocyte cross-sectional area. Subcutaneous injection of fsANP, a long-lived ANP analog, increased plasma cGMP and decreased aldosterone in male GC-AWT/WT and GC-A8E/8E mice at 15min, but only GC-A8E/8E mice had elevated levels of plasma cGMP and aldosterone at 60min. fsANP reduced ventricular ERK1/2 phosphorylation to a greater extent and for a longer time in the male mutant compared to WT mice. Finally, ejection fractions were increased in male but not female hearts from GC-A8E/8E mice. We conclude that increased phosphorylation-dependent GC-A activity decreases cardiac ERK activity, which results in smaller male hearts with improved systolic function.

Elevated Circulating Stem Cells Level is Observed One Month After Implantation of Carmat Bioprosthetic Total Artificial Heart.

The Aeson® total artificial heart (A-TAH) has been developed as a total heart replacement for patients at risk of death from biventricular failure. We previously described endothelialization of the hybrid membrane inside A-TAH probably at the origin of acquired hemocompatibility. We aimed to quantify vasculogenic stem cells in peripheral blood of patients with long-term A-TAH implantation. Four male adult patients were includedin this study. Peripheral blood mononuclear cells were collected before A-TAH implantation (T0) and after implantation at one month (T1), between two and five months (T2), and then between six and twelve months (T3). Supervised analysis of flow cytometry data confirmed the presence of the previously identified Lin-CD133+CD45- and Lin-CD34+ with different CD45 level intensities. Lin-CD133+CD45-, Lin-CD34+CD45- and Lin-CD34+CD45+ were not modulated after A-TAH implantation. However, we demonstrated a significant mobilization of Lin-CD34+CD45dim (p = 0.01) one month after A-TAH implantation regardless of the expression of CD133 or c-Kit. We then visualized data for the resulting clusters on a uniform manifold approximation and projection (UMAP) plot showing all single cells of the live Lin- and CD34+ events selected from down sampled files concatenated at T0 and T1. The three clusters upregulatedat T1 are CD45dim clusters, confirming our results. In conclusion, using a flow cytometry approach, we demonstrated in A-TAH-transplanted patients a significant mobilization of Lin-CD34+CD45dim in peripheral blood one month after A-TAH implantation. Using a flow cytometry approach, we demonstrated in A-TAH transplanted patients a significant mobilization of Lin-CD34+CD45dim in peripheral blood one month after A-TAH implantation. This cell population could be at the origin of newly formed endothelial cells on top of hybrid membrane in Carmat bioprosthetic total artificial heart.

Investigating the Efficacy of Zingerone on Mesothelioma and the Role of TRPV1 in This Effect

Mesothelioma is a highly lethal cancer developing in the lung, heart, and abdominal membranes. Zingerone, a capsaicin-like bioactive compound, has been shown to have anticancer properties. Transient Receptor Potential Vanilloid 1 (TRPV1) is an ion channel involving in the cytotoxicity of capsaicin. In the present study, we aimed at determining the cytotoxicity of zingerone on a mesothelioma cell line and to evaluate the role of TRPV1 in this effect. For this purpose, H2452 was used as the mesothelioma cell line and MTS was performed to calculate zingerone cytotoxicity. Moreover, TRPV1 was inhibited by capsazepeine while TRPV1 production was reduced through shRNA treatment. Besides, wound healing and clonogenic assays were performed to measure the migration and colony forming abilities, respectively. As a result, IC50 value of zingerone was calculated as 11.49 mM. Capsazepine treatment or lowered TRPV1 gene expression did not appear to affect zingerone cytotoxicity (p > 0.05) even though the migration rate and colony forming abilities of the zingerone treated cells decreased significantly compared to the control (p < 0.05). Therefore, we concluded that zingerone was less cytotoxic to H2452 cells than the most cancer types and TRPV1 did not seem to have a role in its cytotoxicity.

Incidence and clinical outcomes of bleeding complications and acute limb ischemia in STEMI and cardiogenic shock.

Bleeding complications and acute limb ischemia (ALI) are devastating vascular complications in patients with ST-segment elevation myocardial infarction (STEMI). Cardiogenic shock (CS) can further increase this risk due to multiorgan failure. In the contemporary era, percutaneous mechanical circulatory support is commonly used for management of CS. We hypothesized that vascular complications may be an important determinant of clinical outcomes for CS due to STEMI (CS-STEMI). We evaluated 10-year national trends, resource utilization and outcomes of bleeding complications, and ALI in CS-STEMI. We performed a retrospective cohort study of CS-STEMI patients from a large U.S. national database (National Inpatient Sample) between 2005 and 2014. Events were then divided into four different groups: no MCS, with intra-aortic balloon pump, percutaneous ventricular assist device includes Impella or Tandem Heart or extracorporeal membrane oxygenation. Bleeding complications and ALI were observed in 31,389 (18.2%) and 1,628 (0.9%) out of 172,491 admissions with CS-STEMI, respectively. Between 2005 and 2014, overall trends increased for ALI; however, the number of bleeding events decreased. ALI was associated with increased in-hospital mortality in comparison to those without any ALI. However, bleeding complications were not associated with increased in-hospital mortality. Compared to patients without complications, both bleeding and ALI were associated with increased length of stay (LOS) and hospitalization costs. Bleeding and ALI are common complications associated with CS-STEMI in the contemporary era. Both complications are associated with increased hospital costs and LOS. These findings highlight the need to develop algorithms focused on vascular safety in CS-STEMI.

Analyze of Impedance for Water Management in Proton Exchange Membrane Fue Fells Using Neural Networks Methodology

The objective of this work is to define and to implement a simple method to assess the impacts of relative humidity and operating time on the fuel cell impedance. The method is based on the physical model of Randles with CPE and a mathematical tool for identifying various parameters based on the least squares’ method. The objective of the theoretical model development is the model implementation of the control system and water management of predictive diagnostics. Artificial neural networks are used to create the optimum impedance model. The model is applied for the identification of all resistors (internal resistors measured at high frequency, biasing resistors measured at high frequency) which are characterized by a high sensitivity for both cases, the flooding or drying of the cell heart (membrane and electrodes). This model is able to easily generate Nyquist diagram for any condition of relative humidity and operating time, it helped define the stack hydration status. Based on the obtained results, the model demonstrated a best flexible response, accurate and fast. The developed model can be integrated into a water management control system in PEM fuel cells.

Distribution and properties of cardiac and pulmonary β-adrenergic receptors in corn snakes (Pantherophis guttatus) and Boa constrictor (Boa constrictor)

The aim of the present study was to characterize β-adrenergic receptors in the snake heart and lung of corn and Boa constrictor snakes. The β-adrenergic receptor binding sites were studied in purified heart and lung membranes using the specific β-adrenergic receptor antagonist [125J]-iodocyanopindolol (ICYP) and subtypes using selective β1-adrenergic receptor antagonist CGP-20712A and selective β2-adrenergic receptor antagonist ICI-118.551. A saturable and specific β-adrenergic receptor binding site was detected in cardiac membranes with maximal receptor density (Bmax) of 43.99 ± 3.86 fmol/mg protein (corn snake) and 58.07 ± 2.88 fmol/mg protein (Boa constrictor) as well as KD of 24.21 ± 7.38 pM (corn snake) and 21.48 ± 3.85 pM (Boa constrictor) and in lung membranes (Bmax fmol/mg protein: 55.95 ± 16.28 (corn snake) and 107.00 ± 14.21 (Boa constrictor); KD pM: 71.25 ± 21.92 (corn snake) and 55.04 ± 18.68 (Boa constrictor)). Competition-binding studies showed β-adrenergic receptors with low affinities to the β2-selective adrenergic receptor antagonist and high affinity binding to β1-selective adrenergic receptor antagonist in both heart and lung tissues of both snake species, suggesting the presence of high population of the post-synaptic β1-adrenergic receptor subtype. It seems that the presence of the predominant β1-subtype also in lung tissues may indicate the importance of the vascular system in the snake lung.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study.

IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

Safety validation study of the hybrid membrane in the Carmat bioprosthetic Total Artificial Heart

The Carmat bioprosthetic Total Artificial Heart (C-TAH) is a mechanical circulatory support device providing an alternative to the heart transplant for patients in advanced heart failure. One of the most original components of the C-TAH is its biocompatible hybrid membrane made of bovine pericardial tissue and polyurethane separating the actuating liquid of the pump from blood inside ventricles. The aim of our study is to evaluate safety of the hybrid membrane in terms of mechanical aging, risks of calcification, and impact on the hemodynamics inside ventricles on red blood cells, platelet and von willebrand factor (vWF) impairment. Hybrid membranes were aged in a custom-designed endurance bench then evaluated in terms of mechanical proprieties and physical degradation. Erosion areas were explored in electronic microscopy and surface topography. Samples were subcutaneously implanted in Wistar rats as a model for calcification. Hemodynamic simulation using computational model has been used to reproduce shear stress in both ventricles. Compliance of the membrane is not significantly modified from 9 month to a 4 years aging simulating process. Physical proprieties as glass transition temperature and infrared spectrum are not modified. Exploration of erosion areas showed no oil diffusion through the membrane. Implantation in wistar rats demonstrated that anticalcification pretreatment with FET was able to significantly reduce the calcium concentration. We confirmed the absence of calcification in the areas in contact with blood by microtomography in the feasability study. The hemodynamic simulation showed no significant stress able to trigger hemolysis, platelet activation or the degradation of the vWFmultimers, confirmed with the patients implanted in feasibility study. Mechanical aging, calcification model and hemodynamic simulation allowed us to confirm safety of the hybrid membrane used in the C-TAH.

Inorganic mercury and cadmium induce rigidity in eukaryotic lipid extracts while mercury also ruptures red blood cells

Hg and Cd are non-essential toxic heavy metals that bioaccumulate in the tissues of living systems but less is known about their interactions with Eukaryotic lipid bilayers. Microscopy experiments showed that Hg and Cd changed the cell morphology of rabbit erythrocytes while Hg also induced cell rupture. As membranes are one of the first available targets, our study aimed to better understand metal-lipid interactions that could lead to toxic effects. Fluorescence spectroscopy (Laurdan Generalized Polarization) and dynamic light scattering were used to analyze metal-induced changes in membrane fluidity and the size of liposomes composed of Brain (Porcine), Liver (Bovine), Heart (Bovine) and Yeast (S. cerevisiae) lipid extracts. Under physiological chloride and pH levels, Hg irreversibly cleaves plasmalogens resulting in an increase in membrane rigidity. These lipids are enriched in Brain, Heart and Erythrocyte membranes and are important in signalling and the protection against oxidative stress. Interestingly, Hg had a heavily reduced effect on the plasmalogen-free Yeast extract membrane. In contrast, Cd induced rigidity by targeting negatively charged phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and cardiolipin in these extracts. Metal-induced liposome aggregation depended on the proportion of negatively charged lipids/plasmalogen and even the order of metal addition. Our results show that data from model systems correlate with trends observed in complex biological extracts and red blood cells and serve as a predictive tool for analyzing metal-lipid interactions. The determination of the specific lipid targets for Hg and Cd provides new insights how these metals exert toxic effects on cell membranes.

Inhibition of PDE4 by low doses of rolipram induces changes in lipid and protein components of mice heart.

There is significant increasing interest in phosphodiesterase-4 (PDE4) inhibition in treatment of cardiovascular diseases. Related with this, research has focused on cellular, biochemical, molecular and structural changes in heart tissue induced by PDE4s inhibitors. However, for their clinical applicability additional studies are still needed. Fourier transform infrared spectroscopy offers promising approach to contribute such issue due to its ability in detection the changes in biomolecules. By utilizing this method, we examined the effects of PDE4 inhibition by rolipram at 0.05 mg/kg and 0.1 mg/kg doses on content of lipids and proteins, and fluidity, order and packing of membranes in naive mice heart. In treated groups, there was a significant decrease in unsaturated, saturated lipids, cholesterol esters, fatty acids, phospholipids and triacylgylcerols obtained from CH2, C=O, olefinic=CH, and COO- areas, and CH2/lipid, C=O/lipid, olefinic=CH/lipid, and COO-/lipid ratios. Additionally, olefinic=CH area and olefinic=CH/lipid ratio may suggest decreased lipid peroxidation, confirmed by thiobarbituric acid assay. Also, a higher degree of membrane order, slight increase in membrane fluidity and differences in membrane packing were obtained. Amide I and II areas and RNA/protein ratios showed that variation in protein content is not correlated with applied concentration. Analysis of amide I mode predicted alterations in secondary structures like an increase in random coils and decrease in alpha-helices. Moreover, all groups were successfully discriminated by cluster analysis. The corresponding results may help to understand the potential effects of PDE4 inhibition by rolipram.

Canine Fetal Peritonitis

Background: The occurrence of neonatal diseases and neonatal deaths in canines are considered higher in the first days of life of individuals and bacterial diseases are considered the second most common cause. Inflammation of the mesothelial lining of the abdominal cavity is referred peritonitis, which can be classified as primary or secondary and there is no description on the occurrence of canine fetal peritonitis in the veterinary literature. The objective of this study is to describe a case of canine fetus peritonitis, presenting abnormal sonographic findings of the fetus and abnormalities identified after birth that were not yet described in veterinary literature.Case: A female Poodle, six years of age, considered healthy and without clinical complications during the gestational period, was referred for sonographic evaluation at the end of the pregnancy, in order to evaluate of the fetal viability and estimate the gestational period. By ultrasound examination, two canine fetuses were identified and based on its morphogenesis and biparietal diameter, the estimated gestational age was approximately 58 days. On the individual examination of each of the fetus by ultrasound, it was found that one of them had normal sonographic findings and on the second canine fetus it was found an increase of the intra-abdominal echogenicity of tissues, loss of the sonographic definition of the intestinal loops and presence of laminar anechoic areas between the interfaces of organs, consistent with abdominal effusion. Parturition occurred the following day of the ultrasound examination no complications and with the birth of two canine fetuses. About six hours after birth, one of the neonates showed dyspnea, hypothermia, areas of ecchymosis, suffusion in the ventral abdominal region, icteric and pale mucous membrane and the presence of abdominal fluid and came to death about 30 min after. Cytological analysis of the sample revealed exuberant amount of erythrocytes, fibrin and leukocytes. Culture revealed infection by Staphylococccus aureus and Enterococcus sp. Presence of bloody fluid in the abdominal cavity was observed at necropsy, with yellowish parietal surface of the thoracic and abdominal cavities and petechiae on the lungs, heart, stomach surfaces and intestinal mucous membrane. Microscopically, the lungs showed extensive areas of parenchymal consolidation, vascular congestion and intense inflammatory infiltrate in the alveolar wall; the liver was characterized by vascular congestion, areas of degeneration of hepatocytes and presence of calcified foci, necrosis and inflammatory infiltrates. Associating the results and findings of the exams was possible to conclude the diagnostic of fetal peritonitis.Discussion: The visualization of abnormal findings in affected fetus by a gestational ultrasound exam makes this report inedited and relevant for veterinary obstetrics. This demonstrates the importance of ultrasound exam in pregnant bitches for early detection of maternal fetal diseases. Presence of abdominal fluid and increased mesenteric ochogenicity associated with loss of the intestinal loop definition corroborates with the clinical suspicion of peritonitis in fetus. Findings on the anatomopathological examination, cytology and culture allowed conclusion of the cause of death. Bacterial forms in the cytological smear supported the diagnostic suspicion of peritonitis. Gestational ultrasound exam performed in this report was essential for proper conduct of the pregnant bitch and viability of the fetuses, suggesting that the detected fetal abnormalities showed predictive value in the viability of diseased individual. This case report contributes with the description of canine fetal peritonitis.

Impedance model for diagnosis of water management in fuel cells using artificial neural networks methodology

The objective of this work is to define and to implement a simple method to assess the impacts of relative humidity and operating time on the fuel cell impedance. The method is based on the physical model of Randles with CPE and a mathematical tool for identifying various parameters based on the least squares method. The objective of the theoretical model development is the model implementation of the control system and water management of predictive diagnostics. Artificial neural networks are used to create the optimum impedance model. The model is applied for the identification of all resistors (internal resistors measured at high frequency, biasing resistors measured at high frequency) which are characterized by a high sensitivity for both cases, the flooding or drying of the cell heart (membrane and electrodes). This model is able to easily generate Nyquist diagram for any condition of relative humidity and operating time, it helped define the stack hydration status. Based on the obtained results, the model demonstrated a best flexible response, accurate and fast. The developed model can be integrated into a water management control system in PEM fuel cells.

An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that early axon initial segments and epithelial lateral membranes initially were based on spectrin-ankyrin-cell adhesion molecule assemblies and subsequently served as "incubators," where ion transporters independently acquired ankyrin-binding activity through positive selection.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

HCN2 channels: a permanent open state and conductance changes.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the membranes of heart and brain cells can conduct Na(+) and K(+) ions and activate between -30 and -120 mV. We express the α subunit of HCN2 channels in Xenopus laevis oocytes and are confronted with two unexpected problems. First, we observe a rise in membrane conductance at resting potential proportional to the amount of expression. On activation to hyperpolarizing potentials, the instantaneous conductance rises in proportion to the amount of activated current. CsCl reduces the observed effects. This can be explained by the expression in oocytes membranes of a fraction of permanently open HCN2 channels. Second, using TEVC technique, our data show a completely different behaviour in physiological solutions of heterogeneously expressed HCN2 currents from what is observed in wild-type currents in the absence of drugs. During pulse trains, we frequently observe (1) a fast and significant decline of the amplitude of HCN2 current during hyperpolarizing steps, (2) no recovery of this decline after a long period at resting membrane potential, (3) a different behaviour of the tail currents at depolarization with other and slower changes than during activation, (4) recovery of this decline in high K(+)/low Na(+) bath solution. The decline of the HCN2 current in physiological conditions is caused by a reduction of the conductance of the HCN2 channel presumably caused by the mere presence of sodium in the channel, in competition with potassium ions and with a limitative effect on the channel conductance.

A 2014 update on the management of patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, relapsing autoimmune connective tissue disease, primarily affecting the skin, joints, kidneys, heart, lungs, nervous system, blood elements, and serosal membranes. SLE is characterized by cytokine dysregulation, polyclonal B-cell activation, autoantibody production, and increased immune complex formation due to aberrations involving hyperactive B cells, T cells, and cells of the monocytic lineage. The symptoms of SLE are often diverse and nonspecific, and timely identification of SLE and associated comorbidities in patients is critical as aggressive monitoring and therapy may be warranted, especially in patients with poor prognoses. Based on the up-to-date understanding of the pathophysiology of SLE, the first targeted biological agent belimumab has been approved by the US Food and Drug Administration (FDA) in more than 50 years, and many targeted agents are being evaluated in late-stage clinical trials. There is a clear need to discuss how and when to incorporate new and emerging biological agents in managing patients with SLE. Additionally, the potential for increased risk of infections is a factor that heavily influences the rheumatologist׳s decision to use biological agents in managing patients with SLE. Hence, in this roundtable educational activity, expert faculty will review and discuss the strategies for timely diagnosis of SLE and associated comorbidities. They will also discuss the current understanding of the pathophysiology of SLE and how new and emerging biological agents help address the underlying pathophysiological aberrations in patients with SLE. The faculty will also review strategies to minimize the risk of infections and other toxicities in patients with SLE.

G.P.131: Cell membrane integrity in myotonic dystrophy type 1: Implications for therapy

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG∗CAG) n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding of this RNA to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and muscle and brain cell membranes. It has been reported that DM1 patients show elevated levels of muscular markers in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface distribution of dystrophin was normal. Combined, our findings show that cells in DM1 tissues have a normal functional membrane, which forms a barrier that must be overcome in future work towards effective drug delivery.