Different efficiency of auxiliary/chaperone proteins to promote the functional reconstitution of honeybee glutamate and acetylcholine receptors in Xenopus laevis oocytes.

Heterologous expression systems (e.g., Xenopus laevis oocytes) are useful to study the biophysical properties and pharmacology of ionotropic receptors such as ionotropic glutamate (iGLuRs) and nicotinic acetylcholine (nAChRs) receptors. However, insect receptors often require the co-expression of chaperone proteins to be functional. Only few iGluRs and nAChRs have been successfully expressed in such systems. Here, we compared the efficiency of chaperone proteins to promote the functional expression of one Apis mellifera iGluR and several nAChR subunit combinations (α1α8ß1, α7, α2α8ß1 and α2α7α8ß1) in Xenopus oocytes. To this end, we cloned a new iGluR (GluR-1) and potential chaperone proteins (e.g., SOL-1, Neto, NACHO) and tested more than 40 combinations of human, nematode and honeybee proteins. We obtained robust expression of GluR-1 and α1α8ß1 when co-expressed with honeybee chaperone proteins and found that nAChR expression critically depended on the α1 subunit N-terminal sequence. We recorded small ACh-gated currents in few oocytes when the α7 subunit was co-expressed with Caenorhabditis elegans RIC-3, but none of the chaperone proteins allowed efficient expression of α2α8ß1 or α2α7α8ß1. Our results show that only some protein combinations can reconstitute functional receptors in Xenopus oocytes and that protein combination efficient in one species is not always efficient in another species.

Cav2.1 C-terminal fragments produced in Xenopus laevis oocytes do not modify the channel expression and functional properties.

The sequence and genomic organization of the CACNA1A gene that encodes the Cav2.1 subunit of both P and Q-type Ca2+ channels are well conserved in mammals. In human, rat and mouse CACNA1A, the use of an alternative acceptor site at the exon 46-47 boundary results in the expression of a long Cav2.1 splice variant. In transfected cells, the long isoform of human Cav2.1 produces a C-terminal fragment, but it is not known whether this fragment affects Cav2.1 expression or functional properties. Here, we cloned the long isoform of rat Cav2.1 (Cav2.1(e47)) and identified a novel variant with a shorter C-terminus (Cav2.1(e47s)) that differs from those previously described in the rat and mouse. When expressed in Xenopus laevis oocytes, Cav2.1(e47) and Cav2.1(e47s) displayed similar functional properties as the short isoform (Cav2.1). We show that Cav2.1 isoforms produced short (CT1) and long (CT1(e47)) C-terminal fragments that interacted in vivo with the auxiliary Cavß4a subunit. Overexpression of the C-terminal fragments did not affect Cav2.1 expression and functional properties. Furthermore, the functional properties of a Cav2.1 mutant without the C-terminal Cavß4 binding domain (Cav2.1ΔCT2) were similar to those of Cav2.1 and were not influenced by the co-expression of the missing fragments (CT2 or CT2(e47)). Our results exclude a functional role of the C-terminal fragments in Cav2.1 biophysical properties in an expression system widely used to study this channel.

Multiple combinations of RDL subunits diversify the repertoire of GABA receptors in the honey bee parasite Varroa destructor.

In insects, γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and GABA-gated ion channels are the target of different classes of insecticides, including fipronil. We report here the cloning of six subunits (four RDL, one LCCH3, and one GRD) that constitute the repertoire of the GABA-gated ion channel family of the Varroa mite (Varroa destructor), a honey bee ectoparasite. We also isolated a truncated GRD subunit with a premature stop codon. We found that when expressed in Xenopus laevis oocytes, three of the four RDL subunits (VdesRDL1, VdesRDL2, and VdesRDL3) formed functional, homomultimeric anionic receptors, whereas GRD and LCCH3 produced heteromultimeric cationic receptors. These receptors displayed specific sensitivities toward GABA and fipronil, and VdesRDL1 was the most resistant to the insecticide. We identified specific residues in the VdesRDL1 pore-lining region that explain its high resistance to fipronil. VdesRDL4 did not form a functional receptor when expressed alone, but it assembled with VdesRDL1 to form a heteromultimeric receptor with properties distinct from those of the VdesRDL1 homomultimeric receptor. Moreover, VdesRDL1 physically interacted with VdesRDL3, generating a heteromultimeric receptor combining properties of both subunits. On the other hand, we did not detect any functional interaction between VdesLCCH3 and the VdesRDL subunits, an observation that differed from what was previously reported for Drosophila melanogaster In conclusion, this study provides insights relevant to improve our understanding of the precise role of GABAergic signaling in insects and new tools for the development of Varroa mite-specific insecticidal agents that do not harm honey bees.

Honeybee CaV4 has distinct permeation, inactivation, and pharmacology from homologous NaV channels.

DSC1, a Drosophila channel with sequence similarity to the voltage-gated sodium channel (NaV), was identified over 20 years ago. This channel was suspected to function as a non-specific cation channel with the ability to facilitate the permeation of calcium ions (Ca2+). A honeybee channel homologous to DSC1 was recently cloned and shown to exhibit strict selectivity for Ca2+, while excluding sodium ions (Na+), thus defining a new family of Ca2+ channels, known as CaV4. In this study, we characterize CaV4, showing that it exhibits an unprecedented type of inactivation, which depends on both an IFM motif and on the permeating divalent cation, like NaV and CaV1 channels, respectively. CaV4 displays a specific pharmacology with an unusual response to the alkaloid veratrine. It also possesses an inactivation mechanism that uses the same structural domains as NaV but permeates Ca2+ ions instead. This distinctive feature may provide valuable insights into how voltage- and calcium-dependent modulation of voltage-gated Ca2+ and Na+ channels occur under conditions involving local changes in intracellular calcium concentrations. Our study underscores the unique profile of CaV4 and defines this channel as a novel class of voltage-gated Ca2+ channels.

Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

Cav2.1 channels are expressed throughout the brain and are the predominant Ca2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA-A Receptors.

The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-clamp recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α2ß2γ2 and α2ß2γ2). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.

Manipulations of Glutathione Metabolism Modulate IP3-Mediated Store-Operated Ca2+ Entry on Astroglioma Cell Line.

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca2+ homeostasis. IP3-mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by Gq-linked purinergic P2Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P2Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca2+-dependent K+ currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca2+-channels, and thus modify cellular excitability under purinergic stimulation.

The Glutathione Metabolite γ-Glutamyl-Glutamate Partially Activates Glutamate NMDA Receptors in Central Neurons With Higher Efficacy for GluN2B-Containing Receptors.

Gamma-L-glutamyl-L-glutamate (γ-Glu-Glu) was synthetized and further characterized for its activity on cultured neurons. We observed that γ-Glu-Glu elicited excitatory effects on neurons likely by activating mainly the N-methyl-D-aspartate (NMDA) receptors. These effects were dependent on the integrity of synaptic transmission as they were blocked by tetrodotoxin (TTX). We next evaluated its activity on NMDA receptors by testing it on cells expressing these receptors. We observed that γ-Glu-Glu partially activated NMDA receptors and exhibited better efficacy for NMDA receptors containing the GluN2B subunit. Moreover, at low concentration, γ-Glu-Glu potentiated the responses of glutamate on NMDA receptors. Finally, the endogenous production of γ-Glu-Glu was measured by LC-MS on the extracellular medium of C6 rat astroglioma cells. We found that extracellular γ-Glu-Glu concentration was, to some extent, directly linked to GSH metabolism as γ-Glu-Glu can be a by-product of glutathione (GSH) breakdown after γ-glutamyl transferase action. Therefore, γ-Glu-Glu could exert excitatory effects by activating neuronal NMDA receptors when GSH production is enhanced.

Heterogeneous expression of GABA receptor-like subunits LCCH3 and GRD reveals functional diversity of GABA receptors in the honeybee Apis mellifera.

BACKGROUND AND PURPOSE: Despite a growing awareness, annual losses of honeybee colonies worldwide continue to reach threatening levels for food safety and global biodiversity. Among the biotic and abiotic stresses probably responsible for these losses, pesticides, including those targeting ionotropic GABA receptors, are one of the major drivers. Most insect genomes include the ionotropic GABA receptor subunit gene, Rdl, and two GABA-like receptor subunit genes, Lcch3 and Grd. Most studies have focused on Rdl which forms homomeric GABA-gated chloride channels, and a complete analysis of all possible molecular combinations of GABA receptors is still lacking. EXPERIMENTAL APPROACH: We cloned the Rdl, Grd, and Lcch3 genes of Apis mellifera and systematically characterized the resulting GABA receptors expressed in Xenopus oocytes, using electrophysiological assays, fluorescence microscopy and co-immunoprecipitation techniques. KEY RESULTS: The cloned subunits interacted with each other, forming GABA-gated heteromeric channels with particular properties. Strikingly, these heteromers were always more sensitive than AmRDL homomer to all the pharmacological agents tested. In particular, when expressed together, Grd and Lcch3 form a non-selective cationic channel that opens at low concentrations of GABA and with sensitivity to insecticides similar to that of homomeric Rdl channels. CONCLUSION AND IMPLICATIONS: For off-target species like the honeybee, chronic sublethal exposure to insecticides constitutes a major threat. At these concentration ranges, homomeric RDL receptors may not be the most pertinent target to study and other ionotropic GABA receptor subtypes should be considered in order to understand more fully the molecular mechanisms of sublethal toxicity to insecticides.

Tissue localization of Coxiella-like endosymbionts in three European tick species through fluorescence in situ hybridization.

Ticks are commonly infected by Coxiella-like endosymbionts (Coxiella-LE) which are thought to supply missing B vitamin nutrients required for blood digestion.While this nutritional symbiosis is essential for the survival and reproduction of infected tick species, our knowledge of where Coxiella-LE is localized in tick tissues is partial at best since previous studies have focused on a limited number of Asian or American tick species. To fill this gap, we investigated the tissue localization of Coxiella-LE in three European tick species, Ornithodoros maritimus, Dermacentor marginatus and Ixodes hexagonus, using a diagnostic fluorescence in situ hybridization (FISH) assay, combined with PCR-based detection. Specific fluorescent foci were observed in several tick tissues. We visualized a pronounced tissue tropism of Coxiella-LE for tick ovaries and Malpighian tubules, a pattern suggestive of a high degree of lifestyle specialization toward mutualism: infection of the ovaries is indicative of transovarial transmission, whereas infection of the Malpighian tubules suggests a nutritional function. We postulate that Malpighian tubules are key organs for the nutritional symbiosis, notably the synthesis of B vitamins by Coxiella-LE, whereas the infection of the ovaries ensures vertical transmission of the symbionts to future generations. We also detected occasional infections in other organs, such as salivary glands and the midgut. Finally, we discuss the potential significance of the different tissue tropism for tick biology.

Initiation of embryonic cardiac pacemaker activity by inositol 1,4,5-trisphosphate-dependent calcium signaling.

In the adult, the heart rate is driven by spontaneous and repetitive depolarizations of pacemaker cells to generate a firing of action potentials propagating along the conduction system and spreading into the ventricles. In the early embryo before E9.5, the pacemaker ionic channel responsible for the spontaneous depolarization of cells is not yet functional. Thus the mechanisms that initiate early heart rhythm during cardiogenesis are puzzling. In the absence of a functional pacemaker ionic channel, the oscillatory nature of inositol 1,4,5-trisphosphate (InsP3)-induced intracellular Ca2+ signaling could provide an alternative pacemaking mechanism. To test this hypothesis, we have engineered pacemaker cells from embryonic stem (ES) cells, a model that faithfully recapitulates early stages of heart development. We show that InsP3-dependent shuttle of free Ca2+ in and out of the endoplasmic reticulum is essential for a proper generation of pacemaker activity during early cardiogenesis and fetal life.

Tick-Bacteria Mutualism Depends on B Vitamin Synthesis Pathways.

Mutualistic interactions with microbes have facilitated the radiation of major eukaryotic lineages [1, 2]. Microbes can notably provide biochemical abilities, allowing eukaryotes to adapt to novel habitats or to specialize on particular feeding niches [2-4]. To investigate the importance of mutualisms for the exclusive blood feeding habits of ticks, we focused on a bacterial genus of medical interest, Francisella, which is known to include both virulent intracellular pathogens of vertebrates [5, 6] and maternally inherited symbionts of ticks [7-9]. Through a series of physiological experiments, we identified a Francisella type, F-Om, as an obligate nutritional mutualist in the life cycle of the African soft tick Ornithodoros moubata. Francisella F-Om mutualism synthesizes B vitamins that are deficient in the blood meal of ticks. Indeed, experimental elimination of Francisella F-Om resulted in alteration of tick life history traits and physical abnormalities, deficiencies which were fully restored with an oral supplement of B vitamins. We also show that Francisella F-Om is maternally transmitted to all maturing tick oocytes, suggesting that this heritable symbiont is an essential adaptive element in the life cycle of O. moubata. The Francisella F-Om genome further revealed a recent origin from a Francisella pathogenic life style, as observed in other Francisella symbionts [6, 7, 10]. Though half of its protein-coding sequences are now pseudogenized or lost, Francisella F-Om has kept several B vitamin synthesis pathways intact, confirming the importance of these genes in evolution of its nutritional mutualism with ticks.

Is xenotransplantation of embryonic stem cells a realistic option?

To test the purported immune privilege of embryonic stem cells (ESC) in the challenging setting of xenotransplantation, 14 immunocompetent baboons were subjected to a coronary artery occlusion-reperfusion sequence and, two weeks later, randomized to receive in-scar injections of culture medium or cardiac-committed mouse ESC engineered to express fluorescent reporter genes driven by cardiac-specific promoters. Two months after transplantation, left ventricular function, as assessed by echocardiography, deteriorated to a similar extent in control and treated baboons. This correlated with failure to identify the grafted cells by X-gal histology and immunofluorescence. Rejection did not seem to be mediated by xenoantibodies, but rather by T lymphocytes and natural killer cells as suggested by positive immunostaining for CD3 and CD56 early after transplantation. There was no increase in circulating levels of regulatory T cells. These data raise a cautionary note about the immune privilege of ESC and suggest that from a mere immunologic standpoint, ESC xenotransplantation is likely to be an unrealistic challenge.

Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study.

BACKGROUND: Heart failure develops after myocardial infarction and is a major cause of morbidity and mortality. The ability to direct differentiation of embryonic stem cells (ESC) towards a cardiomyogenic phenotype makes them an attractive therapeutic option for cardiac repair, but species-specific and individual-specific immunological imprinting remains a hurdle. Our aim was to ascertain whether the purported immune privilege of ESC allows for their cross-species engraftment in a clinically relevant large-animal model. METHODS: We studied engraftment and differentiation of cardiac-committed mouse ESC in 18 sheep in which a myocardial infarction had been induced; nine controls received medium and nine sheep (five of which were immunosuppressed) received ESC. The gain in myocardial function was measured by echocardiography 1 month after cell transplantation. FINDINGS: Cardiac-committed murine ESC engrafted in infarcted myocardium of immunosuppressed and immunocompetent sheep, and differentiated into mature cardiomyocytes that expressed connexins. Colonisation of the scar area by ESC was accompanied by a functional benefit of the damaged myocardium. Left-ventricular ejection fraction deteriorated in the control group by a median of 9.9% (range -20 to 0.3) relative to baseline (p=0.011) whereas in the treated group it improved by 6.6% (-5.7 to 50.8; comparison between groups p=0.002). INTERPRETATION: These findings obtained in a clinically relevant large-animal model of heart failure strengthen the potential therapeutic use of ESC to regenerate the severely dysfunctional myocardium and bring additional evidence for an immune privilege of these cells.

Cardiac commitment of embryonic stem cells for myocardial repair.

Embryonic stem (ES) cells represent a source for cell-based regenerative therapies of heart failure. The pluripotency and the plasticity of ES cells allow them to be committed to a cardiac lineage following treatment with growth factors of the transforming growth factor (TGF)-beta superfamily. We describe a protocol designed to turn on expression of cardiac-specific genes in undifferentiated murine ES cells stimulated with BMP2 and/or TGF-beta. Cell commitment results in a significant improvement in spontaneous cardiac differentiation of ES cells both in vitro and in vivo.

Regulation of neuronal high-voltage activated Ca(V)2 Ca(2+) channels by the small GTPase RhoA.

High-Voltage-Activated (HVA) Ca(2+) channels are known regulators of synapse formation and transmission and play fundamental roles in neuronal pathophysiology. Small GTPases of Rho and RGK families, via their action on both cytoskeleton and Ca(2+) channels are key molecules for these processes. While the effects of RGK GTPases on neuronal HVA Ca(2+) channels have been widely studied, the effects of RhoA on the HVA channels remains however elusive. Using heterologous expression in Xenopus laevis oocytes, we show that RhoA activity reduces Ba(2+) currents through CaV2.1, CaV2.2 and CaV2.3 Ca(2+) channels independently of CaVß subunit. This inhibition occurs independently of RGKs activity and without modification of biophysical properties and global level of expression of the channel subunit. Instead, we observed a marked decrease in the number of active channels at the plasma membrane. Pharmacological and expression studies suggest that channel expression at the plasma membrane is impaired via a ROCK-sensitive pathway. Expression of constitutively active RhoA in primary culture of spinal motoneurons also drastically reduced HVA Ca(2+) current amplitude. Altogether our data revealed that HVA Ca(2+) channels regulation by RhoA might govern synaptic transmission during development and potentially contribute to pathophysiological processes when axon regeneration and growth cone kinetics are impaired.

Collagen scaffolds with or without the addition of RGD peptides support cardiomyogenesis after aggregation of mouse embryonic stem cells.

Embryonic stem (ES) cell-based cardiac muscle repair using tissue-engineered scaffolds is an attractive prospective treatment option for patients suffering from heart disease. In this study, our aim was to characterize mouse ES cell-derived cardiomyocytes growing on collagen I/III scaffolds, modified with the adhesion peptides arginine-glycine-aspartic acid (RGD). Mouse ES-derived embryoid bodies (EBs) differentiated efficiently into beating cardiomyocytes on the collagen scaffolds. QPCR analysis and immunofluorescent staining showed that cardiomyocytes expressed cardiac muscle-related transcripts and proteins. Analysis of cardiomyocytes by electron microscopy identified muscle fiber bundles and Z bands, typical of ES-derived cardiomyocytes. No differences were detected between the collagen + RGD and collagen control scaffolds. ES cells that were not differentiated as EBs prior to seeding on the scaffold, did not differentiate into cardiomyocytes. These results indicate that a collagen I/III scaffold supports cardiac muscle development and function after EB formation, and that this scaffold appears suitable for future in vivo testing. The addition of the RGD domain to the collagen scaffold did not improve cardiomyocyte development or viability, indicating that RGD signaling to integrins was not a rate-limiting event for cardiomyogenesis from EBs seeded on a collagen scaffold.

Chimeric mutations in the M2 segment of the 5-hydroxytryptamine-gated chloride channel MOD-1 define a minimal determinant of anion/cation permeability.

The ionic selectivity of ligand-gated ion channels (LGICs) determines whether receptor activation produces an excitatory or inhibitory response. The determinants of anion/cation selectivity were investigated for a new member of the LGIC superfamily, MOD-1, a serotonin-gated chloride channel cloned from the nematode Caenorhabditis elegans. In common with other anionic LGICs (glycine receptors and GABA(A) receptors), the selectivity triple mutant in the pore-forming M2 segment (proline insertion, Ala --> Glu substitution at the central ring, and Thr --> Val at the hydrophobic ring) converted the selectivity of MOD-1 from anionic to cationic. Unlike other LGICs, however, this mutant in MOD-1 was highly selective for K+ over other cations. Subsets of this selectivity triple mutant were studied to define the minimal change required for conversion from anion-permeable to cation-permeable. The double mutant at the central ring of charge (deltaPro-269/A270E) produced a non-selective cation channel. Charge reversal at the central ring alone (A270E) was sufficient to convert MOD-1 to cation-permeable. These results refine the determinants of ion-charge selectivity in LGICs and demonstrate the critical role of the central ring of charge formed by the M2 segments.

Interplay between the retinoblastoma protein and LEK1 specifies stem cells toward the cardiac lineage.

The molecular mechanisms governing early cardiogenesis are still largely unknown. Interestingly, the retinoblastoma protein (Rb), a regulator of cell cycle, has recently emerged as a new candidate regulating cell differentiation. Rb-/- mice die at midgestation and mice lacking E2f1/E2f3, downstream components of the Rb-dependent transcriptional pathway, die of heart failure. To gain insight into the function of Rb pathway in early cardiogenesis, we used Rb-/- embryonic stem (ES) cells differentiating into cardiomyocytes. Rb-/- cells displayed a dramatic delay in expression of cardiac-specific transcription factors and in turn in the whole process of cardiac differentiation. The phenotype of Rb-/- ES cell-derived cardiomyocytes was rescued by reintroducing Rb in cardiac progenitors, by stimulating the BMP-dependent cardiogenic pathway or by overexpression of Nkx2.5. ES cells deficient in the recently identified factor LEK1, a murine homolog of the cardiomyogenic factor 1, or specific disruption of Rb-LEK1 interaction into the nucleus of differentiating ES cells recapitulated the delay in cardiac differentiation of Rb-/- ES cells. Thus, we provide evidence for a novel Rb/LEK1-dependent and BMP-independent transcriptional program, which plays a pivotal role in priming ES cells toward a cardiac fate.

Cardiac specification of embryonic stem cells.

Over the past decade, cell transplantation has been recognized as a mean of repairing infarcted myocardium. Both adult stem cells and differentiated cells have yielded encouraging results with regard to engraftment into postinfarction scars. However, these cells now feature serious restrictions. Asan alternative, embryonic stem (ES) cells are particularly attractive, because of their plasticity and the subsequent possibility to drive them towards a cardiomyogenic phenotype after exposure to appropriate growth factors. An additional theoretical advantage of ES cells is their expected immune privilege. In this article, we summarize the findings obtained in cell therapy using ES cells and discuss the molecular mechanisms of cardiac specification of the cells.