Heart Muscle Differentiation Research Articles

MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

Myocyte enhancer factor 2C (MEF2C) is a transcription factor that regulates heart and skeletal muscle differentiation and growth. Several protein-encoding genes were identified as targets of this factor; however, little is known about its contribution to the microtranscriptome composition and dynamics in myogenic programs. In this report, we aimed to address this question. Deep sequencing of small RNAs of human muscle cells revealed a set of microRNAs (miRNAs), including several muscle-specific miRNAs, that are sensitive to MEF2C depletion. As expected, in cells with knockdown of MEF2C, we found mostly downregulated miRNAs; nevertheless, as much as one-third of altered miRNAs were upregulated. The majority of these changes are driven by transcription efficiency. Moreover, we found that MEF2C affects nontemplated 3′-end nucleotide addition of miRNAs, mainly oligouridylation. The rate of these modifications is associated with the level of TUT4 which mediates RNA 3′-uridylation. Finally, we found that a quarter of miRNAs which significantly changed upon differentiation of human skeletal myoblasts is inversely altered in MEF2C deficient cells. We concluded that MEF2C is an essential factor regulating both the quantity and quality of the microtranscriptome, leaving an imprint on the stability and perhaps specificity of many miRNAs during the differentiation of muscle cells.

Genome-wide transcriptomics analysis identifies sox7 and sox18 as specifically regulated by gata4 in cardiomyogenesis

The transcription factors GATA4, GATA5 and GATA6 are important regulators of heart muscle differentiation (cardiomyogenesis), which function in a partially redundant manner. We identified genes specifically regulated by individual cardiogenic GATA factors in a genome-wide transcriptomics analysis. The genes regulated by gata4 are particularly interesting because GATA4 is able to induce differentiation of beating cardiomyocytes in Xenopus and in mammalian systems. Among the specifically gata4-regulated transcripts we identified two SoxF family members, sox7 and sox18. Experimental reinstatement of gata4 restores sox7 and sox18 expression, and loss of cardiomyocyte differentiation due to gata4 knockdown is partially restored by reinstating sox7 or sox18 expression, while (as previously reported) knockdown of sox7 or sox18 interferes with heart muscle formation. In order to test for conservation in mammalian cardiomyogenesis, we confirmed in mouse embryonic stem cells (ESCs) undergoing cardiomyogenesis that knockdown of Gata4 leads to reduced Sox7 (and Sox18) expression and that Gata4 is also uniquely capable of promptly inducing Sox7 expression. Taken together, we identify an important and conserved gene regulatory axis from gata4 to the SoxF paralogs sox7 and sox18 and further to heart muscle cell differentiation.

Bone Marrow-Derived Mesenchymal Cell Differentiation toward Myogenic Lineages: Facts and Perspectives

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are valuable platforms for new therapies based on regenerative medicine. BM-MSCs era is coming of age since the potential of these cells is increasingly demonstrated. In fact, these cells give origin to osteoblasts, chondroblasts, and adipocyte precursors in vitro, and they can also differentiate versus other mesodermal cell types like skeletal muscle precursors and cardiomyocytes. In our short review, we focus on the more recent manipulations of BM-MSCs toward skeletal and heart muscle differentiation, a growing field of obvious relevance considering the toll of muscle disease (i.e., muscular dystrophies), the heavier toll of heart disease in developed countries, and the still not completely understood mechanisms of muscle differentiation and repair.

Sfrp1 promotes cardiomyocyte differentiation in Xenopus via negative-feedback regulation of Wnt signalling.

Wnt signalling is a key regulator of vertebrate heart development, yet it is unclear which specific Wnt signalling components are required to regulate which aspect of cardiogenesis. Previously, we identified Wnt6 as an endogenous Wnt ligand required for controlling heart muscle differentiation via canonical Wnt/β-catenin signalling. Here we show for the first time a requirement for an endogenous Wnt signalling inhibitor for normal heart muscle differentiation. Expression of sfrp1 is strongly induced in differentiating heart muscle. We show that sfrp1 is not only able to promote heart muscle differentiation but is also required for the formation of normal size heart muscle in the embryo. sfrp1 is functionally able to inhibit Wnt6 signalling and its requirement during heart development relates to relieving the cardiogenesis-restricting function of endogenous wnt6. In turn, we discover that sfrp1 expression in the heart is regulated by Wnt6 signalling, which for the first time indicates that sfrp genes can function as part of a Wnt negative-feedback regulatory loop. Our experiments indicate that sfrp1 controls the size of the differentiating heart muscle primarily by regulating cell fate within the cardiac mesoderm between muscular and non-muscular cell lineages. The cardiac mesoderm is therefore not passively patterned by signals from the surrounding tissue, but regulates its differentiation into muscular and non-muscular tissue using positional information from the surrounding tissue. This regulatory network might ensure that Wnt activation enables expansion and migration of cardiac progenitors, followed by Wnt inhibition permitting cardiomyocyte differentiation.

Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Wnt/β‐catenin signaling represses heart muscle development via GATA transcription factors

Inhibition of Wnt/β‐catenin signalling is required in vertebrate embryos for initiation of heart development. The identity of the endogenous Wnt ligand that needs to be inhibited has so far remained unclear.We identified Wnt6 to be expressed close to the heart forming tissue in Xenopus. Endogenous Wnt6 expression is required during organogenesis to prevent differentiation of an abnormally large heart muscle. Even at these remarkably late stages, Wnt6/β‐catenin signalling regulates the pro‐cardiogenic transcription factor network rather than heart muscle differentiation directly.Our loss‐of‐Wnt6‐function experiments suggest a sequential deregulation of cardiogenic markers, with GATA genes deregulated earliest. We find that GATA genes are not only among the cardiogenic genes repressed by Wnt/β‐catenin signalling, but that reinstating GATA function experimentally will rescue subsequent heart muscle development. This result suggests that Wnt/β‐catenin signalling primarily restricts GATA expression in the cardiogenic mesoderm; and that where Wnt signalling does not repress GATA expression; GATA factors drive subsequent heart development.Wnt/β‐catenin is usually associated with transcriptional activation rather than repression. However, our recent experiments with protein‐synthesis inhibitor suggest that Wnt/β‐catenin is capable of repressing GATA gene expression directly.

Wnt/β‐catenin signalling regulates cardiomyogenesis via GATA transcription factors

A functioning heart muscle is required continuously throughout life. During embryonic development the heart muscle tissue differentiates from mesoderm that has heart-forming potential. Heart-forming potential in the embryonic mesoderm is regulated by pro-cardiogenic transcription factors, such as members of the GATA and NK-2 transcription factor families. Subsequent heart muscle differentiation involves the expression of cytoskeletal proteins, including myosins and troponins. Different Wnt signalling pathways have various functions in heart development. So-called 'canonical' (Wnt/beta-catenin-mediated) signalling has a conserved role in vertebrate heart development, regulating and restricting heart development and subsequent heart muscle differentiation. Here we investigated the way in which Wnt/beta-catenin signalling functionally interacts with the GATA family of pro-cardiogenic transcription factors to regulate subsequent heart muscle differentiation. We used whole Xenopus embryos as an accessible experimental model system for vertebrate heart development. Our experiments confirmed that activation of Wnt signalling results in reduced gata gene expression, as well as reduced gene expression of other pro-cardiogenic transcription factors and heart muscle differentiation markers. Remarkably, we discovered that when GATA function is experimentally restored, the expression of other pro-cardiogenic transcription factors and heart muscle differentiation markers is rescued. These findings, obtained from whole-embryo experiments, show that Wnt signalling regulates heart development at the level of GATA factors, confirming earlier results from tissue-culture experiments. Furthermore, our rescue experiments in Xenopus embryos revealed differences in functional activity between the various GATA transcription factors involved in heart development. We discovered that GATA4 is more efficient at reinstating the gene expression of other pro-cardiogenic transcription factors, whereas GATA6 is more potent at promoting the expression of genes associated with terminal heart muscle differentiation. In conclusion, our findings show that the inhibition of heart development by Wnt/beta-catenin signalling during organogenesis is mediated by the loss of expression of GATA pro-cardiogenic transcription factors and reveal functional differences between those GATA factors in heart development.

Interaction of the heart‐specific LIM domain protein, FHL2, with DNA‐binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.

A Novel Drosophila, mef2-Regulated Muscle Gene Isolated in a Subtractive Hybridisation-Based Molecular Screen Using Small Amounts of Zygotic Mutant RNA

It is unknown how the general patterning mechanisms that subdivide the mesoderm initiate different pathways of cell differentiation. One route to understanding these events is to isolate and analyse genes specifically expressed early in this differentiation process. I have therefore undertaken a novel molecular screen in Drosophila in a systematic search for such genes. The approach utilised subtractive hybridisation coupled to directional cDNA library construction. Libraries were made from as little as 20 μg total RNA isolated from hand-picked embryos of defined stage of development and genotype. In a one-step procedure, the subtraction was 6.5- to 7.25-h wild-type embryos minus 6.5- to 7.25-h twist (twi) zygotic mutant embryos. A two-step procedure in which maternally expressed sequences were subtracted from each of these cDNA libraries, before subtracting twi from wild-type, increased the subtraction efficiency. It resulted in a cDNA population enriched more than 100-fold for mesodermal cDNAs. This was screened by determination of the embryonic expression pattern of each clone in a high throughput procedure and then DNA sequencing. The method, which is comprehensive and does not discriminate against rarer cDNAs, is generally applicable and calculations show that it would work for just 10 embryos. Analysis of one clone, Dmeso18E, that encodes a putative nuclear protein and fulfils the screen's aims is described. It is novel and its expression is mesoderm-specific, twi-dependent, and early during somatic, visceral, and heart muscle differentiation. Two pivotal regulators of mesoderm development and gene expression are Dmef2 and tinman (tin). Analysis of Dmeso18E expression revealed new aspects to their roles: there are effects of Dmef2 on developing muscle much earlier than hitherto believed, and there is tin-independent gene expression in, and invagination of, prospective midgut visceral muscle cells. Dmeso18E expression is regulated by Dmef2, although some expression is Dmef2-independent. The tin-independent and Dmef2-independent expression of Dmeso18E indicates that it either occupies a link between twi and genes like tin and Dmef2 or it lies in a parallel pathway of gene activation.

Titin expression as an early indication of heart and skeletal muscle differentiation in vitro. Developmental re-organisation in relation to cytoskeletal constituents.

Established myogenic cell lines of different species and tissue origin have been used to study expression and organisation of muscle-specific proteins during differentiation. Furthermore, primary cultures of rat myocard cells were used to examine these same processes during dedifferentiation. In particular, we were interested in the general mechanism that underlies the changes in the supramolecular organisation of titin during in vitro myogenesis. It became obvious that in the differentiating muscle cell cultures the redistribution of desmin, actin and myosin in a typical, differentiation state dependent fashion, always showed a certain delay when compared to titin. The sequence of changes in the assembly of cytoskeletal and sarcomeric structures observed during differentiation of the cell lines was reversed during the process of dedifferentiation in cultured rat myocard cells. These results all indicate that titin is an early marker of myogenic differentiation, both in vivo and in vitro, and the typical reorganisation of this giant molecule is independent of species or muscle cell type.

Cardiac differentiation induced by dopamine in undifferentiated cells of early chick embryo

Evidence suggests that neurotransmitters can act as possible chemical signals involved in cell division and morphogenetic movements long before neurons appear in the embryo. However, whether they are playing a role in differentiation is now unknown. It was recently observed ( M. Sarasa and S. Climent, 1987, J. Exp. Zool. 241, 181–190) that the neurotransmitter dopamine exerted a stimulating effect on cardiac differentiation in the chick in ovo. We show here that dopamine acts as a specific inducer of heart muscle differentiation in vitro. When cells of the gastrula of embryos treated with dopamine were dissociated and reaggregated, the aggregates obtained almost entirely underwent cardiac muscle differentiation. Also, when small postnodal pieces obtained from the most posterior region of the gastrula were cultivated in the presence of dopamine, they differentiated into myocardic tissue instead of following their fate map. Therefore, dopamine can trigger a process that both causes undifferentiated cells to differentiate into heart muscle and compels cells already determined to another way of differentiation to become myocardic tissue.

A single troponin T gene regulated by different programs in cardiac and skeletal muscle development.

A cloned complementary DNA derived from a messenger RNA transiently present at low abundance levels in early chick embryonic skeletal muscle hybridizes to a messenger RNA present at high abundance levels in cardiac muscle. Genomic DNA hybridization and nucleotide sequence identity of complementary DNA's from both heart and skeletal muscle demonstrate that the messenger RNA's from both sources are encoded by the same gene. The encoded polypeptide is a troponin T sequence which is probably a cardiac isoform. This single copy troponin T isogene is governed by different regulatory programs in heart and skeletal muscle differentiation.

A reexamination of heart muscle differentiation in the postnodal piece of chick blastoderm mediated by exogenous RNA

Previous findings on exogenous RNA-induced heart muscle differentiation in the non-heartforming cultured explants of the chick blastoderm, the postnodal pieces, were reexamined. Some of the changes that characterized the transition in the host tissues were: (i) the formation of highly ordered myofibrils; (ii) the appearance of characteristic cytoplasmic glycogen particles; (iii) a 2.5- and 3.5-fold increase in actin and myosin-like polypetides respectively; (iv) an increase in acetylcholinesterase activity; and (v) the acquisition of spontaneous and rhythmic pulsations. These specific changes appeared only in those explants that received a poly(A)-containing RNA fraction obtained from the 16-day-old chick embryonic heart. Neither synthetic polynucleotides nor a variety of RNA from several sources could replace the RNA from chick embryonic heart as an inducer of heart muscle differentiation.

Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.

“Conditioned” medium and heart muscle differentiation: Contrast between explants and disaggregated cells in chemically defined medium

“Conditioned” medium and heart muscle differentiation: Contrast between explants and disaggregated cells in chemically defined medium