<p class="first" id="P1">Pacemaker cells residing in the sinoatrial node generate
the regular heartbeat. Ca
<sup>2+</sup> signaling controls the heartbeat rate—directly, through the effect on
membrane molecules
(NCX exchange, K
<sup>+</sup> channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA
signaling.
Thus, the physiological role of signaling in pacemaker cells can only be assessed
if the Ca
<sup>2+</sup> dynamics are in the physiological range. Cultured cells that can be
genetically manipulated
and/or virally infected with probes are required for this purpose. Because rabbit
pacemaker cells in culture experience a decrease in their spontaneous action potential
(AP) firing rate below the physiological range, Ca
<sup>2+</sup> dynamics are expected to be affected. However, Ca
<sup>2+</sup> dynamics in cultured pacemaker cells have not been reported before.
We aim to a develop
a modified culture method that sustains the global and local Ca
<sup>2+</sup> kinetics along with the AP firing rate of rabbit pacemaker cells.
</p><p id="P2">We used experimental and computational tools to test the viability
of rabbit pacemaker
cells in culture under various conditions. We tested the effect of culture dish coating,
pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function.
The cells were maintained in culture for 48 h in two types of culture media: one without
the addition of a contraction uncoupler and one enriched with either 10mM BDM (2,3-Butanedione
2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior
to the experiments. Cells were successfully infected with a GFP adenovirus cultured
with either BDM or blebbistatin. Using either uncoupler during culture led to the
cell surface area being maintained at the same level as fresh cells. Moreover, the
phospholamban and ryanodine receptor densities and their phosphorylation level remained
intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing
rate, spontaneous Ca
<sup>2+</sup> kinetics, and spontaneous local Ca
<sup>2+</sup> release parameters were similar in the cultured cells with blebbistatin
as in fresh
cells. However, BDM affects these parameters. Using experimental and a computational
model, we showed that by eliminating contraction, phosphorylation activity is preserved
and energy is reduced. However, the side-effects of BDM render it less effective than
blebbistatin.
</p>