Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells.

Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) (in vitro). We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-myosin heavy chain (alpha-MHC) promoter (palphaMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4.Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of alpha-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of alpha-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

Inactivation of neuregulin-1 by nitration

Nitration is a posttranslational modification that can compromise protein function. We hypothesized that nitration of growth factors secreted in the lung may alter their interaction with their respective receptors and modulate the normal growth and differentiation program induced by ligand-receptor interaction. We tested this hypothesis in vitro by nitration of neuregulin-1's (NRG-1) EGF-like domain and studying the effect on NRG-1's activity. Nitration of NRG-1's (nNRG-1) EGF-like domain resulted in an inability to activate its receptor, the human epidermal growth factor receptors 2 and 3 (HER2/HER3) heterodimer, as defined by loss of HER2 tyrosine phosphorylation induced by nNRG-1 in MCF-7 cells. Receptor activation was not restored with increasing nNRG-1 concentration or exposure times. nNRG-1 did not compete with NRG-1 for HER2/HER3 binding in competition assays. In addition, nNRG-1 no longer induced proliferation of the MCF-7 cell line, as MCF-7 cells exposed to nNRG-1 and NRG-1 concurrently had the same proliferation rate as that induced by NRG-1 alone. Thus nitration of NRG-1's EGF-like domain caused it to lose its ability to bind and activate its receptor with loss of ligand-induced proliferation. Posttranslational nitration of growth factors in states where reactive nitrogen species are increased may be an important means of regulating growth factor receptor effects in the lung.     

Generation and characterization of neuregulin-2-deficient mice

The neuregulins (NRGs) are a family of four structurally related growth factors that are expressed in the developing and adult brain. NRG-1 is essential for normal heart formation and has been implicated in the development and maintenance of both neurons and glia. NRG-2 was identified on the basis of its homology to NRG-1 and, like NRG-1, is expressed predominantly by neurons in the central nervous system. We have generated mice with the active domain of NRG-2 deleted in an effort to characterize the biological function of NRG-2 in vivo. In contrast to the NRG-1 knockout animals, NRG-2 knockouts have no apparent heart defects and survive embryogenesis. Mutant mice display early growth retardation and reduced reproductive capacity. No obvious histological differences were observed in the major sites of NRG-2 expression. Our results indicate that in vivo NRG-2 activity differs substantially from that of NRG-1 and that it is not essential for normal development in utero.     

Activation of SIRT1, a class III histone deacetylase, contributes to fructose feeding-mediated induction of the alpha-myosin heavy chain expression

Fructose feeding has been shown to induce the cardiac alpha-myosin heavy chain (MHC) expression and protect the heart from ischemia- and reperfusion-mediated cell injury. This study was designed to investigate the mechanism involved in the effect of this sugar on MHC gene expression and cardiac protection. Adult mice were fed with a 6-propyl-2-thiouracil (PTU) diet or PTU combined with a fructose-rich diet. PTU treatment made animals hypothyroid and that resulted in total replacement of cardiac alpha-MHC with the beta-MHC isoform. Addition of fructose in the PTU diet led to reexpression of the alpha-MHC isoform to a significant level. Similar induction of alpha-MHC expression was also seen when PTU diet was combined with resveratrol, an agonist of sirtuin (SIRT) 1 deacetylase. Analysis of heart lysate of these animals indicated that fructose feeding augmented the NAD-to-NADH ratio and the cardiac SIRT1 levels, thus suggesting a role of SIRT1 in fructose-mediated activation of alpha-MHC isoform. To analyze a direct effect of SIRT1 on MHC isoform expression, we generated transgenic mice expressing SIRT1 in the heart. Treatment of these transgenic mice with PTU diet did not lead to disappearance of alpha-MHC, as it did in the nontransgenic animals. SIRT1 overexpression also activated the alpha-MHC gene promoter in transient transfection assays, thus confirming a role of SIRT1 in the induction of alpha-MHC expression. Fructose feeding also attenuated the MHC isoform shift and blocked the cardiac hypertrophy response associated with pressure overload, which was again associated with the induction of cardiac SIRT1 levels. These results demonstrate that fructose feeding protects the heart by induction of the SIRT1 deacetylase and highlight its role in the induction of alpha-MHC gene expression.     

Osteoblastic differentiation of monkey embryonic stem cells in vitro

Monkey embryonic stem (ES) cell is a useful tool for preclinical studies of regenerative medicine. In this paper, we investigated whether monkey ES cells can be differentiated into osteoblasts in vitro using factors known to promote osteogenesis. We prepared embryoid bodies (EB) in the presence of retinoic acid (RA) and subsequently differentiated in the medium containing either dexamethasone (DEX) or bone morphogenetic protein (BMP)-2 in addition to osteogenic supplements (OS), specifically ascorbic acid and beta-glycerophosphate. RA treatment during EB formation induced osteoblastic marker genes, such as collagen type 1, osteopontin, and Cbfa1. For the expression of osteocalcin, however, cultivation with medium containing either DEX or BMP-2 in addition to OS was required. These results showed that osteoblasts could be derived from monkey ES cells in vitro and BMP-2 + OS was effective to induce calcification.     

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.     

Efficient cardiomyocyte differentiation of embryonic stem cells by bone morphogenetic protein-2 combined with visceral endoderm-like cells

As the signals required for cardiomyocyte differentiation and functional regulation are complex and only partly understood, the mechanisms prompting the differentiation and specification of pluripotential embryonic stem (ES) cells into cardiomyocytes remain unclear. We hypothesized that a combined technology system, cocultured with a visceral endoderm (VE) - like cell line, END-2, and added cytokine BMP-2, would induce high percentage conversion of murine ES-D3 cell line into cardiomyocytes, and derived cardiomyocytes in this system would exhibit more mature characteristics. It was observed that 92% (P<0.01) ES cell-derived aggregates in this system exhibited rhythmic contractions, and the contractile areas were greater. By contrast, in ES cells cultured alone, on the feeder layer of END-2 cells, or with added BMP-2, the total percentage of beating aggregates was 19, 69 (P<0.01) and 44% (P<0.01), respectively. All the rhythmically contractile cells derived from ES cells expressed cardiac-specific proteins for troponin T. Among them, the combined system resulted in significantly increased cardiac-specific genes (NKx2.5, alpha-MHC). Transmission electron microscopy (TEM) revealed varying degrees of myofibrillar organization, and the combined system resulted in a more mature phenotype such as Z bands, nascent intercalated discs and gap junctions. Before shifting to the cardiomyocyte phenotype, this system could accelerate apoptosis of the cell population (P<0.01). The inductive efficacy of this system can provide an opportunity to facilitate cardiomyocyte differentiation of ES cells. The inducible effects of this system may depend on increasing cardiac-specific gene expression and the induction of apoptosis in cells that are not committed to cardiac differentiation.     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Role of neuregulin-1/ErbB2 signaling in endothelium-cardiomyocyte cross-talk

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, has been shown to be indispensable for the normal function of the adult heart by binding to ErbB4 receptors on cardiomyocytes. In the present study, we have investigated to what extent ErbB2, the favored co-factor of ErbB4 for heterodimerization, participates in the cardiac effects of endothelium-derived NRG-1. In addition, in view of our previously described anti-adrenergic effects of NRG-1, we have studied which neurohormonal stimuli affect endothelial NRG-1 expression and release and how this may fit into a broader frame of cardiovascular physiology. Immunohistochemical staining of rat heart and aorta showed that NRG-1 expression was restricted to the endocardial endothelium and the cardiac microvascular endothelium (CMVE); by contrast, NRG-1 expression was absent in larger coronary arteries and veins and in aortic endothelium. In rat CMVE in culture, NRG-1 mRNA and protein expression was down-regulated by angiotensin II and phenylephrine and up-regulated by endothelin-1 and mechanical strain. CMVE-derived NRG-1 was shown to phosphorylate cardiomyocyte ErbB2, an event prevented by a 24-h preincubation of myocytes with monoclonal ErbB2 antibodies. Pretreating cardiomyocytes with these inhibitory anti-ErbB2 antibodies significantly attenuated CMVE-induced cardiomyocyte hypertrophy and abolished the protective actions of CMVE against cardiomyocyte apoptosis. Accordingly, ErbB2 signaling participated in the paracrine survival and growth controlling effects of NRG-1 on cardiomyocytes in vitro, explaining the cardiotoxicity of ErbB2 antibodies in patients. Cardiac NRG-1 synthesis occurs in endothelial cells adjacent to cardiac myocytes and is sensitive to factors related to the regulation of blood pressure.