(111b) Electrophysiological Rhythms in Red Blood Cells

Coordination of many of the body's behavioural,
metabolic, and physiological processes are controlled

around the 24 hour clock typically controlled through endogenous
mechanisms, but can be re-

synchronised (entrainable) by
external stimuli. For example, the process of the body overcoming jetlag

via rhythms 
resetting.  Circadian rhythms are
observed in most mammalian cells, in vivo and in vitro. From

rhythmic gene expression and cell division, to metabolic
oscillations, biological clocks regulate myriad

aspects of cell biology and physiology. Hence, their
disruption is strongly associated with disease.

Current
models indicate the main drivers of the process are the products of rhythmic
expressions of

?clock genes' over a 24 hour cycle, though the specific
transcriptional component can differ. O'Neill &

Reddy,
2011 [1] showed following temperature entrainment, circadian rhythms present in
human red blood

cells (RBCs). Very little is known about RBC clock
mechanisms, or whether functional consequences exist for

erythrocyte biology. To address this, we have established an
electrophysiological approach to investigate

the causes and consequences of timekeeping in the RBCs.

We
have employed dielectrophoresis through the 3DEP System to simultaneously
measure cellular response

at 20 different frequencies to create a
DEP ?spectrum? of RBCs over a time course [2]. The spectra are then

fit to a single shell dielectric model to
obtain electrophysiological properties of the cells.

Following
the isolation and entrainment methodologies described in [1], we used DEP to
obtain the

electrophysiological properties of RBCs from four male adult
donors. Our preliminary results provided DEP

fingerprints of four donors (Figure 1 A-C), from which the
extracted membrane conductance Geff

 andcytomplasmic
conductivity σcyt fluctuated antiphasically
with near-24-hour periods. The averages of

Geff and σcyt were fit to cosinor curves  peaking at 17.35 h and 5.45 h after the
end of the temperature

cycle, respectively.No significant
variation was observed in Ceff, confirming the
microscopic observation that

cell radius and morphology did not change over the
incubation.

The
near-antiphasic appearance of Geff  and  σcyt suggests a
rhythmic regulation of

transmembrane ion flux. These
results demonstrated, for the first time, a circadian rhythm in the

electrophysiological properties of both red blood cell
membrane and cytoplasm. We interpreted  the

observed changes in electrophysiological parameters as
reflecting alterations in ion channel activity, which

in turn is likely to be mediated by post-translational
modifications, in particular phosphorylation. As a first

step towards elucidating the mechanism underlying
timekeeping in RBCs, we chose to investigate to what

extent it shares key features with the clock mechanism in
nucleated cells, As an initial test, we predicted

that RBC rhythms would be sensitive to the generic kinase
inhibitor, staurosporine. We observed an increase

in the circadian period of the electrophysiological
oscillation.

One
of the main issues with detecting circadian desynchrony
in physiology is the absence of a non-invasive,

rapid method for detecting systemic temporal state. Recent studies
show that forced circadian

desynchrony in humans leads to
large-scale perturbation of circadian rhythmicity of blood mRNA's,

suggesting that blood is a good reflection of the wider internal
desynchrony. Measurement of circadian

rhythms in animals routinely requires the sacrifice of
multiple animals per time-point, meaning that tens of

animals are used in each experiment. DEP has the potential
to provide an accurate and reproducible readout

of circadian status using very small blood volumes, and
significantly reduce the numbers of animals required

for an experiment. Our preliminary data, using 10 µL tail-knick whole blood samples from
voles,

demonstrates, for  the  first
time, an electrophysiological correlate of the ultradian
feeding-fasting cycle in

blood (Figure 1 D&E).

In
the absence of nuclei or other organelles in RBCs, this work potentially
indicates 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.

1.)O'NEILL, J. S. & REDDY,
A. B. 2011. John S.  Circadian clocks in
human red blood cells. Nature,  469,

    498?503

2.) HOETTGES, K.
F., HÜBNER, Y., BROCHE, L. M., OGIN, S. L., KASS, G. E. N. & HUGHES, M. P.
2008.

     Dielectrophoresis-activated multiwell
plate for label-free high-throughput drug assessment.

     Analytical
Chemistry, 80, 2063-2068.

3.) IRIMAJIRI, A., HANAI, T.
& INOUYE, A. 1979. Dielectric theory of multi-stratified shell-model with

      its application to a lymphoma cell. Journal of Theoretical Biology, 78, 251-269.

4.) FRY, C.H., SALVAGE, S.C., MANAZZA, A., DUPONT, E.,
LABEED, F. H., HUGHES, M. P.  &  JABR, R.I.

      2012. Cytoplasm
Resistivity of Mammalian Atrial Myocardium Determined by Dielectrophoresis and

     
Impedance Methods. Biophysical
Journal, 103(11),  2287?2294.

Figure  SEQ Figure \* ARABIC 1: Electrophysiological results for isolated human RBCs.
Circadian variation in A.) Membrane conductance, B.)
Membrane Capacitance, and C.) Cytoplasmic
Conductivity. D.)  Ultradian variation in vole whole blood samples of
cytoplasmic conductivity.