SUPPLEMENTAL MATERIAL
1
SUPPLEMENTAL METHODS
hiPSC derivation and cardiomyocyte differentiation. All the protocols for this study were
approved by the Stanford University Human Subjects Research Institutional Review Board.
Written consent was obtained from the study participant. Dermal fibroblasts were obtained by
incubating freshly isolated skin punch biopsies from a normal, healthy 14-year old male subject
in collagenase II (1 mg/ml in Dulbecco’s modified Eagle medium (DMEM), Invitrogen,
Carlsbad, CA) followed by culture in DMEM supplemented with 10% FBS (Invitrogen).
Cultures were passaged three times with TrypLE (Invitrogen) before lentiviral infection and
characterization as described previously 1. Colonies fulfilling established “stemness” criteria
were induced toward the cardiomyocyte lineage using standard 3D embryoid body (EB)
differentiation protocols and maintained in a 5% CO 2 /air environment as described previously 1.
Differentiation of hiPSC-CMs from iPSC monolayers: Colonies fulfilling established
“stemness” criteria were also differentiated into cardiomyocytes using a 2D iPSC monolayer
differentiation protocol and maintained in a 5% CO 2 /air environment. Briefly, on day 0 hiPSC
colonies were dissociated with Accutase (Sigma) into a single cell suspension and resuspended
in E8 media (Invitrogen). Roughly 100,000 cells were then replated into one well of a 6 well dish
pre-coated in Matrigel (Invitrogen), taking care to ensure that single iPSCs were evenly
dispersed in the dish. iPSC monolayers were cultured for 3-4 days until ~95% confluency was
achieved. For the next 5 days, cells were treated with an activator and then a subsequent inhibitor
of Wnt pathway signaling as described previously 2. Cells were then placed on RPMI + B27 with
insulin (Invitrogen) until beating was observed.
2
Adult heart tissue preparation. Myocardial tissue was obtained from a 21-year old male organ
donor. The tissue was transported to the laboratory within 2 hrs of procurement in ice-cold UW
(University of Wisconsin) solution. The tissue was then immediately transferred to ice-cold
modified Tyrodes solution (MTS) for further processing 3. The tissue samples from all four heart
chambers were cut into 1 cm2 pieces and preserved with RNAlater (Invitrogen).
Single-cell microfluidic RT-PCR. Single beating CMs were picked 30 days post induction
using a 10 uL pipettor and introduced into separate PCR tubes for reverse transcription - cDNA
amplification as previously described
4, 5
. For specific primers, see Supplemental Table 1.
Amplified cDNA was loaded into Biomark 48.48 Dynamic Array chips for analysis with
BioMark Real-Time PCR Analysis software (Fluidigm, South San Francisco, CA).
RNA isolation, reverse transcription, and real-time PCR. Tissues were cut into millimeter
size pieces and incubated in Qiazol (Qiagen, Valencia, CA). Tissue was homogenized with the
TissueRuptor (Qiagen) handheld homogenizer for isolation of total RNA with the miRNeasy kit
(Qiagen). Total RNA was eluted in water and stored at −80°C. RNA concentration was measured
using a Nanodrop (Thermo Scientific). cDNA was synthesized using High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, foster City, CA) and Real-time PCR was done
with Taqman assays (Applied Biosystems) using StepOne (Applied Biosystems).
Immunofluorescence and laser confocal microscopy. Beating EBs were incubated in Accutase
for 1 hr followed by TrypLE for 5 minutes followed by mechanical dissociation using a 200 μL
pipettor and plated on 0.1-0.2% gelatin-coated glass coverslips. Primary antibodies consisted of
mouse anti-human sarcomeric alpha-actinin (Sigma-Aldrich, St. Louis, MO) and rabbit anti3
human TNNT2 (Thermo Scientific, Barrington, IL). Chicken and anti-mouse Alexa 488 chicken
anti-rabbit Alexa 594 (Invitrogen) were used as secondary antibodies. Imaging was performed
using a DM IL LED inverted tissue culture microscope running LEICA LAS software with either
a 5x N-Plan Phase objective and a 10x Plan Fluotar phase objective or an A1-R Resonant
Confocal System running NIS Elements: C software on a Ti-E PFS microscope with a Plan Apo
VC 60x Oil objective.
Video contractility analyses. Movement was quantified using a cross-correlation algorithm
implemented in a custom MATLAB script, which measured pixel displacements of contracting
cells 6. For each video frame, the mean magnitude of displacement was measured. The frame in
which maximum movement was observed was considered the frame of maximum contraction
and used for all subsequent calculations. Regions exhibiting movement above this threshold were
considered to be contracting.
Single cell patch-clamping. Coverslips were transferred to an RC-26C recording chamber
maintained at a constant 36-37°C by a TC-324B heating system (Warner Instruments, Hamden,
CT) mounted on the stage of a TE2000-U inverted microscope (Nikon, Tokyo, Japan). APs were
recorded from single beating CMs using whole-cell current-clamping techniques with an EPC-10
amplifier and PatchMaster software (HEKA, Lambrecht, Germany). Recordings were conducted
in Tyrode’s solution composed of 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 10 mM glucose,
1.8 mM CaCl 2 , and 10 mM HEPES; pH was adjusted to 7.4 with NaOH. The pipette solution
contained 120 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 3 mM Mg-ATP, and 10 mM EGTA; pH
was adjusted to 7.2 with KOH. Data were analyzed using PulseFit (HEKA), Igor Pro
4
(Wavemetrics, Portland, OR), Origin 6.1 (Microcal, Northampton, MA), and Prism (Graphpad,
La Jolla, CA). An APD of 300 ms was used as the cutoff point for distinguishing ventricularlike CMs from atrial- and nodal-like cells. Maximal diastolic potential above -45 mV was the
criterion used to discern nodal-like cells from atrial- and ventricular-like cells. Action potential
amplitude (APA) was used to further classify our hiPSC-CM. Nodal-like cells usually had APAs
below 70 mV and atrial and ventricular-like cells’ APAs were between 95 and 110 mV.
Microelectrode array recordings and analyses. Baseline electrophysiology studies were
performed from 35 days post-differentiation and pharmacological experiments were performed
65-95 days post-differentiation. All experiments were performed in DMEM without FBS or
antibiotics. Microenvironmental control was provided via feedback temperature controllers
powering two resistive heating pads beneath the MEA probe and a humidifying warmer (Warner
Instruments, Hamden, CT) for the carbogen gas supply to keep the preparation at 35.8 to 37.5°C.
Interspike intervals (ISI) and FP waveform data were extracted offline from the channel with the
clearest waveform for determining the FPD in each individual EB quadrant using Mobius QT
and saved as CSV files. Instantaneous beat rate was calculated dividing the ISI (in milliseconds)
by 1000 and multiplying by 60. Waveform data was imported into IGOR Pro for FPD, FPA, and
maximal velocity of depolarization (Vmax, mV/ms) measurement, as well as fitting to the Hill
equation.
Assay media and drug formulations. Pharmacological studies were performed in DMEM
(Invitrogen) without phenol red or FBS. All chemicals were purchased from Sigma-Aldrich.
Stock solutions were prepared daily in water at 1-10 mM (1000x the highest concentration to
5
test) and serially diluted in medium. Dose-response experiments were performed by adding 0.4
to 2 μL of 1000x incremental drug concentrations to the 2 mL volume in the MEA probe for 10
minutes at each dose.
6
SUPPLEMENTAL FIGURE LEGENDS
Supplemental Figure 1: MEA probe, plated embryoid bodies, and MEA system setup. A,
Gelatin-coated MEA probe (left) patterned with four separate 4x4 electrode grids each plated
with a beating EB (right). Arrow points to the probe in the complete MEA setup. B, The MEA
setup consisting of the PC/Monitor, MED64 amplifier, MED connector and AL-MEDP5004A
MEA probe covered by a humidifying chamber. On the left is the microenvironmental control
equipment consisting of feedback temperature controllers and humidifying warmer module,
which is connected to a carbogen tank (not shown). The probe is warmed by two resistive
heating pads underneath. Note the distinct waveforms and independent beating frequency in each
quadrant.
Supplemental Figure 2: Gene expression of sarcomeric proteins. Cardiac-specific structural
genes encoding sarcomeric myofilament proteins expression levels demonstrate presence of
ACTC1 (alpha cardiac muscle actin 1), MYH6 (myosin heavy chain 6), MYH7 (myosin heavy
chain 7) and MYL2 (myosin regulatory light chain 2) at 3 different timepoints representing 20
(1), 30 (2), and 40 (3) days post-induction.
Supplemental Figure 3: Immunostainings of sarcomeric proteins from monolayer-derived
iPSC-CMs. A, Brightfield (BF) imaging of monolayer-derived iPSC-CMs. B- C, Alpha-actinin
(green) and TNNT2 (red) immunostainings demonstrates the presence of cardiac specific
sarcomeric proteins. D, Merged imaging of B and C. The inset shows organized horizontal
myofilaments with parallel red striations indicating the sarcomeric Z-lines. DAPI (blue) indicates
nucleus.
7
Supplemental Figure 4: hiPSC-CM subtypes identified via single-cell patch-clamping.
Representative waveforms for the 3 CM subtypes. Cells with ventricular-like APs were the most
frequently encountered, comprising 67% of all tested hiPSC-CMs, followed by atrial-like cells
(28%), and nodal-like cells (5%).
Supplemental Figure 5: Stability of baseline MEA recordings. A, 10-min baseline MEA
recordings from hiPSC-CMs derived using the EB method. The beating frequencies changed
very little within the 10-min time course. B. Left panel, magnified view of the first minute
recording as indicated by the black line in Panel A. Right panel, magnified view of the final
minute recording as indicated by red line in Panel A. Each field potential (FP) changed little
from the first minute to the last minute within the 10-min baseline recording.
Supplemental Figure 6: Baseline MEA recordings from embryoid body-derived hiPSC-CM
and monolayer-derived hiPSC-CM techniques. A, Example of EB-derived hiPSC-CMs
attached to and cultured on a MEA chip. B, Example of monolayer-derived hiPSC-CMs attached
to and cultured on a MEA chip. C, Representative baseline MEA recording trace from EBderived hiPSC-CMs. D. Representative baseline MEA recording trace from monolayer-derived
hiPSC-CMs.
Supplemental Figure 7: Comparison of key parameters of baseline MEA recording
between EB and monolayer-derived iPSC-CMs. A, Quantification of BPM between EBderived hiPSC-CMs and monolayer-derived hiPSC-CMs. B, Quantification of FPD between EBderived hiPSC-CMs and monolayer-derived hiPSC-CMs. C, Quantification of cFPD between
8
EB-derived hiPSC-CMs and monolayer-derived hiPSC-CMs. There is no significant change in
BPM, FPD or cFPD between EB-derived hiPSC-CMs and monolayer-derived hiPSC-CMs.
Supplemental Figure 8: Isoproterenol causes sustained increases in hiPSC-CM beat
frequency. A, Graph plotting the timecourse of beat frequency response to 5 nM ISO in a
representative EB. B, Steady-state concentration dependent increases on absolute beating
frequency. Note the stability of the response during the last 30 seconds of a 10 minute recording.
C, Rhythmic spontaneous field potentials at baseline during the last 30 seconds of a 10 minute
recording. D, Effect of 1 μM isoproterenol on beat frequency.
Supplemental Figure 9: Isoproterenol induces dose-dependent increases in BPM and
decreases in FPD. A, The dose-dependent effect of ISO on beat rate could be fitted well with
the Hill equation (red line), yielding a half-maximal excitatory concentration of 72.03 ± 16.80
nM (n=4). B, Representative traces illustrating the dose-dependent shortening of FPD secondary
to ISO. C, Graph plotting ISO concentration-FPD response and fit to the Hill equation (red line
connecting the datapoints, n=3) illustrating that beat rate is an important variable affecting the
QT interval. Arrow points to the left-shifting peak of the repolarizing wave to indicate shortening
of the FPD. D, ISO concentration vs FPD corrected according to the Bazett formula (known to
overcorrect at high beat frequencies as can be seen here) and Hill fit.
Supplemental Figure 10: Beat frequency decreases due to cholinergic stimulation. A,
Rhythmic spontaneous field potentials at baseline during the last 30 seconds of a 10 minute
recording. B, Effect of 100 μM carbachol on beat frequency after 9.5 minutes of exposure. C,
9
“Steady-state” concentration dependent decreases on absolute beating frequency. Note the
stability of the response during the last 30 seconds of a 10 minute recording. D, CCh had a dose
dependent effect on beat rate regardless of prior adrenergic stimulation.
Supplemental Figure 11: Carbamyl choline increases field potential duration and
amplitude. A, As expected, CCh also increases FPD in a dose-dependent manner due to the
decrease in beat frequency. Arrow points to the right-shifting peak of the repolarizing wave to
indicate prolongation of the FPD. B, CCh also increases FPA in a dose-dependent manner, as
this zoomed in view of the FPs in Supplemental Figure 6A demonstrates. Arrow points to the
increasing amplitude of the fast depolarizing spike.
Supplemental Figure 12: Sotalol-induced FPD prolongations and EADs in monolayerderived hiPSC-CMs. A. Graph plotting sotalol concentration-FPDc response and fit to the Hill
equation (n=3). B. Representative trace showing 100 μM sotalol-induced EADs (indicated by red
arrow) in monolayer-derived iPSC-CMs. C, Waveform of expanded timescales demonstrating
the EAD indicated by the box in Panel B.
Supplemental Figure 13: hERG block by quinidine prolongs FPD and reduces FPA/Vmax.
A, Dose-dependent prolongation of a representative EB’s field potentials caused by quinidine.
Arrow points to the right-shifting peak of the repolarizing wave to indicate prolongation of the
FPD. B, Quinidine also had a dose-dependent effect on FPDc that was statistically significant at
100 nM and physiologically significant at 300 nM and higher concentrations, as indicated by the
asterisks. C, Quinidine also decreases FPA and Vmax in a dose-dependent manner. Arrows point
10
to the decreasing amplitude of the fast depolarizing spike. D, Hill fits to the dose-dependent
effect of lidocaine on FPA and Vmax.
Supplemental Figure 14: Incidence of drug-induced arrhythmias is consistent with animal
models. A, The incidence of sotalol-induced arrhythmias at a concentration of 100 µM was most
similar to Shimizu & Antzelevitch’s dog left ventricle (LV) wedge model and Abi-Gerges et al.’s
isolated dog mid-myocardial cardiomyocyte data. B, The incidence of quinidine-induced
arrhythmias at a concentration of 1 µM was close to Roden & Hoffman’s and Davidenko et al.’s
dog Purkinje fiber model. Columns in red indicate rabbit studies.
Supplemental Figure 15: Verapamil causes a dose-dependent decrease in BPM and FPA. A,
Verapamil abolished spontaneous electrical activity at high doses. “Older” EBs were more
resistant to verapamil’s effect on spontaneous beating with an IC50 and Hill coefficient 4 times
greater at 82 days post-induction as compared to 30 days post-induction. B, Verapamil
dramatically decreased hiPSC-CM EB’s FP amplitude in a concentration-dependent manner,
suggesting calcium currents have a pivotal role in the generation of spontaneous electrical
activity. Arrow points to the decreasing amplitude of the fast depolarizing spike.
Supplemental Figure 16: Effect of calcium and sodium channel block on FPDc, BPM,
FPA, and Vmax. A, Nifedipine induced a dose-dependent shortening in FPD, as shown in this
representative EB. Arrows point to the reduction of the fast depolarizing spike amplitude and
left-shifting peak of the slow repolarizing wave to indicate shortening of the FPD. B, Nifedipine
also caused a dose-dependent reduction in beat frequency and FPD, similar to verapamil.
11
Asterisks denote the shortening in FPDc was statistically and physiologically significant at 10
nM, consistent with the effect on ESC-CM APD. At 300 μM, all EBs stopped beating. C,
Lidocaine caused reduction of FP amplitude and FPD prolongation with increasing doses.
Arrows point to the reduction of the fast depolarizing spike amplitude and right-shifting peak of
the slow repolarizing wave to indicate prolongation of the FPD. D, Normalized Vmax and FPA
were also reduced by lidocaine in a dose-dependent manner. Asterisks note the reduction in
Vmax was statistically and physiologically significant at concentrations of 10 uM and higher.
Supplemental Figure 17: Dose-dependent FPDc shortening by nifedipine in monolayerderived hiPSC-CMs. Graph plotting nifedipine concentration-FPDc response and fit to the Hill
equation (n=3).
12
Supplemental Movies
Supplemental Movie 1. Movie of a beating EB (left), dynamic heatmap showing the area and
levels of contractility (middle), and graph plotting the contraction over time (right). Videos were
recorded and analyzed at 100fps. In this video, every fourth frame is displayed in real time for
ease of viewing.
Supplemental Movie 2. Movie of the electrical activity recorded from 4 separate EBs plated on
one MEA probe. The screen's 4 quadrants demonstrate independent beating and distinct
waveforms. Recording was approximately 15 fps and playback speed 29 fps.
Supplemental Movie 3. Movie showing EB beating at top left MEA grid. Recording speed was
approximately 15 fps and playback speed is 29 fps.
Supplemental Movie 4. Movie showing EB beating at top right MEA grid. Recording speed was
approximately 15 fps and playback speed is 29 fps.
Supplemental Movie 5. Movie showing EB beating at bottom right MEA grid. Recording speed
was approximately 15 fps and playback speed is 29 fps.
Supplemental Movie 6. Movie showing EB beating at bottom left MEA grid. Recording speed
was approximately 15 fps and playback speed is 29 fps.
Supplemental Movie 7. Movie showing monolayer beating at MEA grid. Recording speed was
approximately 15 fps and playback speed is 29 fps.
13
SUPPLEMENTAL REFERENCES
1.
Sun N, Panetta NJ, Gupta DM, Wilson KD, Lee A, Jia F, Hu S, Cherry AM, Robbins RC,
Longaker MT, Wu JC. Feeder-free derivation of induced pluripotent stem cells from
adult human adipose stem cells. Proc Natl Acad Sci U S A. 2009;106:15720-15725
2.
Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp
TJ, Palecek SP. Robust cardiomyocyte differentiation from human pluripotent stem cells
via temporal modulation of canonical wnt signaling. Proc Natl Acad Sci U S A.
2012;109:E1848-1857
3.
Brandenburger M, Wenzel J, Bogdan R, Richardt D, Nguemo F, Reppel M, Hescheler J,
Terlau H, Dendorfer A. Organotypic slice culture from human adult ventricular
myocardium. Cardiovasc Res. 2012;93:50-59
4.
Narsinh KH, Plews J, Wu JC. Comparison of human induced pluripotent and embryonic
stem cells: Fraternal or identical twins? Mol Ther. 2011;19:635-638
5.
Narsinh KH, Sun N, Sanchez-Freire V, Lee AS, Almeida P, Hu S, Jan T, Wilson KD,
Leong D, Rosenberg J, Yao M, Robbins RC, Wu JC. Single cell transcriptional profiling
reveals heterogeneity of human induced pluripotent stem cells. J Clin Invest.
2011;121:1217-1221
6.
Santiago JG, Wereley ST, Meinhart CD, Beebe DJ, Adrian RJ. A particle image
velocimetry system for microfluidics. Experiments in Fluids. 1998;25:316-319
6.
Shimizu W, Antzelevitch C. Sodium channel block with mexiletine is effective in
reducing dispersion of repolarization and preventing torsade des pointes in lqt2 and lqt3
models of the long-qt syndrome. Circulation. 1997;96:2038-2047
7.
Chen X, Cordes JS, Bradley JA, Sun Z, Zhou J. Use of arterially perfused rabbit
ventricular wedge in predicting arrhythmogenic potentials of drugs. J Pharmacol Toxicol
Methods. 2006;54:261-272
8.
Abi-Gerges N, Ji GJ, Lu ZJ, Fischmeister R, Hescheler J, Fleischmann BK. Functional
expression and regulation of the hyperpolarization activated non-selective cation current
in embryonic stem cell-derived cardiomyocytes. J Physiol 2000;523 Pt 2:377-89.
9.
Roden DM, Hoffman BF. Action potential prolongation and induction of abnormal
automaticity by low quinidine concentrations in canine purkinje fibers. Relationship to
potassium and cycle length. Circ Res. 1985;56:857-867
10.
Davidenko JM, Cohen L, Goodrow R, Antzelevitch C. Quinidine-induced action
potential prolongation, early afterdepolarizations, and triggered activity in canine
purkinje fibers. Effects of stimulation rate, potassium, and magnesium. Circulation.
1989;79:674-686
11.
Wu L, Guo D, Li H, Hackett J, Yan GX, Jiao Z, Antzelevitch C, Shryock JC, Belardinelli
L. Role of late sodium current in modulating the proarrhythmic and antiarrhythmic
effects of quinidine. Heart Rhythm. 2008;5:1726-1734
14
Supplemental Table 1: Genes screened in adult human heart samples and hiPSC-CMs
Gene
Protein
Gene
SCN5A
Nav 1.5 α subunit
ADRA1A β -1 adrenoreceptor
SCN1B
Nav 1.5 β-1 subunit
ADRB1
β -2 adrenoreceptor
CACNA1C
Cav1.2, α-1 subunit
ADRB2
α -1A adrenoreceptor
CACNA1H
Cav 3.2, α-H subunit
HCN1
Pacemaker channel
HCN2
Pacemaker channel
CACNA2D1 Cav1.2, α-2/delta-1 subunit
Protein
CACNB1
Cav1.2, β-1 subunit
HCN4
Pacemaker channel
CACNB2
Cav1.2, β-2 subunit
CFTR
Cystic Fibrosis Transmembrane Regulator
KCNA4
Kv1.4 α subunit
CLCN1
CLC-1
KCNA5
Kv 1.5 α subunit
CLCN2
CLC-2
KCNAB1
Kv 1.5 β -1 subunit
CLCN3
ClC-3
KCNAB2
Kv 1.5 β -2 subunit
GJA1
Connexin 43, gap junction
KCND3
Kv 4.3
GJC1
Connexin 45, gap junction
KCNE1
minK
SLC8A1
Na/Ca exchanger
KCNE2
MiRP1
ATP2A2
SERCA 2A
KCNH2
Kv 11.1 (KvLQT2, hERG)
RYR2
Ryanodine receptor
KCNH2
Variant (3) of hERG1b
TNNT2
Cardiac troponin T type 2
KCNIP1
Kv channel-interacting protein
ACTC1
Actin, α, cardiac muscle 1
KCNIP2
Kv channel-interacting protein
MYH6
Myosin heavy chain, α
KCNJ2
Kir 2.1
MYH7
Myosin heavy chain, β
KCNQ1
Kv 7.1 (KvLQT1)
MYL2
Myosin regulatory light chain 2
15
A
B
Supplemental Figure 1
Relative expression/GAPDH
2
1
0
0.0
1
2
1
Supplemental Figure 2
3
MYH7
1.5
1.0
0.5
2
3
Relative expression/GAPDH
3
Relative expression/GAPDH
Relative expression/GAPDH
ACTC1
MYH6
12
10
8
6
4
0
1
1
2
2
3
MYL2
10
8
6
4
2
3
B
A
50 μM
C
D
Actinin/TNNT2/DAPI
Supplemental Figure 3
Ventricular-like
Atrial-like
Nodal-like
80mV
0mV
200ms
Supplemental Figure 4
A
B
The first 60s baseline recording
Supplemental Figure 5
The last 60s baseline recording
A
hiPSC-CMs, EB
B
hiPSC-CMs, Monolayer
C
hiPSC-CMs, EB
D
hiPSC-CMs, Monolayer
Supplemental Figure 6
A
B
Supplemental Figure 7
C
A
B
5 nM ISO
C
Baseline
Supplemental Figure 8
D
ISO 1µM
A
B
C
D
Supplemental Figure 9
A
Baseline
C
Supplemental Figure 10
B
D
CCh 100µM
A
Supplemental Figure 11
B
A
B
100 μM Sotalol, hiPSC-CMs, monolayer
Supplemental Figure 12
C
A
B
C
D
Supplemental Figure 13
A
B
Sotalol-induced % Arrhythmia
Quinidine-induced % EAD
100
100
90
90
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
Author: Assay
Shimizu & Antzelevitch: dog LV wedge6
Chen et al: rabbit LV wedge7
Abi-Gerges et al: dog midmyocardial CM8
Supplemental Figure 14
Author: Assay
Roden & Hoffman: dog Purkinje fiber9
Davidenko et al: dog Purkinje 10
Wu et al: Langendorrf perfused rabbit heart11
A
Supplemental Figure 15
B
A
B
C
D
Supplemental Figure 16
Supplemental Figure 17