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Interact CardioVasc Thorac Surg 2007;6:293-297. doi:10.1510/icvts.2006.144014
© 2007 European Association of Cardio-Thoracic Surgery
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Institutional report - Experimental

Can erythropoietin improve skeletal myoblast engraftment in infarcted myocardium?{star}

Sylvain Chanséaumea,b,1, Kasra Azarnousha,b,1, Agnès Maurela, Valérie Bellamya, Séverine Peyrardc, Patrick Brunevald,e, Albert A. Hagègea,f and Philippe Menaschéa,g,*

a INSERM U 633, Laboratoire d'Etude des Greffes et Prothèses Cardiaques, Hôpital Broussais; Assistance Publique-Hôpitaux de Paris, Ecole de Chirurgie, Paris, France
b CHU G. Montpied, Department of Cardiology, Clermont-Ferrand, France
c Clinical Investigation Center 9201, Assistance Publique-Hôpitaux de Paris/INSERM, Hôpital Européen Georges Pompidou, Paris, France
d INSERM, U 652, Hôpital Broussais, Paris, France
e Assistance Publique-Hô pitaux de Paris, Hôpital Européen Georges Pompidou, Department of Pathology; Université Paris Descartes, Faculté Médecine, Paris, France
f Assistance Publique-Hô pitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiology; Université Paris Descartes, Faculté de Médecine, Paris, France
g Assistance Publique-Hô pitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; Université Paris Descartes, Faculté de Médecine, Paris, France

Received 9 September 2006; received in revised form 23 February 2007; accepted 26 February 2007

1 These two authors equally contributed to this work. Back

{star} This work was supported by a grant from the Fondation de l'Avenir.

*Corresponding author. Tel.: +33 1 56093622; fax: +33 1 56093261.

E-mail address: philippe.menasche{at}egp.aphp.fr (P. Menasché).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Data analysis
 4. Results
 5. Discussion
 Acknowledgements
 References
 
The benefits of skeletal myoblast transplantation are limited by the high rate of early cell death which is partly of ischemic origin. We, therefore, assessed whether graft survival could be improved by the additional use of the angiogenic cytokine erythropoietin (EPO). Thirty-five Lewis rats underwent coronary artery ligation and, two weeks later, were randomized to receive in-scar injections of control medium, skeletal myoblasts (5x106) or skeletal myoblasts with EPO started the day before transplantation and continued for two weeks (500 U/kg three times a week). A fourth group was treated by EPO alone without injections. Function was assessed by 2D echocardiography before transplantation and one month thereafter. Compared with controls and hearts treated by EPO-alone, those transplanted with myoblasts yielded a significantly better recovery of LV ejection fraction, irrespective of whether they had received EPO or not. Neither the area of myoblast engraftment, nor angiogenesis differed between the myoblast-alone and the myoblast+EPO groups. Apoptosis was hardly detectable and, therefore, unaffected by EPO therapy. In this model, EPO failed to improve myoblast engraftment and postinfarction LV function. These negative findings justify to pursue the search for alternate cell survival-enhancing strategies.

Key Words: Stem cells; Myoblasts; Transplantation; Heart failure; Myocardial infarction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Data analysis
 4. Results
 5. Discussion
 Acknowledgements
 References
 
Despite encouraging proof-of-concept data suggesting the ability of transplanted skeletal myoblasts to improve postinfarct left ventricular (LV) function, the efficacy of the procedure remains hampered by the high rate of cell loss [1] which results from both mechanical leakage during injections and subsequent biologically-induced death of engrafted cells. As one contributing factor to cell death is the poor vascularization of the target scars and the resulting ischemia of the graft, several strategies have been developed to increase vascularization of the injected areas, which have primarily relied on growth factors [2, 3]. The present study was designed to rather assess the effects of erythropoietin (EPO) with the premise that this angiogenic and anti-apoptotic cytokine [4] could increase skeletal myoblast survival and the related functional outcome.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Data analysis
 4. Results
 5. Discussion
 Acknowledgements
 References
 
The experiments complied with the ‘Principles of Laboratory Animal Care’ formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ prepared by the Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council, and published by the National Academy Press, revised 1996.

2.1. Cell cultures

Primary muscle cell cultures were prepared from newborn Lewis male rats (Charles Rivers, Arbresle, France) according to a previously established protocol [3]. The day of transplantation, the cells were thawed and washed three times in modified Eagle's medium with 0.5% bovine serum albumin (fraction V; Sigma, St Louis, MO).

2.2. Erythropoietin

rhEPO (Epoetine Alfa 10000 U/ml [Eprex®], Janssen-Cilag, Issy-les-Moulineaux, France) was administered by intra-peritoneal injections starting the day before cell transplantation. The drug was given at the dose of 500 U/kg three times a week during two weeks.

2.3. Myocardial infarction model

Female Lewis rats (Charles Rivers), were anesthetized with isoflurane (1–3%). The heart was approached through a left thoracotomy and a myocardial infarction was created by ligation of the left coronary artery with a 5/0 polypropylene snare (Ethicon, Somerville, NJ).

2.4. Experimental protocol

Thirteen days after creation of infarction, rats underwent a baseline echocardiographic assessment of left ventricular (LV) function and only those with an ejection fraction (EF) below 45% were selected for the trial. Following a median sternotomy, these animals were randomly allocated to receive intramyocardial injections of culture medium (controls, n=7), skeletal myoblasts (5x106, n=10), EPO (n=8), or skeletal myoblasts (5x106) in combination with EPO (n=10). All injections consisted of a 150-µl volume delivered in three or four sites in the core and at the borders of the scar by using a 29 gauge needle. Immunosuppression was not given because of the syngeneic nature of the experimental animals.

2.5. End points

2.5.1. Hematocrit
Hematocrit was measured the day of infarction and at the time of sacrifice.

2.5.2. Left ventricular function
Left ventricular function was assessed by 2-dimensional echocardiography (Agilent SONOS HP-5500 with a 7.5 MHz probe) shortly before injections (i.e. 13 days after infarction) and one month thereafter, as previously described [3].

2.5.3. Cell engraftment and angiogenesis
After the last echocardiographic assessment, hearts were harvested and separated in two halves by a short-axis section through the midportion of the infarcted area. Histological and immunohistochemical studies were carried out from the two blocks of each heart on 8-µm-thick cryostat sections. Measurements of vascularization and myoblast engraftment were performed by immunolabeling using monoclonal antibodies against rat endothelial cells (RECA, clone HIS52, Serotec, Oxford, UK) and fast skeletal myosin (clone My-32, Sigma, St Louis, MI) both conjugated with a biotinylated anti-mouse IgG secondary antibody (Vector, Burlingame, CA). Examinations were performed with a microscope (Leica DMIL, Wetzlar, Germany) equipped with a digital camera (Qicam, Qimaging, Burnaby, BC, Canada). An average of ten high-power fields (x10 and x5 objective magnification for angiogenesis and cell engraftment, respectively) were used to obtain digital images which were processed with a Metamorph software (Universal Imaging Corporation, Downington, PA).

2.5.4. Cell survival
In a subset of experiments involving transplantation of male myoblasts into female recipients (n=3 for the myoblast-alone and the myoblast+EPO groups), left ventricles removed at the end of the study were snap-frozen in liquid nitrogen and stored at –80 °C. Muscles were thawed on ice, minced, and digested to homogeneity overnight at 4 °C in lysis buffer (Roche, Basel, Switzerland). Desoxyribonucleic acid (DNA) was isolated from whole homogenates by using the Wizard DNA purification kit (Promega, Charbonnieres, France), dissolved in Tris–HCl buffer (5 mmol/l, pH 8.5) and analysed by real-time quantitative polymerase chain reaction, as previously described [5]. A rat Y chromosome specific sequence in the sex-determining region Y (sry) was used to determine the relative quantities of male cells after transplantation.

2.5.5. Apoptosis
Nuclear deoxyribonucleic acid fragmentation was visualized in situ by Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) assay, using the DeadEndTM Fluorometric TUNEL System (Promega).


   3. Data analysis
Top
 Abstract
 1. Introduction
 2. Methods
 3. Data analysis
4. Results
 5. Discussion
 Acknowledgements
 References
 
All functional and histological studies were performed in a blinded fashion. Analyses of variance (ANOVA) including group, time and their interaction were used to compare between-group and within-group differences in hematocrit and left ventricular function. When the global Fisher's test was significant, pairwise comparisons were performed using Student's t-tests. The critical {alpha} level was set at 0.05 and the Bonferroni-Holm step-down procedure was used to adjust for multiple comparisons. Data are reported as mean±one standard deviation (S.D.) or as estimated differences with their associated confidence interval (CI 95%). Differences between means of EF are expressed as percentage points. All analyses were carried out using SAS Statistical Software (Version 8.2, Cary, NC, USA).


   4. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Data analysis
 4. Results
5. Discussion
 Acknowledgements
 References
 
4.1. Animal mortality

The early mortality rate after coronary artery ligation and transplantation was 11% and 5%, respectively, without any difference between the different groups.

4.2. Characterization of the cell injectate

At the time of transplantation, the percentage of myogenic cells, as assessed by desmin positivity, was 54.6%. The post-thawing viability rate, as assessed by exclusion of trypan blue, was 85%.

4.3. Changes in hematocrit

Baseline hematocrits averaged 42±2%, 41±3%, 42±4%, and 41±3% in the control, EPO, myoblast-alone and myoblast+EPO groups, respectively (simple main effects of group at baseline: P=0.90). At the 1-month study point, hematocrits were unchanged from initial values in the control and myoblast-alone groups (41±2% and 41±3%, respectively) whereas they had significantly increased in rats treated with EPO alone (52±5%) or EPO+myoblasts (50±5%). For these two groups, the difference with their drug-untreated counterparts was highly significant (estimated mean differences at the 1-month study point were between 8.3 and 11.5% points, P<0.0001 for each pairwise comparison).

4.4. Functional outcomes

Baseline EFs ranged from 33–38% and were not significantly different among groups (simple main effects of group at baseline: P=0.34). The patterns of changes were then markedly different between groups (Fig. 1). Thus, after 1 month, EF in the two myoblast transplantation groups was significantly higher than in the control group regardless of whether EPO was added (11.4 [CI 95%: 3.7; 19.1]% points, P=0.02) or not (11 [CI 95%: 2.9; 19.1]% points, P=0.03). Differences between the two myoblast-transplanted groups and the EPO-alone group featured similar patterns (14.9 [CI 95%: 7.1; 22.8]% points for myoblast alone vs. EPO, P=0.02 and 15.3 [CI 95%: 7.9; 22.8]% points for myoblast+EPO vs. EPO, P=0.001). However, within the two myoblast-treated groups, the addition of EPO failed to significantly increase EF beyond that seen in myoblast-alone-injected hearts (difference between groups: 0.4 [CI 95%: –7.0; 7.9]% points, P=0.91). Hearts of all groups incurred significant increases in both LVEDV and LVESV (P=0.0003 and P=0.0011, respectively, vs. baseline values) and although those injected with myoblasts, with or without EPO, showed smaller increases in LVESV than non-cell-injected hearts (Table 1), 1-month between-group differences were not statistically significant.


Figure 1
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  		Fig. 1. Changes in left ventricular ejection fraction. *P=0.03 vs. controls; {dagger}P=0.02 vs. EPO alone; #P=0.02 vs. controls; {ddagger}P=0.001 vs. EPO alone; LVEF, left ventricular ejection fraction. Data are given as means±S.D.

 

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  		Table 1 Changes in left ventricular enddiastolic and endsystolic volumes (LVEDV and LVESV, respectively)

 
4.5. Cell engraftment, angiogenesis and survival

The number of RECA-positive vessels found in the infarcted area of myoblast-transplanted rats was not affected by EPO: 1,191±147/mm2 and 1,086±228/mm2 in the myoblast-alone and myoblast+EPO groups, respectively. The drug equally failed to increase angiogenesis in the normal myocardium remote from the necrotic region (2,120±39/mm2 vs. 2,359±386/mm2 in the myoblast-alone group). Myoblast engraftment paralleled these patterns as the area of My32-positive fluorescence (expressed as a percentage of the infarct area) did not differ between hearts injected with myoblasts only (7.1±1.5%) and those in which myoblast injections were combined with EPO administration (11.2±6.9%). Representative sections are shown in Fig. 2. In keeping with these data, the number of donor cells estimated from PCR for the sry chromosome was similar in these two groups and averaged 1x106.


Figure 2
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  		Fig. 2. Myoblast engraftment in a myoblast-alone injected heart (upper panel) and in a myoblast+EPO-treated heart (lower panel). Magnificationx5.

 
4.6. Apoptosis

One month after transplantation, apoptosis events were almost negligible since only very few cells containing TUNEL-positive nuclei were detected on heart sections, irrespective of whether EPO had been combined with myoblasts or not.


   5. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Data analysis
 4. Results
 5. Discussion
Acknowledgements
 References
 
The present study confirms the benefits of skeletal myoblast transplantation but fails to show that an adjunct EPO therapy further enhances cell survival, angiogenesis or functional outcome.

5.1. Rationale for the use of EPO

The major causes of post-transplantation cell death primarily include inflammation, loss of survival signals from the extracellular matrix and graft ischemia. To address this ischemic component, interventions (aside from direct revascularization by angioplasty or bypass surgery) can be categorized into co-injections of angiogenic growth factors [3], cell transfection with some of these factors [2] or co-transplantation of angiogenic bone marrow-derived cells [5]. These strategies have been successful in increasing local neovascularization and postinfarction ventricular function but their clinical applicability may be problematic because of the short half-life of recombinant proteins, the safety concerns raised by viral vectors or the complexity of a combined cell transplantation. We, therefore, tested the hypothesis that administration of EPO could be an alternate effective option.

This cytokine has been shown to improve LV function in animal models of myocardial infarction [6, 7] independently of its effects on erythropoiesis, through its angiogenic [4, 8], and anti-apoptotic effects [6, 7]. In a clinical perspective, EPO is appealing because of its well documented safety record, including that in cardiac surgical patients [9]. The discovery of EPO receptors on human ventricular cardiomyocytes and endothelial cells [10] further supports that the drug might be a useful adjunct to cell transplantation in patients with ischemic heart disease.

5.2. Interpretation of data

The failure of EPO to potentiate the functional benefits of myoblast transplantation is unlikely to be due to an ineffective drug delivery since hematocrit values were significantly higher in the two EPO-treated groups.

These negative findings may look surprising because, except for one study [11], previous reports on EPO have documented its ability to reduce infarct size, LV remodelling and contractile dysfunction in rat and canine models of occlusion-reperfusion [6] or permanent coronary artery ligation [7, 12]. This discrepancy, however, could be due to basic differences in timing of treatment and primary end point. In all but one [8] of these positive studies, EPO was started at the time of infarction [12] or at the onset of reperfusion [6, 12] with the major objective of reducing infarct size. Conversely, in the present study, the administration of the drug was delayed until three weeks after infarction and was primarily intended to improve graft vascularization and survival. At this late stage where infarct healing is largely completed already, the signals required for EPO to be effective may have waned. In particular, apoptosis of the native cardiomyocytes is no longer a predominant event so that a major target for the effects of EPO is missing. Likewise, if the angiogenic properties of EPO are mediated by mobilization of bone marrow-derived endothelial progenitor cells [13], the pathway required for an effective myocardial homing may also be disrupted because SDF-1 is no longer upregulated shortly after infarction. This hypothesis is supported by dose–response studies showing a relatively narrow therapeutic window [14].

5.3. Study limitations

Study limitations include relatively small sample sizes, operator-dependency of the echocardiographic assessment, shortness of follow-up and lack of dose–response relationship. Indeed, although the lack of a direct assay of EPO myocardial levels does not allow to exclude a dosing issue, the drug regimen was selected to match a clinically relevant protocol and actually effective in significantly increasing hematocrits. Rather than the dose, the systemic (intraperitoneal) mode of administration could account for the suboptimal therapeutic efficacy since our permanent coronary artery occlusion model may have impeded an effective drug delivery to the target myocardial areas. This hypothesis is supported by the recent finding of improved function and decreased fibrosis in a porcine model of chronic progressive myocardial ischemia in which EPO was directly delivered in the ischemic region by transendocardial injections [15].

In conclusion, the present results suggest that a clinically relevant protocol of EPO administration in the setting of a non-reperfused myocardial infarction fails to improve myoblast engraftment and postransplantation LV function and, as such, justifies the ongoing search for more effective cell survival-enhancing strategies.


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Methods
 3. Data analysis
 4. Results
 5. Discussion
 Acknowledgements
References
 
We acknowledge the technical assistance of Chantal Mandet, Department of Pathology and INSERM U 652.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Data analysis
 4. Results
 5. Discussion
 Acknowledgements
 References
 

  1. Maurel A, Azarnoush K, Sabbah L, Vignier N, Le Lorc'h M, Mander C, Bissery A, Garcin I, Carrion C, Fiszman M, Bruneval P, Hagege A, Carpentier A, Vilquin JT, Menasche P. Can cold or heat shock improve skeletal myoblast engraftment in infarcted myocardium? Transplantation 2005; 80:660–666.[CrossRef][Medline]
  2. Yau TM, Li G, Weisel RD, Reheman A, Jia ZQ, Mickle DA, Li RK. Vascular endothelial growth factor transgene expression in cell-transplanted hearts. J Thorac Cardiovasc Surg 2004; 127:1180–1187.[Abstract/Free Full Text]
  3. Azarnoush K, Maurel A, Sabbah L, Carrion C, Bissery A, Mandet C, Pouly J, Bruneval P, Hagege AA, Menasche P. Enhancement of the functional benefits of skeletal myoblast transplantation by means of coadministration of hypoxia-inducible factor 1-alpha. J Thorac Cardiovasc Surg 2005; 130:173–179.[Abstract/Free Full Text]
  4. Maiese K, Li F, Chong ZZ. New avenues of exploration for erythropoietin. J Am Med Assoc 2005; 293:90–95.[Abstract/Free Full Text]
  5. Ott HC, Bonaros N, Marksteiner R, Wolf D, Margreiter E, Schachner T, Laufer G, Hering S. Combined transplantation of skeletal myoblasts and bone marrow stem cells for myocardial repair in rats. Eur J Cardio-thorac Surg 2004; 25:627–634.[Abstract/Free Full Text]
  6. Calvillo L, Latini R, Kajstura J, Leri A, Anversa P, Ghezzi P, Salio M, Cerami A, Brines M. Recombinant human erythropoietin protects the myocardium from ischemia-reperfusion injury and promotes beneficial remodeling. Proc Natl Acad Sci USA 2003; 100:4802–4806.[Abstract/Free Full Text]
  7. Moon C, Krawczyk M, Ahn D, Ahmet I, Paik D, Lakatta EG, Talan MI. Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats. Proc Natl Acad Sci USA 2003; 100:11612–11617.[Abstract/Free Full Text]
  8. Van der Meer P, Lipsic E, Henning RH, Boddeus K, Van der Velden J, Voors AA, Van Veldhuisen DJ, Van Gilst WH, Schoemaker RG. Erythropoietin induces neovascularization and improves cardiac function in rats with heart failure after myocardial infarction. J Am Coll Cardiol 2005; 46:125–133.[Abstract/Free Full Text]
  9. Alghamdi AA, Albanna MJ, Guru V, Brister SJ. Does the use of erythropoietin reduce the risk of exposure to allogeneic blood transfusion in cardiac surgery? A systematic review and meta-analysis. J Card Surg 2006; 21:320–326.[CrossRef][Medline]
  10. Depping R, Kawakami K, Ocker H, Wagner JM, Heringlake M, Noetzold A, Sievers HH, Wagner KF. Expression of the erythropoietin receptor in human heart. J Thorac Cardiovasc Surg 2005; 130:877–878.[Free Full Text]
  11. Hale SL, Sesti C, Kloner RA. Administration of erythropoietin fails to improve long-term healing or cardiac function after myocardial infarction in the rat. J Cardiovasc Pharmacol 2005; 46:211–215.[CrossRef][Medline]
  12. Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, Thompson RB, Petrofski JA, Annex BH, Stamler JS, Koch WJ. A novel protective effect of erythropoietin in the infarcted heart. J Clin Invest 2003; 112:999–1007.[CrossRef][Medline]
  13. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rhim C, Martin H, Zeiher AM. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003; 102:1340–1346.[Abstract/Free Full Text]
  14. Moon C, Krawczyk M, Paik D, Lakatta EG, Talan MI. Cardioprotection by recombinant human erythropoietin following acute experimental myocardial infarction: dose response and therapeutic window. Cardiovasc Drugs Ther 2005; 19:243–250.[CrossRef][Medline]
  15. Krause KT, Jaquet K, Geidel S, Schneider C, Mandel C, Stoll HP, Hertting K, Harle T, Kuck KH. Percutaneous endocardial injection of erythropoietin: assessment of cardioprotection by electromechanical mapping. Eur J Heart Fail 2006; 8:443–450.[Abstract/Free Full Text]

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