Conceived and designed the experiments: JY AB WG HSB YY. Performed the experiments: Jy H. Shih RES Y. Zhang Y. Zhou. Analyzed the data: JY AB HSB YY. Wrote the paper: JY AB HSB YY.
The authors have declared that no competing interests exist.
Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.
Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1+CD45- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1+CD45- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells
These studies demonstrate that cloned Sca-1+CD45- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
Growing evidence demonstrates the existence of endogenous cardiac progenitor cells in the adult mammalian heart, which can divide and differentiate into cardiomyocytes, endothelial cells and smooth muscle cells, and potentially play an important role in maintaining normal cardiac homeostasis
During fetal development, the LIM homeodomain transcription factor Islet-1 (Isl1) is expressed in a cell population that gives rise to second heart field structures and the myocardial vasculature, and is accepted as a marker of endogenous cardiac progenitors
Questions remain regarding the behavior and cellular composition of CSs and their response to signals from the myocardial tissue environment, including: 1) whether acute myocardial infarction (MI) effects the generation of CSs; 2) whether CSs derived from injured myocardium have therapeutic potential to repair ischemically damaged hearts
To determine whether myocardial injury influences the generation of CS-forming cells, whole hearts including the infarct area were removed from mice following experimental MI, as well as from sham-operated and non-operated mice, and were cut to small pieces as “explants”. A monolayer of fibroblast-like cells migrated out from the cardiac explants over several weeks in culture. From this monolayer, small, round, phase-bright cells (CS-forming cells) were seen to emerge (
In addition to faster growth rates, the number of putative CS-forming cells harvested from hearts at 1-week (5.12×105±0.45×105/heart) and 2-weeks (3.75×105±0.52×105/heart) post-MI was significantly higher than those from sham-operated (2.20×105±0.70×105/heart) and non-operated hearts (1.67×105±0.26×105 cells/heart) (P<0.045) (
(A) Numbers of CS-forming cells harvested from hearts at 1-week and 2-weeks post-MI was significantly higher than those from sham-operated and non-operated hearts (N = 6). (B) Hearts at 1- and 2-weeks post-MI generated more CSs than sham-operated and non-operated hearts. # P<0.03. Hearts at 4-weeks post-MI produced similar number of CSs to sham-operated hearts (N = 6). Data in Figure shown as mean±SEM.
Consistent with our observation of an increase in CS-forming cell number with injury, we observed that the number of CSs derived from hearts harvested 1-week (354±50/heart) and 2-weeks (213±38/heart) post-MI were significantly higher than from sham-operated hearts (80±33/heart) (p<0.001) and non-operated hearts (18±14/heart) (P<0.01) (
We have demonstrated that at each of the three stages of CS formation (explant outgrowth, CS-forming cell generation and number of CSs), the proliferative was greater at 1–2 weeks post-MI, and this then returned toward baseline by 4 weeks post-MI.
To determine the CS generating potential of different regions of the heart, we separated the hearts 1-week post-MI into five regions: left ventricle (LV) excluding scar, right ventricle (RV), septum, left atrium (LA) and right atrium (RA), and cultured them separately. The number of CSs from each region of the heart was counted and adjusted for tissue weight. We observed no statistically significant regional differences in CS production (LV: 2.6±0.6 CSs/mg; RV: 3.8±1.0; septum: 2.3±0.5; LA: 2.2±0.2; RA: 2.9±0.7; P>0.05) (
We used fluorescence-activated cell sorting (FACS) to determine the cellular composition of CSs derived from control and infarcted hearts after 14 days in culture. CSs from non-operated hearts contained several populations of cells, based on their expression of Sca-1, c-kit, CD45, CD133, CD34, Flk1 and CD31 (
(A) Flow cytometric analysis of Sca-1, CD45 and c-kit expression in disaggregated CS cells. Typical results are shown (N = 6). (B, C) Bar graph showing the profile of progenitor cell markers in CSs by FACS (N = 6). (D) Immunocytochemical staining demonstrates that CS cells express Isl1, Nkx2-5 and GATA4. Arrows point to positive staining cells (red). Nuclei stained with DAPI. Scale bar = 35 µm and 15 µm (NKx2-5). (E) Bar graph shows the profile of Isl1, Nkx2-5, GATA4 positive cells in CSs by immunocytochemical staining. Data in Figure shown as mean±SEM.
Real-time RT-PCR was used to compare Isl1 expression in heart, indicated cells, and liver (negative control). Results show as Isl1 mRNA expression relative to Hypoxanthine phosphoribosyltransferase (HPRT). (A) Isl1 expression in CSs (N = 10) was 17-fold higher than in the hearts (n = 4). (B) Isl1 expression in Sca-1+CD45− cells (N = 6) derived from CSs was 3-fold higher compared to that in whole CSs (N = 10). (C) Isl1 expression in cloned Sca-1+ CD45− cells (n = 5) was similar to primary Sca-1+CD45− cells isolated from CSs (N = 6). Isl1 expression dropped significantly with differentiation (N = 3). CM: cardiomyocytes.
(A) The percentages of Sca-1+CD45− in CSs were not altered by MI (N = 6). (B) The percentages of total CD45+cells in CSs was increased at 1-week post-MI and attenuated at 2-week post-MI (N = 6). (C) Semi-quantitative RT-PCR analysis showed that CSs from all groups expressed similar level of Nkx2-5, GATA4, Flk-1 and SMA. HPRT was used as control. N: Non-surgery. S: Sham-operated. 1W: 1 week post-MI. 2W: 2 weeks post-MI. 4W: 4 weeks post-MI. H: mouse heart (positive control). SM: skeletal muscle (negative control). Data in Figure shown as mean±SEM.
To determine the source of Isl1 enrichment in CSs, we analyzed RNA from Sca-1+CD45− and CD45+ cells sorted from CSs, and found that Isl1 expression in Sca-1+CD45− cells was 3-fold higher than in whole CS cells, and that the CD45+ fraction did not express Isl1 (
We investigated whether Isl1 protein expression was detected in the post-MI hearts of “middle aged” mice using immunohistochemical staining and found Isl1+ cells in the epicardium at the border of the infarct region at 1 week post-MI (
To determine whether CSs from infarcted myocardium differentiate into cardiac cells
CS cells from 1-week post-MI GFP transgenic mice were injected into the peri-infarct zone (PZ) of syngeneic wild type mice 3 days post-MI. Injected cells were detected in the PZ at 25 days after delivery. The surviving cells expressed cardiac Troponin I (A), CD31 (B), and SMA (C). Nuclei were stained with DAPI. Typical results are shown (N = 7). Scale bars = 15 µm in A, B and C.
To determine whether transplantation of CSs promoted angiogenesis in ischemic myocardium, we quantified the capillary and arteriole density in hearts at day 25 post-injection The results showed a greater density of CD31+ vessels in the infarct zone (IZ) (10.0±1.9% vs. 5.3±2.8%, P = 0.002) and PZ (10.3±2.4% vs. 6.0±2.4%, P = 0.0004) in CS-injected versus control hearts (
At 25 days post-injection, engrafted cells resulted in both increased vessel density (A) and numbers of arterioles (B) in the infarct zone and peri-infarct zone, but not remote zone (N = 7). *P = 0.002, #P = 0.0004 (C) LVEF improved with CS cell injection compared to control. Each line represents the mean of one group (N = 6). (D) Mice treated with CS cells had smaller infarct sizes (circumferential extent of scar) compared to control (N = 7).
To determine whether injection of CSs from infarcted myocardium improved cardiac function in a MI mouse model, we evaluated left ventricular ejection fraction (LVEF) by echocardiography and measured infarct size by histochemical staining at 25 days post-injection. LVEF was significantly reduced from an average of 51.2±1.7% before MI to 35.1±2.9% at 2 days post-MI in both groups, with no significant difference between two groups (P>0.05). At 28 days post-MI (25 days post-injection), LVEF was significantly higher in the CS-injected group (37.5±3.5%) compared to control (30.9±6.6%, P = 0.03) (
In our studies, the Sca-1+CD45- subpopulation comprised the largest fraction of CS cells, and contained more Isl1+ cells.
To further investigate the origin of Sca-1+CD45- in CSs, chimeric mice were generated by transplantation of bone marrow cells isolated from GFP transgenic mice to lethally irradiated C57BL/6 mice. Flow cytometric analysis demonstrated that 88±0.5% of peripheral blood mononuclear cells were GFP+ in the chimeric mice 5 months post-transplantation (
To further investigate whether these cells play a key role in restoring cardiac function and reducing infarct size, we sorted Sca-1+CD45− cells from CSs from 1-week post-MI hearts of adult GFP transgenic mice, and clonally expanded these in culture from single cells. After culturing for 14 days, 5.6% (16/288) of the single cells grew to colonies. About 30% (3/10) of clones grew to >106 cells after 30 days in culture. To determine the cellular composition of cloned cells derived from a single Sca-1+CD45− cell, we analyzed the cell-types of cloned cells by FACS after 30 days at passage 4, 15 and 22. FACS analysis showed that 61.8±12.4% of the clonally derived cells was still Sca-1+CD45− (
(A) Flow cytometry showed that ∼60% of cloned cells were Sca-1+CD45−. Typical results are shown (N = 6). (B–D) Immunocytochemical staining showed that cloned Sca-1+CD45− cells expressed Isl-1 (B), Nkx2-5 (C) and GATA4 (D). Nuclei were stained with DAPI. Scale bar = 35 µm in B3; Scale bar = 15 µm in C3 and D3. Typical results are shown (N = 3).
After treatment with 5-azacytidine, transforming growth factor β1 (TGF-β1), and vitamin C
(A–C) After treatment with 5-azacytidine, TGF-β and vitamin C, cloned Sca-1+CD45− cells differentiated
To determine whether Isl1 expression persisted in clonally expanded Sca-1+CD45− cells, we compared Isl1 transcript levels between primary Sca-1+CD45− isolates, clonally expanded Sca-1+CD45− cells, and cardiomyocytes differentiated from Sca-1+CD45− clones by real-time RT-PCR. We found that expression of Isl1 was not significantly different between primary Sca-1+CD45− cells and cloned cells. However, Isl1 expression decreased in cardiomyocytes, consistent with differentiation beyond the progenitor stage (
To determine whether cloned Sca-1+CD45− cells can differentiate after implantation
Cloned Sca-1+CD45−GFP+ cells were injected into the peri-infarct zone (PZ) of infarcted myocardium of syngeneic wild type mice 3 days post-MI. Injected cells were detected by GFP expression 25 days after transplantation, but there was no evidence of cardiac differentiation at this time point (A) (N = 7). At 75 days post-injection (C–D), transplanted cells were detected in the PZ, and expressed CD31 (C) and SMA (D), but not Troponin I (B). Nuclei were stained with DAPI. Scale bar = 35 µm in A, B, C and D. Typical results are shown (N = 2).
To quantify the level of engraftment and persistence of injected cells in infarcted hearts, cloned Sca-1+CD45− (GFP+) cells (106) were injected into infarcted hearts of wild type mice. The injected hearts were harvested at 1 hour and 1, 3, 7, 14 and 25 days post-injection. RNA was isolated from whole heart and mRNA expression of GFP was quantified by real-time RT-PCR as a surrogate for the number of engrafted cells. The expression level of GFP in the heart collected 1 hour post-injection was used to represent 100% of injected cells. Approximately 15% and 4% of injected cells were detected in injected heart 3 and 7 days post-injection respectively. Approximately 3% of injected cells were detected 14 to 25 days post-injection (
To determine whether transplantation of cloned Sca-1+CD45− cells promote angiogenesis in ischemic myocardium, we quantified the capillary and arteriole density in hearts 25 days after cell-injection. The results showed that there were more CD31+ vessels at the IZ (13.2±2.9% vs. 6.2±2.4%, P = 0.001) and PZ (11.7±3.6% vs. 7.2± 1.9%, P = 0.02) in the cell-injected group vs. control (
At 25 days post-injection of cloned Sca-1+CD45− cells, engrafted cells resulted in both increased vessel density (A) and number of arterioles (B) in the infarct zone (IZ) and peri-infarct zone (PZ) but not the remote zone (RZ). *P<0.02, #P<0.009. HPF, high power field at 40X (N = 6). (C) Echocardiography showed that LVEF improved in mice treated with cloned cells compared to control (PBS). Each line represents the mean of one group (N = 6). (D) Left ventricular wall thickness evaluated by the echocardiography showed a significant increase in PZ wall thickness in mice treated with cloned cells versus control (N = 6). (E) Infarct size was determined morphometrically with picosirius red staining (N = 6). (F) Mice treated with cloned cells exhibited smaller infarct sizes than control, as measured by circumferential extent of the scar (N = 6).
To evaluate the effects of Sca-1+CD45− cells injection on cardiomyocyte apoptosis, we used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and co-staining troponin I in the hearts 25 days after cell-injection. Cell injection resulted in significant reduction in the number of TUNEL+/troponin I+ cells in PZ compared to control (0.25±0.17 vs. 1.08±0.35/low power field (LPF), P<0.05) (
To determine whether the cloned Sca-1+CD45− cells alone improve cardiac function in the MI mouse model, we evaluated LVEF by echocardiography and measured infarct size by histochemistry 25 days after cell injection. LVEF was significantly reduced from an average of 51.2±1.5% before MI to 36.3±2.0% at 2 days post-MI in both groups, with no significant difference between the two groups (P = 0.9). At 28 days post-MI (25 days post-injection), LVEF was significantly higher in the cell-injected group compared to control (39.7±3.2% versus 30.9±6.6%, P = 0.02) (
In this study, we have shown that: 1) there is a significant increase in the proliferative capacity of CS-forming cells isolated from the “middle aged” heart following acute MI resulting in a significant rise in the number of CSs
Previous studies
A previous report
It has been previously reported that CSs from non-infarcted hearts could differentiate into cardiac cells and preserve cardiac function
Recent reports have found Isl1+ cells in adult mouse, rat and human hearts
While cloned Sca-1+CD45− cells improved cardiac function post-MI in transplanted mice, we did not find evidence for differentiation of these cells into cardiomyocytes
A recent report has suggested that CSs are composed of fibroblasts, and not cardiac progenitor cells
There are several limitations to this study. First, under the experimental conditions used, we did not find evidence for differentiation of cloned Sca-1+CD45− cells into cardiomyocytes
In summary, our data suggest that the cloned Sca-1+CD45− cells derived from CSs from post-MI hearts are enriched in Isl1+ progenitors, have the characteristics of progenitor cells, and are an attractive source of autologous cells for myocardial therapy post-MI.
C57BL/6J mice and C57BL/6J GFP transgenic mice with chicken α-actin promoter driving EGFP expression were purchased from the Jackson Laboratory (Bar Harbor, Maine). All animals were housed in the animal care facility at The University of California, San Francisco (UCSF), and all experiments were approved by, and conducted in accordance with the guidelines of, the Institutional Animal Care and Use Committee of UCSF (Approval number: AN078431).
Nine month-old, male C57BL/6J mice were used for all experiments to simulate “middle-aged” subjects. Mice underwent total permanent ligation of the left anterior descending coronary artery (LAD) to induce MI, and hearts were collected 1, 2, and 4 weeks post-MI (n = 6/group). Hearts were also harvested from animals that had undergone sham operation or no surgery (n = 6/group). The surgical procedure for MI has been previously described
Bone marrow cells were harvested from 8–10 week old GFP transgenic mice and transplanted into lethally irradiated (9.5 Gy) 2 month -old C57BL/6 mice through tail vein injection (2x106 nucleated unfractionated cells per mouse). The expression of GFP by peripheral blood mononuclear cells was analyzed by FACS 5–6 months later. CSs were isolated from no-surgery or 2-week post-MI hearts of chimeric mice.
Nine month-old, male C57BL/6J GFP transgenic mice (n = 12) were used as CSs donors. One week post-MI, hearts were harvested and used to generate CSs for injection. CSs were dissociated into single cell suspension by Blendzyme 4 and resuspended with phosphate buffered saline (PBS). Alternatively, cloned Sca-1+CD45− cells were dissociated into single cell suspension by trypsin and also resuspended with PBS. These cells in 10 µl PBS were injected into the hearts of 3-day post-MI mice by ultrasound-guided injection, a technique developed and reported by our laboratory
CSs were generated using the method described by Messina et al.
CSs were dissociated into single cell suspension by Blendzyme 4 (5.6u/ml) (Roche). The following phycoerythrin (PE) or allophycocyanin (APC) conjugated rat anti-mouse antibodies and conjugated isotype-matched control antibodies were used: Sca-1-PE, c-kit-PE, CD133-PE, CD34-PE, CD45-APC, Flk-1-APC and CD31-APC (eBioscience). The cells were incubated with antibodies for 25 min on ice, washed with PBS containing 0.2% BSA, and analyzed by FACSCabilur with CellQuest software (BD Biosciences).
For cell sorting, the dissociated CS cells from hearts of 1-week post-MI GFP transgenic mice were stained by the following antibodies: Sca-1-PE and CD45-APC. The Sca-1+CD45− cells were sorted by FACSAria with FACSdiva software (BD Biosciences) and were dropped into a 96-well plate, one cell/well, on top of mitomycin-C treated murine embryonic fibroblast cells (Millipore, New Jersey). The cells were then cultured with CGM at 370C with 5% CO2. For isolating RNA, sorted Sca-1+CD45− cells or CD45+ cells from CSs were collected into a tube with 1 ml of CGM respectively.
Cloned Sca-1+CD45− cells were loaded into chamberslides coated with gelatin at 15,000 cells/cm2 in differentiation medium, treated by 5-Azacytidine (5 mM) for 3 days, and then we added TGF-β1 (1 ng/ml) and vitamin C (0.1mM) for three weeks to induce cardiomyocyte differentiation
The cells cultured in chamberslides were washed with PBS, fixed with cold methanol for 5 min or 4% paraformaldehyde/PBS for 15 min, and blocked with Dako antibody diluent (DakoCytomation, Carpinteria, CA) for 1 hour. When using mouse derived monoclonal antibody, we also used Rodent Block M (Biocare Medical, Concord, CA) blocking for 30 min. The cells were incubated with the following primary antibodies diluted in Dako antibody diluent at 40C overnight: rabbit anti-Nkx2-5, GATA4 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Isl-1 (39.4D5) (Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-α SMA (Sigma), mouse anti-Troponin T (Thermo Fisher Scientific, Fremont, CA), mouse anti-SA (Abcam, Cambridge, MA), rabbit anti-connexin-43 (Sigma), mouse anti-CD31 (Abcam) and rabbit anti-VWF (Abcam). The cells were then incubated with the Alexa Fluor 546 labeled goat anti-rabbit antibody or goat anti-mouse antibody (Invitrogen) at room temperature for 1 hour. The slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and viewed with a Nikon E800 fluorescence microscope using Openlab software (Improvision, Lexington, MA).
Acetylated low density lipoprotein labeled with Dil-ac-LDL (Invitrogen) was added into the medium with cells at 2ug/ml as final concentration and incubated at 370C with 5% CO2 for 1 hour. The medium was removed. The cells were washed with PBS and fixed with 4% paraformaldehyde/PBS for 15 min. The slides were mounted and viewed same as immunocytochemical staining.
The total RNA from CSs and tissues were isolated by TRIzol reagent (Invitrogen). cDNA was generated from 0.3 µg of total RNA by using SuperScript III First-Strand Synthesis kit (Invitrogen). RT-PCR was performed using 1 µl of cDNA and Advantage 2 PCR kit (Clontech, Mountain View, CA) with the following program: 95°C 3 min, (95°C 30 s– 68°C– 3 min)×30 cycles, 68°C 10 min. PCR products were separated on 2% agarose gel. Every pair of PCR primers was designed to span one or several introns to distinguish the signals amplified from genomic DNA contamination. The primers sequence of Nkx2-5, GATA4, Flk-1, SMA and internal control hypoxanthine phosphoribosyltransferase (HPRT) are from previous publications
The total RNA from sorted cells was isolated and the cDNA was generated by Taqman Gene Expression Cells-to-Ct kit (Applied Biosystems, Foster City, CA, USA). The primers and probe for murine Isl1 and HPRT were purchased from Applied Biosystems. The real-time PCR were performed by ABI PISM 7300 (Applied Biosystems) using Taqman Master Mix (Applied Biosystems) in duplicates and the average threshold cycles (CT) of duplicate were used to calculate the relative value of Isl-1 in different cells and tissues. The CT for HPRT was used to normalize the samples. Expression of Isl1 mRNA relative to HPRT mRNA was calculated based on the CT, ΔCTIsl1 = CTIsl1-CTHPRT. The relative values of Isl1 were calculated as 2−ΔCTIsl1.
For studying the retention of injected cloned GFP+ Sca-1+CD45− cells, total RNA from whole heart was isolated by TRIzol, genomic DNA was removed from total RNA by RNeasy Mini Kit with RNase-free DNase (Qiagen) and 25-ng cDNA was used for real-time PCR. The sequences of primers and probes for GFP and histone 3.3A were as previously published
Tissue was analyzed by two blinded reviewers. Mice were sacrificed 25 days post-injection of cells (28 days post-MI) or 75 day post-injection. The hearts were arrested in diastole with KCl, perfusion and fixed with 10% formalin, embedded in paraffin, cut into 5 mm sections and blocked with Dako antibody diluent for 1 hour. When using mouse or rat derived monoclonal antibody, we also incubated the sections with Rodent Block M or R blocking for 30 min. To detect GFP and troponin I double positive cells, sections were stained with anti-troponin I (Abcam) and rabbit anti-GFP (Invitrogen) overnight at 4°C. Alexa Fluor 546 goat anti mouse IgG and Alexa Fluor 660 goat anti rabbit were used as secondary antibodies (Invitrogen). Detection of GFP and CD31 double positive cells were stained with rat anti-CD31 (Biocare Medical) and Alexa Fluor goat anti rat 546 were used secondary antibodies. GFP and α-SMA double positive cells were stained with mouse anti α-SMA and without prior antigen retrieval but otherwise followed the steps described above. The slides were mounted with ProLong Gold antifade reagent with DAPI and viewed with a Nikon E800 fluorescence microscope using Openlab software.
In order to assess vascular density in the hearts, the sections from mid-ventricular level were stained by antibodies of rat anti-CD31 and mouse anti- α-SMA at room temperature for 1–2 hours. A CD31 signal was detected using a Rat on Mouse HRP-Polymer kit (Biocare) and 3,3′Diaminobenzidine (DAB) (Biocare). α-SMA signal was detected by a MM AP-Polymer kit (Biocare) and a Vulcan Fast Red Chromogen kit (Biocare) for color development. The slides were mounted and observed as described above. ImagePro Plus 6.0 software (MediaCybernetics, Bethesda, MD) was used to analyze the percentage area occupied by CD31 positive vessels
For Isl1 staining, the hearts were perfused and fixed with 4% paraformaldehyde overnight, equilibrated with 20–30% sucrose and frozen in OCT for tissue sectioning using a cryostat. The sections were blocked with Rodent Block M and Dako antibody diluent for 30 min respectively, stained with mouse anti-Isl-1 (39.4D5) overnight at 4°C and Alexa Fluor 546 goat anti mouse IgG used as secondary antibody.
TUNEL staining was performed with ApopTag® Plus Peroxidase
In order to assess the size of infarct scar, the sections from mid-ventricular level (mid-papillary) were stained by picosirius red. The scar was stained as dark red. The slides were mounted and viewed same as above. All histological sections were examined with a Nikon Eclipse E800 microscope using a 1x objective with the use of Openlab software (Improvision, Lexington, MA). To assess the circumferential extent of the infarct, the epicardial and endocardial infarct lengths, epicardial and endocardial circumferences of LV were traced manually using the ImagePro Plus 6.0 software. Epicardial infarct ratio was obtained by dividing the epicardial infarct length by the epicardial circumference of LV. Endocardial infarct ratio was calculated by dividing the endocardial infarct length by the endocardial circumference of LV. The circumferential extent of the infarct scar was calculated as [(epicardial infarct ratio + endocardial infarct ratio)/2]×100.
Echocardiography was accomplished under isoflurane anesthesia with the use of a Vevo-660 (VisualSonic, Toronto) equipped with a 30 MHz transducer. Echocardiograms were obtained at baseline, 2 days post-MI (before injection), and day 28 post-MI. We measured LVEF and wall thickness. Wall thickness was measured at the apical anterior wall (infarct wall thickness) and at the mid-anterior segment (peri-infarct wall thickness) separately on the parasternal long-axis view; posterior wall thickness was obtained at the papillary muscle level. Three cycles were measured for each assessment and average values were obtained
One way ANOVA with Fisher's post hoc test was used to analyze the difference among multiple groups. Student's t test was used to analyze differences between two groups. Values were expressed as mean±SD unless otherwise specified, with P<0.05 considered significant. SPSS 15.0 software was used to conduct all statistical analysis.
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We thank Drs. Zhien Wang, Junya Takagawa, Muhammad Khan, Yagai Yang, Meenakshi Gaur and Mr. Brian Lee for technical input and assistance.