Fibroblast Circadian Rhythms of PER2 Expression Depend on Membrane Potential and Intracellular Calcium

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus synchronizes circadian rhythms of cells and tissues throughout the body. In SCN neurons, rhythms of clock gene expression are suppressed by manipulations that hyperpolarize the plasma membrane or lower intracellular Ca²⁺. However, whether clocks in other cells also depend on membrane potential and calcium is unknown. In this study, the authors investigate the effects of membrane potential and intracellular calcium on circadian rhythms in mouse primary fibroblasts. Rhythms of clock gene expression were monitored using a PER2::LUC knockin reporter. Rhythms were lost or delayed at lower (hyperpolarizing) K⁺ concentrations. Bioluminescence imaging revealed that this loss of rhythmicity in cultures was due to loss of rhythmicity of single cells rather than loss of synchrony among cells. In lower Ca²⁺ concentrations, rhythms were advanced or had shorter periods. Buffering intracellular Ca²⁺ by the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) or manipulation of inositol triphosphate (IP₃)-sensitive intracellular calcium stores by thapsigargin delayed rhythms. These results suggest that the circadian clock in fibroblasts, as in SCN neurons, is regulated by membrane potential and Ca²⁺. Changes in intracellular Ca²⁺ may mediate the effects of membrane potential observed in this study. (Author correspondence: [email protected])

Keywords:

• Calcium
• Circadian rhythm
• Fibroblast
• Membrane potential
• Potassium

ACKNOWLEDGMENTS

This work was supported by NIH (R01 MH082945 to DKW) and a V.A. Career Development Award (D.K.W.). We thank Lexie Wang for excellent research assistance, Dr. Gabriella Lundkvist for providing details of her experimental protocol, and Drs. Andrea Meredith and Michael McCarthy for helpful discussions. We also thank Dr. Meredith for attempted patch-clamp experiments.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.