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Cardiovascular Research 2005 68(3):394-404; doi:10.1016/j.cardiores.2005.06.014
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Copyright © 2005, European Society of Cardiology

Host-derived circulating cells do not significantly contribute to cardiac regeneration in heterotopic rat heart transplants

Simonetta Ausonia,*, Tania Zagliaa,1, Arben Dedjad,e,1, Raffaella Di Lisig, Michela Sevesod, Ermanno Anconac,d,e, Gaetano Thienef, Emanuele Cozzic,d,e,2 and Stefano Schiaffinoa,b,g,2,*

aDepartment of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35121 Padova, Italy
bConsiglio Nazionale delle Ricerche Institute of Neurosciences, University of Padua, Italy
cDirezione Sanitaria and Clinica Chirurgica III, Padua General Hospital, Italy
dCORIT (Consorzio per la Ricerca sul Trapianto d'Organi), Italy
eDepartment of Medical and Surgical Sciences, Italy
fInstitute of Pathological Anatomy, University of Padua, Italy
gVenetian Institute of Molecular Medicine (VIMM), Padua, Italy

* Corresponding authors. Ausoni is to be contacted at Department of Biomedical Sciences University of Padua Viale G. Colombo 3, 35121 Padova, Italy. Tel.: +39 049 8276036; fax: +39 049 8276040. Schiaffino, Venetian Institute of Molecular Medicine (VIMM), Via Orus 2, 35129 Padova, Italy. Tel.: +39 049 8276034; fax: +39 049 8276040. Email address: ausoni{at}civ.bio.unipd.it stefano.schiaffino{at}unipd.it

Received 17 February 2005; revised 22 June 2005; accepted 23 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objectives: The aim of this study was to investigate the contribution of host-derived circulating cells to cardiac repair after tissue damage using the model of heterotopic heart transplantation between transgenic recipient rats expressing green fluorescent protein (GFP) and wild-type donors.

Methods: Unlabeled donor rat hearts, some of which underwent prolonged cold ischemia pretreatment, were transplanted into the abdominal cavity of GFP+ transgenic recipient rats and were analyzed 15 and 90 days after surgery. An additional experimental group underwent heart transplantation following administration of granulocyte-colony stimulatory factor (G-CSF) to mobilize bone marrow cells.

Results: Most transplants contained GFP+ mature cardiomyocytes. However, systematic counting in the transplants showed that the proportion of GFP+ cardiomyocytes was only 0.0005% to 0.008% of all cardiomyocytes. These relative proportions did not change after G-CSF treatment, despite evidence for sustained marrow cell mobilization. Confocal image analysis showed that the majority of GFP+ cardiomyocytes contained a high number of nuclei, suggesting that these cells may derive from fusion events. Very rarely, small GFP+ undifferentiated cells, expressing GATA-4, were also identified. Occasionally, GFP+ endothelial cells, but not smooth muscle cells, were detected in blood vessels of some transplants.

Conclusions: Our results demonstrate that cardiomyocytes expressing a host transgenic marker are detectable in heterotopic heart transplants; however, they do not significantly contribute to repopulation of the damaged myocardium.

KEYWORDS Cardiac regeneration; Adult stem cells; Heterotopic heart transplantation; Trangenic rats

This article is referred to in the Editorial by Zimmermann and Eschenhagen (pages 344–346) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
The mammalian heart has a very limited regenerative capacity and responds to tissue injury by scar formation and tissue remodeling. The ability of adult bone marrow-derived stem cells to regenerate cardiomyocytes has been verified in multiple experimental settings in the last few years. Direct injection of bone marrow cells into the infarcted myocardium has been reported to induce extensive regeneration through transdifferentiation [1,2]. However, this conclusion has been challenged by studies showing lack of transdifferentiation into cardiomyocytes following intracardiac injection of bone marrow cells [3,4] or transfusion of these cells into the circulation of recipient mice [5,6]. Extensive myocardial regeneration from extra-cardiac stem cells in human sex-mismatched heart transplants has likewise been described [7], although other studies reported only modest [8] or negligible [9,10] contribution of host-derived cells to cardiomyocytes. Here, we used for the first time an experimental model of heterotopic heart transplantation in rats to investigate engraftment and differentiation of host-derived cells in heart transplants both spontaneously and following administration of granulocyte-colony stimulatory factor (G-CSF) to mobilize bone marrow stem cells. Using a green fluorescent protein (GFP) reporter transgene we were able to identify host-derived cells engrafting the donor heart genetically, a stringent approach that provides very low background and adequate sensitivity for positive cell detection.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1 Animals
Thirty-one adult male or female Sprague Dawley wild type rats were used as heart donors. The grafts were transplanted into male or female GFP+ rats, a gift from Dr. Okabe (University of Osaka, Japan) [11]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). The study was conducted under supervision of the internal Ethics Committee.

2.2 Cardiac allotransplantation
A heterotopic cardiac transplant was performed. The surgical procedure was undertaken according to the method described by Ono and Lindsey [12], slightly modified at our center. Briefly, both donors and recipients were anesthetized with Isoflurane (FORANE®, Abbott SpA) at 1–1.5% with oxygen supplemented with 5 mg/kg of intraperitoneal tramadol. After donor heparinization, heart harvesting was performed flushing the organ with CELSIOR® (CS) (Imtix Sangstat) cardioplegic preservation solution. The hearts were immersed in CS for a few minutes or 4 h (depending on the experimental groups) and the graft was transplanted under magnification into the abdomen of the recipient. An end-to-side anastomosis was performed between the graft aorta and the abdominal aorta and the graft pulmonary artery with the abdominal vena cava of the recipient.

As analgesia, 5 mg/kg tramadol were injected intramuscularly twice daily in the first two postoperative days. A long-acting antibiotic, 60 mg/kg of TERRAMYCIN® L. A. (Pfizer) in 5 ml of saline, was administered subcutaneously for infection prophylaxis. Ten milligrams per kilogram intramuscular (in saline solution) or oral (in olive oil) Cyclosporine A were administered to the recipient once daily, throughout all the postoperative period, starting on the day of surgery. The cardiac grafts were monitored once daily by direct palpation through the abdominal wall. In a first experimental group heterotopic hearts were explanted at day 15 (n=15) and at day 90 (n=10). In an additional group (n=6) heterotopic heart transplantation was performed and, about 3 h later, the rats started to be treated with human recombinant G-CSF (250 µg/kg/day subcutaneously for 6 days consecutively) to induce bone marrow cell mobilization. Heterotopic hearts were explanted at day 15. At four different times, namely before surgery (T=0) and 1, 6 and 14 days after surgery (T=1, T=6 and T=14) blood samples of G-CSF treated rats were drawn and neutrophyl counts were performed.

2.3 Histopathological evaluation
Grafts were removed 15 or 90 days following transplantation, fixed in 2% paraformaldehyde at room temperature for 2 h, equilibrated in sucrose gradient and frozen in liquid nitrogen. Transverse 10 µm sections from the midportion of the hearts were cut and stained with hematoxilin-eosin for standard histology. Degree of tissue damage was scored according to a scale ranging from 0 to 2, in which grade 0 described no infiltrate and no rejection, grade 1 a moderate perivascular and interstitial infiltrate with focal cell necrosis and grade 2 a severe multifocal infiltrate accompanied by widespread tissue damage. A Leica DMR Optical Microscope with a digital LEICA DC300 and Image Software IM1000 were used for the analysis.

2.4 Immunofluorescence and confocal image analysis
Analyses were restricted to the ventricular myocardium. Detection of GFP+ cells and cell characterization by immunofluorescence were performed on 10 µm sections, using a panel of antibodies: anti-alpha/beta myosin heavy chain [13]; anti-cardiac troponin I; anti-smooth muscle actin (clone 1A4, Sigma); anti-rat CD-31 (Pharmingen); anti-RECA-1 (AbCAM, ab9774); anti-rat CD-45 (Pharmingen); anti-CD-163, specific for macrophages (clone ED2, Serotec); anti-GFP (Molecular Probes, A-11122); anti-connexin-43 (Chemicon, 4E6.2); anti-von Willebrand factor (Dako); anti-Ki67 (Novocastra); anti-c-kit and anti-GATA-4 (Santa Cruz, c-19 and H-112, respectively). Indirect immunofluorescence was performed according to standard procedures using appropriate secondary antibodies conjugated to TRITC (Dako) or Alexa Fluor 405 for blue emission (Molecular Probes). All antibodies were diluted in 1% bovine serum albumin, 0.2% Tween 20 in PBS and incubations were performed on fresh cryosections either overnight at 4 °C or at 37 °C for a maximum of 2 h. Sections were post-fixed for 5 min with 2% paraformaldehyde and examined within a short interval from preparation (1–3 days). This protocol was found to give the better GFP preservation in combination with optimal antibody staining. In some sections, we counterstained with propidium iodide (PI) to visualize the nuclei. Confocal image analysis was performed using the Biorad MRC 1024ES Laser equipped with Nikon fluorescence and Biorad Software.

2.5 Number of GFP+ cardiomyocytes
Heart transplants were cryosectioned at a thickness of 10 µm from the apex to the base. Sections were collected and every 100 µm two consecutive sections were analyzed. The first section was used to count GFP+ cardiomyocytes. The second consecutive section was stained with hematoxilin-eosin and used to count the total number of cardiomyocytes present in the section. This value did not change if cardiomyocytes were identified and counted on the first section counterstained with an anti-troponin I antibody. The density of cardiomyocytes per section was determined by enumerating 16 square fields of 1.5 x 105 µm2. The area of the section was quantitated using the IMAGE J Software. The proportion of GFP+ cardiomyocytes in each transplant was calculated by dividing the total number of GFP+ cardiomyocytes, counted in all sections, by the total number of counted cardiomyocytes present in the sections, obtained by multiplying cell density times section area.

2.6 Number of nuclei in GFP+ cardiomyocytes
The number of nuclei in GFP+ cardiomyocytes was determined as follows: 50-µm sections from different hearts were collected and stained by PI (25 min at room temperature to ensure complete staining throughout the section). To simplify the analysis, only longitudinally sectioned GFP+ cardiomyocytes were considered and only those cells that were entirely represented within the sections were examined further. Images were collected and analyzed by scanning 2-µm thick optical sections using confocal laser microscopy. For 3D-reconstruction and animation, images were elaborated by IMAGE J Volume J 1.7b Software.

2.7 FACS analysis of GFP+ circulating cells
Peripheral blood samples from wild type and GFP+ rats were obtained from the tail vein using a heparin filled syringe. Red blood cells were lysed with BD PharM lyse buffer (Beckton Dickinson). After hemolysis, the cells were scored using a FACScalibur analyzer (BD Biosciences – Immunocytometry Systems) with a 488-nm argon laser. Data were processed using the CellQuest software (BD).

2.8 Determination of nuclear DNA content of cardiomyocytes in heterotopic heart transplants
Nuclear DNA content in GFP– cardiomyocytes of heterotopic heart transplants was determined on tissue sections using PI staining as previously described [14]. PI staining was combined with labeling with anti-Ki67 antibody to distinguish between cycling (Ki67-positive) and non-cycling (Ki67-negative) cells. PI fluorescence intensity was determined as follows: sections were analyzed by confocal microscope and only those nuclei that were entirely represented within the cells analyzed were examined further. Images of PI staining were captured and the DNA content of each nucleus was calculated as fluorescence arbitrary units generated by PI using the IMAGE J Volume J 1.7b Software. A total number of 6 hearts were analyzed for this purpose. Rat lymphocytes (n=1000) and neonatal cardiomyocytes (n=304) were used for baseline 2n and 4n DNA content and for optimization of Ki67 immunological detection. PI intensity was measured by fluorescence microscope. Tissue sections of neonatal rat heart were processed as above. Peripheral blood lymphocytes were purified by Ficoll gradient, counted and smeared on slides, dried at room temperature, fixed with 2% paraformaldehyde and processed with the anti-Ki67 antibody and PI staining.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
We used transgenic rats, expressing the enhanced GFP under the control of the cytomegalovirus enhancer and the chicken beta actin promoter [11], as recipients in heart transplantation. In this transgenic line GFP was highly expressed and homogeneously distributed in cardiomyocytes, endothelial and smooth muscle cells of the coronary vessels and in interstitial cells (Fig. 1A,B). Marked and uniform GFP expression was also observed in circulating cells by FACS analysis of native GFP (Fig.1C) and by immunohistochemistry using an anti-GFP antibody (Fig. 1D-F). Homogeneous GFP expression in peripheral blood cells differs from the data reported for another GFP+ transgenic rat line [15].


Figure 1
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  		Fig. 1 GFP expression in cardiomyocytes, vascular and circulating cells of adult GFP+ transgenic rats. Tissue sections from an adult rat heart show homogeneously high GFP expression in cardiomyocytes (A) and intimal, medial and adventitial layer of a coronary artery (B). Expression of native GFP in peripheral blood cells was analyzed by FACS (C). Grey and white histograms show mean cell fluorescence from control rats and GFP rats, respectively. GFP expression was also examined in peripheral blood cells (D–F) by direct visualization of native GFP (D) and immunostaining with anti-GFP antibody (E). Cells were counterstained with DAPI (F). Bar=(A, B; D–F) 50 µm.

 
GFP- hearts transplanted abdominally into GFP+ recipients showed variable degree of tissue damage, inflammatory infiltration with granulation tissue and scar remodeling (Table 1). In a few hearts only focal areas of tissue damage were observed (Fig. 2A,B), whereas in most cases necrotic areas were distributed throughout the left and right ventricular wall and the septum. The degree of tissue damage was not affected by prolonged cold ischemia pretreatment. In the 90-day hearts the left ventricular wall was usually thinner and contained abundant scar tissue (Fig. 2C,D). Thus, if extensive tissue damage is an essential prerequisite for stem cell recruitment, heterotopic heart transplants represent a valuable model to investigate.


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  		Table 1 Heterotopic heart transplants examined in this study and percentage of identified GFP+ cardiomyocytes

 

Figure 2
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  		Fig. 2 Histopathological analysis of heart transplants 15 and 90 days after surgery. (A, B) 15-day heart transplant with focal areas of necrosis and inflammatory cells. The boxed area in (A) is shown at higher magnification in (B). (C, D) 90-day heart transplant with severe tissue damage, granulation tissue with abundant lymphocytes and tissue remodeling. The boxed area in (C) is shown at higher magnification in (D). Bar (A, C)=1 mm; bar (B, D)=200 µm.

 
A large number of GFP+ cells were found in the 15-day transplants, mainly in the subepicardial regions and around the coronary vessels (Fig. 3A), while at 90 days after surgery, GFP+ cell infiltrates were, in general, less abundant. Most but not all GFP+ cells stained positive for the CD-45 pan marker of hematopoietic cells (Fig. 3B,C) and a large majority of this population expressed the macrophage marker CD-163 (Fig. 3D–F). No GFP+ cells carried the stem cell marker c-kit, although the specific antibody used in immunofluorescence analysis identified c-kit positive cells in the rat bone marrow (not shown).


Figure 3
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  		Fig. 3 GFP+ cells infiltrate the interstitium of heart transplants. (A) GFP+ cells clustered around a coronary artery. (B, C) GFP+ cells (B) are reactive with an anti-CD-45 antibody (C). (D–F) Most GFP+ cells (D) co-express CD-45 (red) (E) and the macrophage marker CD-163 (blue) (F). Note that rare GFP+ cells (D, arrowheads) do not express either CD-45 (E) or CD-163 (F). Bar=50 µm.

 
GFP+ cells were also occasionally observed in the vascular endothelium of some 90-day, but not 15-day, transplanted hearts. These cells were mostly located in those vessels proximal to the epicardium, where inflammatory GFP+ cells mainly accumulated. In some vessels the whole endothelial layer contained GFP+ cells whereas in other vessels GFP+ and GFP- cells were intermingled. The endothelial nature of these GFP+ cells was confirmed by co-expression of the lineage markers von Willebrand factor (Fig. 4A,B), CD-31 and RECA-1 (not shown). Laser scanning and confocal image elaboration were used to exclude the possibility that circulating inflammatory cells, adhering to the vessels, could be mistaken for endothelial cells. The GFP+ endothelial cell population was present in large and small coronary arteries and veins but not in the capillary network (not shown). No labeled cells were ever detected in the medial and adventitial layers of larger vessels (Fig. 4C).


Figure 4
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  		Fig. 4 GFP+ cardiomyocytes and endothelial cells in heart transplants. (A, B) Confocal images of GFP+ endothelial cells in a coronary artery of a 90-day heart transplant. Endothelial cells co-express GFP (A) and the von Willebrand factor detected with a specific antibody (B). (C) GFP is not expressed in the medial layer of a coronary artery, stained by the anti-alpha smooth muscle actin antibody (red). (D–I) Confocal images of GFP+ cardiomyocytes in 15- and 90-day heart transplants. (D) GFP+ cardiomyocyte with nuclei counterstained by PI. (E, F) Cardiomyocyte co-expressing GFP (E) and sarcomeric myosin heavy chain (F). (G, H) Cardiomyocyte co-expressing GFP (G) and cardiac troponin I (H). (I) GFP+ cardiomyocyte expressing connexin-43 at the intercalated disks (arrowheads). (L, M) GFP+ cells in a cryosection of a 15-day heart transplant visualized by native GFP fluorescence (L) and by anti-GFP antibody revealed with a secondary TRITC conjugate (M). A GFP+ cardiomyocyte and numerous GFP+ inflammatory cells are visible in this field. Bar (A–I)=30 µm; bar (L, M)=100 µm.

 
In 21 out of 25 transplanted hearts which underwent surgical treatment but no G-CSF administration, we found occasional GFP+ cells showing the typical morphology and cross-striation of mature cardiomyocytes and staining for sarcomeric myosin heavy chain and cardiac troponin I (Fig. 4D–H). These cells were localized close to areas of tissue damage and were always close to, or surrounded by, GFP- cardiomyocytes. Labeled cardiomyocytes showed connection with adjacent unlabeled cardiomyocytes through gap junctions, as demonstrated by connexin-43 expression at the intercalated disks (Fig. 4I). All transplanted hearts were sectioned from the apex to the base to count systematically GFP+ cardiomyocytes. Values ranged from 0.0005% to a maximum of 0.008% of all cardiomyocytes (Table 1). The relative frequency of GFP+ cardiomyocytes was not related to the degree of tissue damage, did not differ significantly between 15- and 90-day transplants and was not affected by cold ischemia pretreatment. It is unlikely that the direct visualization of GFP fluorescence underestimated the count of GFP+ cardiomyocytes because similar results were obtained by immunological detection using an anti-GFP antibody (Fig. 4L,M). Specifically, in random sections from 5 different transplants, first examined for GFP fluorescence and subsequently processed with anti-GFP antibody, we never observed cardiomyocytes and inflammatory cells stained by the antibody but with undetectable native GFP fluorescence.

To determine whether cardiomyocyte repopulation in heterotopic heart transplants may be increased by bone marrow cell mobilization, we examined a group of 6 rats that underwent cardiac transplantation followed by administration of G-CSF (250 µg/kg/day) for 6 consecutive days. Such treatment promotes sustained marrow cell mobilization. Neutrophyl number, expressed as mean values (± SE), during the first 6 days of treatment raised from 1330 ± 520 (T=0) to 8722 ± 6377 (T=1) and 10225 ± 5789 (T=6). At day 14 the neutrophyl number decreased to 4295 ± 3265 (T=14). A similar increase in neutrophyl number was observed in control rats (n=3) that underwent G-CSF administration but no heart transplantation. These rats had values of 777 ± 115 (T=0), 3140 ± 2979 (T=1), 11930 ± 1881 (T=6) and 1677 ± 1087 (T=14). Immunohistochemical analysis of the treated rats showed that the relative frequency of GFP+ cardiomyocytes did not increase in this group (Table 1) and no increase in GFP+ endothelial cells was likewise found.

We also looked for two early markers of cardiac lineage, Nkx2.5 and GATA-4, with the aim to identify possible immature, undifferentiated cardiomyocytes in the transplants. Nkx-2.5 was never observed. Very rare cells expressing GATA-4 were detected in 8 out of 12 transplanted hearts analyzed with anti-GATA-4 antibody. These cells, often intermingled with inflammatory cell infiltrates and located close to mature cardiomyocytes, were small in size (Fig. 5) and did not express either cardiac-specific or endothelial-specific markers of maturation, such as sarcomeric myosin heavy chain or CD-31 (not shown).


Figure 5
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  		Fig. 5 Rare GFP+ cells express GATA-4. (A) A representative histological section of a 15-day heart transplant shows focal cell necrosis and preserved cardiomyocytes. The boxed region in (A) is magnified and examined by confocal microscopy in B–D. Note one cell co-expressing GFP (B) and GATA-4 (C). The merge is shown in (D). Bar (A)=50 µm; bar (B–D)=12 µm.

 
We observed that GFP+ cardiomyocytes often contained a high number of nuclei. Therefore we counted the nuclei in GFP+ cardiomyocytes using thick cryosections (50 µm) counterstained with PI. Confocal microscope analysis and serial image reconstruction showed that in a sample of randomly chosen GFP+ cardiomyocytes (n=60) from different transplants, 85% contained four to six nuclei, often variable in size (Fig. 6A; B and Supplementary data for animated top and bottom images) and 15% had three nuclei. In contrast, in a random sample of cardiomyocytes (n=60) from normal control hearts, we estimated that 29% of cardiomyocytes was mononucleated, 66% binucleated and 5% three-nucleated. To determine whether multinucleation could be induced in the context of transplantation, we counted the number of nuclei in GFP- cardiomyocytes present in heterotopic heart transplants (n=90): mononucleated, binucleated and three-nucleated cells accounted for 25%, 66% and 9% of cells, respectively. In conclusion, a higher number of nuclei was a distinctive feature of GFP+ cardiomyocytes in heart transplants.


Figure 6
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  		Fig. 6 GFP+ cardiomyocytes contain a high number of nuclei identified by confocal scanning laser microscopy and 3D-reconstruction. (A) Serial optical sections of a GFP+ cardiomyocyte showing GFP (top row), PI staining of nuclei (middle row) and the merge (bottom row). This cell contains four nuclei indicated with white, blue, yellow and white open arrowheads. (B) 3D-reconstruction of the cell shown in A (top) and of another GFP+ cardiomyocyte that contains five nuclei (bottom). Images in (B) can be also visualized using animation (supplementary file: animation B, top.AVI and animation B, bottom.AVI). Bar=15 µm.

 
The lack of GFP+ mono or binucleated cardiac myocytes in heart transplants may have resulted from GFP silencing after nuclear fusion. To address this issue, we measured DNA content per nucleus in a randomly chosen sample of cardiac nuclei of GFP- cardiomyocytes from 15-day (450 nuclei) and 90-day heterotopic hearts (550 nuclei). A total number of 6 hearts were analyzed for this purpose. All the adult cardiac nuclei examined, both in 15- and 90-day transplants, had a 2n DNA content and none of these nuclei stained positive with anti-Ki67 antibody (Fig. 7). On the other hand, neonatal cardiomyocytes and lymphocytes had both 2n Ki67-negative cells and 4n Ki67-positive cells and displayed corresponding levels of PI intensity staining. In conclusion, it is unlikely that counts of GFP+ cardiomyocytes in transplanted hearts were underestimated due to nuclear fusion between circulating cells and donor cardiomyocytes, followed by GFP silencing.


Figure 7
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  		Fig. 7 Nuclei of GFP- cardiomyocytes in heterotopic heart transplants have 2n DNA content. DNA content was determined in GFP- cardiomyocytes of heterotopic heart transplants by staining with PI, labeling with anti-Ki 67 antibody, and quantification of PI fluorescence intensity. The diagrams indicate value distributions of PI fluorescence, expressed as arbitrary units, in (A) cardiomyocytes from 15 and 90 days heart transplants; (B) cardiomyocytes from neonatal rat heart and (C) adult lymphocytes. Diagrams represent Ki-67 negative (continuous line) and Ki-67-positive (dotted line) cells.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
Previous experimental studies, aimed to establish the potential of circulating cells to repopulate and repair the damaged heart, were performed in mice and most of them used the acute ischemic damage induced by coronary artery ligation as the paradigm to test the hypothesis [1–6]. This is the first study in which the model of heterotopic heart transplantation is used to track the fate of genetically targeted circulating cells, derived from GFP+ transgenic rats, in cardiac repair. Heterotopic heart transplantation has been extensively used in a variety of acute and chronic studies, ranging from immunological analyses to gene and cell therapy approaches [16]. The perfused but non-functional transplanted heart has a reduced heart rate and performs essentially no stroke work. Cardiac unloading leads to remodeling and atrophy of the ventricular myocardium with re-activation of fetal genes, with a switch from alpha to beta myosin heavy chain gene expression [17,18]. However, isolated myocytes and papillary muscles from transplanted hearts maintain an intact contractile function when contractile indexes are normalized to account for reductions in cell size [19]. For the purpose of the present investigation, heterotopic heart transplantation seems to provide an appropriate experimental model because the slower progression of tissue damage and remodeling, compared to the infarction model, might favor stem cell recruitment and homing.

Here we show that, following transplantation, a large number of GFP+ host-derived cells, predominantly inflammatory cells, invaded the heterotopic hearts. GFP+ cardiomyocytes were also present in most transplants and showed shape and protein profile typical of mature, structurally integrated cardiac muscle cells. In the wall of arteries and veins of some 90-day transplants, we also occasionally observed GFP+ endothelial cells, but not smooth muscle cells.

The overall number of GFP+ cardiomyocytes was very low in all the grafts and did not increase over time, at least during the time window of our study. It seems unlikely that poor chimerism in heterotopic hearts results from an unfavorable environment of these transplants. Indeed, in a heterotopic heart transplantation model of doxorubicin-induced myocardial injury followed by intracoronary infusion of skeletal myoblasts, Suzuki et al. [20] demonstrated survival and differentiation of skeletal myoblasts and improved cardiac function.

It seems also unlikely that poor chimerism derived from scarce bone marrow stem cell mobilization. Treatment with G-CSF, a procedure that has been reported to enhance tissue regeneration and to improve cardiac function in myocardial infarction [21], did not increase the number of GFP+ cardiomyocytes in the transplants. Recent findings demonstrated that favourable effects of G-CSF in the infarcted heart are due to reduced apoptosis of pre-existing cardiomyocytes, rather than increased cardiac homing of bone marrow cells [22]. Our results show that, in a different biological context, bone marrow cell mobilization neither potentiates GFP+ cell recruitment nor increases cardiac chimerism.

The sequence of events leading to the formation of GFP+ cardiomyocytes in the transplanted hearts is unclear. Two possibilities can be envisaged. One possibility is that these cells migrate to the graft and subsequently differentiate into mature cardiomyocytes. These cells could be either pluripotent stem cells that become committed to the cardiac lineage in the context of the damaged myocardium or tissue-committed circulating cells, similar to those expressing specific cardiac muscle markers and increasing after experimental and human myocardial infarction [23,24]. Our finding of small undifferentiated cells expressing GATA-4, but lacking cardiac markers of differentiation, such as sarcomeric myosin, might support this interpretation. However, GATA-4-positive cells were extremely rare and, more importantly, we never observed cells expressing Nkx2.5, a more specific marker of cardiac commitment. In addition, we never observed intermediate stages, such as small-size GFP+ cardiomyocytes containing sarcomeric proteins, which could resemble newly formed cardiac muscle cells similar to those present in the embryonic and fetal heart. Labeled cardiomyocytes identified in the grafts were all without exception large-size cells similar to surrounding unlabeled cardiomyocytes.

An alternative possibility is that circulating cells migrate to the graft and fuse with mature cardiomyocytes. At the moment we cannot provide a genetic demonstration that GFP+ cardiomyocytes in the transplanted hearts derive from cell fusion. However, the large size of all these cells and the evidence that most of them contain four to six nuclei, whereas adult rat cardiomyocytes, including the unlabeled cells in the grafts, are binucleated and never contain more than three nuclei, support the hypothesis that cell body fusion accounts for the majority of the GFP+ cardiomyocytes. Moreover, the finding that GFP- cardiomyocytes in the heterotopic hearts had diploid nuclei, as it is in normal adult hearts [25], rules out the possibility that cell fusion between GFP+ circulating cells and resident cardiomyocytes was followed by nuclear fusion and GFP silencing. Cell fusion, as opposed to transdifferentiation, has been demonstrated for bone marrow-derived and cardiac stem cells in multiple experimental conditions, including the infarcted heart. However, for the moment only cell body fusion has been demonstrated unambiguously [4,26,27].

In conclusion, our results suggest that circulating cells are involved in two distinct processes taking place in heart transplants. One is the homing to the graft of circulating cells that express the cardiac marker GATA-4 but do not appear to undergo further differentiation along the cardiac lineage. The second is the fusion of circulating cells with cardiomyocytes present in the transplanted hearts. Both events are extremely rare and do not appear to support generation of mature cardiomyocytes de novo.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2005.06.014.


   Acknowledgements
 
This work was supported by MIUR (Ministero dell–Istruzione, dell–Università e della Ricerca, FIRB project RBNE015AX4) to S.A. and S.S, by PRIN 2003 2003067092-001 to G.T and by CORIT. We are grateful to Dr. Claudia Minotto, Department of Oncology and Hematoncology, Noale (VE) and Dr. Licia Ravarotto, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), for help in neutrophyl counting.


   Notes
 
1 These authors contributed equally to the work. Back

2 These authors contributed equally to the work. Back

Time for primary review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

  1. Orlic D., Kajstura J., Chimenti S., Jakoniuk I., Anderson S.M., Li B., et al. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410:701–705.[CrossRef][Medline]
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