This thesis investigates the origin of spontaneous activity in the uterus. This organ does not show any activity until shortly before delivery, where fast and efficient contractions are generated. The aim of this work is to provide insight into the origin of spontaneous oscillations and into the transition from asynchronous to synchronized activity in the pregnant uterus. One intriguing aspect in the uterus is the absence of any pacemaker cell. The organ is composed of muscular cells, which are excitable, and connective cells, whose behavior is purely passive; None of these cells, taken in isolation, spontaneously oscillates. We develop an hypothesis based on the observed strong increase in the electrical coupling between cells in the last days of pregnancy. The study is based on a mathematical model of excitable cells, coupled to each other on a regular lattice, and to a fluctuating number of passive cells, consistent with the known structure of the uterus. The two parameters of the model, the coupling between excitable cells, and between excitable and passive cells, grow during pregnancy.Using both a model based on measured electrophysiological properties, and a generic model of excitable cell, we demonstrate that spontaneous oscillations can appear when increasing the coupling coefficients, ultimately leading to coherent oscillations over the entire tissue. We study the transition towards a coherent regime, both numerically and semi-analytically, using the simple model of excitable cells. Last, we demonstrate that, the realistic model reproduces irregular action potential propagation patterns as well as the bursting behavior, observed in the in-vitro experiments.

Previous studies have suggested that phosphodiesterase (PDE) hydrolysis of cyclic adenosine monophosphate (cAMP) is important in the generation of specific and segregated cAMP signals within cells. The purpose of this study was to determine if PDE compartmentation was important in cardiac ventricular myocytes. Therefore, we investigated the effects of β-adrenergic (β-AD) stimulation with isoproterenol in the presence of cilostamide, a PDE3 inhibitor, or Ro 20-1724, a PDE4 inhibitor, on unloaded cell shortening, L-type calcium currents and intracellular calcium levels in freshly dissociated adult rat ventricular myocytes. PDE3 inhibition resulted in a 216 ± 17 % (n=8) increase in unloaded cell shortening after ten minutes of isoproterenol exposure, whereas isoproterenol produced a statistically smaller increase of 155 ± 12 % (n=8) in the presence of PDE4 inhibition. There was a non-significant trend for PDE4 inhibition to produce larger increases in calcium currents (179 ± 17 % (n=4) of controls) than PDE3 inhibition (155 ± 10 % (n=6) of controls). Both PDE3 and PDE4 inhibitors had similar effects on isoproterenol-stimulated increases of calcium transient amplitude with values of 209 ± 14 % (n=8) and 185 ± 12 % (n=8), respectively. Determination of sarcoplasmic reticulum (SR) calcium load using caffeine pulse experiments demonstrated that PDE4 inhibition and isoproterenol superfusion produced a statistically larger increase in SR-calcium loading (139 ± 9 % (n=6)) than PDE3 inhibition and isoproterenol superfusion (113 ± 9 % (n=6)). These results suggest that PDE3 may be active in proximity to the contractile apparatus of cardiac myocytes, whereas PDE4 may be localized in a domain consisting of the L-type calcium channel and junctional SR. Consequently, our study provides functional evidence for differential localization of PDE isoforms in cardiac myocytes. / Thesis (Master, Physiology) -- Queen's University, 2008-01-18 10:14:29.671 / CIHR OGS OGSST

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS. / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.

Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

Orientador: Orientador : Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T12:58:21Z (GMT). No. of bitstreams: 1 Cardoso_AlissonCampos_D.pdf: 4487510 bytes, checksum: eec48cf16c52d89f7324a6a56476ca59 (MD5) Previous issue date: 2012 / Resumo: Durante os primeiros dias do desenvolvimento pós-natal, os miócitos cardíacos perdem a capacidade de proliferação, sendo o crescimento adicional do coração decorrente de hipertrofia e não hiperplasia dos miócitos cardíacos. No entanto, em situações de estresse os miócitos cardíacos diferenciados podem apresentar desdiferenciação e reestabelecimento do ciclo celular. Os mecanismos envolvidos nesse fenômeno são ainda pouco compreendidos. No presente estudo, demonstramos que a ativação do fator de transcrição MEF2C (Myocyte Enhancer Factor 2-C) tem papel crítico no processo de desdiferenciação de miócitos cardíacos. Essa conclusão foi obtida por meio de experimentos de ganho de função pela superexpressão de MEF2C em miócitos ventriculares de ratos neonatos em cultura (MVRNs). Demonstramos que a superexpressão de MEF2C em MVRNs induziu a desdiferenciação e a ativação de mecanismos envolvidos na progressão do ciclo celular. Esses resultados foram obtidos por meio de experimentos de microarranjo de DNA, PCR em tempo real, western blotting e análise do fenótipo celular por microscopias de luz, confocal e eletrônica de transmissão. Esses fenômenos foram atenuados pela superexpressão da quinase de adesão focal (FAK), uma proteína que reconhecidamente exerce efeitos pró-hipertróficos em miócitos cardíacos adultos. Experimentos in vivo e in vitro demonstraram a interação direta entre o fator de transcrição MEF2C e a FAK. Estudos com base em ensaios de reação cruzada associada à espectrometria de massas, dinâmica molecular, espalhamento de raios-X a baixos ângulos e mutação sítio dirigida, demonstraram que as hélices 1 e 4 do domínio FAT da FAK interagem diretamente com a domínio de ligação ao DNA do dímero de MEF2C. Estudos de afinidade e de gel shift demonstraram que a porção FAT da FAK desloca a interação MEF2C/DNA in vitro. Ensaios de gene repórter demonstraram que a FAK, mediada pela região C-terminal, diminui a atividade transcricional de MEF2C em células C2C12. O conjunto de dados demonstra que a ativação do fator de transcrição MEF2C em MVRNs induz a desdiferenciação e ativação de mecanismos de progressão do ciclo celular e que a FAK impede esses efeitos através da interação inibitória no domínio de ligação de MEF2C ao DNA / Abstract: During the first days of postnatal development, cardiac myocytes lose their ability to proliferate, and the further growth of the heart is due to hypertrophy and not hyperplasia of cardiac myocytes. However, in response to stress, cardiac myocytes may have dedifferentiation and re-establishment of the cell cycle. The mechanisms involved in this phenomenon are still poorly understood. In the present study, we demonstrated that activation of the transcription factor MEF2C (myocyte enhancer factor 2-C) plays a critical role in the process of dedifferentiation of cardiac myocytes. This conclusion was obtained by gain-of-function experiments through overexpressing MEF2C in neonatal rat ventricular myocytes in culture (NRVMs). We also showed that overexpression of MEF2C in NRVMs induced the dedifferentiation and activation of mechanisms involved on cell cycle progression. These results were obtained by DNA microarray experiments, real time PCR, western blotting and cell phenotype analysis by light microscopy, confocal and electronic transmission. These effects were attenuated by overexpression of focal adhesion kinase (FAK) protein known to exert pro-hypertrophic effects on adult cardiac myocytes. In vivo and in vitro experiments demonstrated the direct interaction between the transcription factor MEF2C and FAK. A model based on crosslinking technology coupled with mass spectrometry, small angle X-ray scattering and the site directed mutation analyses indicated that alpha-helices 1 and 4 of FAK FAT domain interacts directly with the DNA binding domain of MEF2C dimer. Affinity studies and gel shift assay demonstrated that the FAK FAT domain displaces the MEF2C/DNA interaction in vitro. Reporter gene assays demonstrated that FAK, mediated by the C-terminal region, decreases the transcriptional activity of MEF2C in C2C12 cells. The data set shows that the activation of the transcription factor MEF2C in MVRNs induces dedifferentiation and activation of cell cycle progression and that FAK prevents these effects by inhibitory interaction with DNA binding domain of MEF2C / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Ciências

[EN] Heart failure, a cardiomyopathy that produces mechanical dysfunction and sudden cardiac death following fatal arrhythmias, is one of the main causes of mortality worldwide that also causes elevated morbidity rates. Current clinical therapies are challenged by the complexity of this cardiac pathology, in which many factors are involved in the electrical instabilities that lead to an altered function. The electrical activity of the heart comprises a wide range of spatial and temporal scales. Ion transport across transmembrane proteins initiate the cellular depolarization that is propagated cell to cell through the myocardium depolarizing and then repolarizing the entire heart in an orchestrated manner. The electrical excitation of cardiomyocytes triggers the cellular contraction, a process in which Ca2+ ions are the main mediators. Ca2+ dynamics plays a relevant role in controlling excitation-contraction coupling and consequently, investigations have focused on Ca2+-handling proteins and the regulation of Ca2+ homeostasis to elucidate the causes of impaired contractility and pro-arrhythmic conditions in cardiac diseases. This thesis takes advantage of the existence of mathematical models with detailed representation of the subcellular processes to perform computational simulations of cardiac electrophysiology and understand the altered mechanisms that govern heart failure, especially those related with intracellular Ca2+ cycling. It is known that failing myocytes undergo a specific remodeling of ion channels and Ca2+-handling proteins that lead to an impaired excitation-contraction coupling. Initially, it was analyzed, in the human action potential model of ventricular myocytes selected for the whole study, the effects of modulating ionic mechanisms on the electrical activity and Ca2+ dynamics. In tissue, heart failure induces additional changes affecting cellular coupling. The development of fibroblasts and impact on myocyte electrophysiology was investigated, including the vulnerability to generate alternans, a common precursor to arrhythmogenesis. Finally, the beta-adrenergic signaling model was integrated with the action potential model because of the electrophysiological modulation exerted by the sympathetic nervous system, which is aggravated under heart failure conditions. Results highlighted the need of studying heart failure therapies on failing cells because of the different response of ion channels and membrane proteins to drugs. Functional Ca2+ proteins were important to maintain Ca2+ homeostasis and to avoid malignant electrical consequences, being SERCA pump the most critical factor. Apart from the electrophysiological remodeling, fibroblast interaction contributed to alter Ca2+ dynamics in myocytes and, when analyzing Ca2+ alternans, spatial electrical discordances predominated in failing tissues. The inclusion of beta-adrenergic stimulation showed that the inotropic response was diminished in heart failure as well as the antiarrhythmic benefits provided by catecholamines in the normal heart. These findings contribute to gain insight into the pathophysiology of heart failure and the development of new pharmacological agents targeted to restore Ca2+ dynamics. The control of intracellular Ca2+ cycling is crucial to ensure both the mechanical force and the electrical activity that lead to a rhythmic contraction of the heart. / [ES] La insuficiencia cardíaca, una cardiomiopatía que provoca disfunción mecánica y muerte súbita tras arritmias cardíacas letales, es una de las principales causas de mortalidad en todo el mundo que además causa tasas de morbilidad elevadas. Las terapias usadas actualmente en la clínica están comprometidas por la complejidad de esta patología cardíaca, ya que son muchos los factores que están implicados en las inestabilidades eléctricas que conllevan a alteraciones funcionales. La actividad eléctrica del corazón abarca un amplio rango escalas espaciales y temporales. El transporte de iones a través de las proteínas transmembrana inicia la despolarización celular que se propaga de célula en célula a través del miocardio, despolarizando y luego repolarizando todo el corazón de manera sincronizada. La excitación eléctrica de los cardiomiocitos desencadena la contracción celular, un proceso en el que los iones de Ca2+ son los principales intermediarios. La dinámica de Ca2+ tiene un papel relevante en el control del acoplamiento excitación-contracción y, como consecuencia, las investigaciones se han centrado en las proteínas que controlan el ciclo del Ca2+ y la regulación homeostática para encontrar las causas que empeoran la contractilidad y conducen a condiciones proarrítmicas en casos de insuficiencia cardíaca. Esta tesis hace uso de la existencia de modelos matemáticos con una representación detallada de los procesos subcelulares para realizar simulaciones computacionales de electrofisiología cardíaca y comprender los mecanismos que están alterados y predominan en insuficiencia cardíaca, especialmente aquellos relacionados con el ciclo intracelular de Ca2+ . Se sabe que los miocitos dañados por insuficiencia cardíaca experimentan un remodelado específico en los canales iónicos y en las proteínas partícipes en el ciclo de Ca2+, ocasionando fallos en el acoplamiento excitación-contracción. Inicialmente, se analizaron, en el modelo de potencial de acción humano de miocitos ventriculares seleccionado para todo el estudio, los efectos de la modulación de los mecanismos iónicos sobre la actividad eléctrica y la dinámica de Ca2+. En los tejidos, la insuficiencia cardíaca induce cambios adicionales que afectan el acoplamiento celular. Se ha investigado la presencia de fibroblastos y su impacto en la electrofisiología de los miocitos, incluida la vulnerabilidad para generar alternantes, un precursor común de la arritmogénesis. Finalmente, se ha incluido el modelo de señalización -adrenérgica integrado con el modelo de potencial de acción debido a la modulación electrofisiológica ejercida por el sistema nervioso simpático, que se agrava en condiciones de insuficiencia cardíaca. Los resultados han destacado la necesidad de estudiar las terapias de insuficiencia cardíaca en células de estos corazones debido a la diferente respuesta de los canales iónicos y las proteínas de membrana a los medicamentos. El buen funcionamiento de las proteínas reguladoras del Ca2+ es importantes para mantener la homeostasis del Ca2+ y evitar consecuencias eléctricas malignas, siendo la bomba SERCA el factor más crítico. Además del remodelado electrofisiológico, la interacción con fibroblastos contribuye a alterar la dinámica de Ca2+ en los miocitos y, al analizar los alternantes de Ca2+, predominan las discordancias eléctricas espaciales en los tejidos de corazones con insuficiencia cardíaca. La inclusión de la estimulación -adrenérgica ha mostrado que la respuesta inotrópica disminuye en insuficiencia cardíaca, así como los beneficios antiarrítmicos proporcionados por las catecolaminas en un corazón normal. Estos hallazgos contribuyen a obtener información sobre la fisiopatología de la insuficiencia cardíaca y el desarrollo de nuevos agentes farmacológicos destinados a restaurar la dinámica de Ca 2+. El control del ciclo de Ca2+ intracelular es crítico para garantizar tanto la fuerza mecánica como la actividad eléctrica que conducen a una contracción rítmica del corazón. / [CA] La insuficiència cardíaca, una cardiomiopatia que provoca disfunció mecànica i mort sobtada després d'arrítmies cardíaques letals, és una de les principals causes de mortalitat a tot el món que a més causa taxes de morbiditat elevades. Les teràpies utilitzades actualment en la clínica estan compromeses per la complexitat d'aquesta patologia cardíaca, ja que són molts els factors que estan implicats en les inestabilitats elèctriques que comporten a alteracions funcionals. L'activitat elèctrica del cor abasta un ampli rang d'escales espacials i temporals. El transport d'ions a través de les proteïnes transmembrana inicia la despolarització cel·lular que es propaga de cèl·lula en cèl·lula a través del miocardi, despolaritzant i després repolaritzant tot el cor de manera sincronitzada. L'excitació elèctrica dels cardiomiòcits desencadena la contracció cel·lular, un procés en el qual els ions de Ca2+ són els principals intermediaris. La dinàmica de Ca2+ té un paper rellevant en el control de l'acoblament excitació-contracció i, com a conseqüència, les investigacions s'han centrat en les proteïnes que controlen el cicle del Ca2+ i la regulació homeostàtica per a trobar les causes que empitjoren la contractilitat i condueixen a condicions proarrítmiques en casos d'insuficiència cardíaca. Aquesta tesi fa ús de l'existència de models matemàtics amb una representació detallada dels processos subcel·lulars per a realitzar simulacions computacionals de l'electrofisiologia cardíaca i comprendre els mecanismes que estan alterats i predominen en insuficiència cardíaca, especialment aquells relacionats amb el cicle intracel·lular de Ca2+. Se sap que els miòcits danyats per insuficiència cardíaca experimenten un remodelat específic en els canals iònics i en les proteïnes partícips en el cicle de Ca2+, ocasionant fallades en l'acoblament excitació-contracció. Inicialment, es van analitzar, en el model de potencial d'acció humà de miòcits ventriculars seleccionat per a tot l'estudi, els efectes de la modulació dels mecanismes iònics sobre l'activitat elèctrica i la dinàmica de Ca2+. En els teixits, la insuficiència cardíaca indueix canvis addicionals que afecten l'acoblament cel·lular. S'ha investigat la presència de fibroblasts i el seu impacte en l'electrofisiologia dels miòcits, inclosa la vulnerabilitat per a generar alternants, un precursor comú de l'arritmogènesi. Finalment, s'ha inclòs el model de senyalització beta-adrenèrgica integrat amb el model de potencial d'acció a causa de la modulació electrofisiològica exercida pel sistema nerviós simpàtic, que s'agreuja en condicions d'insuficiència cardíaca. Els resultats han destacat la necessitat d'estudiar les teràpies d'insuficiència cardíaca en cèl·lules d'aquests cors a causa de la diferent resposta dels canals iònics i les proteïnes de membrana als medicaments. El bon funcionament de les proteïnes reguladores del Ca2+ és importants per a mantindre l'homeòstasi del Ca2+ i evitar conseqüències elèctriques malignes, sent la bomba SERCA el factor més crític. A més del remodelat electrofisiològic, la interacció amb fibroblasts contribueix a alterar la dinàmica de Ca2+ en els miòcits i, en analitzar els alternants de Ca2+, predominen les discordances elèctriques espacials en els teixits de cors amb insuficiència cardíaca. La inclusió de l'estimulació beta-adrenèrgica ha mostrat que la resposta inotròpica disminueix en insuficiència cardíaca, així com els beneficis antiarrítmics proporcionats per les catecolamines en un cor normal. Aquestes troballes contribueixen a obtindre informació sobre la fisiopatologia de la insuficiència cardíaca i el desenvolupament de nous agents farmacològics destinats a restaurar la dinàmica de Ca2+. El control del cicle de Ca2+ intracel·lular és crític per a garantir tant la força mecànica com l'activitat elèctrica per a una contracció rítmica del cor. / Mora Fenoll, MT. (2020). In silico study of calcium handling in the human failing heart [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153143 / TESIS

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

Increased osteopontin (OPN) expression associates with increased myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify the receptor for OPN and get insight into the mechanism by which OPN induces cardiac myocyte apoptosis. Adult rat ventricular myocytes (ARVMs) and transgenic mice expressing OPN in a myocyte-specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral-mediated OPN expression induced apoptosis in ARVMs. OPN co-immunoprecipitated with CD44 receptors, not with β1 or β3 integrins. Proximity ligation assay confirmed interaction of OPN with CD44 receptors. Neutralizing anti-CD44 antibodies inhibited OPN-stimulated apoptosis. OPN activated JNKs and increased expression of Bax and levels of cytosolic cytochrome c, suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase-12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase-12 inhibitor significantly reduced OPN-stimulated apoptosis. Expression of OPN in adult mouse heart in myocyte-specific manner associated with decreased left ventricular function and increased myocyte apoptosis. In the heart, OPN expression increased JNKs and caspase-12 activities, and expression of Bax and Gadd153. Thus, OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of mitochondrial death pathway and ER stress.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during 'metabolic' preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U·ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the A1/A3 antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.