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Programme
Contents
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Recent studies have demonstrated that intracellular Ca2+ plays
an important role in cardiac pacemaking (Ju & Allen, 2001).
However,
the mammalian sinoatrial node (SAN) is a heterogeneous structure and
studies on the centre leading pacemaker region suggest that central
pacemaker cells do not require the sarcoplasmic reticulum (SR) calcium
release for spontaneous activity (Lancaster et al., 2004).
In order to study
the Ca2+ dependent pacemaker mechanisms in different regions
of mammalian preparation, we developed a new technique to image Ca2+
from intact mouse sino-atrial preparations. Mice (7 -10 weeks)
were deeply anaesthetized. The right atrium was opened under a dissecting
microscope to expose the crista terminalis, the intercaval area and
the interatrial septum as described by Verheijck et al. (2001).
The preparation
was pinned into a sylgard block with a 3 × 5mm open window that allowed
microscopic imaging. The SAN preparation was loaded with the fluorescent
Ca2+ indicator Fluo-4 AM (10 µM) by incubation in Tyrode
solution at 4°C for 5 h. The SAN area was recognized by its anatomic
landmarks. 10 mM 2,3–butanedione monoxime (BDM) was used to reduce
the motion artifact. The loading of dye into the central region normally
was weaker than for peripheral regions for reasons that are unclear. It
is known that Na+ channels are required for peripheral but
not for the central pacemaker activity. Therefore 100 µM lidocaine
was added to the perfusate to block any pacemaker type activity from
the periphery of the SAN. Spontaneous Ca2+ signals from the
central SAN were imaged using a confocal microscope (LSM410) in XY and
XT modes. We found that the SAN Ca2+ signal was modulated
by the SR Ca2+ release channel modulator caffeine and by
ATP. These results show that it is possible to record Ca2+
signals from the centre of leading pacemaker region in mouse heart.
Ju, Y.K. & Allen D.G. (2001) Clinical and Experimental Pharmacology and Physiology 28, 703-8.
Lancaster, M.K., Jones, S.A., Harrison, S.M. & Boyett, M.R. (2004) Journal of Physiology 556, 481-494.
Verheijck, E.E., van Kempen, M.J.A., Veereschild, M., Lurvink, J., Jongsma, H.J. & Bouman, L.N. (2001) Cardiovascular Research 52, 40-50.