Cellular Biology
Cellular Mechanism of Calcium-Mediated Triggered
Activity in the Heart
Rodolphe P. Katra, Kenneth R. Laurita
Abstract—Calcium overload due to enhanced calcium entry is a mechanism for spontaneous calcium release (SCR) from
the sarcoplasmic reticulum, delayed-afterdepolarizations (DAD), and triggered activity. However, the exact mechanistic
relationship between elevated intracellular calcium levels and triggered activity originating from a specific location
remains unclear. We hypothesize that under conditions of enhanced calcium entry, elevation of intracellular calcium will
result in multiple calcium release events of which only one is more likely to initiate a triggered beat. We used optical
mapping of action potentials and ratiometric calcium transients in an electromechanically-uncoupled canine wedge
model of enhanced calcium entry, using IKs blockade with ␤-adrenergic stimulation. Under conditions of enhanced
calcium entry, the rate of calcium uptake was faster compared with control conditions; however, during rapid pacing,
cytoplasmic calcium elevation at the endocardium was significantly increased (15⫾4%) compared with control (10⫾3,
P⬍0.04). Rapid pacing induced multiple simultaneous SCR events with largest amplitude and earliest onset near the
endocardium compared with the epicardium. Furthermore, SCR events with largest amplitude and earliest onset served
as a focus for DAD-mediated triggered activity. Interestingly, polymorphic VT occurred in some experiments when
multiple SCR events occurred. In conclusion, multiple, simultaneous SCR events occur over a broad region of relatively
slower calcium uptake and elevated diastolic calcium levels. However, SCR events closer to the endocardium have the
largest amplitude and earliest onset and are, thereby, more likely to initiate DAD-mediated triggered activity. Finally,
multiple SCR events may be a mechanism of polymorphic VT under calcium overload conditions. (Circ Res. 2005;96:
535-542.)
Key Words: intracellular calcium 䡲 delayed afterdepolarization 䡲 triggered activity 䡲 optical mapping 䡲 arrhythmias
D
elayed afterdepolarization (DAD)–mediated triggered activity is believed to play an important role in arrhythmias
associated with catecholamine-excess,1,2 heart failure,3–5 and
mutations of the ryanodine receptor.6 – 8 DADs are caused by
membrane depolarization in response to nonelectrically driven
(ie, spontaneous) calcium release from the sarcoplasmic reticulum.9 –11 Calcium overload caused by enhanced calcium entry is
one mechanism for spontaneous calcium release (SCR) from the
sarcoplasmic reticulum.12–16 Previously, we and others have
shown that myocytes near the endocardium and midmyocardium
are more prone to elevated diastolic intracellular calcium levels,17 calcium overload, and thereby triggered activity18,19 compared with myocytes near the epicardium. However, preliminary
data from our laboratory suggests that only one triggered beat
typically occurs from a relatively broad area of diastolic calcium
elevation.18 Therefore, the exact mechanistic relationship between elevated calcium levels and the origin of triggered activity
from a specific site is not clear. For example, it is possible that
calcium overload occurs at only one unique site, and this is
where an SCR event and triggered activity occur. Alternatively,
it is possible that calcium overload and SCR events occur at
multiple regions simultaneously; however, only the largest SCR
causes a DAD that is sufficient to trigger a beat. The exact
mechanism is difficult to determine due, in part, to the difficulty
associated with mapping relative calcium levels simultaneously
from intact tissue with high temporal and spatial resolution. We
hypothesize that under conditions of enhanced calcium entry,
elevation of intracellular calcium will result in multiple SCR
events of which only one is more likely to initiate a triggered
beat. To test this hypothesis, novel ratiometric optical mapping
techniques were used to measure the magnitude and kinetics of
calcium release events across the transmural wall. In addition,
dual calcium and voltage optical mapping was performed in the
same preparation to simultaneously measure membrane potential and intracellular calcium.
Materials and Methods
Experimental Preparation
Experiments were performed in accordance with Public Health
Service guidelines for the care and use of laboratory animals.
Original received August 9, 2004; revision received January 31, 2005; accepted February 3, 2005.
From The Heart and Vascular Research Center, MetroHealth Campus, and the Department of Biomedical Engineering, Case Western Reserve
University, Cleveland, Ohio.
Presented in part at the 76th Annual Scientific Sessions of the American Heart Association, Orlando, Fla, November 9 –12, 2003.
Correspondence to Kenneth R. Laurita, PhD, MetroHealth Campus, Case Western Reserve University, 2500 MetroHealth Dr, Rammelkamp, 6th fl,
Cleveland, OH 44109-1998. E-mail [email protected]
© 2005 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000159387.00749.3c
535
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Mongrel dogs (20 to 25 Kg) (LBL Kennels; Reelsville, Ind) were
anticoagulated with heparin (2 mL) and anesthetized with sodium
pentobarbital (30 mg/Kg intravenous), and hearts were removed by
a left lateral thorocatomy and placed in cold (4°C) cardioplegia
solution. Transmural wedges of cardiac tissue (average dimensions:
20⫻10⫻10 mm) surrounding and parallel to branches of the left
anterior descending coronary artery and left circumflex artery were
dissected from the left ventricular free wall (n⫽12).20 All preparations were taken near the base of the left ventricle and any free
running Purkinje fibers were removed from the endocardial surface.
The coronary artery of each wedge was cannulated and perfused with
oxygenated (95% O2, 5% CO2) Tyrode solution containing
(in mmol/L) NaCl 135, NaH2PO4 0.9, MgSO4 0.492, KCl 4.03,
dextrose 5.5, CaCl2 1.8, and HEPES 10 (pH 7.40). Perfusion pressure
was maintained between 50 to 70 mm Hg by regulating coronary
flow. Wedges were stained with the calcium-sensitive indicator
indo-1AM dissolved in 1 mL solution of DMSO and Pluronic
(20%wt/vol) at a final concentration of 10 ␮mol/L for 45 minutes at
room temperature. The dye-loading period was followed by a
15-minute washout period to remove unhydrolyzed or partially
hydrolyzed dye.21 In addition, a subset of wedges (n⫽5) was also
stained with the voltage-sensitive dye di-4-ANEPPS (15 ␮mol/L). In
all experiments, 10 to 15 ␮mol/L of Cytochalasin-D was used to
ensure that motion artifact, if present, did not influence our result.
The absence of motion artifact was confirmed by CCD video
imaging and ratiometric bH plots described previously.21
The perfused wedge was placed in a Lexan chamber where the
transmural surface was gently positioned against an imaging window
using a movable piston. To avoid surface cooling and desiccation,
the wedge was immersed in the coronary effluent, which was
maintained at a temperature equal to the perfusion temperature
(37⫾1°C). The volume-conducted ECG (ECG) was monitored using
three silver disk electrodes fixed to the chamber. A fine gauge
(0.003-inch diameter) PTFE-coated silver unipolar electrode was
inserted into the endocardial surface to stimulate at twice-diastolic
threshold current. Physiological stability of the preparation was
ensured by monitoring the ECG, coronary pressure, coronary flow,
and perfusion temperature continuously throughout each experiment.
Preparations remained viable for 2 to 3 hours, but the entire
experiment lasted no more than 2 hours. All optical recordings were
performed 1 hour after cannulation to allow healing of the cut
transmural surface.
Optical Mapping System
To determine the amplitude of calcium transients, ratiometric imaging of intracellular calcium was performed.22 Briefly, excitation light
was filtered at 350⫾10 nm and directed to the preparation. Fluorescent light from the preparation was collected by a tandem lens
assembly. A 445-nm dichroic long-pass mirror was positioned in the
tandem lens assembly at a 45° angle to transmit all wavelengths
above 445 nm to a 16⫻16 element photodiode array and reflect all
wavelengths below 445 nm to another 16⫻16 element photodiode
array. All optical components were aligned with an accuracy of
35 ␮m.23 For all experiments before dye loading, background
fluorescence was recorded at both emission wavelengths (485 and
405 nm). Ratiometric calcium transients were calculated by dividing
the background-subtracted calcium transients at 405 nm by the
background-subtracted calcium transients at 485 nm. Although
ratiometric calcium transients can be used to accurately compare
relative changes in intracellular calcium,22 under certain conditions,
absolute calcium levels may be difficult to infer.
To measure intracellular calcium and transmembrane voltage
simultaneously, we also used optical mapping techniques described
previously.23 Briefly, in addition to the excitation light used for
calcium imaging, light at 514⫾10 nm was directed to the heart. The
longer wavelength calcium emission filter (485 nm) was replaced by
a long-pass emission filter (⬎695 nm) for simultaneous voltage
imaging.
All signals recorded from each photodiode and ECG signals were
multiplexed and digitized with 12-bit precision at a sampling rate of
1000 Hz per channel. For the present study, an optical magnification
of 1.24⫻ was used, resulting in a total mapping field of 14⫻14 mm
with 0.9 mm spatial resolution and 0.8 mm2 pixel size. For wedges
with a transmural length ⬍14 mm, some edge pixels were omitted
from the analysis. To view, digitize, and store the position of the
mapping array relative to anatomical features, the dichroic mirror
was rotated to reflect an image of the preparation to a CCD video
camera.
Experimental Protocol
In 12 wedges from 12 animals, HMR1556 (7␮mol/L), a selective IKs
channel blocker,24 and Isoproterenol (0.2 ␮mol/L), a ␤-adrenergic
stimulant, were administered simultaneously to enhance calcium
entry. This model has been shown previously to be associated with
triggered activity in single cells and tissue slices.19,25,26 Ratiometric
calcium transients and simultaneous action potentials and calcium
transients were recorded at constant baseline pacing (600 ms) and
during a momentary (5 second) step increase to 110 –200 ms.
Recordings were also made during rapid pacing, at the fastest cycle
length that would capture the tissue 1-to-1 (100 –150 ms, as determined by 10 ms decrements near refractoriness), followed by a halt
in pacing to elicit ectopic activity. Before optical recordings were
made, all preparations were electrically quiescent, and 2 of 12
wedges demonstrated a slight increase in automaticity due to
␤-adrenergic stimulation. This increase in automaticity was abolished by rapid pacing.
Data Analysis
To quantify the rate of decrease of intracellular calcium to diastolic
levels, the decay portion of the calcium transient (from 30% to 100%
of the decline phase) was measured by the time constant (␶) of a
single exponential fit using an automated Nelder-Mead simplex
iterative algorithm (MatLab, Mathworks Inc). When minimum
diastolic calcium level alternated from beat-to-beat during rapid
pacing, the average diastolic calcium level from 4 consecutive
minima was used. To account for differences in baseline diastolic
calcium levels, the elevation in diastolic calcium during rapid pacing
(⌬Cadias) was measured as the difference in diastolic levels before
and at the end of rapid pacing. Action potential depolarization times
were determined from the maximum first derivative during the
upstroke. Spontaneous calcium release (SCR) events were defined as
a spontaneous increase in calcium levels during diastole (ie, nonelectrically driven) that exceeded 10% of baseline calcium transient
amplitude. Spontaneous calcium release amplitude (SCRamp) was
measured as the difference between minimum and maximum calcium levels during the SCR event at the center pixel. SCR onset was
calculated as the time of peak SCR level relative to the earliest SCR
onset. All measurements were made using automated algorithms
with visual inspection by an experienced investigator. Levels of
significance were determined using a Student t test, ANOVA, and
Fisher exact test where a value of P⬍0.05 was considered statistically significant.
Results
Transmural Heterogeneity of Calcium Handling
Under Conditions of Enhanced Calcium Entry
Shown in Figure 1A are representative normalized ratiometric calcium transients recorded near the endocardium (Endo)
and epicardium (Epi) under conditions of enhanced calcium
entry. The decay time constant (␶) of the calcium transient
recorded near the endocardium (102 ms) was slower compared with that near the epicardium (78 ms). Over all
experiments (Figure 1B), ␶ was faster, in general, under
conditions of enhanced calcium entry (Endo⫽110⫾30 ms,
Epi⫽84⫾25 ms; P⬍0.002) compared with control conditions
(Endo⫽204⫾26 ms, Epi⫽140⫾13 ms; P⬍0.001, data not
shown). Figure 2A shows ratiometric calcium transients
recorded during the last beat of baseline pacing (CL 600 ms)
Katra and Laurita
Calcium-Mediated Arrhythmogenesis
537
Figure 1. Transmural heterogeneity of calcium handling under
conditions of enhanced calcium entry. Normalized ratiometric
calcium transients (A) recorded near the endocardium (ENDO)
and epicardium (EPI). Uptake time constant (␶), as calculated
from a single exponential fit to the decay portion of the calcium
transient (gray line), was slower at the endocardium (102 ms)
compared with the epicardium (78 ms) under conditions of
enhanced calcium entry. Over all experiments (B), ␶ was significantly slower (P⬍0.002) at regions near the endocardium compared with regions near the epicardium.
followed by a period of rapid pacing. During rapid pacing, the
apparent increase in mean diastolic intracellular calcium level
was higher near the endocardium (51 RU, 8%) compared with
the epicardium (42 RU, 7%), and was further increased under
conditions of enhanced calcium entry (Endo⫽71 RU, 13%
and Epi⫽49 RU, 8%). This occurred even though cytosolic
calcium removal is faster under conditions of enhanced
calcium entry compared with control. Calcium alternans was
also greater near the endocardium, especially under conditions of enhanced calcium entry. On average (Figure 2B),
pharmacological enhancement of calcium entry caused a
significant augmentation of the pacing-induced increase in
the mean diastolic ratio near the endocardium (15⫾4 versus
10⫾3%, P⬍0.04), but not the epicardium (8⫾3 versus
7⫾2%, P⫽NS). These data suggest that regions exhibiting
relatively slower uptake of intracellular calcium by the SR are
more prone to elevated diastolic calcium levels in response to
rapid pacing under conditions of enhanced calcium entry.
Triggered Activity Under Condition of Enhanced
Calcium Entry
Under conditions of enhanced calcium entry, ectopic activity
was observed immediately on termination of rapid pacing.
Shown in Figure 3A are membrane potential (Vm) and
intracellular calcium (Ca) signals from a representative experiment under conditions of enhanced calcium entry, recorded from a site near the endocardium (baseline pacing, left;
rapid pacing, middle) and a site near the epicardium (rapid
pacing, right). Immediately after halting rapid pacing, an
ectopic beat (asterisk) occurred, followed by a DAD and a
SCR ⬇250 ms after final repolarization, only at the endocardium. Furthermore, just preceding the ectopic beat, Vm and
Ca2⫹ levels at the endocardial site slowly increased (arrow)
and had a similar initial time course and amplitude compared
with the DAD and SCR after the ectopic beat. In contrast,
there was no slow increase in Vm and Ca2⫹ during baseline
pacing (left) or at the epicardial site (right). This is consistent
with an ectopic beat triggered by an SCR-mediated DAD at
Figure 2. Relative changes in diastolic calcium ratio levels under
conditions of enhanced calcium entry. A, Representative calcium transients recorded near the endocardium (ENDO) and epicardium (EPI) during rapid pacing (180 ms) after a period of
baseline pacing (600 ms), under control and conditions of
enhanced calcium entry. Complete 1-to-1 capture was confirmed by activation contour maps (data not shown). Relative
increase in diastolic intracellular calcium level during rapid pacing (⌬Cadias) was measured as the difference between minimum
diastolic level before and during rapid pacing (dotted lines) and
expressed in ratio units (RU). Mean diastolic calcium was used
when alternans was present. The relative increase in diastolic
calcium was greater near the endocardium compared with the
epicardium under control (51 and 42 RU, respectively) and conditions of enhanced calcium entry (71 and 49 RU, respectively).
Over all experiments (B), ⌬Cadias (normalized to baseline levels)
was greater under conditions of enhanced calcium entry compared with control near the endocardium but not the epicardium
(see text for details). Under both conditions, ⌬Cadias was greater
near the endocardium compared with the epicardium.
the endocardium. Figure 3B shows the activation pattern of
the ectopic beat in Figure 3A. Earliest activation of the
ectopic beat occurred near the endocardium (asterisk) at the
site of DAD and SCR activity shown in Figure 3A, remote
from the pacing site (stimulus symbol). Over all experiments,
triggered beats occurred most frequently (P⬍0.001) closer to
the endocardium (Figure 3C), where diastolic calcium levels
were highest under conditions of enhanced calcium entry.
In several experiments (n⫽3), optical mapping of voltage
and calcium was performed sequentially from the transmural
surface and the endocardial surface. When episodes of triggered activity occurred reproducibly at the same rate of rapid
pacing with similar ECG morphology, transmural and endocardial contour maps were combined. Contour maps were
time-aligned by the pacing artifact and aligned in space using
images captured with the CCD camera. Shown in Figure 4 is
an example of triggered activity with earliest activation (ie,
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March 18, 2005
Figure 3. Triggered activity under conditions of enhanced calcium entry. A, Representative action potentials (Vm) and
calcium transients (Ca) recorded during
baseline pacing (600 ms) from the endocardium (left) and rapid pacing (180 ms)
followed by an abrupt halt in pacing from
the same endocardial site (middle) and
an epicardial site (right). After cessation
of rapid pacing, an ectopic beat is evident (*). In contrast to the epicardium, a
delayed-afterdepolarization (DAD) and a
spontaneous calcium release (SCR)
occurred at the endocardium that did not
elicit an ectopic beat. Activation map of
the ectopic beat (B) shows its origin (*)
close to the endocardium yet away from
the site of stimulation (stimulus symbol).
Over all experiments (C), triggered activity (TA) occurred more frequently at the
endocardium compared with other
regions (P⬍0.001).
breakthrough) occurring on the endocardial surface. In this
episode, triggered activity probably originated closer to the
endocardial surface rather than the cut surface of the transmural wall. Such episodes of triggered activity were excluded
from analysis because the origin was far from the transmural
surface. However, these data suggest that triggered activity is
not necessarily caused by injury to the cut surface.
Mechanisms of Calcium-Mediated Triggered
Activity and Arrhythmogenesis
Using ratiometric imaging techniques, we were able to map
the amplitude of spontaneous calcium release events. Shown
in Figure 5A is a contour map of SCR amplitude in the
absence of a triggered beat. Regions of largest calcium
Figure 4. Triggered activity from the endocardial surface under
conditions of enhanced calcium entry. Temporally and spatially
aligned endocardial and transmural maps were recorded
sequentially from a representative experiment where triggered
activity occurred consistently from the same site of origin. Earliest activation (*) occurred in the middle of the endocardial surface rather than the cut surface of the transmural wall.
release are indicated by lighter shades of gray. Interestingly,
we observed multiple simultaneous SCR events in a single
recording. In the example shown, two significant release
events occurred, one near the endocardium (site a) and a
second in the midmyocardium (site c). Figure 5B shows the
ECG and calcium transients recorded from sites a, b, c, and d.
SCR amplitude and time-to-peak at site a (endocardium) was
largest and earliest compared with b and c. No SCR event was
observed at site d (epicardium). Furthermore, a much smaller
SCR amplitude at site b suggests that the SCR events at a and
c are independent. Another interesting finding was that each
SCR event did not occur from a single cell but from a region
of myocardium comprised of many cells, as indicated by the
SCRs occurring over more than one pixel. These data indicate
that calcium release does not occur from a single cell but from
a large group of cells.
To explain the preferential occurrence of a single ectopic
beat near the endocardium in the presence of multiple
Figure 5. Multiple simultaneous SCR events under conditions of
enhanced calcium entry. A, Representative transmural map of
SCR amplitude (SCRamp) reveals 2 “hot spots” of high SCR
amplitude near the endocardium (a) and midmyocardial region
(c). B, ECG and calcium transients recorded at a, b, c, and d.
SCR event at site a had an earlier occurrence and larger amplitude compared with b and c. Note that no SCR was observed
at d (epicardium).
Katra and Laurita
Figure 6. SCR kinetics across the transmural wall under conditions of enhanced calcium entry. A, Histogram of transmural
SCR occurrence over all experiments. SCRs occurred more frequently near the endocardium compared with other regions
(P⬍0.001). B and C, SCR amplitude and SCR onset (normalized
to the earliest SCR onset). SCR events had significantly larger
amplitude and earlier onset at the endocardium compared with
other regions (P⬍0.01).
calcium release events, the timing and amplitude of all
calcium release events from each experiment were analyzed.
Figure 6A indicates that most SCR events occurred near the
endocardium (P⬍0.001). This pattern closely matches that of
triggered activity occurrence (Figure 3C). In addition, SCR
events near the endocardium occurred with larger amplitude
(Figure 6B) and earlier onset (Figure 6C) compared with
SCRs at other myocardial regions. These data suggest that
SCR events (and thereby DADs) are more likely to occur near
the endocardium with larger amplitude and earlier onset, all
of which may explain why a single triggered beat occurs
preferentially near the endocardium.
In several experiments (n⫽3), ectopic activity and SCR
events occurred reproducibly from the same location across
the transmural wall, under conditions of enhanced calcium
entry. Shown in Figure 7 are contour maps of ␶, ⌬Cadias,
SCRamp, and activation time of the ectopic beat from one such
experiment. The map of ␶ (Figure 7A) reveals a distinct
region of slow decay of the calcium transient near the
endocardium (white spot). During rapid pacing, mean diastolic calcium levels (Figure 7B) were, in general, greater near
the endocardium but particularly high around the same region
of slow ␶. After rapid pacing (Figure 7C), two SCR events
occurred; however, the SCR with earliest onset and largest
amplitude (asterisk) occurred where ␶ and ⌬Cadias were
slowest and largest, respectively. Finally, from the same
Calcium-Mediated Arrhythmogenesis
539
Figure 7. Slow calcium uptake kinetics as a mechanism of triggered activity under conditions of enhanced calcium entry.
Shown from a representative experiment, where triggered activity consistently occurred from the same site, are transmural
maps of ␶ (A), pacing-induced ratiometric ⌬Cadias (B), SCR
amplitude (C), and the origin of triggered activity (D). In A, the
region of slowest calcium uptake (white) coincided with the
region of largest diastolic calcium level in B. Immediately after
cessation of rapid pacing, the SCR amplitude map (C) revealed
two SCRs; however, the triggered beat (D) occurred at the site
where SCR amplitude was greatest and the onset was earliest (*).
preparation, when an ectopic beat occurred, the site of earliest
activation occurred where the largest and earliest SCR event
occurred (Figure 7D). These data suggest that the mechanism
of ectopic activity in this model is calcium overload where
calcium uptake kinetics are slowest.
It is possible that multiple regions of calcium release
events may produce ectopic beats originating from several
regions. Figure 8A shows a representative example of polymorphic ventricular tachycardia under conditions of enhanced calcium entry. On termination of rapid pacing (S1),
multiple ectopic beats were observed (V1 to V4). The first
beat (V1) is likely to be triggered activity occurring near the
transmural surface, similar to that shown in Figures 3 and 7.
V2 is preceded by a relatively long isoelectric interval (⬇350
ms) that also suggests triggered activity. The remaining beats
(V3 and V4) may or may not be triggered activity. The exact
mechanisms are unknown because the activation sequence
from deeper layers could not be determined. These data
suggest that multiple SCR events may be a mechanism of
polymorphic tachycardia in the intact heart.
Discussion
In this study, we investigated the cellular mechanisms of
calcium-mediated triggered activity under conditions of enhanced calcium entry in an intact cardiac preparation. We
found spontaneous calcium release (SCR) events occurring
over broad regions of relatively slower calcium uptake and
elevated diastolic calcium levels. Interestingly, we observe
multiple SCR events occurring across the transmural wall;
however, they occurred more frequently and had largest
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March 18, 2005
Mechanism of Triggered Activity and DADs
Figure 8. Polymorphic ventricular tachycardia under conditions
of enhanced calcium entry. A, ECG with two paced beats (S1)
and four ectopic beats (V1 to V4) recorded from a representative experiment. Note the difference in V1, V2, V3, and V4 QRS
morphology, indicating a different focal source of ectopic activity for each beat. Transmural activation maps of the last S1
paced beat (B) and V1-V4 ectopic beats (C, D, E, and F) confirm
the different focal origin of ventricular tachycardia.
amplitude and earliest onset near the endocardium. These
SCR events are also responsible for DAD-mediated triggered
activity and may represent an important mechanism of
arrhythmogenesis associated with abnormal calcium cycling.
Heterogeneity of Calcium Handling Under
Condition of Enhanced Calcium Entry
We have shown previously that under normal conditions,
myocytes near the endocardium exhibit a slow intracellular
calcium decay (␶) and elevated diastolic calcium levels
during rapid pacing.17 In the present study, using ratiometric
optical mapping techniques, we were able to compare calcium transients over long time periods (ie, across interventions) and across experiments.22 Under conditions of enhanced calcium entry, ␶ was faster, in general, compared with
control conditions, presumably due to phospholamban phosphorylation. However, as under control conditions, ␶ near the
endocardium was still relatively slower (ie, larger) than ␶ near
the epicardium. We have previously shown in the guinea pig
that Isoproterenol increases the rate of calcium uptake overall, but it does not change the base to apex gradient of ␶ on the
epicardial surface of the heart.22 Interestingly, although uptake was faster overall under conditions of enhanced calcium
entry, mean diastolic calcium level during rapid pacing was
greater. This may reflect increased calcium entry through
L-type calcium channels.27,28
Under conditions of enhanced calcium entry, we and others19
have observed triggered activity after brief periods of rapid
pacing. The triggered activity we observed is most likely due
to DADs, considering the following. Near the origin of
triggered activity, action potentials were preceded by a slow
depolarization in membrane potential: the hallmark of a
DAD-mediated beat. Such slow depolarization was absent
during baseline pacing and at sites remote to the origin of
triggered activity. The amplitude of DADs we measured
reached ⬇25 mV (based on a 100 mV action potential and
⫺80 mV resting potential29), which is sufficient to activate
some sodium channels29 and trigger an action potential.30
DADs of comparable amplitude (7 to 15 mV) were measured
in vivo and in single cells.1,30 Finally, DAD activity was
always accompanied by a simultaneous SCR event, suggesting that DADs were mediated by spontaneous calcium
release.
Interestingly, we also found that SCR and DAD events
occurred from a group of cells over several pixels, suggesting
that these events do not occur in just a single cell. In fact, if
DADs and SCRs had occurred only in a single cell, it would
be unlikely that our optical mapping system could detect
them. However, the exact size (ie, number of cells) at the site
of origin is unknown because its location may be slightly
below the transmural surface. Nevertheless, from a biophysical stand point, our finding makes sense because to electrically capture and stimulate well-coupled myocardium, a large
“source” is required.31 Therefore, our data are consistent with
the notion of a spatially large afterdepolarization, potentially
explaining why DAD-mediated triggered activity can occur
in well-coupled myocardium.
Mechanism of SCR Events
In this study and others,32–34 triggered activity occurs preferentially near the endocardium. Our data suggests that under
conditions of enhanced calcium entry, mean elevated diastolic calcium levels and calcium overload (as with digitalis and
Ouabain) occurred preferentially near the endocardium where
the rate of calcium uptake was relatively slower compared
with the epicardium. Furthermore, SCRs near the endocardium had the largest amplitude, greatest occurrence rate, and
earliest time of onset. In cases of triggered activity originating
consistently from one site (n⫽3), we observed a distinct
region of large ␶ (slower uptake), elevated diastolic calcium
level, corresponding to the “strongest” (ie, largest and earliest) SCR and origin of triggered activity. Taken together,
these findings suggest a causal relationship between regions
of slower calcium uptake, diastolic calcium elevation, and the
location of triggered activity. It is possible that local calcium
alternans35 is a mechanism for increased diastolic calcium
levels and SCR activity in this model. However, although
calcium alternans was greater near the endocardium, it did not
correlate with mean elevated diastolic calcium levels and
SCR activity (data not shown).
The exact mechanism by which elevated calcium initiates
an SCR event remains unclear; it could be due to elevated
cytoplasmic calcium,36,37 increased SR calcium,38,39 or
both.40,41 Our data suggest that elevated cytoplasmic levels
Katra and Laurita
directly trigger SCR events. However, we did not measure SR
calcium content and cannot completely rule out the contribution of SR calcium load. It is also unknown why we observed
a relatively large (several pixels) region of calcium release. It
is possible that overload and, hence, an SCR event occurs
simultaneously from many cells.31 Alternatively, an SCR can
occur from a single cell, which then initiates a calcium wave
that diffuses from cell-to-cell across gap junctions, similar to
calcium waves in Purkinje cell aggregates.42 Further studies
are needed to elucidate the exact mechanism of SCR events in
intact tissue. We do know, however, that in our model, SCR
events cannot be explained by local calcium release and
propagation due to nonuniform contraction,42 because the
myocardium was electromechanically uncoupled. We predict
that in freely contracting myocardium under similar conditions, the kinetics and occurrence of SCR events may be
enhanced.
Multiple Simultaneous Calcium Release Events
One interesting finding was the presence of multiple simultaneous SCR events, confirming our hypothesis and suggesting that ectopic activity may originate from different locations and could explain the occurrence of polymorphic
ventricular tachycardia. In our study, nonsustained polymorphic ventricular tachycardia occurred in one third of all
preparations and exhibited multifocal activity. Theoretically,
such activity could be sustained and occur more frequently in
an intact heart where the ventricular mass is much greater,
and may represent a mechanism of TdP based on multiple
ectopic foci.32 Nonetheless, our findings suggest a mechanistic link between abnormal calcium homeostasis and the onset
of polymorphic ventricular tachycardia. Ultimately, this study
may help in devising better pharmacological or gene therapies for calcium-mediated arrhythmogenesis associated with
atrial fibrillation,43 heart failure,3,4 mutations of calcium
regulatory proteins,6,7 and long QT syndrome.25
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Acknowledgments
This work was supported by National Institutes of Health grant
HL68877 and American Heart Association, Ohio Valley Affiliate
predoctoral fellowship 0415213B (to R.P.K.). The authors thank
Aventis Pharma for their generous donation of HMR1556 for
this study.
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