Cardiac Muscle Differentiation Research Articles

Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-β induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-β stimulation at nucleus, suggesting that TGF-β facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

The role of histone deacetylases in embryonic development.

The basic units of chromatin are nucleosomes, that are made up of DNA wrapped around histone cores. Histone lysine residue is a common location for posttranslational modifications, with acetylation being the second most prevalent. Histone acetyltransferases (HATs/KATs) and histone deacetylases (HDACs/KDACs) regulate histone acetylation, which is important in gene expression control. HDACs/KDACsregulate gene expressions through the repression of the transcription machinery. HDAC/KDAC isoforms play a major role during various stages of embryo development and neurogenesis. In specific, class I and II HDACs/KDACs are involved in cardiac muscle differentiation and development. An insight into different pathways and genes associated with embryonic development, the effect of HDAC/KDAC activity during the embryonic stem cell differentiation, preimplantation, embryo development, gastrulation, and the role of different HDAC/KDAC inhibitors during the process of embryogenesis is summarized in the present review article.

From Spheroids to Organoids: The Next Generation of Model Systems of Human Cardiac Regeneration in a Dish.

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.

Secretome signature of cardiopoietic cells echoed in rescued infarcted heart proteome.

Stem cell paracrine activity is implicated in cardiac repair. Linkage between secretome functionality and therapeutic outcome was here interrogated by systems analytics of biobanked human cardiopoietic cells, a regenerative biologic in advanced clinical trials. Protein chip array identified 155 proteins differentially secreted by cardiopoietic cells with clinical benefit, expanded into a 520 node network, collectively revealing inherent vasculogenic properties along with cardiac and smooth muscle differentiation and development. Next generation RNA sequencing, refined by pathway analysis, pinpointed miR‐146 dependent regulation upstream of the decoded secretome. Intracellular and extracellular integration unmasked commonality across cardio‐vasculogenic processes. Mirroring the secretome pattern, infarcted hearts benefiting from cardiopoietic cell therapy restored the disease proteome engaging cardiovascular system functions. The cardiopoietic cell secretome thus confers a therapeutic molecular imprint on recipient hearts, with response informed by predictive systems profiling.

In This Issue

HomeCirculation ResearchVol. 127, No. 2In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Jul 2020https://doi.org/10.1161/RES.0000000000000419Circulation Research. 2020;127:204is related toIn Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered OrganoidGlobal Reach 2018 Heightened α-Adrenergic Signaling Impairs Endothelial Function During Chronic Exposure to Hypobaric Hypoxiais related toIdentification of Functional Variant Enhancers Associated With Atrial FibrillationIn Situ Differentiation to Produce Cardiac Tissues (p 207)Kupfer et al use 3D-printing to create beating human heart organoids.Download figureDownload PowerPointCultured human cardiac muscle is valuable for testing the efficacy and toxicity of drugs, studying diseases, assessing injury responses, and more. But growing such muscle in the lab is tricky. For one thing, adult cardiomyocytes don’t generally proliferate. For another, cells in a dish don’t recapitulate the forces and functions of a 3D heart. Generating cardiomyocytes from stem cells enables researchers to largely circumvent the issue of proliferation, so Kupfer and colleagues have focused their attention on the issue of 3D structure. They developed a photocrosslinkable bioink containing extracellular matrix proteins and pluripotent stem cells that can be printed within a supportive gel bath, and then stabilized with UV-induced crosslinking. After removal of the printed structure from the support gel, the stem cells proliferate to high density and are differentiated into cardiomyocytes. Using this approach, the team printed a chambered human heart organoid—approximately the size of a mouse heart—showing that it could propagate action potentials as well as beat and pump fluid. Furthermore, beat rate was pharmacologically modifiable. By showing the mechanical and electrical activities of this mini-heart, the work sets the stage for using such organoids in all manner of drug and disease studies.Functional Variant Elements in Atrial Fibrillation Models(p 229)van Ouwerkerk et al discover gene regulatory elements at atrial fibrillation associated loci.Download figureDownload PowerPointAtrial fibrillation (AF) is the most common form of arrhythmia and a major risk factor for heart failure, dementia, stroke, and sudden death. Genome-wide association studies have revealed more than 100 genetic loci linked to the condition, many of which are in intergenic or intronic regions and are enriched for transcription factor binding sites and epigenetic modifications suggesting potential gene regulatory roles. To test this idea, Ouwerkerk and colleagues used a method called self-transcribing active regulatory region sequencing (or STARR-Seq) to screen 12 of the strongest AF-linked regions of the genome—together containing more than 1600 AF-linked variants—for their gene regulatory activity in cultured rat atrial myocytes. From this screen, they found ≈400 regulatory elements, of which 24 exhibited variant-specific differences in regulatory activity. For one of these elements—upstream of the gene HCN4—deletion of the orthologous element in mice caused diminished transcriptional activity of the gene. Moreover, the mice had brachycardia and sinus node dysfunction. This proof-of-principle study confirms that such a regulatory element screen can provide insight into the consequences of variants associated with AF or, for that matter, other diseases.Vascular Function in Chronic Hypoxia(p e1)Tymko et al, examine how low oxygen at high altitudes affects vascular endothelium function.Download figureDownload PowerPointChronic hypoxia, whether due to disease or time spent at high altitude, is associated with increased sympathetic nerve activity and poor endothelial function—marked by reduced endothelial-dependent vasodilation. But whether the heightened nerve activity actually causes endothelial dysfunction was unclear. To investigate, Tymko and colleagues analyzed heart rate, nerve activity, and hemodynamics—including blood pressure, flow, and arterial diameter—in 9 people who normally live in lowlands after they spent 2-to-3 weeks at 4300 meters (14 000 feet) in the Andean highlands. The team found that, at high altitude, lowlanders had increased sympathetic nerve activity and reduced endothelial function—assessed by their vasodilatory responses to acetylcholine infusion. But also that administration of adrenergic blockers, which reduce sympathetic nerve activity, improved the subjects’ endothelial function. Similar results were seen in native Andean highlanders with excess red blood cell counts—a mark of endothelial dysfunction—compared with their healthy counterparts. The work provides the first direct link between sympathetic nerve activity and endothelial function as well as insights into the pathophysiological processes associated with low-oxygen conditions. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesIn Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered OrganoidMolly E. Kupfer, et al. Circulation Research. 2020;127:207-224Identification of Functional Variant Enhancers Associated With Atrial FibrillationAntoinette F. van Ouwerkerk, et al. Circulation Research. 2020;127:229-243Global Reach 2018 Heightened α-Adrenergic Signaling Impairs Endothelial Function During Chronic Exposure to Hypobaric HypoxiaMichael M. Tymko, et al. Circulation Research. 2020;127:e1-e13 July 3, 2020Vol 127, Issue 2Article InformationMetrics Download: 200 © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000419 Originally publishedJuly 2, 2020 PDF download

MiR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.

miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid.

One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes-a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell-laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

Effects of Cardiovascular Risk Factors on Cardiac STAT3.

Nuclear, mitochondrial and cytoplasmic signal transducer and activator of transcription 3 (STAT3) regulates many cellular processes, e.g., the transcription or opening of mitochondrial permeability transition pore, and its activity depends on the phosphorylation of Tyr705 and/or Ser727 sites. In the heterogeneous network of cardiac cells, STAT3 promotes cardiac muscle differentiation, vascular element formation and extracellular matrix homeostasis. Overwhelming evidence suggests that STAT3 is beneficial for the heart, plays a role in the prevention of age-related and postpartum heart failure, protects the heart against cardiotoxic doxorubicin or ischaemia/reperfusion injury, and is involved in many cardioprotective strategies (e.g., ischaemic preconditioning, perconditioning, postconditioning, remote or pharmacological conditioning). Ischaemic heart disease is still the leading cause of death worldwide, and many cardiovascular risk factors contribute to the development of the disease. This review focuses on the effects of various cardiovascular risk factors (diabetes, aging, obesity, smoking, alcohol, depression, gender, comedications) on cardiac STAT3 under non-ischaemic baseline conditions, and in settings of ischaemia/reperfusion injury with or without cardioprotective strategies.

Injectable silk fibroin hydrogels functionalized with microspheres as adult stem cells-carrier systems

Hydrogels are good candidate materials for cell delivery scaffolds because they can mimic the physical, chemical, electrical and biological properties of most of the native tissues. In this study, composite biosynthetic hydrogels were produced by combining the bio-functionality of silk fibroin (SF) with the structural versatility of polyethylene-glycol-diacrylated (PEGDa). The formation of a photopolymerizable PEGDa-SF hydrogel (PSFHy) was optimized for 3D-cell culture. Functionalization of the 3D-PSFHy with protein microspheres (MS) was required to increase the porosity and cell-adhesive properties of the material. Cardiac mesenchymal stem cells, which were cultured within the MS-embedding PSFHy, exhibited good viability and expression of proteins that are characteristic of the initial phases of the cardiac muscle differentiation process. Further, the addition of chondroitin sulfate into the scaffolds improved the cell viability. A cell-preconditioning of the scaffold was also performed, suggesting a potential application of these sponge-like scaffolds for analysing the effects of several extracellular microenvironments, produced by different kinds of cells, on the stem cells fate. The results presented herein highlight on the possibility to use the PSFHys functionalized with MS as stem cell-carrier systems with sponge-like properties, potential ultrasound-imaging contrast agents and controlled biochemical factor delivery.

Actin isoform expression patterns in adult extracardiac and cardiac rhabdomyomas indicate a different cell of origin.

Rhabdomyomas are rare striated muscle-type tumors arising in the heart or in soft tissues. Using a monoclonal antibody specific for the cardiac isoform of α-actin (α-cardiac actin, α-CAA), differential expression patterns in striated muscle tissues were reported previously. The purpose of the present study was to determine whether the α-actin isoform specificity is maintained in rhabdomyomas according to their origin, comparing extracardiac to cardiac rhabdomyomas. We immunohistochemically investigated adult extracardiac (pharyngeal) rhabdomyomas (n=4) and cardiac rhabdomyomas (n=7) employing isoform-specific monoclonal antibodies. The extracardiac rhabdomyomas revealed only a few scattered α-CAA-positive tumor cells (antibody cAc1-20.42) while the cardiac rhabdomyomas exhibited abundant expression of α-CAA, indicating a close relatedness to cardiac muscle fibers. The α-skeletal actin (α-SKA) specific monoclonal antibody (3B3) produced the reverse results. General sarcomeric antibodies (HHF35 and Alpha Sr-1) displayed strong positivity in all rhabdomyomas studied. Alpha-smooth muscle actin (α-SMA) was negative or heterogeneously positive in extracardiac and cardiac rhabdomyomas. Our results suggest that despite similar morphology, the intrinsic differential alpha-actin isoform specificity of mature skeletal vs. cardiac muscle is maintained in extracardiac and cardiac rhabdomyomas. Thus, adult extracardiac rhabdomyomas differentiate towards mature skeletal muscle although they may exhibit centrally placed nuclei like cardiac muscle cells, while cardiac rhabdomyomas reflect true cardiac muscle differentiation. Our findings appear to indicate a different biological nature of cardiac and extracardiac rhabdomyomas, probably related to a different cell of origin. To our knowledge, this is the first report suggesting a derivation of extracardiac and cardiac rhabdomyomas from skeletal and cardiac muscle cells, respectively.

心外膜下脂肪由来脱分化脂肪細胞の心筋分化能の検討

成熟脂肪細胞から調製される脱分化脂肪細胞(DFAT) は，高い増殖能と間葉系幹細胞に類似した多分化能を有することが明らかにされている．本研究では，ブタ心外膜下脂肪組織から調製したDFAT (EC-DFAT) の心筋細胞への分化指向性を検討した．In vitro 分化誘導実験では，EC-DFAT は皮下脂肪由来DFAT (SC-DFAT) と同様に脂肪，骨，軟骨，平滑筋への分化能を示した．EC-DFAT はSC-DFAT に比べ， 心筋特異的転写因子GATA4 の発現が有意に高く，ラット新生仔心筋細胞との共培養により，心筋特異的蛋白質であるNkx2.5 および心筋型トロポニンI や，ギャップ結合蛋白質であるコネキシン43 の発現が強く認められた．以上よりECDFAT は心筋細胞への高い分化指向性を示すことが明らかになった．EC-DFAT は心筋再生用の細胞源として有用である可能性が示唆された．

The Cytomegalovirus Enhancer Induces an Immediate Response to the Myosin Light Chain 2v Promoter during P19CL6 Cell Differentiation

The P19CL6 mouse embryonic carcinoma cells efficiently differentiate into cardiac muscle cells in the presence of DMSO. A reporter plasmid for cardiac muscle differentiation was constructed by connecting the CMV enhancer and a 250 bp MLC-2v promoter in front of the GFP gene to further evaluate the role of the CMV enhancer. This plasmid (pCBVenh/MLC-2vpro/EGFP) was stably introduced into P19CL6 cells, and the transfectant differentiated into cardiomyocytes with DMSO. Upon DMSO addition, GFP was immediately transcribed (within 2 days) and the amount of the transcript increased with cultivation. Concomitantly, GFP fluorescence was detected in the cells under a microscope. However, native MLC-2v was transcribed later on day 4. This expression time course is different from that of GFP. Clearly the CMV enhancer responded immediately to DMSO. Since GATA DNA-binding proteins play crucial roles in the initiation of cardiomyocyte differentiation, such a response could be ascribed to the presence of multiple GATA motifs in the enhancer sequence but not in the native MLC-2v promoter. Thus the CMV enhancer may be not only useful for gene therapy and monitoring cell differentiation but also the study of the role of GATA transcription factors expressed in P19CL6 cells.

New Trends in Heart Regeneration: A Review

In this review, we focus on new approaches that could lead to the regeneration of heart muscle and the restoration of cardiac muscle function derived from newly-formed cardiomyocytes. Various strategies for the production of cardiomyocytes from embryonic stem cells, induced pluripotent stem cells, adult bone marrow stem cells and cardiac spheres from human heart biopsies are described. Pathological conditions which lead to atherosclerosis and coronary artery disease often are followed by myocardial infarction causing myocardial cell death. After cell death, there is very little self-regeneration of the cardiac muscle tissue, which is replaced by non-contractile connective tissue, thus weakening the ability of the heart muscle to contract fully and leading to heart failure. A number of experimental research approaches to stimulate heart muscle regeneration with the hope of regaining normal or near normal heart function in the damaged heart muscle have been attempted. Some of these very interesting studies have used a variety of stem cell types in combination with potential cardiogenic differentiation factors in an attempt to promote differentiation of new cardiac muscle for possible future use in the clinical treatment of patients who have suffered heart muscle damage from acute myocardial infarctions or related cardiovascular diseases. Although progress has been made in recent years relative to promoting the differentiation of cardiac muscle tissue from non-muscle cells, much work remains to be done for this technology to be used routinely in translational clinical medicine to treat patients with damaged heart muscle tissue and return such individuals to pre-heart-attack activity levels.

Generation of a mef2aa:EGFP transgenic zebrafish line that expresses EGFP in muscle cells.

Transgenesis is an important tool for exploring gene expression and function. The myocyte enhancer factor 2a (mef2a) gene encodes a member of the Mef2 protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to studies on human and animal models, mef2a is highly expressed in the heart and somites. To explore the potential of mef2a as a tool for selective labeling of muscle cells in living zebrafish embryos, we constructed a transgene mef2aa:EGFP to induce the expression of green fluorescent protein (GFP) under the control of mef2a promoter. A ~2-kb DNA fragment, upstream of the translational start site of mef2aa, was identified to drive muscle-specific expression of EGFP in zebrafish embryos. Interestingly, the cranial muscles, abductor muscle, and adductor muscle were clearly labeled with EGFP in the established line Tg(mef2aa:EGFP) ntu803 . In addition, we showed that mef2aa mRNA was highly present in adult zebrafish heart, but not the skeleton muscle, whereas it was expressed in both embryonic heart and myotome, suggesting that mef2a is vital to the function of adult heart in vertebrates.

Pm-miR-133 hosting in one potential lncRNA regulates RhoA expression in pearl oyster Pinctada martensii

Long non-coding RNAs (LncRNAs) are abundant in the genome of higher forms of eukaryotes and implicated in regulating the diversity of biological processes partly because they host microRNAs (miRNAs), which are repressors of target gene expression. In vertebrates, miR-133 regulates the differentiation and proliferation of cardiac and skeletal muscles. Pinctada martensii miR-133 (pm-miR-133) was identified in our previous research through Solexa deep sequencing. In the present study, the precise sequence of mature pm-miR-133 was validated through miR-RACE. Stem loop qRT-PCR analysis demonstrated that mature pm-miR-133 was constitutively expressed in the adductor muscle, gonad, hepatopancreas, mantle, foot, and gill of P. martensii. Among these tissues, the adductor muscle exhibited the highest pm-miR-133 expression. Target analysis indicated that pm-RhoA was the potential regulatory target of pm-miR-133. Bioinformatics analyses revealed that a potential LncRNA (designated as Lnc133) with a mature pm-miR-133 could generate a hairpin structure that was highly homologous to that of Lottia gigantea. Lnc133 was also highly expressed in the adductor muscle, gill, hepatopancreas, and gonad. Phylogenetic analysis further showed that the miR-133s derived from chordate and achordate were separated into two classes. Therefore, Lnc133 hosting pm-miR-133 could be involved in regulating the cell proliferation of adductor muscles by targeting pm-RhoA.

Molecular characterization and expression regulation of Smyd1a and Smyd1b in skeletal muscle of Chinese perch (Siniperca chuatsi).

Smyd1 is a SET and MYND domain-containing protein, which functions as a histone methyltransferase for control of gene expression and regulates the skeletal and cardiac muscle differentiation. In this study, the full-length cDNA sequences of Smyd1a and Smyd1b were cloned from Chinese perch, and their molecular structure and expression profile in response to nutrition supply and in vivo IGF treatments were also analyzed. The cDNA sequence of Smyd1a and Smyd1b consists of 1862 and 1802 base pairs (bp), encoding 479 and 476 amino acids, respectively. The SET domains of the two proteins were split into two segments by the MYND domain. Furthermore, the amino acid sequence of Smyd1a contains an extra 13-aa insertion in the SET domain in comparison with Smyd1b. The two genes apparently exhibited temporal and spatial differential expression status. In adults, the two genes showed the higher expression level in the muscle and heart than in other testing tissues. During the post-embryonic developmental stages, the higher expression of Smyd1a was detected at 150 days post-hatching (dph), whereas the expression of Smyd1b peaked at 50 dph. It was indicated that they have potential function in muscle developmental regulation. The mRNA levels of Smyd1a and Smyd1b were sharply up-regulated within one day after refeeding in the Chinese perch juveniles following fasting for a week. Moreover, IGF-1 treatments in vivo significantly stimulated their expression in the skeletal muscle. Together, these data provide us with further understanding of the molecular characterization and expression regulation of the two genes upon internal and external stimuli.

Development of Bipotent Cardiac/Skeletal Myogenic Progenitors from MESP1+ Mesoderm.

SummaryThe branchiomeric skeletal muscles co-evolved with new chambers of the heart to enable predatory feeding in chordates. These co-evolved tissues develop from a common population in anterior splanchnic mesoderm, referred to as cardiopharyngeal mesoderm (CPM). The regulation and development of CPM are poorly understood. We describe an embryonic stem cell-based system in which MESP1 drives a PDGFRA+ population with dual cardiac and skeletal muscle differentiation potential, and gene expression resembling CPM. Using this system, we investigate the regulation of these bipotent progenitors, and find that cardiac specification is governed by an antagonistic TGFβ-BMP axis, while skeletal muscle specification is enhanced by Rho kinase inhibition. We define transcriptional signatures of the first committed CPM-derived cardiac and skeletal myogenic progenitors, and discover surface markers to distinguish cardiac (PODXL+) from the skeletal muscle (CDH4+) CPM derivatives. These tools open an accessible window on this developmentally and evolutionarily important population.

Mef2d is essential for the maturation and integrity of retinal photoreceptor and bipolar cells.

Mef2 transcription factors play a crucial role in cardiac and skeletal muscle differentiation. We found that Mef2d is highly expressed in the mouse retina and its loss causes photoreceptor degeneration similar to that observed in human retinitis pigmentosa patients. Electroretinograms (ERGs) were severely impaired in Mef2d-/- mice. Immunohistochemistry showed that photoreceptor and bipolar cell synapse protein levels severely decreased in the Mef2d-/- retina. Expression profiling by microarray analysis showed that Mef2d is required for the expression of various genes in photoreceptor and bipolar cells, including cone arrestin, Guca1b, Pde6h and Cacna1s, which encode outer segment and synapse proteins. We also observed that Mef2d synergistically activates the cone arrestin (Arr3) promoter with Crx, suggesting that functional cooperation between Mef2d and Crx is important for photoreceptor cell gene regulation. Taken together, our results show that Mef2d is essential for photoreceptor and bipolar cell gene expression, either independently or cooperatively with Crx.

RBM4-MEF2C network constitutes a feed-forward circuit that facilitates the differentiation of brown adipocytes

Myocyte enhancer factor 2c (MEF2C) is the MADS-box type transcription factor involved in the differentiation of cardiac and skeletal muscle and synaptic formation. Alternatively spliced transcripts of the MEF2C gene were proven to encode isoforms which exert distinct functions in transcriptional regulation. During the differentiation of brown adipocytes, upregulated RBM4 enhanced skipping of the MEF2Cγ region which functions as a transcriptional repressor. The presence of an overexpressed MEF2Cγ- isoform in turn induced transcriptional activity of the RBM4 promoter, constituting a positive feedback circuit in differentiating brown adipocytes. The RBM4-MEF2Cγ- network induced the expression of "myogenic" miR-1 to a greater extent than did PRDM17, BMP7 C/EBPβ, or UCP1 transcripts in C3H10T1/2 cells. Overexpression of miR-1 independently exerted the same activity as RBM4 and the MEF2Cγ- isoform of upregulating brown adipocyte-specific factors in C3H10T1/2 cells, which suggests a potential effect of miR-1 on brown adipocytes. These results indicated that the RBM4-MEF2C-miR-1 network constitutes a novel mechanism which programs the gene expression profile toward the development of brown adipocytes.