Development of cardiomyocytes expressing cardiac-specific genes, action potentials, and ionic channels during embryonic stem cell-derived cardiogenesis.
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Action Potentials↗
Biomedical subjects
J Rohwedel
Publications and source records attributed to J Rohwedel.
M-twist expression inhibits mouse embryonic stem cell-derived myogenic differentiation in vitro.
The mouse M-twist gene codes for a basic helix-loop-helix protein which was shown to be inhibitory for differentiation of myogenic cells in culture. Mouse embryonic stem (ES) cells of line BLC6 efficiently differentiating into skeletal muscle cells when cultivated as embryo-like aggregates (embryoid bodies) were stably transfected with the plasmid pME18s-twist containing the M-twist gene under the control of the modified SV40 early promoter SR alpha. Two pME18s-twist-expressing clones showed delayed and reduced skeletal muscle cell differentiation depending on the level of exogenous M-twist expression compared to control cells. By morphological analysis using phase contrast microscopy and hematoxylin-eosin staining, the development of first myocytes and formation of myotubes in embryoid body outgrowths of these clones were found to be delayed for about 3 days in comparison to control cells. Immunofluorescence studies with a monoclonal antibody against sarcomeric myosin heavy chain revealed that myogenic cells appeared in so-called myogenic centers showing a reduced number of myocytes and myotubes in the M-twist-expressing clones. Using RT-PCR analysis the expression of the skeletal muscle determination genes myf5, myogenin, and MyoD as well as muscle-specific genes coding for the gamma-subunit of the nicotinic acetylcholine receptor and the cell adhesion molecule M-cadherin were found to appear with a delay of at least 1 to 4 days in the pME18s-twist-transfected cells during the development of embryoid bodies. We conclude that the constitutive expression of the mouse M-twist gene during ES-cell-derived differentiation has an inhibitory effect on skeletal muscle cell development depending on the level of exogenous M-twist expression.
Animals↗
Expression of M-cadherin protein in myogenic cells during prenatal mouse development and differentiation of embryonic stem cells in culture.
Molecules regulating morphogenesis by cell-cell interactions are the cadherins, a class of calcium-dependent adhesion molecules. One of its members, M-cadherin, has been isolated from a myoblast cell line (Donalies et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 88:8024-8028). In mouse development, expression of M-cadherin mRNA first appears at day 8.5 of gestation (E8.5) in somites and has been postulated to be down-regulated in developing muscle masses (Moore and Walsh [1993] Development 117:1409-1420). Affinity-purified polyclonal M-cadherin antibodies, detecting a protein of approximately 120 kDa, were used to study the cell expression pattern of M-cadherin protein. It was first visualized in somites at E10 1/3 and could be confined to desmin positive, myotomal cells. At all subsequent prenatal stages, M-cadherin was only found in myogenic cells of somitic origin. The detection of the protein at E10 1/3 suggests a translational delay of M-cadherin mRNA of 1 to 2 days (E8.5 vs. E10 1/3). This was further supported by the finding that during differentiation of ES cell line BLC6 into skeletal muscle cells in culture, expression of M-cadherin mRNA can be detected 2 days prior to M-cadherin protein. During prenatal development, the pattern of M-cadherin expression changes: In E10 1/3 embryos and also in myotomal cells of later stages, M-cadherin is evenly distributed on the cell surface. In developing muscle masses (tested at E16 to E18), however, M-cadherin protein becomes clustered most likely at sites of cell-cell contact as indicated by double-labelling experiments: M-cadherin-staining is the positive image of laminin negative areas excluding the presence of a basal lamina at M-cadherin positive sites. Furthermore, M-cadherin is coexpressed with the neuronal cell adhesion molecule N-CAM which has been shown to mediate cell-cell contact in myogenic cells. In summary, our results are in line with the idea that M-cadherin might play a central role in myogenic morphogenesis.
Amino Acid Sequence↗
Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents.
The mouse blastocyst-derived embryonic stem cell (ES cell) line BLC6 efficiently differentiates into myosin heavy chain-, desmin- and myogenin-positive skeletal muscle cells when cultivated in embryo-like aggregates (embryoid bodies). Here, we show that the muscle-specific determination genes myf5, myogenin, myoD, and myf6 are expressed in these embryoid bodies in a characteristic temporal pattern which precisely reflects the sequence observed during mouse development in vivo. Myf5 is the first gene to be expressed followed by myogenin, myoD, and myf6, in this order. In situ hybridization demonstrates transcripts for myogenin and myoD accumulating in mono- and multinucleated myogenic cells, while myf5 mRNA is already found in mononucleated myoblasts. The myocytes also express functional nicotinic cholinoceptors and exhibit T-type Ca2+ currents and later L-type Ca2+ currents, demonstrating physiological properties of skeletal muscle cells. During myocyte differentiation the density of L-type Ca2+ channels significantly increases while the density of T-type Ca2+ channels decreases. The effect of external signals on myogenic differentiation of BLC6 cells was demonstrated by cocultivation with visceral endodermal END-2 cells and the activin A-secreting WEHI-3 cells. END-2 cells essentially prevent skeletal muscle differentiation, whereas basic fibroblast growth factor, transforming growth factor-beta, and WEHI-3 cells have no or an attenuating effect, respectively. Our results suggest that ES cells recapitulate closely the early steps of muscle development in vivo and may serve as an excellent in vitro system to study this process.
Animals↗
Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents.
Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.
Animals↗
Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types.
Pluripotent embryonic stem cells (ESC, ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique, alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC, as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinusnodal, atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state, action potentials of very long duration (0.3-1 s) were found, which may represent developmentally controlled transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.
Action Potentials↗
Different modes of hypervariability in (GATA)n simple sequence repeat loci.
Only a few prominent simple sequence repeat (SSR) loci of the type (GATA)n are found in the genome of the mealmoth Ephestia kuehniella Zeller. Therefore this moth was chosen as a model organism for the genetic and molecular analysis of hypervariability of SSR loci. We characterized alleles of (GATA)n loci in different Ephestia strains by cloning and genomic restriction mapping. Some variants appeared to be mere variable number of tandem repeat (VNTR) alleles, others showed considerable changes in the sequence neighbourhood of the GATA repeats. These may be produced by major rearrangements or by transposition of the (GATA)n block together with flanking sequences into a different sequence environment.
Alleles↗