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Cellular Plasticity in the Cardiovascular System
1Instituto de Biofísica Carlos Chagas Filho UFRJ, Rio de Janeiro RJ, CEP-21949-900; 2Ecodata Exames Médicos LTDA, Rio de Janeiro RJ, CEP-22020-120; 3Departamento de Ciências Fisiológicas da UFES, Vitória ES, CEP-29040-090; 4Hospital Pró-Cardíaco, Rio de Janeiro RJ, CEP-22251-150; and 5Centro de Pesquisa Gonçalo Muniz, Fundação Oswaldo Cruz, Salvador BA, CEP-40295-00, Brazil; and 6Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 1 December 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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myocardial infarction; cell therapy
Despite its limitations, such as shortage of donors, problems with immunosuppression, and graft failure, cardiac transplantation was, until recently, the only available solution for treatment of severe terminal heart failure. New therapeutic approaches such as cellular cardiomyoplasty (CCM), in which appropriate donor cells are delivered to the injured myocardium, target the pathophysiological basis of congestive heart failure by attempting to regenerate the damaged myocardium through the transplantation of healthy cells. Many cell types have been transplanted into injured myocardium: embryonic (15), fetal (16, 21), and adult cardiomyocytes (9), skeletal myoblasts (24), immortalized myoblasts (14), smooth muscle cells (8), fibroblasts (5), bone marrow-derived stromal (27) and hematopoietic (12) stem cells, and human bone marrow-derived endothelial precursors (7).
The efficiency of CCM therapy to improve cardiac function in models of ischemia-reperfusion (18), cryoinjury (9, 24), and dilated cardiomyopathy (29) has been reported. CCM has also been extensively performed in models of permanent ligation of the left anterior descending coronary artery (LAD), but mainly within a short period of time after the ischemic injury: 7 days (2), 2 days (7), 3–5 h (12), 1 h (23), 30 min (11), and immediately after surgery (19).
Thus the main purpose of this study was to investigate whether CCM can improve the performance of healed infarcted rat hearts when a fibrous scar is present in the wall of the left ventricle and heart failure has developed.
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METHODS |
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Animals
Male inbred Wistar rats (200–250 g) were obtained from the Centro de Pesquisa Gonçalo Muniz (Fiocruz-Bahia/Brazil). Animals were housed at controlled temperature (23°C) with daily exposure to a 12:12-h light-dark cycle and free access to water and standard rat chow. These isogenic rats were used as donors and recipients of bone marrow-derived stromal cells (BMSC) to simulate clinical autologous implantation.
Cell Isolation and Culture Procedures
The isolation and primary culture of BMSC from femoral and tibial bones of donor rats were performed as described by Caplan (1). BMSC were cultured in DMEM supplemented with 20% fetal bovine serum (GIBCO-BRL), 2 mM L-glutamine (Sigma), and antibiotics (100 U/ml penicillin G and 100 µg/ml streptomycin; GIBCO-BRL) and maintained at 37°C in a 5% CO2 incubator. The cultures were maintained for 1 wk, during which medium was changed at least twice, which washed away all floating hematopoietic cells. At 
80–90% confluence, the cells were detached from the culture flasks with 0.25% trypsin-EDTA (Sigma), resuspended in DMEM, and labeled with 4',6-diamidino-2-phenylindole (DAPI; Sigma) for 30 min. The morphology of the cells just before labeling with DAPI is shown in Fig. 1A. The cells were rinsed six times in balanced salt solution to remove unbound DAPI and kept in warm DMEM (1–3 x 105 cells) for a few minutes before transplantation. This labeling procedure was very efficient, ensuring 100% labeling of cell nuclei.
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On the day of injection, BMSC were detached from culture flasks with ice. Briefly, 106 cells were stained with anti-CD34-phycoerythrin (ICO 115, Santa Cruz Biotechnology) and anti-CD45-FITC (Caltag Laboratories, Burlingame, CA) for 30 min at 4°C and washed twice before flow cytometry (FACSCalibur, Becton-Dickinson). Figure 1B shows that >99% of the cells were negative for both hematopoietic markers. Mouse isotype controls IgG1-phycoerythrin and IgG1-FITC were also used. All antibody dilutions were 1:100.
Myocardial Infarction and Cell Transplantation
Left ventricular myocardial infarction was induced following the procedure described by Johns and Olson (6). Briefly, male Wistar rats (200–250 g) were anesthetized with halothane (Merck), and a 2-cm incision was made on the left side of the thorax, parallel to the sternum. The fifth and sixth ribs were separated, exposing the heart, and the LAD was permanently occluded as it passes under the left atrial appendage by ligation with a 6-0 silk thread. The chest was then closed with continuous silk stitch, and the rats were allowed to recover.
The infarcted rats were randomized for injection with BMSC or culture medium alone. For the injection procedure, the animals were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg ip) and maintained under positive-pressure ventilation with room air supplemented with oxygen (2 l/min; model 680 rodent respirator, Harvard Apparatus) at 60–80 cycles/min with a tidal volume of 1 ml/100 g body wt. The heart was exposed, the macroscopic scar area was visualized, and 200 µl of a BMSC suspension containing a total of 1–3 x 105 cells were injected with a tuberculin syringe into two to three sites along the borders of the left ventricular wall scar tissue. The same volume of DMEM was injected into two to three points on corresponding areas in the hearts of the medium-treated animals. After treatment with cells or medium, the chest was stitched closed, and the animals were carefully monitored for 4 h postoperatively.
The mortality resulting from our experimental procedures was 20% within 24 h after infarction surgery and 35% within 24 h after BMSC or medium injection.
Experimental Groups
Animals were divided into three experimental groups: nonoperated (normal, n = 34), sham-operated (sham, n = 33), and infarcted (infarcted, n = 30) rats. The nonoperated, normal group was subjected to electro- and echocardiographic analysis at least twice during the study. The infarcted group was analyzed 29–30 days after ligation of the LAD by electro- and echocardiography. The sham group was subjected to the same surgical procedure as the infarcted group, but the LAD was not ligated. In this group, functional analysis was also performed 29–30 days after surgery. After electro- and echocardiographic parameters were determined for the three groups, to control for interanimal variability and because of mortality during the delivery procedure, we used a smaller number of infarcted rats (n = 20), in which we determined functional parameters in a serial manner. Electro- and echocardiograms were recorded before and 14 days after treatment with BMSC (n = 10) or culture medium alone (medium, n = 10).
Functional Assessment
Electrocardiographic study. Rats anesthetized with ketamine and xylazine were placed in the supine position for electrocardiographic recording (Cardimax FX-2111, Fukuda Denshi) in classic configuration: six limb leads (L1, L2, L3, aVR, aVL, and aVF) and two chest leads (Vant, recorded at half the distance from the sternal manubrium and the xyphoid process, and Vlat, recorded at the midaxillary line, at the same height of the 1st chest lead). To obtain reproducible electrocardiographic recordings in the rat, the anesthetized animals were fixed on a board with their anterior paws in the orthogonal direction with respect to the body and their posterior limbs kept free. Additionally, all animals were carefully examined at the time of medium or cell injection (in the infarcted group) and postmortem (in all groups) to detect alterations of the anatomic relations of the heart in the thoracic cage that might lead to misleading results in the evaluation of the ventricular depolarization vector. Electrocardiograms were recorded for 3 min, and the anesthetized animals were then subjected to echocardiographic examination. During the 5- to 15-min echocardiographic examination, we continuously recorded an L2 lead electrocardiogram.
The electrocardiogram parameters were as previously described (17): heart rate, presence of a P wave >0.1 mV, P-R interval, QRS duration, Q-T interval, frontal QRS axis, QRS amplitude index (i.e., the sum of QRS complex voltage in L1, L2, and L3), and the presence of a Q wave in L1, L2, L3, aVF, Vant, and Vlat. In all infarcted animals (n = 30), the presence of a frontal QRS axis >90° (rightward deviation) was used as evidence of an extensive infarction as demonstrated by postmortem pathological analysis that confirmed the presence of transmural scar tissue in an area corresponding to 30% of the left ventricle (data not shown). Thus the data for frontal QRS axis are represented as frequency of occurrence: animals with frontal QRS axis >90° (rightward) or between 0 and 90° (leftward).
Echocardiographic study. We used an echocardiographic color system (Megas/Esaote) equipped with a 10-MHz electronic phased-array transducer. Under ketamine and xylazine anesthesia, the chests of the animals were shaved, and they were maintained in a left lateral decubitus or a supine position. All echocardiographic analysis were performed blind by the same echocardiographer and included morphological and functional parameters. Images were obtained from the left parasternal and apical windows. Short-axis two-dimensional views of the left ventricle were taken at the level of the papillary muscles to obtain the M-mode recordings. Anterior and posterior end-diastolic and end-systolic wall thickness, left ventricle, left atrium, and aorta internal dimensions, and relative wall thickness (2 x posterior end-diastolic wall thickness ÷ left ventricle internal end-diastolic dimension) were measured following the American Society of Echocardiography leading-edge method. The systolic function was expressed by the fractional shortening obtained from the M-mode traces. The pulsed-wave Doppler spectra of mitral inflow were recorded from the apical four-chamber view with the guidance of the color Doppler. All Doppler spectra [mitral flow velocity pattern: peak early diastolic filling velocity (E velocity), peak filling velocity at atrial contraction (A velocity), and their ratio (E/A)] were recorded, and morphological parameter values were measured during the echocardiographic exam.
Immunohistochemistry.
At 3 wk after CCM, BMSC-treated (n = 3) and medium-treated (n = 3) animals were anesthetized with ether and killed by cervical dislocation, and their hearts were perfused with 4% paraformaldehyde in phosphate buffer. The tissues were cryoprotected and cooled in liquid nitrogen, and 10-µm consecutive cryostat sections were obtained. Thereafter, the sections were processed for immunofluorescence using a monoclonal anti-
-smooth muscle actin antibody (clone 1A4, Dako) and a monoclonal anti--sarcomeric actin (clone 5C5, Sigma). Both antibodies were used at 1:100 dilutions. Goat secondary antibodies were used as follows: anti-mouse IgG FITC conjugate (catalog no. F-2012, Sigma) and anti-mouse IgM FITC conjugate (catalog no. F-9259, Sigma), both at 1:50 dilutions. The grafted BMSC were identified by DAPI-labeled nuclei. An Axiovert 135 microscope coupled to a high-resolution Axiocam HR charge-coupled device videocamera (Zeiss) was used to obtain differential interference contrast and fluorescence images from regions of the infarcted left ventricle displaying nuclear and cytoskeletal markers. Images were overlaid and processed using Photoshop 7 software (Adobe).
Statistical Analysis
Values are means ± SE. Differences between electro- and echocardiographic measurements before and after medium or BMSC treatment and between groups were evaluated by paired and unpaired Student's t-test, respectively. Values were considered different when P < 0.05 after Bonferroni's correction. When frequency data were compared, the nonparametric Fisher's exact test with the 0.05 level of probability was used.
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RESULTS |
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Electrocardiographic study. Electrocardiographic parameters from nonoperated (normal) and sham-operated groups were not statistically different. However, interesting differences were detected between electrocardiographic parameters from infarcted and normal or sham animals (Table 1). The most outstanding difference is the significant rightward shift of the frontal QRS axis in all 30 animals from the infarcted group. This group also differed from the sham-operated and normal groups in relation to the presence of the Q wave in L1 and Vlat and the significant decrease in the QRS amplitude index, confirming previous reports (17).
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All other electrocardiographic parameters that are not listed in Tables 1 and 2 were similar in all groups.
Echocardiographic study. Heart rate was constantly monitored during echocardiography and was not statistically different between all groups studied, ranging from 250 to 300 beats/min under anesthesia (data not shown). As shown in Table 3, 4 wk after infarction, a significant increase in left atrium-to-aorta ratio, left ventricular end-diastolic dimension, and E/A velocity ratio (restrictive mitral flow) and a significant decrease in relative wall thickness and fractional shortening were observed in the infarcted group compared with the other two groups. These differences point to the fact that 4 wk after infarction the animals have already developed heart failure. No major changes were detected in other echocardiographic parameters.
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Doppler analysis of the BMSC-treated infarcted hearts shows that, 2 wk after treatment, the mitral flow pattern measured by the E/A velocity ratio had decreased from 7.49 ± 2.37 to 2.66 ± 0.91 (P < 0.05). In contrast, in the medium-injected animals, the E/A velocity ratio increased from 6.43 ± 2.64 to 10.52 ± 2.61 (P < 0.05). The values attained 2 wk after medium or BMSC injection are statistically significant (P < 0.01).
Representative M-mode traces of one infarcted rat before (Fig. 3A) and 2 wk after (Fig. 3B) CCM show clear improvement in left ventricular contractility, which in this animal, as in most animals studied, was particularly striking in the anterior wall.
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DAPI-labeled transplanted cells could be easily identified in consecutive sections obtained from the middle portion of the left ventricle of the BMSC-treated rat hearts. Figure 4 shows representative images of positive immunostaining for smooth muscle and sarcomeric actin colocalized with DAPI-labeled nuclei in individual smooth muscle cells from the tunica media of coronary vessels and in cardiomyocytes (Fig. 4, A–E). These double-labeled vessels and myocardium seemed to be structurally integrated with the healthy ventricular wall. However, not all the cells presenting nuclear and cytoskeletal fluorescent labels seemed to have achieved a fully differentiated state (Fig. 4F). When this was the case, cell shape was round, and the cells were frequently found in groups or distributed along the cardiomyocytes. By counting the numbers of DAPI-labeled nuclei in an 6-mm2 area around the scar (which should account for the localization of most of the injected cells), we determined an average of 3 x 104 nuclei, suggesting that 
6–10% of the injected cells were still present in the heart 3 wk after cell injection.
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DISCUSSION |
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BMSC are a good choice of cell type to be used in the regeneration of the damaged myocardium. These cells have the potential to differentiate into cardiomyocytes "in vitro" (10) and express functional adrenergic (
1 and 2) and muscarinic (M1 and M2) receptors (4) after treatment with 5-azacytidine. In addition, BMSC can also exhibit a cardiomyogenic phenotype when implanted into myocardium (27). Moreover, from a clinical point of view, the use of BMSC in CCM would be extremely convenient, inasmuch as these cells can easily be harvested from patients during bone marrow aspiration and then expanded in culture. Although the therapeutic effects of BMSC have been recognized (3, 20, 25, 26, 28), it is still not known whether a specific subpopulation in the BMSC is actually responsible for these effects. Phenotypic analysis by flow cytometry showed that the BMSC used in our transplants are mainly CD34– and CD45– and extremely homogeneous from a morphological standpoint. Many groups isolate the so-called "mesenchymal stem cells" from the bone marrow by the adherence protocol we used. The mesenchymal stromal cells were originally isolated by Caplan (1) using such a method. Because classical markers for bone marrow stem cells are scant for rats, we opted to call our cells stromal cells and used the absence of CD34 and CD45 markers to prove their nonhematopoietic lineage. Further phenotypic analysis is rendered difficult by the lack of availability of specific markers for rat bone marrow cells.
The functional data presented here provide strong evidence that use of BMSC in CCM can improve cardiac performance even after the healing process has occurred. Electro- and echocardiographic parameters of BMSC-treated animals improved significantly compared with medium-treated animals, suggesting the participation of BMSC in the chain of events that ultimately led to the improvement of cardiac function. Additionally, the histological examination suggests that implanted BMSC engrafted in the myocardium give rise to new myocardium and blood vessels. However, one limitation of our study is that we cannot rule out that the colocalization in the fluorescent images results from structures that are not in the same focal plane, because we did not use confocal microscopy. Furthermore, because our histological studies were conducted 3 wk after cell injection, we cannot speculate about the mechanisms involved in new myocardial and blood vessel formation. Investigating the responsible mechanisms for new muscle and vessel formation would require sequential histological evaluation at much earlier time points. Our study was clearly not designed to address this point but, rather, to investigate the therapeutic value of bone marrow cell transplantation in postischemic heart failure.
Sakakibara et al. (16), using the same infarction model and fetal cardiomyocyte transplantation, did not report improvement in cardiac function 4 wk after treatment. It is possible that the different cell types used for transplantation may account for the diverse results. This hypothesis is supported by work developed in pig models where Fuchs and colleagues (3) showed that bone marrow cells secrete angiogenic factors that induce endothelial cell proliferation and, when injected transendocardially, augment collateral perfusion and myocardial function of ischemic myocardium. More recently, three other groups (20, 25, 28) reported that bone marrow mesenchymal stem cells do engraft, generate cardiomyocytes, and restore cardiac function in porcine hearts subjected to ischemic injury 2–4 wk before cell therapy.
If it is considered that BMSC treatment prevented additional left atrial enlargement, restored normal mitral flow pattern, and substantially increased fractional shortening, it is tempting to hypothesize that cellular therapy with BMSC can reverse or at least delay the evolution of the processes that, after myocardial infarction, lead to heart failure. Whether a reversion of the heart failure associated with myocardial infarction can be sustained by CCM deserves further investigation with long-term follow-up. The use of mononuclear bone marrow cells (MBMC) has proven efficient in patients with acute myocardial infarction (22), and our group has reported improvement in heart function after MBMC transplantation in patients with healed infarcts (13). MBMC contain the stromal cells. The percentage of BMSC in a mononuclear fraction of bone marrow cells varies from 0.5 to 1%.
Data presented here suggest that improvement of cardiac function may result from new heart vessel and muscle formation even after scar tissue has formed. These findings are extremely relevant clinically, because postischemic congestive heart failure is the major cause of death in patients who survive myocardial infarction, and there are no effective therapies to stop the progression of this disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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