Bruno D Stuyvers

Henk E D J Ter Keurs

Penelope A Boyden

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Calcium Waves
Physiological Relevance in Cardiac Function
Bruno D. Stuyvers, Penelope A. Boyden, Henk E.D.J. ter Keurs
Modulation of the cytosolic Ca
21 concentration
([Ca21]i) constitutes a fundamental mechanism of
signal transduction in excitable cells. Generally,
this phenomenon is triggered by an excitation process of the
external membrane and subsequently leads to activation of
functional proteins specific to the cell type in which the
increase of [Ca21]i has occurred. However, in addition to the
normal excitation-activation coupling providing Ca21 influx,
fluctuations in [Ca21]i can occur spontaneously, without
external electrical or hormonal stimulation. A spontaneous
increase in Ca21concentration can occur at a single focus or
multiple foci within a cell and can lead to propagation of an
elevation of [Ca21]i throughout the cytosol in a wave-like
pattern. Although these Ca21 waves have been observed
widely in both excitable and inexcitable cells,1 their role in
the cardiac cell function remains unclear.
In the last 2 decades, a substantial body of literature
documenting Ca21 waves in cardiac tissues has been created.
Studies on waves in cardiac cells provided fundamental
information about the role of the sarcoplasmic reticulum (SR)
in excitation-contraction coupling.2 Local contractions were
reported in cells with disrupted sarcolemma,2 clearly demon-
strating that Ca21 waves were not related to depolarization of
the membrane. Interest in cardiac Ca21 waves was further
stimulated by a study that suggested a role for them in the
generation of abnormal, nondriven electrical activity of the
heart.3 Initially, Ca21 waves in cardiac muscle were assessed
indirectly from observations of local sarcomere contraction
and waves of propagating sarcomere shortening,4,5 and direct
evidence of spontaneous fluctuations in [Ca21]i was obtained
from analysis of aequorin photoluminescence.6,7 Progress in
the development of digital-imaging techniques and fluores-
cent Ca21-sensitive dyes8 enabled investigators to provide a
more precise, quantitative spatiotemporal analysis of the
dynamics and propagation of Ca21 waves in and between
cardiac myocytes.9–12 More recently, laser confocal micros-
copy has brought this analysis to the submicrometer scale,
revealing that like the stimulated action potential–elicited
Ca21 transient, spontaneous Ca21 waves result from Ca21
sparks.13 It has been shown that Ca21 waves are based on the
localized Ca21 release occurring during a spark that couples to
release by adjacent SR sites by diffusion of the released Ca21
ions.13
Early consideration of a role for Ca21 waves in cardiac
electrical activity arose from studies showing that spontane-
ous increase in [Ca21]i could activate transient inward cur-
rents.14 The transient inward current is generally assumed to
be arrhythmogenic15–17and is likely carried by the Na1-Ca21
exchanger and other Ca21-activated channels.18 Furthermore,
Capogrossi et al3 showed that when spontaneous Ca21 release
occurred simultaneously at several subcellular loci, mem-
brane depolarization occurred and under some circumstances
could induce an action potential. Subsequently, several inves-
tigators, using different methods, have studied the spatiotem-
poral aspects of spontaneous increases in [Ca21]i, [Ca21]i
propagation, and resulting membrane depolarizations. For
example, using ouabain or potassium-free solutions in single
myocytes, Miura et al19 showed the concomitant occurrence
of propagating Ca21 waves, cell shortening, and membrane
depolarization, which is referred to as a delayed
afterdepolarization.
These studies together have shown convincingly that spon-
taneous release of Ca21 in a myocyte constitutes a potential
arrhythmogenic process in the heart. Importantly, additional
evidence showed that Ca21 waves with similar characteristics
are present in the working heart in situ. A recent study by
Wier et al20 revealed that cardiac multicellular preparations
such as trabeculae exhibit similar properties of intracellular
Ca21 cycling compared with isolated single myocytes. The
challenge of applying our knowledge of Ca21 waves from cell
studies to the more integrated system is now being addressed
in intact heart studies, as presented by Kaneko et al21 in this
issue of Circulation Research. Kaneko et al21 confirm an
earlier study by Minamikawa et al22 and extend their findings
by providing a descriptive and quantitative analysis of 3
different types of Ca21waves in the intact heart using a
Langendorff perfusion system. Importantly, their results are
clearly consistent with data obtained from single myocytes,
aggregates of myocytes, or isolated trabeculae. No spontane-
ous fluctuations of [Ca21]i could be detected in regions that
had no apparent damage when the healthy heart was paced at
a physiological rate, similar to earlier reports in stimulated
myocytes. When pacing was terminated, Ca21 waves ap-
peared sporadically, each having limited intercellular propa-
gation. The observations by Kaneko et al21 re also consistent
with the confocal studies of cardiac trabeculae, in which Ca21
waves were shown to propagate at low velocity ('30 mm/s)
and over a limited distance throughout the muscle.20 It is
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Department of Pharmacology (P.A.B.), Columbia Univer-
sity, New York, NY; and the Departments of Medicine, Physiology, and
Biophysics (B.D.S., H.E.D.J.t.K.), University of Calgary, Calgary, Al-
berta, Canada.
Correspondence to Dr Penelope A. Boyden, Department of Pharma-
cology, Columbia College of Physicians and Surgeons, 630 W 168th St,
New York, NY 10032.
(Circ Res. 2000;86:1016-1018.)
© 2000 American Heart Association, Inc.
Circulation Researchis available at http://www.circresaha.org
1016
generally assumed that under physiological conditions, the
probability of spontaneous local Ca21 release is low. Hence,
the probability that Ca21 waves propagate to adjacent cells is
likely to be small.23 Kaneko et al21 found that in regions
where higher basal levels of Ca21 were suspected, Ca21 waves
occurred more frequently and propagated at a higher velocity
(116 mm/s). Again, this finding is consistent with observa-
tions in Ca21-loaded myocytes.19 Finally, Kaneko et al21
report the presence of a third pattern of spontaneous Ca21
waves (agonal waves) in regions of the intact heart that had
been overtly damaged by microelectrode impalement. Agonal
waves occur at high frequency over a small extent throughout
cells. As described by Kaneko et al,21 agonal waves are
similar to waves observed in Ca21-overloaded myocytes
during the hypercontraction that precedes cell death.14 As
concluded by the authors and consistent with data obtained in
isolated myocytes, the occurrence of each type of Ca21 w ve
depends on the degree of cellular Ca21 load, ie, SR load and
cytosolic Ca21 load.24 This dependence, as well as the
fundamental properties of Ca21 waves observed in isolated
myocytes, has been reproduced in the study by Kaneko et al21
on the intact heart. Thus, the study establishes an important
link between isolated cells and the working heart. Therefore,
the assumption that the fundamental mechanisms of Ca21
cycling in myocytes constitute a potential substrate for certain
types of arrhythmias is applicable to the myocardium in situ.
Nevertheless, some aspects of the conclusions formulated
in this study should be considered with caution and merit
additional exploration. The authors claim that “the spatiotem-
poral properties of Ca21 waves in the heart are diverse and
modulated by the Ca21-loading state” (page 1093). It is
important to consider the extent of the damage inherent to the
experimental model used by the authors. First, as mentioned
by the authors, manipulation of the heart during the mounting
in the Langendorff apparatus and positioning of the elec-
trodes are significant mechanical (and thus electrical and
biochemical) perturbations that may damage the preparation.
Hence, one may suspect the presence of Ca21-overloaded
cells in the epicardial area because this is the region directly
exposed to this type of injury. Second, the isolated hearts
were perfused through coronary arteries with a protein-free
solution. In the absence of plasma proteins, exudation of
water into the interstitial space occurs simultaneously with an
increase of the geometrical resistance of the coronary bed.25
These latter factors may affect supply of O2, nutrients, and
Ca21 of the myocytes. More importantly, edema is bound to
occur during perfusion in the absence of colloid osmotic
particles. Edema leads unavoidably to the stretching of
intercellular structures, including the collagen meshwork
attached to the surface of the cardiac myocytes. Strain of the
myocyte membrane is therefore likely and may contribute to
increased Ca21 entry into the cells in this model.
Ca21overload of cardiac myocytes is accelerated in metabol-
ically deficient regions.26 It is possible that the 3 distinctive
patterns in Ca21 waves described by Kaneko et al21 reflect the
response to incremental Ca21 overload of the myocytes in this
preparation. The velocity of propagation and the incidence of
Ca21 waves may reveal an increased extent of damage. It
would be reasonable to assume that in the heart in vivo, the
probability of occurrence of Ca21 waves is near the lower
limits of the observations by Kaneko et al.21
Furthermore, questions about quantitative analysis of the
dynamics of intracellular Ca21 transients may arise from other
technical limitations of the Langendorff method of perfusion
of the heart. Analysis of the effects of dynamic changes in
extracellular ion concentrations, such as quantitative analysis
of velocity or frequency of waves in response to changes in
the extracellular milieu, will be hampered by the presence of
edema. Another limitation of the study is the lack of infor-
mation about uniformity of the loading of myocytes with
fluorescent dye. This problem gains critical importance when
the fluorescence of a nonratiometric dye such as fluo 3 is used
in the estimate of Ca21 load of the cells, as was used in the
present study. The same problem leads to questions about the
extent of propagation of Ca21 waves in the multicellular
preparation. When dye is not uniformly distributed among
cells, it is difficult to determine whether the disappearance of
a wave is due to termination of propagation or to the low
concentration of dye in the sampling area. An additional
concern, applying to all observations of processes that extend
in 3 dimensions under the confocal microscope, is that Ca21
waves may seem to be limited in extent in the plane of
observation simply because they have disappeared from that
confocal plane. Ca21 waves are 3-dimensional dynamic
events. Thus, full evaluation of their properties requires
3-dimensional spatial as well as temporal analyses.
In summary, Kaneko et al21 have presented challenging
data suggesting that intracellular Ca21 waves do occur in the
intact heart and thereby confirm concepts proposed from
studies of structures at a simpler level of organization. The
incidence of Ca21 waves may be at the lower limits of this
study in the heart in vivo, but this study suggests that the
mechanisms of Ca21 cycling derived from work on isolated
cells are present in the intact organ and may underlie
arrhythmogenicity. This notion may be specifically relevant
to the heart with acute damage, and it warrants additional
exploration of the effects of damage shown in isolated muscle
preparations in which Ca21 waves are initiated near identified
damaged regions of the tissue.27 The results of Kaneko et al21
are consistent with the model underlying damage-induced
Ca21 waves in which cellular-Ca21 load is one of the main
determinants of the velocity of propagation of Ca21 waves in
cardiac muscle.24
Acknowledgments
This work was supported by grants from the National Heart, Lung,
and Blood Institute of Health (HL-58860), the Medical Research
Council of Canada, and the Alberta Heritage Foundation for Medical
Research and NATO Collaborative Research Grant.
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KEY WORDS: Ca21 wavesn Cai transientsn arrhythmias
1018 Circulation Research May 26, 2000


Calcium Waves Physiological Relevance in Cardiac Function

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Calcium Waves Physiological Relevance in Cardiac Function

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