Regulation of Connexin40 Gap Junctions

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 β-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 β-adrenergic receptor-adenylyl cyclaseProtein 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 Ca concentration ([Ca]i ). [Ca]i was increased by addition of the ionophore ionomycin and elevating extracellular calcium [Ca]o from 1.8 mM to 21.8 mM. This resulted in an elevation of [Ca]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) β-adrenergic receptor activation increases Cx40-mediated cell-to-cell dye transfer which requires the activation of PKA; (2) A sustained elevation in [Ca]i causes a partial inhibition of Cx40 gap junction-mediated cell-to-cell dye transfer which was Ca-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. Index words: Connexin40, Atria, β-adrenergic receptor, Intracellular calcium, Chemical ischemia. REGULATION OF CONNEXIN40 GAP JUNCTIONS by