Electrophysiology of human erythrocytes exhibits circadian variation

Coordination of many of the body’s behavioral, metabolic, and physiological processes are controlled around the 24-hour clock through endogenous circadian rhythms which can be synchronised (entrained) by external stimuli, which allows for gradual readjustments with the natural changes in photoperiod, and for abrupt adjustments such as following transmeridian travel. Disruption of these circadian rhythms can lead to negative health outcomes related to areas such as metabolism, cardiovascular health, and mental health. Understanding these rhythms can help determine optimal timing of physical and mental activities as well as drug delivery. There is ample evidence that the main drivers of the process are specific clock genes which drive rhythmic expression over a near-24-hour cycle. Disruption of these genes in mammals leads to disruptions in circadian rhythms. This paradigm was recently challenged by the finding that following temperature entrainment, circadian rhythms in redox status are present in human red blood cells (RBCs) [1]. RBCs have no nucleus and thus no transcription. What other mechanisms are controlling rhythms present in RBCs, and how are they generated? Although the finding of transcription-independent circadian rhythmicity has attracted considerable interest, it has not been corroborated in the published literature. Here, we used dielectrophoresis (DEP), an electrostatic technique used to examine cellular electrophysiology, to human erythrocytes cultured under the same paradigm, to demonstrate rhythmic perturbations in the electrophysiological properties of human RBCs. DEP well chips [2], which consist of an array of 20 wells, have been designed along with the 3DEP reader to simultaneously measure cellular response at 20 different frequencies. The reader collects images of the wells every 0.5 s and tracks changes in light due to cell movement. The changes in light intensity are related to DEP force, and after each experiment a DEP “spectrum” is obtained as a function of frequency [2]. The spectra are then fit to a single-shell dielectric model to obtain electrophysiological properties of the cells. Four male donors, free of self-reported health problems including sleep disorders, provided 10-ml blood samples in heparin/EDTA tubes. RBCs were purified and entrained in two consecutive cycles of 12 h at 37°C and 12 h at 32°C, after which cells were left to free-run at 37°C for the duration of the incubation [1]. Starting at Zeitgeber time 0h, 500µl samples were removed at four hourly intervals for DEP analysis over a total period of 48 h. Samples were washed in isoosmotic DEP medium [4] adjusted to a conductivity of 0.043 S/m using phosphate-buffered saline (PBS), and resuspended at a final cell concentration of 106 cells/ml. Cell suspensions were pipetted in DEP well chips (DEPtech, Uckfield, East Sussex, UK) for analysis. Each time point was repeated at least 3 times. Raw data were fitted to a single-shell model [3] (R<0.9 rejected), through which values for the effective membrane conductance (describing ion transport across the membrane) and capacitance (reflective of membrane morphology), and cytoplasmic conductivity (numerating ionic strength) were obtained [2]. One-way ANOVA was carried out in MATLAB to examine the effect of time on these parameters. Membrane conductance and cytoplasmic conductivity both fluctuated with a near 24-hour period (p<0.001 for peak values). A standard method in fitting circadian patterns is through a cosinor model. Membrane conductance fit a cosinor model of a 22.00 h period with an R-square value of 0.9113 while cytoplasmic conductivity best fit the cosinor curve at a period of 21.75 h with an R-square value of 0.9538. The near antiphasic appearance of the membrane capacitance and cytoplasmic conductivity suggests a cyclic ion movement from the cytoplasm across the cell membrane. No significant variation was observed in capacitance which is expected since there should be no membrane morphology changes across time points. These observations indicate an underlying cyclic activity of ion transport across the membrane. In the absence of nuclei or other organelles in RBCs, these finding suggest membrane mechanisms involving differential ion channel activities and renders DEP a new technology to enhance the understanding of chronobiology. Future work will investigate the underlying mechanisms behind these changes and the effects of experimental conditions.