myocardial infarction treated with cardiac stem cells

Improvements of cardiac electrophysiologic stability and ventricular fibrillation threshold in rats with myocardial infarction treated with cardiac stem cells *Zheng, Shaoxin MD; Zhou, Changqing MD; Weng, Yinlun MD; Huang, Hui MD; Wu, Hao MD; Huang, Jing MD; Wu, Wei MD; Sun, Shijie MD; Wang, Jingfeng MD; Tang, Wanchun MD; Wang, Tong MDAuthor InformationFrom the Sun Yat-sen Memorial Hospital of Sun Yat-sen University, (SZ, CZ, YW, HH, WW, JW, WT, TW), Guangzhou, China; Weil Institute of Critical Care Medicine (YW, SS, WT), Rancho Mirage, CA; and the Second Affiliated Hospital of Guangzhou Medical College (HW, JH), Guangzhou, China.Supported, in part, by the National Natural Science Foundation of China (81070125, 30700304, and 30973207) and the Natural Science Foundation of Guangdong Province (8151008901000119).The authors have not disclosed any potential conflicts of interest.For information regarding this article, E-mail: tongwang163@Back to TopAbstractObjectives: Arrhythmia is of concern after cardiac stem celltransplantation in repairing infarcted myocardium. However,whether transplantation improved the ventricular fibrillationthreshold and whether severe malignant ventricular arrhythmiais induced in the myocardial infarction model are still unclear.We sought to investigate the electrophysiologic characteristicsand ventricular fibrillation threshold in rats with myocardialinfarction by treatment with allogeneic cardiac stem cells.Design: Prospective, randomized, controlled study.Setting: University-affiliated hospital.Subjects: Male Sprague-Dawley rats.Interventions: Myocardial infarction was induced in 20 maleSprague-Dawley rats. Two weeks later, animals wererandomized to receive 5 × 106 cardiac stem cells labeled withPKH26 in phosphate buffer solution or a phosphate buffersolution-alone injection into the infarcted anteriorventricular-free wall.Measurements and Main Results: Six weeks after the cardiac stem cell or phosphate buffer solution injection, electrophysiologic characteristics and ventricular fibrillation threshold were measured at the infarct area, infarct marginal zone, and noninfarct zone. Labeled cardiac stem cells were observed in 5-µm cryostat sections from each harvested heart. The unipolar electrogram activation recovery time dispersions were shorter in the cardiac stem cell group compared with those at the phosphate buffer solution group (15.5 ± 4.4 vs. 38.6 ± 14.9 msecs, p = .000177). Malignant ventricular arrhythmias were significantly (p = .00108) less inducible in the cardiac stem cell group (one of ten) than the phosphate buffer solution group (nine of ten). The ventricular fibrillation thresholds were greatly improved in the cardiac stem cell group compared with the phosphate buffer solution group. Labeled cardiac stem cells were identified in the infarct zone and infarct marginal zone and expressed Connexin-43, von Willebrand factor,[alpha]-smooth muscle actin, and [alpha]-sarcomeric actin.Conclusions: Cardiac stem cells may modulate the electrophysiologic abnormality and improve the ventricular fibrillation threshold in rats with myocardial infarction treated with allogeneic cardiac stem cells and cardiac stem cell express markers that suggest muscle, endothelium, and vascular smooth muscle phenotypes in vivo.In recent years, cell therapy has emerged as a strategy for improving contractility of the diseased heart (1, 2). Restorative therapies consider the use of exogenous multipotent stem cells capable of differentiating into cardiomyocytes. These include embryonic and bone marrow-derived stem cells (3, 4) as well as intrinsic cardiac stem cells (CSCs) that reside in the heart (5–7)and are programmed to reconstitute cardiac tissue (8). In vitro, CSCs can differentiate into cardiomyocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts (9), while in vivo, CSCs can differentiate into cardiomyocytes (10), and several animal studies have shown promising results on improvement of left ventricular function (11–13). Nevertheless, clinical application of stem cells remains controversial (14, 15); therefore, additional preclinical investigations to further enhance the beneficial effects of stem cell therapy are advocated (16).As with other stem cell therapies, safety and proarrhythmia concerns have arisen in relation to the potential risks of cardiac stem cell therapy (17, 18) and more specifically after intramyocardial administration (19). This is related to the formation of distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone. Until now, no study has focused on cardiac electrophysiologic characteristics and ventricular fibrillation threshold (VFT) after CSC transplantation. Safety and significant improvement in VFT are important aspects for CSCs treatment in ischemic heart disease and dysfunction. In the present study, we investigated safety, proarrhythmic effects and effects on VFT of allogeneic CSCs used to treat myocardial infarction induced by left anterior descending coronary artery ligation in rats. Our hypothesis was that cardiac electrophysiologic stability and VFT would be improved significantly in a rat model of myocardial infarction treated with allogeneic CSCs.Back to TopMETHODSAdult male Sprague-Dawley rats weighing 350–450 g were obtained from the animal experimental center of our SunYat-Sen University and were housed in a standard animal facility with 12-hr on/off light conditions. All animals were acclimatized for at least 1 wk before surgery and allowedstandard food and water ad libitum. All procedures were in compliance with the guidelines for the care and use of laboratory animals and the ethical review process of our institution and were approved by the institutional animal care committee.Back to TopIsolation and Culture of Cardiac Stem Cells.The 3-day-old Sprague-Dawley rats were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg) and then dipped into 75% ethanol for 30 seconds for sterilization. Under sterile conditions, the hearts were excised, put into the aseptic culture dish, and minced into 1-mm3 pieces. The pieces were then washed twice with phosphate buffer solution (PBS) to remove impurities. Then, 2–3 mL of 0.2% trypsin was added for 5 min of digestion, followed by 2–3 mL of 0.1% collagenase II for 5 min for additional digestion, and subsequent elimination of the digestive liquid. The procedure was then repeated for an additional three times. The pieces were then washed twice with complete explant medium (supplemented with Iscove's modified Dulbecco's medium, 10% fetal calf serum, 100units/mL penicillin G, 100 µg/mL streptomycin, 2 mmol/LL-glutamine, and 0.1 mmol/L 2-mercaptoethanol), then transferred and dispatched with pipette into a 25-cm2 flask, and incubated with 1 mL of complete explant medium at37°C-humidified atmosphere with 5% CO2 for 12 hrs. An additional complete explant medium (3–4 mL) was then added. At 90% confluence, the cells were trypsinized (0.25%trypsin-ethylenediaminetriacetic acid, catalog number25–053-CI, Mediatech, Herndon, VA) and were passed into25-cm2 flasks at 1:2 ratios. Third-passage CSCs were used in all experiments.Cultured CSCs were analyzed by fluorescence-activated cell sorting (FACScan flow cytometer, Becton Dickinson, Sparks, MD) as reported previously (7, 11).To monitor cell distribution in the heart, on the day of implantation, suspended CSCs were labeled with fluorescent dyes with the use of a PKH26 red fluorescent cell linker kit (Sigma Aldrich, St. Louis, MO), as reported previously (20).Back to TopRat Myocardial Infarction Model Preparation.Twenty male Sprague-Dawley rats weighing 350–450 g were fasted overnight except for free access to water. The animals were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg). Additional doses (10 mg/kg) were administered at intervals of approximately 1 hr, or as required to maintain anesthesia. The trachea was orally intubated with a 14-g cannula mounted on a blunt needle with a 145° angled tip (Abbocath-T, Abbott Critical Care Systems, North Chicago, IL) as previously described (20). The animals were mechanically ventilated with room air at a tidal volume of 0.65 mL/100 g of body weight and a frequency of 100 breaths/min. The electrocardiogram lead II was continuously monitored. A thoracotomy was performed via the left fourth intercostal space. The pericardium was incised, and the left atrial appendage was elevated to expose the left anterior descending coronary artery. The left anterior descending coronary artery was ligated by using a 5/0 nylon suture. The chest was closed with a soft tube in the cavity to withdraw air or blood. Successful occlusion was confirmed electrocardiographically by the elevation of the ST segment (20, 21). Animals were allowed to recover from anesthesia and then were returned into their cages for 2 wks. Postoperation pain was controlled with intramuscular injections of ketorolac (0.4 mg/kg).Back to TopExperimental Procedures.Two weeks after surgical intervention, the animals were anesthetized and orally intubated as previously described (20, 21). The animals were mechanically ventilated with room air, and a new thoracotomy was performed as described above. Theanimals were randomized to be subjected to injection of either 5 × 106/0.1 mL of CSCs labeled with PKH26 in PBS or PBS alone as a placebo into the infarcted anterior ventricular-free wall. Successful injection was typified by the formation of a bleb covering the infarct zone. The animals were allowed to recover from anesthesia and returned to their cages for another 6 wks. Postoperative pain was controlled as described above.Six weeks after CSCs or PBS injection, the animals were reanesthetized. Additional doses of pentobarbital were administered at hourly intervals but not within 30 min preceding the start of measurements. The trachea was orally intubated as previously described. A thoracotomy was performed via the left fifth intercostal space, and the heart was exposed. Three precurved guide wires were then attached to the infarct area, infarct marginal zone, and noninfarct zone. The electrocardiogram lead II was continuously recorded. A heat lamp was used to maintain body temperature at 36.8°C ± 0.2°C throughout the experiment as we described previously (22).Back to TopMeasurementsAll electrophysiologic testing in the current study was performed by a blinded investigator who did not know to which group the rat belonged.Back to TopEffective Refractory Period.The ventricular effective refractory period of the infarct marginal zone and the noninfarct zone were assessed by the programmed electrical stimulation technique (DF-6A, Jiangsu, China). The pacing protocol consisted of S1S2 and S1S2S3 (S1S2: Eight beats of basal stimuli in a cycle length of 120 msecs and up to one extra stimuli; S1S2S3: Eight beats of basal stimuli in a cycle length of 120 msecs and up to two extra stimuli). Starting in late diastole, the last extrastimulus-coupling interval wasshortened in 10-msec decrements until refractoriness occurred. Electrocardiograms were recorded with a frequency range from 0.05 to 500 Hz by using a multichannel electrocardiogram amplifier and stored in a computer (Biopac Systems, Goleta, CA).Back to TopUnipolar Electrograms.The unipolar epicardial electrograms were obtained from electrodes placed on the ventricular epicardium (the infarct area, the infarct marginal zone, and the noninfarct zone) and standard bipolar limb leads and recorded simultaneously. Electrocardiograms were recorded with the application of subcutaneous needle electrodes. The signals were isolated, amplified, multiplexed, and recorded by a computerized recording system (Biopac Systems, Goleta, CA) with a frequency range from 0.05 Hz to 500 Hz. Activation time, repolarization time, and activation recovery time (ART), as a marker of local repolarization duration, were determined in each epicardial lead as the minimum of the potential time derivative during the QRS, maximum of the potential time derivative during ST-T, and the period between the two, respectively. The ART dispersion (ARTd) was defined as the difference between the maximal and minimal ART durations in the infarct area, the infarct marginal zone, and the noninfarct zone. The correct ART (ARTc) were calculated by using Bazett's formula (ARTc =ART/square root from RR). The ARTc dispersion (ARTcd) was the difference between the maximal and minimal ARTc durations (23).Back to TopMalignant Ventricular Arrhythmias Induction.The inducibility of the malignant ventricular arrhythmias, including ventricular tachycardia and ventricular fibrillation (VF), was calculated as a ratio between the number of induced arrhythmias and the number of attempts for arrhythmia induction. The programmed electrical stimulation includedS1S2, S1S2S3, and the burst pacing (performed at cycle lengths of 100/90/80/70/60/50 msecs for 1 sec) on the infarct area, the infarct marginal zone, and the noninfarct zone. The end point was the induction of ventricular tachycardia or VF or completion of the protocol.Back to TopVFT.VFTs of the infarct area, the infarct marginal zone, and the noninfarct zone were determined by a homemade stimulator, using a previously reported method (24). Briefly, the hearts were stimulated with rectangular pulses at a frequency of 30 Hz, an impulse length of 10 msecs, and a stimulation duration of 500 msecs. Current intensity was increased in increments of 1 mA until VF was attained. Two minutes after stabilization, another stimulus was given with the same current intensity. VFT was determined as the lowest current intensity at which two consecutive stimuli precipitated VF.Back to TopImmunohistochemistry.The animals were euthanized, and the hearts were harvested. Five-micrometer sections (HM 500 OM, Micron, Walldorf, Germany) were obtained from each heart and fixed in acetone overnight at 4°C, blocked with 10% goat serum in PBS, and used for immunohistochemistry with the primary antibody, Connexin-43 (Santa Cruz Biotech, Santa Cruz, CA), von Willebrand factor (Santa Cruz Biotech, Santa Cruz, CA), [alpha]-smooth muscle actin (Abcam, San Francisco, CA), and [alpha]-sarcomeric actin. The primary antigen-antibody reaction was detected with goat anti-rabbit IgG conjugated with Alexa Fluor-488 (Invitrogen, Carlsbad, CA) forConnexin-43, von Willebrand factor, and [alpha]-smooth muscle actin and goat anti-mouse IgM conjugated with fluorescein isothiocyanate (Santa Cruz Biotech, Santa Cruz, CA) for [alpha]-sarcomeric actin. Nuclei were counterstained with4',6-diamidino-2-phenylindole. The samples were examined byusing a confocal laser scanning microscope (TCS SP2 AOBS,Leica, Mannheim, Germany).Back to TopStatistical Analysis.All quantitative data are described as mean ± sd. Basiccomparative statistics were performed by using Student'stwo-tailed t test. Differences in the frequency of inducibility ofventricular arrhythmias were tested by Fisher's exact test. Avalue of p < .05 was considered to be statistically significant.Back to TopRESULTSHigh efficiency of staining (>90%) was observed after PKH26staining, as shown in Figure 1. Figure 1No differences in ventricular effective refractory period(programmed electrical stimulation S1S2) were observedbetween the CSC group and PBS group on the infarct marginalzone (62.0 ± 6.3 msecs vs. 64.0 ± 7.0 msecs, p = .75) and thenoninfarct zone (65.0 ± 5.3 msecs vs. 63.0 ± 6.7 msecs, p= .14).No differences in ventricular effective refractory period(programmed electrical stimulation S1S2S3) were observedbetween the CSC group and PBS group on the infarct marginalzone (66.0 ± 5.2 msecs vs. 63.0 ± 6.7 msecs, p = .39) and thenoninfarct zone (64.0 ± 8.4 msecs vs. 66.0 ± 8.4 msecs, p= .75).There were also no significant differences in unipolarelectrogram ART on the infarct area between the CSC group and PBS group. However, the ART was significantly longer on the infarct marginal zone and the noninfarct zone in PBS group compared with that in CSC group (Table 1).Table 1 Table 2Coincidentally, unipolar electrogram ARTd were significantlygreater in the PBS group compared with that in CSC group(Table 1). After correction, the effects on the ARTc andARTcd were similar to those before correction (Table 2).Malignant ventricular arrhythmias were significantly (p= .00108) less inducible in the CSC group (one of ten) thanthe PBS group (nine of ten). VFTs were higher in the CSCgroup compared with those in the PBS group (Table 3).Table 3In the CSC group, CSCs were found to be surviving in theinfarct zone and the infarct marginal zone, expressing theConnexin-43 (Fig. 2), [alpha]-sarcomeric actin (Fig. 3), von Willebrand factor (Fig. 4), and [alpha]-smooth muscle actin (Fig. 5). CSCs differentiated into cardiomyocytes, endothelial cells, and vascular smooth muscle cells in the cardiac microenvironment of the infarct zone and the infarct marginal zone and connected with native myocardium byConnexin-43.Figure 2 Figure 3Figure 4 Figure 5Back to TopDISCUSSIONThis study demonstrated the antiarrhythmic effects of intramyocardial allogeneic CSC transplantation after myocardial infarction. Local injection of CSCs can significantly improve cardiac electrophysiologic stability and increase VFT in rats with myocardial infarction treated with CSCs.Despite major improvements in medical therapy, a significant proportion of patients with ischemic heart disease remain symptomatic (25). The disease processes lead to myocardial contractile dysfunction and heterogeneouselectrical instability that are associated with ventricular arrhythmogenesis (15). Malignant arrhythmias are, in fact, a major concern in patients with advanced heart disease. In recent years, cell therapy has emerged as a potential new strategy for patients with ischemic heart disease. However, the major safety issue raised by the use of stem cells for cardiac repair has been the occurrence of ventricular arrhythmias, including ventricular tachycardia and/or VF (17). The proarrhythmic condition following stem cell therapy might be attributed to one or more of the following: 1) intrinsic electrophysiologic properties of stem cells; 2) modulated graft-host or graft-graft electromechanical coupling (or both); 3) changes in ion channel function; 4) induced heterogeneity; 5) altered myocardial tissue architecture, and 6) local injury or edema induced by intramyocardial injection (18, 26). Accordingly, particularly after skeletal myoblast transplantation (17), even though bone marrow cells seem less arrhythmogenic than skeletal myoblasts, their implantation could produce local delays in propagation because of the interposition of electrically coupled but unexcitable cells with normal host cardiomyocytes (18). On the other hand, embryonic stem cells showed spontaneous activity, low dV/dt, prolonged action potential duration, and easily inducible triggered arrhythmias, which may act as an unanticipated arrhythmogenic source from any of the three classic mechanisms (reentry, automaticity, or triggered activity) (27).A functional integration of the grafted cells such that they can safely and efficiently contribute to increased pump function requires that their phenotype supports electrical propagation, electromechanical coupling, and contraction (18). There is compelling evidence that cells recapitulating the cardiogenic differentiation program are the best suited for achieving this goal. CSCs, therefore, represent a logical source to be investigated in cardiac regeneration therapybecause, unlike other adult stem cells, they are intrinsically programmed to generate cardiac tissue and to couple with host cardiomyocytes (16). In support of the above discussion, our study showed that CSC injection increased the inducibility threshold of malignant ventricular arrhythmias and significantly improved cardiac electrophysiologic stability. These CSCs have also the advantage of being able to be isolated and expanded in vitro from small samples of myocardium (6). Not only can the CSCs be transplanted through intravascular injection (10, 12), which is the clear prerequisite for clinical translation, but they can also contribute to improving the hemodynamics in rats with myocardium infarction (10, 12, 28).Increased heterogeneity of ventricular repolarization facilitates and provides a substrate for reentry and the development of ventricular arrhythmias (29, 30). The ART from unipolar electrograms is a valuable evaluation of global sequence and dispersion of ventricular repolarization (23). In our study, ART, ARTd, ARTc, and ARTcd were significantly shorter on the infarct marginal zone and the noninfarct zone in the CSC group compared with that in PBS group. Furthermore, there were similar effects and tendency on the infarct zone, although there were no statistical significances. The results suggest that CSCs transplanted in the infarct zone may replace the fibroblasts and therefore decrease the electrical heterogeneity between the infarct zone and the noninfarct zone. In addition, VFTs on the infarct zone, the infarct marginal zone, and the noninfarct zone were improved significantly in the CSC-treated group compared with those in the PBS-treated group. The results of the VFT measurement were consistent with the measurement of unipolar electrograms and the ventricular arrhythmias induced by PBS.After myocardial infarction occurs, there are several changesin the infarct zone and the infarct marginal zone: 1) the number of myocardiocytes decreases due to necrosis and apoptosis; 2) inflammatory cells infiltrate combined with inflammatory factor exudation, and 3) myocardial fibrosis following myocardial infarction (31, 32). These changes lead to ventricular heterogeneity, which plays an important role on cardiac electrophysiologic instability and ventricular arrhythmias. The mechanisms of the significant improvement in cardiac electrophysiologic stability and VFT after CSC transplantation are still unclear. The following several mechanisms may be considered. First of all, the CSCs, which reside in the heart itself, can differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells in vitro and in vivo (6, 7, 16) and reconstitute cardiac tissue and electromechanical coupled with host cardiomyocytes. In the current study, CSCs expressed the markers, including muscle, endothelium, and vascular smooth muscle in the infarct zone and infarct marginal zone, which means that CSCs may differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells in the cardiac microenvironment, rather than phagocytosed by the inflammatory cells. de Boer et al (33) proved CSC-derived cardiomyocytes had a homogeneous and rather mature electrical phenotype: Stable resting membrane potential and near mature I Na, I ca-L, and I K1 current densities. Therefore, electrical heterogeneity and conduction disturbances could be attenuated by effective cardiac regeneration strategies, such as grafting of CSCs with suitable electrophysiologic properties. Connexin-43 plays an important role in integrating the electrical characteristics and engrafting for the transplanted cells (33, 34). Rat models have shown that loss of Connexin-43 expression greatly increases the risk of ventricular tachycardia and sudden death (35). Another study showed that in an infarcted rat model, myoblast transplantation induces electrical ventricular instability (36). The in vitro study demonstrated that coculture of myoblasts and cardiomyocytes resulted in reentrant arrhythmias and that such arrhythmias could belimited by overexpression of Connexin-43 (37). Connexin-43 also contributes to mesenchymal stem cell survival and improves therapeutic efficacy in myocardial infarction (34). Recent study demonstrated that mesenchymal stem cells (MSCs) implanted into the rats with myocardial infarction reduce vulnerability to ventricular arrhythmias; and abnormal alterations of Connexin-43, including reduction and lateralization in the infarct marginal zone and noninfarct zone were significantly attenuated by MSC treatment (38). All of the above studies demonstrated that Connexin-43 plays an important role in integrating the electrical characteristics and engrafting for cell transplantation. The CSC-derived cardiomyocytes had a homogeneous and rather mature electrical phenotype, and Connexin-43 was translocated to the cell border (33). Our study showed that the CSCs survived in the infarct zone and the infarct marginal zone, and expressed the Connexin-43 coupling to each other and the neighboring cardiac myocytes. CSC transplantation improved the electrical heterogeneity, and the reentry in the infarct zone, the marginal infarct zone, and noninfarct zone would be blocked. The results of the Connexin-43 measurement were consistent with the measurement of unipolar electrograms, the ventricular arrhythmias induction, and the VFT. Another antiarrhythmic effect of CSCs therapy stems may be related to its potential ability to lessen ischemia, limit infarct size, and favorably affect left ventricular remodeling, through neovascularization and paracrine mechanisms, thereby controlling endogenous arrhythmic substrates.PKH26-marked CSCs can express [alpha]-sarcomeric actin, von Willebrand factor, and [alpha]-smooth muscle actin in the CSC group means CSCs can differentiate into cardiomyocytes, endothelial cells, and vascular smooth muscle cells, rather than CSCs, which were phagocytosed by the inflammatory cells. Neovascularization in the infarct area and the infarct marginal zone after CSC transplantationplays an important role in CSCs and CSC-derived cardiomyocytes surviving in the infarct area and the infarct marginal zone. Furthermore, the surviving CSC-derived cardiomyocytes lead to reduction in scar area (28), reduction of electrical heterogeneity, and improvement of myocardial contractility (28).Back to TopCONCLUSIONSCSCs can modulate the electrophysiologic abnormalities and increase VFT in rats with myocardial infarction treated with allogeneic CSCs, and CSCs express markers that suggest muscle, endothelium, and vascular smooth muscle phenotypes in vivo.Back to TopREFERENCES1. Herrmann JL, Abarbanell AM, Weil BR, et al: Cell-based therapy for ischemic heart disease: A clinical update. Ann Thorac Surg 2009; 88:1714–1722 Bibliographic Links Library Holdings[Context Link]2. LaPar DJ, Kron IL, Yang Z: Stem cell therapy for ischemic heart disease: Where are we? Curr Opin Organ Transplant 2009; 14:79–84 [Context Link]3. Menasché P: [Cell therapy: results in cardiology]. Bull Acad Natl Med 2009; 193:559–568; discussion 568–569 [Context Link]4. 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