L'objectif de ce travail a été de comprendre comment les canaux ioniques sont adressés, organisés et régulés dans des domaines spécialisés de la membrane plasmique des cardiomyocytes. Parmi les partenaires des canaux, les protéines MAGUK (Membrane Associated GUanylate Kinase) sont spécialisées dans l'ancrage, l'agrégation et la formation de complexes macromoléculaires à la membrane. J'ai caractérisé pour la première fois au niveau cardiaque une de ces protéines MAGUK, la protéine CASK. CASK est localisée à la membrane latérale des cardiomyocytes et exclue des disques intercalaires, zones de conduction privilégiée dans l'axe longitudinal. À la membrane latérale, la protéine CASK est exprimée au sein du complexe costamérique dystrophine/glycoprotéines. L'inhibition de CASK entraîne l'augmentation du courant sodique INa dans les HEK293 et les myocytes cardiaques. Dans les HEK293, la microscopie à onde évanescente (TIRF) et des expériences de biotinylation ont mis en évidence que cette augmentation du courant INa est associée à une augmentation du nombre de canaux NaV1.5 à la membrane. La microscopie de conductance ionique (SICM) couplée au patch clamp en configuration cellule attachée a permis de montrer que CASK retient les canaux sodiques au niveau des crêtes et prévient leur agrégation en clusters dans les T-tubules. Enfin, l'inhibition de CASK in vivo par une stratégie reposant sur l'utilisation de virus adéno-associés (AAV) est responsable d'un allongement de la durée de dépolarisation ventriculaire et de l'apparition d'une cardiopathie dilatée. / The aim of the thesis was to understand how ion channels are addressed, organized and regulated in specialized domains of the plasma membrane of cardiac myocytes. Among these partners, the MAGUK proteins (Membrane Associated GUanylate Kinase) are specialized in anchoring, aggregation and clustering of macromolecular complexes at the plasma membrane. In particular, characterized for the first time at the level of the hearth, one of these MAGUK proteins is the CASK protein. CASK is localized at the lateral membrane of cardiomyocytes, but excluded from intercalated disks which are privilege zones of the longitudinal axial conduction. At the lateral membrane, CASK protein is expressed among the costameric dystrophin/glycoproteins complex. CASK inhibition leads to the increase in sodium current density in HEK293 cells and in cardiomyocytes. In HEK293, evanescent wave microscopy (TIRF) and biotinylation experiments pointed out that the INa increase is associated to an increase in the number of NaV1.5 channels at the plasma membrane. Scanning ion conductance microscopy (SICM) coupled to cell-attached patch-clamp has demonstrated that CASK holds together sodium channels at the crest level and prevents their aggregation into clusters in the T-tubules. Finally, inhibition of CASK, in vivo, using an adeno-associated virus strategy resulted to an increase in duration of ventricular depolarization and to the appearance of dilated cardiomyopathy.

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

<p>Evidence has shown that hypoxic hearts have greater heart/fetus mass ratio. However, it is still unclear if either hyperplasia or hypertrophy causes the relatively increased heart mass. Furthermore, the genes that might be involved in the process have not yet been identified. In the present study, the cardiac transcriptome was analyzed to identify differentially expressed genes related to hypoxia. Eggs were incubated for 15 and 19 days in two different environments, normoxic and hypoxic. Normalized microarray results were analyzed to isolate differentially expressed probes using the Affymetrix chip. Total RNA was also isolated from another set of fetuses incubated in the same conditions and used to perform a qPCR in order to confirm the microarray results. In the four groups (15N, 15H, 19N, 19H), some probes were differentially expressed. From the eggs incubated for 15 days, the microarray revealed five probes that were differentially expressed according to the criteria (p<0.01 and absolute fold change FC>2) in the two programs (PLIER & RMA) used to normalize the data. From the eggs incubated up to 19 days, eight probes were differentially expressed in both programs. No further tests were performed on the 19 days fetuses since there was no significant difference in that group after incubation for the heart/fetus mass ratio. Apolipoprotein-A1, p22, similar to ENS-1 and b2 adrenergic receptor were further tested in qPCR (15 days sample). The differently expressed genes are linked to cell division and should be further studied to identify their function, especially the similar to ENS-1.</p>

Evidence has shown that hypoxic hearts have greater heart/fetus mass ratio. However, it is still unclear if either hyperplasia or hypertrophy causes the relatively increased heart mass. Furthermore, the genes that might be involved in the process have not yet been identified. In the present study, the cardiac transcriptome was analyzed to identify differentially expressed genes related to hypoxia. Eggs were incubated for 15 and 19 days in two different environments, normoxic and hypoxic. Normalized microarray results were analyzed to isolate differentially expressed probes using the Affymetrix chip. Total RNA was also isolated from another set of fetuses incubated in the same conditions and used to perform a qPCR in order to confirm the microarray results. In the four groups (15N, 15H, 19N, 19H), some probes were differentially expressed. From the eggs incubated for 15 days, the microarray revealed five probes that were differentially expressed according to the criteria (p&lt;0.01 and absolute fold change FC&gt;2) in the two programs (PLIER &amp; RMA) used to normalize the data. From the eggs incubated up to 19 days, eight probes were differentially expressed in both programs. No further tests were performed on the 19 days fetuses since there was no significant difference in that group after incubation for the heart/fetus mass ratio. Apolipoprotein-A1, p22, similar to ENS-1 and b2 adrenergic receptor were further tested in qPCR (15 days sample). The differently expressed genes are linked to cell division and should be further studied to identify their function, especially the similar to ENS-1.

The fabrication of biomimetic heart muscle suitable for pharmaceutical compound evaluation and disease modeling is hindered by limitations in our understanding of how to guide and assess the maturity of engineered myocardium in vitro. We hypothesized that tissue architecture serves as an important cue for directing the maturation of engineered heart tissues and that reliable assessment of maturity could be performed using a multi-parametric rubric utilizing cardiomyocytes of known developmental state as a basis for comparison. Physical micro-environmental cues are recognized to play a fundamental role in normal heart development, therefore we used micro-patterned extracellular matrix to direct isolated cardiac myocytes to self-assemble into anisotropic sheets reminiscent of the architecture observed in the laminar musculature of the heart. Comparison of global sarcomere alignment, gene expression, and contractile stress in engineered anisotropic myocardium to isotropic monolayers, as well as, adult ventricular tissue revealed that anisotropic engineered myocardium more closely matched the characteristics of adult ventricular tissue, than isotropic cultures of randomly organized cardiomyocytes. These findings support the notion that tissue architecture is an important cue for building mature engineered myocardium. Next, we sought to develop a quality assessment strategy that utilizes a core set of 64 experimental measurements representative of 4 major categories (i.e. gene expression, myofibril structure, electrical activity, and contractility) to provide a numeric score of how closely stem cell-derived cardiac myocytes match the physiological characteristics of mature, post-natal cardiomyocytes. The efficacy of this rubric was assessed by comparing anisotropic engineered tissues fabricated from commercially-available murine ES- (mES) and iPS- (miPS) derived myocytes against neonatal mouse ventricular myocytes. The quality index scores calculated for these cells revealed that the miPS-derived myocytes more closely resembled the neonate ventricular myocytes than the mES-derived myocytes. Taken together, the results of these studies provide valuable insight into the fabrication and validation of engineered myocardium that faithfully recapitulate the characteristics of mature ventricular myocardium found in vivo. These engineered tissue design and quality validation strategies may prove useful in developing heart muscle analogs from human stem cell-derived myocytes that more accurately predict patient response than currently used animal models. / Engineering and Applied Sciences

Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and an increase in AMP:ATP activates the energy sensor and putative insulin-sensitizer AMP-dependent protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defense pathway involving AMPK-dependent, insulin-independent activation of the insulin signaling pathway. Results from my work showed that the central insulin signaling pathway is rapidly activated when cardiac and skeletal myocytes are subjected to conditions of glucose starvation. The effect occurred independently of insulin receptor ligands (insulin and IGF-1). There was a >10-fold increase in the activity of Akt as determined by phosphorylation on both Thr308 and Ser473. Phosphorylation of glycogen synthase 3 beta (GSK3b) increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase (S6K) and the mammalian target of rapamycin complex 1 (mTORC1) decreased. We identified AMPK as an intermediate in this signaling network; AMPK was activated by glucose starvation and many of the effects were mimicked by the AMPK-selective activator aminoimidazole carboxamide ribonucleotide (AICAR) and blocked by AMPK inhibitors. Glucose starvation increased the phosphorylation on IRS-1 on Ser789, but phosphomimetics revealed that this conferred negative regulation. Glucose starvation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. Insulin receptor kinase activity was essential for cardiac myocytes to survive gluose starvation as inhibition of the IR led to increased cell death in glucose-starved myocytes. Selective activation of mTORC2 by glucose starvation to increase Akt-Ser473 phosphorylation was dependent on the presence of rictor. SIN1 also seemed to be instrumental in the activation of mTORC2 as its levels and binding to rictor increased under glucose starvation. AMPK-mediated activation of the insulin signaling pathway conferred significant protection against the stresses of glucose starvation. Glucose starvation promoted energy conservation, augmented glucose uptake and enhanced insulin sensitivity in an AMPK- and Akt-dependent manner. My results describe a novel ligand-independent and AMPK-dependent activation of the insulin signaling pathway via direct phosphorylation and activation of the IR followed by activation of PI3K and Akt. These results may be relevant in conditions of myocardial ischemia superimposed with type 2 diabetes where AMPK could directly modify the IR to promote cell survival and confer protection.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Orientador: Pedro Xavier de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-24T14:24:09Z (GMT). No. of bitstreams: 1 Prado_LuizaNaiaraSiqueirado_M.pdf: 1448858 bytes, checksum: 14894261e1f5acfa5c112a5bd731af13 (MD5) Previous issue date: 2014 / Resumo: Apesar de a aplicação de campos elétricos de alta intensidade ser atualmente a única terapia disponível para interromper a fibrilação ventricular, esse processo pode causar lesões às células cardíacas, prejudicando sua contratilidade. Neste estudo, aplicamos pulsos elétricos de alta intensidade a miócitos isolados de ratos Wistar adultos. Obtivemos as curvas de letalidade por meio de análise de sobrevivência, que foram usadas para determinar a intensidade de campo necessária para matar 50% das células (EL50) e com esses valores obtivemos a curva de intensidade-duração (IxD) para letalidade para 10 durações diferentes: 0,1; 0,2; 0,5; 1; 3; 5; 10; 20; 35 e 70 ms. Também obtivemos a curva IxD para excitação celular, por meio dos valores de média ± erro padrão da média para a intensidade de campo limiar de excitação para todas as durações, e obtivemos uma relação entre letalidade e excitação em função da duração do pulso, chamada de Fator de Segurança (FS), um indicador de segurança estimulatória. Essa curva foi determinada a partir da divisão entre os pontos das curvas IxD de letalidade e excitação. Observamos que quanto me-nor a duração de pulso, maior a intensidade de campo que causa morte celular. Ao contrário do que se esperava, o maior valor de FS não correspondeu à menor duração utilizada (0,1 ms), mas sim à duração de 0,5 ms. Como o limiar de desfibrilação foi descrito como dependente da duração do pulso aplicado, nossos resultados indicam que o uso de estímulos com duração mais curta - em vez da duração tipicamente usada na clínica, de 10 ms - pode diminuir as lesões celulares, e, portanto, aumentar a efetividade da desfibrilação / Abstract: Although high intensity electric fields application is currently the only effective therapy available to terminate ventricular fibrillation, it may cause injury to cardiac cells, impairing their contractility. In this study we applied high electric field pulses with different durations to isolated rat ventricular myocytes. We obtained lethality curves by survival analysis, which were used to determine the value of applied electric field necessary to kill 50% of cells (EL50) and plotted a strength-duration (IxD) curve for lethality with 10 different durations: 0.1, 0.2, 0.5, 1, 3, 5, 10, 20, 35 and 70 ms. For the same durations we also obtained an IxD curve for excitation and established an indicator for stimulatory safeness, named Safety Factor (FS), as the ratio between the points on the IxD curve for lethality and the one for excitation. We found that the lower the pulse duration, the higher the electric field intensity required to cell death. Contrary to expecta-tions, the highest FS value does not correspond to the lowest pulse duration but to the one of 0.5 ms. As defibrillation threshold has been described as duration dependent, our results imply that the use of shorter stimulus duration - instead of the one typically used in the clinic (10 ms) - may decrease electric cell damage, therefore increasing defibrillation effectiveness / Mestrado / Engenharia Biomedica / Mestra em Engenharia Elétrica

Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-19T11:34:49Z (GMT). No. of bitstreams: 1 Monteiro_MarinaCarneiro_M.pdf: 5677929 bytes, checksum: 5c42afe705a43b66a4505a6ecded7b7c (MD5) Previous issue date: 2011 / Resumo: A fração do conteúdo de Ca2+ do retículo sarcoplasmático (RS) liberada a cada contração (Fractional Release - FR) em miócitos cardíacos é regulada pela corrente de entrada de Ca2+ através da membrana celular pelos canais de Ca2+ tipo-L (ICa,L) e pelo conteúdo de Ca2+ do RS ([Ca2+]RS). Em trabalho anterior foi desenvolvido, no nosso laboratório, um modelo experimental denominado de modelo do RS funcionalmente isolado (MRSFI). Neste modelo, cardiomiócitos são perfundidos em solução sem Na+ e sem Ca2+, o que torna as suas membranas eletricamente inexcitáveis e inibe o transporte do íon pelo trocador Na+/Ca2+. As variações (transientes) da concentração intracelular de Ca2+ ([Ca2+]i) medidas com o indicador fluorescente Fluo-3 AM (5 ?M, 20 min, 24ºC) são evocadas por aplicação de pulsos rápidos (100 ms) de cafeína (10 mM). No presente trabalho, o MRSFI foi usado para estudo da relação entre FR e [Ca2+]RS na ausência do gatilho fisiológico (ICa,L) para liberação reticular de Ca2.... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (Fractional Release - FR) in cardiac myocytes is regulated by the transmembrane inward Ca2+ current through the L-type Ca2+ channel (ICa,L) and by the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR model (FISRM), previously developed in this laboratory, cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells electrically unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Variations in the intracellular Ca2+ concentration ([Ca2+]i) was measured with the Ca2+ indicator fluo-3 and Ca2+ transients due to SR release are evoked by pulse-like (100 ms duration) application of 10 mM caffeine. In the present work, the FISRM was used to study the relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for the release of Ca2+ from the SR.... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Abstract Background – The heart cell is a prime example of a system, in which numerous interconnected regulatory mechanisms affect the dynamic balance of cellular function. The function of the system emerges from the interactions of its components rather than from their individual properties. Thus, it is a challenging task to understand the causal relations within such a system, based on the analysis of experimental results. Facing this complexity, the systems biological approach has gained interest during recent years, since with using it we can make an effort to observe, quantitatively and simultaneously, multiple components and their interdependencies in biological networks. Methods and aims – One of the most important tools in systems biology is mathematical modelling. In this thesis, novel model components have been developed and existing components integrated to describe mathematically the calcium dynamics in cardiac myocytes with improved physiological accuracy. Special attention was paid to both the activity-dependent and automatic regulation of the dynamics. This enabled the quantitative analysis of the regulation’s role in both physiological and pathophysiological conditions. Results – Validation of the novel model components that describe the calcium transport mechanisms indicates that the developed schemes are accurate and applicable also beyond the normal physiological state of the cardiac myocyte. Results also highlight the importance of autoregulation of calcium dynamics in the excitation-contraction coupling. Furthermore, the analysis indicates that the CaMK-dependent regulation of the calcium uptake to and release from the sarcoplasmic reticulum calcium stores could have substantial roles as downstream effectors in beta-adrenergic stimulation. Conclusions – Results emphasize mathematical modelling as a valuable complement to experiments in understanding causal relations within complex biological systems such as the cardiac myocytes. That is, rigorous data integration with mathematical models can provide significant insight to the quantitative role of both the individual model components and the interconnected regulatory loops. This is especially true for the analysis of genetically engineered animal models, in which the intended modification is always accompanied by compensatory changes that can mask to a varying degree the actual phenomenon of interest.