iPSC-cardiomyocyte models of Brugada syndrome : achievements, challenges and future perspectives

Brugada syndrome (BrS) is an inherited cardiac arrhythmia that predisposes to ventricular fibrillation and sudden cardiac death. It originates from oligogenic alterations that affect cardiac ion channels or their accessory proteins. The main hurdle for the study of the functional effects of those variants is the need for a specific model that mimics the complex environment of human cardiomyocytes. Traditionally, animal models or transient heterologous expression systems are applied for electrophysiological investigations, each of these models having their limitations. The ability to create induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), providing a source of human patient-specific cells, offers new opportunities in the field of cardiac disease modelling. Contemporary iPSC-CMs constitute the best possible in vitro model to study complex cardiac arrhythmia syndromes such as BrS. To date, thirteen reports on iPSC-CM models for BrS have been published and with this review we provide an overview of the current findings, with a focus on the electrophysiological parameters. We also discuss the methods that are used for cell derivation and data acquisition. In the end, we critically evaluate the knowledge gained by the use of these iPSC-CM models and discuss challenges and future perspectives for iPSC-CMs in the study of BrS and other arrhythmias

The Effects of Arrhythmogenic Right Ventricular Cardiomyopathy-Causing Proteins on the Mechanical and Signaling Properties of Cardiac Myocytes

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by a high incidence of lethal ventricular arrhythmias, fibrofatty replacement of myocardium, and can account for up to 20% of sudden cardiac death (SCD) cases in the young. Typically involving autosomal dominant transmission, germline mutations in genes encoding desmosomal proteins have been identified as a cause of ARVC, although the pathogenesis of the disease is still unclear. While early detection and treatment can provide a normal life expectancy for the majority of patients, with less than 10% progressing to overt right ventricular failure, low genetic penetrance and epigenetic modifiers (such as endurance exercise) can make the condition difficult to diagnose. Addressing this clinical challenge requires a better understanding of the defective molecular mechanisms that underlie the disease. To that end, the goal of this dissertation is to provide insight into the effects of ARVC-causing mutant proteins on the mechanical and signaling properties of cardiac myocytes. Using elastography and histological techniques, we begin by characterizing the structural and mechanical properties of the native right ventricular myocardium, particularly the right ventricular apex (RVA). Because the RVA is a key site for development of arrhythmias and a potential pacing target, a careful characterization of its structure and mechanical properties are essential for understanding its role in cardiac physiology. In the first section of this dissertation, we perform a systematic analysis of the structural features and mechanical strains in the heart, focusing on the RVA region. More than half of ARVC patients exhibit one or more mutations in genes encoding desmosomal proteins. This has led many investigators to suggest that ARVC is a "disease of the desmosome" in which defective cell-cell adhesion plays a critical pathogenic role, although direct evidence for this hypothesis is lacking. To gain greater insights into potential mechanisms by which desmosomal mutations cause ARVC, we next characterize biomechanical properties and responses to shear stress (motivated by our results in the previous section) in neonatal rat ventricular myocytes expressing two distinct mutant forms of the desmosomal protein plakoglobin which have been linked to ARVC in patients. We show that ARVC-causing mutations in plakoglobin lead to altered cellular distribution of plakoglobin, without alterations in cell mechanical properties or certain early signaling pathways. The identification of defective molecular mechanisms that are common across ARVC-patients remains a strategic area of research. Specifically, recent studies have investigated the mechanistic basis for different ARVC-causing mutations in hopes of identifying common defects in a signaling pathway - information that could be used to develop diagnostic tests or identify therapeutic targets. In the last section of this dissertation, we investigate the effects of mutant plakophilin-2 expression, and repeat key experiments performed in the previous section to identify common defects in mechanical and signaling properties. We identify a common, underlying defect in ARVC pathogenesis. Specifically, we show that disease-causing mutations across different desmosomal proteins can cause the cell to respond abnormally to mechanical shear stress with respect to plakoglobin trafficking

Identification and functional validation of genomic boundaries in mammals

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 30-06-2014Eukaryotic genomes are divided into expression domains, which contain DNA coding sequences together with all the regulatory elements needed for their correct spatio-­‐ temporal expression pattern. Genomic boundaries, also known as insulators, flank these domains preventing undesirable crosstalk between the regulatory elements of neighboring domains. They employ various mechanisms and thus, are functionally rather than structurally defined. For this reason, in an attempt to find boundaries in a genome-­‐ wide unbiased fashion in mammals, we focused on identifying those loci where the presence of boundary function would be required to satisfy a biological need. For example, we hypothesized that adjacent genes with opposite expression patterns would need to be separated by boundaries to maintain the independency of their different expression domains. Also, boundaries could be found partitioning the chromatin into inactive heterochromatic and active euchromatic domains, impeding the deleterious effects the spread of the former would have on the latter. Finally, boundaries could also bracket clusters of co-­‐expressed genes to ensure their co-­‐regulation and co-­‐expression. Different algorithms, based on the analysis of gene expression data, were developed in order to explore these scenarios. The resulting evolutionarily conserved non-­‐coding putative insulator sequences were functionally validated using a number of assays. Their enhancer-­‐ blocking properties were evaluated in vitro in human cells in culture, and then in vivo by using transgenic zebrafish. Additionally, one of the most powerful elements was further tested for its ability to protect from chromosomal position effects in transgenic mice. The description and characterization of new genomic boundaries would shed some light into the way mammalian genomes are organized, as well as expand the repertoire of genetic tools that can be incorporated in heterologous constructs to improve the gene transfer technologies by preventing chromosomal position effects.Los genomas de eucariotas están divididos en dominios de expresión, que se definen como aquellas porciones del genoma que contienen uno o varios genes y todos los elementos reguladores necesarios para que que se expresen de acuerdo con un patrón espacio-­‐temporal concreto. Los aisladores genómicos, también llamados insulators, flanquean estos dominios y los protegen de la influencia no deseada de los elementos reguladores contenidos en los dominios vecinos. Existen diversos mechanismos de aislamiento, por lo que los insulators no se definen por una secuencia de ADN concreta, sino porque comparten una misma función. Así, para encontrar aisladores en el genoma de mamíferos de una forma no sesgada, nos propusimos identificar aquellas posiciones del genoma donde se requiere la presencia de función aisladora para satisfacer un problema biológico. Por ejemplo, genes adyacentes con perfiles de expresión completamente distintos deberían estar separados por aisladores que mantuviesen dominios de expresión independientes. Asimismo, cabe esperar la presencia de aisladores entre dominios silentes de heterocromatina y dominios activos de eucromatina. Aquí, impedirían los efectos perjudiciales que el avance de los primeros tendrían sobre los segundos. Finalmente, también podrían encontrarse aisladores flanqueando grupos de genes co-­‐expresados para asegurar su co-­‐regulación y, por tanto, co-­‐expresión. Basándonos en estos escenarios, se desarrollaron diversos algoritmos que usaban datos de expresión génica para predecir la presencia de aisladores. Como resultado de estos algoritmos, se obtuvo una serie de secuencias conservadas evolutivamente y no codificantes que se validaron funcionalmente empleando varios tests. La capacidad de bloqueo de enhancers se evaluó mediante ensayos in vitro en células humanas en cultivo primero, y luego in vivo mediante el uso de peces cebra transgénicos. Además, se analizó la capacidad de uno de los elementos más potentes para proteger de efectos de posición cromosomales en ratones transgénicos. La descripción y caracterización de nuevos aisladores genómicos no sólo sirve para entender mejor cómo se organizan los genomas de mamíferos. También es útil para ampliar el abanico de herramientas disponibles que se pueden usar en construcciones heterólogas para bloquear los efectos de posición cromosomales que se dan comúnmente en experimentos de transferencia genética

The Story of Redundant Catenins & Their Roles in Cell-cell Adhesion in the Liver

β-Catenin is important in liver homeostasis as a part of Wnt signaling and adherens junctions (AJs); however, aberrant β-catenin activation is observed in a subset of hepatocellular carcinomas (HCC). Since therapeutic targeting of β-catenin in HCC is inevitable, it is important to investigate the implications of such interventions on AJs. We address this important issue both in vivo and in vitro. We observed that hepatocyte-specific β-catenin knockout (βKO) mice have no noticeable adhesive defects. We identified an increase in γ-catenin at AJs in βKO livers. We further observed that γ-catenin was unable to translocate to the nucleus in absence of β-catenin after partial hepatectomy in βKO mice. Since γ-catenin is a desmosomal protein, we next investigated if γ-catenin changes in βKOs were at the expense of desmosomes. We did not observe any differences on desmosomal structure or composition in βKO livers. Similarly, as some tight junction components are β-catenin target genes, we observed only minor changes in tight junctions, such that the function of the junctions was not affected in vivo. To further determine the role and regulation of γ-catenin in absence of β-catenin we established an in vitro model. Hep3B human HCC cells transfected with siRNA to β-catenin led to γ-catenin increase. Using this model we showed γ-catenin was unable to rescue decreased Wnt reporter activity. Scratch-wound assays showed β- and γ-catenin single knockdowns did not affect cell migration, but double knockdown significantly increased wound closure. Centrifugal assay for cell adhesion and hanging-drop assays, measuring hetero- and homotypic cell-cell interactions, showed significant decreases in adhesive strength with double knockdown only. Lastly, we showed the increased γ-catenin with β-catenin loss appears to be regulated by the serine/threonine phosphorylation of γ-catenin by protein kinase A, introducing the possibility of a catenin sensing mechanism. In conclusion, β-catenin loss is compensated by γ-catenin at AJs without negatively affecting other junctions; however, the function of β-catenin as part of the Wnt pathway remains unfulfilled by γ-catenin. Thus, proposed anti-β-catenin therapies for HCC may be able to target aberrant β-catenin in Wnt signaling specifically without negatively affect HCC prognosis, as long as γ-catenin is spared

Mechanisms of Autoantibody-Induced Pathology

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves' disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein

Arrhythmogenic cardiomyopathy - beyond monogenetic disease

Interpreting genetic variants, describing their associated clinical characteristics, and identifying new genetic loci involved in arrhythmogenic cardiomyopathy (ACM) is the focus of this thesis. By investigating various aspects of these genetic variants, we were able to correctly classify two variants occurring in the lamin A/C (LMNA) and titin (TTN) gene. We demonstrated that the reduced force generation seen in cardiomyocytes with the LMNA variant (LMNA c.992G>A) is due to remodelling within the cardiomyocytes and that patients with this specific variant have a milder phenotype compared to what is known from other pathogenic LMNA variants. By extensive phenotyping of carriers of a truncating TTN variant (TTN c.59926+1G>A) we were the first to show that (paroxysmal) atrial fibrillation is an important clinical feature in carriers of truncated TTN variants, even in the absence of dilated cardiomyopathy, atrial enlargement or generally accepted risk factors for atrial fibrillation. Thanks to extensive international collaboration it was possible to compile one of the largest cohorts of patients carrying truncating variants in desmoplakin (DSP). We showed that the location of such a genetic variant within the gene is associated with disease severity. Moreover, these studies show that enrichment of truncating genetic variants in specific regions of DSP variants in ACM patients, when compared to controls, facilitating interpretation of such variants. The multifactorial nature of ACM was underscored in a systematic analysis of the clinical outcome of patients from ACM cohorts carrying multiple variants in ACM related genes, showing that carrying multiple variants influences disease severity. Finally, by analysing genes encoding the sarcomere, the contractile unit of the heart muscle and the plectin (PLEC) gene for rare variants in ACM patients, we showed that these genes do not have a major role in the development of ACM

DEVELOPMENT OF A MICROFLUIDICS INTEGRATED MICROVASCULARISED HUMAN SKIN-ON-A-CHIP TISSUE MODEL

Tissue engineered skin constructs have been under development since the 1980s as a replacement for cadaverous human skin and animal models. These have evolved from simple confluent single cell-type arrangements to models with integrated dermal equivalents and often multiple cell types. Concomitantly, formation of stable self-assembled nanofibrous peptide amphiphile (PA) membranes upon contact with hyaluronic acid (HA) in aqueous solution, with comparable ultrastructure to the apical skin basement membrane (BM) have been reported. With the rise of microfluidic cell culture, scientists have scaled down these technologies; however no group to our knowledge has yet published a full thickness microfluidic skin equivalent with a physiologically mimetic tubular microvasculature or investigated integration of a skin model PA-based BM equivalent. This project aimed to integrate these features into a full thickness human skin-on-a-chip model, with a focus on microvasculature. We report the formation of interfacial self-assembled nanofibrous membranes between fibrin and collagen hydrogels and various PAs including C16V3A3K3. We exploit this in the stabilisation of the dermoepidermal interface in a macroscale fibrin-based skin equivalent model. We then design a novel skin-on-a-chip model integrating human-derived cells in a full-thickness arrangement. The model features a stratified epidermal equivalent featuring correct spatial localisation of keratinocyte basal and differentiation markers, dermoepidermal interfacial deposition of laminin 332 and a dermal compartment populated with human fibroblasts expressive of vimentin. The extent of expression of basal and corneal keratinocyte markers, and the sizes of dermally localised vimentin positive bodies, are comparable between our model and ex vivo human skin. The vascularised model incorporates either HUVECs alone or alongside primary pericytes and displays formation of tubular microvessels positive for CD31 surrounded by a laminin positive basement membrane. Microvessel diameters are comparable to those reported in literature for ex vivo human skin vasculature. The skin-equivalent is also tested in 3D extrusion bioprinted devices. We believe that this is a novel development in the field of microfluidic skin models, with a complex microvascular component displaying anastomosis and lumen formation unlike other vascularised skin-chip models. This model has potential utility in the fields of preclinical drugs testing and disease modellin

The Snakeskin-Mesh Complex of Smooth Septate Junction Restricts Yorkie to Regulate Intestinal Homeostasis in Drosophila

The work presented in this thesis provides insights into the Drosophila smooth septate junction complex Ssk-Mesh that regulates ISC proliferation and tissue homeostasis in addition to the well-known barrier function in the epithelial integrity. With CRISPR-generated tag knockin alleles of Ssk and Mesh, I characterized the intracellular expression pattern of Ssk and Mesh. Ssk and Mesh had low but detectable expression in punctate format in the cytoplasm of enteroblasts (EBs). The protein expression profile of Ssk and Mesh correlated with their ability to regulate the ISC proliferation even though the septate junctions in EBs had not fully formed. Along with further differentiation into mature enterocytes (ECs), Ssk and Mesh gradually localized to the epithelial apical domain, where they coordinated with other junction proteins, such as Tsp2A and Coracle, to form the septate junction. RNAi-conducted genetic assays and mutant clonal analyses by knockout mutant alleles of Ssk and mesh further revealed that Ssk and Mesh restricted the activity of the transcription coactivator Yki, which governs the production of the cytokine Upd3 along the EB-EC differentiation lineage in adult midgut. Loss of Ssk or Mesh activated Yki to elevate the upd3 expression and thereby to induce the robust ISC proliferation non-autonomously. Although the total number of EBs in midgut is much fewer than that of ECs, surprisingly, knockdown Ssk or mesh in EBs resulted in a comparable upd3 upregulation and ISC proliferation as knockdown their expression in ECs. Leaky midgut caused by knockdown of Ssk or mesh in ECs activated the stress-responding mechanisms to repair the damaged intestinal epithelium, and was eventually associated with death of animals. The reduction of Ssk and Mesh in EBs displayed much milder gut leakage and lower lethality further confirmed that Ssk and Mesh in the two distinct cell types had their own roles in governing ISC proliferation

Mechanobiology of the basement membrane

The microenvironment (ME) of epithelial cells lies at the heart of the function and architecture of most organs. Epithelial cells define the function of the organ. The stroma ensures that the highly specialized MEs within an organ are correctly assembled. A key organising element of the ME is the rather stiff basement membrane (BM), which is the structural part that passively, through its mechanical rigidity and actively through signalling via its components separates the luminal epithelia from the stromal fibroblasts, creating and maintaining tissue polarity to ensure proper function of the organ. The primary goal of this work is to find universal features of the BM and epithelial cells across organs and to understand how the cells mechanobiology is influenced by the BM. The cells mechanobiology is defined by the interaction of the cells integrins with the BMs proteins, notably laminin, perlecan and collagen IV. Disturbing either of the interaction partners quickly leads to distinct new mechanobiological phenotypes of the epithelial cells, without the need for genetic modifications. Further, I will show that changes to the BM by cancer cells are a key step in forming invasive carcinomas which goes together with recent research that shows that the mechanics of epithelial cells and extra-cellular matrix (ECM) are fundamentally altered. In the case of BMs and the extracellular matrix in general, the mechanical properties are governed by the type of fibres and the state of cross-linking. The only tool available for characterizing this physical properties of cells and ECM components under physiological conditions is the atomic force microscope. It gives insight about stiffness and E-modulus on a sub-micrometer scale and is very sensitive to changes in the low Pascal-range. The combination with confocal light microscopy allows one to correlate changes in ridigity to cytoskeletion localisation, to understand what part of the cytoskeleton defines the mechanics and how this is changed by disturbing cell-BM interactions