Involvement of ubiquitin-proteasome system in icariin-induced cardiomyocyte differentiation of embryonic stem cells using two-dimensional gel electrophoresis

Icariin has been shown to significantly facilitate the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. However, the mechanism underlying the icariin-induced cardiomyocyte differentiation is still not fully understood. In the present study, 52 differentially displayed proteins selected from two-dimensional electrophoresis gels were identified by MALDI-TOF mass spectrometry analysis. More than half of proteins could be assigned to six main categories: (1) protein synthesis, metabolism, processing and degradation, (2) stress response, (3) cytoskeleton proteins, (4) energy metabolism, (5) carbohydrate metabolism/transport, and (6) RNA/other nucleic acids metabolisms and transport, nuclear proteins. MALDI-TOF/MS showed that icariin treatment resulted in the induction of five ubiquitin-proteasome system (UPS)-related proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), ubiquitin-conjugating enzyme E2N, proteasome 26S, proteasome subunit-alpha type 6, and proteasome subunit-alpha type 2 in the differentiated cardiomyocytes. These results implied that UPS might play an important role in the control of cardiomyocyte differentiation. Epoxomicin (a proteasome inhibitor) significantly reduced the cardiomyocyte differentiation rate of ES cells and proteasome activities, as well as inhibited NF-κB translocation into the nucleus, which were evidently reversed by presence of icariin. Meanwhile, icariin could significantly reverse the reduction of four proteins (proteasome subunit-alpha type 6, proteasome subunit-alpha type 2, UCH-L1, and ubiquitin-conjugating enzyme E2N) expressions owing to application of epoxomicin. These suggest UPS could be a means by which icariin may regulate expressions of key proteins that control cardiomyocyte differentiation. Taken together, these results indicated that UPS played an important role in ES cell differentiate into cardiomyocytes induced by icariin.     

Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells

Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus.     

Involvement of androgen receptor in nitric oxide production induced by icariin in human umbilical vein endothelial cells

Icariin, a flavonoid isolated from Epimedii herba, stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser1177, Akt (Ser473) and ERK1/2 (Thr202/Tyr204). The icariin-induced eNOS phosphorylation was abolished by an androgen receptor (AR) antagonist, nilutamide in human umbilical vein endothelial cells (HUVECs). Furthermore, it was also reduced in the cells transfected with small interfering RNA in which the expression of AR was broken down. The icariin-induced eNOS phosphorylation was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. These data suggest that icariin stimulates release of NO by AR-dependent activation of eNOS in HUVECs. PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells

Background  

We reported previously that the cardiac troponin I (cTnI) promoter drives cardiac-specific expression of reporter genes in cardiac muscle cells and in transgenic mice, and that disruption of GATA elements inactivates the cTnI promoter in cultured cardiomyocytes. We have now examined the role of cTnI promoter GATA elements in skeletal muscle cells.     

Fabrication of Mouse Embryonic Stem Cell-Derived Layered Cardiac Cell Sheets Using a Bioreactor Culture System

Bioengineered functional cardiac tissue is expected to contribute to the repair of injured heart tissue. We previously developed cardiac cell sheets using mouse embryonic stem (mES) cell-derived cardiomyocytes, a system to generate an appropriate number of cardiomyocytes derived from ES cells and the underlying mechanisms remain elusive. In the present study, we established a cultivation system with suitable conditions for expansion and cardiac differentiation of mES cells by embryoid body formation using a three-dimensional bioreactor. Daily conventional medium exchanges failed to prevent lactate accumulation and pH decreases in the medium, which led to insufficient cell expansion and cardiac differentiation. Conversely, a continuous perfusion system maintained the lactate concentration and pH stability as well as increased the cell number by up to 300-fold of the seeding cell number and promoted cardiac differentiation after 10 days of differentiation. After a further 8 days of cultivation together with a purification step, around 1×10⁸ cardiomyocytes were collected in a 1-L bioreactor culture, and additional treatment with noggin and granulocyte colony stimulating factor increased the number of cardiomyocytes to around 5.5×10⁸. Co-culture of mES cell-derived cardiomyocytes with an appropriate number of primary cultured fibroblasts on temperature-responsive culture dishes enabled the formation of cardiac cell sheets and created layered-dense cardiac tissue. These findings suggest that this bioreactor system with appropriate medium might be capable of preparing cardiomyocytes for cell sheet-based cardiac tissue.     

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.     

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.