Europace Advance Access originally published online on October 18, 2007
Europace 2007 9(12):1218-1221; doi:10.1093/europace/eum224
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EXPERIMENTAL STUDIES
Electrophysiological consequence of adipose-derived stem cell transplantation in infarcted porcine myocardium
1 Department of Medicine, Section of Cardiology, Tulane University Health Sciences Center, New Orleans, LA 70112, USA; 2 Department of Molecular Pathology, M.D. Anderson Cancer Center, University of Texas, Box 951 7435 Fannin Street, Houston, TX 77030, USA
Manuscript submitted 6 August 2007. Accepted after revision 12 September 2007.
* Corresponding author. Tel: +1 713 834 6106; fax: +1 713 834 6105. E-mail address: ealtmd{at}aol.com
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Abstract |
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Aims: Aim of this study was to investigate the effect of intracoronary administration of freshly isolated adipose-derived mononuclear cells (ADMCs) on myocardial vulnerability to arrhythmia induction after infarction.
Methods and results: A transmural myocardial infarction in an experimental porcine model was induced by occlusion of the mid-left anterior descending artery with an angioplasty balloon for 3 h. Upon reperfusion, a cellular suspension with freshly isolated ADMCs (1.5 x 106 cells/kg BW) or vehicle alone was injected into the infarct artery. All animals underwent a programmed ventricular stimulation at 8 weeks follow-up for possible induction of ventricular arrhythmias using a train of 8 S1 stimuli. Cell injections did not cause acute ventricular arrhythmia, bradycardia, or conduction block. The cycle length of the ventricular arrhythmia was compared at 1 and 10 s following its induction. Despite comparable infarct size in both groups, we found that the cycle length of the induced ventricular arrhythmia in the ADMC-treated group was significantly longer compared with control animals (P < 0.05). We also found that extra-stimuli were required for arrhythmia induction in the ADMC-treated group compared with control animals.
Conclusion: Freshly isolated autologous stem cell therapy is not proarrhythmic in pigs.
Key Words: Stem cell, Arrhythmia, Myocardial vulnerability
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Introduction |
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Stem cell transplantation has been successfully used for repair of infarcted myocardium,1
–5 but concerns have been raised regarding its pro-arrhythmic potential. Studies of skeletal myoblast therapy have shown incidence of ventricular arrhythmias.6–8 In contrast, recent studies using bone marrow-derived cell therapy8–10 have not reported a significant incidence of arrhythmias. Mills et al.11 showed that intravenous (i.v.) infusion of mesenchymal stem cells after myocardial infarction (MI) trended towards being less vulnerable to arrhythmias than controls although the difference did not reach statistical significance. In this report, we performed a programmed stimulation 8 weeks after cell therapy and additionally implanted an ECG loop/event recorder to determine the electrophysiological consequence of cell therapy in the porcine infarction model. |
Methods |
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Animal care and acute myocardial infarction
All experiments were performed in accordance with guidelines published in Guide for the Care and Use of Laboratory Animals (NIH publication No. 86–23, revised 1985), and under the protocol reviewed and approved by the Institutional Animal Care and Use Committee at Tulane University.
Pigs were sedated with 2.75 mg/kg Telazol® and 2.47 mg/kg Xylazine. Anaesthesia was maintained by isoflurane and propofol. Fentanyl was administered i.v. every 30 min (0.017 mg per bolus) until the end of the surgical procedure.
Anti-coagulant therapy was administered i.v. as follows: acetylsalicylate 500 mg; enoxaparin 1 mg/kg bolus, then 0.5 mg/kg every 4 h, then 1 mg/kg s.c. at the end of the MI induction procedure; eptifibatide prior to balloon occlusion in two 180 µg/kg boli, 10 min apart followed by a 2 µg/kg per min infusion during balloon occlusion. Intravenous anti-arrhythmic medication during the MI procedure consisted of 50 mg magnesium sulfate every 60 min, supplemented by amiodarone at 25–75 mg per bolus during ventricular arrhythmias.
Following the baseline coronary angiogram (Advantx LC with DLx3-C1024 digital system, GE Medical System, USA, and Heartlab Inc, Data Storage), an angioplasty balloon (length 9 mm, diameter 2.5–3.5 mm, Maverick OTW, Boston Scientific, USA) was selected for occlusion of the left anterior descending (LAD) artery. The balloon was inserted through a standard technique and inflated for 3 h between the first and second diagonal branch under the lowest possible inflation pressure that occluded the vessel (Figure 1).
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Adipose-derived mononuclear cell isolation
Using a scalpel, 5–15 g of subcutaneous adipose tissue was harvested, bilaterally, from inguinal fat pads. After mincing, the tissue was transferred to T-75 cm2 flasks to which saline and collagenase were added as described previously.12
,13 The viable cell yield was determined with an automated cell counter (Casy®, Schärfe System, Reutlingen, Germany), which measured the viable cell concentration within a cell diameter range set at 7–35 µm.
Flow cytometric analysis of fresh pig adipose-derived mononuclear cells
Antibodies against pig CD90, CD44, CD31, CD29, CD45, and CD11 were purchased from BD Bioscience. Isotype-matched mouse IgGs were used as controls. After incubation for 20 min and washing twice with PBS, the cells were analysed by flow cytometry (Cytomics FC 500 FACS; Beckman Coulter, Miami, FL, USA).
Adipose-derived mononuclear cell or vehicle injection
Adipose-derived mononuclear cell (ADMC) or control solution (Plasmalyte®) was injected through the central lumen of the balloon catheter under balloon occlusion in the mid-LAD, concentrating the injections to the infarct-affected area. Repetitive injection cycles of 3 ml cell suspension containing 7.5 million cells or Plasmalyte® were performed within 30–60 s and the inflated position was maintained for 
90 s during each cycle, allowing the cells to settle in the capillary bed. The balloon was then deflated for 1 min to avoid further ischemic damage to the heart. This procedure was repeated until the target amount of cells (1.5 x 106 cells/kg) or the corresponding volume of Plasmalyte® was infused.
Ventricular vulnerability testing
An ECG loop/event recorder (Reveal Plus, Medtronic Inc, Minneapolis, MN, USA) was implanted subcutaneously on the left side of the thorax over the 5–6 rib. The device continuously monitors a single-lead ECG. In the case of a bradycardia or tachycardia event, the ECG preceding and following the triggering event is recorded and stored in the memory of the device for (3 min per event). A total of 14 events of 3 min duration each can be stored. The device was interrogated and the data were downloaded to a computer via transcutaneous telemetry every 1–2 days.
Analogue to clinical practice, at 8 weeks follow-up, all animals underwent a programmed stimulation protocol for the induction of ventricular arrhythmias using a train of 8 S1 stimuli (interval 500 and 400 ms) and up to three extra-stimuli applied from the right ventricular apex and right ventricular out-flow tract. After induction, ventricular cycle length was measured at 1 and 10 s via the right ventricular mapping catheter.
Statistical analysis
Results are shown as mean ± SD. Student's t-test was used. A P-value of 
0.05 was considered statistically significant.
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Results |
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Cell characterization
On average of 9 ± 3 g adipose tissue was removed from the inguinal region yielding 92.1.0 ± 48.3 x 106 cells per animal. ADMC samples from all animals expressed CD90 (70.0 ± 8.9%), CD31 (8.1 ± 3.0), CD44 (3.9 ± 0.31), CD29 (6.8 ± 2.25), CD45 (13.6 ± 7.1%), and CD11 (4.9 ± 0.37%). To assess the percentage of mesenchymal cells present in our freshly isolated cells, the cells were cultured for 1 day (n = 10) and 12.1 ± 4.8% of these cells were found to be plastic adherent.
Myocardial infarction in pigs
Twenty-nine pigs were randomized to either cell therapy or only control plasmalite (carrier for cells solution) injection. The acute MI resulted in a similar reduction of 15–17% in LVEF in both groups. Six of the 29 animals did not survive on the initial MI induction, 2 animals had to be excluded (congenital ventricular septal defect, only small perfusion defect of <15%) after MI induction. Four animals died in the postoperative period: two animals (one each group) died on day 3 of ventricular arrhythmias; one (control) died at day 46, another (control) had to be sacrificed due to an anal prolaps. Complete follow-up data were obtained from 16 animals: ADMC treated (n = 9) and control group (n = 7), with a mean follow-up period of 56 ± 3 days. One animal died during the final, 8-week follow-up angiographic procedure.
Arrhythmia monitoring and programmed ventricular stimulation
Arrhythmia was monitored for 592 days in 16 pigs (ADMC mean of 42.6 ± 13.1 and control 30 ± 16.6 days). Two animals (one in each group) died from ventricular fibrillation 3 days after MI as documented by the device (Figure 2). The device did not record any other sustained arrhythmias (e.g. bradycardia <45 min–1 or tachycardia >165 min–1) during the recording period. Cell injections did not cause acute ventricular arrhythmia, bradycardia, or conduction block.
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Table 1 depicts the refractory period data. There is a significantly longer ERP for the cell group compared with control for S2 (P < 0.01) and a trend towards longer ERP for S3 (P = 0.09). S4 was required only in one animal in the control; however, the cell-treated animals were more stable and required an S4 in five out of seven cases. As shown in Table 1, the percentage of animals in which VT and VF was induced in the control group is 33 and 67% in the control and 43 and 57% in the cell treated group, respectively.
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The programmed ventricular stimulation was performed in eight animals in the ADMC-treated group and in seven animals in the control group. Arrhythmias were not inducible in one animal from each experimental group. Among the remaining ADMC animals (n = 7), ventricular tachycardia or ventricular fibrillation was induced in two animals using two extra-stimuli, whereas in the remaining five ADMC animals three extra-stimuli were required for the induction. Among the controls, malignant arrhythmias were induced in five animals already with two extra-stimuli and in only one animal three extra-stimuli were required. The number of stimuli and the cycle length of the stimuli required for arrhythmic induction are shown in Table 2.
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The cycle length of the ventricular arrhythmia was compared at 1 and 10 s following its induction. The arrhythmia type and cycle lengths observed at these two time points are shown in Table 3. A significantly (P < 0.05) longer cycle length of the induced ventricular arrhythmia at 1 s was found in the ADMC-treated group compared with control animals.
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Discussion |
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Our present study, for the first time, demonstrated MI in a large animal model, the acute treatment with autologous adipose tissue-derived mononuclear cells has no detrimental effect on electrical myocardial stability over the course of 8 weeks.
It has been suggested that the delivery method of cell therapy (intramyocardial injection vs. i.v. infusion) may have affected the arrhythmia vulnerability. It has been shown that intramyocardially injected skeletal muscle myoblasts (SKMB) tend to cluster near injection sites14,15 and do not fully electrically couple with native myocardium in vivo.16 The clustering of directly injected SKMB may have formed a local zone of slow conduction,14 which is a substrate for re-entrant excitation. In contrast, mesenchymal stem cells delivered by i.v. infusion have been shown to form gap junctions with host cardiomyocytes in vivo.17,18 Mill et al.11 suggested that the expression of connexin protein of MSCs in the border zone of hearts may explain the observed reduction in arrhythmia vulnerability. In addition, the refractory period in skeletal myoblasts is very short in the range of 30 ms, whereas in cardiomyocytes it—dependent on heart rate—is 300 ms. Previous patch clamp studies from our groups have shown that adipose tissue-derived stem cells show a full functional range of ion channels even in an early undifferentiated stage19 and that these cells when further differentiated are capable of expressing a more cardiogenic electrical phenotype.12
A recent study by Katritsis et al.20 evaluated the possible proarrhythmic potential of stem cell transplantation. They showed that intracoronary transplantation of autologous mesenchymal and endothelial progenitor cells does not appear to be arrhythmogenic in humans. ADMCs behave similarly compared with the bone marrow-derived cells in this regard.
Limitations and future studies
Electrophysiology (EP) study shortly after cellular injection would have added very helpful information to our understanding of electrophysiological changes. However, the infarct itself caused a considerable electrical instability, which was counteracted by β-blockers, magnesium, and Amiodarone. Additionally, the risk of not being able to rescue the animal when inducing a VT/VF out-weighted the benefits of EP study after cell injection.
This initial study design was aimed at evaluating the feasibility and safety. We cannot prove homing of the injected cells based on this study design, which is a clear limitation of the present study. Continuing studies are currently ongoing to define further the condition of engraftment and the molecular electrical cell–cell interaction after engraftment.
Conflict of interest: none declared.
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Funding |
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Alliance of Cardiovascular Researchers (Metairie, LA, USA) with support from Cytori Therapeutics, Inc (543102) to A.E.; American Heart Association Southeast Affiliate Award (St Petersburg, FL, USA) (0555331B) to S.Y.H.
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[1] 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.[CrossRef][Web of Science][Medline]
[2] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature (2001) 410:701–5.[CrossRef][Medline]
[3] Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M, Duboc D, et al. Myoblast transplantation for heart failure. Lancet (2001) 357:279–80.[CrossRef][Web of Science][Medline]
[4] Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation (2002) 106:1913–8.
[5] Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet (2003) 361:45–6.[CrossRef][Web of Science][Medline]
[6] Menasche P, Hagege AA, Vilquin JT, Desnos M, Abergel E, Pouzet B, et al. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol (2003) 41:1078–83.
[7] Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onderwater EE, Lee CH, et al. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol (2003) 42:2063–9.
[8] Fernandes S, Amirault JC, Lande G, Nguyen JM, Forest V, Bignolais O, et al. Autologous myoblast transplantation after myocardial infarction increases the inducibility of ventricular arrhythmias. Cardiovasc Res (2006) 69:348–58.
[9] Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation (2002) 106:3009–17.
[10] Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet (2004) 364:141–8.[CrossRef][Web of Science][Medline]
[11] Mills WR, Mal N, Kiedrowski MJ, Unger R, Forudi F, Popovic ZB, et al. Stem cell therapy enhances electrical viability in myocardial infarction. J Mol Cell Cardiol (2007) 42:304–14.[CrossRef][Web of Science][Medline]
[12] Bai X, Pinkernell K, Song YH, Nabzdyk C, Reiser J, Alt E. Genetically selected stem cells from human adipose tissue express cardiac markers. Biochem Biophys Res Commun (2007) 353:665–71.[CrossRef][Web of Science][Medline]
[13] Song YH, Gehmert S, Sadat S, Pinkernell K, Bai X, Matthias N, et al. VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes. Biochem Biophys Res Commun (2007) 354:999–1003.[CrossRef][Web of Science][Medline]
[14] Fouts K, Fernandes B, Mal N, Liu J, Laurita KR. Electrophysiological consequence of skeletal myoblast transplantation in normal and infarcted canine myocardium. Heart Rhythm (2006) 3:452–61.[CrossRef][Web of Science][Medline]
[15] Thompson RB, Emani SM, Davis BH, van den Bos EJ, Morimoto Y, Craig D, et al. Comparison of intracardiac cell transplantation: autologous skeletal myoblasts versus bone marrow cells. Circulation (2003) 108:II264–II271.[Medline]
[16] Reinecke H, MacDonald GH, Hauschka SD, Murry CE. Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. J Cell Biol (2000) 149:731–40.
[17] Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, et al. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol (2004) 555:617–26.
[18] Potapova I, Plotnikov A, Lu Z, Danilo P Jr, Valiunas V, Qu J, et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res (2004) 94:952–9.
[19] XM, Bai J, Pan Z, Song YH, Freyberg S, Yan Y, Vykoukal D, et al. Electrophysiological properties of human adipose tissue-derived stem cells. Am J Physiol (2007) in press.
[20] Katritsis DG, Sotiropoulou P, Giazitzoglou E, Karvouni E, Papamichail M. Electrophysiological effects of intracoronary transplantation of autologous mesenchymal and endothelial progenitor cells. Europace (2007) 9:167–71.
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