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149 Raised intracellular ca2+ concentration by persistent na+ channels activation disrupts cellular circadian rhythmicity of bmal1 clock gene in cultured atrial myocytes
  1. Andria Siakalli,
  2. Daan van der Veen,
  3. Rita Jabr
  1. University of Surrey

Abstract

One of the main contributor to atrial arrhythmias (AA) is the activation of the persistent Na channels (NaP) 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 NaP and Bmal1 rhythmicity is unknown. The aim of this study is to assess the effects of 1)NaP opener, ATXII, Anemonia Sulcata toxin II and 2)raised [Ca2+]i induced by NaP 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; NaP 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 NaP activation channels in the disruption of atrial circadian rhythm.

  • bmal1 circadian rhythm
  • persistent Na channels
  • atrial myocyte

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