Omentopexy enhances graft function in myocardial cell sheet transplantation.

Myocardial cell sheets (MCS) are a potentially valuable tool for tissue engineering aimed at heart regeneration. Several methods have recently been established for the fabrication of MCS. However, the lack of a sufficient blood supply has inhibited functional recovery of the MCS. To address this challenge, we combined MCS transplantation with omentopexy (OP), which utilizes omental tissue as a surgical flap. Rats were divided into five groups: sham, myocardial infarction (MI), MCS transplantation, OP, and MCS+OP. Histologic analysis revealed that MCS+OP drastically reversed MI-induced cardiac remodeling. Echocardiography revealed that MCS increased cardiac function, while OP had a synergistic beneficial effect with MCS transplantation. Immunofluorescence imaging showed that OP increased the survival of transplanted cardiomyocytes, and increased the blood supply through enhancement of angiogenesis and migration of small arteries into the MCS. Taken together, we concluded that OP is a promising strategy for the enhancement of graft function in MCS transplantation.

Prostaglandin E2 induces hypertrophic changes and suppresses alpha-skeletal actin gene expression in rat cardiomyocytes.

Prostaglandin E2 (PGE2) is a potent lipid mediator in a diverse range of biological processes. This study examined the hypertrophic effect of PGE2 in primary cultured rat neonatal cardiomyocytes. PGE2 increased total protein synthesis in a dose-dependent manner, as measured by [3H]-phenylalanine uptake. PGE2 increased the cell size and surface area and induced the reorganization of myofilaments. Phosphorylation of the p42/44 and p38 mitogen-activated protein kinases (MAPK) was also induced by PGE2, and U0126 [a mitogen-activated extracellular signal regulated kinase kinase (MEK) 1/2 inhibitor] significantly inhibited the PGE2-induced protein synthesis. Expression of the hypertrophic marker genes, atrial natriuretic peptide and brain natriuretic peptide, was increased by PGE2, but expression of the alpha-skeletal actin gene was significantly attenuated. Transcripts for all 4 PGE2 receptor subtypes (EP1, EP2, EP3, and EP4) were detected in the cardiomyocytes. AE3-208 (an EP4-selective antagonist) significantly inhibited the alpha-skeletal actin gene suppression induced by PGE2, whereas SC51322 (an EP1-selective antagonist) did not. In conclusion, PGE2 induced hypertrophic changes in cardiomyocytes and attenuated alpha-skeletal actin gene expression in part via EP4.

Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair.

The administration of granulocyte colony-stimulating factor (G-CSF) after myocardial infarction (MI) improves cardiac function and survival rates in mice. It was also reported recently that bone marrow (BM)-derived c-kit(+) cells or macrophages in the infarcted heart are associated with improvement of cardiac remodeling and function. These observations prompted us to examine whether BM-derived hematopoietic cells mobilized by G-CSF administration after MI play a beneficial role in the infarct region. A single hematopoietic stem cell from green fluorescent protein (GFP)-transgenic mice was used to reconstitute hematopoiesis in each experimental mouse. MI was then induced, and the mice received G-CSF for 10 days. In the acute phase, a number of GFP(+) cells showing the elongated morphology were found in the infarcted area. Most of these cells were positive for vimentin and alpha-smooth muscle actin but negative for CD45, indicating that they were myofibroblasts. The number of these cells was markedly enhanced by G-CSF administration, and the enhanced myofibroblast-rich repair was considered to lead to improvements of cardiac remodeling, function, and survival rate. Next, G-CSF-mobilized monocytes were harvested from the peripheral blood of GFP-transgenic mice and injected intravenously into the infarcted mice. Following this procedure, GFP(+) myofibroblasts were observed in the infarcted myocardium. These results indicate that cardiac myofibroblasts are hematopoietic in origin and could arise from monocytes/macrophages. MI leads to the recruitment of monocytes, which differentiate into myofibroblasts in the infarct region. Administration of G-CSF promotes this recruitment and enhances cardiac protection.

Dominant negative suppression of Rad leads to QT prolongation and causes ventricular arrhythmias via modulation of L-type Ca2+ channels in the heart.

Disorders of L-type Ca2+ channels can cause severe cardiac arrhythmias. A subclass of small GTP-binding proteins, the RGK family, regulates L-type Ca2+ current (I(Ca,L)) in heterologous expression systems. Among these proteins, Rad (Ras associated with diabetes) is highly expressed in the heart, although its role in the heart remains unknown. Here we show that overexpression of dominant negative mutant Rad (S105N) led to an increase in I(Ca,L) and action potential prolongation via upregulation of L-type Ca2+ channel expression in the plasma membrane of guinea pig ventricular cardiomyocytes. To verify the in vivo physiological role of Rad in the heart, a mouse model of cardiac-specific Rad suppression was created by overexpressing S105N Rad, using the alpha-myosin heavy chain promoter. Microelectrode studies revealed that action potential duration was significantly prolonged with visible identification of a small plateau phase in S105N Rad transgenic mice, when compared with wild-type littermate mice. Telemetric electrocardiograms on unrestrained mice revealed that S105N Rad transgenic mice had significant QT prolongation and diverse arrhythmias such as sinus node dysfunction, atrioventricular block, and ventricular extrasystoles, whereas no arrhythmias were observed in wild-type mice. Furthermore, administration of epinephrine induced frequent ventricular extrasystoles and ventricular tachycardia in S105N Rad transgenic mice. This study provides novel evidence that the suppression of Rad activity in the heart can induce ventricular tachycardia, suggesting that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

A new method for manufacturing cardiac cell sheets using fibrin-coated dishes and its electrophysiological studies by optical mapping.

We developed a novel simple method for making functional myocardial cell sheets that may be used as transplants. Polymerized human fibrin-coated dishes were prepared with fibrinogen monomers mixed with thrombin. Neonatal rat cardiomyocytes cultured on these dishes formed myocardial cell sheets within 4 days. These cell sheets were easily dissociated intact from the polymerized fibrin layer, because the fibrin had been digested by intrinsic protease. Two overlaid myocardial cell sheets exhibited synchronized spontaneous beating and captured artificial pacing. Optical mapping confirmed that the conduction of the action potential between two partially overlaid myocardial cell sheets was established, and the action potential propagated across the junction without any delay. Transplanted three-layered myocardial cell sheets exhibited strong spontaneous beating and showed well-differentiated striations and an increase in cell size. This simple method of cell sheet engineering may also be applicable for various other cell types.

Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice.

OBJECTIVE: We have previously isolated cardiomyogenic cells from murine bone marrow (CMG cells). Regenerated cardiomyocytes are important candidates for cell transplantation, but as they are stem cell derived, they can be contaminated with various cell types, thereby requiring characterization and purification. Our objectives were to increase the efficiency of cell transplantation and to protect the recipients from possible adverse effects using an efficient and effective purification process as well as to characterize regenerated cardiomyocytes. METHODS: Noncardiomyocytes were eliminated from a mixture of stem-cell-derived cells using a fluorescence-activated cell sorter to specifically isolate CMG cells transfected with a recombinant plasmid containing enhanced green fluorescent protein (EGFP) cDNA under the control of the myosin light chain-2v (MLC-2v) promoter. Gene expression and the action potential were investigated, and purified cells were transplanted into the heart of adult mice. RESULTS: Six percent to 24% of transfected CMG cells expressed EGFP after differentiation was induced, and a strong EGFP-positive fraction was selected. All the sorted cells began spontaneous beating after 3 weeks. These cells expressed cardiomyocyte-specific genes such as alpha-skeletal actin, beta-myosin heavy chain, MLC-2v, and CaV1.2 and incorporated bromodeoxyuridine for 5 days. The isolated EGFP-positive cells were expanded for 5 days and then transplanted into the left ventricle of adult mouse hearts. The transplanted cells survived for at least 3 months and were oriented in parallel to the cardiomyocytes of the recipient heart. CONCLUSIONS: The purification and transplantation of differentiated cardiomyocytes from adult stem cells provides a viable model of tissue engineering for the treatment of heart failure.

Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats.

Dividing cardiomyocytes are observed in autopsied human hearts following recent myocardial infarction, however there is a lack of information in the literature on the division of these cells. In this study we used a rat model to investigate how and when adult mammalian cardiomyocytes proliferate by cell division after myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. The rats were sacrificed periodically up to 28 days following induced myocardial infarction, and the hearts subjected to microscopic investigation. Cardiomyocytes entering the cell cycle were assayed by observation of nuclear morphology and measuring expression of Ki-67, a proliferating cell marker. Ki-67 positive cardiomyocytes and dividing nuclei were observed initially after 1 day. After 2 days dividing cells gradually increased in number at the ischemic border zone, reaching a peak increase of 1.12% after 3 days, then gradually decreasing in number. Dividing nuclei increased at the ischemic border zone after 3 days, peaked by 0.14% at day 5, and then decreased. In contrast, Ki-67 positive cells and dividing nuclei were limited in number in the non-ischemic area throughout all experiments. In conclusion, mitogenic cardiomyocytes are present in the adult rat heart following myocardial infarction, but were spatially and temporally restricted.

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.