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Cardiovascular Research Advance Access originally published online on April 23, 2008
Cardiovascular Research 2008 79(3):395-404; doi:10.1093/cvr/cvn097
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation

Seyedhossein Aharinejad1,2,*, Dietmar Abraham2, Patrick Paulus2, Karin Zins2, Michael Hofmann2, Wolfgang Michlits2, Mariann Gyöngyösi3, Karin Macfelda4, Trevor Lucas2, Karola Trescher4, Michael Grimm1 and E. Richard Stanley5

1 Department of Cardio-Thoracic Surgery, Medical University of Vienna, Waehringerguertel 18–20, A-1090 Vienna, Austria
2 Laboratory for Cardiovascular Research, Department of Anatomy and Cell Biology, Medical University of Vienna, Waehringerstrasse 13, A-1090 Vienna, Austria
3 Department of Cardiology, Medical University of Vienna, Waehringerguertel 18–20, A-1090 Vienna, Austria
4 Center for Biomedical Research, Medical University of Vienna, Währingerguertel 18–20, A-1090, Vienna, Austria
5 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, New York, NY 10461, USA

* Corresponding author. Tel: +431 4277 61119; fax: +431 4277 61120. E-mail address: seyedhossein.aharinejad{at}meduniwien.ac.at

Received 22 January 2008; revised 4 April 2008; accepted 10 April 2008

Time for primary review: 15 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure.

Methods and results: Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT–PCR (reverse transcription–polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias.

Conclusion: Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

KEYWORDS Heart failure; Myoblasts; Growth factor; Gene therapy


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
For end-stage heart failure patients, cardiac transplantation is the only accepted treatment being offered and only to a small percentage of patients. Therefore, approaches that bypass heterologous organ transplantation are highly desired. Myoblast transplantation has recently received considerable attention as a means of replacing lost cardiomyocytes. Skeletal myoblasts possess clinically attractive characteristics, including ease of procurement, growth potential in vitro, and strong resistance to ischaemia.1 The ability of engrafted skeletal myoblasts to improve post-infarction cardiac function and to withstand long-term cardiac-type workload has been shown in both animal and human studies.1–9

Although efficacy data resulting from early results of phase-I clinical trials involving small patient cohorts are encouraging, the large multicentre MAGIC trial, which featured a randomized, double-blind, placebo-controlled dose-ranging design will assess the effect of myoblast transplantation on heart function and will also provide comprehensive safety data and thus allow a more objective assessment of the risk-benefit ratio.10 A major concern in myoblast transplantation, besides the caveat of arrhythmic events, is the loss of a significant fraction of grafted myoblasts upon engraftment. In a long-term follow-up study in heart failure patients treated with myoblast transplantation, >50% of grafted segments exhibiting systolic thickening recovery in the short-term was reduced to 33% at long-term. This was consistent with a high rate of cell death, which was incompletely compensated for by proliferation within the scar.11 Consequently, the efficacy of myoblast transplantation is hampered by these facts and the clinical use of cell therapy based upon skeletal myoblasts is still far from being a successful therapeutic measure.

Thus, this study focused on myoblast manipulations that enhance graft survival and proliferation on the one hand and increase blood vessels in the fibrous tissue within the infarcted myocardium on the other.10 We hypothesized that myoblasts genetically engineered to overexpress Colony- stimulating factor-1 (CSF-1) prior to transfer to the failing heart following myocardial infarction would improve graft survival and cardiac function. The rationale for this hypothesis is two-fold: First, CSF-1 accelerates angiogenesis,12 and affects macrophage-induced production of matrix metalloproteases (MMPs) that leads to extracellular matrix remodelling.13,14 Secondly, proliferation of L6{alpha}1 rat myoblasts is regulated in an autocrine fashion by CSF-1 and this regulation is lost as they differentiate to myotubes.15


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Experimental design and animals
A total number of 270 male Sprague Dawley rats (Harlan, Borchen, Germany) were coded for experiments. Due to haemodynamic compromise, ventricular fibrillation (VF), myocardial rupture, and bleeding following myocardial infarction, the number of surviving animals with comparable base-line cardiac function was 170. These animals were randomly divided into groups of n = 20 and treated with intramyocardial injection of: mouse CSF-1-overexpressing autologous rat myoblasts (MB-CSF-1), DiIC18-labelled CSF-1-overexpressing autologous rat myoblasts (DiI MB-CSF-1), autologous rat myoblasts (MB), DiIC18-labelled autologous rat myoblasts (DiI-MB), recombinant human CSF-1 (recCSF-1), mouse pCIneo-CSF-1 plasmid (pl-CSF-1), or culture medium (control). Sham operated rats (n = 20) were additional controls.

Rats were anaesthetized,14 intubated tracheally and ventilated with an oxygen/isoflurane mixture before undergoing a left lateral thoracotomy. The branch of the left coronary artery was either ligated with a 7-0 polypropylene snare (Ethicon, Somerville, NJ, USA) or left intact in sham procedure. At day (d) 7, left ventricular (LV) function was assessed by echocardiography. At d21, re-thoracotomy was performed, and 150 µL DMEM culture medium, 150 µL DMEM containing 5x106 MB, DiI MB, MB-CSF-1, DiI MB-CSF-1, 150 µL Ringer's solution containing 106 IU recCSF-1 or 10 µg pl-CSF-1 and Effectene (Qiagen, Hilden, Germany) were injected into the infarcted myocardium using a 30-gauge needle.16 The volume delivered was evenly divided between the central (two injections) and bordering (five injections) areas of infarcted myocardium. On d52 and d86, the LV function was assessed, and animals were sacrificed on d88. For measuring CSF-1 expression levels in vivo, additional animals (n = 6) of the control, MB, recCSF-1, pl-CSF-1, and MB-CSF-1 groups were sacrificed on d52. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.2 Myoblast culture and characterization
The right anterior tibial muscle was isolated, minced in phosphate buffered saline containing collagenase, and passed through a 100 µm sieve (BD, Franklin Lakes, NJ, USA). Cells were cultured in DMEM containing 10% foetal calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). Cultured cells were screened using myocyte specific anti-desmin (Neomarkers, Fremont, CA, USA) and fibroblast specific antibody (Harlan/Sera-Lab, Sussex, UK). The proportion of myoblasts was calculated (SIS image analysis, Münster, Germany) by dividing the number of desmin positive cells corrected to the total number of cells and ranged from 60 to 75%.

2.3 Myoblast proliferation assay
Myoblasts cell proliferation was determined using WST-1 Reagent (Roche Diagnostics, Indianapolis, IN, USA).

2.4 Echocardiography and electrocardiography
In anaesthetized rats, maximal LV long-axis lengths (L) and endocardial area tracing were measured using a 15 MHz linear array scan head to calculate LV end-diastolic (LVEDV) and end-systolic (LVESV) volume and ejection fraction (LVEF = LVEDV – LVESV/LVEDV). Measurements utilized three consecutive cardiac cycles. A telemetry device (DataSciences, Arden Hills, MN, USA) recorded ECG signals continuously.17

2.5 Heart-to-body weight ratio, histology, immunocytochemistry
Heart-to-body weight ratios were estimated.18 Heart tissue was partly formalin fixed, embedded in paraffin, or prepared for cryo-sectioning for performing histology and immunocytochemistry.

2.6 Fluorescence optical imaging
In experiments employing DiIC18-labelled myoblasts, three-dimensional cellular integration was assessed on an imaging station (IS2000MM, Kodak, Eastman Kodak Company, New Haven, CT, USA). The ventricles were then dissected, mechanically dissociated in collagenase and the DiIC18-labelled myoblasts detected on an inverted fluorescent microscope. Images were obtained and analysed using morphometry software (Lucia G, Optoteam, Vienna, Austria). The percentage of DiIC18-labelled myoblasts was determined for DiIC18-labelled MB and MB-CSF-1 groups.

2.7 Quantitative real-time reverse transcription–polymerase chain reaction and western blotting
Real-time RT–PCR (reverse transcription–polymerase chain reaction) and western blotting were performed as described.14,19

2.8 Statistical analysis
The Wilcoxon rank test and analysis of variance with Bonferroni's t-test were used to compare the data between the groups. Statistical tests were performed using SPSS software (version 10.0.7, SPSS, Chicago, IL, USA). Data are expressed as means ± SD. P-values of <0.05 were considered to indicate statistical significance.

Supplementary Materials and Methods are available online.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Colony-stimulating factor-1-transfected myoblasts improved myocardial function
Baseline d7 LVEF was not different between controls (27.6%), MB (29.5%), recCSF-1 (29%), pl-CSF-1 (27.7%) or MB-CSF-1 (28%), although it was significantly lower vs. the sham group (68%). LVEF in all treated animals on d52 did not differ significantly from LVEF on d7, although the average LVEF in the MB-CSF-1 group was the only one increased. LVEF on d86 was significantly lower in all treatment groups vs. sham; however, LVEF for MB-CSF-1 group (49%) was significantly increased vs. d7 and vs. d52. Importantly, LVEF was significantly elevated in MB-CSF-1 as compared with MB group on d52 and d86 (Figure 1A).


Figure 1
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  		Figure 1 Functional analysis of hearts. Left ventricular ejection fraction (LVEF; A) and left ventricular end-diastolic volume (LVEDV; B) within each group at different time points. Only colony-stimulating factor-1 (CSF-1)-transfected myoblasts (MB-CSF-1) improved cardiac LVEF and LVEDV significantly over time, while LVEF and LVEDV of untreated myoblasts (MB), the recombinant human CSF-1 (recCSF-1), CSF-1 expression plasmid (pl-CSF-1), and untreated control groups vs. sham operated rats (Sham) had lesser or no effects. Data are expressed as means ± SD. (Asterisk) significantly different from sham on days (d) 7, 52, and 86, respectively; A: P < 0.001; B: P < 0.001 for controls, MB, recCSF-1; P < 0.001, P < 0.001, and P = 0.004 for pl-CSF-1; and P < 0.001 and P = 0.004 vs. sham group on d7 and d52, respectively, for MB-CSF-1. (Dagger) significantly different within an individual group; A: P < 0.001 and P = 0.007, MB-CSF-1 vs. d7 and d52, respectively; B: P < 0.001, Control d52 vs. d7; P < 0.001 and P = 0.004, MB d52 and d86, respectively, vs. d7; P < 0.001, recCSF-1 d52 vs. d7; P < 0.001, pl-CSF-1 d52 vs. d7. (Double dagger) significantly different from MB (A: P < 0.001 for d52, and P < 0.001 for d86 vs. d52, and d86; B: P = 0.007 for d52, and P < 0.005 for d86 vs. d52 and d86.

 
The LVEDV was significantly elevated in controls, MB, recCSF-1, and pl-CSF-1 vs. sham. In contrast, LVEDV improved significantly in the MB-CSF-1 group on d86, although LVEDV on d7 and d52 was still significantly higher in MB-CSF-1 vs. sham. Of note, the LVEDV was significantly improved in MB-CSF-1 as compared with MB group on d52 and d86 (Figure 1B). ECG recordings showed that ventricular tachycardia (VT) or VF were absent in the sham group. Within 24 h following infarction, however, the animals developed at least one episode of VT or VF that terminated spontaneously. Short episodes of VT or VF were observed in 5 and 2 animals of the MB and MB-CSF-1 groups, respectively; whereas no ventricular arrhythmias were observed in other treatment groups. The arrhythmia episodes in MB and MB-CSF-1 groups occurred within 7 days after myoblast engraftment, were self-limited, and haemodynamically well tolerated. These data indicated that CSF-1-overexpressing myoblasts significantly improved LVEF and attenuated LV dilation associated with heart failure.

3.2 Colony-stimulating factor-1-transfected myoblasts attenuate myocardial remodelling
Despite similar initial reductions in LVEF by 1 week in the non-sham groups, the mean proportion of scar tissue/normal LV myocardium on d88 post-myocardial infarction was 34, 32, 36, and 33% in control, MB, recCSF-1, and pl-CSF-1 groups, respectively, and tended to be less in rats receiving MB-CSF-1 (29%) (Figures 2A and B). The thickness of the LV infarct wall was increased significantly in the MB-CSF-1 (1.76 ± 0.41 mm) group, but not in the MB (1.35 ± 0.4 mm), recCSF-1 (1.1 ± 0.29 mm) or pl-CSF-1 (1.29 ± 0.3 mm) groups as compared with the controls (1.04 ± 0.23 mm). In addition, the LV wall thickness increased significantly in the MB-CSF-1 compared with the MB group (Figure 2C). All groups had a significantly reduced wall thickness vs. sham (2.68 ± 0.22 mm). Heart-to-body weight ratio increased significantly in controls, in MB, in recCSF-1, and in pl-CSF-1 groups, but not in rats receiving MB-CSF-1 as compared with sham (Figure 2D). These results further indicated that by 12 weeks, CSF-1-overexpressing myoblasts attenuated myocardial remodelling and reduced ventricular dilation.


Figure 2
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  		Figure 2 Morphometric analysis of hearts. Representative images (A) and quantitative morphometric analysis (B) of collagenous scar tissue in left ventricular (LV) myocardium by Goldner trichrome stain on day 88 after infarction in the control, myoblasts (MB), the recombinant human colony-stimulating factor-1 (recCSF-1), CSF-1 expression plasmid (pl-CSF-1), and CSF-1-transfected myoblasts (MB-CSF-1) groups. LV infarct wall thickness (C) and heart-to-body weight ratio (D). Data are expressed as means ± SD. (Asterisk) significantly different from sham (C: P < 0.001; D: P = 0.002, P = 0.012, P = 0.003, and P = 0.029 for controls, MB, recCSF-1, and pl-CSF-1, respectively); (Dagger) significantly different from control, MB, recCSF-1, and pl-CSF-1 (P < 0.014).

 
3.3 Colony-stimulating factor-1 enhances myoblast survival and angiogenesis
To track the grafted myoblasts in the heart, unmodified (DiI-MB) and CSF-1-transfected myoblasts (DiI MB-CSF-1) were labelled with DiIC18. Cardiac function of the DiI-labelled MB group was comparable with the unlabelled MB group (data not shown). We employed both fluorescence optical imaging of the hearts and fluorescence microscopy to analyse changes in myoblast number on d88 post-myocardial infarction. Visual comparison of the DiIC18 images revealed marked differences in the size of the area containing myoblasts between DiI-MB and DiI MB-CSF-1 groups. Treatment with DiI MB-CSF-1 clearly increased the area of fluorescent myoblasts being found in both the infarction and the zone bordering the infarction region compared with DiI-MB-treated animals (Figure 3A). DiIC18–labelled myoblasts in both MB and MB-CSF-1 groups had not formed an organized tissue within the myocardium as shown by fluorescence microscopy, visualization of the nuclei by DAPI staining, and HE-staining in heart tissue sections of the same rats used for optical imaging (Figure 3B). The grafted myoblasts in MB and MB-CSF-1-treated animals were found in-between the cardiac myofibers and expressed skeletal muscle myosin heavy chain (Figure 3C). No fibrotic reaction was seen surrounding these areas of grafted and integrated cells (Figures 3B and C). The number of surviving myoblasts was significantly higher in the DiI MB-CSF-1 group as compared with the DiI-MB group (Figure 3D). To understand whether CSF-1 gene transfer affects myoblast proliferation, we first compared unmodified and CSF-1-transfected myoblasts in an in vitro proliferation experiment. At 72 h post-transfection, the CSF-1-transfected myoblasts had a significantly higher proliferation rate than control untreated myoblasts (Figure 3E). The proliferation enhancing effect of CSF-1 on myoblasts in vitro, however, was not detectable in vivo on d88, assessed by PCNA staining in both DiI MB and DiI MB-CSF-1 groups (Figure 3F).


Figure 3
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  		Figure 3 Localization of DiIC18-labelled autologous rat myoblasts (DiI-MB) and DiIC18-labelled colony-stimulating factor-1 (CSF-1)-overexpressing autologous rat myoblasts (DiI MB-CSF-1) in the heart, obtained 88 days after infarction. (A) Representative white-light images (left), colour-coded fluorescent map of DiIC18-signal (middle), and image overlays of the heart produced in Adobe Photoshop (right) showing the localization of DiIC18-labelled myoblasts (red/yellow) in the infarction area of the heart (Asterisk). The fluorescence signals from the implanted myoblasts in the heart are strongest in the DiI MB-CSF-1 group. LV, left ventricle; RV, right ventricle; the atriums are removed. (B) Representative red-channel fluorescence images of DiIC18-signal (left), image overlay of the red fluorescence images with the corresponding section counterstained with DAPI (middle) and HE-staining of the same section (right) in the border zone of the DiI-MB and DiI MB-CSF-1 groups. The HE stain (right) defines the morphology in the border zone of the infarction area with integrated myoblasts. The myoblasts are easily identified on the fluorescent optical images from the surrounding tissue, because of their bright fluorescence. (C) Representative images of myocytes stained by skeletal muscle myosin heavy chain specific antibody in both the MB (upper image) and MB-CSF-1 (lower image) groups, showing integration of transplanted myoblasts (arrows) into neighboring myocardial tissue Bar = 50 µm. (D) Quantitative analysis of DiIC18-labelled grafted myoblasts in the heart. Data are expressed as means ± SD. (Asterisk) significantly different from control and MB (P < 0.02). (E) CSF-1 gene transfer increases proliferation of myoblasts in vitro. Values are expressed as means ± SD. (Asterisk) significantly different from MB (P = 0.003) at 72 h. (F) PCNA stain (green) of DiIC18-labelled (red) MB and MB-CSF-1 transplanted groups. PCNA-positive MBs are not detected. Occasional PCNA positive proliferative events of non-MB cells are detected (insert).

 
In both the MB and MB-CSF-1 groups a large number of vessels was found in areas with transplanted myoblasts (Figures 4A–D). Consistent with these observations, the density of vWF-positive vessels and CD-31 protein expression were significantly higher in the border zone of the MB and MB-CSF-1 groups, while vascular density and CD-31 levels in the recCSF-1 group and vascular density in the pl-CSF-1 group were unchanged as compared with controls (Figures 4E and F). Importantly, the density of vWF-positive vessels in MB-CSF-1 group was significantly higher compared with the MB group (Figure 4E). In the myocardial infarction zone, the average vascular density and CD-31 expression levels in the MB, pl-CSF-1, and MB-CSF-1 groups were not significantly different from those in control and recCSF-1 groups (Figures 4E and F). These findings indicated that CSF-1 overexpression enhances long-term survival of myoblasts associated with increased angiogenesis.


Figure 4
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  		Figure 4 Analysis of angiogenesis. Representative vWF-stained sections in the myoblasts (MB) (A, B) and MB-colony-stimulating factor-1 (CSF-1) (C, D) groups, reveal a large number of vessels (arrows) in areas with transplanted myoblasts. Bar = 50 µm. Quantification of vWF-positive vessels in myocardial tissue of the border zone (BZ) and the myocardial infarction zone (MI) 88 days after infarction per high-power field (HPF; E). Representative western blot images and quantification of protein levels of CD-31 western blots in BZ and the MI zone 88 days after infarction. Actin was used as a loading control (LC) (F). Data are expressed as means ± SD. (Asterisk) significantly different vs. sham (E: P < 0.025; F: P < 0.001); (Dagger) significantly different vs. control [E: P = 0.001; F: P < 0.001 for MB and MB-CSF-1, respectively, and P = 0.01 for CSF-1 expression plasmid (pl-CSF-1)]; (Double dagger) significantly different from MB, recombinant human CSF-1 (recCSF-1), and pl-CSF-1 (P < 0.001).

 
3.4 Colony-stimulating factor-1 increases macrophage recruitment
The transfection efficiency in cultured myoblasts determined in preliminary experiments with a green fluorescent protein reporter plasmid on d2 after transfection was ~70% declining to 45% and 20% on d7 and d14, respectively (data not shown). CSF-1 mRNA was overexpressed significantly on d2, d7, and d14 following transfection as compared with untransfected control myoblasts, respectively (Figure 5A). In vivo gene expression analyses indicated that CSF-1 gene expression was still significantly increased on d52 post-myocardial infarction in the MB-CSF-1 group compared with others. On d88, however, no significant changes were detectable for CSF-1 mRNA levels (Figure 5B).


Figure 5
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  		Figure 5 Analysis of colony-stimulating factor-1 (CSF-1) expression and macrophages. (A) CSF-1 mRNA expression levels following transfection of myoblasts with CSF-1 expression plasmids in vitro, normalized to GAPDH. (Asterisk) significantly different from control P < 0.001). (B) Analysis of myocardial CSF-1 mRNA expression on days (d) 52 and 88 after myocardial infarction normalized to GAPDH. Data are expressed as means ± SD. (Asterisk) significantly different from other groups (P < 0.05). (C) Quantitative histo-morphometric analysis of myocardial tissue sections of the border zone on d88 after infarction stained with the macrophage specific RM-4 antibody. Data are expressed as means ± SD. (Asterisk) significantly different from control (A: P < 0.001; B: P = 0.047). Representative immunocytochemistry images of myocardial tissue sections of the border zone on d88 after infarction in MB (D, E) and MB-CSF-1 (F, G) group stained with the macrophage specific RM-4 antibody. Arrowheads indicate macrophages stained positively with RM-4 antibody inside the myocardial tissue. Calibration bar = 50 µm.

 
After MB-CSF-1 treatment, the number of macrophages in the border zone increased significantly on d88 post-myocardial infarction compared with the control group (Figures 5C–G). In contrast, neither treatment with recCSF-1, pl-CSF-1 nor with MB resulted in significant changes in the number of macrophages. These experiments indicated that the transfected myoblasts retain the ability to overexpress CSF-1 for at least 2 weeks and that CSF-1 expressing myoblasts are able to attract macrophages.

3.5 Colony-stimulating factor-1 affects myocardial matrix metalloprotease expression
MMP-2 protein levels on d67 post-treatment were significantly increased in the border zone of the MB-CSF-1 compared with the control group (Figure 6A). No significant changes compared with controls were found in the MB, recCSF-1, and pl-CSF-1 groups in the border zone. In contrast, in the myocardial infarction zone, no significant changes were observed between the groups. MMP-9 protein expression remained unchanged in the border zone, but was significantly reduced in the myocardial infarction zone for the MB-CSF-1 and MB groups vs. controls. MMP-1 protein expression was not significantly altered for these groups in both border and myocardial infarction zones, while MMP-13 levels, similar to MMP-9, were found to be significantly reduced within the myocardial infarction zone in the MB-CSF-1 as well as the MB group vs. control. Macrophage-specific MMP-12 protein expression was significantly increased in the border zone of the MB-CSF-1 group compared with controls, but was not altered in the myocardial infarction zone. In contrast to these changes in MMP levels, protein expression of tissue inhibitors of metalloproteinases 1–4 did not significantly change in the border or myocardial infarction zone of any group (data not shown). In line with western blot analyses, immunocytochemistry revealed significantly higher density of both MMP-2- and MMP-12-positive cells in the border zone of MB-CSF-1-treated animals compared with the MB group (Figure 6B). These data indicated that a specific expression pattern of MMPs is displayed in the myocardial border zone of CSF-1-transfected myoblasts.


Figure 6
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  		Figure 6 Analysis of matrix metalloproteases (MMP) expression. (A) Quantification of protein expression levels of MMP-2, MMP-9, MMP-1, MMP-13, and MMP-12 and representative western blot images in myocardial tissue of the border zone (BZ) and the myocardial infarction zone (MI) 88 days after myocardial infarction. Actin was used as a loading control (LC); SP, specific protein. Data are expressed as means ± SD. MMP-2: (Asterisk) significantly different from control (P = 0.006); MMP-9: (Asterisk) significantly different from control [P < 0.0001 for myoblasts (MB) group; P = 0.001 for MB-colony-stimulating factor-1 (CSF-1) group]; MMP-13: (Asterisk) significantly different from control (P = 0.045 for MB group; P = 0.003 for MB-CSF-1 group); MMP-12: (Asterisk) significantly different from control (P = 0.039). (B) Representative immunocytochemistry images of MMP-2- (left panels) and MMP-12- (right panels) stained myocardial sections in MB (upper panels) and MB-CSF-1 (lower panels). Both MMP-2- and MMP-12-positive cells (arrows) reveal a higher density in the MB-CSF-1 group. Bar = 50 µm.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
This study was designed to enhance the survival of grafted myoblasts and improve the angiogenesis following myoblast transfer to the failing heart. It is evident that myosin heavy chain is not expressed in myoblasts early after engraftment; however, with time, cells fuse into myotubes, differentiate into myofibers, and express myosin heavy chain.4 Therefore, those areas that stained positively for myosin heavy chain within the grafts reflect myoblasts that have survived and gone on to fuse and form myofibers. This process took place in both the MB and MB-CSF-1 groups. However, the percentage of myoblasts that survived was significantly higher in the MB-CSF-1 group. Myoblast transfer 7 days after myocardial infarction was reported to improve cardiac function.16 However, the same procedure failed in our setting of ischaemic heart failure, involving myoblast transfer at 21 days after myocardial infarction. In contrast, we found that myoblasts overexpressing CSF-1 did improve the cardiac function, indicating the benefit of CSF-1-transfected myoblasts in the treatment of ischaemic heart failure. Collectively, these results might be explained by the timing of therapy initiation. At 3 weeks following infarction, the scar tissue is more matured than at 1 week, rendering rescue by myoblasts more difficult. CSF-1 expression, besides increasing long-term survival, might enable the grafted cells to more efficiently recruit macrophages and endothelial cells, thereby facilitating angiogenesis for their further survival. In concert with this hypothesis, CSF-1 mRNA was overexpressed in the MB-CSF-1 group on d52, associated with increased LVEF, which further improved on d88. The higher density of myoblasts and macrophages and the higher vascular density in the MB-CSF-1 group in vivo, further support the positive role of CSF-1.

Previous studies utilizing rat L6{alpha}1 myoblasts showed that CSF-1 regulates myoblast proliferation in an autocrine fashion, but that expression of both CSF-1 and the CSF-1R is lost upon differentiation of the myoblasts to myotubes. Indeed, the induced expression of CSF-1R antisense RNA in these cells blocked myoblast proliferation and accelerated myogenic differentiation.15 Whether the proliferative effect of CSF-1 on myoblasts observed in vitro had contributed to the results observed in our in vivo studies, however, remains uncertain.

The mechanism for autocrine regulation of myoblast proliferation in vitro by CSF-1 is apparently intracellular,15 thus we would not expect an effect of concomitant recombinant CSF-1 administration and myoblast transplantation on myoblast numbers. Importantly, CSF-1 plasmid application was not as efficient as CSF-1 overexpressing cells to maintain CSF-1 gene expression levels. Myoblasts can be transfected much more efficiently in vitro prior to transplant as compared with the protocol utilizing in vivo systemic plasmid administration, resulting in higher CSF-1 expression levels in grafted cells, which explains the failure of pl-CSF-1 in improving angiogenesis and macrophage recruitment.

Local production of CSF-1 has been shown to recruit and sustain many tissue macrophage populations which possess trophic and scavenger functions that are important for normal tissue development.20–22 In addition, macrophages can act as supportive cells for myoblasts through delivery of both soluble mitogenic factors and cell contact-mediated survival signals.23 These functions of the macrophages are also quite relevant to the changes observed during recovery from myocardial infarction (discussed later). Thus, another reason for the effectiveness of the MB-CSF-1 group is that ectopic expression of CSF-1 in myoblasts increases the initial recruitment and survival of macrophages.

Relevant to the important role played by macrophages in the repair of injured muscle, recent studies indicate that macrophages recruited from the blood to sites of muscle injury exhibit a non-proliferating, pro-inflammatory expression profile, but once within muscle switch to become proliferating, anti-inflammatory macrophages. In vitro, in contrast to the pro-inflammatory macrophages, which stimulate myoblast proliferation, the anti-inflammatory macrophages stimulate myoblast differentiation.24 Furthermore, these same authors demonstrate that depletion of circulating monocytes in CD11b-diptheria toxin receptor mice at the time of injury totally prevented muscle regeneration.

It is noteworthy that the number of surviving myoblasts was significantly higher in the MB-CSF-1 group compared with the MB group. Additional evidence for the long-term survival of myoblasts in the failing heart is that the LV infarction wall was thickest in the MB-CSF-1 group; even significantly thicker than in the MB group. Moreover, the ejection fraction was improved and the LV dilation ameliorated significantly in the MB-CSF-1 group. Recent studies indicate that there is a lack of functional electromechanical coupling between the majority of grafted myoblasts and cardiomyocytes.25,26 This suggests that it is the enhanced number of grafted myoblasts and the increased angiogenesis induced by CSF-1 that contributes to the improved cardiac function we have observed with CSF-1-transfected myoblasts.

The capacity of myoblast transplants to regenerate myocardial tissue following infarction depends on nourishment and spatially appropriate growth of myoblasts. This requires a potent neovasculature, appropriate MMP activity, and proper fibrillar collagen scaffolding. In the border zone, the number of vWF-positive vessels and expression of endothelial cell-specific CD31 was increased in both myoblast transplant groups. However, the vascular network formed at the infarction border zone was highest for the MB-CSF-1 group, indicating an additional beneficial angiogenic effect of myoblast-derived CSF-1. Consistent with these angiogenic effects, CSF-1 stimulates monocytes, progenitors of macrophages, to secrete VEGF27 and in tumour settings CSF-1 through its action on macrophages has been shown to regulate angiogenesis.14,28

The myocardial MMP profile in the MB-CSF-1 group suggests that CSF-1-induced recruitment of macrophages contributes to cardiac tissue remodelling and scar and collagen areas were smaller in the MB-CSF-1 group than in the other groups. In line with this, MMP-2 and macrophage specific MMP-12 were overexpressed in the border zone of MB-CSF-1-treated animals. Additionally, the expression of both MMP-9 and MMP-13 was reduced in the infarction zone in both myoblast groups 12 weeks post-infarction. Previous studies reported that MMPs were increased in post-infarction heart failure models, leading to LV dilation and that MMP-inhibitors beneficially affected cardiac remodelling and function.29,30 However, the excessive fibrosis without contractility or relaxation could accelerate cardiac remodelling and decrease cardiac function, as seen in ischaemic or idiopathic-dilated cardiomyopathy. In such cases, an increase in the MMP family and proteolysis of excess collagen may be a protective mechanism. In fact, targeted deletion of MMP-9 attenuated LV remodelling and collagen accumulation via overexpression of MMP-2 and MMP-13.31 Furthermore, the importance of appropriate expression of MMPs has been demonstrated in mice by showing that acute myocardial infarction resulted in cardiac rupture in mice lacking MMP-3 or MMP-12, while lack of MMP-9 partially protected against rupture.32

Consistent with these observations, our data indicate that alteration of region- and type-specific distribution of MMPs within the myocardium after infarction by CSF-1-producing myoblasts is associated with improved cardiac function. CSF-1 is likely to exert its effects on MMP-expression via enhanced recruitment of macrophages and regulation of their MMP production. This assumption is supported by previous findings in tumours in which CSF-1 was shown to enhance MMP expression and macrophage recruitment.33 Moreover, macrophage recruitment may be related to increased absorption of necrotic tissues by phagocytosis. The failure of the recombinant CSF-1 group to beneficially affect remodelling and cardiac function compared with the MB-CSF-1 group may be due to prolonged expression of CSF-1 by myoblasts, resulting in the maintenance of increased local CSF-1 concentrations for several weeks. The fact that MB-CSF-1 improved cardiac function, resulting in CSF-1 over-expression for at least 14 days, supports this hypothesis.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This study was supported by a grant from the Herzfelder Foundation and the CARDIOWORKBENCH EU-grant # PL 018671 to S.A. and NIH grants CA26504 and CA32551 to E.R.S.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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