Embryonic Cardiomyocyte, but Not Autologous Stem Cell Transplantation, Restricts Infarct Expansion, Enhances Ventricular Function, and Improves Long-Term Survival

and Improves Long-Term Survival. PLoS ONE 8(4): e61510. doi:10.1371/journal.pone.0061510 Embryonic Cardiomyocyte, but Not Autologous Stem Cell Transplantation, Restricts Infarct Expansion, Enhances Ventricular Function, and Improves Long-Term Survival Leonie E. Paulis 0 Alexandra M. Klein 0 Alexander Ghanem 0 Tessa Geelen 0 Bram F. Coolen 0 Martin Breitbach 0 Katrin Zimmermann 0 Klaas Nicolay 0 Bernd K. Fleischmann 0 Wilhelm Roell 0 Gustav J. Strijkers 0 Patrick C.H. Hsieh, Institute of Clinical Medicine, National Cheng Kung University, Taiwan 0 1 Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven , The Netherlands , 2 Institute of Physiology I, Life and Brain Centre, University of Bonn , Bonn, Germany , 3 Department of Medicine/Cardiology, University of Bonn , Bonn, Germany , 4 Department of Pharmacology and Toxicology, Biomedical Center, University of Bonn , Bonn, Germany , 5 Department of Cardiac Surgery, University of Bonn , Bonn , Germany Aims: Controversy exists in regard to the beneficial effects of transplanting cardiac or somatic progenitor cells upon myocardial injury. We have therefore investigated the functional short- and long-term consequences after intramyocardial transplantation of these cell types in a murine lesion model. Methods and Results: Myocardial infarction (MI) was induced in mice (n = 75), followed by the intramyocardial injection of 1226105 luciferase- and GFP-expressing embryonic cardiomyocytes (eCMs), skeletal myoblasts (SMs), mesenchymal stem cells (MSCs) or medium into the infarct. Non-treated healthy mice (n = 6) served as controls. Bioluminescence and fluorescence imaging confirmed the engraftment and survival of the cells up to seven weeks postoperatively. After two weeks MRI was performed, which showed that infarct volume was significantly decreased by eCMs only (14.862.2% MI+eCM vs. 26.761.6% MI). Left ventricular dilation was significantly decreased by transplantation of any cell type, but most efficiently by eCMs. Moreover, eCM treatment increased the ejection fraction and cardiac output significantly to 33.462.2% and 22.361.2 ml/min. In addition, this cell type exclusively and significantly increased the end-systolic wall thickness in the infarct center and borders and raised the wall thickening in the infarct borders. Repetitive echocardiography examinations at later time points confirmed that these beneficial effects were accompanied by better survival rates. Conclusion: Cellular cardiomyoplasty employing contractile and electrically coupling embryonic cardiomyocytes (eCMs) into ischemic myocardium provoked significantly smaller infarcts with less adverse remodeling and improved cardiac function and long-term survival compared to transplantation of somatic cells (SMs and MSCs), thereby proving that a cardiomyocyte phenotype is important to restore myocardial function. - Funding: This work was supported by the Dutch Technology Foundation STW, applied science division of NWO and the Technology Program of the Ministry of Economic Affairs (grant number 07952) (Dr. Gustav J. Strijkers), EU FP7 consortium grant CardioCell (grant No223372) (Dr. Bernd K. Fleischmann), and EC-FP6project DiMI (grant number LSHB-CT-2005-512146). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Myocardial infarction is characterized by extensive necrosis of cardiomyocytes, which causes a non-reversible loss of rhythmic contractile abilities [1]. The low proliferative capacity of terminally differentiated cardiomyocytes has led to the exploration of stem cell-based therapies to regenerate myocardium and reduce the occurrence of heart failure [2]. Intramyocardial injection of embryonic cardiomyocytes (eCMs) in mice has previously been shown to result in electrical coupling to native myocardium, improved ventricular function and reduction of ventricular arrhythmias [36]. The combination of these key functional characteristics of eCMs is unique compared to already clinically applied somatic cells such as skeletal myoblasts (SMs) and mesenchymal stem cells (MSCs). For these cell types neither electromechanical integration into the heart nor transdifferentiation into cardiomyocytes has been unequivocally proven [7,8]. Therefore, beneficial effects seen in some animal studies and human trials after cardiac SM or MSC transplantation [9,10] have been preferentially attributed to passive mechanisms such as the preservation of cardiac compliance or paracrine effects [11]. Nevertheless, this area is still controversial and the best-suited cell type has not been identified so far. Therefore, the goal of this study was to investigate in detail global and regional cardiac contractile function following cellular cardiomyoplasty. In particular we aimed to address, if observed changes were dependent on the ability of the cells to contract and couple electrically to native myocardium. Therefore, contractile and electrically coupling eCMs were compared to, on the one hand, contractile but not electrically coupling SMs and, on the other hand, neither contractile nor electrically coupling MSCs. The cells were transplanted into infarcted syngeneic hearts of two different mouse strains (CD1 and C57BL/6). To evaluate myocardial function, we applied a unique set of imaging and analysis tools to investigate local and global cardiac contractile function and geometry, cell survival, and long-term outcome. High spatial- and temporal-resolution in vivo magnetic resonance imaging (MRI) was performed to characterize global cardiac function [12]. Moreover, to investigate active enhancement of local contractility by the transplanted cells throughout the cardiac cycle, data were evaluated by use of the AHA 16-segment model, where for each segment 4550 radial chords were analyzed. Furthermore, using late gadolinium enhancement (LGE) MRI, infarcted and remote myocardium could be discriminated clearly [13]. Additionally, survival of transplanted cells, cardiac morphology and function were monitored up to 7 weeks by bioluminescence imaging (BLI) and three-dimensional echocardiography. All animal experiments were performed in accordance with the declaration of Helsinki and were approved by the local ethical committees for animal experiments of Nordrhein-Westfalen (LANUV) and the University of Maastricht. Cell isolation and culture eCMs and SMs were obtained from transgenic CD-1 and C57BL/6 mouse embryos, expressing green fluorescent protein (GFP) under the a-actin promoter as described [4,14,15]. eCMs were isolated from embryonic hearts at E13.5, whereas for SM isolation the diaphragm was used at E18.5. Transgenic MSCs were aspirated from the femur and tibia of adult CD-1 and C57BL/6 mice, with GFP expression under control of t (...truncated)