TISSUE ENGINEERING: Part A Volume 16, Number 6, 2010 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2009.0537
Adenylate-Cyclase VI Transforms Ventricular Cardiomyocytes into Biological Pacemaker Cells Arjang Ruhparwar, M.D.,1 Klaus Kallenbach, M.D.,1 Gunnar Klein, M.D.,2 Christoph Bara, M.D.,3 Ali Ghodsizad, M.D.,1 Daniel C. Sigg, M.D., Ph.D.,4 Matthias Karck, M.D.,1 Axel Haverich, M.D.,3 and Michael Niehaus, M.D.2
Introduction: When sinus node or atrioventricular (AV) node cells are damaged by disease, the implantation of an artificial cardiac pacemaker becomes necessary. In search for a biological alternative, the objective of this study was to demonstrate whether in vivo adenoviral gene transfer of Adenylate-Cyclase type VI (AC-VI) can create biological pacemaker activity in a porcine AV node block model. Genetic therapy of arrhythmic disorders of the heart has been subject of extensive studies. Cyclic AMP is generated in response to Beta-adrenergic receptor stimulation and also binds to HCN channels, where it regulates spontaneous rhythmic activity in the sinus node. Materials and Methods: Adenoviruses encoding either AC-VI or Beta-Galactosidase (lacZ) gene were injected into the lateral wall of the left ventricle of adult pigs via anterolateral thoracotomy at a dose of 1010 virus particles each. After 12 days, the AV node was ablated and three-dimensional electrophysiological cardiac mapping was performed using the Ensite electro-anatomical system. Results: After rapid ventricular pacing and administration of Isoprenalin, all animals of the AC-VI group exhibited an escape rhythm originating from the area of the left ventricular injection site at a rate of 100 þ 7 beats/min (n ¼ 5), whereas the escape rhythms in the control group (n ¼ 4) originated from the right ventricle. Western blot analysis of the injection sites revealed significantly higher expression of AC-VI in the respective group as compared with the control group. Conclusions: Our study demonstrates that AC-VI gene transfer has the potential to create a biological pacemaker system.
Introduction
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he heart is endowed with specialized excitatory and conducting myocytes that are responsible for the generation and conduction of rhythmic impulses and contractions throughout the heart. If these cells are damaged by disease, the implantation of an artificial cardiac pacemaker becomes necessary with emphasis on rapid restoration of physiologic cardiac rhythm or creation of the best physiologic alternative.1 As to the classic indications for cardiac pacemaker implantation, several limitations and problems have emerged during the past decades such as electrode fracture or damage to insulation, infection, finite longevity with requirement for re-operations for battery exchange, and vein thrombosis. In
children, including premature newborn babies, with congenital or acquired arrhythmia such as atrioventricular (AV) block, initial size mismatch of the children and growth affecting the position of leads and generators can pose a problem. Lead extraction has additional, potentially lifethreatening risks such as perforation of the right ventricle, atrium, superior vena cava, or the subclavian vein.2 Apart from potential advancements of current pacemaker systems, it would therefore be worthwhile to search for biological alternatives for cardiac pacing. Conceivable approaches are transplantation of pacemaker-competent cells, in vivo gene therapy, or in vitro gene transfer with subsequent transplantation of cells into the myocardium.3 In the first study we were able to show that transplanted fetal cardiomyocytes can function as a biological cardiac
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Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany. Department of Cardiology and Angiology, and 3Division of Thoracic, Transplantation and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany. 4 Medtronic, Inc., Mounds View, Minnesota. 2
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pacemaker. However, the main drawback of cell therapy is extensive donor cell death or apoptosis early after implantation in the diseased myocardium.4,5 Also, it is unclear whether a cardiac cell therapy product derived from allogeneic human embryonic stem cells can be successfully commercialized, aside from significant ethics challenges. As to the possible use of hormones, Neuregulin-1 and cyclic AMP (cAMP) possess the capacity to cause significant transformation of a mixed population of fetal cardiomyocytes into cardiac pacemaker-like cells only during a restricted period of fetal development.6 Gene therapeutic studies have aimed at specific inhibition of Ik1, which in turn inhibits latent pacemaker capability in nonconduction system cardiomyocytes, and overexpression of HCN2, an isoform of the gene encoding the wild-type pacemaker current If.7,8 Potapova et al. demonstrated that transplantation of HCN2-transfected human mesenchymal stem cells leads to expression of functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes.9 However, this approach may be hampered by possible heteromultimerization with endogenous HCN family members. An interesting approach has been the transfer of synthetic pacemaker channel genes into the heart.10 In vitro experiments have demonstrated the ability of HCN1 fibroblasts to fuse with ventricular cardiomyocytes.11 In the presented novel approach we chose to boost intracellular cAMP production to activate two different mechanisms to increase the heart rate and therefore create a biological pacemaker system:
N52.E6 cell line (primary human aminocytes) was used for homologous recombination of adenoviral vectors to avoid replication-competent adenoviral vectors. Success of transfection into the cells was verified by Western blot analysis. Surgical technique and gene injection About 1010 virus particles containing the AC-VI gene, suspended in 200 mL cell culture medium, were obliquely injected into the free wall of the left ventricle of the isofluraneanesthetized domestic pigs (n ¼ 5, weight: 40–44 kg) via antero-lateral thoracotomy. The epicardial injection site was labeled with Titanium clips for later identification of the injection site by fluoroscopy. The control group (n ¼ 4) received the same number of viruses carrying the lac-Z gene. Additionally, an epimyocardial pacemaker system with a minimum rate of 30 beats/min (VVI-mode) was implanted at the same time to guarantee survival after AV node ablation. Electrophysiological studies and radiofrequency catheter ablation
During all experiments the Principles of Laboratory Animal Care (NIH publication No. 86-23, revised 1985) as well as the specific German Law on the Protection of Laboratory Animals were followed.
Twelve days after gene injection, the animals were anesthetized and introducer sheaths were placed in the right femoral artery and vein as well as in the right jugular vein and carotid artery. After administration of Heparin, the multielectrode array balloon (Ensite; St. Jude Medical, St. Paul, MN) was introduced over a 0.035-inch exchange guidewire into the left ventricle in a retrograde fashion under fluoroscopic guidance. Creation of an adequate, threedimensional image of the left ventricle could be completed in several minutes, and all catheters could be freely maneuvered and observed within the left ventricle. By projecting the fluoroscopy image onto the Ensite image, the labeled area of gene injection, apex, and basis of the heart could be transferred and identified on the Ensite image. A 7F hexapolar catheter was placed at the His bundle region, and a 7F steerable ablation catheter was positioned at the presumed compact AV node based on anatomical and electrophysiological guidance. As previously described,3 after 1–3 temperature-controlled radiofrequency applications, total AV-block was induced. As described by Alison et al.,15 subsequent rapid ventricular pacing (120 beats/min.) was performed and 0.5 mg Isoproterenol was administered to provoke the initiation of a left ventricular escape rhythm. In our hands this method has been the best way to elicit an escape rhythm and it also ensures comparability of the escape rhythms.3 Additionally, it avoids long periods of bradycardia with subsequent ventricular fibrillation in the sensitive porcine model. Detailed activation mapping of the left ventricle was performed, monitored by the Ensite system. All data obtained with the Ensite system were stored on optical disks for further off-line evaluation. The animals were sacrificed, and specimens of the myocardium were obtained for histology and Western blot analysis.
Adenoviral constructs and expression
Western blot analysis
1. Clinical studies have shown that Amrinone and Milrinone, which are phosphodiesterase inhibitors and thus inhibit the cleavage of cAMP in cardiomyocytes, significantly increase heart rate in patients.12 2. cAMP binds to the cytoplasmatic site of HCN and therefore permits the channels to open more rapidly and completely after repolarization of the action potential with the result of accelerating rhythmogenesis.13 Lai et al. were able to demonstrate that Adenylate-Cyclase VI (AC-VI) enhances intracellular cAMP level in a porcine model.14 Therefore, we hypothesized that using ACVI leads to overproduction of cAMP in the cell and would also have a positive chronotropic effect on cardiomyocytes in the porcine heart. The aim of our study was the in vivo adenoviral transfection of ventricular cardiomyocytes in a large animal model (porcine) with the gene encoding for ACVI to increase the intracellular concentration of cAMP and thus the intrinsic rhythmic rate of the transfected cells, creating an ectopic biological pacemaker at the injection site in the left ventricle. Materials and Methods
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As described by Lai et al., we also used an E1-deleted, replication-incompetent adenovirus vectors encoding murine or nuclear-tagged bacterial b-galactosidase (lacZ), driven by a cytomegalovirus promoter. For animal experiments,
The production of the AC-VI protein in the myocardium at the injection site was again verified by Western blotting. A myocardial cube of approximately 1.5 cm3 was excised at the injection site in both groups. Equally loaded total cell lysates
GENE THERAPY OF CARDIAC ARRHYTHMIA
FIG. 1. Western blot shows successful transfection of N52 cells with the AC-VI gene as indicated by a significantly higher production of the AC-VI protein in the transfected cell group (Orig. N52) compared with control cells (D9N52). Color images available online at www.liebertonline.com/ten. (50 mg/lane, measured by photometry and comparison with standard loading dose) of each sample were fractionated on 10% sodium dodecyl sulfate–polyacrylamide gels. The proteins were transferred electrophoretically to a polyvinylidene difluoride membrane. Equal transfer was verified by staining with Ponceau Red. Membranes were blocked using 5% bovine serum albumin (BSA) in TTBS (20 mM Tris, 150 mM
1869 NaCl, 0.05% Tween 20, pH 6.8) for 1 h. Membranes were then incubated overnight in primary antibody solutions (Adenylatcyclase V/VI, anti-rabbit; dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) in 5% BSA in TTBS at 48C. After washing three times in TTBS, membranes were incubated with horseradish peroxidase–conjugated antimouse IgG-antibody (Amersham, Little Chalfont, United Kingdom) in 5% BSA in TTBS for 1 h at room temperature. Then, the membranes were washed three times in TTBS before performing antibody detection by chemiluminescence. Results Successful adenoviral expression and virus purification Successful transfection of the genes into cells designed for virus amplification and subsequent production of AC VI was verified by AC-VI protein production using Western blot analysis. The result is depicted in Figure 1.
FIG. 2. Representative ECG and activation map of left ventricle showing the transition from atrioventricular block and junctional rhythm in the AC VI group after rapid ventricular pacing and administration of Isoproterenol as reconstructed by the Ensite system. Color images available online at www.liebertonline.com/ten.
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FIG. 3. ECG and activation map of left ventricle during the right ventricular escape rhythm in the control group after rapid ventricular pacing and administration of Isoproterenol as reconstructed by the Ensite system. No left ventricular escape rhythm is visible. Color images available online at www.liebertonline.com/ten.
Successful initiation of a left ventricular escape rhythm around the gene injection site by AC-VI AV node ablation was successful in all animals as representatively depicted in Figure 2. All animals of the AC-VI group presented a stable escape rhythm whose origin was around the left ventricular injection site as demonstrated by the Ensite system. In the control group, only right ventricular escape rhythms were observed (Fig. 3). Figure 4 shows a representative three-dimensional image of myocardial activation during the escape rhythm from an animal of the ACVI group. The image could be viewed from any angle and at any magnification by rotation (also see cine loop, Supplemental Movie S1; available online at www .liebertonline.com/ten). The rate of the escape rhythm ranged from 90 to 110 beats/min after rapid ventricular pacing and infusion of Isoproterenol (mean rate: 100 þ 7 beats/min). The results of the electrophysiology studies are summarized in Table 1.
Successful expression of AC-VI at the injection site The production of the AC-VI protein in the myocardium at the injection site was again verified by Western blotting. A cube of 1.5 cm3 was excised at the injection site. The result was a significant higher amount of AC-VI present in the ACVI group as compared with the control group (Fig. 5). Discussion Our results indicate that AC-VI overexpression in the left ventricle can create a left ventricular biological pacemaker rhythm (escape rhythm) sufficient to drive the heart when injected into a localized region of left ventricle, offering a promising gene therapy for bradyarrhythmia. In this experimental setting, the essential result is that the origin of the escape rhythm was the left ventricle, where we had injected our gene, whereas none of the control animals had a left ventricular escape rhythm. If this effect can be
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FIG. 4. Activation map of left ventricle during the escape rhythm with a three-dimensional posterior projection of the left ventricle as reconstructed by the Ensite system. The multielectrode array balloon and the icon of the ablation/pacing catheter are observed within the left ventricle in an animal injected with adenovirus encoding AC-VI. The injection site is labeled by a white color on the left side. The electrophysiological information was color-coded. The earliest site of activation is (red) located in the anterior wall. The ECG pattern of the stable escape rhythm is presented at the bottom of the screen. As the cursor moves through the QRS complex, the activation then spreads onto the rest of the myocardium (yellow and blue). AV, atrioventricular node; ABL, ablation catheter. Color images available online at www.liebertonline.com/ten. maintained in a long-term setting and a positic chronotropic response to physical exertion is observed, ideally biological pacemakers may eventually replace artificial cardiac pacemakers or at least support these in a tandem application to reduce energy consumption of the batteries, leading to less frequent exchange of pacemakers. The fact that application of Isoprenalin helped provoke the initiation of the escape Table 1. In All Animals of the AC-VI Group, the Escape Rhythm Originated From the Left Ventricle as Opposed to the Control Group, Where in All Animals the Source of the Escape Rhythm was the Right Ventricle Animal number 1 2 3 4 5 6 7 8 9
(control) (control (control) (control) (þAC-VI) (þAC-VI) (þAC-VI) (þAC-VI) (þAC-VI)
Escape rhythm present
Location
Mean heart rate
þ þ þ þ þ þ þ þ þ
RV RV RV RV LV LV LV LV LV
110 100 90 100 100
Mean rate of left ventricular escape rhythm: 100 7/min. RV, right ventricle; LV, left ventricle; AC-VI, Adenylate-Cyclase type VI.
rhythm is encouraging with regard to partial simulation of physical activity. As to broader clinical application, it would be desirable to use a gene transfection system that allows for long-term expression of the transfected genes. Adenoviral gene expression used in our study usually lasts for approximately 3 weeks. The use of retroviruses or adeno-associated viruses for clinical application that would enable a stable long-term gene transfection is still controversial due to possible malignant transformation of transfected cells. For this reason, the large size of the gene would need to be trimmed down. An alternative approach would be a combination of cell and gene therapy as already demonstrated by Potapova et al., who used mesenchymal stem cells as a gene delivery system for the HCN gene.9 However, the extensive donor cell death or apoptosis early after implantation in the myocardium is
FIG. 5. Myocardial samples obtained from the AC-VI group (þþ ) showed a significantly higher production of Adenylate-Cyclase VI as compared with the control group (c). Color images available online at www.liebertonline .com/ten.
1872 still an unsolved problem. The use of growth factors might improve the environment for transplanted cells and therefore reduce the rate of cell death after transplantation.16 Another possibility is the transfection of an anti-apoptotic gene such as Bcl-2 to reduce apoptosis of transplanted cells within the myocardium as recently demonstrated by Li et al.17 Finally, future systematic studies are necessary to assess the potential ability of injected genes to induce arrhythmia that must be excluded in long-term studies with mapping experiments at various time points and with a larger cohort of animals. Study Limitations The spatial resolution of the Ensite system may not be high enough to pinpoint the exact location of the escape rhythm on an mm-scale. Correct LV mapping by the Ensite system is dependent on the correct labeling of topographic landmarks during placement of the catheter tip in the apex, mitral valve, and septum, which may not always be congruent with the anatomic axis and can result in vector deviations. Respiration of the animals can also influence the geometry of the left ventricle. Additionally, the medium containing the genes was displaced away from the injection site between different layers of myocardium. This phenomenon is well known from cell transplant studies,18 where myocardial motion of the various muscle layers with different muscle fiber orientations displaced cells or nano-particles far from the injection sites.
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Acknowledgments We would like to specially thank H.K. Hammond, University of California, San Diego, for providing the original virus containing the AC-VI gene. We also thank Martin Bonerath for expert technical assistance with the Ensite system. This study was financially supported by a research grant from Medtronic, Inc., Minneapolis.
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Disclosure Statement Daniel Sigg, one of the co-authors, was a senior manager at Medtronic, Inc. References 1. Ruhparwar, A., and Haverich, A. Prospects for biological cardiac pacemaker systems. Pacing Clin Electrophysiol 26, 2069, 2003. 2. Friedman, R.A., Fenrich, A.L., and Kertesz, N.J. Congenital complete atrioventricular block. Pacing Clin Electrphysiol 24, 1681, 2001. 3. Ruhparwar, A., Tebbenjohanns, J., Niehaus, M., Mengel, M., Irtel, T., Kofidis, T., Pichlmaier, A.M., and Haverich, A. Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg 21, 853, 2002. 4. Reinecke, H., Zhang, M., Bartosek, T., and Murry, C.E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, 193, 1999. 5. Muller-Ehmsen, J., Whittaker, P., Kloner, R.A., Dow, J.S., Sakoda, T., Long, T.I., Laird, P.W., and Kedes, L. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J Mol Cell Cardiol 34, 107, 2002. 6. Ruhparwar, A., Er, F., Martin, U., Radke, K., Gruh, I., Niehaus, M., Karck, M., Haverich, A., and Hoppe, U.C.
17.
18.
Enrichment of cardiac pacemaker-like cells: Neuregulin-1 and cyclic AMP increase If-current density in fetal cardiomyocytes. Med Biol Eng Comput 45, 221, 2007. Miake, J., Marban, E., and Nuss, H.B. Gene therapy: biological pacemaker created by gene transfer. Nature 419, 132, 2002. Plotnikov, A.N., Sosunov, E.A., Qu, J., Shlapakova, I.N., Anyukhovsky, E.P., Liu, L., Janse, M.J., Brink, P.R., Cohen, I.S., Robinson, R.B., Danilo, P., Jr., and Rosen, M.R. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation 109, 506, 2004. Potapova, I., Plotnikov, A., Lu, Z., Danilo, P., Jr., Valiunas, V., Qu, J., Doronin, S., Zuckerman, J., Shlapakova, I.N., Gao, J., Pan, Z., Herron, A.J., Robinson, R.B., Brink, P.R., Rosen, M.R., and Cohen, I.S. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res 94, 952, 2004. Kashiwakura, Y., Cho, H.C., Barth, A.S., Azene, E., and Marba´n, E. Gene transfer of a synthetic pacemaker channel into the heart: a novel strategy for biological pacing. Circulation 114, 1682, 2006. Cho, H.C., Kashiwakura, Y., and Marban, E. Creation of a biological pacemaker by cell fusion. Circ Res 100, 1112S, 2007. Shibata, T., Suehiro, S., Sasaki, Y., Hosono, M., Nishi, S., and Kinoshita, H. Slow induction of milrinone after coronary artery bypass grafting. Ann Thorac Cardiovasc Surg 7, 23, 2001. Wainiger, B.J., Dehennaro, M., Santoro, B., Siegelbaum, S.A., and Tibbs, G.R. Molecular mechanism of cAMP modulation of HCN pacemaker channels. Nature 411, 805, 2001. Lai, N.C., Roth, D.M., Gao, M.H., Fine, S., Head, B.P., Zhu, J., McKirnan, M.D., Kwong, C., Dalton, N., Urasawa, K., Roth, D.A., and Hammond, H.K. Intracoronary delivery of adenovirus encoding adenylyl cyckase VI increases left ventricular function and cAMP generating capacity. Circulation 102, 2396, 2000. Alison, J.F., Yeung-Lai-Wah, J.A., Schulzer, M., and Kerr, C.R. Characterization of junctional rhythm after atrioventricular node ablation. Circulation 91, 84, 1995. Kofidis, T., de Bruin, J.L., Yamane, T., Tanaka, M., Lebl, D.R., Swijnenburg, R.J., Weissman, I.L., and Robbins, R.C. Stimulation of paracrine pathways with growth factors enhances embryonic stem cell engraftment and host-specific differentiation in the heart after ischemic myocardial injury. Circulation 111, 2486, 2005. Li, W., Ma, N., Ong, L.L., Nesselmann, C., Klopsch, C., Ladilov, Y., Furlani, D., Piechaczek, C., Moebius, J.M., Lu¨tzow, K., Lendlein, A., Stamm, C., Li, R.K., and Steinhoff, G. Bcl-2 engineered MSCs inhibited apoptosis and improved heart function. Stem Cells 11, 798, 2007. Soonpaa, M.H., Koh, G.Y., Klug, M.G., and Field, L.J. Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264, 98, 1994.
Address correspondence to: Arjang Ruhparwar, M.D. Department of Cardiac Surgery University of Heidelberg Im Neuenheimer Feld 110 69120 Heidelberg Germany E-mail:
[email protected] Received: August 4, 2009 Accepted: January 11, 2010 Online Publication Date: April 19, 2010
