The Role of MicroRNAs in Dilated Cardiomyopathy: New Insights for an Old Entity.

Dilated cardiomyopathy (DCM) is a clinical diagnosis characterized by left ventricular or biventricular dilation and systolic dysfunction. In most cases, DCM is progressive, leading to heart failure (HF) and death. This cardiomyopathy has been considered a common and final phenotype of several entities. DCM occurs when cellular pathways fail to maintain the pumping function. The etiology of this disease encompasses several factors, such as ischemia, infection, autoimmunity, drugs or genetic susceptibility. Although the prognosis has improved in the last few years due to red flag clinical follow-up, early familial diagnosis and ongoing optimization of treatment, due to its heterogeneity, there are no targeted therapies available for DCM based on each etiology. Therefore, a better understanding of the mechanisms underlying the pathophysiology of DCM will provide novel therapeutic strategies against this cardiac disease and their different triggers. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play key roles in post-transcriptional gene silencing by targeting mRNAs for translational repression or, to a lesser extent, degradation. A growing number of studies have demonstrated critical functions of miRNAs in cardiovascular diseases (CVDs), including DCM, by regulating mechanisms that contribute to the progression of the disease. Herein, we summarize the role of miRNAs in inflammation, endoplasmic reticulum (ER) stress, oxidative stress, mitochondrial dysfunction, autophagy, cardiomyocyte apoptosis and fibrosis, exclusively in the context of DCM.

Regulation of Epicardial Cell Fate during Cardiac Development and Disease: An Overview.

The epicardium is the outermost cell layer in the vertebrate heart that originates during development from mesothelial precursors located in the proepicardium and septum transversum. The epicardial layer plays a key role during cardiogenesis since a subset of epicardial-derived cells (EPDCs) undergo an epithelial-mesenchymal transition (EMT); migrate into the myocardium; and differentiate into distinct cell types, such as coronary vascular smooth muscle cells, cardiac fibroblasts, endothelial cells, and presumably a subpopulation of cardiomyocytes, thus contributing to complete heart formation. Furthermore, the epicardium is a source of paracrine factors that support cardiac growth at the last stages of cardiogenesis. Although several lineage trace studies have provided some evidence about epicardial cell fate determination, the molecular mechanisms underlying epicardial cell heterogeneity remain not fully understood. Interestingly, seminal works during the last decade have pointed out that the adult epicardium is reactivated after heart damage, re-expressing some embryonic genes and contributing to cardiac remodeling. Therefore, the epicardium has been proposed as a potential target in the treatment of cardiovascular disease. In this review, we summarize the previous knowledge regarding the regulation of epicardial cell contribution during development and the control of epicardial reactivation in cardiac repair after damage.

MiRNAs and Muscle Regeneration: Therapeutic Targets in Duchenne Muscular Dystrophy.

microRNAs (miRNAs) are small non-coding RNAs required for the post-transcriptional control of gene expression. MicroRNAs play a critical role in modulating muscle regeneration and stem cell behavior. Muscle regeneration is affected in muscular dystrophies, and a critical point for the development of effective strategies for treating muscle disorders is optimizing approaches to target muscle stem cells in order to increase the ability to regenerate lost tissue. Within this framework, miRNAs are emerging as implicated in muscle stem cell response in neuromuscular disorders and new methodologies to regulate the expression of key microRNAs are coming up. In this review, we summarize recent advances highlighting the potential of miRNAs to be used in conjunction with gene replacement therapies, in order to improve muscle regeneration in the context of Duchenne Muscular Dystrophy (DMD).

The prefoldin bud27 mediates the assembly of the eukaryotic RNA polymerases in an rpb5-dependent manner.

The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.

Identification of regulatory elements directing miR-23a-miR-27a-miR-24-2 transcriptional regulation in response to muscle hypertrophic stimuli.

MiRNAs are small non-coding RNAs that significantly regulate the translation of protein coding genes in higher organisms. MicroRNAs are involved in almost every biological process, including early development, lineage commitment, growth and differentiation, cell death, and metabolic control. Misregulation of miRNAs belonging to the intergenic miR-23a-miR-27a-miR-24-2 cluster has been recently associated to cardiac and skeletal muscle diseases, and they are up-regulated in hypertrophic cardiomyopathy and skeletal muscle atrophy. Despite these facts, the basal transcriptional regulation of miR-23a/miR-27-a/miR-24-2 cluster and how it is altered under pathological conditions remain unclear. In this study, we identified and functionally characterized conserved upstream and downstream regulatory sequences from the miR-23a-miR-27a-miR-24-2 locus that are implicated on its transcriptional control. Our data demonstrate that Srf plays a pivotal role in modulating miR-23a-miR-27a-miR-24-2 cluster proximal promoter activity. Importantly, pro-hypertrophic signalling pathways such as those driven by angiotensin II and norepinephrine also regulate miR-23a-miR-27a-miR-24-2 cluster proximal promoter activity. Taking together, our results provide new insights into the regulatory networks driving miR-23a-miR-27a-miR-24-2 cluster expression in cardiac and skeletal muscles.

Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients.

Myotonic dystrophy type 1 (DM1) is caused by the expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Several missplicing events and transcriptional alterations have been described in DM1 patients. A large number of these defects have been reproduced in animal models expressing CTG repeats alone. Recent studies have also reported miRNA dysregulation in DM1 patients. In this work, a Drosophila model was used to investigate miRNA transcriptome alterations in the muscle, specifically triggered by CTG expansions. Twenty miRNAs were differentially expressed in CTG-expressing flies. Of these, 19 were down-regulated, whereas 1 was up-regulated. This trend was confirmed for those miRNAs conserved between Drosophila and humans (miR-1, miR-7 and miR-10) in muscle biopsies from DM1 patients. Consistently, at least seven target transcripts of these miRNAs were up-regulated in DM1 skeletal muscles. The mechanisms involved in dysregulation of miR-7 included a reduction of its primary precursor both in CTG-expressing flies and in DM1 patients. Additionally, a regulatory role for Muscleblind (Mbl) was also suggested for miR-1 and miR-7, as these miRNAs were down-regulated in flies where Mbl had been silenced. Finally, the physiological relevance of miRNA dysregulation was demonstrated for miR-10, since over-expression of this miRNA in Drosophila extended the lifespan of CTG-expressing flies. Taken together, our results contribute to our understanding of the origin and the role of miRNA alterations in DM1.

Role of miRNA-mRNA Interactome in Pathophysiology of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy is an inherited entity characterized by irregular cell-cell adhesion, cardiomyocyte death and fibro-fatty replacement of ventricular myocytes, leading to malignant ventricular arrythmias, contractile dysfunction and sudden cardiac death. Pathogenic variants in genes that encode desmosome are the predominant cause of arrhythmogenic cardiomyopathy. Moreover, signalling pathways such as Wnt/ß-catenin and transforming growth factor-ß have been involved in the disease progression. However, still little is known about the molecular pathophysiological mechanisms that underlie arrhythmogenic cardiomyopathy pathogenesis. We used mRNA and small RNA sequencing to analyse the transcriptome of health and arrhythmogenic cardiomyopathy of autopsied human hearts. Our results showed 697 differentially expressed genes and eight differentially expressed miRNAs. Functional enrichment revealed mitochondrial respiratory-related pathways, impaired response to oxidative stress, apoptotic signalling pathways and inflammatory response-related and extracellular matrix response pathways. Furthermore, analysis of the miRNA-mRNA interactome identified eleven negatively correlated miRNA-target pairs for arrhythmogenic cardiomyopathy. Our finding revealed novel arrhythmogenic cardiomyopathy-related miRNAs with important regulatory function in disease pathogenesis, highlighting their value as potential key targets for therapeutic approaches.

Unraveling the Etiology of Dilated Cardiomyopathy through Differential miRNA-mRNA Interactome.

Dilated cardiomyopathy (DCM) encompasses various acquired or genetic diseases sharing a common phenotype. The understanding of pathogenetic mechanisms and the determination of the functional effects of each etiology may allow for tailoring different therapeutic strategies. MicroRNAs (miRNAs) have emerged as key regulators in cardiovascular diseases, including DCM. However, their specific roles in different DCM etiologies remain elusive. Here, we applied mRNA-seq and miRNA-seq to identify the gene and miRNA signature from myocardial biopsies from four patients with DCM caused by volume overload (VCM) and four with ischemic DCM (ICM). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were used for differentially expressed genes (DEGs). The miRNA-mRNA interactions were identified by Pearson correlation analysis and miRNA target-prediction programs. mRNA-seq and miRNA-seq were validated by qRT-PCR and miRNA-mRNA interactions were validated by luciferase assays. We found 112 mRNAs and five miRNAs dysregulated in VCM vs. ICM. DEGs were positively enriched for pathways related to the extracellular matrix (ECM), mitochondrial respiration, cardiac muscle contraction, and fatty acid metabolism in VCM vs. ICM and negatively enriched for immune-response-related pathways, JAK-STAT, and NF-kappa B signaling. We identified four pairs of negatively correlated miRNA-mRNA: miR-218-5p-DDX6, miR-218-5p-TTC39C, miR-218-5p-SEMA4A, and miR-494-3p-SGMS2. Our study revealed novel miRNA-mRNA interaction networks and signaling pathways for VCM and ICM, providing novel insights into the development of these DCM etiologies.

Evaluation of pro-regenerative and anti-inflammatory effects of isolecanoric acid in the muscle: Potential treatment of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating degenerative disease of skeletal muscles caused by loss of dystrophin, a key protein that maintains muscle integrity, which leads to progressive muscle degeneration aggravated by chronic inflammation, muscle stem cells' (MuSCs) reduced regenerative capacity and replacement of muscle with fibroadipose tissue. Previous research has shown that pharmacological GSK-3ß inhibition favors myogenic differentiation and plays an important role in modulating inflammatory processes. Isolecanoric acid (ILA) is a natural product isolated from a fungal culture displaying GSK-3ß inhibitory properties. The present study aimed to investigate the proregenerative and anti-inflammatory properties of this natural compound in the DMD context. Our results showed that ILA markedly promotes myogenic differentiation of myoblasts by increasing ß-Catenin signaling and boosting the myogenic potential of mouse and human stem cells. One important finding was that the GSK-3ß/ß-Catenin pathway is altered in dystrophic mice muscle and ILA enhances the myofiber formation of dystrophic MuSCs. Treatment with this natural compound improves muscle regeneration of dystrophic mice by, in turn, improving functional performance. Moreover, ILA ameliorates the inflammatory response in both muscle explants and the macrophages isolated from dystrophic mice to, thus, mitigate fibrosis after muscle damage. Overall, we show that ILA modulates both inflammation and muscle regeneration to, thus, contribute to improve the dystrophic phenotype.

Intron retention and transcript chimerism conserved across mammals: Ly6g5b and Csnk2b-Ly6g5b as examples.

BACKGROUND: Alternative splicing (AS) is a major mechanism for modulating gene expression of an organism, allowing the synthesis of several structurally and functionally distinct mRNAs and protein isoforms from a unique gene. Related to AS is the Transcription Induced Chimerism (TIC) or Tandem Chimerism, by which chimeric RNAs between adjacent genes can be found, increasing combinatorial complexity of the proteome. The Ly6g5b gene presents particular behaviours in its expression, involving an intron retention event and being capable to form RNA chimera transcripts with the upstream gene Csnk2b. We wanted to characterise these events more deeply in four tissues in six different mammals and analyse their protein products. RESULTS: While canonical Csnk2b isoform was widely expressed, Ly6g5b canonical isoform was less ubiquitous, although the Ly6g5b first intron retained transcript was present in all the tissues and species analysed. Csnk2b-Ly6g5b chimeras were present in all the samples analysed, but with restricted expression patterns. Some of these chimeric transcripts maintained correct structural domains from Csnk2b and Ly6g5b. Moreover, we found Csnk2b, Ly6g5b, and Csnk2b-Ly6g5b transcripts that present exon skipping, alternative 5' and 3' splice site and intron retention events. These would generate truncated or aberrant proteins whose role remains unknown. Some chimeric transcripts would encode CSNK2B proteins with an altered C-terminus, which could affect its biological function broadening its substrate specificity. Over-expression of human CSNK2B, LY6G5B, and CSNK2B-LY6G5B proteins, show different patterns of post-translational modifications and cell distribution. CONCLUSIONS: Ly6g5b intron retention and Csnk2b-Ly6g5b transcript chimerism are broadly distributed in tissues of different mammals.

Pitx2c modulates Pax3+/Pax7+ cell populations and regulates Pax3 expression by repressing miR27 expression during myogenesis.

Pitx2 is a paired-related homeobox gene that is expressed in muscle progenitors during myogenesis. We have previously demonstrated that overexpression of Pitx2c isoform in myoblasts maintained these cells with a high proliferative capacity and completely blocked terminal differentiation by inducing high Pax3 expression levels (Martinez et al., 2006). We now report that Pitx2c-mediated proliferation vs. differentiation effect is maintained during in vivo myogenesis. In vivo Pitx2c loss of function leads to a decrease in Pax3+/Pax7- cell population in the embryo accompanied by an increase of Pax3+/Pax7+ cells. Pitx2c transient-transfection experiments further supported the notion that Pitx2c can modulate Pax3/Pax7 expression. Pitx2c but not Pitx3 controls Pax3/Pax7 expression, although redundant roles are elicited at the terminal myoblast differentiation. Contrary to Pitx2c, Pitx3 does not regulate cell proliferation or Pax3 expression, demonstrating the specificity of Pitx2c mediating these actions in myoblasts. Furthermore we demonstrated that Pitx2c modulates Pax3 by repressing miR27 expression and that Pax3-miR-27 modulation mediated by Pitx2c is independent of Pitx2c effects on cell proliferation. Therefore, this study sheds light on previously unknown function of Pitx2c balancing the different myogenic progenitor populations during myogenesis.

Pitx2 Differentially Regulates the Distinct Phases of Myogenic Program and Delineates Satellite Cell Lineages During Muscle Development.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors' fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

miR-106b is a novel target to promote muscle regeneration and restore satellite stem cell function in injured Duchenne dystrophic muscle.

Satellite cells (SCs), muscle stem cells, display functional heterogeneity, and dramatic changes linked to their regenerative capabilities are associated with muscle-wasting diseases. SC behavior is related to endogenous expression of the myogenic transcription factor MYF5 and the propensity to enter into the cell cycle. Here, we report a role for miR-106b reinforcing MYF5 inhibition and blocking cell proliferation in a subset of highly quiescent SC population. miR-106b down-regulation occurs during SC activation and is required for proper muscle repair. In addition, miR-106b is increased in dystrophic mice, and intramuscular injection of antimiR in injured mdx mice enhances muscle regeneration promoting transcriptional changes involved in skeletal muscle differentiation. miR-106b inhibition promotes the engraftment of human muscle stem cells. Furthermore, miR-106b is also high in human dystrophic muscle stem cells and its inhibition improves intrinsic proliferative defects and increases their myogenic potential. This study demonstrates that miR-106b is an important modulator of SC quiescence, and that miR-106b may be a new target to develop therapeutic strategies to promote muscle regeneration improving the regenerative capabilities of injured dystrophic muscle.

Pitx2c modulates cardiac-specific transcription factors networks in differentiating cardiomyocytes from murine embryonic stem cells.

AIM: The knowledge of the molecular signals that control cell differentiation into cardiomyocytes is critical to apply cell-based therapies and repair an injured heart. The transcription factor Pitx2 has essential roles in the development of different organs including the heart. Although a direct role of Pitx2 in the developing myocardium has recently been reported, the molecular pathways driven by Pitx2 as well as its cardiac target genes remain largely unexplored. The aim of this study was to unravel the molecular mechanisms driven by Pitx2 during the process of cardiomyocyte differentiation in vitro in mouse embryonic stem cell-derived cardiomyocytes. METHODS AND RESULTS: Pitx2c was overexpressed in the R1-embryonic stem cell line. mRNA levels and protein distribution of several specific cardiac genes were analyzed by real-time PCR and immunohistochemistry experiments in R1-embryonic stem cell-derived beating areas at different stages of in vitro differentiation. Our results show that overexpression of Pitx2c in embryonic stem cell-derived cardiomyocytes is able to dynamically upregulate several cardiac-enriched transcription factors such as Isl1, Mef2c and Gata4. Additionally, Pitx2c induces the expression of chamber-specific cardiac genes such as Tbx5, Nppa and Cx40. These data were validated in an in vivo model of Pitx2 loss of function. CONCLUSION: Taken together, these results demonstrate that Pitx2 plays a major role reinforcing the transcriptional program of cardiac differentiation.

Muscle Satellite Cell Heterogeneity: Does Embryonic Origin Matter?

Muscle regeneration is an important homeostatic process of adult skeletal muscle that recapitulates many aspects of embryonic myogenesis. Satellite cells (SCs) are the main muscle stem cells responsible for skeletal muscle regeneration. SCs reside between the myofiber basal lamina and the sarcolemma of the muscle fiber in a quiescent state. However, in response to physiological stimuli or muscle trauma, activated SCs transiently re-enter the cell cycle to proliferate and subsequently exit the cell cycle to differentiate or self-renew. Recent evidence has stated that SCs display functional heterogeneity linked to regenerative capability with an undifferentiated subgroup that is more prone to self-renewal, as well as committed progenitor cells ready for myogenic differentiation. Several lineage tracing studies suggest that such SC heterogeneity could be associated with different embryonic origins. Although it has been established that SCs are derived from the central dermomyotome, how a small subpopulation of the SCs progeny maintain their stem cell identity while most progress through the myogenic program to construct myofibers is not well understood. In this review, we synthesize the works supporting the different developmental origins of SCs as the genesis of their functional heterogeneity.

Differential Spatio-Temporal Regulation of T-Box Gene Expression by microRNAs during Cardiac Development.

Cardiovascular development is a complex process that starts with the formation of symmetrically located precardiac mesodermal precursors soon after gastrulation and is completed with the formation of a four-chambered heart with distinct inlet and outlet connections. Multiple transcriptional inputs are required to provide adequate regional identity to the forming atrial and ventricular chambers as well as their flanking regions; i.e., inflow tract, atrioventricular canal, and outflow tract. In this context, regional chamber identity is widely governed by regional activation of distinct T-box family members. Over the last decade, novel layers of gene regulatory mechanisms have been discovered with the identification of non-coding RNAs. microRNAs represent the most well-studied subcategory among short non-coding RNAs. In this study, we sought to investigate the functional role of distinct microRNAs that are predicted to target T-box family members. Our data demonstrated a highly dynamic expression of distinct microRNAs and T-box family members during cardiogenesis, revealing a relatively large subset of complementary and similar microRNA-mRNA expression profiles. Over-expression analyses demonstrated that a given microRNA can distinctly regulate the same T-box family member in distinct cardiac regions and within distinct temporal frameworks, supporting the notion of indirect regulatory mechanisms, and dual luciferase assays on Tbx2, Tbx3 and Tbx5 3' UTR further supported this notion. Overall, our data demonstrated a highly dynamic microRNA and T-box family members expression during cardiogenesis and supported the notion that such microRNAs indirectly regulate the T-box family members in a tissue- and time-dependent manner.

The Role of Bmp- and Fgf Signaling Modulating Mouse Proepicardium Cell Fate.

Bmp and Fgf signaling are widely involved in multiple aspects of embryonic development. More recently non coding RNAs, such as microRNAs have also been reported to play essential roles during embryonic development. We have previously demonstrated that microRNAs, i.e., miR-130, play an essential role modulating Bmp and Fgf signaling during early stages of cardiomyogenesis. More recently, we have also demonstrated that microRNAs are capable of modulating cell fate decision during proepicardial/septum transversum (PE/ST) development, since over-expression of miR-23 blocked while miR-125, miR-146, miR-223 and miR-195 enhanced PE/ST-derived cardiomyogenesis, respectively. Importantly, regulation of these microRNAs is distinct modulated by Bmp2 and Fgf2 administration in chicken. In this study, we aim to dissect the functional role of Bmp and Fgf signaling during mouse PE/ST development, their implication regulating post-transcriptional modulators such as microRNAs and their impact on lineage determination. Mouse PE/ST explants and epicardial/endocardial cell cultures were distinctly administrated Bmp and Fgf family members. qPCR analyses of distinct microRNAs, cardiomyogenic, fibrogenic differentiation markers as well as key elements directly epithelial to mesenchymal transition were evaluated. Our data demonstrate that neither Bmp2/Bmp4 nor Fgf2/Fgf8 signaling is capable of inducing cardiomyogenesis, fibrogenesis or inducing EMT in mouse PE/ST explants, yet deregulation of several microRNAs is observed, in contrast to previous findings in chicken PE/ST. RNAseq analyses in mouse PE/ST and embryonic epicardium identified novel Bmp and Fgf family members that might be involved in such cell fate differences, however, their implication on EMT induction and cardiomyogenic and/or fibrogenic differentiation is limited. Thus our data support the notion of species-specific differences regulating PE/ST cardiomyogenic lineage commitment.

Deletion of the Wilms' Tumor Suppressor Gene in the Cardiac Troponin-T Lineage Reveals Novel Functions of WT1 in Heart Development.

Expression of Wilms' tumor suppressor transcription factor (WT1) in the embryonic epicardium is essential for cardiac development, but its myocardial expression is little known. We have found that WT1 is expressed at low levels in 20-25% of the embryonic cardiomyocytes. Conditional ablation of WT1 using a cardiac troponin T driver (Tnnt2 Cre ) caused abnormal sinus venosus and atrium development, lack of pectinate muscles, thin ventricular myocardium and, in some cases, interventricular septum and cardiac wall defects, ventricular diverticula and aneurisms. Coronary development was normal and there was not embryonic lethality, although survival of adult mutant mice was reduced probably due to perinatal mortality. Adult mutant mice showed electrocardiographic anomalies, including increased RR and QRS intervals, and decreased PR intervals. RNASeq analysis identified differential expression of 137 genes in the E13.5 mutant heart as compared to controls. GO functional enrichment analysis suggested that both calcium ion regulation and modulation of potassium channels are deeply altered in the mutant myocardium. In summary, together with its essential function in the embryonic epicardium, myocardial WT1 expression is also required for normal cardiac development.

Isolation and Culture of Quiescent Skeletal Muscle Satellite Cells.

It has been shown that freshly isolated satellite cells from adult muscle constitute a stem cell-like population that exhibits more efficient engraftment and self-renewal activity in regenerating muscle than myoblast. Thus, purification of pure populations of quiescent satellite cells from adult skeletal muscle is highly necessary, not only for understanding the biology of satellite cells and myoblasts but also for improving cell-based therapies for muscle regeneration. This chapter describes a basic protocol used in our laboratory to isolate quiescent muscle satellite cells from adult skeletal muscle by enzymatic dissociation followed by a sequential magnetic-activated cell sorting (MACS). This method is cheap and fast providing and alternative procedure to other purification methods that require fluorescence-activated cell sorting (FACS) machines. Freshly isolated quiescent satellite cells purified by this method can be used in a broad range of experiments including cell transplantation for satellite cell self-renewal experiments or cell therapies.

MiR-195 enhances cardiomyogenic differentiation of the proepicardium/septum transversum by Smurf1 and Foxp1 modulation.

Cardiovascular development is a complex developmental process in which multiple cell lineages are involved, namely the deployment of first and second heart fields. Beside the contribution of these cardiogenic fields, extracardiac inputs to the developing heart are provided by the migrating cardiac neural crest cells and the proepicardial derived cells. The proepicardium (PE) is a transitory cauliflower-like structure located between the cardiac and hepatic primordia. The PE is constituted by an internal mesenchymal component surrounded by an external epithelial lining. With development, cells derived from the proepicardium migrate to the neighboring embryonic heart and progressive cover the most external surface, leading to the formation of the embryonic epicardium. Experimental evidence in chicken have nicely demonstrated that epicardial derived cells can distinctly contribute to fibroblasts, endothelial and smooth muscle cells. Surprisingly, isolation of the developing PE anlage and ex vivo culturing spontaneously lead to differentiation into beating cardiomyocytes, a process that is enhanced by Bmp but halted by Fgf administration. In this study we provide a comprehensive characterization of the developmental expression profile of multiple microRNAs during epicardial development in chicken. Subsequently, we identified that miR-125, miR-146, miR-195 and miR-223 selectively enhance cardiomyogenesis both in the PE/ST explants as well as in the embryonic epicardium, a Smurf1- and Foxp1-driven process. In addition we identified three novel long non-coding RNAs with enhanced expression in the PE/ST, that are complementary regulated by Bmp and Fgf administration and well as by microRNAs that selectively promote cardiomyogenesis, supporting a pivotal role of these long non coding RNAs in microRNA-mediated cardiomyogenesis of the PE/ST cells.