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Molecular Cardiology
Sca-1ⴙ Stem Cell Survival and Engraftment in the
Infarcted Heart
Dual Role for Preconditioning-Induced Connexin-43
Gang Lu, MD, PhD; Husnain K. Haider, MPharm, PhD; Shujia Jiang, MD; Muhammad Ashraf, PhD
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Background—We report that elevated connexin-43 (Cx-43) in stem cells preconditioned with insulin-like growth factor-1
(IGF-1) is cytoprotective and reprograms the cells for cardiomyogenic differentiation.
Methods and Results—Sca-1⫹ cells were preconditioned with 100 nmol/L IGF-1 for 30 minutes followed by 8 hours of
oxygen glucose deprivation to assess the cytoprotective effects of preconditioning. LDH release assay, cytochrome c
release, and mitochondrial membrane potential assay showed improved survival of preconditioned Sca-1⫹ cells under
oxygen glucose deprivation compared with nonpreconditioned Sca-1⫹ cells via PI3K/Akt-dependent caspase-3
downregulation. We observed PI3K/Akt-dependent upregulation of cardiac-specific markers including MEF-2c
(2.5-fold), GATA4 (3.1-fold), and Cx-43 (3.5-fold). Cx-43 inhibition with specific RNA interference reduced cell
survival under oxygen glucose deprivation and after transplantation. In vivo studies were performed in a female rat
model of acute myocardial infarction (n⫽78). Animals were grouped to receive intramyocardially 70 ␮L Dulbecco
modified Eagles medium without cells (group 1) or containing male 1⫻106 nonpreconditioned Sca-1⫹ cells (group 2)
or preconditioned Sca-1⫹ (group 3) cells labeled with PKH26. Survival of the preconditioned Sca-1⫹ cells was 5.5-fold
higher in group 3 compared with group 2 at 7 days after transplantation. Confocal imaging after actinin and Cx-43
specific immunostaining showed extensive engraftment and myogenic differentiation of preconditioned Sca-1⫹ cells.
Compared with group 2, group 3 showed increased blood vessel density (22.3⫾1.7 per microscopic field; P⬍0.0001)
and attenuated infarction size (23.3⫾3.6%; P⫽0.002). Heart function indices including ejection fraction (56.2⫾3.5;
P⫽0.029) and fractional shortening (24.3⫾2.1; P⫽0.03) were improved in group 3 compared with group 2.
Conclusions—Preconditioning with IGF-1 reprograms Sca-1⫹ for prosurvival signaling and cardiomyogenic differentiation with an important role for Cx-43 in this process. (Circulation. 2009;119:000-000.)
Key Words: angiogenesis 䡲 apoptosis 䡲 connexin 43 䡲 insulin-like growth factor-1 䡲 stem cells
T
he electric and mechanical integrity of the heart is
compromised after myocardial infarction because of
massive loss of functioning myocytes. Heart cell therapy
provides an unconventional corrective measure to compensate for myocyte loss in the infarcted heart.1– 4 Nevertheless,
poor survival of donor cells is one of the major concerns that
hampers a better prognosis.5 Additionally, poor engraftment
and lack of functional coupling of donor cells with the viable
host tissue greatly impede cell-to-cell signaling and electric
communication. Most previous strategies have addressed the
issue of cell survival alone, although with limited success.6 – 8
Because functional improvement of the heart is proportional
to the number of injected cells, we therefore proposed that a
strategy that concurrently addresses both of these issues
would increase the effectiveness of the procedure.
Connexin-43 (Cx-43), with its dual role as an antiapoptotic
and as a gap-junctional protein, can effectively resolve both
of these issues.9 –11
Clinical Perspective on p ●●●
The connexin family of genes encodes for ⬎20 proteins, of
which Cx-30, Cx-37, Cx-40, Cx-43, and Cx-45 have been
studied extensively for their role in the heart.12 Cx-43 is
predominantly synthesized in the plasma membrane of cardiomyocytes and forms intercellular channels to link cytoplasmic compartments of the adjacent myocytes. As an
alternative to the paracrine mechanism of intercellular communication, Cx-43 ensures a direct transfer of ions and signaling
molecules that regulates intracellular calcium and cell survival
via releasing ATP, NAD⫹, or glutamate and propagation of
electric impulses.13,14 Gap-junctional intercellular communication is also important for cellular proliferation and differentiation.15 Under physiological conditions, Cx-43 participates in cellular response to ischemia.16,17 Moreover,
localization of Cx-43 in intracellular structures such as the
Received October 14, 2008; accepted March 20, 2009.
From the Department of Pathology and Laboratory Medicine, 231 Albert Sabin Way, University of Cincinnati, Cincinnati, Ohio.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.827691/DC1.
Correspondence to Professor Muhammad Ashraf, Department of Pathology and Laboratory Medicine, 231 Albert Sabin Way, University of Cincinnati,
Cincinnati, OH 45267-0529. E-mail [email protected]
© 2009 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.108.827691
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May 19, 2009
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Figure 1. Flow cytometry of purified
mouse Sca-1⫹ cells for surface markers.
Shown are unlabeled Sca-1 cells (as a
control) (A1) and labeled cells showing
92.9% Sca-1⫹ (A2), 0.7% c-kit⫹ (A3), and
4.3% CD45 (A4) cell populations. B, Immunostaining of cells for Sca-1 antigen
(red⫽Sca-1 antigen; blue⫽DAPI; magnification ⫻200). C1, LDH release assay
showed 31.4% cell death in bovine serum
albumin–treated non-PCSca-1⫹ compared
with 13.7% in PCSca-1⫹. C2, TUNEL staining showed that 8-hour OGD caused
higher TUNEL positivity in non-PCSca-1⫹
compared with PCSca-1⫹. JC-1 staining of
non-PC
Sca-1⫹ (D1) and PCSca-1⫹ (D2) for
mitochondrial membrane potential after
OGD. D3, Significantly lower PCSca-1⫹
(13.8%) showed depolarized mitochondrial membrane potential compared with
non-PC
Sca-1⫹ (47.6%). Cytochrome c–specific immunostaining of PCSca-1⫹ (E1) and
non-PC
Sca-1⫹ (E2) after OGD treatment. A
typical punctuate distribution of cytochrome c (green) was observed in
PC
Sca-1⫹ compared with the diffused
appearance in non-PCSca-1⫹ (red boxed
areas in E1 and E2 have been enlarged
for clarity). Cell nuclei were observed by
propidium iodide staining (red). Phase
contrast microscopy showed better preserved cell morphology in PCSca-1⫹ (E3)
compared with non-PCSca-1⫹ (E4). F,
Annexin-V–fluorescein isothiocyanate/propidium iodide staining showed reduced
apoptosis in PCSca-1⫹ (50.5%) compared
with non-PCSca-1⫹ (69.7%).
Lu et al
Role of Preconditioning-Induced Connexin-43
3
mitochondria seems to be cardioprotective.18,19 Any reduction
in Cx-43 renders the heart more susceptible to electric
instability. Cell-based delivery of the Cx-43 transgene prevented ventricular arrhythmia after infarction.19 The functional versatility of Cx-43 supports our study rationale that
pharmacological targeting of Cx-43 in stem cells may improve their survival and integration in the infarcted heart.
Different growth factors potentiate Cx-43 expression.20 –22
Insulin-like growth factor-1 (IGF-1) increases intracellular
Cx-43.22 Our results emphasize the importance of IGF-1/
IGF-1 receptor (IGF-1R) interaction to initiate downstream
survival signaling involving Cx-43, which primarily curtailed
stem cell apoptosis and promoted their survival, factors
crucial for subsequent engraftment of donor cells in the
infarcted heart. These are novel findings that underscore the
need to exploit a dual role for the preconditioning-induced
Cx-43 to promote stem cell survival and their electromechanical coupling after transplantation for better prognosis.
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Methods
The experimental protocols are described in the expanded Methods
in the online-only Data Supplement. The lists of antibodies and
primer sequences used are given in Tables I and II in the online-only
Data Supplement.
Statistical Analysis
All experiments were performed at least 3 times to assess reproducibility of the results. The data were expressed as mean⫾SEM.
Student t test or 1-way or 2-way ANOVA was performed to analyze
statistical differences in each response variable. Prespecified comparisons between groups were made, and Bonferroni or Tukey
adjustment for multiple comparisons was done when appropriate. A
value of Pⱕ0.05 was considered statistically significant.
The authors had full access to and take full responsibility for the
integrity of the data. All authors read and agreed to the manuscript as
written.
Results
Flow cytometry showed that 92.9% of cells expressed Sca-1
and were very low in c-kit (0.7%) and CD45 (4.3%) expression (Figure 1A1 to 1A4). The homogeneity of the purified
cells was confirmed by fluorescent immunostaining for Sca-1
antigen (Figure 1B). These cells were later used in all of the
in vitro and in vivo experiments.
Cytoprotection by IGF-1 Pretreatment
The percent cell viability under oxygen glucose deprivation
(OGD) was significantly higher in preconditioned Sca-1⫹ cells
(PCSca-1⫹) compared with nonpreconditioned Sca-1⫹ cells
(non-PCSca-1⫹). Preconditioning reduced the nonviable cells from
5.9% to 1.7% under 4-hour OGD and from 31.4% to 13.7%
under 8-hour OGD (P⫽0.001 by 2-way ANOVA; Figure
1C). These results were confirmed by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling
(TUNEL), which showed that 8-hour OGD caused higher
TUNEL positivity in non-PCSca-1⫹ compared with PCSca-1⫹
(Figure 1C2). JC-1, a cationic dye that exhibits membrane
potential– dependent accumulation in the mitochondria, was
used to detect the early stage of apoptosis (Figure 1D1 to
1D3). IGF-1 pretreatment significantly reduced the percentage of early apoptotic cells (green) under OGD (Figure 1D3).
Moreover, cytochrome c–specific immunostaining revealed a
Figure 2. Representative blots showing higher phosphorylation
of IGF-1R (A) and Akt (B) in PCSca-1⫹ compared with non-PCSca1⫹. Treatment of the cells with PI3K inhibitor (LY294002; 40
␮mol/L) before preconditioning abolished pAkt under normoxia
and after 8-hour OGD. Total Akt remained unchanged. C, LDH
release assay showed that cell death was attenuated in
PC
Sca-1⫹ compared with non-PCSca-1⫹ under 8-hour OGD. Pretreatment of cells with LY294002 abolished the cytoprotective
effects of preconditioning under OGD. D1, Downstream of pAkt,
caspase-3 cleavage (active form) was significantly prevented in
PC
Sca-1⫹, which otherwise was increased in non-PCSca-1⫹ under
8-hour OGD. Pretreatment of cells with LY294002 increased
caspase-3 cleavage. D2, Cells treated with caspase-3 inhibitor
(Z-DEVD-FMK; 20 ␮mol/L) showed lower cell death compared
with the cells treated with dimethyl sulfoxide (DMSO) (vehicle of
caspase inhibitor).
typical punctuate distribution of fluorescence in the mitochondria of PCSca-1⫹ (Figure 1E1), whereas release of cytochrome c from the mitochondria after OGD was indicated by
diffused fluorescence in the cytoplasm (Figure 1E2). Phase
contrast microscopy revealed well-preserved cell morphology
in PCSca-1⫹ (Figure 1E3) compared with non-PCSca-1⫹ (Figure
1E4). Annexin V–fluorescein isothiocyanate/propidium iodide staining coupled with flow cytometry showed that
preconditioning reduced apoptotic cells from 69.7% in
non-PC
Sca-1⫹ to 50.5% in PCSca-1⫹ (Figure 1F). Taken together,
IGF-1 preconditioning offered cytoprotection against OGD in
vitro.
Cell Signaling in IGF-1 Initiated Cytoprotection
Sca-1⫹ cells expressed IGF-1 receptors that were phosphorylated by 30-minute pretreatment with 100 nmol/L IGF-1
(Figure 2A). PI3K/Akt signaling plays a significant role
downstream of IGF-1/IGF-1R ligand/receptor interaction.23
We observed higher pAkt in PCSca-1⫹ compared with
non-PC
Sca-1⫹ (Figure 2B). OGD for 8 hours significantly
reduced pAkt in non-PCSca-1⫹, whereas it still remained high in
PC
Sca-1⫹ (Figure 2B). Pretreatment with PI3K inhibitor
(LY294002; 40 ␮mol/L) abolished pAkt in PCSca-1⫹. The
effect of LY294002 was more pronounced in the cells
subjected to 8-hour OGD compared with normoxia. LDH
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Figure 3. A, Real-time PCR of Sca-1⫹ after 1-day
and 7-day IGF-1 treatment in culture. The cells
cultured for 1 day and 7 days without IGF-1 were
used as controls. IGF-1 treatment for 7 days
induced higher mRNA expression of GATA-4 (3.1fold), Cx-43 (3.5-fold), and MEF2c (2.5-fold),
whereas troponin-I (Tn-I) upregulation was statistically insignificant. B, Western blot on cell lysate
from these treatment groups confirmed these findings. Whereas GATA-4 protein expression
remained undetectable, both Cx-43 and MEF-2c
protein levels were significantly increased after
7-day IGF-1 treatment under normoxia. C, Cx-43
and MEF-2c mRNA induction by IGF-1 treatment
was abolished by LY294002 (IGF-1 without LY,
MEF2C P⫽0.0003 and Cx-43 P⫽0.0021; IGF-1
with LY, MEF2C P⫽0.99 and Cx-43 P⫽1; 2-way
ANOVA). D, The expression of Cx-43 and MEF-2c
mRNA was sensitive to anoxia and was abolished
in Sca-1⫹ after 12-hour OGD. However, the effect
of OGD was less pronounced in Sca-1⫹ with continuous IGF-1 presence during 12-hour OGD treatment. E, LDH release assay showed that continuous treatment with IGF-1 during OGD greatly
reduced cell death from 35.8% to 12.4%. F, Representative blots of Sca-1⫹ after 8-hour OGD
showing enhanced IGF-1 protein expression compared with Sca-1⫹ grown under normoxia. G, Representative blots of protein samples from rat heart
on 1 day and 7 days after cell engraftment.
PC
Sca-1⫹ showed higher IGF-1 expression at 1 day
and 7 days compared with non-PCSca-1⫹.
release assay showed higher PCSca-1⫹ survival under 8-hour
OGD compared with non-PCSca-1⫹ (Figure 2C). However,
cytoprotection was abolished in PCSca-1⫹ pretreated with
LY294002 (17.9% in PCSca-1⫹ versus 31.2% in IGF1⫹LY294002–treated cells). Western blotting showed that
8-hour OGD increased caspase-3 cleavage (activation of
caspase-3) in non-PCSca-1⫹ compared with PCSca-1⫹. Contrarily, pretreatment with LY294002 showed a higher level of
caspase-3 cleavage in PCSca-1⫹, thus showing an inverse
relation between the dynamics of caspase-3 cleavage and Akt
phosphorylation (Figure 2D1). Treatment with caspase-3
inhibitor and subsequent exposure to 8-hour OGD showed
enhanced cell survival under OGD (31.1%) compared with
inhibitor untreated cells (64.9%) (Figure 2D2).
Preconditioning With IGF-1
Favored Cardiomyogenesis
Although IGF-1 treatment for 1 day and 7 days significantly
induced mRNA expression of cardiac marker proteins, 7-day
IGF-1 treatment was more effective in upregulating GATA-4
(3.1-fold), Cx-43 (3.5-fold), and MEF-2c (2.5-fold), whereas
troponin-I upregulation remained insignificant (Figure 3A).
Western blotting confirmed these findings except for
GATA-4, which remained undetectable (Figure 3B).
LY294002 pretreatment reversed the effect of IGF-1 on cells
grown in normoxia (Figure 3C). We observed that cardiac-
specific gene expression was sensitive to anoxia. Cells
cultured with 0.5% bovine serum albumin for 12 hours under
OGD showed abrogation of Cx-43 and MEF2c, whereas the
presence of IGF-1 during OGD significantly enhanced their
gene expression (Figure 3D). The presence of IGF-1 during
OGD also offered cytoprotection in addition to promoting
cardiac-specific proteins (Figure 3E). These data suggested
that OGD not only imposed a survival challenge for cells but
also impeded their differentiation. Even though downregulation of cardiac markers under OGD was not seen to be fully
reversed from our 12-hour study, the presence of IGF-1
supported Sca-1⫹ cells toward cardiac differentiation. Hence,
it is reasonable to project a more pronounced effect of IGF-1
in vivo when one considers the kinetics of its expression in
the infarcted heart, which significantly affects the transplanted cells. In accordance with Western blotting results
(Figure 3F), PCSca-1⫹ transplanted hearts showed higher
IGF-1 on day 1 and day 7 (Figure 3G).
Cx-43 Confers Cytoprotection
Because we observed that IGF-1 preconditioning increased
Cx-43 in Sca-1⫹, we established its role in cytoprotection.
Cx-43 was abolished in Cx-43 small interfering RNA
(siRNA) transfected cells grown under normoxia or OGD
compared with scramble siRNA (Sc siRNA) transfected cells
(Figure I in the online-only Data Supplement). These results
Lu et al
Role of Preconditioning-Induced Connexin-43
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were confirmed by Western blotting (Figure 4A1). LDH
assay showed higher cell death in Cx-43 siRNA transfected
cells under 4-hour and 8-hour OGD compared with Sc siRNA
transfected cells (P⫽0.0075 by 2-way ANOVA; Figure 4A2).
Additionally, Sca-1⫹ transfected with Cx-43 siRNA showed
shrunken and rounded morphology (Figure 4A3) compared
with Sc siRNA transfected cells (Figure 4A4). Elucidating the
mechanism for increased death in Cx-43 siRNA transfected
cells under 8-hour OGD, we observed that caspase-3 cleavage
was significantly increased (Figure 4A5). Double fluorescence immunostaining revealed punctate and colocalized
distribution of Cx-43 (red) and cytochrome c (green) in the
mitochondria (Figure 4A6). Figure 4A7 shows a magnified
image of an Sca-1⫹ cell from Figure 4A6 (white box) to show
colocalization of Cx-43 (red) and cytochrome c (green).
Western blotting on subcellular fractions confirmed a higher
presence of Cx-43 in the mitochondrial fraction in PcSca-1⫹
(Figure 4B). Voltage-dependent anion channel protein was
used as an indicator of purity of the mitochondrial fraction.
Real-time polymerase chain reaction (PCR)– based gene array showed that 10 genes from the apoptotic cascade had
⬎2-fold higher expression in Cx-43 siRNA transfected cells
(Figure II in the online-only Data Supplement). The expression
of caspase recruitment domain family member-10 (Card-10),
which increased 6.02-fold in Cx-43 siRNA transfected cells
under normoxia, led to higher sensitivity to anoxia and
increased to ⬎10-fold (Figure III in the online-only Data
Supplement). We observed that elevation of Cx-43 in response to IGF-1 treatment abrogated Card-10 with a concomitant increase in cell survival under OGD (Figure 4C).
Sca-1⫹ cells from male mice transfected with Cx-43 siRNA
(n⫽4) or Sc siRNA cells (n⫽4) were transplanted in a female
rat heart model of acute myocardial infarction. Real-time
PCR for sry gene on day-7 myocardial samples showed that
survival of Cx-43 siRNA transfected cells was significantly
lower compared with Sc siRNA transfected cells (Figure 4D).
Figure 4. A1, Western blot of Sc siRNA (control) or Cx-43 siRNA
transfected Sca-1⫹ cells grown under normoxia or OGD. Cx-43
siRNA successfully abolished Cx-43 expression. A2, LDH release
assay showing higher cell death in Cx-43 siRNA transfected cells
compared with Sc siRNA transfected cells under 4-hour and
8-hour OGD. Similarly, Cx-43 siRNA transfected cells (A3) showed
more obvious rounded and shrunken morphology under 8-hour
OGD compared with Sc siRNA transfected cells (A4). A5, Western
blot showing elevated caspase-3 cleavage in Cx-43 siRNA transfected cells compared with Sc siRNA cells. A6, Double fluorescence immunostaining showing distribution of colocalized Cx-43
(red) and cytochrome c (green) in mitochondria as punctate bodies
in Sca-1⫹. A7, Magnified image of a Sca-1⫹ cell from A6 (white
box; magnification ⫻63) shows clear apposition of red (Cx-43) and
cytochrome c (green). B, Western blot showing higher level Cx-43
in the mitochondrial fraction of Sca-1⫹ after IGF-1 treatment compared with cells without IGF-1 treatment. VDAC indicates voltagedependent anion channel. C, IGF-1 treatment significantly
increased Cx-43 level with concomitant suppression of Card-10
compared with bovine serum albumin (BSA)–treated cells. D, Realtime PCR for mice sry gene in female rat hearts (n⫽4 per group)
on day 7 after transplantation of male Sca-1⫹. The cells were
transfected with Sc siRNA or Cx-43 siRNA. Cx-43 siRNA significantly reduced the survival of Sca-1⫹.
In Vivo Proliferation and Survival of
PC
Sca-1ⴙ
Immunostaining showed pronounced pAkt at the site of cell
graft in PCSca-1⫹ transplanted animals (group 3) (Figure 5A1
to 5A3) compared with non-PCSca-1⫹ transplanted animals
(group 2) (Figure 5A4 to 5A6). The number of TUNEL⫹ cells
was significantly higher in group 2 compared with group 3 on
day 7 after engraftment (Figure 5B1 to 5B5). We also
observed a higher presence of Ki67⫹ cells in group 3
(P⬍0.001 versus group 2), especially in the cell-engrafted
regions (Figure 5C1 to 5C2). For an overall estimation of
donor cell survival, the entire left ventricle (LV) was used for
mice sry gene estimation in female recipient rat heart at 1 day,
4 days, and 7 days after engraftment of male donor cells.
Significantly higher cell survival was observed in group 3
compared with group 2 at all the studied time points (Table III
in the online-only Data Supplement and Figure 5D). Dulbecco modified Eagles medium–injected female animal
hearts (group 1) were used as a control.
Angiomyogenic Fate of
PC
Sca-1ⴙ
Confocal imaging after immunostaining for ␣-sarcomeric
actinin 7 days after cell engraftment depicted extensive
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Figure 5. Confocal microscopy images of rat
heart tissue sections 1 day after transplantation
of PKH26-labeled PCSca-1⫹ (A1 to A3) and non⫹
PCSca-1 (A4 to A6) (red fluorescence). A3,
Merged image showing higher pAkt levels
(green) in group 3 animal heart compared with
group 2 (A6). B1 to B5, TUNEL staining on histological sections from various treatment
groups on day 7 showed that the number of
TUNEL⫹ cells was lower in group 3 compared
with group 2. B2 to B5 are representative figures of the TUNEL-stained histological sections
on day 7 after cell engraftment in group 3 (B2,
red⫽PKH26; B3, green⫽TUNEL staining; B4,
merged image of TUNEL staining [green] with
DAPI [blue] to visualize all the cell nuclei; B5,
B2 to B4 merged image). C1, Graph showing
higher number of Ki67⫹ cells in group 3 compared with group 2 on day 7 after cell engraftment as determined by Ki67-specific immunostaining. C2, Representative confocal images of
Ki67⫹ cells (green) in infarction area 7 days
after PKH26-labeled PCSca-1⫹ engraftment
(red). DAPI was used for visualization of the
nuclei (blue). D, Real-time PCR for mouse sry
gene to determine donor cell survival in the
female recipient heart. PCSca-1⫹ survival was
significantly higher compared with non-PCSca-1⫹
at all time points of engraftment.
neomyogenesis in the PCSca-1⫹ transplanted area (Figure 6A1
to 6A4). Colocalization of PKH26-labeled cells (red) with
cardiac actinin (green) was observed in the infarct and
peri-infarct regions, indicating myogenic differentiation of
the engrafted cells. However, the propensity of differentiating
non-PC
Sca-1⫹ was obviously low compared with PCSca-1⫹
(Figure 6B1 to 6B4). These results suggest that Sca-1⫹ has
inherent differentiation potential that is accentuated by preconditioning. Immunostaining of histological sections from
group 3 for myosin heavy chain (slow isoform) and Cx-43
showed that neofibers also expressed Cx-43 and were well
engrafted in the host tissue (Figure 6C1 to 6C4). Electron
microscopy confirmed the presence of tight junctions and
myofilaments in the newly differentiating Sca-1⫹ (Figure 6D1
to 6D4).
To evaluate angiogenesis and maturation index at the
6-week time point, histological sections were immunostained
for von Willebrand factor VIII alone (Figure 7A1 to 7A7) or
with smooth muscle actin (Figure 7B1 to 7B4). The highest
angiogenic activity was observed in group 3 (Figure 7A7). By
2-way ANOVA, blood vessel density in infarct and peri-
infarct regions (22.3⫾1.7 and 32⫾2.2, respectively) was
higher compared with group 2 (16.9⫾1.5, P⬍0.0001;
24.3⫾1.4, P⬍0.0001) and group 1 (11.3⫾1.6, P⬍0.0001;
19.3⫾2.1, P⬍0.0001). No significant difference in maturation index was found among the 3 groups in the infarct
region, but group 3 showed higher maturation index in the
peri-infarct region compared with group 2 (P⫽0.11 by 2-way
ANOVA; Figure 7B1 to 7B4). However, the total number of
mature blood vessels was higher in group 3 compared with
the other groups.
Infarction Size and Heart Function
Cross sections at the midpapillary muscle level showed
transmural infarction in all the animals. Marked LV wall
thinning was observed at 6 weeks in group 1 with 51.9⫾5.6%
infarction of the LV (Figure 8A1). Comparatively, infarction size at 6 weeks was attenuated in group 2 (38.8⫾1.2%)
and group 3 (23.3⫾3.6%) (P⫽0.002 versus group 2)
(Figure 8A2).
The indices of systolic function including LV ejection
fraction and LV fractional shortening were higher in group 2
Lu et al
Role of Preconditioning-Induced Connexin-43
7
(43.3⫾2.8%, P⫽0.024 and 17.3⫾1.4%, P⫽0.024) and group
3 (56.2⫾3.5%, P⫽0.001 and 24.3⫾2.1%, P⫽0.001) compared with group 1 (28.3⫾4.7%; 10.5⫾2%) (Figure 8B and
8C). LV ejection fraction (P⫽0.029) and LV fractional
shortening (P⫽0.03) between group 3 and group 2 also
showed a significant difference. LV end-systolic dimension
(in centimeters) was smaller in group 3 (0.57⫾0.05;
P⫽0.0003 versus all other groups by 2-way ANOVA) compared with group 1 (0.71⫾0.03) and group 2 (0.7⫾0.01)
(Figure 8D). LV wall thickness improved from 0.07 cm in
group 1 to 0.08⫾0.01 cm in group 2 (group 2 versus group 1,
P⫽0.59) and 0.11⫾0.01 cm in group 3 (group 3 versus group
1, P⫽0.02) (Figure 8E).
Discussion
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IGF-1 acts through the IGF-1/IGF-1R ligand/receptor system
and significantly affects LV remodeling.24 The IGF-1 autocrine and paracrine systems in the viable cardiomyocytes are
altered transiently in response to myocardial infarction.25
Therefore, strategies were designed to manipulate intrinsic
IGF-1 for its continuous participation in myocardial repair.
To this end, IGF-1 protein and transgene delivery either by
direct DNA injection or by ex vivo cell-based delivery has
been studied.26,27 We have recently shown that IGF-1 transgene overexpression in the mesenchymal stem cells promoted
their survival after transplantation.28 Similar beneficial effects
were achieved by preconditioning to activate IGF-1–mediated signaling in the stem cells. Our preconditioning approach
with IGF-1 protein is simpler and much safer to follow from
a clinical perspective compared with IGF-1 transgene overexpression. The results of preconditioning also implied that
IGF-1 was not required to be carried by stem cells. Preconditioning improved cell survival through classic survival
signaling involving Akt activation, which preserved mitochondrial integrity and prevented cytochrome c release, thus
preventing caspase-3 cleavage with a possible role for Cx-43.
Multiple cellular and physiological functions of IGF-1 have
been reported through activation of PI3K-Akt.27,29,30 Interestingly, 30-minute preconditioning in this study generated
pAkt, which remained elevated after 8-hour OGD in vitro and
for 24 hours after transplantation. We infer that better
engraftment of PcSca-1⫹ after transplantation in this study was
due to improved cell survival, reduced cell apoptosis, and
increased cell proliferation.
Another interesting observation in this study was elevation
of IGF-1 in the infarcted heart after PCSca-1⫹ engraftment.
Besides adoption of cardiac phenotype, the beneficial effect
Figure 6. Confocal images of histological sections immunostained for ␣-sarcomeric actinin on day 7 after cell engraftment;
A1 to A4, group 3; B1 to B4, group 2. C1 to C4 shows immunostaining of the histological sections for myosin heavy chain at 6
weeks after PCSca-1⫹ transplantation. Primary antigen-antibody
reaction in all the panels was detected by secondary antibody
conjugated with Alexa Fluor 488 (green; A1, B1, C1). The cells
were labeled with PKH26 (red; A2, B2, and C2). The nuclei were
visualized with DAPI (blue; A3, B3, C3). C3 is a merged image
of myosin heavy chain (green) counterstained for Cx-43 expression
Figure 6 (Continued). (cyan) and DAPI (blue). Merged images
(A4 and C3 to C4) showed extensive myogenesis in group 3
animal hearts compared with group 2 (B4) in the center of the
infarct region. C4 shows colocalization of myosin heavy chain
(green) and Cx-43 (cyan) in the newly formed myofibers in the
cell-engrafted region (red). Large yellow and red boxes are magnified in C4 to show colocalization of Cx-43 (cyan), myosin
heavy chain (green), and PKH26 (red). D1 through D4, Ultrastructure studies of the rat heart tissue at 6 weeks in group 3.
D1 shows tight junctions between 2 adjacent differentiating cells
(yellow box). D3 shows typical newly differentiating myofibers
with visible filaments (red box). D2 and D4 represent magnified
images of D1 and D3, respectively.
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May 19, 2009
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Figure 7. Immunostaining of rat heart tissue sections for von Willebrand factor (vWFactor) VIII
(green) in the infarct (A1 to A3) and peri-infarct (A4
to A6) areas at 6 weeks in group 1 (A1 and A4),
group 2 (A2 and A5), and group 3 (A3 and A6). The
nuclei were visualized with DAPI (blue). A7, Blood
vessel density was highest in group 3 in the infarct
and peri-infarct areas. B1 to B4, Double immunostaining of rat heart tissue samples for von Willebrand factor VIII (green) and smooth muscle actin
(blue) for determination of maturation index of the
newly formed blood vessels in group 3. Blood vessel maturation index changed insignificantly
between the 3 treatment groups. Magnification
⫻400.
of heart cell therapy is attributed to release of paracrine
factors that are accentuated by their genetic and pharmacological manipulation.6,31 Western blotting showed copious
expression of IGF-1 from Sca-1⫹ under OGD in vitro and in
the infarcted heart until day 7 of observation. Additionally,
we observed that short-term IGF-1 treatment for preconditioning and the continuous presence of IGF-1 during OGD
had distinct effects on stem cells in terms of cardiomyogenic
gene expression. First, the timing of IGF-1 paracrine release
was optimal to provide a second window of preconditioning
when the effects of initial in vitro preconditioning vanished.
Second, in agreement with our in vitro results from supplementation of recombinant IGF-1 for 7 days in culture under
normoxia as well as anoxia, the duration of IGF-1 paracrine
release in vivo spanning 7 days prompted expression of
cardiac-specific marker proteins including Cx-43. Therefore,
PC
Sca-1⫹ manifested enhanced cardiomyogenesis compared
with their non-PCSca-1⫹ counterparts. This was evident from
immunohistological studies in which ␣-sarcomeric actinin–
and myosin heavy chain (slow isoform)–positive structures
were colocalized with Cx-43 expression, thus signifying stem
cell engraftment and coupling with existing fibers. We
therefore inferred that the cardiomyogenesis observed in this
study was due to extensive transdifferentiation of PCSca-1⫹.
Although the neofibers were connected by gap junctions,
their real contribution to contractility was not determined.
Additional studies are therefore needed to demonstrate that
neofibers were functionally competent and contributed to
improved heart function. On the basis of the present data, it is
safe to say that improvement in global heart function was due
to implantation of mesenchymal stem cells forming neofibers
and release of paracrine factors.1,2
We also discovered that Cx-43 expression indeed played
an important role in the cytoprotective effects of preconditioning. As discussed earlier, Cx-43 is responsible for electric
coupling and maintenance of homeostasis between the adjacent cardiomyocytes. In other cells such as astrocytes, gap
junctions composed of Cx-43 reduced apoptotic neuronal
damage in cerebral ischemia.32 Moreover, Cx-43 hemichannels are capable of responding to extracellular cues and
induce survival signals via extracellular signal–regulated
kinase activation.33 Cx-43 is also located in the mitochondrial
membrane of cardiomyocytes and is upregulated in response
to ischemic preconditioning.34 Additionally, Cx-43 translocates into the mitochondrial membrane in response to ischemic stress.35 In both cases, Cx-43 becomes a modulator of
mitochondrial functions during cell apoptosis. We observed
that abrogation of Cx-43 in Sca-1⫹ caused poor cell survival
under OGD and in the infarcted heart. Although the mechanism of Cx-43 in conferring cytoprotection after preconditioning requires more in-depth studies, our data showing
punctate and colocalized distribution of Cx-43 and cytochrome c in Sca-1⫹ cells confirm its mitochondrial distribution. Supporting an antiapoptotic role for Cx-43, further
studies will be required to delineate the mechanism by which
Cx-43 prevents cytochrome c translocation from the mito-
Lu et al
Role of Preconditioning-Induced Connexin-43
9
ularly interested in the Sca-1⫹ cell population because of the
association of Sca-1 antigen with cell growth activity and
differentiation potential.38 Sca-1⫹ cells have wide distribution
in the body tissues including skeletal muscle, heart, and bone
marrow and have been manipulated to adopt cardiac phenotypes.39 High-level Sca-1 cells expressing mesenchymal multipotent stem cells differentiate into cardiomyocytes.40 Further studies should focus on defining Sca-1⫹ with multiple
surface markers to select sublineages with greater cardiomyogenic capacity and on assessing functional coupling. The
cytoprotective role of IGF-1–induced Cx-43 could implicate
Bcl10 and nuclear factor-␬B signaling. Despite these study
limitations, the overwhelmingly beneficial effects of IGF-1
preconditioning, as well as the simplicity, reproducibility,
easy adoptability, and lack of safety issues, make this
approach highly appealing for clinical applications.
Sources of Funding
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
This work was supported by National Institutes of Health grants
R37-HL074272, HL-080686, HL087246 (Dr Ashraf), and
HL087288 and HL089535 (Dr Haider).
Disclosures
None.
References
Figure 8. A1 to A2, Measurement of the infarction size by Masson’s trichome staining on formalin-fixed, paraffin-embedded
histological sections. Infarction size was significantly attenuated
in group 3 compared with group 1 and group 2. B to E, Indices
of heart function including ejection fraction (EF), fractional shortening (FS), LV chamber dimensions (end-diastolic dimension
[EDD] and end-systolic dimension [ESD]), and wall thickness
improved significantly in cell-transplanted groups 2 and 3 compared with group 1 at 6 weeks after their respective treatment.
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CLINICAL PERSPECTIVE
The hostile microenvironment of ischemic myocardium causes extensive cell death. Preconditioning of stem cells by
short-term treatment with growth factor is shown here as a simple, safe, effective, and potentially therapeutic intervention
to promote their survival after engraftment. Insulin-like growth factor-1 preconditioning also promoted connexin-43
translocation to the mitochondria. Studies are being performed to investigate a possible role for the mitochondrial
connexin-43 in cytoprotection. The potential benefits of preconditioning the stem cells before transplantation are enormous
and will revolutionize our thinking about strategies for cell-based therapies.
Sca-1+ Stem Cell Survival and Engraftment in the Infarcted Heart. Dual Role for
Preconditioning-Induced Connexin-43
Gang Lu, Husnain K. Haider, Shujia Jiang and Muhammad Ashraf
Circulation. published online May 4, 2009;
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CIRCULATIONAHA/2008/827691/R3
SUPPLEMENTAL MATERIAL
Sca-1+ stem cell survival and engraftment in the infarcted heart: dual role for
preconditioning induced connexin-43
Gang Lu M.D. PhD, Husnain Kh Haider MPharm PhD., Shujia Jiang MD., Muhammad
Ashraf PhD. Department of Pathology and Laboratory Medicine, 231-Albert Sabin Way,
University of Cincinnati, Ohio-45267-0529
Supplemental Methods.
Selection of bone marrow derived Sca-1+. Bone marrow was harvested from young (68 weeks old) male C57Black-6 mice by flushing the cavities of femurs and tibias with
DMEM supplemented with 20% fetal bovine serum (FBS). Bone marrow from each
mouse was plated into one 150 mm2 culture dish and cultured in DMEM plus 20% FBS.
Fresh medium was added every 3 days. Adherent cell cultures at 70% confluence were
harvested and selected for Sca-1+ cell population using mouse Sca-1 cells selection
cocktail (Stem Cell Technologies Inc, Vancouver, Canada) as per manufacturer’s
instructions. The selected Sca-1+ cells were maintained and expanded for no more than 5
passages for later studies.
Flowcytometry. The cells were harvested with cell dissociation solution (Sigma, Saint
Louis, MI). 1x106 cells were then re-suspended with 100μl FACS solution (PBS
containing 1% bovine serum albumin (BSA) and 1mM EDTA). After blocking for nonspecific binding, the cells were incubated with 0.5μg PE-conjugated anti-Sca-1
(Cedarlane Laboratories Ltd, Hornby, Canada) at 4°C for 15-min. After washing twice
with FACS solution, cells were resuspended in 500μl FACS solution and analyzed by
1
CIRCULATIONAHA/2008/827691/R3
flow cytometry (FACSCalibur, BD Bioscience, San Jose, CA). Unlabelled cells were
used as reference and whole bone marrow stem cells or unrelated cells were incubated
with anti Sca-1 to confirm the specificity of the labeling.
In Vitro studies
Cytoprotection under Oxygen and Glucose Deprivation (OGD). Sca-1+ cells were
grown in DMEM supplemented with 20% FBS. The day before OGD, they were 70~80%
confluent and the medium was switched to DMEM containing 0.5% FBS. Recombinant
IGF-1 (R&D System, Minneapolis, MN) was used to precondition the cells at 100 nM for
30 min whereas in control group, the cells were treated with 0.1% BSA in PBS. After
three washes with glucose and sodium pyruvate free DMEM (Invitrogen, Calsbad, CA),
the cells were subjected to anoxia (95% N2/5% CO 2 ; Forma Scientific Corp) for 8-h.
Cell viability was measured with multiple readouts. Intracellular lactate dehydrogenase
(LDH) release was measured using Homogeneous Membrane Integrity Assay (Promega,
Madison, WI) as an indicator of cell membrane integrity and viability. The percentage of
apoptotic cells was determined with the annexin-V/propidium iodide (PI) Apoptosis
Detection kit (Sigma, Saint Louis, MI) according to manufacture’s procedure. JC-1
staining (Invitrogen, Calsbad, CA) was performed as an indicator of early apoptotic cells
to assess change in mitochondrial membrane potential. Cytochrome-c (BD Pharmingen,
San Jose, CA) staining was performed to evaluate the integrity of mitochondrial
membrane.
Western blotting. Protein lysates from cultured cells were lysed with ice cold lysis
buffer (50mM Tris-HCl, pH 7.6, 120mM NaCl, 0.5% NP-40, 1mM EDTA, 0.1mM
2
CIRCULATIONAHA/2008/827691/R3
PMSF, 2µg/ml Leupeptin, 2µg/ml Aprotinin) on ice for 30 min. Protein lysates from
heart tissues were harvested with Tissue Protein Extraction Reagent (Pierce, Rockford,
IL) according to manufacturer’s protocol. Samples were centrifuged at 14,000 rpm for
10-min and the supernatants were collected. Protein concentrations in the samples were
quantified with Bio-Rad DC-Protein Assay Reagent (Bio-Rad, Hercules, CA).
Sub-cellular fractionation was carried out to obtain mitochondrial protein fraction and
cytoplasmic protein fraction for Westrn blotting using ProteoExtract
Cytosol/Mitochondria Fractionation kit (Calbiochem, USA) per instructions of the
manufacturer.
The protein samples were electrophoresed on a precast 4~12% gradient gel
(Invitrogen, Calsbad, CA) using 20~50µg protein samples and electroimmunoblotted as
described earlier.1 The primary and secondary antibodies used for detection of the desired
antigens have been listed in Online Table-I.
RNA interference. Small interfering RNA (siRNA) duplexes for mouse Cx-43 were
purchased from QIAGEN (Valencia, CA). The sequences were: sense, r(GAAUUCACA
CAGUGUUCAA) dTdT antisense, r(UUGAACACUGUGUGAAUUC) dGdA. Scramble
siRNA (Sc RNAi) was purchased from Dharmacon (Chicago, IL). Sca-1+ cells were
plated in 6-well plates at 1.3x105 per well for 24-h. Cx-43 specific siRNA transfection of
the cells was performed with HiPerfect Transfection Reagent (QIAGEN) per instruction
of the manufacturer. Cells transfected with scramble siRNA were used as a control. Cell
lysate samples were collected from the transfected cells at 48-h after transfection for
Western blotting. Parallel studies were performed under OGD using cells transfected with
Cx-43 siRNA and scramble siRNA to evaluate cell viability.
3
CIRCULATIONAHA/2008/827691/R3
To study the effect of Cx-43 knock-down on in vivo cell survival, 1x106 male
Sca-1+ cells transfected with Cx-43 specific siRNA or scramble siRNA were injected
intramyocaridally in the female rat heart model of acute myocardial infarction (n=4 per
group) (the details of acute myocardial infarction models have been described elsewhere
in the manuscript). The animals were harvested on day-7 and the tissue samples were
processed for sry gene analysis by real-time PCR for quantification of the surviving cells
in the infarcted heart.1
Experimental animal model and cell transplantation. The study conformed to the
Guidelines for the Care and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised 1985) and protocols approved by
the Institutional Animal Care and Use Committee, University of Cincinnati.
Young female Fischer-344 rats (6-8 weeks) were anesthetized by intraperitoneal
injection of Ketamine/Xylazine (87mg/kg and 13mg/kg respectively). An experimental
model of acute myocardial infarction was developed in 78 animals by the left anterior
coronary artery ligation as described earlier.2 The animals received intramyocardial
injections of 70μl basal DMEM without cells (DMEM, group-1) or containing 1x106
male Sca-1+ cells, either non-preconditioned (non-PCSca-1+ cells; group-2) or
preconditioned by IGF-1 treatment (100 nM) (PCSca-1+; group-3). The cells were labeled
with PKH26 cell tracker dye to identify and study their fate post engraftment. The
injections were performed at multiple sites (4-5 sites/ animal heart) in the free wall of the
left ventricle (LV). The chest of the animals was sutured and the animals were allowed to
recover in an oxygen perfused chamber. The animals were harvested at different time
points for use in respective experiments.
4
CIRCULATIONAHA/2008/827691/R3
Donor cell survival and cardiac marker expressions. The DNA of the heart tissue from
groups-2 and 3 (n=4 per group per time point) was extracted on day 1, 4, and 7 using
Genomic DNA extraction Kit (QIAGEN). DMEM injected group-1 (n= 4) served as a
negative control. The amount of DNA was quantified with a spectrophotometer. The
primers used to amplify mice sry were purchased from QIAGEN (Cat#: QT01038555).
Real-time PCR was performed with SYBR Green Master Mix (QIAGEN), 200 ng
genomic DNA, 10x sry primers and PCR grade water. The reaction was run with Bio-Rad
iQ-5 i-cycler and the program was run; denaturing at 95oC for 15-min, 45 cycles of 95oC
15-seconds (s), 55oC 30-s and 72oC 30-s. Upon completion of PCR, a melting curve was
run from 55oC to 95oC with an increment of 1oC and a dwelling time of 15-s to verify the
purity of amplification product. To assess the quantification efficiency, different numbers
of cells (1x103, 1x104, 1x105) were mixed with the same amount of female heart tissue
and their DNA was isolated for sry-gene amplification. To measure the expression of
cardiac specific markers, RNA isolation and reverse transcription were performed with
Trizol Reagent and Cloned AMV First-Strand cDNA Synthesis Kit according to
manufacturer’s protocol (Invitrogen, CA). The sequence of primers used for real-time
PCR has been shown in Online Table-II. The primary curve method was used to calculate
the threshold cycle (C t ) which is defined as the cycle at which the fluorescence level
reaches a predetermined threshold.2 C t was measured for each reaction and used to
calculate the fold change of each experimental sample as compared with the control
according to the equation:
fold change = 2–
Ct
, where C t = (C tTarget ) Experimental – (C tTarget ) Control .
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CIRCULATIONAHA/2008/827691/R3
Histological studies. Briefly, 5 μm thin cryosections were fixed in 4% paraformaldehyde,
blocked with 5% goat serum in phosphor buffered saline (PBS), and used for
immunoreactivity studies with specific primary antibodies at 4oC overnight as described
earlier.3 The antibodies used have been listed in Online Table-I. The primary antigen–
antibody reaction was detected with goat anti-mouse or anti-rabbit IgG conjugated with
Alexa Fluor-488 or Alexa Fluor-546 (Invitrogen, Calsbad, CA). Nuclei were
counterstained with DAPI. The samples were examined using fluorescence microscope
(BX41, Olympus, Tokyo, Japan) or confocal microscopy (Leica, Germany).
Blood vessel density was assessed with von Willebrand factor VIII (vWF-VIII)
and/or smooth muscle actin (SMA) immunostaining as described earlier.1 The blood
vessels positive for vWF-VIII and/or SMA were counted in the infarct and peri-infarct
regions. At least 5 high power microscopic fields (x400) each in infarct and peri-infarct
regions, were randomly selected and counted from at least 3 sections from each animal
(n=4 animals per group). Blood vessel density was expressed as the number of vessels
per microscopic field (x400). Blood vessel maturation was assessed by the ratio of SMA
positive blood vessels to vWF-VIII positive.
Electron microscopy was on rat heart tissue samples for ulltrastructural studies as
described earlier.1 Infarct size and area of fibrosis were measured (n=4 per group) after
hematoxylin/eosin staining and Masson’s trichome staining using computer based
planimetry with Image J analysis software (version 1.6065; NIH).3
Heart function studies. Transthoracic echocardiography was performed to evaluate heart
function at 6-weeks after cells engraftment as previously described.1 The hearts (n= 8 per
group) were imaged in 2D and M-Mode, and recordings were obtained from the
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parasternal long axis view at the papillary muscles level using Compact Linear Array
probe CL10–5 on an HDI-5000 SONOS CT. Anterior and posterior end-diastolic and
end-systolic wall thickness and LV end-systolic (LVESD) and end-diastolic (LVEDD)
dimensions were measured from at least 3 consecutive cardiac cycles. Indices of LV
systolic functions, including LV fractional shortening (LVFS) and LV ejection fraction
(LVEF) were calculated using the following equations:
LVFS= (LVEDD-LVESD)/LVEDD*100 and LVEF= [(LVEDD3-LVESD3)/LVEDD3] *100
Supplemental References
1-
Niagara MI, Haider H, Jiang S, Ashraf M. Pharmacologically preconditioned
skeletal myoblasts are resistant to oxidative stress and promote angiomyogenesis
via release of paracrine factors in the infarcted heart. Circ Res. 2007; 100:545-55.
2.
Shujia J, Haider HK, Idris NM, Lu G, Ashraf M. Stable therapeutic effects of
mesenchymal stem cell-based multiple gene delivery for cardiac repair.
Cardiovasc Res. 2008; 77:525-33.
3.
Jiang S, Haider H, Idris NM, Salim A, Ashraf M. Supportive interaction between
cell survival signaling and angiocompetent factors enhances donor cell survival
and promotes angiomyogenesis for cardiac repair. Circ Res. 2006; 99:776-84.
Supplemental Figure Legends
Supplemental Figure-I. Real time PCR showing significantly abrogated CX-43
expression in Cx-43 siRNA transfected cells grown under normoxia and anoxia as
compared with Sc siRNA transfected cells grown under similar culture conditions.
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CIRCULATIONAHA/2008/827691/R3
Supplemental Figure-II. Real-time PCR based Gene Array analysis for a set of 86 genes
involved in apoptosis (Cell Signaling) showing 10 genes from the apoptotic cascade with
more than two fold increased expression in Cx-43 siRNA transfected cells as compared
with Sc siRNA transfected cells.
Supplemental Figure-III. Real-time PCR showed that the expression of Caspase
recruitment domain family, member 10 (Card-10) was sensitive to anoxia in the absence
of Cx-43. The expression of Card-10 was elevated in Cx-43 siRNA transfected cells to
more than 10-fold as compared with Sc siRNA transfected cells.
Supplemental Tables
Online Table-I. Primary antibodies for immunostaining.
Antibody
Source
Dilution used
Ki67
BD Pharmigen
1: 50
Cytochrome-c
BD Pharmigen
1: 200
Cardiac myosin heavy chain
AbCam
1: 50
Actinin
Sigma
1: 50
Connexin 43
Santa Cruz Biotech.
1: 50
vWillebrand factor VIII
Chemicon
1: 50
phspho-Akt
Cell Signaling Tech.
1: 100
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CIRCULATIONAHA/2008/827691/R3
Supplemental Table-II. Sequence of primers used for PCR studies.
Gene
Primer sequence
Product size (bp)
Connexin 43 forward 5’-GAACACGGCAAGGTGAAGAT
reverse 5’-GAGCGAGAGACACCAAGGAC
247
cTnI
forward 5’-AGGGCCCACCTCAAGCA-3’
reverse 5’-GGCCTTCCATGCCACTCA-3’
103
GAPDH
forward 5'-CCAAGGCTGTGGGCAAGGTC-3'
reverse 5'-GGCAGGTTTCTCCAGGCGG-3'
119
GATA 4
forward 5’-CCGGGCTGTCATCTCACTAT
reverse 5’-GCCTGCGATGTCTGAGTGAC
151
MEF2c
forward 5’-CATCAGCCATTTCAACAACA-3’
reverse 5’-CGGCATGTTATGTAGGTGCT-3’
140
Supplemental Table-III. Ct and delta Ct values to support the fold changes observed in
donor cell survival at various time points (as shown in Figure-5D) post-engraftment in
the infarcted rat heart.
Day 1
41.12
40.36
Non-PC
40.05
39.17
37.04
37.27
PC
36.9
36.87
Ct Value
Day 4
41.69
40.88
39.77
39.47
38.57
38.06
37.35
36.58
Day 7
43.09
42.24
41.73
40.54
39.66
39.35
39.09
38.66
9
Day 1
0.945
0.185
-0.125
-1.005
-3.135
-2.905
-3.275
-3.305
Delta Ct Value
Day 4
Day 7
1.515
2.915
0.705
2.065
-0.405
1.555
-0.705
0.365
-1.605
-0.515
-2.115
-0.825
-2.825
-1.085
-3.595
-1.515
Cx-43 (fold change)
1.5
1
0.5
0
Sc siRNA Cx-43 Sc siRNA
siRNA
Normoxia
Cx-43
siRNA
Anoxia
Supplemental Figure-I
Well ID
Apoptotic Gene Names
Fold change
A02
Apoptotic protease activating factor 1
2.37
C02
Caspase recruitment domain family, member 10
6.02
C03
Caspase recruitment domain 4
2.41
C06
Caspase 12
2.28
D02
Caspase 9
2.53
F03
PYD and CARD domain containing protein
2.59
F11
Tumor necrosis factor receptor superfamily, member 5
4.83
F12
Tumor necrosis factor (lignad) superfamily, member 10
2.21
Supplemental Figure-II
Card 10 (fold change)
15
10
5
0
Scramble Cx-43 Scramble Cx-43
siRNA siRNA siRNA siRNA
Normoxia
Anoxia
Supplemental Figure-III