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Am J Physiol Heart Circ Physiol 302: H2321–H2330, 2012.
First published March 23, 2012; doi:10.1152/ajpheart.00031.2012.
Innovative Methodology
A novel, minimally invasive, segmental myocardial infarction with a clear
healed infarct borderzone in rabbits
Ohad Ziv,1 Lorraine Schofield,1 Emily Lau,1 Lenny Chaves,1 Divyang Patel,1 Paul Jeng,1 Xuwen Peng,2
Bum-Rak Choi,1* and Gideon Koren1*
1
Cardiovascular Research Center, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence,
Rhode Island; and 2Pennylvania State Hershey College of Medicine, Hersey, Pennsylvania
Submitted 13 January 2012; accepted in final form 20 March 2012
arrhythmia; optical mapping; ventricular tachycardia
(SCD) remains a major public health
issue constituting an estimated 20% of deaths in industrialized
countries (31, 40, 49). In autopsy series, nearly 50% of SCD
victims have had healed myocardial infarction (MI; Refs. 24,
69). The majority of sudden deaths after MI occur due to
ventricular tachyarrhythmias (26). Post-MI ventricular arrhythmias have been studied extensively in animal models. The
animal studies suggest the epicardial borderzone as a key
player in the formation of reentrant ventricular tachyarrhythmias due to changes in tissue structure, ion channels, and gap
junctions that slow conduction and generate anisotropy (6, 21,
SUDDEN CARDIAC DEATH
* B.-R. Choi and G. Koren contributed equally to this work.
Address for reprint requests and other correspondence: G. Koren, Rhode
Island Hospital, Aldrich Bldg. 307, Providence, RI (e-mail: gideon_koren
@brown.edu).
http://www.ajpheart.org
22, 35, 47, 65, 67). While these studies have greatly enhanced
our understanding of post-MI arrhythmias, our understanding
of the arrhythmia mechanisms in healed infarct remains limited
because most of studies used 5 days postinfarction, not a fully
healed post-MI heart. In addition, the use of pericardial incision and coronary ligation in these models may result in a
healing process that deviates from post-MI healing in humans
(17). With larger animal models, this concern has been addressed by development of closed-chest models (7, 37).
Investigations of postinfarction remodeling specifically using the rabbit may further our understanding of action potential
(AP) dynamics related to arrhythmia formation. The rabbit
heart shares with the human heart ion currents responsible for
depolarization and repolarization, demonstrates an AP shape
similar to that of the human, and has been successfully used in
transgenic models (3, 42). Furthermore, the rabbit heart appears to most resemble the human heart with respect to wave
dynamics during complex arrhythmia such as ventricular fibrillation (46, 64). Complex wave dynamics of ventricular
fibrillation are most suitably investigated with high resolution
optical mapping techniques using charged-coupled device
cameras or photodiode arrays (9 –11, 34, 44, 56).
Previous studies of arrhythmia formation in rabbit models of
healed MI have used an open-chest method of infarction using
a thorocotomy for epicardial access followed by ligation of one
of the three coronary arteries. This technique, however, has
several drawbacks; 1) the use of coronary ligation in an
open-chest model results in pericardial fibrosis that may limit
the use of optical mapping of electrical activation and repolarization due to signal attenuation (12, 18, 19); 2) perioperative
mortality with this model has traditionally been high, ranging
from 30% to 35% during the perioperative period (41, 50, 51);
and 3) epicardial visualization of the left circumflex is difficult,
with 10 –20% failures to induce infarction (39). These limitations reduce the efficiency of generating the MI model, increasing the costs of the MI model and potentially introducing a
selection bias. A minimally invasive, closed-chest model in the
rabbit, using endovascular techniques that target a segmental
artery, would therefore theoretically reduce perioperative mortality and avoid limitations listed above.
Given the purported importance of the borderzone and the
potential usefulness of the rabbit postinfarction model, our
laboratory set out to develop a novel, minimally invasive,
closed-chest model of chronic healed myocardial infarction in
the rabbit with improved periprocedural survival. We developed a coil embolization technique with a two-catheter system
that allows for targeted coil embolization into the obtuse
marginal coronary circulation. Our two objectives were to
obtain a consistent segmental myocardial infarction with low
0363-6135/12 Copyright © 2012 the American Physiological Society
H2321
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Ziv O, Schofield L, Lau E, Chaves L, Patel D, Jeng P, Peng X,
Choi BR, Koren G. A novel, minimally invasive, segmental myocardial infarction with a clear healed infarct borderzone in rabbits. Am
J Physiol Heart Circ Physiol 302: H2321–H2330, 2012. First published March 23, 2012; doi:10.1152/ajpheart.00031.2012.—Ventricular arrhythmias in the setting of a healed myocardial infarction have
been studied to a much lesser degree than acute and subacute infarction, due to the pericardial scarring, which results from the traditional
open-chest techniques used for myocardial infarction (MI) induction.
We sought to develop a segmental MI with low perioperative mortality in the rabbit that allows optimal visualization and therefore
improved study of the infarction borderzone. Rabbits underwent MI
using endovascular coil occlusion of the first obtuse marginal artery.
Three weeks postprocedure, we evaluated our model by echocardiography and electrophysiology studies, optical mapping of isolated
hearts, and histological studies. Seventeen rabbits underwent the
protocol (12 MI and 5 sham) with a 92% survival to completion of the
study (11 MI and 5 sham). MI rabbits demonstrated wall motion
abnormalities on echocardiography while shams did not. At electrophysiological study, two MI rabbits had inducible ventricular tachycardia and one had inducible ventricular fibrillation. Isolated hearts
demonstrated no pericardial scarring with a smooth, easily identifiable
infarct borderzone. Optical mapping of the borderzone region showed
successful mapping of peri-infarct reentry formation, with ventricular
fibrillation inducible in 11 of 11 MI hearts and 1 of 5 sham hearts. We
demonstrate successful high resolution mapping in the borderzone,
showing delayed conduction in this region corresponding to late
deflections in the QRS on ECG. We report the successful development
of a minimally invasive MI via targeted coil delivery to the obtuse
marginal artery with an exceptionally high rate of procedural survival
and an arrhythmogenic phenotype. This model mimics human
post-MI on echocardiography, gross pathology, histology, and electrophysiology.
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perioperative mortality and to generate a model that allows
optimal visualization and therefore improved study of the
infarction borderzone and periborderzone regions.
METHODS
Minimally Invasive In Vivo Electrophysiological Study
The protocol was modified from a previously published protocol
from our laboratory (45). Rabbits were anesthetized and monitored as
described for MI induction. A steerable 4-Fr decapolar EP catheter
(Irvine Biomedical, Irvine, CA) was inserted in the right femoral vein
through a 4-Fr sheath and placed in the right ventricle (RV), guided by
fluoroscopy and pacing thresholds (Fig. 1A). Signals from the Hisbundle were obtained with the basal electrodes on the ventricular EP
catheter (RV base; Fig. 1B). During the procedure, 12-lead surface, 5
intracardiac ECG signals were recorded continuously using the EPBard-System Software OS2/warp (kindly provided by Bard, Lowell,
MA), filtered with a bandwidth of 30 –250 Hz (intracardiac signals)
and 0.01–100 Hz (surface ECG).
Electrophysiological study (EPS) was performed at a stimulation
cycle length (CL) of 240 and 200 ms. The following parameters were
Echocardiography
Transthoracic echocardiography was performed in sedated animals
immediately before EPS. Two-dimensional ECG images (Hewlett
Packard 5500) were obtained with a 7.5-mHz probe, and long axis and
short axis views were used mimicking those used in human ECGs.
Analysis included dimensions of the LV, left atrium, wall thickness,
valve function, and LV ejection fraction (calculated using Simpson’s
Rule) and was performed by an experienced echocardiographer
blinded to the protocol used and animal group.
Isolated Heart Preparations
Rabbits were injected with buprenorphene (0.03 mg/kg im),
acepromazine (0.5 mg/kg im), xylazene (15 mg/kg im), ketamine (60
mg/kg im), pentothal (35 mg/kg iv), and heparin (200 U/kg). After the
appropriate level of anesthesia was obtained as determined by corneal
reflex and response to painful stimuli, rabbits were euthanized via
beating heart harvest. The hearts were retrogradely perfused through
the aorta with the following (in mmol/l): 130 NaCl, 24 NaHCO3, 1.0
MgCl2, 4.0 KCl, 1.2 NaH2PO4, 5 dextrose, 25 mannitol, and 1.25
CaCl2, at pH 7.4, gassed with 95% O2-5% CO2. Temperature was
maintained at 37.0 ⫾ 0.2°C, and perfusion pressure was adjusted to
⬃60 mmHg with a peristaltic pump (Radnoti Glass Technology,
Monrovia, CA). Hearts were placed in a chamber to maintain temperature and to reduce movement artifact, and 5 ␮mol/l blebbistatin
were added to the perfusate (25). At the end of optical mapping, the
LV free wall was dissected and fixed with a 10% solution of formalin
in PBS for 24 h, embedded in paraffin, and then cut serially from
endocardium to epicardium. Sections were stained with Masson’s
trichrome stain for histopathological analysis.
Optical Mapping
The optical apparatus has been previously described (10). Fluorescence images from the anterior surface and LV free wall of heart were
focused on a CMOS camera (100 ⫻ 100 pixels, Ultima-L; Scimedia)
with a 50-mm Nikon f/1.2 lens. Excitation light was illuminated
through a dichroic box located between the camera lens and the
camera. Sampling rate was set to 1,000 frames/s, and data were
analyzed with a custom built software using Interactive Data Language (ITT Visual Information Solutions, Boulder, CO). Hearts were
monitored for adequate perfusion throughout the study by visual
inspection for pink hue, homogeneous fluorescence, and AP shape
(with prominent plateau phase).
We used two fields of view. For studies of arrhythmia initiation, a
1.5 ⫻ 1.5 cm2 (with a spatial resolution of 150 ⫻ 150 ␮m2) field of
view was used. For high resolution mapping of the borderzone region,
a 1.5 ⫻ 1.5 mm2 (with a spatial resolution of 15⫻15 ␮m2) field of
view was used. Hearts were stained with the voltage-sensitive dye di-4
ANEPPS (Invitrogen, Carlsbad, CA) using 25 ␮l of stock solution (1
mg/ml DMSO) delivered through a bubble trap above the aortic
cannula. ECG and perfusion pressure were continuously monitored
(Powerlab, ADinstrument, Colorado Springs, CO). All hearts were
initially challenged with our standard protocol (2, 3, 29) of decrement
in pacing CL consisting of 10-ms steps with progressively shorter CL
until loss of 1:1 capture or ventricular fibrillation induction. A second
protocol was also used mimicking the extra stimuli at in vivo EPS
(above). Ventricular fibrillation was terminated by injection of KCL
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This investigation conformed to the current Guide for Care and
Use of Laboratory Animals published by the National Institutes of
Health (NIH Publication No. 85–23, revised 1996), as well as the
standards recently delineated by the American Physiological Society
(16), and was approved by the Animal Welfare Committee at Rhode
Island Hospital. Adult male rabbits (3.5–5.5 kg) ages 6 –12 mo were
used. Rabbits were anesthetized with intramuscular ketamine/xylazine
(25 mg/kg/3.75 mg/kg) and buprenorphine (0.03 mg/kg), intubated,
and ventilated with isoflurane (1–2%, FiO2 0.5). Blood pressure and
peripheral oxygen saturation were measured continuously. A neck cut
down was performed, and the right carotid artery was canalized. A
4-Fr guide catheter (Cook, Slip-Cath Beacon Tip with custom alteration for use in a rabbit) was advanced retrogradely to the ascending
aorta using fluoroscopy and angiography of the left main coronary
artery was performed. A continuous 12-lead ECG was recorded
during the induction of myocardial infarction. Lidocaine (1 mg/kg
load followed by 20 mcg·kg⫺1·min infusion⫺1) was infused during
the procedure to reduce periinfarct ventricular arrhythmias. Heparin
was administered to achieve a target activation clotting time of 300 s.
With the use of fluoroscopic guidance with the guide at the left main
coronary ostium, a 0.014-in. floppy wire was advanced into the
coronary. Based on the angiogram performed, the wire was advanced
into the left circumflex artery and then distally into the obtuse
marginal branch coursing toward the apex. Over the wire, a 2.5-Fr
delivery catheter (Boston Scientific, FastTracker18) was tracked into
the obtuse marginal until the tip reached two-thirds the distance from
the left main coronary ostium to the left ventricular (LV) apex. After
removal of the 0.014-in. floppy wire, a platinum 0.018-in. Hilal
microcoil (Cook) cut to a length of 1.5–2 mm was pushed out the tip
of the delivery catheter using an 0.018-in. floppy wire. The microcoil
has a synthetic thrombogenic fiber (30), which induces clot in the
targeted artery during the first 2–5 min of coil delivery. MI was
confirmed postcoil embolization using ST elevations on two consecutive ECG leads. The rabbits were continued on a Lidocaine infusion
and monitored for 1-h postcoil embolization. Sustained ventricular
arrhythmias during this period were treated with external defibrillation
(Medtronic Biphasic Defibrillator) using 6 J.
Sham rabbits underwent the exact same protocol, but the procedure
was terminated after coronary angiography. Two rabbits undergoing
MI were killed 2 h after coil embolization for measurement of infarct
size. These hearts were cut in the coronal plane into four disks from
apex to base and placed in 1% triphenyltetrazolium chloride for 15
min. Surfaces were scanned, and percentage of infarct was measured
by calculating percentage of area not stained brick red.
analyzed as previously described (36). Atrium to His (AH) and His to
ventricle (HV) intervals were measured. Programmed ventricular
stimulation was performed with one, two, and three extra stimuli in
apical and basal positions to examine inducibility of monomorphic
ventricular tachycardia or ventricular fibrillation (45). We strictly
defined all ventricular tachycardia or ventricular fibrillation as sustained ventricular arrhythmias requiring cardioversion or defibrillation
respectively.
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NOVEL MI IN RABBITS
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solution (0.3 mM, 100 ␮l to the bubble trap). As with in vivo studies,
during whole heart experiments we defined ventricular tachycardia
and ventricular fibrillation as sustained ventricular arrhythmia requiring intervention for termination.
Statistical Analysis
Normally distributed continuous variables were compared using a
Student unpaired t-test. Categorical values were compared using a
Fisher’s exact test. Statistical significance was set at P ⬍ 0.05.
Data Analysis
RESULTS
The activation and repolarization time-points at each site were
determined from fluorescence (F) signals by calculating (dF/dt)max
and (d2F/dt2)max. Data were filtered using a spatial Gaussian filter
(3 ⫻ 3 pixel) and first/second derivatives (dF/dt, d2F/dt2) were
calculated using polynomial filter (3rd order, 13 points). Pixels with
low signal-to-noise ratio determined by (dF/dt)max (⬍3⫻␴ of baseline) and outliers of pixels determined by Grubbs’ test were removed
from the analysis (typically ⬍1% of total pixels). Action potential
duration (APD) dispersion was defined as APD max-min across the
field of view. Reproducibility of infarct size was analyzed further by
calculating the surface area of the infarct on the epicardium of the LV
free wall. Digital photographs of the LV free wall were analyzed using
ImageJ (1) to report average infarct surface area.
A total of 17 rabbits entered the study with 5 receiving sham
interventions and 12 receiving true coil embolization. Angiograms of the left coronary artery were successfully performed
in all rabbits studied (Fig. 1). In all angiograms performed, the
left circumflex coronary was dominant, supplying the left free
wall and apex via obtuse marginal branches. In two animals,
postembolization angiogram of the left coronary artery was
performed, both demonstrating total occlusion of the first
obtuse marginal branch. All sham animals received angiograms successfully. No complications were seen with angiograms alone.
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Fig. 1. Technique of targeted coil embolization. A: schematic of coil, not to scale- see dimensions in METHODS. Coil length is 1. B: fluoroscopic image of coil
embolization. Right anterior oblique caudal projection view, showing the inner catheter is successfully inserted in mid-region of the first obtuse marginal branch
of the left circumflex artery. C: myocardial infarction (MI) image from Langendorff perfused heart. Note that MI region can be identified as white fibrosis region,
and the surface of heart including MI regions is clean without blood clots or scars from surgery. D: triphenyltetrazolium chloride (TTC) staining to verify survival
tissue and fibrotic tissue. Infarct size was measured from TTC staining. E: histological staining with Masson’s trichrome in postoptical mapping hearts of the
periphery zone (PZ) demonstrated areas of scar with strands of surviving myocardial tissue invading into this region of scar. LV, left ventricular.
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Table 1. Coil embolization procedure results
MI
Sham
Echocardiograms
Total
Successful
Deployment
Acute
VF
Acute
Death
3-wk
Survival
12
5
12
0
4
0
1
0
11
5
VF, ventricular fibrillation; MI, myocardial infarction.
Electrophysiology Study
EPS was successfully performed with survival of the rabbit
postprocedure in all 11 MI rabbits and 5 sham rabbits. Programmed stimulation with up to three extra stimuli induced
sustained ventricular arrhythmias in 3 of 11 MI rabbits but
none of the shams (P ⫽ NS). Sustained ventricular fibrillation
with two extra stimuli was induced in one MI rabbit and
sustained monomorphic ventricular tachycardia was induced in
two rabbits, each requiring triple extra stimuli for induction.
Ventricular tachycardia was terminated with over-driving pacing in one rabbit and with delivery of external shock in another.
One-to-one ventricular arrhythmias conduction was seen in
both cases (Table 2). Ventricular tachycardia was verified in
each instance with His activation onset subsequent to the initial
deflection of the QRS and altered QRS morphology and axis
from baseline (Fig. 3). Ventricular tachycardia morphology
was right-bundle with a left-superior axis and a CL of 157 ms
in one case and right-bundle with a right-superior axis and a
CL of 172 ms in the other.
Mapping Activation and Repolarization in MI Region
Hearts were visually inspected after isolation from the chest.
In both sham and MI hearts, the pericardium appeared pristine
upon isolation and was easily removed from the epicardium
(Fig. 4, A and B). No pericardial adhesions or fibrosis was seen
in either group. In all 11 postinfarction rabbits, MI at the
apical-lateral region of the LV free wall was easily identifiable.
The infarct size was consistent with a mean infarct surface area
of 4.3 ⫾ 0.94 cm2 (n ⫽ 8) and a minimum and maximum
infarct area of 3.4 and 5.9 cm2, respectively. Infarction was
absent in all sham hearts. The periphery of the infarct was
easily identifiable in all infarct hearts and demonstrated an
irregular pink margin along the LV free wall between the
infarct region and the remote, preserved region. We defined
Fig. 2. ECG 2-chamber view demonstrating
systole and diastole in a sham animal (A) and
MI animal (B). Note the apical wall motion
abnormality (WMA in yellow) seen in the MI
rabbit. LA, left arterial.
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Twelve rabbits underwent coil embolization (Fig. 1, A and
B). Eleven survived the procedure (92%; Table 1). The single
death occurred during attempted coil deployment. Failure to
successfully load the coil into the delivery catheter resulted in
several passes of the delivery catheter and wire. During these
attempts, ventricular fibrillation storm developed, with electrical-mechanical dissociation seen after defibrillation attempts.
The rabbit died likely from left main spasm. With the 11
rabbits that survived the procedure, ventricular fibrillation
occurred within 15 min of coil deployment in 3 of 11 (27%). In
each of these three cases, successful defibrillation and circulatory stabilization was achieved with a single application of
external defibrillation. All 11 rabbits that survived the remainder of the 3 wk protocol and 5 sham rabbits underwent ECG,
EPS, and isolated heart preparation optical mapping. These
results are presented below. When inspected before optical
mapping, hearts demonstrated a healed infarct appearing white
with a pink rim borderzone region over the apex and apical
lateral walls (Fig. 1C). With perfusion, akinesis was visually
seen in this region. Additionally, fluorescence was absent from
this region with optical mapping. Two additional rabbits that
underwent the protocol were killed acutely for triphenyltetrazolium chloride staining, which revealed a segmental infarction that
included the lateral and posterior aspect of the apex through the
mid LV free wall (Fig. 1D). The infarct size was 20 ⫾ 2% (n ⫽
2) of the entire LV. All infarcts had a transmural section. Transmurality of the infarct was reduced at the borderzones. A pink rim
of surviving myocardium was seen at the epicardial borderzone as
with other MI models (14, 15, 20, 22, 23, 48, 66, 68) (Fig. 1D).
We also noted a well-defined junction of normal myocardium
basal to the scar comprising a basal periphery of the scar (see Fig.
4). Histological staining with Masson’s trichrome in postoptical
mapping hearts of this periphery zone (PZ) demonstrated areas of
scar with strands of surviving myocardial tissue invading into
regions of scar (Fig. 1E).
All hearts with MI demonstrated wall motion abnormality (Fig.
2), while no sham demonstrated wall motion abnormality (11/11
vs. 0/5; P ⬍ 0.05). Significantly lower LV ejection fraction was
seen in post-MI rabbits compared with shams (37 ⫾ 9 vs. 59 ⫾
7%; P ⬍ 0.05). All other measurements were not significantly
different between the sham and MI groups (Table 2).
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Table 2. Inducibility of ventricular arrhythmias
In Vivo EP Study
Sham
MI
Ex Vivo Extra Stimuli
Protocol
Ex Vivo Ramp
Protocol*
VT/VF
No VT/VF
VT/VF
No VT/VF
VT/VF
No VT/VF
0
3
5
8
0
4
5
7
1
11
4
0
EP, electrophysiological; VT, ventricular tachycardia. *P ⬍ 0.05 for sham
vs. MI.
Fig. 3. Induction of monomorphic ventricular tachycardia (VT) from programmed
stimulation in in vivo electrophysiological
study. A: sinus rhythm. His activation (arrow) indicates His activation followed by
ventricular activation. B: programmed
stimulation caused VT. VT was induced by
S1-S2-S3-S4 protocol. Note that the ventricular depolarization on ECGs (dotted
line) precedes His activation (arrow), demonstrating a ventricular origin to the wide
complex monomorphic tachycardia.
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this region as the PZ in our optical mapping studies. This PZ is
distinct from the epicardial border zone seen in this model and
traditional models. We focused on the periphery of the infarct
and our defined PZ since in our optical mapping studies
described below, this region demonstrated conduction abnormality and reentry formation that was of interest.
Optical mapping of voltage and calcium transients using a
1.5 ⫻ 1.5 cm field of view showed excellent signal quality due
to the clean surface of the epicardium (Fig. 4). The remote zone
signals have normal appearing upstroke and calcium transient
similar to the signals seen in the LV free wall of the sham
hearts. However, PZ signals demonstrate lower amplitude and
lower signal to noise ratio, with slower rise in activation
upstroke. Signals were absent in the infarct zone (Fig. 4C).
Examples of epicardial activation maps are shown in Fig. 4D.
The infarct zone introduces new curvature to the activation
wavefront as demonstrated in the activation map (Fig. 5B,
black arrow). The extra-stimuli protocol mimicking in vivo
programmed stimulation was used and 4 of 11 hearts were
inducible for sustained ventricular arrhythmias. The arrhythmias began with single reentry beats that then typically lead to
ventricular fibrillation that was sustained. Three of the four
hearts were also inducible during in vivo EPS. With the ramp
protocol, all 11 of the post-MI hearts were inducible for
ventricular fibrillation. Only one of the five sham hearts was
inducible with the ramp protocol and none of the five were
inducible by the extra-stimuli protocol (Table 2). Reentry
formation appeared to occur at the basal border between the
normal LV and the infarct territory. We therefore focused on
this region in optical mapping studies rather than the traditional
epicardial rim borderzone. Figure 5 demonstrates reentry induction in a postinfarct heart. The map in Fig. 5A demonstrates
a peri-infarct region where a third extra stimulus generates
reentry formation with activation proceeding from region 1 to
region 4, with conduction block occurring between region 1
and 4. Optical signals in Fig. 5, right, show APs in positions
1– 4 during the train of S1-S2-S3-S4 stimuli (stimulus artifact at
the Fig. 5, top). Traces in Fig. 5, bottom right, are optical signal
from positions 1– 4. S4 correlates to the activation map in Fig.
5, left. S4 in traces is the fourth upstroke seen, indicated by the
gray arrow on traces. Activation progresses from position 1 to
position 4 and reenters position 1 again. The following beat is
a beat of ventricular tachycardia, followed quickly by degeneration to ventricular fibrillation in the following beats in the
traces. In Fig. 5B, the activation and AP duration maps for the
stimulation are shown. Activation between regions 1 and 4 are
initially smooth, without block. During S3, a large repolarization gradient occurs which allows for functioning line of block
to develop between position 1 and 4 and reentry. Notably,
maps of the complex activation and repolarization dynamics in
the peri-infarct zone are clearly delineated.
High resolution field of view optical mapping of the borderzone was performed to investigate detail activation patterns
along the heterogeneous cell population shown in the histology
images (see Fig. 1E) at the MI periphery. Due to amplification
of motion artifact, this mapping was performed with higher
concentration of blebbistatin (10 ␮M). An example is shown in
Fig. 6 of borderzone activation from epicardial pacing at
BCL ⫽ 350 ms from 3 ⫻ 3 mm2 field of view. ECG recordings
(Fig. 6A) near this region of borderzone demonstrated a late
fractionation in the QRS. A local activation map of the region
is shown in Fig. 6B. Activation in the region appears to have
two components, an initial rapid (estimated conduction velocity of 0.75 mm/ms) component (labeled 1 in yellow) from top
right to bottom left, aspect of the map (red to orange) and a
second late component (labeled 2 and 3 in yellow) at the center
of the map (white to green) progressing right to left after a
large delay initially (40 ms) requiring 38 ms to completely
traverse the 2-mm central area (estimated 0.05 mm/ms). Fluorescence recordings (Fig. 6C) from the regions 1– 4 exhibit a
multiple component upstroke, as shown in two peaks in the
first derivatives (red). Since the fluorescence signals are the
sum of multiple cells from the spatial resolution of 30 ⫻ 30
␮m2 and depth resolution of ⬃120 ␮m, multiple peaks suggest
multiple activations in different depth of heterogeneous tissue
in the border zone of MI. Figure 6D shows overlapped first
derivative of fluorescence signal in the region. Activation
wavefronts labeled 1, 2, and 3 are labeled demonstrating the
time lag between each activation wavefront.
Figure 1E shows histology images from an MI border zone.
The heterogeneous MI borderzone seen may be responsible for
the complex optical signals and the fractionated wave form in
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ECG. Understanding the activation and repolarization dynamics of these borderzone late activation sites may greatly enhance our understanding of post-MI arrhythmias.
DISCUSSION
We report the successful development of a minimally
invasive MI via targeted coil delivery to the obtuse marginal
artery with an exceptionally high rate of procedural survival
and an arrhythmogenic phenotype. Furthermore, we have
documented that this model successfully mimics echocardiographic, gross pathology, histology, and electrophysiology of human postinfarction heart. Of specific interest, is the
fact that using the same stimulation protocol and definitions
of arrhythmia initiation used in human trials, our cohort
demonstrated similar results as seen human trials of postinfarction risk stratification for SCD (5). The simultaneous use
of a 12-lead ECG and a decapolar intracardiac catheter, with
the ability to capture the His, allows us to demonstrate
without question the induction of monomorphic ventricular
tachycardia despite the 1:1 ventricular arrhythmias activation in these post-MI rabbits using the same stimulation
protocol used in humans. Finally, our technique leaves the
pericardium untouched, which enables access to an easily
identifiable infarct borderzone. The clean epicardium in our
model would allow for high quality optical images and high
resolution borderzone optical mapping. Much of the previous work on post-MI arrhythmias focuses on the epicardial
rim of healthy tissue remaining after a total occlusion of a
coronary artery (15, 21, 23, 48, 68). This same rim is present
in human transmural infarcts (57) as well. Still, from catheter ablation experience in humans, substrate allowing for
ventricular tachyarrhythmias is highly complex and likely
involve endocardial as well as epicardial rim borderzones
(53, 54, 59, 61, 63). The MI border with normal tissue at the
periphery of the infarct territory may also play an important role
in these arrhythmias. Noncontact mapping in human hearts has
suggested that functional reentry at the periphery of the MI may
play a role in early reentry formation and the initiation of these
arrhythmias (13, 58). The electrophysiology at this junction of
normal and infarcted tissue at this periphery is not as well studied.
Our model is well suited to study this region where noninfarcted
tissue meets a border with scar and surviving muscle fibers and
may therefore increase the knowledge base of ventricular arrhythmias post-MI. Further detailed analysis of the endocardial surface
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Fig. 4. Simultaneous optical mapping of voltage and calcium transients. A and B: isolated
heart MI seen in anterior and lateral view (LV
free wall). C: fluorescence signals from the LV
free wall of sham hearts and post-MI hearts.
Vm-CaT, voltage and calcium; BCL, pacing
basic cycle length of 190 ms. D: corresponding
activation and action potential duration (APD)
maps from C. Note that APD dispersion in MI
heart increased dramatically and the amplitude
of fluorescence signals are diminished in the
MI region. RZ, remote zone; IZ, infarct zone.
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NOVEL MI IN RABBITS
needs to be performed in this model along with transmural
investigations, as these surfaces may play a significant role in
arrhythmogenesis in this model as well.
Comparison to Previous Studies
We found one other report of coil embolization into a large
coronary artery in a closed-chest model of MI in a rabbit (17).
This prior study attempted to deliver the coil with a catheter sitting
at the ostium of the left main artery. In our hands, reproduction of
this protocol yielded a large perioperative mortality and difficulty
in delivering the coil into the left coronary. Of the four animals
attempted, two died during the procedure, one animal failed two
attempts at coil embolization (coil embolized into peripheral
circulation rather than the coronary), and one rabbit received a
successful embolization. This coil lodged in a small obtuse marginal branch that resulted in a small linear infarction at the lateral
border of the LV. We surmised that the prior closed-chest model,
not currently used by any group to our knowledge, lacks depend-
able results. In our experience, full catheter engagement into the
left main ostium predisposed the animal to left main artery spasm
and occlusion during the procedure and an intra-procedure death.
On the other hand, when the catheter sits just outside the ostium,
the coil can fail to embolize into the coronary. Finally, even if the
coil embolizes into the artery, its final position is left up to chance.
Our targeted delivery method is a novel method that targets
segmental artery. In this procedure, a guide catheter sits outside
the left main ostium and a floppy wire is advanced into a branch
of the left circumflex artery that is chosen to induce a reproducible
size infarction. A delivery inner catheter is then advanced to the
location of coil embolization, and there, the coil is delivered. This
method is associated with low perioperative mortality and ease of
coil delivery, potentially making this method of targeted coil
delivery more reproducible by multiple labs.
We also found one previous description of induction of
ventricular tachycardia in vivo in the rabbit postepicardial
coronary ligation MI (43). However, this study used up to five
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Fig. 5. Activation maps of reentry formation in
post-MI heart at the MI PZ. A, left: activation
map of functional reentry formation in the PZ
within the region of optical mapping. A, right,
top: we demonstrate the S1-S2-S3-S4 protocol
used to induce this arrhythmia. Traces at right
are optical signal from positions 1– 4. S4 correlates to the activation map at left. S4 in traces
is the fourth upstroke seen, indicated by the
gray arrow on traces. Activation progresses
from position 1 to position 4 and reenters position 1 again. The following beat is a beat of
VT, followed quickly by degeneration to ventricular fibrillation in the following beats in the
traces. B: activation (top) and APD (bottom)
maps of premature stimulation followed leading up to the functional reentry seen in the
activation map in A. Changes in activation and
APD in this PZ of the MI with introduction of
extra stimuli is seen. APD dispersion increased
dramatically in S3 premature beat, followed by
functional conduction block in S4 that causes
reentry formation.
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NOVEL MI IN RABBITS
extra stimuli in the inductions that resulted in ventricular
tachycardia with significantly shorter CL. Compared with human ventricular tachycardias (5, 33, 36, 52, 60, 62), this model
more closely resembles ventricular flutter. Here we report
monomorphic ventricular tachycardia with a CL similar to that
of humans post-MI with induction method that exactly duplicate human EPS. Therefore, this model of healed MI in the
rabbit is more arrhythmogenic than other healed MI rabbit
models. Furthermore, our rate of induction of ventricular arrhythmias is similar to rates seen in humans, ranging from
20 –30% with EPS using this same protocol (4). Finally, the
pericardium in previous investigations has had demonstrable
effects on hemodynamics and therefore its instrumentation
may result in ventricular remodeling (27, 28). Such remodeling
would add a confounder to investigations of effects of remodeling on arrhythmias in the healed MI. Thus we believe that the
study of arrhythmias seen in our model is more likely applicable to the human postinfarct condition compared with the
open-chest models.
Previous optical mapping studies of post-MI rabbit hearts
have been published (12, 38, 55, 56) all using epicardial
coronary ligation open-chest techniques. However, high resolution detailed analyses of activation and repolarization at the
site of reentry formation and high resolution optical mapping
of late activation correlating with fractionated ECGs have not
been reported,. While earlier studies have clearly improved our
Table 3. Echocardiographic findings
Sham
MI
LV Septal Wall Thickness
in Diastole
LV Posterior Wall Thickness
in Diastole
LV End-Diastole
Diameter
LV End-Systole
Diameter
LV EF, %*
2.7 ⫾ 0.1
2.7 ⫾ 0.1
2.6 ⫾ 0.2
2.5 ⫾ 0.7
15 ⫾ 2.2
16 ⫾ 2.2
11 ⫾ 1.7
12 ⫾ 1.7
59 ⫾ 7
37 ⫾ 9
Values are means ⫾ SE. EF, ejection fraction. *P ⬍ 0.05 for sham vs. MI.
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Fig. 6. High resolution mapping of late activation from the MI. A: fractionated ECG indicating multiple late activations through MI region. B: activation maps
of late activation in MI border zone. Field of view was set to 3 ⫻ 3 mm2, 30 ⫻ 30 ␮m2 pixel resolution, at 5,000 f/s. C: fluorescence recordings from single
pixel shows multiple component of depolarization indicating multiple activation through the border zone (see text for details). First derivatives of fluorescence
recordings are shown in red. D: superimposed first derivatives along the activation pathways exhibit rapid, normal activation initially through the region followed
by 2 distinct late activations, which correlates with timing of deflections in the corresponding ECG recordings in the A. E: histology images of MI border zone.
Finger like fibrosis into ventricular tissue may be responsible for complex activation along the border zone.
Innovative Methodology
NOVEL MI IN RABBITS
4.
5.
6.
7.
8.
9.
10.
11.
12.
CONCLUSION
We present here a novel technique of targeted coil embolization closed-chest MI in the rabbit, which demonstrates low
mortality periprocedure, applicability to human post-MI arrhythmias, and is ideal for high resolution optical mapping
techniques due to the pristine nature of the epicardium after MI
healing. Use of this technique by this group and other investigators may further our understanding of the crucial activation
and repolarization dynamics leading to post-MI arrhythmias
and SCD Table 3.
13.
14.
15.
16.
ACKNOWLEDGMENTS
We thank Zachary Pfeiffer for work on data gathering and analysis for this
manuscript.
Present address for O. Ziv: Case Western Reserve, MetroHealth Medical
Center, Cleveland, Ohio.
17.
18.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
19.
AUTHOR CONTRIBUTIONS
20.
Author contributions: O.Z., B.-R.C., and G.K. conception and design of
research; O.Z., L.S., E.L., L.C., D.P., P.J., X.P., and B.-R.C. performed
experiments; O.Z., L.S., E.L., L.C., D.P., P.J., and B.-R.C. analyzed data; O.Z.,
E.L., B.-R.C., and G.K. interpreted results of experiments; O.Z., L.C., P.J., and
B.-R.C. prepared figures; O.Z. drafted manuscript; O.Z., B.-R.C., and G.K.
edited and revised manuscript; O.Z., B.-R.C., and G.K. approved final version
of manuscript.
21.
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