The past 20 years have experienced tremendous progress in the medical treatment of cardiac diseases and decreased cardiovascular morbidity and mortality in clinical practice.1 However, evidence accumulated shows that interventions intended to slow down the progression of chronic heart diseases have approached their limit. Recent progress in stem cell biology has attracted much attention. Human embryonic stem cell lines nowadays can be generated from human blastocysts at a high success rate and have generated human cardiac myocytes.2-4 But there are still many open questions and some controversies in the field.5,6 The most straightforward strategy is to infuse or inject these cells into the injured heart and to develop methods to increase the fraction of cells that home and survive in the heart and differentiate to the desired functional elements. This strategy has already been clinically evaluated, but gave mixed results,7-10 potentially because the homing rate after infusion and the survival rate after intramyocardial injections are very low.11 Given the creativity and technical expertise of invasive cardiology, significant progress in the efficacy of such delivery technique is to be expected.
Several groups have demonstrated effectiveness in improving cardiac function through the injection of the cells in saline, cell culture medium, or bovine serum albumin (BSA) into ischemic myocardium.12,13 In the study we examined whether the neonatal cardiomyocytes plus fibrin sealant, as an injectable biopolymer, could improve cardiac function after myocardial infarction (MI).
Sixty adult female Sprague-Dawley (SD) rats weighing (210±10)g and neonatal male SD rats were purchased from the Experimental Animal Center of Lanzhou University of China (Lanzhou, Certificate No. SCXK2002-0010). All experimental procedures were performed according to the Regulation to the Care and Use of Experimental Animals (1996) of the Lanzhou Council on Animal Care.
Rat model of MI
Female SD rats were anesthetized with 5% isoflurane by an airtight mask. The animals were incubated and ventilated at a rate of 60 cycles/minute with a tidal volume of 2.5 ml under room air supplemented with oxygen (0.2 L/min) and 1% to 2.5% isoflurane. Lead I of the electrocardiogram (ECG) was monitored throughout the experiment. The chest was opened under sterile conditions by left thoracotomy through the fourth intercostals space. The pericardium was removed. A single stitch of 6-0 prolene suture was placed through the myocardium at a depth slightly greater than the perceived level of the left anterior descending (LAD) portion of the left coronary artery while taking care not to enter the ventricular chamber. The suture was tightened to occlude the LAD. Myocardial ischemia was confirmed by typical ST-segment changes on the ECG, regional myocardial color changes and ventricular arrhythmias. The air was expelled from the chest and the incision was closed. The rats recovered from anesthesia and were returned to their cages.
Cell isolation and culture
Ventricular cardiomyocytes of one-day-old SD neonatal SD rats (male sex) were isolated and cultured according to the following described procedure. After the atria and great vessels were removed from the hearts, the ventricles were minced into small fragments (1 mm3). The minced tissue was incubated in digestion buffer (0.08% trypsin) and digested for 8-10 circles (8 minutes per circle). To reduce non-myocyte contamination, cells were preplated in cell culture flasks (Corning Life Science, NY, USA) for 30 minutes at 37°C. After preplating, to facilitate the identification of the transplanted cells after their implantation, the non-attached cells were resuspended in working dyeing solution with 2 μg/ml of Chlorome-thylbenzamido (CellTrackerTM CM-DiI, C-7001, Molecular Probe, OR, USA) in phosphate buffer solution (PBS) at 37°C for 5 minutes and at 4°C for 15 minutes. After labeling, the cells were plated in 150-mm culture flasks (Corning Life Science, NY, USA) and cultured in the presence of 5% CO2 and 95% air at 37°C for 1 to 2 days.
Fibrin sealant (BD Biosciences, Bedford, MA, USA) was used in this study. Two component systems remained liquid for several seconds before solidifying into a semirigid gel matrix. The first component consisted of concentrated fibrinogen and aprotinin, a fibrinolysis inhibitor, whereas the second was a mixture of thrombin and CaCl2. It was delivered through the supplied applicator, which held the two components in separate syringes and provided simultaneous mixing and delivery. The ratio of fibrinogen to thrombin components was 1:1.
Three weeks after MI, animals were screened by echocardiography for heart function as estimated by fraction shortening (FS) at the mid-papillary level of the left ventricle (LV) in short-axis 2-dimensional images. Animals with FS less than 25% were randomized into 4 groups (40 SD rats). The first group was neonatal ventricular cardiomyocytes plus fibrin sealant (group CF, n=10). Neonatal ventricular cardiomyocytes (4×106) were suspended in 50 μl of the thrombin component of the fibrin sealant. The thrombin-cell mixture was simultaneously injected into the myocardium with 50 μl of the fibrinogen component. The second group was cardiomyocytes alone (group C, n=10). Cardiomyocytes (4×106) were suspended in 100 μl of 0.5% BSA and injected into the myocardium. The third group was fibrin sealant alone (group F, n=10). Thrombin (50 μl) and 50 μl of fibrinogen were simultaneously injected into ischemic myocardium. The fourth group was control group (n=10). BSA (0.5%) in 100 μl of PBS was injected into the ischemic LV. Using a median sternotomy, one injection was made by a needle into the area under the epicardium of the left ventricular scar. Approximately 100 μl of liquid was injected per heart for one minute. Once injection was completed, the puncture holes were closed by sutures, which served as a marker for the area of transplantation at follow-up thoracotomy. The incision was closed by suturing sternum, muscle and skin continuously. Analgesics were given by oral injection of tramadol for three days after surgery. Cyclosporin A (Sandimmun, Novartis Pharma AG, Switzerland) was administed orally in both groups by injection into the mouth daily, starting on the day of transplantation at a dose of 50 mg/kg.
Transthoracic echocardiography was performed on all animals 3 weeks after myocardial infarction (baseline echocardiogram). A follow-up echocardiogram was performed 4 weeks after control or treatment injection. Rats were anesthetized as described above and transthoracic echocardiography was performed on all rats. The left parasternal images were taken in the right lateral decubitus position using Vivid 7 (GE Vingmed Ultrasound, Horten, Norway) with a 10-MHz multiphase array probe. After adequate short-axis two-dimensional images at the mid-papillary level of left ventricle were obtained, the M-mode cursor was positioned perpendicular to the ventricular anteroseptal wall (at the site of infarction) and the posterior wall. Wall thickness and left ventricular internal dimensions were measured according to the leading edge method of the American Society of Echocardiography. FS as a measure of systolic function was calculated as ((LVIDd-LVIDs) / LVIDd) ×100%, where LVID was the left ventricular internal dimension, d was diastole, and s was systole. Two echocardiographer blinded to the experimental treatment acquired the images and performed the data analysis.
Isolated Langendorff heart perfusion
One to three days after performing echocardiography, the global heart function was evaluated using a non-working constant pressure Langendorff preparation. The rats were anesthetized, and sodium heparin (100 U) was administered intravenously. The heart was quickly isolated and perfused in a Langendorff apparatus with filtered Krebs-Henseleit buffer (mmol/L: NaCl 118; KCl 4.7; KH2PO4 1.2; CaCl2 2.5; MgSO4 1.2; NaHCO3 25; Glucose 11; pH 7.4) at a pressure of 65 mmHg equilibrated with 5% carbon dioxide and 95% oxygen. A latex balloon was passed into the left ventricle through the mitral valve and connected to a pressure transducer (model ML110, AD Instruments, Australia) and differentiator amplifier (SP8520, Powerlab/8SP, AD Instruments Pty Ltd, Castle Hill, Australia). After 20 minutes of stabilization, the balloon size was increased by 0.02 ml increments from 0.04 ml by the stepwise addition of saline. LV peak systolic and end-diastolic pressures were measured at ventricular volumes from 0.04 ml and in 0.02 ml increments until the end-diastolic pressure was over 30 mmHg. Developed pressure was calculated as the difference between the peak systolic and end-diastolic pressures at each ventricular volume. Passive diastolic pressures were recorded at each balloon volume in 0.04 ml increments until the diastolic pressure was over 30 mmHg. Hearts were then arrested with 10 ml of KCl solution (20 mmol) for histological studies.
Histology and immunohistochemistry
The site of the injected area was identified and dissected. Four to five specimens were used, and each of these was cut into 2 pieces. The first one was fixed in 10% phosphate-buffered formalin embedded in paraffin, and sectioned to yield five-μm-thick slices. The second piece was frozen in liquid nitrogen for 15 seconds and then transferred to -70°C refrigerator to be stored until it was processed on a cryostat. Paraffin sections were stained with hematoxylin and eosin (HE). Five-μm-thick cryostat sections were used for immunohistochemistry. Primary antibodies included mouse monoclonal anti-α-actinin (Sarcomeric). Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were used against the primary antibodies. Stained tissue was examined with an inverted fluorescent microscope (IX70, Olympus Cooperation, Japan). HE stains were used to confirm ensuing scarring. α-actinin accompanying CM-DiI was used to show implanted cells. 4,6-diamidino-2-phenylindole (1 μg/ml, DAPI, Sigma-Aldrich, USA) was used to stain nuclear material in cryostat section
Polymerase chain reaction for identifying Y chromosome
Neonatal cardiomyocytes from male rats were implanted into female rats. The polymerase chain reaction (PCR) amplification was used to identify the Y chromosome to detect the surviving male cardiomyocytes. Left ventricular tissue from the injection site was isolated and refrigerated in liquid nitrogen. Genomic DNA for PCR template was extracted. Genomic DNA templates (100 ng) from different groups were used in PCR reactions (50 μl) with rat SRY primers according to the protocols published by Muller-Ehmsen et al.14 Forward primer sequence is as follows: 5′-AGTGTTCAGCCCTACAGCCTGAGGAC-3′, and reverse sequence: 5′-GCTGCAATGGGACAACAACCTACACAC-3′. PCR assays were performed in triplicate. Control reactions were performed using the reduced nicotinamide adenine dinucleotide phosphate primers and the same templates. PCR products were separated by electrophoresis on a 2% agarose gel with 1× tris-aminomethane tis-acetate-ethylenediaminete-traacetic acid buffer, stained with EtBr, and photographed in ultraviolet light.
All data were expressed as mean ± standard deviation (SD). The software of SPSS 11.5 for Windows (SPSS Inc., IL, USA) was used for analysis. Comparisons of continuous variables among the 4 groups were performed by a one-way analysis of variance (ANOVA). The LSD method was used to specify differences among groups. P value less than 0.05 was considered statistically significant.
Thirteen of sixty rats died during ligation of the left coronary artery. Three weeks after myocardial infarction, seven of forty seven rats were excluded because the FS was more than 25%. The remaining forty rats were randomized into 4 groups as described above.
Echocardiographic evaluation of cardiac function
The FS in each group was as follows: group CF, (27.80±6.32)%; group C, (22.29±4.54)%; group F, (19.24±6.29)%; control group, (20.36±3.29)%. The FS of group CF was significantly different compared with other groups (P=0.028, 0.001, 0.005 respectively (post hoc LSD test)). The group CF showed significantly higher FS compared with other groups (Fig. 1).
Langendorff perfusion assessment of cardiac function
Cardiac function assessed by Langendorff perfusion revealed significant differences among four groups when the left ventricular balloon volume was at the level of 0.10 ml. The left ventricular high developed pressure (LVDPmax, mmHg) in each group was as follows: group CF, 104.81±17.05; group C, 80.97±21.60; group F, 72.07±26.17; control group, 71.42±17.55. The LVDPmax of group CF was significantly different compared with other groups (P=0.015, 0.001, 0.001 respectively (post hoc LSD test)). The group CF showed significantly higher LVDPmax compared with other groups (Fig. 2).
The left coronary artery ligation led to a transmural infarction in the anterior wall of all examined rats. The anterior wall in group CF was the thickest among all groups (Fig. 3). The results of immunohistochemical staining for α-actinin are showed in Fig. 4. CM-DiI was used to trace implanted surviving cells.
Survival of transplanted cells by PCR analysis
Left ventricular tissue from three rats in each group was used for detecting the SRY gene by PCR. All tissues from animals of group CF and group C, who received neonatal heart cells, were positive, confirming the presence and survival of transplanted cells, and all tissues from group F and control group which did not receive neonatal heart cells were negative (Fig. 5).
The results of heart function assessment indicated transplantation of neonatal cardiomyocytes plus fibrin sealant preserved left ventricle fraction shortening and the left ventricular peak developed pressure most effectively. The present study indicated also that fibrin sealant may be used as a support and tissue-engineering scaffold to improve cardiac function after MI in rats.
The optimal type of cells to restore cardiac function after myocardial infarction is still controversial. New hope focuses on embryonic stem cells (ESCs) and adult stem cells, especially bone marrow stem cells (BMSCs). But for ESCs, transplantation of undifferentiated ESCs may lead to teratoma and it is still difficult to obtain enough and purified cardiac-like cells from ESCs in vitro.15 However, only when 100% ESCs differentiated into cardiomyocytes, can tumor formation be excluded. For BMSCs, Orlic and colleagues16 reported that BMSCs could differentiate into cardiac-like cells. But the transdifferentiation ability of BMSCs has come under suspicion recently.17 Jackson18 indicated that BMSCs may have the ability to transdifferentiate into cardiac-like cells, but the transdifferentiation rate is very low (<0.02%). If this is true, choosing BMSCs as a new cell source for myocardium regeneration is not practical. Though human fetal or neonatal cardiomyocytes cannot be obtained in sufficient numbers because of practical and ethical reasons, neonatal cardiomyocytes are the most reliable and easily available cardiac cells in an experimental situation. Therefore, we used rat neonatal heart cells as the cell source for myocardial regeneration.
Transplantation of cultured cardiomyocytes to treat heart failure has been investigated for more than a decade. Koh and associates19 firstly transplanted AT-1 cardiomyocytes (a cell line isolated from subcutaneous tumors derived from the left atrium of transgenic mice) into syngeneic mouse hearts and found that the transplanted cells survived and proliferated. They also found that cardiomyocytes formed gap junctions with host cardiomyocytes in the host myocardium,20 and angiogenesis occurred in the transplant.21 Several years later, Li and colleagues22 transplanted cultured fetal cardiomyocytes and autologous heart cells,23 and got similar results. Despite these initial encouraging results, however, cardiomyocytes transplantation has several considerable obstacles, including a poor cellular surviving rate, uncontrollable graft properties and inadequacy of graft vascularity. In our study the ventricular cardiomyocytes of one-day-old SD neonatal rats (male sex) were isolated and cultured, and the transplanted cardiomyocytes were found to have survived, proliferated and formed gap junctions with host cardiomyocytes.
The system of engineering heart tissue introduced by Eschenhagen and co-workers was a crucial milestone in the path to restoring injured myocardium by tissue engineering techniques. For the first time, they cultured viable artificial tissue that can contract in vitro as an integrative tissue, and such construction allowed for preconditioning before implantation.24-26 The same research team also performed the constructions for myocardial restoration.27 However because engineering heart tissue is fragile and limited because of its narrow circular shape, it is very difficult for this approach to gain acceptance on a practicing scale. In our study, we injected neonatal cardiomyocytes plus fibrin sealant in myocardial scar tissue directly, and observed that the transplanted neonatal cardiomyocytes survived in the scar tissue. The transplanted neonatal cardiomyocytes plus fibrin sealant improved heart function more than other groups.
Fibrin sealant may act as an internal support to preserve cardiac function. Injection of myoblasts in fibrin sealant prevented infarct wall thinning and preserved cardiac function. The benefit of using fibrin sealant as a scaffold is that it is injectable, thus requiring only a minimally invasive procedure in humans. Injection of cells in fibrin sealant increases cell viability, presumably by increasing arteriole density. Injection in the fibrin scaffold also results in viable grafts distributed within the infarct scar instead of simply in the border zone. As a scaffold, fibrin sealant is also capable of delivering proteins, plasmids, growth factors and other cells to the myocardium.28 Further modification to fibrin sealant may be performed to enhance its functionality in the myocardium. As a support, fibrin sealant may be modified to tailor its mechanical properties. An increase in thrombin or fibrinogen concentration results in an increase in tensile strength. An increase in fibrinogen concentration will also decrease the degradation rate of the biopolymer.
1. MacIntyre K, Capewell S, Stewart S, Chalmers JW, Boyd J, Finlayson A, et al. Evidence of improving prognosis in heart failure: trends in case fatality in 66,547 patients hospitalized between 1986 and 1995. Circulation 2000; 102: 1126-1131.
2. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein A, et al. Human embryonic stem cells can differentiate into myocyres with structural and functional properties of cardiomyocytes. J Clin Invest 2001; 108: 407-414.
3. Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R, van den Brink S, Hassink R, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003; 107: 2733-2740.
4. Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 2002; 91: 501-508.
5. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundation of cardiac repair. J Clin Invest 2005; 115: 572-583.
6. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol 2005; 23: 845-856.
7. Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, et al. Transplantation
of progenitor cells and regeneration enhancement in acute myocardial infarction
: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004; 44: 1690-1699.
8. Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C,. Intracoronary autologous bone-marrow cell transfer after myocardial infarction
: the BOOST randomised controlled clinical trial. Lancet 2004; 364: 141-148.
9. Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction
. N Engl J Med 2006; 355: 1199-1209.
10. Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction
: double-blind, randomized controlled trial. Lancet 2006; 367: 113-121.
11. Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L, Hertenstein B. Monitoring of bone marrow cell homing into the infracted human myocardium. Circulation 2005; 111: 2198-2202.
12. Li RK, Jia ZQ, Weisel RD, Mickle DA, Zhang J, Mohabeer MK, et al. Cardiomyocyte transplantation
improves heart function. Ann. Thorac Surg 1996; 62: 654-660.
13. Christman KL, Fok HH, Sievers RE, Fang Q, Lee RJ. Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction
. Tissue Engineering 2004; 10: 403-409.
14. Muller-Ehmsen J, Whittaker P, Kloner PA. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol 2002; 34:107-116.
15. Heng BC, Haider HKh, Sim EK, Cao T, Ng SC. Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro
. Cardiovasc Res 2004; 62: 34-42.
16. Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701-705.
17. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischemic myocardium. Nature 2004; 428: 668-673.
18. Jackson KA, Majka SM, Wang H. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: 1395-1402.
19. Koh GY, Soonpaa MH, Klug MG, Field LJ. Long-term survival of AT-1 cardiomyocyte grafts in syngeneic myocardium. Am J Physiol 1993; 264(5 Pt 2): H1727- H1733.
20. Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994; 264: 98-101.
21. Koh GY, Kim SJ, Klug MG, Park K, Soonpaa MH, Field LJ. Targeted expression of transforming growth factor-beta 1 in intracardiac grafts promotes vascular endothelial cell DNA synthesis. J Clin Invest 1995; 95: 114-121.
22. Li RK, Jia ZQ, Weisel RD, Mickle DA, Zhang J, Mohabeer MK, et al. Cardiomyocyte transplantation
improves heart function. Ann Thorac Surg 1996; 62: 654-660.
23. Sakai T, Li RK, Weisel RD, Mickle DA, Kim EJ, Tomita S, et al. Autologous heart cell transplantation
improves cardiac function after myocardial injury. Ann Thorac Surg 1999; 68: 2074-2080.
24. Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J, et al. Three dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J 1997; 11: 683-694.
25. Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T. Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol Bioeng 2000; 68: 106-114.
26. Zimmermann WH, Schneiderbanger K, Schubert P, Didie M, Munzel F, Heubach JF, et al. Tissue engineering of a differentiated cardiac muscle construct. Circ Res 2002; 90: 223-230.
27. Zimmermann WH, Didie M, Wasmeier G, Nixdorff U, Hess A, Melnychenko I, et al. Cardiac grafting of engineered heart tissue in syngenic rats. Circulation 2002; 106(12 Suppl 1): I151-I157.
28. Christman KL, Fang Q, Yee MS, Johnson KR, Sievers RE, Lee RJ. Enhanced neovasculature formation in ischemic myocardium following delivery of pleiotrophin plasmid in a biopolymer. Biomaterials 2005; 26: 1139-1144.
Keywords:© 2007 Chinese Medical Association
transplantation; myocytes, cardiac; myocardial infarction; cell regeneration; fibrin sealant