Three-Dimensional Structure of the Intercalated Disc Reveals Plicate Domain and Gap Junction Remodeling in Heart Failure

The intercalated disc (ICD) orchestrates electrochemical and mechanical communication between neighboring cardiac myocytes, properties that are perturbed in heart failure (HF). Although structural data from transmission electron microscopy two-dimensional images have provided valuable insights into the domains forming the ICD, there are currently no three-dimensional (3D) reconstructions for an entire ICD in healthy or diseased hearts. Here, we aimed to understand the link between changes in protein expression in an ovine tachypacing-induced HF model and ultrastructural remodeling of the ICD by determining the 3D intercalated disc architecture using serial block face scanning electron microscopy. In the failing myocardium there is no change to the number of ICDs within the left ventricle, but there is an almost doubling of the number of discs with a surface area of <1.0 × 10⁸μm² in comparison to control. The 3D reconstructions further revealed that there is remodeling of the plicate domains and gap junctions with vacuole formation around and between the contributing membranes that form the ICDs in HF. Biochemical analysis revealed upregulation of proteins involved in stabilizing the adhesive and mechanical properties consistent with the morphological changes. Our studies here have shown that in tachypacing-induced HF mechanical stresses are associated with both structural and molecular alterations. To our knowledge, these data together provide novel, to our knowledge, insights as to how remodeling at the molecular and structural levels leads to impaired intercellular communication.     

Coxsackievirus-Induced miR-21 Disrupts Cardiomyocyte Interactions via the Downregulation of Intercalated Disk Components

Intercalated disks (ICDs) are substantial connections maintaining cardiac structures and mediating signal communications among cardiomyocytes. Deficiency in ICD components such as desmosomes, fascia adherens and gap junctions leads to heart dysfunction. Coxsackievirus B3 (CVB3) infection induces cardiac failure but its pathogenic effect on ICDs is unclear. Here we show that CVB3-induced miR-21 expression affects ICD structure, i.e., upregulated miR-21 targets YOD1, a deubiquitinating enzyme, to enhance the K48-linked ubiquitination and degradation of desmin, resulting in disruption of desmosomes. Inhibition of miR-21 preserves desmin during CVB3 infection. Treatment with proteasome inhibitors blocks miR-21-mediated desmin degradation. Transfection of miR-21 or knockdown of YOD1 triggers co-localization of desmin with proteasomes. We also identified K108 and K406 as important sites for desmin ubiquintination and degradation. In addition, miR-21 directly targets vinculin, leading to disturbed fascia adherens evidenced by the suppression and disorientation of pan-cadherin and α-E-catenin proteins, two fascia adherens-components. Our findings suggest a new mechanism of miR-21 in modulating cell-cell interactions of cardiomyocytes during CVB3 infection.     

Dutch outcome in implantable cardioverter-defibrillator therapy (DO-IT): registry design and baseline characteristics of a prospective observational cohort study to predict appropriate indication for implantable cardioverter-defibrillator

Background

Implantable cardioverter-defibrillators (ICDs) are widely used for the prevention of sudden cardiac death. At present, both clinical benefit and cost-effectiveness of ICD therapy in primary prevention patients are topics of discussion, as only a minority of these patients will eventually receive appropriate ICD therapy.

Methods/design

The DO-IT Registry is a nationwide prospective cohort with a target enrolment of 1,500 primary prevention ICD patients with reduced left ventricular function in a setting of structural heart disease. The primary outcome measures are death and appropriate ICD therapy for ventricular tachyarrhythmias. Secondary outcome measures are inappropriate ICD therapy, death of any cause, hospitalisation for ICD related complications and for cardiovascular reasons. As of December 2016, data on demographic, clinical, and ICD characteristics of 1,468 patients have been collected. Follow-up will continue up to 24 months after inclusion of the last patient. During follow-up, clinical and ICD data are collected based on the normal follow-up of these patients, assuming ICD interrogations take place every six months and clinical follow-up is once a year. At baseline, the mean age was 66 (standard deviation [SD] 10) years and 27% were women.

Conclusion

The DO-IT Registry represents a real-world nationwide cohort of patients receiving ICDs for primary prevention of sudden cardiac death with reduced left ventricular function in a setting of structural heart disease. The registry investigates the efficacy of the current practice and aims to develop prediction rules to identify subgroups who will not (sufficiently) benefit from ICD implantation and to provide results regarding costs and budget impact of targeted supply of primary preventions ICDs.

     

Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold

Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides.     

Clinical implementation of guidelines for cardioverter defibrillator implantation: lost in translation?

Purpose: Guidelines for implantation of cardioverter defibrillators (ICD) are increasingly including indications for primary prevention of sudden cardiac death in high-risk groups, where ICDs were traditionally implanted for secondary prevention. We performed a single-centre audit to evaluate adherence to the recent Dutch guidelines. Methods: All 1886 patients visiting a large regional Dutch teaching hospital (attending 1.8 to 2.0% of the Dutch population) in November 2005 were screened using the recently updated Dutch guidelines. Patients fulfilling these criteria were categorised as having an ICD indication for primary or secondary prevention. Results: 135 patients had an indication for ICD, 19 of whom had one or received one. Of the remaining 116 patients, 14 were ‘new’ to the department of cardiology. The 102 ‘known’ patients had 466 doctor-patient contacts in the previous year, which averages 4.57 cardiology contacts per patient per year. Patients were more likely to receive an ICD for the secondary prevention of SCD (10/11, 91%) than for primary prevention (9/124, 7%). Conclusion: In a large regional teaching hospital in the Netherlands, only a small proportion of patients eligible for ICD implantation actually receive one. Cardiologists tend to implant ICDs for secondary prevention of SCD. The low ICD implantation rate for primary prevention of SCD may relate to logistics (e.g. permission to implant ICDs, the presence of an electrophysiology lab) or the perceived low cost-benefit ratio. Our results indicate that once the substantial backlog (13,500 ICDs) has been addressed, the annual implantation of new ICDs should rise from the current 125 to at least 510 per million inhabitants per year in the Netherlands. (Neth Heart J 2007;15:129-32.)     

Investigation of Human Albumin-Induced Circular Dichroism in Dansylglycine

Induced circular dichroism (ICD), or induced chirality, is a phenomenon caused by the fixation of an achiral substance inside a chiral microenvironment, such as the hydrophobic cavities in proteins. Dansylglycine belongs to a class of dansylated amino acids, which are largely used as fluorescent probes for the characterization of the binding sites in albumin. Here, we investigated the ICD in dansylglycine provoked by its binding to human serum albumin (HSA). We found that the complexation of HSA with dansylglycine resulted in the appearance of an ICD band centred at 346 nm. Using this ICD signal and site-specific ligands of HSA, we confirmed that dansylglycine is a site II ligand. The intensity of the ICD signal was dependent on the temperature and revealed that the complexation between the protein and the ligand was reversible. The induced chirality of dansylglycine was susceptive to the alteration caused by the oxidation of the protein. A comparison was made between hypochlorous acid (HOCl) and hypobromous acid (HOBr), and revealed that site II in the protein is more susceptible to alteration provoked by the latter oxidant. These findings suggest the relevance of the aromatic amino acids in the site II, since HOBr is a more efficient oxidant of these residues in proteins than HOCl. The three-dimensional structure of HSA is pH-dependent, and different conformations have been characterised. We found that HSA in its basic form at pH 9.0, which causes the protein to be less rigid, lost the capacity to bind dansylglycine. At pH 3.5, HSA retained almost all of its capacity for binding to dansylglycine. Since the structure of HSA at pH 3.5 is expanded, separating the domain IIIA from the rest of the molecule, we concluded that this separation did not alter its binding capacity to dansylglycine.     

Assessing conservation value of stream communities: a comparison of approaches based on centres of density and species richness

1. We used a recently developed Index of Centres of Density (ICD) to assess conservation value of thirty-nine sites in the upper Clinch River drainage of Virginia and Tennessee, U.S.A. The ICD incorporates information on the population density of each species at a site relative to sites throughout the region.
2. Value assessments based on the ICD were compared to those based on species richness. Species richness at sites was not related to ICD scores, but collective species lists from high-ranking sites were similar for both approaches. All sites with rare species had either a high ICD score or high species richness.
3. Four community types (defined by physiography and stream size) were better represented in sites with high-ranking ICDs than in sites with high-ranking species richness. Sites with high ICD scores were distributed uniformly throughout the region.
4. The ICD is a more powerful tool than species richness for assessing conservation value because the ICD identifies areas with regionally rare species, especially viable populations, or distinctive communities, all of which are key components of a region's biodiversity.     

Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme.

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzyme of the intermediary metabolism, as it controls the carbon flux within the citric acid cycle and supplies the cell with 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independent of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD gene, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showed up to 10-fold-higher specific ICD activities. The nucleotide sequence of a 3,595-bp DNA fragment containing the icd gene was determined. The predicted gene product of icd consists of 739 amino acids (M(r) = 80.091) and showed 58.5% identity with the monomeric ICD isozyme II from Vibrio sp. strain ABE-1 but no similarity to any known ICD of the dimeric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesting that only a single ICD is present in C. glutamicum. From an icd-overexpressing C. glutamicum strain, ICD was purified and biochemically characterized. The native ICD was found to be a monomer; to be specific for NADP+; to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxylate. The data indicate that ICD from C. glutamicum is structurally similar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium, and Azotobacter but different from all other known procaryotic and eucaryotic ICDs.     

Shape and volume of craniofacial cavities in intentional skull deformations

Intentional cranial deformations (ICD) have been observed worldwide but are especially prevalent in preColombian cultures. The purpose of this study was to assess the consequences of ICD on three cranial cavities (intracranial cavity, orbits, and maxillary sinuses) and on cranial vault thickness, in order to screen for morphological changes due to the external constraints exerted by the deformation device. We acquired CT‐scans for 39 deformed and 19 control skulls. We studied the thickness of the skull vault using qualitative and quantitative methods. We computed the volumes of the orbits, of the maxillary sinuses, and of the intracranial cavity using haptic‐aided semi‐automatic segmentation. We finally defined 3D distances and angles within orbits and maxillary sinuses based on 27 anatomical landmarks and measured these features on the 58 skulls. Our results show specific bone thickness patterns in some types of ICD, with localized thinning in regions subjected to increased pressure and thickening in other regions. Our findings confirm that volumes of the cranial cavities are not affected by ICDs but that the shapes of the orbits and of the maxillary sinuses are modified in circumferential deformations. We conclude that ICDs can modify the shape of the cranial cavities and the thickness of their walls but conserve their volumes. These results provide new insights into the morphological effects associated with ICDs and call for similar investigations in subjects with deformational plagiocephalies and craniosynostoses. Am J Phys Anthropol 151:110–119, 2013. © 2013 Wiley Periodicals, Inc.     

Rate and predictors of electrical failure in non-recalled defibrillator leads

BackgroundImplantable cardioverter defibrillator (ICD) leads are considered as the ‘weakest link’ in defibrillator systems due to FDA recalls and advisories involving popular lead models from major manufacturers. The rate of electrical failure of ICD leads not implicated in a recall is however not well determined.MethodsMedical records of patients implanted with ICDs at hospitals of the University of Pittsburgh Medical Center between 2002 and 2014 were analyzed. Leads were classified as having electrically failed if removed or replaced for reasons other than infection or heart transplantation. Patients were followed to endpoint of death or electrical lead failure.Results2410 consecutive ICD recipients (mean age 66 ± 13 years, women 22%, single/dual/biventricular-ICD 20%/44%/36%) were included. During a mean follow-up of 3.9 ± 3.3 years, 1272 patients (53%) died, 55 patients (2.3%) had ICD lead electrical failure, and 1052 (44%) patients were alive with functional leads at the time of last follow-up. Patients with failed leads had higher BMI (p = 0.07), better functional status (p = 0.04), higher serum creatinine (p = 0.004), wider QRS complex (p = 0.01), higher number of implanted leads (p = 0.06) and were more likely to have ischemic cardiomyopathy (p = 0.03). After adjusting for these variables in a binary logistic regression model, only a lower BMI, presence of non-ischemic cardiomyopathy, and a better functional status remained independently predictive of electrical failure.ConclusionsOnly 2.3% of non-recalled ICD leads experience electrical failure (annual failure rate of 0.6%). A higher patient functional status, lower BMI, and non-ischemic etiology of cardiomyopathy are independently associated with higher rates of ICD lead failure.     

Dynamics of heteropentameric nicotinic acetylcholine receptor: implications of the gating mechanism

The dynamics characteristics of the currently available structure of Torpedo nicotinic acetylcholine receptor (nAChR), including the extracellular, transmembrane, and intracellular domains (ICDs), were analyzed using the Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). We found that a symmetric quaternary twist motion, reported previously in the literature in a homopentameric receptor (Cheng et al. J Mol Biol 2006;355:310-324; Taly et al. Biophys J 2005;88:3954-3965), occurred also in the heteropentameric Torpedo nAChR. We believe, however, that the symmetric twist alone is not sufficient to explain a large body of experimental data indicating asymmetry and subunit nonequivalence during gating. Here we report our results supporting the hypothesis that a combination of symmetric and asymmetric motions opens the gate. We show that the asymmetric motion involves tilting of the TM2 helices. Furthermore, our study reveals three additional aspects of channel dynamics: (1) loop A serves as an allosteric mediator between the ligand binding loops and those at the domain interface, particularly the linker between TM2 and TM3; (2) the ICD can modulate the pore dynamics and thus should not be neglected in gating studies; and (3) the F loops, which are peculiarly longer and poorly-conserved in non-alpha-subunits, have important dynamical implications.     

Mammalian enabled (Mena) is a critical regulator of cardiac function

Mammalian enabled (Mena) of the Drosophila enabled/vasodilator-stimulated phosphoprotein gene family is a cytoskeletal protein implicated in actin regulation and cell motility. Cardiac Mena expression is enriched in intercalated discs (ICD), the critical intercellular communication nexus between adjacent muscle cells. We previously identified Mena gene expression to be a key predictor of human and murine heart failure (HF). To determine the in vivo function of Mena in the heart, we assessed Mena protein expression in multiple HF models and characterized the effects of genetic Mena deletion on cardiac structure and function. Immunoblot analysis revealed significant upregulation of Mena protein expression in left ventricle tissue from patients with end-stage HF, calsequestrin-overexpressing mice, and isoproterenol-infused mice. Characterization of the baseline cardiac function of adult Mena knockout mice (Mena(-/-)) via echocardiography demonstrated persistent cardiac dysfunction, including a significant reduction in percent fractional shortening compared with wild-type littermates. Electrocardiogram PR and QRS intervals were significantly prolonged in Mena(-/-) mice, manifested by slowed conduction on optical mapping studies. Ultrastructural analysis of Mena(-/-) hearts revealed disrupted organization and widening of ICD structures, mislocalization of the gap junction protein connexin 43 (Cx43) to the lateral borders of cardiomyoycytes, and increased Cx43 expression. Furthermore, the expression of vinculin (an adherens junction protein) was significantly reduced in Mena(-/-) mice. We report for the first time that genetic ablation of Mena results in cardiac dysfunction, highlighted by diminished contractile performance, disrupted ICD structure, and slowed electrical conduction.     

Identification of similar regions of protein structures using integrated sequence and structure analysis tools

Background  

Understanding protein function from its structure is a challenging problem. Sequence based approaches for finding homology have broad use for annotation of both structure and function. 3D structural information of protein domains and their interactions provide a complementary view to structure function relationships to sequence information. We have developed a web site and an API of web services that enables users to submit protein structures and identify statistically significant neighbors and the underlying structural environments that make that match using a suite of sequence and structure analysis tools. To do this, we have integrated S-BLEST, PSI-BLAST and HMMer based superfamily predictions to give a unique integrated view to prediction of SCOP superfamilies, EC number, and GO term, as well as identification of the protein structural environments that are associated with that prediction. Additionally, we have extended UCSF Chimera and PyMOL to support our web services, so that users can characterize their own proteins of interest.     

Molecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANA

LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANA_DBD) to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANA_DBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.     

Computational approaches for protein function prediction: A combined strategy from multiple sequence alignment to molecular docking-based virtual screening

The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein–ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.     

The cytoplasmic sequence of E-cadherin promotes non-amyloidogenic degradation of A beta precursors

The presenilin (PS)/gamma-secretase system promotes production of the A beta (A beta) peptides by mediating cleavage of amyloid precursor protein (APP) at the gamma-sites. This system is also involved in the processing of type-I transmembrane proteins, including APP, cadherins and Notch1 receptors, at the epsilon-cleavage site, resulting in the production of peptides containing the intracellular domains (ICDs) of the cleaved proteins. Emerging evidence shows that these peptides have important biological functions, raising the possibility that their inhibition by gamma-secretase inhibitors may be detrimental to the cell. Here, we show that peptide E-Cad/CTF2, produced by the PS1/gamma-secretase processing of E-cadherin, promotes the lysosomal/endosomal degradation of the transmembrane APP derivatives, C99 and C83, and inhibits production of the APP ICD (AICD). In addition, E-Cad/CTF2 decreases accumulation of total secreted A beta. These data suggest a novel method to promote the non-amyloidogenic degradation of A beta precursors and to inhibit A beta production.     

Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex

Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.     

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.