Abstract
Atrial fibrillation (AF) cardiac arrhythmia is associated with [Ca2+]i overload contributing in disturbed action potential prolongation, and in alteration of gap junctional connexin protein expression and phosphorylation status. Episodes of AF are more frequent during daytime, suggesting a possible involvement of circadian rhythms (and the clock genes driving it, in particular Bmal1) contributing to its occurrence. Also, Bmal1 reported to directly regulate Ca2+ and Na+ ionic currents, as well as, Ca2+-related release channels, however, the relationship between AF-mediated pathways, Bmal1 clock gene expression and circadian rhythmicity is far from clear. This study aimed to explore the involvement of Bmal1 in driving the AF occurrence via changes in Ca2+ homeostasis through sustained activation of NaP channels and/or alteration in connexin expression and phosphorylation status by Ca2+-dependent protein phosphatase calcineurin (CaN). HL-1 clone 6 (HL1-6) cells were used as an in vitro model and synchronised with 50 % fetal bovine serum for the interpretation of circadian characteristics. HL1-6 cells were treated with anemonia sulcata toxin II (ATXII; 1 nM) in presence and/or absence of Ranolazine (10 µM), BAPTA-AM (5 µM) or CAIP (25 µM). Bmal1 gene circadian rhythmicity was observed under control conditions (26.09±0.78 hrs). Such pattern was highly disturbed under raised [Ca2+]i by ATXII (17.05±1.07 hrs) and was restored in presence of Ranolazine (25.32±0.97 hrs) and BAPTA-AM (25.91±0.54 hrs). This suggests that Ca2+ mediated changes in the cellular circadian clock are an integral part of the aetiology of atrial arrhythmia’s, and that the NaP/raised Ca2+ arrhythmogenic pathway directly affects Bmal1 circadian expression in atria; hence strengthens its association with AF occurrence. BMAL1 oscillatory protein expression observed under ATXII treatment was similarly reversed in presence of Ranolazine, proposing the involvement of Nav1.5. channel subtype driving such mechanisms. BMAL1 protein expression was also affected by CAIP (in presence of ATXII), suggesting a possible action of CaN on Ser42 residue. Interestingly, the pCx43-Ser368 show a similar protein expression pattern as BMAL1, proposing an immediate association of circadian mechanisms driving Cx43 (but not Cx40) expression in AF occurrence. Finally, the phosphorylated Cx43-Ser368 protein expression levels and oscillatory pattern were restored in presence of CAIP, proposing CaN involvement in mediating the ATXII-effects under AF occurrence. This further implies that the Ca2+/CaN signalling pathway is driving BMAL1 protein expression and localisation in atria.