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Regulation of Connexin40 Gap JunctionsSheela, Thomas Vinaya 31 August 2008 (has links)
Gap junctions provide direct electrical and biochemical communication between cardiomyocytes in the heart. Connexin40 (Cx40) is the major connexin in the atria of the heart and little is known regarding its regulation. Thus, the goal was to investigate the regulation of Cx40 in both physiological and pathophysiological conditions. The first objective of this thesis was to determine whether Cx40 gap junctions were regulated by â-adrenergic receptor activation. Cx40 has previously been shown to be acutely activated by cAMP, this cAMP-induced increase in Cx40-mediated cell-to-cell dye transfer has been shown to be effected through the â-adrenergic receptor-adenylyl cyclase- Protein Kinase A (PKA) pathway in Cx40-transfected HeLa cells. The second objective of this thesis was to determine whether Cx40 gap junctions were regulated by intracellular Ca2+ concentration ([Ca2+]i ). [Ca2+]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca2+]o from 1.8 mM to 21.8 mM. This resulted in an elevation of [Ca2+]i and effected an inhibition of Cx40-mediated cell-to-cell dye transfer (IC50 of 500 ± 0.72 nM) which was Calmodulin-dependent. The third objective of this thesis was to determine whether Cx40 gap junctions were regulated by ischemia. Inducing ischemia chemically by inhibiting the electron transport chain with sodium cyanide and glycolysis with iodoacetate and 2-deoxyglucose effected an inhibition of Cx40-mediated cell-to-cell dye transfer that was shown to be Calmodulin dependent. The main conclusions of this thesis were: (1) â-adrenergic receptor activation increases Cx40-mediated cell-to-cell dye transfer which requires the activation of PKA; (2) A sustained elevation in [Ca2+]i causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was Ca2+-and Calmodulin dependent; (3) Chemical ischemia causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was shown to be Calmodulin-dependent.
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Avalia??o do efeito do 17?-estradiol sobre a express?o g?nica da conexina40 e suas implica??es na propaga??o da atividade el?trica card?aca / Evaluation of the effect of 17 ?-estradiol on the gene expression of Connexin40 and its implications for the spread of cardiac electrical activity.Amarante, D?bora Barbosa 26 February 2016 (has links)
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Previous issue date: 2016-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The 17?-estradiol (E2) regulate many cardiac genes via its estrogen receptor (ER). The propagation of electrical activity in the myocardium depends on the current transfer at gap junctions. Connexins 40 (Cx40) and 43 are the predominant junctional proteins. In mice, Cx40 is restricted to the atrium and conduction system. Alterations of Cx40 expression or activity are associated with atrial fibrillation. Here we evaluate the effect of the E2 onCx40 mRNA expression, in vitro, and in vivo the effect on atrium expression of Cx40 mRNA in correlation to ECG studies. We treated A7r5 cells (smooth muscle cells from rat thoracic aorta) with low and high doses ofestradiol benzoate, EB, (10-8 M and 10-6M) for 24h and rat neonatal cardiomyocytes with EB 10-6 M for 2, 4 and8 hours. A7r5 cells were trasiently transfected with 0.5 microgramas of the test plasmid (-1190/+121Cx40Luc pGL3) and treated with 10-6M of EB for 24 hours. Female mice were subjected to ovariectomy (OVX) and then, treated with a low (2?g) and a high dose (20?g) of estradiol benzoate (EB; OVX+EB2 and OVX+EB20) for 15 days. ECG recordings were obtained from mice and atrium Cx40 mRNA expression were evaluated by RT-PCR real time. First, we observed that high doses of EB down regulated Cx40 mRNA in vitro (A7r5 cells and rat atrial cardiomyocytes). The transcriptional activity of Cx40 promoter was inhibited by 10-6M of EB in A7r5 cells. We observed lower heart rate for OVX animals when to compared to control animals, FO. In this regard, no difference was observed between OVX+EB2 and OVX+BE20 heart rate. The OVX group showed decrease P wave duration when to compared to FO group, but no difference in the P wave duration was observed among the others groups. No difference was observed in another ECG intervals among the experimental groups. The atrial Cx40mRNA level was reduced in OVX+BE20group when to compared to FO group. Our data indicate, for the first time, high doses of estradiol benzoate reduce Cx40 mRNA in vitro and ovariectomized mice. We proposed that when E2 levels are high, the complex ER-E2 acts directly from the DNA, inhibiting the transcriptional activity of Cx40 promoter / O 17?-estradiol (E2), regula muitos genes card?acos atrav?s de seu receptor (receptor para estr?geno, RE). A propaga??o da atividade el?trica no mioc?rdio depende da transfer?ncia de corrente atrav?s das jun??es comunicantes. As conexinas 40 (Cx40) e 43 s?o as principais conexinas que formam as jun??es expressas no cora??o. Em camundongos, a Cx40 ? restrita ao ?trio e sistema de condu??o. Altera??es na express?o ou atividade da Cx40 est?o associadas a fibrila??o atrial. Aqui n?s avaliamos o efeito do E2 in vitro e in vivo sobre a express?o do RNAm da Cx40 nos ?trios e correlacionamos aos estudos eletrocardiogr?ficos (ECG). N?s tratamos a linhagem A7r5 (derivada de m?sculo liso a?rtico de rato embrion?rio) com alta e baixa concentra??es de benzoato de estradiol, BE, (10-6 M e 10-8 M) durante 24 horas e cultura prim?ria de cardiomi?citos atriais de ratos neonatos foram incubados com BE 10-6 M durante 2, 4 e 8 horas. Ensaios de transfec??o transiente foram realizados na linhagem A7r5 com um plasm?deo contendo um segmento da regi?o promotora da Cx40 (-1190/+121Cx40LucpGL3) e tratadas com 10-6 M de BE durante 24 horas. Camundongos f?meas foram ovariectomizadas (OVX) e ent?o tratadas com baixa (2 microgramas) e alta (20 microgramas) doses de benzoato de estradiol (OVX+BE2 e OVX+BE20) durante 15 dias, sendo que o grupo controle sofreu apenas estresse cir?rgico (FO). Foram realizados registros ECG e a express?o atrial do RNAm da Cx40 foi avaliada por RT-PCR em tempo real. N?s observamos que altas doses de BE diminui a express?o do RNAm da Cx40 in vitro (na linhagem A7r5 e na cultura de cardiomi?citos). A atividade transcricional do promotor foi inibida por BE10-6 M. N?s observamos diminui??o da frequ?ncia card?aca dos animais do grupo OVX em rela??o ao controle, grupo FO. Nenhuma diferen?a foi observada na frequ?ncia card?aca entre os grupos OVX+BE2 e OVX+BE20. Os animais do grupo OVX apresentaram diminui??o na dura??o da onda P em rela??o ao grupo FO, no entanto nenhuma diferen?a foi observada na dura??o da onda P entre os outros grupos. Nenhuma diferen?a foi observada nos outros intervalos eletrocardiogr?ficos entre os grupos experimentais. A express?o atrial do RNAm da Cx40 estava reduzida nos animais do grupo OVX+BE20 em rela??o ao grupo FO. Nossos dados demonstram que altas doses de benzoato de estradiol reduzem a express?o do RNAm da Cx40 in vitro e em camundongos ovariectomizados. N?s propomos que altas doses de E2 ativa o seu receptor, e o complexo RE-E2 age diretamente no DNA, inibindo a atividade transcricional do promotor da Cx40
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