Supplemental Material
Expanded Methods and Results
Statistical analysis
All results are presented as mean±SEM. Normality and equality of variances of data sets were
first tested using Kolmogorov-Smirnov and Levene's tests, respectively. If both were confirmed,
t-test or analysis of variance followed by Bonferroni 's post hoc test were used for determination
of statistical significance; if either normality or equality of variances was not assured,
nonparametric tests (Wilcoxon test or Kruskal-Wallis test followed by Dunn's post-test) were
applied (SPSS II, SPSS Inc., Chicago, Illinois). No preliminary data were available for a power
analysis. Experiments were planned with a sample size of 4 animals per group as an initial pilot
project. Results from the pilot project allowed us to power subsequent studies.
Echocardiography
Echocardiographic studies were performed two days before (Baseline) and 3 weeks, 2 and 3
months after surgery using the Vevo 770 Imaging System (VisualSonics, Toronto, Canada).1
After induction of light general anesthesia, the heart was imaged at the level of the greatest LV
diameter. LV ejection fraction (LVEF) was measured with VisualSonics version 1.3.8 software
from 2-dimensional long-axis views. Echocardiographic studies were read independently by two
investigators blinded to treatment allocation, and the averages of results in each mouse were used
for statistical analysis. Changes in left ventricular (LV) end diastolic and systolic volumes:
CDC transplantation resulted in a sustained improvement of LV end-diastolic (LV EDV) and
end-systolic (LV ESV) volumes in Gαq mice, relative to placebo. This was true whether CDCs
were injected at 6, 8 or 11 weeks of age (Suppl. Fig. 1). Preservation of LV EDV and LV ESV
1
was sustained in CDC-treated animals for at least 3 months. CDCs also resulted in sustained
preservation of LV anterior and posterior wall thickness for at least 3 months, in contrast to the
findings in placebo-treated animals (Suppl. Table 1 & Suppl. Fig. 2).
For measurement of baseline LV anterior and posterior wall thickness, harvested hearts from 6-,
8-, and 11-week old control (CTL; wild type; n= 4-5) and Gαq mice (n= 4-5) were paraffin
embedded and sectioned transversely at middle part (2 mm from apex; 100 µm interval). 10
collected sections were, subsequently, H&E stained and applied for measurement of wall
thickness using Leica SCN 400 F Slide Scanner and SlidePath Gateway Software (Leica
Biosystems, Buffalo Grove, IL). Measurements were averaged for each heart. The same method
was applied for measurement of month 3 LV anterior and posterior wall thickness in vehicle- and
CDC-treated Gαq mice (n=6-8) 3 months after treatment and in control mice (n=6-8) at 18-, 20and 23- weeks of age (3 months after 6-, 8-, and 11-weeks of age). CDC- and vehicle-treated
Gαq mice were treated at 6-, 8-, and 11-weeks of age.
Expansion of CDCs
Briefly, ventricular tissues were minced into ~1 mm explants, partially digested enzymatically
and plated on adherent (fibronectin-coated) culture dishes. These explants spontaneously yield
outgrowth cells (explant-derived cells) which were harvested once confluent and plated in
suspension culture (105 cells/mL on poly-D-lysine–coated dishes) to enable the self-assembly of
three-dimensional cardiospheres. Subsequent replating of cardiospheres on adherent culture
dishes yielded CDCs which were used in all experiments at passage one.
2
CDC transplantation
To optimize the process of CDC transplantation, preliminary dose-response experiments were
performed, which identified 1x105 cells as an effective dose, consistent with prior dose-ranging
experiments in an ischemic mouse model2. A total of 1x105 cells/40µL phosphate-buffered saline
(PBS) or PBS alone was injected into left ventricular (LV) myocardium at 4 points as described3
with some modifications. The LV was visually divided into three zones: basal, middle, and
apical, with one injection in the basal, two in the middle and one in the apical zone. Six-, eightand eleven-week old Gαq mice were injected with CDCs (Gq+CDC, n=6-8) or vehicle (placebo:
Gq+PBS, n=6-8) during open-chest thoracotomy via a 28 ½ gauge-needle. All surgical
procedures were carried out while the animals were under general anesthesia (Dexmedetomidine
(0.5mg/kg)/Ketamine (75mg/kg); IP; once before surgery). Six-, eight- and eleven-week old
FVB/NJ wild type mice served as control (CTL, n=6-8). At the conclusion of each observation
period (3 weeks or 3 months after injection), the animals were sacrificed and the hearts
harvested.
Assessment of engraftment by real-time polymerase chain reaction: Female Gαq mice were
enrolled for this assessment. Quantitative polymerase chain reaction (PCR) was performed 1, 3
and 6 weeks after cell injection to assess cell engraftment. Male CDCs were injected to enable
detection of the SRY gene located on the Y chromosome as a marker of engraftment using the
TaqMan assay (Applied Biosystems, Foster City, CA).4 The whole mouse heart was harvested,
weighed, and homogenized. A standard curve was generated with multiple dilutions of genomic
DNA isolated from the injected CDCs. All samples were spiked with equal amounts of genomic
DNA from non-injected mouse hearts as a control. For each reaction, 50 ng of genomic DNA
was used. Real-time PCR was performed in triplicate. Engraftment was quantified from the
3
standard curve. Percentage engraftment of CDCs at 1 week was ~10% and <1% at 3 weeks. By 6
weeks, no surviving CDCs could be detected (Suppl. Fig. 3). For assessment of distribution of
CDCs within the heart after injection, male CDCs were injected into apex of female hearts to
enable detection of the SRY gene, as a marker of the injected CDCs, in the upper half of the
heart (basal part). The harvested hearts were bisected in short axis and the upper and lower
halves (basal and apical, respectively) were weighed, homogenized and used for real-time PCR.
Real-time PCR was performed 1, 3 and 7 days after cell injection. CDCs were detectable in the
basal part of heart at both days 1 and 3 after cell injection, denoting spread of CDCs within the
heart. The quantity of CDCs detected in the basal half of the heart was roughly equal to that in
the apical half, where the CDCs had been injected. At day 3 the quantity of detectable CDCs was
lower at both basal and apical parts compared to day 1. While there were still detectable CDCs in
the apical part of the heart at day 7, none survived in the basal part, highlighting the indirect
nature of the mechanisms of action.
Cardiomyocyte isolation, hydrogen peroxide stress and conditioned media:
Neonatal rat cardiomyocytes (NRCMs) were isolated from 1- to 2-day-old pups and cultured as
monolayers as described previously5. A monolayer of NRCMs was incubated with serum-free
basal media (IMDM; Life Technologies, Grand Island, NY) and CDC- and cardiac fibroblastconditioned media for 24 hours before exposure to oxidizing agent, hydrogen peroxide (10µM;
30 minutes). CDCs and cardiac fibroblasts were isolated from Sprague-Dawley rat hearts and
passaged for preparation of conditioned media. Conditioned Media: 1 million CDCs or cardiac
fibroblasts were incubated with serum-free basal media (1 mL, Iscove's Modified Dulbecco's
Medium: IMDM; Life Technologies, Grand Island, NY) for three days and, subsequently, were
applied for preconditioning of NRCMs (Suppl. Fig. 5).
4
MMP9 activity assay:
The activity level of endogenous active MMP9 was measured in the heart tissues of wild type
mice (n=4) and CDC- and vehicle-treated Gαq mice 1 day and 1 week after treatment (n=6),
Suppl. Fig. 6. The enrolled mice were at 8 weeks of age. Whole heart tissues were washed with
cold PBS and homogenized in extraction buffer (pH 5.0) containing 10 mM cacodylic acid, 150
mM NaCl, 20 mM ZnCl2, 1.5 mM NaN3, and 0.01% Triton X-100, as described.6 The
homogenates were centrifuged at 800 X g for 10 min at 4 °C, and the supernatants were collected
and stored at -20 until used. The final protein concentrations of myocardial extracts were
determined using Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL). The
activity level of endogenous active MMP9 in myocardial extracts (300 µg of protein) was
measured using the QuickZyme Mouse MMP-9 activity assay (QuickZyme BioSciences,
Netherlands).
In vitro cytokine release
Among the assessed cytokines, there was marked expression/release of VEGF, VEGF R2,
VEGF R3, SDF-1α, HGF, and IL-10 in CDC conditioned media relative to cardiac fibroblast
conditioned media. Given the major role of these cytokines in angiogenesis, vasculogenesis,7, 8
lymphangiogenesis, neurogenesis,9 stem cell recruitment,10 inhibition of inflammation and
cardiac remodeling,11 prevention of cardiac fibrosis and improvement of heart function,12, 13 this
result heightens the paracrine effects of CDCs as one of their indirect mechanisms for induction
of their effects (Suppl. Fig. 7). Conditioned Media and cytokine array: 1 million CDCs
(passage one) and mouse cardiac fibroblasts (ScienCell research Laboratories, Carlsbad, CA)
were incubated with serum-free basal media (1 mL, Iscove's Modified Dulbecco's Medium:
5
IMDM; Life Technologies, Grand Island, NY) for three days. Conditioned media (600 µl),
subsequently, were collected and applied for mouse cytokine-array analysis according to the
manufacturer's instructions (RayBiotech, Norcross, GA). In analysis of mouse cytokine arrays,
positive control was used to normalize the result from different membranes.
3-Nitrotyrosine level 3 months after treatment: Cardiac 3-nitrotyrosine levels in CDC-treated
mice were higher 3 months after treatment than at 3 weeks, although they still remained lower
than in vehicle–treated mice (Suppl. Fig. 8). This finding hints that the beneficial effects of
CDCs may wear off over time.
Histology
Mice were sacrificed 3 weeks or 3 months after injection. Paraffin-embedded sections from
apical, middle and basal parts of each heart were used for histology. Masson's trichrome staining
(HT15 Trichrome Stain [Masson] Kit; Sigma-Aldrich, St. Louis, MO) and immunostaining with
antibody against mouse collagen I were performed for evaluation of fibrotic changes. T cells, B
cells, total macrophages, M2 subtype of macrophages and apoptotic cells were assessed by
immunostaining with antibody against mouse CD3, CD20, CD68, CD206 and cleaved caspase 3,
respectively, and the average number of cells in each heart was calculated from counting cells in
10 fields (20x magnification) from each of 10 sections selected randomly from the apical (3
sections; 50µm interval), middle (4sections; 50µm interval) and basal (3 sections; 50µm interval)
regions of each heart. Actively-cycling and proliferating (Ki67+ & aurora B+) cardiomyocytes
and c-kit+Nkx2.5+ cells were counted in the same manner, and the cycling and proliferating
fractions were expressed as the number of Ki67+ and aurora B+ cardiomyocytes divided by the
total number of cardiomyocytes per high-power field (HPF), respectively. The portion of c-
6
kit+Nkx2.5+ cells was calculated as the number of c-kit+Nkx2.5+ cells divided by the total
number of cardiomyocytes per HPF. Measurements were averaged for each heart.
Immunofluorescence staining: Heat-induced epitope retrieval in low pH buffer (DAKO,
Carpinteria, CA) was followed by 2 hours permeabilization/blocking with Protein Block Solution
(DAKO, Carpinteria, CA) contained 1% saponin (Sigma, St. Louis, MO; Protein Block Solution
contained 3% saponin was applied for immunofluorescence staining of Ki67). Subsequently,
primary antibodies in Protein Block Solution were applied overnight in 4 C° for
immunofluorescence staining of 5-µm sections from apical, middle and basal parts of each heart.
After 3x wash with PBS, each 10 minutes, Alexa Fluor secondary antibodies (Life Technologies,
Grand Island, NY) were used for detection. Images were taken by a Leica TCS SP5 X confocal
microscopy system. Immunofluorescence staining was conducted using antibodies against mouse
3-Nitrotyrosine (1:400), FSP (1:2000), collagen I (1:500), KLF5 (1:50), AT-R1 (1:50), c-kit (2
µg/ml) and α-SA (1:600) (Sigma, St. Louis, MO), gp91phox (0.5 µg/ml; Abcam, Cambridge,
MA), Ki-67 (SP6; 1:50; Thermo Fisher Scientific, Fremont, CA), WGA (Wheat germ agglutinin;
1:200; Life Technologies, Grand Island, NY), Nkx2.5 (N-19; 1:50) and CD206 (C-20; 1:30;
Santa Cruz Biotechnology, Santa Cruz, CA), aurora B (1:250; BD Biosciences, San Jose, CA)
and phospho-CREB (Ser133; 87G3; 1:800; Cell Signaling Technology, Denver, CO).
Immunoperoxidase staining: Immunohistochemical detection of CD3, CD20, CD68 and
cleaved caspase 3 (large fragment; 17/19 kDa) was performed on 5-µm sections using predilute
rabbit monoclonal antibodies from Ventana Medical System (Tuscon, AZ; CD68), Cell Marque
(Rocklin, CA; CD3, CD20) and Biocare Medical (Concord, CA; cleaved caspase 3). Staining
was conducted on the Leica Bond-Max Ventana automated slide stainer (Chicago, IL) using
onboard heat-induced epitope retrieval method in high pH ER2 buffer (Leica Biosystems,
7
Buffalo Grove, IL). The staining was visualized using the Dako Envision+ rabbit detection
System and Dako DAB (Carpinteria, CA). The slides were subsequently counterstained with
mayer’s hematoxylin for 1 minute and coverslipped.
Western blot analyses
Western blot analysis was performed to compare myocardial abundance of target proteins
contributing to oxidative/nitrosative stress (subunits of NAD(P)H oxidase enzyme: NOX4,
P47phox, p67phox and gp91phox ; iNOS), inflammation (MCP-1, COX-2 and NF-κB), PKC-PKDCREB signaling (PKCδ, PKD and CREB and their phosphorylated/activated form), proapoptosis (JNK), pro-survival (Akt), fibrosis (collagen I and III), and cell anti-oxidant-defense
signaling (Nrf2-Keap1 and its downstream gene targets: Cu-Zn SOD and catalase). 3Nitrotyrosine protein density, a marker of oxidative/nitrosative stress was also measured in the
myocardium by western blotting at 3 weeks and at 3 months after cell/vehicle injections.
Samples from apical, middle and basal parts of each heart (each 1 mm-thick transverse section)
were mixed and homogenized, and nuclear and cytoplasmic fractions were extracted per
manufacturer's instructions (CelLytic NuCLEAR Extraction Kit, Sigma- Aldrich, St. Louis,
MO). Western blot analysis was performed and the abundance of target protein bands was
normalized to GAPDH or Histone H1.Preparation of heart homogenates and nuclear extracts
for Western blot analyses: All solutions, tubes, and centrifuges were maintained at 0-4C°.
Briefly, 50 mg of heart tissue from apical, middle and basal parts of each heart were mixed and
homogenized, and nuclear and cytoplasmic fractions were extracted per manufacturer's
instructions (CelLytic NuCLEAR Extraction Kit, Sigma- Aldrich, St. Louis, MO). The
cytoplasmic and nuclear extracts were stored at −80C°. The protein concentrations in
cytoplasmic and nuclear extracts were determined by the Micro BCA Protein Assay Kit (Thermo
8
Fisher Scientific, Rockford, IL). Target proteins in the cytoplasmic or nuclear fractions of the
heart tissue were measured by Western blot analysis using the following antibodies: antibodies
against mouse NOX4, p47phox, p67phox, gp91phox, iNOS, 3-Nitrotyrosine, COX-2, CREB, CREBpS133, catalase, collagen I, and collagen III were purchased from Abcam (Abcam, Cambridge,
MA). AT-R1, KLF5, cleaved caspase 3, activated JNK, Akt-pT308, Akt, MCP-1, NF-κB p65, CuZn SOD and GAPDH antibodies (Sigma-Aldrich, St. Louis, MO), and p-IκB-α, PKCδ, PKCδpT505, PKD, PKD-pS744/S748 and PKD-pS916 antibodies (Cell Signaling Technology, Denver, CO)
and Nrf2, Keap1 and histone H1antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were
purchased from the cited sources. Antibodies to histone H1 and GAPDH were used for
measurements of the housekeeping proteins for nuclear and cytosolic target proteins,
respectively. Western blot analyses: Briefly, aliquots containing 60 μg proteins were
fractionated on 8, and 4% to 20% tris-glycine gel (Novex, San Diego, CA) at 120 V for 2 h and
transferred to a hybond-ECL membrane (Amersham Life Science, Arlington Heights, IL). The
membrane was incubated for 1 h in blocking buffer (1 × TBS, 0.05% Tween-20 and 5% nonfat
milk) and then overnight in the same buffer containing the given antibodies at optimal dilutions
listed in Suppl. Table 2. The membrane was washed 3 times for 5 min in 1 × TBS, 0.05%
Tween-20 before a 2-h incubation in a buffer (1 × TBS, 0.05% Tween-20 and 3% nonfat milk)
containing horseradish peroxidase-linked anti-rabbit IgG, anti-mouse IgG (Cell Signaling
Technology, Denver, CO) and anti-goat IgG (Sigma-Aldrich, St. Louis, MO) at 1:3000 dilution.
The membrane was washed 3 times for 5 min in 1 × TBS, 0.05% Tween-20 and developed by
autoluminography using the ECL chemiluminescent agents (Super Signal West Pico
Chemiluminescent Substrate; Thermo Fisher Scientific, Rockford, IL). GAPDH and histone H1
were used as housekeeping proteins against which expressions of the proteins of interest were
9
normalized. Phosphorylated PKCδ, PKD, CREB and Akt were normalized to total PKCδ, PKD,
CREB and Akt. Western blot analyses of collagen I and collagen III: Western blot analyses of
collagen I and collagen III under reducing, denaturing condition14 depicted bands of
approximately 70-150 kDa (Suppl. Fig. 9; Predicted band size for collagen I and III is
approximately 120 and 138, respectively).
Supplemental References:
10
1.
Smith RR, Barile L, Cho HC, Leppo MK, Hare JM, Messina E, Giacomello A, Abraham
MR, Marbán E. Regenerative potential of cardiosphere-derived cells expanded from
percutaneous endomyocardial biopsy specimens. Circulation. 2007;115:896-908.
2.
Shen D, Cheng K, Marbán E. Dose-dependent functional benefit of human cardiosphere
transplantation in mice with acute myocardial infarction. J Cell Mol Med. 2012;16:21122116.
3.
Nagaya N, Kangawa K, Itoh T, Iwase T, Murakami S, Miyahara Y, Fujii T, Uematsu M,
Ohgushi H, Yamagishi M, Tokudome T, Mori H, Miyatake K, Kitamura S.
Transplantation of mesenchymal stem cells improves cardiac function in a rat model of
dilated cardiomyopathy. Circulation. 2005;112:1128-1135.
4.
Li TS, Cheng K, Malliaras K, Smith RR, Zhang Y, Sun B, Matsushita N, Blusztajn A,
Terrovitis J, Kusuoka H, Marban L, Marbán E. Direct comparison of different stem cell
types and subpopulations reveals superior paracrine potency and myocardial repair
efficacy with cardiosphere-derived cells. J Am Coll Cardiol. 2012;59:942-953.
5.
Kapoor N, Liang W, Marbán E, Cho HC. Direct conversion of quiescent cardiomyocytes
to pacemaker cells by expression of Tbx18. Nat Biotechnol. 2013;31:54-62.
6.
Vellaichamy E, Khurana ML, Fink J, Pandey KN. Involvement of the NF-kappa B/matrix
metalloproteinase pathway in cardiac fibrosis of mice lacking guanylyl cyclase/natriuretic
peptide receptor A. J Biol Chem. 2005;280:19230-42.
7.
Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in
control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359-371.
8.
Payne TR, Oshima H, Okada M, Momoi N, Tobita K, Keller BB, Peng H, Huard J. A
relationship between vascular endothelial growth factor, angiogenesis, and cardiac repair
after muscle stem cell transplantation into ischemic hearts. J Am Coll Cardiol.
2007;50:1677-1684.
11
9.
Calvo CF, Fontaine RH, Soueid J, Tammela T, Makinen T, Alfaro-Cervello C, Bonnaud
F, Miguez A, Benhaim L, Xu Y, Barallobre MJ, Moutkine I, Lyytikka J, Tatlisumak T,
Pytowski B, Zalc B, Richardson W, Kessaris N, Garcia-Verdugo JM, Alitalo K,
Eichmann A, Thomas JL. Vascular endothelial growth factor receptor 3 directly regulates
murine neurogenesis. Genes Dev. 2011;25:831-844.
10.
Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived
factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial
infarction but is not sufficient to induce homing in the absence of injury. Circulation.
2004;110:3300-3305.
11.
Verma SK, Krishnamurthy P, Barefield D, Singh N, Gupta R, Lambers E, Thal M,
Mackie A, Hoxha E, Ramirez V, Qin G, Sadayappan S, Ghosh AK, Kishore R.
Interleukin-10 treatment attenuates pressure overload-induced hypertrophic remodeling
and improves heart function via signal transducers and activators of transcription 3dependent inhibition of nuclear factor-κB. Circulation. 2012;126:418-429.
12.
Jin H, Yang R, Li W, Ogasawara AK, Schwall R, Eberhard DA, Zheng Z, Kahn D, Paoni
NF. Early treatment with hepatocyte growth factor improves cardiac function in
experimental heart failure induced by myocardial infarction. J Pharmacol Exp Ther.
2003;304:654-660.
13.
Okayama K, Azuma J, Dosaka N, Iekushi K, Sanada F, Kusunoki H, Iwabayashi M,
Rakugi H, Taniyama Y, Morishita R. Hepatocyte growth factor reduces cardiac fibrosis
by inhibiting endothelial-mesenchymal transition. Hypertension. 2012;59:958-965.
14.
Kreger ST, Bell BJ, Bailey J, Stites E, Kuske J, Waisner B, Voytik-Harbin SL.
Polymerization and matrix physical properties as important design considerations for
soluble collagen formulations. Biopolymers. 2010;93:690-707.
Supplemental Tables
Suppl. Table 1. Left ventricular anterior and posterior wall thickness in 6-, 8- and 11-week-old control and Gαq mice (Baseline) and in
control mice at 18-, 20- and 23- weeks of age and in vehicle (Gαq+Vehicle) and CDC (Gαq+CDC)-treated Gαq mice 3 months after
treatment (Month 3). Gαq mice were treated at 6, 8 and 11 weeks of age.
Ant. wall
Post. wall
Baseline
Month 3
Baseline
Month 3
CTL(WT)
1.60±0.013
1.59±0.012
6 wks old
Gαq+Vehicle
1.29±0.056
1.07±0.034*
Gαq+CDC
1.29±0.056
1.23±0.023
CTL(WT)
1.61±0.021
1.59±0.025
8 wks old
Gαq+Vehicle
1.27±0.054
1.05±0.026*
Gαq+CDC
1.27±0.054
1.19±0.027
CTL(WT)
1.62±0.020
1.60±0.022
11 wks old
Gαq+Vehicle
1.25±0.056
1.02±0.022*
Gαq+CDC
1.25±0.056
1.19±0.015
1.60±0.018
1.60±0.019
1.55±0.023
1.48±0.013*
1.55±0.023
1.52±0.031
1.61±0.021
1.59±0.030
1.53±0.014
1.46±0.013*
1.53±0.014
1.47±0.021
1.61±0.030
1.58±0.024
1.51±0.028
1.43±0.016*
1.51±0.028
1.43±0.016
Data are means ± SEM; *<0.05 vs Baseline (Gq+Vehicle); n=4-5 and n=6-8 for Baseline and Month 3 measurements, respectively; CTL: control
(Wild type); Measurement unit: mm.
Suppl. Table 2. Antibodies and the optimal dilutions applied in Western blot analyses.
Antibody
NOX4
p47phox
p67phox
gp91phox
3-NT
iNOS
COX2
MCP-1
NF-κB p65
Dilution
1:1000
1 µg/ml
1:1000
0.5 µg/ml
1:500
1µg/ml
1:300
1:100
1:1000
Antibody
p-IκB-α
CREB-pS133
CREB
PKD-pS744/S748
PKD-pS916
PKD
PKCδ-pT505
PKCδ
Keap1
Dilution
1:200
1:100
1:200
1:100
1:100
1:200
1:100
1:200
1:200
Antibody
JNK
Akt-pT308
Akt
Collagen III
Collagen I
KLF5
AT-R1
Nrf2
Catalase
Dilution
5 µg/ml
1:200
1:1000
1:2000
1:3000
1:50
1:1000
1:200
1:3000
Antibody
Cu-Zn SOD
Caspase 3
Dilution
1:250
1:500
12
Supplemental Figures
Suppl. Fig. 1. Functional benefits after cardiosphere-derived cell (CDC) transplantation. CDC
transplantation resulted in a sustained improvement of LV end-diastolic (LV EDV; upper row)
and end-systolic (LV ESV; lower row) volumes in the Gαq mice, relative to placebo. This was
true whether CDCs were injected at 6 (Left column), 8 (Middle column) or 11 (Right column)
weeks of age. Preservation of LV EDV and LV ESV was sustained in CDC-treated animals for
at least 3 months. Data are means ± SEM; n = 6-8 in each group. * P<0.05 vs Gq+Vehicle; **
P< 0.01 vs Gq+ Vehicle; *** P<0.005 vs Gq+Vehicle.
13
Suppl. Fig. 2. CDC treatment preserved left ventricular anterior and posterior wall thickness for
at least 3 months. Representative cardiac sections (H&E stained) from control (CTL; Wild type)
and Gαq mice at 11 weeks of age (A) and from control (CTL; Wild type) and vehicle and CDCtreated Gαq mice at 23 weeks of age (B). Vehicle and CDC-treated Gαq mice were treated at 11
weeks of age. Scale bars: 1mm.
14
Suppl. Fig. 3. Percentage engraftment of CDCs 1, 3 and 6 weeks after transplantation.
Percentage engraftment of CDCs at 1 week was ~10% and <1% at 3 weeks. By 6 weeks, no
surviving CDCs could be detected. n = 3 at each time point.
15
A
B
Suppl. Fig. 4. Average number of CD68+ macrophages, CD3+ T cells and CD20+ B cells (A)
and representative immunohistochemical images and pooled data (B; CTL [wild type], vehicle
and CDC-treated Gαq mouse hearts stained for CD206) from Gαq mice treated at 8 weeks of
age. CDC treatment markedly reduced infiltration of CD68+ macrophage and CD3+ T cell into
the heart 3 weeks after treatment. Number of CD20+ B cells was not significantly different
16
between experimental groups (A). The number of reparative anti-inflammatory macrophages
(M2 subtype; CD206+), and their percentage, were markedly increased with CDC treatment (B).
Data are means ± SEM; n = 6-8 in each group. †P < 0.005 vs. Gαq+CDC and control (CTL;
wild type); ‡P < 0.05 vs. Gαq+CDC and control (CTL; wild type); #P < 0.05 vs. Gαq+Vehicle
(CD206) and control (CTL; wild type; CD206). Scale bars: 10µm.
17
A
B
Suppl. Fig. 5. Representative immunocytochemistry images of neonatal rat cardiomyocytes
(NRCMs) stained for cleaved caspase 3 (A) and the western blot and pooled blot data (B) from
three experimental repeats, depicting protein abundance of cleaved caspase 3 in the NRCMs
incubated with IMDM (basal media), CDC-conditioned media and cardiac fibroblast (CF)conditioned media for 24 hours before exposure to oxidizing agent, hydrogen peroxide (10µM;
30 minutes). Protein abundance of active apoptotic executor, cleaved caspase 3 was markedly
lower in NRCMs treated with CDC-conditioned media compared with the NRCMs treated with
18
CF-conditioned media or IMDM. Data are means ± SEM; †P < 0.05 vs. CF and IMDM. Scale
bars: 20µm.
19
Suppl. Fig. 6. Cardiac activity level of endogenous active MMP9 in the wild-type mice (n=4)
and in the CDC- and vehicle-treated Gαq mice 1 day and 1 week after treatment (n=6 in each
group). The enrolled mice were at 8 weeks of age. Data are means ± SEM; †P < 0.05 vs.
Gαq+Vehicle (Day1) and control (CTL; wild type); ‡P < 0.05 vs. Gαq+Vehicle (Wk1) and
control (CTL; wild type); #P < 0.001 vs. Gαq+CDC (Day1).
20
A
B
Suppl. Fig. 7. Marked release of cytokines mediating angiogenesis, vasculogenesis,
lymphangiogenesis, neurogenesis, stem cell recruitment, inhibition of inflammation and cardiac
remodeling, prevention of cardiac fibrosis and improvement of cardiac function by CDCs.
Representative dot blot images and group data (A) from three experimental repeats depicting
mouse cytokine-array analysis of CDC and cardiac fibroblast conditioned media. Figure B shows
cytokine-array positions. Data are means ± SEM; † p< 0.01 vs cardiac fibroblast conditioned
media. ICAM-1:Intercellular Adhesion Molecule 1; HGF: Hepatocyte Growth Factor; HGF R:
Hepatocyte Growth Factor Receptor; IL-10: Interleukin 10; SDF-1α: Stromal Cell-derived
Factor-1α; SCF: Stem Cell Factor; VEGF-D: Vascular Endothelial Growth Factor-D; VEGF-R3:
Vascular Endothelial Growth Factor Receptor 3; VEGF-R2: Vascular Endothelial Growth Factor
21
Receptor 2; VEGF-R1: Vascular Endothelial Growth Factor Receptor 1; VEGF: Vascular
Endothelial Growth Factor.
22
A
B
Suppl. Fig. 8. Representative western blot and pooled blot data (A) depicting protein abundance
of 3-Nitrotyrosine (3-NT) in the cardiac tissues of control (CTL; wild type) mice and Gαq mice 3
months after treatment. Figure B represents cardiac protein density of 3-NT at 3 weeks and 3
23
months after treatment. Cardiac 3-nitrotyrosine levels in CDC-treated mice were higher 3 months
after treatment than at 3 weeks, although they still remained lower than in vehicle–treated mice.
24
Collagen I
CTL (WT)
Gq+Vehicle
Gq+CDC
Collagen III
150 kDa
~ 120 kDa
80 kDa
CTL (WT)
Gq+Vehicle Gq+CDC
130 kDa
~ 100 kDa
Suppl. Fig. 9. Western blot analyses of collagen I and collagen III. Western blot analyses of
collagen I and collagen III under reducing, denaturing condition depicted bands of approximately
70-150 kDa. Predicted band size for collagen I and III was approximately 120 and 138,
respectively.
25
Suppl. Fig. 10. 95% confidence interval of difference
NOX4
*
p47
phox
p67
phox
p91
phox
iNOS
3NT
MCP-1
1 (0.26 , 0.52)
3 (0.13 , 0.50)
1 (0.74 , 0.80)
3 (0.55 , 0.78)
1 (0.30 , 0.41)
3 (0.22 , 0.39)
1 (0.16 , 0.35)
3 (0.12 , 0.34)
1 (0.23 , 0.46)
3 (0.22 , 0.45)
1 (0.44 , 0.55)
3 (0.42 , 0.52)
1 (0.24 , 0.29)
3 (0.20 , 0.28)
COX2
p-IκB
p65
PKCδ
1 (0.34 , 0.60)
3 (0.30 , 0.50)
1 (0.30 , 0.36)
3 (0.11 , 0.33)
1 (0.16 , 0.26)
3 (0.10 , 0.24)
1 (0.24 , 0.44)
3 (0.22 , 0.44)
1 (0.33 , 0.54)
3 (0.16 , 0.51)
1 (0.25 , 0.43)
3 (0.24 , 0.43)
1 (0.24 , 0.37)
3 (0.21 , 0.36)
JNK1
JNK2
Akt
Collagen I
Collagen III
Collagen I/III
KLF5
1 (0.24 , 0.31)
3 (0.14 , 0.29)
1 (0.09 , 0.19)
3 (0.08 , 0.20)
1 (-0.24 , -0.08)
3 (-0.45 , -0.27)
1 (0.38 , 0.50)
3 (0.31 , 0.48)
1 (0.08 , 0.16)
3 (0.08 , 0.13)
1 (0.29 , 0.98)
3 (0.15 , 0.72)
1 (0.24 , 0.65)
3 (0.21 , 0.63)
ATR1
Keap1
Nrf2
NT3Month
SOD
Catalase
Caspase 3 cells
1 (0.40 , 0.71)
3 (0.31 , 0.65)
1 (0.24 , 0.46)
3 (0.20 , 0.42)
1 (-0.10 , -0.07)
3 (-0.09 , -0.06)
1 (0.71 , 0.83)
3 (0.25 , 0.49)
1 (-0.42 , -0.28)
3 (-0.23 , -0.10)
1 (-0.36 , -0.19)
3 (-0.26 , -0.13)
1 (1.39 , 3.65)
3 (0.79, 3.16)
+
cKit Nkx2.5
+
1 (0.0009 , 0.0063)
2 (0.0001 , 0.0058)
*
Ki67
+
1 (0.0016 , 0.0069)
2 (0.0003 , 0.0064)
*
Aurora B
S744/S748
PKD-p
S916
PKD-p
CREB
+
+
1 (0.0012 , 0.0066)
2 (0.0001, 0.0061)
1: Wild type 2: Gαq+Vehicle 3: Gαq+CDC
* 95% confidence interval (CI) for the difference between 3 vs 1; 3 vs 2. The rest denotes 95% CI for the difference
between 2 vs 1, 2 vs 3.
26