Abstract

One of the main contributor to atrial arrhythmias (AA) is the activation of the persistent Na channels (Na_P) leading to raised intracellular Ca²⁺ concentration ([Ca²⁺]_i). Incidences of AA exhibit diurnal patterns suggestive of possible correlation between circadian rhythms and atrial electrophysiology. Circadian rhythms are mediated by cellular circadian clocks, made up of transcriptional-translational feedback loops consisting of several genes, in particular Bmal1. So far, the correlation between raised [Ca²⁺]_i induced by activated Na_P and Bmal1 rhythmicity is unknown. The aim of this study is to assess the effects of 1)Na_P opener, ATXII, Anemonia Sulcata toxin II and 2)raised [Ca²⁺]_i induced by Na_P on Bmal1 gene circadian rhythmicity in cultured mouse atrial (HL-1–6) cells and mouse embryonic fibroblasts (MEFs, positive control).

HL-1–6 were grown in Claycomb medium with 10% FBS, 0.1 mM norepinephrine and 2 mM l-glutamine, whereas, MEFs were maintained in DMEM with 10% FBS. The cells were transduced with Bmal1::luciferase (BMAL1::LUC) probe to determine bioluminescence rhythmicity through long-term bioluminescence recording over several circadian cycles (4–5 days). Cells were serum shocked (2 hours) using 50% FBS to synchronise all cellular clocks. Cells were then incubated in control media alone or with 1 nM ATXII in presence/absence of either 10μM ranolazine (Ran; Na_P blocker) or 5µM BAPTA-AM. All data were expressed as mean±SEM. Differences among groups were tested by ANOVA followed by Bonferroni post-hoccomparison. The null hypothesis was rejected at p<0.05.

Under control conditions, HL-1–6 cells and MEFs exhibited 24 hours Bmal1 circadian rhythms with a period of 26.09±0.77 hours (n=8) and 25.22±0.26 hours (n=8), respectively. The Bmal1 rhythmicity was highly disrupted in HL-1–6 cells only when incubated with ATXII, with a 10 hours shortening (17.06±1.07 hours; n=8; p<0.05) in their period. The effect of ATXII on HL1-6 cells was reversed in presence of Ran (25.32±0.97; n=8; p<0.05 vs ATXII). In fura2-AM loaded cells, [Ca²⁺]_i was significantly increased with ATXII (2.30±0.05) when compared to control (0.96±0.08; n=3; p<0.01) and Ran fully reversed ATXII effect (0.94±0.02; n=3; p<0.01). Then, the effects of raised [Ca²⁺]_i by ATXII on Bmal1 rhythmicity was assessed using BAPTA-AM. Indeed, addition of BAPTA-AM prevented ATXII-induced Bmal1 period shortening (ATXII 17.09±1.21; ATX+BAPTA-AM 29.91±0.54; n=8; p<0.05).

This study showed that in HL-1–6 cells only but not MEFs:1)ATXII treatment leads to a significant shortening of Bmal1 driven bioluminescence by almost 9 hours; 2)ATXII effect is fully reversed by ranolazine; 3)ATXII effects are mediated by raised [Ca²⁺]_i and hence was blocked by BAPTA-AM. In conclusion, this study suggests a possible correlation between ATXII-induced atrial arrhythmias and Bmal1 gene and hence proposing a role for Na_P activation channels in the disruption of atrial circadian rhythm.