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Eur J Cardiothorac Surg 2007;31:444-451. doi:10.1016/j.ejcts.2006.12.023
Copyright © 2007, European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved
IMM Recherche, 42 bd Jourdan, 75014 Paris, France
Received 26 May 2006; received in revised form 30 November 2006; accepted 4 December 2006.
* Corresponding author. Tel.: +33 1 56 61 68 21; fax: +33 1 56 61 67 40. (Email: nicolas.borenstein{at}imm.fr).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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Key Words: Cell transplantation • Cell culture • Satellite cells • Cardiomyopathy • Heart failure
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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The concept of myogenic cell transplantation into the myocardium, known as cellular cardiomyoplasty (CCM), is based on the contribution of exogenous cells to replace lost or altered cardiomyocytes in order to restore functional performance of the heart. There is a rich body of evidence showing that CCM can improve cardiac function in ischemic heart disease—see review [2]. Little is known, however, on the impact of cell transplantation in a large animal model of non-ischemic dilated cardiomyopathy. The aim of the present study was therefore to explore whether an ovine model of toxin-induced heart disease could benefit from myogenic cell transplantation. Our group in a previous report [3] showed that non-cultured skeletal muscle cells can be a cell source for cellular cardiomyoplasty. We speculated that autologous cellular transplantation with this non-cultured muscle cell preparation would engraft into injured myocardium and translate into functional benefit.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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2.2 Anesthesia
For doxorubicin injections, echocardiography, muscle biopsy, and thoracotomy, animals were anesthetized with an IV injection of 0.5 mg/kg midazolam and then an IV injection of 0.5 mg/kg etomidate allowing endo-tracheal intubation and maintenance with isoflurane in 100% oxygen, delivered with a veterinary ventilator (HALLOWEL® Pittsfield, MA). A constant rate infusion of lidocaine (50 µg/(kg min)) was started at the same time as intracoronary injections of doxorubicin or imtramyocardial injections. Monitoring included invasive blood pressure (auricular artery), end tidal CO2, oxymetry, and core body temperature.
After surgery, the animals were left to recover with the required analgesic regimen (morphine 0.5 mg/kg IM BID, flunixin 1 mg/kg IM once) and the following day as needed.
2.3 Experimental design
The experimental design was the following: Sequential intracoronary injections of doxorubicin (0.75 mg/kg) were carried out every 2 weeks until echocardiographic detection of signs of systolic myocardial dysfunction (conventional and tissue Doppler imaging echocardiography). One month after the last injection, animals were randomly assigned to either one of the two following groups:
Hemodynamic assessment was then again performed at day 0 (just before intramyocardial injections) and at day 60 (just before sacrifice).
2.4 Heart failure model
In order to establish a reliable heart failure model, we developed a toxin-induced heart failure model in the adult sheep, as previously described [4]. The basic principle relies on sequential percutaneous intracoronary injections of doxorubicin. Doxorubicin is a potent, broad-spectrum chemotherapeutic agent effective against solid tumors and malignant disease [5]. However, the use of doxorubicin is associated with cumulative, dose-related, progressive myocardial damage that may lead to irreversible congestive heart failure through DNA intercalation, oxidative stress, and endothelial cell apoptosis [5]. Our previously published material in the ovine model showed that we could systematically and reliably obtain LV dilation, increased plasma levels of Atrial Natriuretic Factor, and echocardiographic signs of global and regional contractile dysfunction [4].
To avoid the inflammatory pericardial effusion induced by the doxorubicin injections (observed in a pilot study), a small pericardial window was created prior to the first injection. For this purpose, the sheep were placed in left lateral recumbency and were clipped and prepared for standard thoracoscopy. One 10-mm cannula and two 5-mm cannulae were inserted into the thoracic cavity. A 0° 10-mm telescope connected to a video camera and monitor was inserted through the 10-mm cannula; atraumatic tissue forceps as well as scissors connected to cautery were inserted through the 5-mm cannula. A 3-cm window was created. The three portals were closed in two layers and a chest tube was left in place for 1 h postoperatively. The sheep were then placed in dorsal recumbency for left ventricular catheterization.
The left or right femoral artery was percutaneously cannulated with a 5-Fr introducer. After 0.25 mg/kg of an IV bolus of heparin, an Amplatz guiding catheter (AL 2, CORDIS®) was used to catheterize the left coronary ostium. Adequate placement of the catheter's tip was checked with small boluses of contrast media. A constant rate intracoronary infusion of 0.75 mg/kg doxorubicin (Adriblastine, Pharmacia & Upjohn®, St. Quentin en Yvelines, France) diluted in 50 ml of saline was started with a syringe pump and maintained over 60 min. Doses were determined in a pilot study. Animals were closely monitored in the immediate postoperative period for signs of arrhythmia or ventricular dysfunction. One month after the last injection, animals were assessed functionally and assigned to either cell therapy or placebo injections.
2.5 Functional assessment: echocardiography and TDI examinations
Transthoracic conventional echocardiography and TDI examinations were performed by a single experienced operator in a blinded fashion, with continuous ECG monitoring, using a Vingmed system 5 (General Electric medical system, Waukesha, WI, USA) equipped with a 2.2–3.5 MHz phased-array transducer. Two months after the surgery, the scar of the biopsy on the hind leg was completely covered by wool, so the cardiologist was blinded to the treatment group all along.
The animals were placed in ventral recumbency and hair was clipped between the right fourth and seventh intercostal spaces. All transthoracic echocardiographic and TDI measurements included a mean of three consecutive beats.
Left ventricular dimension measurements were performed using 2D-guided M-mode on the right parasternal ventricular short-axis view [6]. Left ventricular end-systolic and end-diastolic diameters were measured and the left ventricular ejection fraction (EF) was then calculated.
Two-dimensional (2D) color TDI was performed after conventional echocardiographic examination. Measurement of myocardial velocities resulting from the radial left ventricular motion was carried out using the right parasternal ventricular short-axis view between the two papillary muscles as previously described [4]. Real time color Doppler was superimposed on grayscale with a frame rate > 100 frames per second. Doppler velocity range was set as low as possible to avoid occurrence of aliasing. Digital images were obtained and stored for later assessment using an offline measuring system (Echo Pac for System 5). Two millimeters sampling was used and a tissue velocity profile was displayed in each sample location, i.e. the endocardial and epicardial segments of the left ventricular free wall. Endocardial and epicardial velocity profiles were obtained simultaneously during the offline analysis. The peak values of myocardial velocities were determined for each segment in systole (S) and the corresponding myocardial velocity gradient (MVG) was then calculated (difference between simultaneous endocardial and epicardial velocities).
2.6 Skeletal muscle biopsy
Animals of group 1 were anesthetized 3 h before thoracotomy in order to perform the muscle biopsy. Approximately 10 g of muscle were explanted from the left femoral biceps under sterile conditions. The biopsy tissue was kept at room temperature until mechanical and enzymatic digestions were started. The operative wound was closed in a routine fashion.
2.7 Muscle cell extraction
In animals of group 1, the non-cultured cell preparation was performed as described by our group [3]. This technique is based on the extraction of satellite cells from a skeletal muscle with mechanical and enzymatic digestion and reimplantation of the cell preparation approximately 3 h after muscle biopsy. Briefly, the muscle was minced with scissors to a slurry. In order to release satellite cells, the muscle fragments were then incubated at 37 °C under agitation, in 10 ml of DMEM supplemented with 0.2% (w/v) crude collagenase. After 20 min, the fragments were centrifuged at 300 rpm for 2 min.
The supernatant containing isolated cells was stored in DMEM; the pellet was then subjected to four more rounds of digestion. The extracted cells were then filtered through a nylon cell strainer (Polylabo®). Cells were then labeled with a chloromethylbenzamido derivative of DiI (fluorescent carbocyanine 1-1'-dioctadecyl-3-3-3'-3'-tetramethylindocarbocyanine perchlorate, CellTrackerTM CM-DiI). The DiI-labeled cell preparation was then resuspended in 2 ml of serum-free DMEM and kept at 4 °C until implantation. A previous study allowed to determine that 1.7 ± 0.3 x 107 mononucleated cells were injected per animal [3].
2.8 Cell culture controls
In order to confirm the presence of muscle precursor cells in the cell preparation, a 100-µl aliquot of the cell suspension was plated in a flask containing fetal calf serum completed-DMEM. The cells were then grown as a control in humid air with 5% CO2. One week after plating, cells were processed for immunodetection of myogenin and troponin as previously described [7].
2.9 Cell implantation or placebo injection into the left ventricular myocardium
A constant rate infusion of lidocaine (50 µg/(kg min)) was started immediately after cutting. A left fifth intercostal thoracotomy was performed and the heart was suspended in a pericardial cradle. Landmarks were made with 5/0 polypropylene suture material on the left ventricular free wall. Twenty epicardial 100-µl injections of the cell preparation (group 1, n
= 8) or medium (group 2, n
= 5) were carried out with a 27G infusion set (VYGON) about 5 mm apart. A 0.1 mg/kg IM injection of dexamethasone was performed before closing in order to reduce the inflammation associated with the numerous intramyocardial punctures (pilot study, data not shown). The thorax was closed in a routine manner and the animals were left to recover with the required analgesic regimen.
2.10 Histology
All sheep were euthanized 60 ± 2 days after intramyocardial injections with the cellular preparation or placebo. After a final contractility assessment using conventional echocardiography and TDI, heparin (10,000 IU) and 60 mg/kg of sodium pentobarbital were injected intravenously.
The heart was exposed through a left thoracotomy. The site of the myocardial injury was identified and dissected free of adhesions. A small fragment of left ventricular free wall was snap-frozen in liquid nitrogen. Serial 5-µ sections from the snap-frozen area were processed and watched under fluorescent microscopy for DiI identification. The rest of the heart was then fixed with 3% formalin for histological evaluation. Formalin-fixed, paraffin-embedded blocks were processed. Serial 5-µ sections from the harvested area were prepared for conventional HES staining and immunostained by an automated immunoperoxidase technique (Ventana Medical Systems-ES, Tucson, AZ) using monoclonal antibodies to a fast skeletal-specific isoform of myosin heavy chain (MY32–SIGMA) and to connexin-43 (SIGMA), a component of gap junctions.
Briefly, deparaffinized sections were blocked for endogenous peroxidase activity and subjected to antigen retrieval. Sections were exposed to a biotinylated anti-mouse secondary antibody. Sections were developed with diaminobenzidine (DAB) or alkaline phosphatase, counterstained with hematoxylin and post-counterstained with a bluing reagent.
2.11 Statistics
Values were expressed as mean ± SD. Each conventional and TDI parameter was compared between time points A (baseline) and B (post-doxorubicin) using a paired Student's t-test. Those parameters were also compared between time points B and C and between both groups at these time points using a one-way ANOVA with repeated measures followed, if necessary, by a Student's t-test. Significant differences were determined as p
< 0.05.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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3.1 Echocardiography and TDI examinations
As expected, intracoronary infusions of doxorubicin resulted in a statistically significant decrease in EF from day 0 (75 ± 7%) to the last injection (51 ± 11%) (p
< 0.05), and increase (p
< 0.05) in both systolic (28 ± 6 mm vs 40 ± 8 mm) and diastolic (45 ± 7 mm vs 51 ± 8 mm) left ventricular diameters (see Table 1
).
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Two months after intramyocardial grafting, TDI and echocardiographic assessment of the groups showed a marked improvement in the non-cultured cell therapy group (see Table 1), whereas systolic myocardial function remained altered and stable in the control group. A significant increase in both the ejection fraction (p < 0.05) and the systolic MVG (p < 0.01) was observed in the grafted group but not in the placebo group. In the grafted group, the systolic MVG returned to similar values to those obtained at baseline, whereas it remained low in the control group with comparable values to those measured after doxorubicin injections (Fig. 1 ). The MVG improvement in the grafted group was related to a significant increase in the systolic endocardial velocities (p < 0.01).
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Aliquots of each grafted cell preparation were analyzed to confirm cell viability and myogenic differentiation. Control culture flasks were inspected daily. When the cells were left to fuse, all flasks were covered with numerous myotubes after 1 week of culture (3-a). Immunodetection with myogenin and troponin showed that cultured cells were indeed highly myogenic (Fig. 2b and c).
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Vast areas of replacement fibrosis or focii of degenerative myocardium made of vacuolated degenerative myocytes were also present. These lesions are characteristic of this heart failure model and were not attributed to the numerous punctures or the presence of skeletal muscle cells.
The myocardium of control animals without implantation or implanted with medium alone did not show any MY32 expression or cmDiI-positive cells under fluorescence microscopy.
Immunohistochemistry with an antibody specific to connexin-43 was performed on myocardial slices. Non-grafted areas were positive, but grafted areas were negative for this antibody, thereby suggesting the absence of electromechanical coupling between grafted cells and their host counterparts.
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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4.1 Heart failure model
Achieving a reliable large animal model of dilated cardiomyopathy (DCM) is a critical component of successful research on new CHF treatment modalities.
DCM is a well-recognized cause of spontaneous heart failure in large and giant breed dogs. We have previously reported the use of this idiopathic model in a preliminary veterinary cell therapy clinical trial for DCM [8]. However, canine DCM is a relatively rare condition, and there is no readily available colony for CHF studies.
Doxorubicin has been used to induce heart failure in several large animal models. Mainly dogs, but also ruminants and monkeys, have been used in doxorubicin-induced heart failure studies [9–14]. Several groups have addressed the devastating systemic effects of doxorubicin in the dog using transcatheter infusion directly into the coronary artery, delivering higher peak concentrations to the myocardial cells while reducing the systemic effects [11,12,15,16]. Fatal arrhythmia remains a serious side effect of this model [9]. Goats and sheep have also been used as a doxorubicin-induced heart failure model [14,17]. Only intravenous methods have been reported so far. In the present model, we have systematically obtained LV dilation, increased plasma levels of ANF, signs of global as well as regional contractile dysfunction.
4.2 Cell source
Optimal cell type is yet undetermined. Each cell source bears advantages and limitations. Important aspects are ease of procurement, cell robustness (tolerance to ischemia), and eventually potential muscular commitment. In a previous report, our group [3] showed that non cultured muscle cells can be a cell source for cellular cardiomyoplasty. Advantages of using non-cultured cells as opposed to cultured satellite cells are the lower cost, the reduced time lag between harvest and grafting, and the diminished risk of contamination. Further, culturing the cells shifts them from their state of quiescence to a state of activated precursors. This might reduce their regeneration capacity unlike freshly isolated cells which are not activated at the time of grafting [18]. This technique requires a biopsy in the morning and a grafting procedure 3 h later. We, therefore, believe that his technique is clinically relevant and could be an interesting alternative to cultured skeletal muscle cells.
4.3 Delivery
Targeted administration implies either transmyocardial or intracoronary – arterial or venous – delivery. Some cell types may not be ideal candidates for transcoronary delivery. The very small muscle fragments still present in our cell preparation precluded intracoronary administration. Indeed, a pilot study carried out by us on this dilated cardiomyopathy model yielded acute myocardial ischemia and death in all animals exposed to intracoronary non-cultured cells (unpublished data). Further, in an experimental setting, knowing precisely where the injections were performed is of paramount importance while avoiding the intercostal thoracotomy with percutaneous means of delivery is mostly relevant in human patients. Therefore, epicardial injections through a thoracotomy were carried out.
4.4 Mechanism of action
Basic and clinical research on cell transplantation have mainly focused on ischemic heart disease since myocardial infarction is the leading cause for heart failure and death in developed countries. Our project was to assess the potential benefit of grafting non-cultured cells in dilated cardiomyopathy in a large animal model.
Successful engraftment translated into a functional benefit, which we could demonstrate with TDI. To the best of the authors’ knowledge, this is the first study to conclude to a beneficial functional effect of satellite cells in a large animal model of non-ischemic heart failure. One largely unanswered question is the causative mechanism by which improvement of function is observed.
Cellular cardiomyoplasty has been proposed as an alternative strategy for augmenting the function of ischemic myocardium. Several mechanisms have been proposed as an explanation for the observed improvement in ischemic heart failure, some of which can be extended to dilated cardiomyopathy. The presence of transplanted cells might ignite some degree of angiogenesis or deliver cytokines, which could contribute to the reparative mechanisms of diseased myocardium [2]. Inotropic support and direct mechanical cellular contribution to contraction and relaxation could also be involved. Considering that we did not find the presence of connexin-43 between grafted myosatellite cells and resident cardiomyocytes and that it is generally accepted that there is no intercalation between grafted myoblasts and their host counterparts [19,20], it is likely that other phenomena are involved. The improvement could also be ascribed to the cells establishing an organized interstitial matrix, which limits dilatation and thinning (remodelling). We do not provide evidence to support this assumption, but our study was not meant to address specifically these questions.
At any rate, the results of the current study are in agreement with previously published papers as regards the potential improvement of function on a global non-ischemic form of heart failure [21–25].
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5. Limitations |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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In conclusion, our data indicate that non-cultured muscle cell transplantation is feasible and may translate into a functional benefit in an ovine model of dilated heart failure.
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Acknowledgments |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. Discussion5. LimitationsReferences |
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