Deciphering ryanodine receptor array operation in cardiac myocytes.

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J. Gen. Physiol. Vol. 136 No. 2 129–133
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129
Perspective
Elemental calcium signals from RYR arrays operating in
cardiac myocytes have been extensively characterized
with ever-improving optical methods and other innova-
tive techniques. However, the exact nature of elemental
calcium signals in terms of RYR gating in situ remains
an enigma. Here, we synthesize insights gleaned from
recent developments in single-channel resolution of
cardiac RYR organization and in visualization of cal-
cium release events that are much smaller than calcium
sparks. This synthesis leads to the proposal of a concep-
tual framework that promises to unify diverse observa-
tions in sparkology.
Introduction
Ever since the discovery of calcium sparks some 17 years
ago (Cheng et al., 1993), the characterization of ele-
mental calcium signals has unveiled a new paradigm of
intracellular calcium signaling. It is now appreciated
that nearly all cells use discrete microscopic calcium sig-
nals to build up exquisite hierarchical calcium dynamics
in space, time, and concentration (Berridge et al., 2000;
Cheng and Lederer, 2008). Elemental calcium signals
are relevant not only in striated muscle contraction
(Cheng et al., 1993; Tsugorka et al., 1995; Klein et al.,
1996), smooth muscle relaxation (Nelson et al., 1995),
and neuronal secretion (Ouyang et al., 2005), but also
in understanding calcium signaling abnormalities in
major diseases such as heart failure and calcium-
dependent arrhythmias (Litwin et al., 2000; Sipido
et al., 2002; Song et al., 2006).
In cardiac myocytes, calcium sparks reflect the gating
of RYR calcium release channels in the ER/SR. Because
much has been learned about how single RYRs behave
in vitro (Fill and Copello, 2002), one might have ex-
pected few surprises to originate from the pursuit of
sparkology. However, an enduring enigma has been
the exact nature of elemental calcium signals in terms
of RYR gating in intact cells. Or, stated differently,
how can one infer the in situ RYR gating mechanism
Correspondence to Heping Cheng: chengp@­pku.edu.cn
Abbreviations used in this paper: CICR, calcium-induced calcium release;
CRU, calcium release unit.
concealed in the spatiotemporal kinetics of elemental
calcium signals?
At the heart of the question lie generic differences
between RYRs in cells and those in artificial lipid bilay-
ers or other cell-free preparations. In the first place,
RYRs form two-dimensional quasi-crystalline arrays in
the membrane of terminal cisterns of the SR. Such
array formation imposes physical constraints and en-
ables rich interactions between and among RYRs, cre-
ating a new entity that differs from RYRs acting solo.
New properties such as the coupled gating of RYRs
emerge (Marx et al., 1998, 2001); some behaviors may
be abrogated, and still others may be retained, but not
without modification. Furthermore, RYR array opera-
tion in situ is entangled with extravagant swings of cis
(the junctional cleft) and trans (the cisternal lumen)
calcium concentration, which may enact positive and
negative feedback regulation, including local calcium-
induced calcium release (CICR), calcium-dependent
channel inactivation/adaptation (Györke and Fill,
1993; Wang et al., 2004), and RYR desensitization by
SR calcium depletion (Györke and Györke, 1998;
Györke and Terentyev, 2008). Apart from these, generic
differences also include decoration of the RYR arrays
with molecular partners on the cytosolic side (e.g.,
calmodulin, calstabin, kinases, and phosphatases),
in the lipid membrane (e.g., triadin and junctin), and
inside the lumen (e.g., calsequestrin). As such, we
should resist the temptation to oversimplify and rede-
fine the laws underlying RYR array operation, includ-
ing its activation, coordination, termination, and even
its pharmacology.
Here, we synthesize insights gleaned from recent de-
velopments, particularly in the single-channel resolu-
tion of RYR organization in situ (Baddeley et al., 2009)
and in visualization of calcium release events that are
much smaller than calcium sparks (Brochet et al., 2009).
This synthesis leads to a conceptual framework that
promises to unify diverse observations in sparkology.
Perspectives on: Local calcium signaling
Deciphering ryanodine receptor array operation in cardiac myocytes
Wenjun Xie,1 Didier X.P. Brochet,2 Sheng Wei,1 Xianhua Wang,1 and Heping Cheng1
1Institute of Molecular Medicine, Peking University, Beijing 100871, China
2Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, MD 21201
© 2010 Xie et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publi-
cation date (see http://www.rupress.org/terms). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
The Journal of General Physiology

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130 RYR array operation in situ
Calcium quark or “invisible” SR release. During cardiac ex-
citation–contraction coupling, calcium sparks are read-
ily resolved on the premise that only a small fraction of
release sites are active (to ensure adequate signal con-
trast). However, calcium transients are spatially uniform
when the release is activated by low-intensity photolysis
of calcium-caged compounds (Lipp and Niggli, 1996)
or by the calcium influx of reverse sodium–calcium
exchange (Lipp et al., 2002). It has been hypothesized
that the sparkless calcium transient results from smaller
fundamental events, “calcium quarks,” which represent
the independent gating of individual RYRs. Likewise,
local calcium release that is smaller than a calcium spark
can be triggered photolytically with focal two-photon ex-
citation (Lipp and Niggli, 1998).
An increase of the SR calcium leak can occur with no
observable increase in the rate of calcium sparks under
certain physiological (Marx et al., 2000; Prestle et al.,
2001; Li et al., 2002) and pathological conditions (Gómez
et al., 1997; Ono et al., 2000; Shannon et al., 2003). To ac-
count for this “invisible” SR calcium efflux, Sobie et al.
(2006) postulated the existence of “rogue RYRs,” one
or a few RYRs in a cluster that are physically uncoupled
from the RYR cluster that underlies spark production.
Differing from RYRs in large arrays, rogue RYRs are
thought to behave in ways more like the descriptions
of single RYRs in terms of gating kinetics and CICR sen-
sitivity. However, ultrastructural evidence for the exis-
tence of such rogue RYRs was lacking until recently
(see below).
Quarky calcium release
Direct visualization of subspark local release events in
resting cells has been made possible by recent technical
innovations. Detection of small signals against noise is
confounded by the dilemma of rejecting false positive
Diversity of elemental calcium signals
RYR array operation in cardiac myocytes has been
extensively characterized with ever-improving optical
methods and other innovative techniques. Steadfast
efforts by many investigators have unraveled rich sub-
structures in a seemingly atomic calcium spark and,
more importantly, distinct modes underlying local cal-
cium release.
Quantal calcium release unit (CRU) in a spark. Although it
was initially suggested that calcium sparks are stereo-
typical, strong evidence indicates that they display
remarkable polymorphism. Shen et al. (2004) used the
loose-seal patch clamp to acquire calcium sparks at well-
defined subsurface locations and demonstrated very
broad distributions in spark amplitude and rate of rise.
Considerable polymorphism can be found even among
events originating from the same release site. Wang et al.
(2004) suggested that calcium release flux in a spark con-
sists of quantal units, stochastic recruitment of which
gives rise to the polymorphic appearance of the spark.
This surprising conclusion was based on the ability to
measure calcium release flux in a calcium spark (Ispark )
with a novel procedure: (a) measuring calcium “spark-
lets” produced by unitary L-type calcium currents (iCa)
when the SR calcium release is abolished pharmacologi-
cally and iCa is enhanced by FPL64176 and high extra-
cellular calcium; (b) determining the iCa under identical
experimental conditions with the giga-seal cell-attached
patch clamp technique; and (c) estimating Ispark with the
sparklet of known iCa as an optical yardstick. The resul-
tant histogram of Ispark exhibits regular or quantal sub-
structures with the quantal unit of 1.24 pA and, on
average, 2–3 quanta per calcium spark. The inclusion of
tetracaine in the patch pipette reduces the number of
quanta per spark while the quantal unit is unchanged.
Figure 1. Distinct modes of elemental calcium release. On a terminal cistern, a main RYR cluster consists of two subdomains with an
isthmic connection and is surrounded by a few rogue RYR groups (indicated by arrows in A). (A) Quarky calcium release from rogue
RYRs only. (B) Calcium spark with two quantal units. Either or both of the subdomains of the main cluster can be activated in a spark.
(C) Calcium spark with quarky substructure. During sparks, the neighbor rogue RYRs are also activated in succession, resulting in pro-
longing calcium release. Cytosol calcium concentration gradients are roughly displayed by colors (top to bottom of the look-up table
corresponds to the calcium concentration from resting to sparked). LCC, L-type calcium channel; SR, sarcoplasmic reticulum.

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Xie et al.131
of clusters within 100 nm edge-to-edge. In contrast, pre-
vious estimates, based on thin-section electron micros-
copy and the intuitive assumption of circular geometry,
place 128, 267, and 90 RYRs in a dyad of mouse, rat,
and dog ventricular myocytes, respectively (Franzini-
Armstrong et al., 1999). Second, there are indeed rogue
RYRs as proposed by Sobie et al. (2006), and the cluster
size varies over a wide, continuous spectrum. Quantita-
tively, the curve follows a steep decaying exponential
distribution, indicating that there are greater numbers
of rogue RYR groups than large RYR arrays. Theoretical
analysis suggests that rogue RYRs are simply in transi-
tion into the stochastic assembly of large RYR arrays.
These newly revealed morphological features of cardiac
RYR organization provide a structural framework to
unify the perplexing diversity of elemental calcium sig-
nals discussed above.
A unifying model
By synthesizing these recent advances in structural and
functional studies of RYR arrays in cardiac myocytes, we
propose a working model for cardiac elemental calcium
release events with the following features (Fig. 1). Struc-
turally, we should treat all RYRs on a terminal cistern or
CRU, associated junctional cleft, and cisternal lumen as
an integrated nano-assembly. Of these, a CRU contains
a main cluster and several surrounding rogue RYRs,
and the main cluster may be further divided into subdo-
mains delimited by isthmic or defective connectivity be-
tween them. Mechanistically, CICR acts across all RYRs
in a CRU, whereas the coupled gating of RYRs is con-
fined to those residing in the same cluster. RYR desensi-
tization by calcium depletion in the shared cisternal
lumen or calcium-dependent inhibition in the junc-
tional cleft provides yet another means for inter-RYR
and intercluster communication in a CRU, manifesting
as use-dependent refractoriness to CICR.
With this rather complex (but seemingly irreducible)
model, we can explain the coexistence of distinct
modes of elemental calcium release in a single CRU and
predict interactions and interconvertibility of these
elemental release modes. The activation of the rogue
RYRs gives rise to invisible or quarky calcium release;
events while preserving high sensitivity to prevent false
negatives. Brochet et al. (2009) implemented simulta-
neous dual imaging of both cytosolic and SR lumenal
calcium to cope with this challenge. This approach en-
hances the detectability by orders of magnitude com-
pared with signal detection in only one imaging channel.
In rabbit ventricular myocytes, regular calcium sparks
(measured by rhod-2 loaded in the cytosol) are mir-
rored by “calcium blinks” (measured by fluo-5N loaded
in the SR) (Brochet et al., 2005, 2009). Remarkably,
there are also tiny events that are only one fifth to one
seventh the size of a regular spark–blink pair. This quark-
like or “quarky” calcium release can occur at the same
sites that support the generation of regular spark–blink
pairs. More surprisingly, virtually all regular calcium
sparks include quarky calcium release components that
last beyond the peak of the spark. Calcium buffering by
EGTA exerts little effect on the initial, high-flux release
that determines spark amplitude, but it suppresses low-
flux, highly variable quarky calcium release, indicating
that the latter is sustained by local CICR during an on-
going calcium spark. Together with the polymorphism
of calcium sparks noted above, we conclude that dis-
tinct modes of elemental calcium release coexist at a
single release site.
RYR arrays in cardiac myocytes
Array formation appears to be a property intrinsic to
the calcium release channel because purified RYRs can
self-assemble into large two-dimensional arrays either in
solution (Yin and Lai, 2000) or on the surface of posi-
tively charged lipid membrane (Yin et al., 2005). In skel-
etal muscles of adult mammals, RYRs (type 1) in the
junctional cleft or triad are organized in double rows
with alternate direct coupling to the sarcolemmal volt-
age sensor or the dihydropyridine receptor (Franzini-
Armstrong et al., 1998). With the advent of optical
superresolution microscopy (30-nm resolution), novel
and interesting details have been revealed in the sub-
surface layer of rat ventricular myocytes (Baddeley
et al., 2009). First, cardiac RYR clusters are much smaller
than previously thought. The mean number of RYRs in
a cluster is 13.6, or 21.6 in a “supercluster” consisting
Figure 2. Stimulus intensity dependence of elemental
calcium release mode. A weak stimulus preferentially
activates rogue RYRs to produce quarky calcium release
(A), whereas a strong stimulus activates the main cluster
as well as the rogue RYRs (B).

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132RYR array operation in situ
This Perspectives series includes articles by Gordon,
Parker and Smith, Prosser et al., Santana and Navedo,
and Hill-Eubanks et al.
The authors would like to thank Iain C. Bruce for manuscript
editing.
This work was supported by the Major State Basic Research Devel-
opment Program of China (2007CB512100) and the National Natu-
ral Science Foundation of China (30628009 and 30900264).
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ment of subdomains produces a calcium spark with
quantal units; and the main cluster activation triggers
surrounding rogue RYRs in a stochastic manner, re-
sulting in a calcium spark with trailing quarky sub-
structures. That quarky calcium release does not
always activate the main cluster is because of the neg-
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cluster, via lumenal calcium depletion-induced de-
sensitization or a cytosolic calcium-dependent inhibi-
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In addition to enabling the coupled gating mecha-
nism, RYR array formation also confers strong use de-
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at a low-level calcium stimulus (Sobie et al., 2006).
As such, RYR arrays of different size within the same
CRU are functionally heterogeneous with respect to
CICR and use-dependent refractoriness. This feature
can also account for the stimulus intensity dependence
of the mode of calcium release: a strong stimulus (iCa
injected into the cleft) preferentially evokes a calcium
spark (with trailing quarky substructures), whereas a
weak stimulus (uncaging calcium, calcium influx via
NCX, iCa at near reversal potentials, and calcium at the
front of an aborting calcium wave) promotes quarky
calcium release (Fig. 2).
If it can survive future validation, this model bears
important ramifications with regard to physiological
and pathophysiological modulation of calcium signal-
ing. First, it predicts that there are many more quarky
calcium release events than calcium sparks in resting
cardiac myocytes (with the weak stimulus afforded by
low-level cytosolic calcium); i.e., spontaneous calcium
sparks are merely the tip of the iceberg of the SR leak.
Second, a multitude of new parameters comes into
play in determining the mode of elemental calcium
release and hence cellular calcium signaling. These in-
clude parameters related to the formation, organiza-
tion, and turnover of CRUs (size of the main cluster,
defects in the main cluster, and proportion of rogue
RYRs). For instance, a smaller main cluster and a
greater number of rogue RYRs would generate a greater
SR leak, even if the total RYRs in a CRU remain constant.
A mismatch between L-type calcium currents and
CRUs will also alter the strength of a stimulus sensed
by RYRs and favor a spark-to-quarky switch of the ele-
mental release mode. Furthermore, the model pre-
dicts that depletion of the SR tends to shift the release
toward the quarky release mode, whereas SR overload
tends to shift it in the opposite direction. Understand-
ing the multiplicity and modulation of the modes of
elemental calcium signals not only affords promising
new directions to explore the still mysterious “invisi-
ble” SR leak in heart failure, but also sheds new light
on how the seemingly plain calcium ion acts as a uni-
versal and versatile second messenger.

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