- Email: [email protected]
Transplantation of neonatal cardiomyocytes after permanent coronary artery occlusion increases regional blood flow of infarcted myocardium
Journal of Molecular and Cellular Cardiology 35 (2003) 607–613 www.elsevier.com/locate/yjmcc
Original Article
Transplantation of neonatal cardiomyocytes after permanent coronary artery occlusion increases regional blood flow of infarcted myocardium Thorsten Reffelmann, Joan S. Dow, Wangde Dai, Sharon L. Hale, Boris Z. Simkhovich, Robert A. Kloner * The Heart Institute, Good Samaritan Hospital, University of Southern California, 1225 Wilshire Boulevard, Los Angeles, CA 90017-2395, USA Received 26 December 2002; received in revised form 30 January 2003; accepted 19 February 2003
Abstract Background. – Cellular cardiomyoplasty is a promising approach for rebuilding scar tissue after acute myocardial infarction. However, the angiogenic potential of transplanted immature cardiomyocytes and their effect on regional myocardial blood flow (RMBF) after coronary artery occlusion remain to be evaluated. Methods and results. – Intramyocardial injection of cultured neonatal cardiomyocytes (4 × 106 cells/50-70 µl) into the scar 1 week after permanent coronary occlusion in rats resulted in improved RMBF in the infarct 4 weeks after transplantation (radioactive microspheres, 0.97 ± 0.18 ml/min/g) in comparison to medium-injected hearts (0.61 ± 0.11 ml/min/g, P < 0.047). The macroscopic perfusion defect after in vivo staining with the blue dye 50% Uniperse blue was significantly smaller in the cell transplantation group (1.5 ± 0.3% of the heart) compared to the medium group (3.0 ± 0.6%, P < 0.017). Clusters of engrafted cells within the scar demonstrated a high capillary density (1217 ± 114 perfused (blue) capillaries/mm2); however, in the scar tissue itself capillary density in the cell group (156 ± 62/mm2) did not significantly differ from the medium group (125 ± 10/mm2), suggesting that neo-angiogenesis was confined to regions of successful engraftment (non-infarcted tissue: 1924 ± 114 perfused capillaries/mm2). The transplantation group was characterized by smaller diastolic and systolic left ventricular volumes, as assessed by intravenous ventriculography, along with thickened infarcts (0.93 ± 0.07 vs. 0.75 ± 0.04 mm, P < 0.020) and lower infarct expansion indices (0.64 ± 0.07 vs. 0.83 ± 0.06, P < 0.023), as determined by post-mortem morphometry of histologic slides. Conclusions. – Transplantation of neonatal cardiomyocytes induced neo-angiogenesis in zones of successful cell engraftment within the scar, which effectively enhanced tissue perfusion. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Capillaries; Transplantation; Cells; Regional blood flow
1. Introduction The quantitatively insufficient capacity of the mammalian heart to replace irreversibly damaged myocardium is the pivotal cause of akinetic scar formation and aneurysmal left ventricular (LV) dilation after acute myocardial infarction [1,2]. Cellular cardiomyoplasty, i.e. the exogenous supply of cells with the inherent potential of forming contractile elements, has become a promising experimental therapeutic approach to rebuild, or in part replace scar tissue. Favorable effects on LV geometry and performance were demonstrated in several animal investigations using immature cardiomyocytes [3–5], skeletal myoblasts [6,7], and stem cells of vari* Corresponding author. Tel.: +1-213-977-4050; fax: +1-213-977-4107. E-mail address: [email protected] (R.A. Kloner). © 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00081-6
ous origin [8,9], that might also translate into improved survival and outcome of heart failure [10]. In addition, bone marrow-derived stem cells were shown to enhance angiogenesis in infarcted myocardium, and might even participate in the formation of a new vasculature [11]. However, the question of whether transplantation of immature cardiomyocytes also results in a vasculature, sufficient to nourish the graft, is crucial, as on the one hand infarcted myocardium is characterized by compromised tissue perfusion, and on the other hand a substantial number of transplanted cardiomyocytes might be at risk for necrotic or apoptotic cell death [12]. Additionally, increasing tissue perfusion of the infarcted myocardium itself may be beneficial with respect to infarct healing and the remodeling process, independent from guaranteeing survival and function of cardiomyocytes [13].
608
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
Several studies, investigating different approaches of cardiomyocyte transplantation reported an increased number of vascular structures on histologic sections after successful engraftment. However, to date no study has demonstrated that transplantation of immature cardiomyocytes into scar tissue, created by coronary artery occlusion, effectively increases perfusion of the infarcted tissue. Therefore, we chose a rat model of permanent coronary occlusion, in which neonatal cardiomyocytes were transplanted into the scar by intramyocardial injection, to investigate the effects of cellular cardiomyoplasty on tissue perfusion.
2. Methods The experiments were conducted in accordance with the national and institutional Guide for the Care and Use of Laboratory Animals. Good Samaritan Hospital’s Heart Institute is an AAALAC-accredited institution. 2.1. Surgical procedures and induction of myocardial infarction in recipients Seventy-four female Fischer rats (body weight: 165–208 g) were anesthetized by ketamine (75 mg/kg i.p.) and xylazine (5 mg/kg i.p.). After intubation and initiation of ventilation (room air, Harvard Apparatus Rodent Ventilator, Model 683, South Natick Mass) the chest muscles were infiltrated by bupivacaine (0.1 mg/kg), the thorax opened (fourth intercostal space), and the pericardium excised using sterile technique. The left coronary artery was ligated, and the chest was re-closed. After application of buprenorphine (0.02 mg/kg SQ), and weaning from the respirator, the rats were placed on a heating pad while recovering from anesthesia. 2.2. Isolation of neonatal cardiomyocytes Neonatal cardiomyocytes were isolated from hearts of 2-d-old neonates (either sex) of Fischer 344 rats, after digestion with collagenase II (95 U/ml) and pancreatin (0.6 mg/ml) in HEPES-20-/phosphate buffer (pH 7.4). To reduce the number of non-cardiomyocytes, they were preplated at 37 °C for 30 min in DMEM, supplemented with 10% horse serum and 5% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Thereafter, the cells were plated and incubated in medium for 1-2-d until used. 2.3. Cardiomyocyte transplantation Before transplantation, the medium was removed from the plates, the cells detached by trypsinization (5 min, 37 °C), and resuspended in serum-free DMEM at a concentration of 4 × 106 cells/ml. At the time of injection, 1 ml of the suspension was centrifuged, and the cells resuspended in 50-70 µl.
One week after coronary artery ligation, the rats were re-anesthetized as described above. After an intercostal incision, 50-70 µl of cell suspension (approximately 4 × 106 cells) (n = 31) or 50-70 µl medium (n = 27) were injected into the center of the infarct (one site), as described previously (6). The rats were allowed to recover for 1 month. 2.4. Ventriculography, hemodynamics, regional myocardial blood flow, and staining of perfused tissue One month after cell transplantation or medium injection, the rats were again anesthetized. Catheters were inserted into the jugular vein and into the carotid artery. Left ventriculography was performed using 1.0–1.5 ml non-ionic contrast medium (Optiray® 320, Mallinckrodt Inc., St Louis, MO), injected intravenously, under constant fluoroscopy with the XiScan 1000 C-arm X-ray system (XiTec Inc., 3-in. field of view). Images were acquired in an anterior-posterior and lateral projection at 30 frames/s on a VHS-videotape. LV volumes were measured off-line in diastole and systole, and averaged over three consecutive cycles in both projections, using the area-length method. Ejection fraction (%) was calculated as 100 × (Voldiastole–Volsystole)/Voldiastole. All parameters were then averaged over both projections. Thereafter, systolic and diastolic blood pressure, and heart rate were measured from the carotid artery catheter, and averaged over three cardiac cycles. For measurement of regional myocardial blood flow (RMBF), approximately 500,000 radioactive microspheres, labeled with 103Ruthenium, were injected into the LV. Simultaneously, a reference blood sample was withdrawn from an arterial catheter (0.443 ml/min). Thereafter, 0.6 ml 50% Uniperse blue (Ciba Geigy, Hawthorne, NY), a suspension of blue particles, was injected intravenously to stain perfused tissue. The deeply anesthetized animals were killed by 2 mEq KCl intravenously. After pressure fixation of the hearts and slicing the heart into five transverse sections (see below), the macroscopically visible infarcted tissue and a non-infarcted tissue piece from the septum were carefully dissected from the apical and second slice. Tissue and blood sample radioactivity was counted in a multi-channel pulse-height analyzer (model ND62, Nuclear Data Schaumburg, IL), and RMBF calculated after correction for background as the ratio of counts in the tissue and the reference blood sample multiplied by 0.443 ml/min and divided by the weight of the tissue. 2.5. Cell survival Survival of transplanted cardiomyocytes was analyzed using polymerase chain reaction (PCR) analysis of the Y-chromosomal Sry gene as an indicator of surviving male cells. Three hearts, that had received an intramyocardial injection of neonatal cardiomyocytes, and—as control for specifity of PCR—four hearts, that had received medium, were frozen in liquid nitrogen and stored at –80 °C after angiography. The minced heart tissue was digested using
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
609
proteinase K (20 h, 60 °C), and after phenol/chloroform extraction, DNA was precipitated in the aqueous phase with 100% ethanol (–20 °C). After centrifugation, the pellet was washed with 70% ethanol, and after drying dissolved in water. Concentration of DNA was measured sphectrophotometrically. PCR reaction contained 2 µg DNA, and 20–25 pmol (each) of a forward and reverse primer for the rat Sry gene (forward: 5’-AGTGTTCAGCCCTACAGGCTGAGGAC-3’, reverse: 5’-GTGTGTAGGTTGTTGTCCCATTGCAGC-3’) [5], along with deoxynucleoside triphosphates, MgCl2, and Tag-DNA-polymerase (Life Technologies, Rockville, MD). Reaction was carried out with 35 cycles (95, 72, and 72 °C, 1 min each), followed by 10 min at 72 °C, and the PCR product was identified using 1% agarose gel electrophoresis.
power field equivalent to 96,163 µm2) within the scar tissue, within the non-infarcted tissue, and within clusters of engrafted cardiomyocytes, which were identified by their marked disarray and round nuclei (n = 5). Counts are expressed as perfused capillaries/mm2.
2.6. Left ventricular volume measurements
3. Results
Following excision of the heart from the thorax, the LV was cannulated retrogradely via the ascending aorta. By connecting the cannula to a column of 10% formalin (pressure equal to 13 cm water column), the heart was fixed at a constant intraventricular pressure. LV volumes were assessed by filling the cavity with water and weighing (average of three measurements).
3.1. Mortality and exclusions
2.7. Histology and post-mortem analyses: macroscopic perfusion defect, density of perfused capillaries, infarct size, wall thickness, and infarct expansion index The formalin-fixed hearts were transversely cut into five slices and photographed under water. The epicardial and endocardial contour, and the area not stained by the blue dye were traced manually from projected slides. The area, not stained by the blue dye as visible macroscopically, was defined as the macroscopic perfusion defect. After computerized planimetry, it was expressed as a percentage of the weight of the whole heart. Histological sections (5 µm), cut from the apical part of the third slice after processing and paraffin embedding, were stained with hematoxylin and eosin (HE) and picrosirius red. The following parameters were determined after manual tracing of projected HE slides and computerized planimetry [14]: • Infarct size: total length of the scar as a percentage of the LV circumference, averaged over the endocardial and epicardial tracing. • The thickness of the infarct and the septum, averaged from three measurements (margins and center of infarct and septum, respectively). • The ratio of LV-cavity area to total LV area, to quantify LV dilation. • Infarct expansion index: ratio of septum-to-scar thickness multiplied by this ratio of LV dilation. As an estimate of perfused capillaries, the number of capillaries containing blue particles was counted in three high-power fields (HE slides, 40-fold magnification, 1 high-
2.8. Statistical analyses Continuous parameters were compared by Student’s t-test, categorical values by Fisher’s exact test. Linear regression analysis was performed using Pearson’s minimal square method with subsequent Anova testing for significance. Values are expressed as mean ± 1 standard error. A P-value < 0.05 was considered statistically significant.
Seventy-four animals were subjected to coronary artery ligation, and 16 of them died, mainly within the first hours after surgery. Six out of 31 animals, that received cell injections 1 week later, died shortly after surgery, while four out of 27 rats with medium injection died. Four medium-injected and three cell-injected hearts were used for PCR analysis after ventriculography, and analyzed separately. Of the remaining animals, one in each group was excluded because of only negligible infarct size, and three cell-injected animals and one medium-injected heart because of technical difficulties. Blood flow, ventriculographic, and morphometric data are based on 18 animals in the cell group and 17 animals in the medium group. 3.2. Cell survival and histology A transmural scar with a small rim of approximately 5–7 endocardial layers of surviving myocytes had developed in all of the included animals. In several hearts of the cell group, clusters of engrafted cardiomyocytes were visible within the scar, which were characterized by rounder nuclei and a marked degree of disarray in comparison with non-infarcted myocardium (Fig. 1a,b). These zones of grafted cells were surrounded by connective tissue, leading to separation from non-infarcted tissue (Fig. 1c). The PCR results indicated survival of transplanted cells over 1 month, as the Y-chromosomal Sry gene was detected in all three hearts that had received an injection of neonatal cardiomyocytes, while all four medium-injected hearts were negative 4 weeks after transplantation. 3.3. Regional myocardial blood flow, macroscopic perfusion defect, and number of perfused capillaries RMBF in the infarcted tissue was markedly reduced in comparison with non-infarcted myocardium (Table 1). In the group that had received injection of neonatal cardiomyo-
610
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
Fig. 1. (a) HE stain (40-fold): The engrafted cells form a cluster with a substantial degree of disarray. Their nuclei appear rounder in comparison to non-infarcted tissue. Note the high number of perfused capillaries, defined as capillaries containing blue particles after in vivo staining with 50% Uniperse blue. (b) HE stain (40-fold): Non-infarcted myocardium is characterized by a more organized pattern, and a high capillary density. (c) Picrosirius-red stain (4-fold): Cardiomyocytes appear yellow, collagen red. A cluster of engrafted cells (center) is surrounded by collagen and scar tissue. (d) HE stain (40-fold): Scar tissue of a heart 4 weeks after medium injection. The scar is characterized by very few capillaries containing blue particles. Table 1 Parameters of LV function, geometry, post-mortem morphometry, and RMBF in both groups Angiographic analysis LV volume (µl) Diastole Systole LV-EF (%) Post-mortem analysis Infarct size (%) Volume (µl) Scar thickness (mm) Septum thickness (mm) Scar-to-septum thickness LV-cavity area/total LV area Infarct expanison index Macroscopic perfusion defect (% of heart) Regional myocardial blood flow Apical slice RMBFscar (ml/min/g) RMBFseptum (ml/min/g) RMBFscar/RMBFseptum Second slice RMBFscar (ml/min/g) RMBFseptum (ml/min/g) RMBFscar/RMBFseptum Perfused capillaries/mm_ Non-infarcted tissue Scar tissue Clusters of engrafted cells
Medium group
Cell group
P-value
300.9 ± 10.5 174.2 ± 11.1 43.2 ± 1.7
256.0 ± 10.4 146.2 ± 8.7 42.6 ± 2.0
< 0.003* < 0.027* < 0.416
38.2 ± 2.2 322.5 ± 13.5 0.75 ± 0.04 1.07 ± 0.04 0.72 ± 0.05 0.55 ± 0.02 0.83 ± 0.06 3.0 ± 0.6
36.0 ± 2.5 274.0 ± 15.1 0.93 ± 0.07 1.05 ± 0.05 0.91 ± 0.08 0.51 ± 0.02 0.64 ± 0.07 1.5 ± 0.3
< 0.262 < 0.012* < 0.020* < 0.381 < 0.025* < 0.025* < 0.023* < 0.017*
0.72 ± 0.14 2.22 ± 0.41 0.40 ± 0.09
1.25 ± 0.43 1.70 ± 0.20 0.73 ± 0.16
< 0.128 < 0.130 < 0.041*
0.61 ± 0.11 2.73 ± 0.39 0.26 ± 0.05
0.97 ± 0.18 2.90 ± 0.48 0.44 ± 0.10
< 0.047* < 0.396 < 0.051
1778 ± 83 125 ± 10 1217 ± 114
1924 ± 114 156 ± 62
< 0.163 < 0.300
* Statistically significant.
cytes, RMBF in the infarct was increased compared with medium-injected hearts. For the apical slice of the heart, differences in absolute blood flow did not reach statistical significance, but the ratio of blood flow in the scar to blood
flow in the septum was significantly higher in the treated group (0.73 ± 0.16 vs. 0.40 ± 0.09; P < 0.041). In the second slice, absolute RMBF was significantly higher in the transplantation group (0.97 ± 0.18 vs. 0.61 ± 0.11 ml/min/g,
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
P < 0.047), with a higher ratio of flow in the scar to flow in the septum in the treated group (0.44 ± 0.10) vs. the medium group (0.26 ± 0.05; P < 0.051) (Table 1). A macroscopic perfusion defect in the scar tissue, defined as tissue not stained by the blue dye, was visible in 89% of the cellinjected hearts and 100% of the medium-injected hearts (not significant). The average size of the perfusion defect was 3.0 ± 0.6% of the heart weight in the control group, which was significantly more than in the cell-treated group (1.5 ± 0.3%, P < 0.017). The number of perfused capillaries, defined as capillaries containing blue particles after in vivo staining, was substantially lower in scar tissue (Fig. 1d) compared with noninfarcted myocardium (Fig. 1b), but there was no significant difference in capillary density of the scar tissue itself between the medium and the cell groups. Clusters of transplanted cardiomyocytes within the scar, however, were characterized by a high number of perfused capillaries (1217 ± 114/mm2) (Fig. 1a). 3.4. Hemodynamics, ventriculography, and left ventricular volumes Hemodynamics did not significantly differ between the two groups after 4 weeks (systolic/diastolic blood pressure: 125 ± 8/95 ± 3 mmHg in the cell-group vs. 130 ± 5/99 ± 2 mmHg in the control group, heart rate: 190 ± 10/min in the cell group vs. 203 ± 9/min in the control group). LV volumes in systole and diastole were significantly smaller in the treated group, while calculated ejection fraction was comparable (Table 1). Concomitantly, calculated stroke volume was slightly, but significantly smaller in the cell-treated group (126.7 ± 20.8 vs. 109.9 ± 22.9 µl, P < 0.05). LV volumes, measured post-mortem, were also significantly smaller in the cell group compared with the control group (P < 0.012). Diastolic volumes, as assessed by ventriculography, tended to be slightly smaller than volumes determined post-mortem, but correlated significantly with each other (r = 0.46, P < 0.005), confirming the validity of the technique. 3.5. Post-mortem morphometry Infarct size, expressed as a percentage of the total LV circumference was similar in both groups (Table 1). Scar thickness, as well as the ratio of scar-to-septum thickness, in the cell group was significantly higher than in the control group. In addition, the ratio of LV dilation was significantly increased in the control group. This ratio correlated significantly with volumes measured post-mortem (r = 0.73, P < 0.0001), confirming the validity of both parameters for describing LV dilation. The differences in infarct thickness and LV dilation resulted in a significantly reduced infarct expansion index in the cell-treated group (0.64 ± 0.07 vs. 0.83 ± 0.06, P < 0.023). Fig. 2 illustrates an example of a transverse section of a heart after cell transplantation in comparison with a control heart.
611
Fig. 2. Cross section of heart slices (picrosirius-red stain). Left: Heart 4 weeks after transplantation of neonatal cardiomyocytes: The infarct (stained red), contains regions of cardiomyocyocytes (stained yellow), leading to thickening of the scar. Right: Heart 4 weeks after medium injection: the hearts were characterized by thinning of the scar and more LV dilation.
4. Discussion The results of the present investigations demonstrate for the first time that transplantation of neonatal cardiomyocytes into 1-week old infarcts, created by coronary artery occlusion, effectively increases RMBF in the infarct after 4 weeks. In addition, cardiomyocyte transplantation resulted in reduced LV dilation, thickening of the infarcted wall, and lower indices of infarct expansion. Survival of transplanted cells in this model was confirmed by PCR analysis. On histology, areas of grafted cells within the scar demonstrated a high number of perfused capillaries, however, the capillary density in the scar tissue itself did not differ between control and transplanted hearts, suggesting that increased tissue perfusion and angiogenesis were mainly confined to regions of successful cell engraftment. 4.1. Cellular cardiomyoplasty and left ventricular remodeling The effects of transplantation of neonatal cardiomyocytes on LV geometry and function, as observed in this study, parallel recent investigations that emphasized a favorable effect of cellular cardiomyoplasty on the remodeling process after myocardial infarction: Etzion et al. [4] reported that transplantation of fetal cardiomyocytes in the scar tissue created by permanent coronary occlusion, prevented scar thinning and progressive LV dilation over a period of 53 d after transplantation. In a follow-up over 6 months after transplantation of neonatal cardiomyocytes, Müller-Ehmsen et al. [5] found significantly thickened scars containing lumps of grafted cells, largely surrounded by collagen. Using quantitative analysis of the Sry gene, similar to the approach chosen in the present study, they estimated a survival rate of approximately 65% of the number of initially injected cells over 6 months. In contrast to the present study, LV dilation was only slightly attenuated, and ejection fraction and regional wall motion in the scar was significantly improved in the cell-injected group. After skeletal myoblast transplantation, Jain et al. [7] reported similar effects on LV remodeling in a temporary coronary occlusion model. They described
612
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
reduced LV dilation, increased ex vivo systolic pressures, and, interestingly, improved in vivo exercise capacity in the transplanted group. In the present study, the main effect of cellular cardiomyoplasty was apparent as attenuated LV dilation, thickening of the infarcted wall of the myocardium, and reduced infarct expansion at 4 weeks after transplantation. Similar to MüllerEhmsen’s report the grafts were surrounded by collagen, resulting in a certain degree of separation from the noninfarcted myocardium [5]. Thus, thickening and stiffening of the scar, leading to reduced systolic wall stress, and subsequently, a beneficial influence on the remodeling process, appear to be the primary effect of cardiomyocyte transplantation in the present study. A certain degree of active systolic force development by the graft cannot be excluded, but—if present—appears to be relatively small, as global ejection fractions did not differ between the two groups. Given the substantial amount of disarray and morphological signs of incomplete differentiation on histology 4 weeks after transplantation (Fig. 1), one might speculate that a longer duration for the neonatal cardiomyocytes to differentiate is required to improve global ejection fraction and regional wall motion. 4.2. Potential of transplanted cardiomyocytes to enhance angiogenesis Early studies, using transplantation of heart tissue into a non-physiological, poorly vascularized environment, such as the pinna of the ear or the anterior chamber of the eye, emphasized that development and growth of the heart tissue was always accompanied by a newly developed vasculature [15,16]. Li et al. [17] created a spontaneously beating tissue after injection of immature cardiomyocytes into the rat hind limb, which demonstrated evidence for neo-angiogenesis on histology. Four weeks after engraftment of fetal cardiomyocytes into myocardial scar tissue in another study, the same research group found a significantly increased number of arterioles and venules in comparison to control hearts [18]. Similarly, Etzion et al. [4] reported a non-significant trend of increased vascularization in their permanent occlusion model. To date no experimental study has demonstrated an increase in absolute RMBF in the scar in a permanent coronary occlusion model after cellular cardiomyoplasty, which is the relevant parameter for effective blood supply to the graft. Indeed, a recent study, which used a model of myocardial cryoinjury, reported a higher vascular density, as assessed by counting factor-VIII-positive vascular structures in the scar, in transplanted hearts in comparison with mediuminjected hearts. But, relative blood flow in this study, expressed as a percentage of flow in the normal myocardium, was not significantly increased (10.4 ± 0.7% in cell group vs. 8.8 ± 0.8% in control hearts) [20]. In the present study, it was possible to demonstrate that the morphologic finding of increased vascular structures results in directed blood flow and effective improvement of regional tissue perfusion, using the microsphere technique.
Thus, transplantation of neonatal cardiomyocytes bears the potential of effectively increasing perfusion of the infarcted tissue. We did not investigate the mechanisms of how transplantation of neonatal cardiomyocytes enhances regional myocardial flow. But one might speculate that the transplanted cells are able to secrete local factors that stimulate angiogenesis, or that the inflammatory stimulus of cell engraftment triggers the induction of new vessel formation. A third possibility, that cannot be absolutely excluded, is that the preparation of neonatal cardiomyocytes also contained a second population of cells with the ability to form vessels after transplantation, since even in the post-natal heart pluripotent stem cells might be present [19]. Absolute blood flow data, as well as blood flow expressed as a percentage of normal flow demonstrated effective improvement of myocardial perfusion after cell transplantation in the present study. However, the absolute blood flow data were also characterized by a substantial variability, which (apart from reasons related to the technique) might reflect the amount of successfully engrafted cells. In contrast to absolute blood flow data in the second heart slice, the difference in absolute blood flow, measured in the apical slices, did not reach statistical significance. This is probably in part related to the fact that the suspension of neonatal cardiomyocytes was mainly injected into the center of the scar, which (given the limited amount of migration or proliferation after transplantation) might lead to a lower number of engrafted cells in the apical parts of the heart. 5. Conclusions Transplantation of neonatal cardiomyocytes effectively improved tissue perfusion of infarcted myocardium 4 weeks after transplantation into an infarct created by permanent coronary artery occlusion. The engrafted cells survived, and formed clusters surrounded by collagen in the scar, leading to thickening of the infarcted wall and less infarct expansion. In addition, LV dilation was significantly attenuated. Capillary density was high in regions of engrafted cells within the scar, but in scar tissue itself the number of perfused capillaries did not differ from controls. These results suggest that transplanted cardiomyocytes had the angiogenic potential to induce a vasculature to nourish them, which effectively enhanced tissue perfusion of the infarcted myocardium after 4 weeks. Acknowledgements Supported in part by NHLBI grant (HL61488). References [1]
[2]
Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737–46. Anversa P, Leri A, Kajstura J, Nadal-Ginard B. Myocyte growth and cardiac repair. J Mol Cell Cardiol 2002;34:91–105.
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613 [3]
Scorsin M, Hagege AA, Marotte F, Mirochnik N, Copin H, Barnoux M, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation 1997;96(Suppl II):II-18893.
[4]
Etzion S, Battler A, Barbash IM, Cagnano E, Zarin P, Granot Y, et al. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 2001;33:1321–30.
[5]
Müller-Ehmsen J, Peterson KL, Kedes L, Whittaker P, Dow JS, Long TI, et al. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:1720–6.
[6]
Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC, Hutcheson KA, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4: 929–33.
[7]
Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920–7.
[8]
Min JY, Yang Y, Converso KL, Liu L, Huang Q, Morgan JP, et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 2002;92:288–96.
[9]
Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100(Suppl II):II-247-56.
[10] Roell W, Lu ZJ, Bloch W, Siedner S, Tiemann K, Xia Y, et al. Cellular cardiomyoplasty improves survival after myocardial injury. Circulation 2002;105:2435–41.
613
[11] Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, et al. Neovascularisation of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7: 430–6. [12] Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001;33:907–21. [13] Kim CB, Braunwald E. Potential benefits of late reperfusion of infarcted myocardium. The open artery hypothesis. Circulation 1993;88: 2426–36. [14] Hochman JS, Choo H. Limitation of myocardial infarct expansion by reperfusion independent of myocardial salvage. Circulation 1987;75: 299–306. [15] Rossi MA. Chronic hemodynamic unloading regulates the morphologic development of newborn mouse hearts transplanted into the ear of isogeneic adult mice. Am J Pathol 1992;141:183–91. [16] Bishop SP, Anderson PG, Tucker DC. Morphological development of the rat heart growing in oculo in the absence of hemodynamic work load. Circ Res 1990;66:84–102. [17] Li RK, Mickle DA, Weisel RD, Zhang J, Mohabeer MK. In vivo survival and function of transplanted rat cardiomyocytes. Circ Res 1996;78:283–8. [18] Li RK, Mickle DAG, Weisel RD, Mohabeer MK, Zhang J, Rao V, et al. Natural historyof fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997;96(Suppl II):II-179-87. [19] Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett 2002;530:239–43. [20] Yau TM, Fung K, Weisel RD, Fujii T, Mickle DA, Li RK. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation 2001;104(Suppl I):I-218-22.
Original Article
Transplantation of neonatal cardiomyocytes after permanent coronary artery occlusion increases regional blood flow of infarcted myocardium Thorsten Reffelmann, Joan S. Dow, Wangde Dai, Sharon L. Hale, Boris Z. Simkhovich, Robert A. Kloner * The Heart Institute, Good Samaritan Hospital, University of Southern California, 1225 Wilshire Boulevard, Los Angeles, CA 90017-2395, USA Received 26 December 2002; received in revised form 30 January 2003; accepted 19 February 2003
Abstract Background. – Cellular cardiomyoplasty is a promising approach for rebuilding scar tissue after acute myocardial infarction. However, the angiogenic potential of transplanted immature cardiomyocytes and their effect on regional myocardial blood flow (RMBF) after coronary artery occlusion remain to be evaluated. Methods and results. – Intramyocardial injection of cultured neonatal cardiomyocytes (4 × 106 cells/50-70 µl) into the scar 1 week after permanent coronary occlusion in rats resulted in improved RMBF in the infarct 4 weeks after transplantation (radioactive microspheres, 0.97 ± 0.18 ml/min/g) in comparison to medium-injected hearts (0.61 ± 0.11 ml/min/g, P < 0.047). The macroscopic perfusion defect after in vivo staining with the blue dye 50% Uniperse blue was significantly smaller in the cell transplantation group (1.5 ± 0.3% of the heart) compared to the medium group (3.0 ± 0.6%, P < 0.017). Clusters of engrafted cells within the scar demonstrated a high capillary density (1217 ± 114 perfused (blue) capillaries/mm2); however, in the scar tissue itself capillary density in the cell group (156 ± 62/mm2) did not significantly differ from the medium group (125 ± 10/mm2), suggesting that neo-angiogenesis was confined to regions of successful engraftment (non-infarcted tissue: 1924 ± 114 perfused capillaries/mm2). The transplantation group was characterized by smaller diastolic and systolic left ventricular volumes, as assessed by intravenous ventriculography, along with thickened infarcts (0.93 ± 0.07 vs. 0.75 ± 0.04 mm, P < 0.020) and lower infarct expansion indices (0.64 ± 0.07 vs. 0.83 ± 0.06, P < 0.023), as determined by post-mortem morphometry of histologic slides. Conclusions. – Transplantation of neonatal cardiomyocytes induced neo-angiogenesis in zones of successful cell engraftment within the scar, which effectively enhanced tissue perfusion. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Angiogenesis; Capillaries; Transplantation; Cells; Regional blood flow
1. Introduction The quantitatively insufficient capacity of the mammalian heart to replace irreversibly damaged myocardium is the pivotal cause of akinetic scar formation and aneurysmal left ventricular (LV) dilation after acute myocardial infarction [1,2]. Cellular cardiomyoplasty, i.e. the exogenous supply of cells with the inherent potential of forming contractile elements, has become a promising experimental therapeutic approach to rebuild, or in part replace scar tissue. Favorable effects on LV geometry and performance were demonstrated in several animal investigations using immature cardiomyocytes [3–5], skeletal myoblasts [6,7], and stem cells of vari* Corresponding author. Tel.: +1-213-977-4050; fax: +1-213-977-4107. E-mail address: [email protected] (R.A. Kloner). © 2003 Elsevier Science Ltd. All rights reserved. DOI: 10.1016/S0022-2828(03)00081-6
ous origin [8,9], that might also translate into improved survival and outcome of heart failure [10]. In addition, bone marrow-derived stem cells were shown to enhance angiogenesis in infarcted myocardium, and might even participate in the formation of a new vasculature [11]. However, the question of whether transplantation of immature cardiomyocytes also results in a vasculature, sufficient to nourish the graft, is crucial, as on the one hand infarcted myocardium is characterized by compromised tissue perfusion, and on the other hand a substantial number of transplanted cardiomyocytes might be at risk for necrotic or apoptotic cell death [12]. Additionally, increasing tissue perfusion of the infarcted myocardium itself may be beneficial with respect to infarct healing and the remodeling process, independent from guaranteeing survival and function of cardiomyocytes [13].
608
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
Several studies, investigating different approaches of cardiomyocyte transplantation reported an increased number of vascular structures on histologic sections after successful engraftment. However, to date no study has demonstrated that transplantation of immature cardiomyocytes into scar tissue, created by coronary artery occlusion, effectively increases perfusion of the infarcted tissue. Therefore, we chose a rat model of permanent coronary occlusion, in which neonatal cardiomyocytes were transplanted into the scar by intramyocardial injection, to investigate the effects of cellular cardiomyoplasty on tissue perfusion.
2. Methods The experiments were conducted in accordance with the national and institutional Guide for the Care and Use of Laboratory Animals. Good Samaritan Hospital’s Heart Institute is an AAALAC-accredited institution. 2.1. Surgical procedures and induction of myocardial infarction in recipients Seventy-four female Fischer rats (body weight: 165–208 g) were anesthetized by ketamine (75 mg/kg i.p.) and xylazine (5 mg/kg i.p.). After intubation and initiation of ventilation (room air, Harvard Apparatus Rodent Ventilator, Model 683, South Natick Mass) the chest muscles were infiltrated by bupivacaine (0.1 mg/kg), the thorax opened (fourth intercostal space), and the pericardium excised using sterile technique. The left coronary artery was ligated, and the chest was re-closed. After application of buprenorphine (0.02 mg/kg SQ), and weaning from the respirator, the rats were placed on a heating pad while recovering from anesthesia. 2.2. Isolation of neonatal cardiomyocytes Neonatal cardiomyocytes were isolated from hearts of 2-d-old neonates (either sex) of Fischer 344 rats, after digestion with collagenase II (95 U/ml) and pancreatin (0.6 mg/ml) in HEPES-20-/phosphate buffer (pH 7.4). To reduce the number of non-cardiomyocytes, they were preplated at 37 °C for 30 min in DMEM, supplemented with 10% horse serum and 5% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. Thereafter, the cells were plated and incubated in medium for 1-2-d until used. 2.3. Cardiomyocyte transplantation Before transplantation, the medium was removed from the plates, the cells detached by trypsinization (5 min, 37 °C), and resuspended in serum-free DMEM at a concentration of 4 × 106 cells/ml. At the time of injection, 1 ml of the suspension was centrifuged, and the cells resuspended in 50-70 µl.
One week after coronary artery ligation, the rats were re-anesthetized as described above. After an intercostal incision, 50-70 µl of cell suspension (approximately 4 × 106 cells) (n = 31) or 50-70 µl medium (n = 27) were injected into the center of the infarct (one site), as described previously (6). The rats were allowed to recover for 1 month. 2.4. Ventriculography, hemodynamics, regional myocardial blood flow, and staining of perfused tissue One month after cell transplantation or medium injection, the rats were again anesthetized. Catheters were inserted into the jugular vein and into the carotid artery. Left ventriculography was performed using 1.0–1.5 ml non-ionic contrast medium (Optiray® 320, Mallinckrodt Inc., St Louis, MO), injected intravenously, under constant fluoroscopy with the XiScan 1000 C-arm X-ray system (XiTec Inc., 3-in. field of view). Images were acquired in an anterior-posterior and lateral projection at 30 frames/s on a VHS-videotape. LV volumes were measured off-line in diastole and systole, and averaged over three consecutive cycles in both projections, using the area-length method. Ejection fraction (%) was calculated as 100 × (Voldiastole–Volsystole)/Voldiastole. All parameters were then averaged over both projections. Thereafter, systolic and diastolic blood pressure, and heart rate were measured from the carotid artery catheter, and averaged over three cardiac cycles. For measurement of regional myocardial blood flow (RMBF), approximately 500,000 radioactive microspheres, labeled with 103Ruthenium, were injected into the LV. Simultaneously, a reference blood sample was withdrawn from an arterial catheter (0.443 ml/min). Thereafter, 0.6 ml 50% Uniperse blue (Ciba Geigy, Hawthorne, NY), a suspension of blue particles, was injected intravenously to stain perfused tissue. The deeply anesthetized animals were killed by 2 mEq KCl intravenously. After pressure fixation of the hearts and slicing the heart into five transverse sections (see below), the macroscopically visible infarcted tissue and a non-infarcted tissue piece from the septum were carefully dissected from the apical and second slice. Tissue and blood sample radioactivity was counted in a multi-channel pulse-height analyzer (model ND62, Nuclear Data Schaumburg, IL), and RMBF calculated after correction for background as the ratio of counts in the tissue and the reference blood sample multiplied by 0.443 ml/min and divided by the weight of the tissue. 2.5. Cell survival Survival of transplanted cardiomyocytes was analyzed using polymerase chain reaction (PCR) analysis of the Y-chromosomal Sry gene as an indicator of surviving male cells. Three hearts, that had received an intramyocardial injection of neonatal cardiomyocytes, and—as control for specifity of PCR—four hearts, that had received medium, were frozen in liquid nitrogen and stored at –80 °C after angiography. The minced heart tissue was digested using
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
609
proteinase K (20 h, 60 °C), and after phenol/chloroform extraction, DNA was precipitated in the aqueous phase with 100% ethanol (–20 °C). After centrifugation, the pellet was washed with 70% ethanol, and after drying dissolved in water. Concentration of DNA was measured sphectrophotometrically. PCR reaction contained 2 µg DNA, and 20–25 pmol (each) of a forward and reverse primer for the rat Sry gene (forward: 5’-AGTGTTCAGCCCTACAGGCTGAGGAC-3’, reverse: 5’-GTGTGTAGGTTGTTGTCCCATTGCAGC-3’) [5], along with deoxynucleoside triphosphates, MgCl2, and Tag-DNA-polymerase (Life Technologies, Rockville, MD). Reaction was carried out with 35 cycles (95, 72, and 72 °C, 1 min each), followed by 10 min at 72 °C, and the PCR product was identified using 1% agarose gel electrophoresis.
power field equivalent to 96,163 µm2) within the scar tissue, within the non-infarcted tissue, and within clusters of engrafted cardiomyocytes, which were identified by their marked disarray and round nuclei (n = 5). Counts are expressed as perfused capillaries/mm2.
2.6. Left ventricular volume measurements
3. Results
Following excision of the heart from the thorax, the LV was cannulated retrogradely via the ascending aorta. By connecting the cannula to a column of 10% formalin (pressure equal to 13 cm water column), the heart was fixed at a constant intraventricular pressure. LV volumes were assessed by filling the cavity with water and weighing (average of three measurements).
3.1. Mortality and exclusions
2.7. Histology and post-mortem analyses: macroscopic perfusion defect, density of perfused capillaries, infarct size, wall thickness, and infarct expansion index The formalin-fixed hearts were transversely cut into five slices and photographed under water. The epicardial and endocardial contour, and the area not stained by the blue dye were traced manually from projected slides. The area, not stained by the blue dye as visible macroscopically, was defined as the macroscopic perfusion defect. After computerized planimetry, it was expressed as a percentage of the weight of the whole heart. Histological sections (5 µm), cut from the apical part of the third slice after processing and paraffin embedding, were stained with hematoxylin and eosin (HE) and picrosirius red. The following parameters were determined after manual tracing of projected HE slides and computerized planimetry [14]: • Infarct size: total length of the scar as a percentage of the LV circumference, averaged over the endocardial and epicardial tracing. • The thickness of the infarct and the septum, averaged from three measurements (margins and center of infarct and septum, respectively). • The ratio of LV-cavity area to total LV area, to quantify LV dilation. • Infarct expansion index: ratio of septum-to-scar thickness multiplied by this ratio of LV dilation. As an estimate of perfused capillaries, the number of capillaries containing blue particles was counted in three high-power fields (HE slides, 40-fold magnification, 1 high-
2.8. Statistical analyses Continuous parameters were compared by Student’s t-test, categorical values by Fisher’s exact test. Linear regression analysis was performed using Pearson’s minimal square method with subsequent Anova testing for significance. Values are expressed as mean ± 1 standard error. A P-value < 0.05 was considered statistically significant.
Seventy-four animals were subjected to coronary artery ligation, and 16 of them died, mainly within the first hours after surgery. Six out of 31 animals, that received cell injections 1 week later, died shortly after surgery, while four out of 27 rats with medium injection died. Four medium-injected and three cell-injected hearts were used for PCR analysis after ventriculography, and analyzed separately. Of the remaining animals, one in each group was excluded because of only negligible infarct size, and three cell-injected animals and one medium-injected heart because of technical difficulties. Blood flow, ventriculographic, and morphometric data are based on 18 animals in the cell group and 17 animals in the medium group. 3.2. Cell survival and histology A transmural scar with a small rim of approximately 5–7 endocardial layers of surviving myocytes had developed in all of the included animals. In several hearts of the cell group, clusters of engrafted cardiomyocytes were visible within the scar, which were characterized by rounder nuclei and a marked degree of disarray in comparison with non-infarcted myocardium (Fig. 1a,b). These zones of grafted cells were surrounded by connective tissue, leading to separation from non-infarcted tissue (Fig. 1c). The PCR results indicated survival of transplanted cells over 1 month, as the Y-chromosomal Sry gene was detected in all three hearts that had received an injection of neonatal cardiomyocytes, while all four medium-injected hearts were negative 4 weeks after transplantation. 3.3. Regional myocardial blood flow, macroscopic perfusion defect, and number of perfused capillaries RMBF in the infarcted tissue was markedly reduced in comparison with non-infarcted myocardium (Table 1). In the group that had received injection of neonatal cardiomyo-
610
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
Fig. 1. (a) HE stain (40-fold): The engrafted cells form a cluster with a substantial degree of disarray. Their nuclei appear rounder in comparison to non-infarcted tissue. Note the high number of perfused capillaries, defined as capillaries containing blue particles after in vivo staining with 50% Uniperse blue. (b) HE stain (40-fold): Non-infarcted myocardium is characterized by a more organized pattern, and a high capillary density. (c) Picrosirius-red stain (4-fold): Cardiomyocytes appear yellow, collagen red. A cluster of engrafted cells (center) is surrounded by collagen and scar tissue. (d) HE stain (40-fold): Scar tissue of a heart 4 weeks after medium injection. The scar is characterized by very few capillaries containing blue particles. Table 1 Parameters of LV function, geometry, post-mortem morphometry, and RMBF in both groups Angiographic analysis LV volume (µl) Diastole Systole LV-EF (%) Post-mortem analysis Infarct size (%) Volume (µl) Scar thickness (mm) Septum thickness (mm) Scar-to-septum thickness LV-cavity area/total LV area Infarct expanison index Macroscopic perfusion defect (% of heart) Regional myocardial blood flow Apical slice RMBFscar (ml/min/g) RMBFseptum (ml/min/g) RMBFscar/RMBFseptum Second slice RMBFscar (ml/min/g) RMBFseptum (ml/min/g) RMBFscar/RMBFseptum Perfused capillaries/mm_ Non-infarcted tissue Scar tissue Clusters of engrafted cells
Medium group
Cell group
P-value
300.9 ± 10.5 174.2 ± 11.1 43.2 ± 1.7
256.0 ± 10.4 146.2 ± 8.7 42.6 ± 2.0
< 0.003* < 0.027* < 0.416
38.2 ± 2.2 322.5 ± 13.5 0.75 ± 0.04 1.07 ± 0.04 0.72 ± 0.05 0.55 ± 0.02 0.83 ± 0.06 3.0 ± 0.6
36.0 ± 2.5 274.0 ± 15.1 0.93 ± 0.07 1.05 ± 0.05 0.91 ± 0.08 0.51 ± 0.02 0.64 ± 0.07 1.5 ± 0.3
< 0.262 < 0.012* < 0.020* < 0.381 < 0.025* < 0.025* < 0.023* < 0.017*
0.72 ± 0.14 2.22 ± 0.41 0.40 ± 0.09
1.25 ± 0.43 1.70 ± 0.20 0.73 ± 0.16
< 0.128 < 0.130 < 0.041*
0.61 ± 0.11 2.73 ± 0.39 0.26 ± 0.05
0.97 ± 0.18 2.90 ± 0.48 0.44 ± 0.10
< 0.047* < 0.396 < 0.051
1778 ± 83 125 ± 10 1217 ± 114
1924 ± 114 156 ± 62
< 0.163 < 0.300
* Statistically significant.
cytes, RMBF in the infarct was increased compared with medium-injected hearts. For the apical slice of the heart, differences in absolute blood flow did not reach statistical significance, but the ratio of blood flow in the scar to blood
flow in the septum was significantly higher in the treated group (0.73 ± 0.16 vs. 0.40 ± 0.09; P < 0.041). In the second slice, absolute RMBF was significantly higher in the transplantation group (0.97 ± 0.18 vs. 0.61 ± 0.11 ml/min/g,
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
P < 0.047), with a higher ratio of flow in the scar to flow in the septum in the treated group (0.44 ± 0.10) vs. the medium group (0.26 ± 0.05; P < 0.051) (Table 1). A macroscopic perfusion defect in the scar tissue, defined as tissue not stained by the blue dye, was visible in 89% of the cellinjected hearts and 100% of the medium-injected hearts (not significant). The average size of the perfusion defect was 3.0 ± 0.6% of the heart weight in the control group, which was significantly more than in the cell-treated group (1.5 ± 0.3%, P < 0.017). The number of perfused capillaries, defined as capillaries containing blue particles after in vivo staining, was substantially lower in scar tissue (Fig. 1d) compared with noninfarcted myocardium (Fig. 1b), but there was no significant difference in capillary density of the scar tissue itself between the medium and the cell groups. Clusters of transplanted cardiomyocytes within the scar, however, were characterized by a high number of perfused capillaries (1217 ± 114/mm2) (Fig. 1a). 3.4. Hemodynamics, ventriculography, and left ventricular volumes Hemodynamics did not significantly differ between the two groups after 4 weeks (systolic/diastolic blood pressure: 125 ± 8/95 ± 3 mmHg in the cell-group vs. 130 ± 5/99 ± 2 mmHg in the control group, heart rate: 190 ± 10/min in the cell group vs. 203 ± 9/min in the control group). LV volumes in systole and diastole were significantly smaller in the treated group, while calculated ejection fraction was comparable (Table 1). Concomitantly, calculated stroke volume was slightly, but significantly smaller in the cell-treated group (126.7 ± 20.8 vs. 109.9 ± 22.9 µl, P < 0.05). LV volumes, measured post-mortem, were also significantly smaller in the cell group compared with the control group (P < 0.012). Diastolic volumes, as assessed by ventriculography, tended to be slightly smaller than volumes determined post-mortem, but correlated significantly with each other (r = 0.46, P < 0.005), confirming the validity of the technique. 3.5. Post-mortem morphometry Infarct size, expressed as a percentage of the total LV circumference was similar in both groups (Table 1). Scar thickness, as well as the ratio of scar-to-septum thickness, in the cell group was significantly higher than in the control group. In addition, the ratio of LV dilation was significantly increased in the control group. This ratio correlated significantly with volumes measured post-mortem (r = 0.73, P < 0.0001), confirming the validity of both parameters for describing LV dilation. The differences in infarct thickness and LV dilation resulted in a significantly reduced infarct expansion index in the cell-treated group (0.64 ± 0.07 vs. 0.83 ± 0.06, P < 0.023). Fig. 2 illustrates an example of a transverse section of a heart after cell transplantation in comparison with a control heart.
611
Fig. 2. Cross section of heart slices (picrosirius-red stain). Left: Heart 4 weeks after transplantation of neonatal cardiomyocytes: The infarct (stained red), contains regions of cardiomyocyocytes (stained yellow), leading to thickening of the scar. Right: Heart 4 weeks after medium injection: the hearts were characterized by thinning of the scar and more LV dilation.
4. Discussion The results of the present investigations demonstrate for the first time that transplantation of neonatal cardiomyocytes into 1-week old infarcts, created by coronary artery occlusion, effectively increases RMBF in the infarct after 4 weeks. In addition, cardiomyocyte transplantation resulted in reduced LV dilation, thickening of the infarcted wall, and lower indices of infarct expansion. Survival of transplanted cells in this model was confirmed by PCR analysis. On histology, areas of grafted cells within the scar demonstrated a high number of perfused capillaries, however, the capillary density in the scar tissue itself did not differ between control and transplanted hearts, suggesting that increased tissue perfusion and angiogenesis were mainly confined to regions of successful cell engraftment. 4.1. Cellular cardiomyoplasty and left ventricular remodeling The effects of transplantation of neonatal cardiomyocytes on LV geometry and function, as observed in this study, parallel recent investigations that emphasized a favorable effect of cellular cardiomyoplasty on the remodeling process after myocardial infarction: Etzion et al. [4] reported that transplantation of fetal cardiomyocytes in the scar tissue created by permanent coronary occlusion, prevented scar thinning and progressive LV dilation over a period of 53 d after transplantation. In a follow-up over 6 months after transplantation of neonatal cardiomyocytes, Müller-Ehmsen et al. [5] found significantly thickened scars containing lumps of grafted cells, largely surrounded by collagen. Using quantitative analysis of the Sry gene, similar to the approach chosen in the present study, they estimated a survival rate of approximately 65% of the number of initially injected cells over 6 months. In contrast to the present study, LV dilation was only slightly attenuated, and ejection fraction and regional wall motion in the scar was significantly improved in the cell-injected group. After skeletal myoblast transplantation, Jain et al. [7] reported similar effects on LV remodeling in a temporary coronary occlusion model. They described
612
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613
reduced LV dilation, increased ex vivo systolic pressures, and, interestingly, improved in vivo exercise capacity in the transplanted group. In the present study, the main effect of cellular cardiomyoplasty was apparent as attenuated LV dilation, thickening of the infarcted wall of the myocardium, and reduced infarct expansion at 4 weeks after transplantation. Similar to MüllerEhmsen’s report the grafts were surrounded by collagen, resulting in a certain degree of separation from the noninfarcted myocardium [5]. Thus, thickening and stiffening of the scar, leading to reduced systolic wall stress, and subsequently, a beneficial influence on the remodeling process, appear to be the primary effect of cardiomyocyte transplantation in the present study. A certain degree of active systolic force development by the graft cannot be excluded, but—if present—appears to be relatively small, as global ejection fractions did not differ between the two groups. Given the substantial amount of disarray and morphological signs of incomplete differentiation on histology 4 weeks after transplantation (Fig. 1), one might speculate that a longer duration for the neonatal cardiomyocytes to differentiate is required to improve global ejection fraction and regional wall motion. 4.2. Potential of transplanted cardiomyocytes to enhance angiogenesis Early studies, using transplantation of heart tissue into a non-physiological, poorly vascularized environment, such as the pinna of the ear or the anterior chamber of the eye, emphasized that development and growth of the heart tissue was always accompanied by a newly developed vasculature [15,16]. Li et al. [17] created a spontaneously beating tissue after injection of immature cardiomyocytes into the rat hind limb, which demonstrated evidence for neo-angiogenesis on histology. Four weeks after engraftment of fetal cardiomyocytes into myocardial scar tissue in another study, the same research group found a significantly increased number of arterioles and venules in comparison to control hearts [18]. Similarly, Etzion et al. [4] reported a non-significant trend of increased vascularization in their permanent occlusion model. To date no experimental study has demonstrated an increase in absolute RMBF in the scar in a permanent coronary occlusion model after cellular cardiomyoplasty, which is the relevant parameter for effective blood supply to the graft. Indeed, a recent study, which used a model of myocardial cryoinjury, reported a higher vascular density, as assessed by counting factor-VIII-positive vascular structures in the scar, in transplanted hearts in comparison with mediuminjected hearts. But, relative blood flow in this study, expressed as a percentage of flow in the normal myocardium, was not significantly increased (10.4 ± 0.7% in cell group vs. 8.8 ± 0.8% in control hearts) [20]. In the present study, it was possible to demonstrate that the morphologic finding of increased vascular structures results in directed blood flow and effective improvement of regional tissue perfusion, using the microsphere technique.
Thus, transplantation of neonatal cardiomyocytes bears the potential of effectively increasing perfusion of the infarcted tissue. We did not investigate the mechanisms of how transplantation of neonatal cardiomyocytes enhances regional myocardial flow. But one might speculate that the transplanted cells are able to secrete local factors that stimulate angiogenesis, or that the inflammatory stimulus of cell engraftment triggers the induction of new vessel formation. A third possibility, that cannot be absolutely excluded, is that the preparation of neonatal cardiomyocytes also contained a second population of cells with the ability to form vessels after transplantation, since even in the post-natal heart pluripotent stem cells might be present [19]. Absolute blood flow data, as well as blood flow expressed as a percentage of normal flow demonstrated effective improvement of myocardial perfusion after cell transplantation in the present study. However, the absolute blood flow data were also characterized by a substantial variability, which (apart from reasons related to the technique) might reflect the amount of successfully engrafted cells. In contrast to absolute blood flow data in the second heart slice, the difference in absolute blood flow, measured in the apical slices, did not reach statistical significance. This is probably in part related to the fact that the suspension of neonatal cardiomyocytes was mainly injected into the center of the scar, which (given the limited amount of migration or proliferation after transplantation) might lead to a lower number of engrafted cells in the apical parts of the heart. 5. Conclusions Transplantation of neonatal cardiomyocytes effectively improved tissue perfusion of infarcted myocardium 4 weeks after transplantation into an infarct created by permanent coronary artery occlusion. The engrafted cells survived, and formed clusters surrounded by collagen in the scar, leading to thickening of the infarcted wall and less infarct expansion. In addition, LV dilation was significantly attenuated. Capillary density was high in regions of engrafted cells within the scar, but in scar tissue itself the number of perfused capillaries did not differ from controls. These results suggest that transplanted cardiomyocytes had the angiogenic potential to induce a vasculature to nourish them, which effectively enhanced tissue perfusion of the infarcted myocardium after 4 weeks. Acknowledgements Supported in part by NHLBI grant (HL61488). References [1]
[2]
Li F, Wang X, Capasso JM, Gerdes AM. Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development. J Mol Cell Cardiol 1996;28:1737–46. Anversa P, Leri A, Kajstura J, Nadal-Ginard B. Myocyte growth and cardiac repair. J Mol Cell Cardiol 2002;34:91–105.
T. Reffelmann et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 607–613 [3]
Scorsin M, Hagege AA, Marotte F, Mirochnik N, Copin H, Barnoux M, et al. Does transplantation of cardiomyocytes improve function of infarcted myocardium? Circulation 1997;96(Suppl II):II-18893.
[4]
Etzion S, Battler A, Barbash IM, Cagnano E, Zarin P, Granot Y, et al. Influence of embryonic cardiomyocyte transplantation on the progression of heart failure in a rat model of extensive myocardial infarction. J Mol Cell Cardiol 2001;33:1321–30.
[5]
Müller-Ehmsen J, Peterson KL, Kedes L, Whittaker P, Dow JS, Long TI, et al. Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function. Circulation 2002;105:1720–6.
[6]
Taylor DA, Atkins BZ, Hungspreugs P, Jones TR, Reedy MC, Hutcheson KA, et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4: 929–33.
[7]
Jain M, DerSimonian H, Brenner DA, Ngoy S, Teller P, Edge AS, et al. Cell therapy attenuates deleterious ventricular remodeling and improves cardiac performance after myocardial infarction. Circulation 2001;103:1920–7.
[8]
Min JY, Yang Y, Converso KL, Liu L, Huang Q, Morgan JP, et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J Appl Physiol 2002;92:288–96.
[9]
Tomita S, Li RK, Weisel RD, Mickle DA, Kim EJ, Sakai T, et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100(Suppl II):II-247-56.
[10] Roell W, Lu ZJ, Bloch W, Siedner S, Tiemann K, Xia Y, et al. Cellular cardiomyoplasty improves survival after myocardial injury. Circulation 2002;105:2435–41.
613
[11] Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, et al. Neovascularisation of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001;7: 430–6. [12] Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001;33:907–21. [13] Kim CB, Braunwald E. Potential benefits of late reperfusion of infarcted myocardium. The open artery hypothesis. Circulation 1993;88: 2426–36. [14] Hochman JS, Choo H. Limitation of myocardial infarct expansion by reperfusion independent of myocardial salvage. Circulation 1987;75: 299–306. [15] Rossi MA. Chronic hemodynamic unloading regulates the morphologic development of newborn mouse hearts transplanted into the ear of isogeneic adult mice. Am J Pathol 1992;141:183–91. [16] Bishop SP, Anderson PG, Tucker DC. Morphological development of the rat heart growing in oculo in the absence of hemodynamic work load. Circ Res 1990;66:84–102. [17] Li RK, Mickle DA, Weisel RD, Zhang J, Mohabeer MK. In vivo survival and function of transplanted rat cardiomyocytes. Circ Res 1996;78:283–8. [18] Li RK, Mickle DAG, Weisel RD, Mohabeer MK, Zhang J, Rao V, et al. Natural historyof fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 1997;96(Suppl II):II-179-87. [19] Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett 2002;530:239–43. [20] Yau TM, Fung K, Weisel RD, Fujii T, Mickle DA, Li RK. Enhanced myocardial angiogenesis by gene transfer with transplanted cells. Circulation 2001;104(Suppl I):I-218-22.