Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells

Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells

Developmental changes in contractility and sarcomeric proteins from the early embryonic to the adult stage in the mouse heart

Developmental changes in force-generating capacity and Ca2+ sensitivity of contraction in murine hearts were correlated with changes in myosin heavy chain (MHC) and troponin (Tn) isoform expression, using Triton-skinned fibres. The maximum Ca2+-activated isometric force normalized to the cross-sectional area (FCSA) increased mainly during embryogenesis and continued to increase at a slower rate until adulthood. During prenatal development, FCSA increased about 5-fold from embryonic day (E)10.5 to E19.5, while the amount of MHC normalized to the amount of total protein remained constant (from E13.5 to E19.5). This suggests that the development of structural organization of the myofilaments during the embryonic and the fetal period may play an important role for the improvement of force generation. There was an overall decrease of 0.5 pCa units in the Ca2+ sensitivity of force generation from E13.5 to the adult, of which the main decrease (0.3 pCa units) occurred within a short time interval, between E19.5 and 7 days after birth (7 days pn). Densitometric analysis of SDS-PAGE and Western blots revealed that the major switches between troponin T (TnT) isoforms occur before E16.5, whereas the transition points of slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) and of β-MHC to α-MHC both occur around birth, in temporal correlation with the main decrease in Ca2+ sensitivity. To test whether the changes in Ca2+ sensitivity are solely based on Tn, the native Tn complex was replaced in fibres from E19.5 and adult hearts with fast skeletal Tn complex (fsTn) purified from rabbit skeletal muscle. The difference in pre-replacement values of pCa50 (−log([Ca2+]m−1)) required for half-maximum force development) between E19.5 (6.05 ± 0.01) and adult fibres (5.64 ± 0.04) was fully abolished after replacement with the exogenous skeletal Tn complex (pCa50= 6.12 ± 0.05 for both stages). This suggests that the major developmental changes in Ca2+ sensitivity of skinned murine myocardium originate primarily from the switch of ssTnI to cTnI

Embryonic cardiomyocyte, but not autologous stem cell transplantation, restricts infarct expansion, enhances ventricular function, and improves long-term survival

Contains fulltext : 118371.pdf (publisher's version ) (Open Access)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 1-2x10(5) 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 (14.8+/-2.2% MI+eCM vs. 26.7+/-1.6% 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 33.4+/-2.2% and 22.3+/-1.2 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

Potential risks of bone marrow cell transplantation into infarcted hearts

Cellular replacement therapy has emerged as a novel strategy for the treatment of heart failure. The aim of our study was to determine the fate of injected mesenchymal stem cells (MSCs) and whole bone marrow (BM) cells in the infarcted heart. MSCs were purified from BM of transgenic mice and characterized using flow cytometry and in vitro differentiation assays. Myocardial infarctions were generated in mice and different cell populations including transgenic MSCs, unfractionated BM cells, or purified hematopoietic progenitors were injected. Encapsulated structures were found in the infarcted areas of a large fraction of hearts after injecting MSCs (22 of 43, 51.2%) and unfractionated BM cells (6 of 46, 13.0%). These formations contained calcifications and/or ossifications. In contrast, no pathological abnormalities were found after injection of purified hematopoietic progenitors (0 of 5, 0.0%), fibroblasts (0 of 5, 0.0%), vehicle only (0 of 30, 0.0%), or cytokine-induced mobilization of BM cells (0 of 35, 0.0%). We conclude that the developmental fate of BM-derived cells is not restricted by the surrounding tissue after myocardial infarction and that the MSC fraction underlies the extended bone formation in the infarcted myocardium. These findings seriously question the biologic basis and clinical safety of using whole BM and in particular MSCs to treat nonhematopoietic disorders

Activation of Endocannabinoid System Is Associated with Persistent Inflammation in Human Aortic Aneurysm

Human aortic aneurysms have been associated with inflammation and vascular remodeling. Since the endocannabinoid system modulates inflammation and tissue remodeling, we investigated its components in human aortic aneurysms. We obtained anterior aortic wall samples from patients undergoing elective surgery for aortic aneurysm or coronary artery disease as controls. Histological and molecular analysis (RT-qPCR) was performed, and endocannabinoid concentration was determined using LC-MRM. Patient characteristics were comparable between the groups except for a higher incidence of arterial hypertension and diabetes in the control group. mRNA level of cannabinoid receptors was significantly higher in aneurysms than in controls. Concentration of the endocannabinoid 2-arachidonoylglycerol was significantly higher, while the second endocannabinoid anandamide and its metabolite arachidonic acid and palmitoylethanolamide were significantly lower in aneurysms. Histology revealed persistent infiltration of newly recruited leukocytes and significantly higher mononuclear cell density in adventitia of the aneurysms. Proinflammatory environment in aneurysms was shown by significant upregulation of M-CSF and PPARγ but associated with downregulation of chemokines. We found comparable collagen-stained area between the groups, significantly decreased mRNA level of CTGF, osteopontin-1, and MMP-2, and increased TIMP-4 expression in aneurysms. Our data provides evidence for endocannabinoid system activation in human aortic aneurysms, associated with persistent low-level inflammation and vascular remodeling