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After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are now recognized for taking part in fine-tuning such gene programs. We identified and characterized the cardiomyocyte specific lncRNASweetheart RNA(Swhtr), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding geneNkx2-5. We show thatSwhtris dispensable for normal heart development and function, but becomes essential for the tissue adaptation process after myocardial infarction. Re-expressingSwhtrfrom an exogenous locus rescues theSwhtr nullphenotype. Genes depending onSwhtrafter cardiac stress are significantly occupied, and therefore most likely regulated by NKX2-5. Our results indicate a synergistic role forSwhtrand the developmentally essential transcription factor NKX2-5 in tissue adaptation after myocardial injury.

Cardiovascular disorders such as heart failure are leading causes of mortality. Patient stratification via identification of novel biomarkers could improve management of cardiovascular diseases of complex etiologies. Long-noncoding RNAs (lncRNAs) are highly tissue-specific in nature and have emerged as important biomarkers in human diseases. In this study, we aimed to identify cardiac-enriched lncRNAs as potential biomarkers for cardiovascular conditions. Deep RNA sequencing and quantitative PCR identified differentially expressed lncRNAs between failing and non-failing hearts. An independent dataset was used to evaluate the enrichment of lncRNAs in normal hearts. We identified a panel of 3233 lncRNAs, named FIMICS, that were either cardiac-enriched or differentially expressed between failing and non-failing hearts. Expression of lncRNAs in blood samples differentiated patients with myocarditis and acute myocardial infarction. We hereby present the FIMICS panel, a readily available tool to provide insights into cardiovascular pathologies and which could be helpful for diagnosis, monitoring and prognosis purposes.

Background IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described. Methods In vivo MI was performed by coronary artery occlusion in the absence or presence of a neutralizing IL-17 antibody for blocking IL-17 actions in vivo. IL-17 signaling was also assessed in isolated primary cardiomyocytes by Western blot, mRNA expression and immunostaining. Results Expression of IL-17A, IL-17F and the IL-17 receptor (IL-17RA) were all increased following MI. Expression of several IL-17 target genes, including Cxcl1, Cxcl2, IL-1β, iNOS and IL-6 was also upregulated following MI. In addition, IL-17A promoted the expression of Cxcl1 and IL-6 in isolated cardiomyocytes in a MAPK and PI(3)K-dependent manner. IL-17A and ischaemia/reperfusion (I/R) injury were found to have an additive effect on Cxcl1 expression, suggesting that IL-17 may enhance myocardial neutrophil recruitment during MI. Moreover, protein levels of both IL-17R and IL-17A were enhanced following in vivo MI. Finally, blocking IL-17 signaling in vivo reduced the levels of apoptotic cell death markers following in vivo MI. Conclusions These data imply that the expression of IL-17 cytokines and their receptor are elevated during myocardial I/R injury and may play a fundamental role in post infarct inflammatory and apoptotic responses.

The mechanisms controlling differentiation in adult cardiac precursor cells (CPCs) are still largely unknown. In this study, CPCs isolated from the human heart were found to produce predominantly smooth muscle cells but could be redirected to the cardiomyocyte fate by transient activation followed by inhibition of NOTCH signaling. NOTCH inhibition repressed MIR-143/145 expression, and blocked smooth muscle differentiation. Expression of the microRNAs is under control of CARMEN , a long noncoding RNA associated with an enhancer located in the MIR-143/145 locus and target of NOTCH signaling. The CARMEN / MIR-145/143 axis represents, therefore, a promising target to favor production of cardiomyocytes in cell replacement therapies.

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.

Recent advances in sequencing and genomic technologies have resulted in the discovery of thousands of previously unannotated long noncoding RNAs (lncRNAs). However, their function in the cardiovascular system remains elusive. Here we review and discuss considerations for cardiovascular lncRNA discovery, annotation and functional characterization. Although we primarily focus on the heart, the proposed pipeline should foster functional and mechanistic exploration of these transcripts in various cardiovascular pathologies. Moreover, these insights could ultimately lead to novel therapeutic approaches targeting lncRNAs for the amelioration of cardiovascular diseases including heart failure.

The exquisite transcriptional control of developmental gene programs is critical for hardwiring the complex expression patterns that govern cell-fate determination and differentiation during heart development. During the past several years, studies have illuminated our understanding of a complex noncoding transcriptional landscape, primarily associated with long noncoding RNAs (lncRNAs), that is implicated in these developmental processes and has begun to reveal key functions of these transcripts. In this review, we discuss the expanding roles for lncRNAs in the earliest points of cardiac development and through differentiation and maturation of multiple cell types within the adult heart. We go on to outline the diverse mechanisms by which cardiovascular lncRNAs orchestrate these transcriptional programs, explore the challenges linked to the study of lncRNAs in developmental phenotypes, and summarize the implications for these molecules in human cardiovascular disorders.

Mammalian genomes are pervasively transcribed generating thousands of long noncoding RNAs (lncRNAs) with emergent regulatory roles. Many of these lncRNAs exhibit highly specialised expression patterns during development and typically flank and regulate key developmental factors. In this review, we discuss and summarise the latest advances in our understanding of the roles of lncRNAs during mesendoderm (ME) specification, a key step during gastrulation and the formation of the primitive streak (PS).

Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. Here, we evaluate remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. We sort mesendodermal progenitors from differentiating embryonic stem cells (ESCs) according to Eomes expression, and find that enhancer usage is coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. Many of these enhancers are associated with the expression of lncRNAs. Examination of ESC-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identifies MesEndoderm Transcriptional Enhancer Organizing Region (Meteor). Genetic and epigenetic manipulation of the Meteor enhancer reveal its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and -dependent mechanisms. Interestingly, Meteor-deleted ESCs are epigenetically redirected towards neuroectodermal lineages. Loci, topologically associating a transcribed enhancer and its cognate protein coding gene, appear to represent therefore a class of genomic elements controlling developmental competence in pluripotency., Long noncoding RNAs (lncRNAs) are key regulators of lineage specification during development. Here, the authors investigate remodeling of enhancers and regulation of the lncRNA transcriptome during mesendoderm specification, and identify a pluripotent stage-specific transcribed enhancer controlling adoption of the mesendodermal cell fate.

© 2014 . The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue specific noncoding RNAs yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis we identified and characterized for the first time a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA sequencing demonstrates that many of these transcripts are polyadenylated multi exonic long noncoding RNAs. Moreover knockdown of two enhancer associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly the reactivation of the fetal gene program a hallmark of the stress response in the adult heart is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer derived lncRNAs. •Fetal cardiac enhancers are transcribed generating enhancer derived lncRNAs.

Background IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described.

Heart failure is a life-threatening and costly ailment characterized by structural and functional impairment of the heart. Despite major advances in understanding protein-mediated transcriptional control and signaling pathways that underlie the cellular and interstitial alterations of heart failure, significant therapeutical breakthroughs for innovative treatments of this disease are still missing. The recent extensive profiling of the mammalian transcriptome has revealed a large number of long non-coding RNAs (lncRNAs) that play a diversity of important regulatory roles in gene expression. In here, we focus on a recent work by Ounzain and colleagues comprising genome-wide profiling of the cardiac transcriptome after myocardial infarction with an emphasis on the identification of novel heart-specific lncRNAs.

Aims The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish. Methods and results Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart. Conclusions This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart.

The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance (IR), normally associated with type 2 diabetes (T2D), whereas Brn-3b is reduced in tissues taken from obese mice fed on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3β and reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly transactivated by this transcription factor in cotransfection assays, whereas chromatin immunoprecipitation assays confirm that Brn-3b binds to this promoter in vivo. In addition, correlation between GLUT4 and Brn-3b in KO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4 expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to abnormal glucose uptake.

Cardiac development, function and pathological remodelling in response to stress depend on the dynamic control of tissue specific gene expression by distant acting transcriptional enhancers. Recently, super-enhancers (SEs), also known as stretch or large enhancer clusters, are emerging as sentinel regulators within the gene regulatory networks that underpin cellular functions. It is becoming increasingly evident that long noncoding RNAs (lncRNAs) associated with these sequences play fundamental roles for enhancer activity and the regulation of the gene programs hardwired by them. Here, we review this emerging landscape, focusing on the roles of SEs and their derived lncRNAs in cardiovascular development and disease. We propose that exploration of this genomic landscape could provide novel therapeutic targets and approaches for the amelioration of cardiovascular disease. Ultimately we envisage a future of ncRNA therapeutics targeting the SE landscape to alleviate cardiovascular disease. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Long noncoding RNAs (lncRNAs) comprise a class of regulatory molecules that may control diverse stem cell properties. Now in Cell Stem Cell, Luo et al. (2016) show that a specific group of lncRNAs, those transcribed divergently from protein coding genes, activate key developmental genes to control embryonic stem cell fate.

Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease.

IL-17A and IL-17F are pro-inflammatory cytokines which induce the expression of several cytokines, chemokines and matrix metalloproteinases (MMPs) in target cells. IL-17 cytokines have recently attracted huge interest due to their pathogenic role in diseases such as arthritis and inflammatory bowel disease although a role for IL-17 cytokines in myocardial infarction (MI) has not previously been described.In vivo MI was performed by coronary artery occlusion in the absence or presence of a neutralizing IL-17 antibody for blocking IL-17 actions in vivo. IL-17 signaling was also assessed in isolated primary cardiomyocytes by Western blot, mRNA expression and immunostaining.Expression of IL-17A, IL-17F and the IL-17 receptor (IL-17RA) were all increased following MI. Expression of several IL-17 target genes, including Cxcl1, Cxcl2, IL-1β, iNOS and IL-6 was also upregulated following MI. In addition, IL-17A promoted the expression of Cxcl1 and IL-6 in isolated cardiomyocytes in a MAPK and PI(3)K-dependent manner. IL-17A and ischaemia/reperfusion (I/R) injury were found to have an additive effect on Cxcl1 expression, suggesting that IL-17 may enhance myocardial neutrophil recruitment during MI. Moreover, protein levels of both IL-17R and IL-17A were enhanced following in vivo MI. Finally, blocking IL-17 signaling in vivo reduced the levels of apoptotic cell death markers following in vivo MI.These data imply that the expression of IL-17 cytokines and their receptor are elevated during myocardial I/R injury and may play a fundamental role in post infarct inflammatory and apoptotic responses.

POU4F2/Brn-3b transcription factor (referred to as Brn-3b) is elevated in >60% of breast cancers and profoundly alters growth and behaviour of cancer cells by regulating distinct subsets of target genes. Previous studies showed that Brn-3b was required to maximally transactivate small heat shock protein, HSPB1/Hsp-27 (referred to as Hsp-27), and consequently, Brn-3b expression correlated well with Hsp27 levels in human breast biopsies. In these studies, we showed that Brn-3b is increased in MCF7 breast cancer cells that survive following treatment with chemotherapeutic drug doxorubicin (Dox) with concomitant increases in Hsp-27 expression. Targeting of Brn-3b using short interfering RNA reduced Hsp-27 in Dox-treated cells, suggesting that Brn-3b regulates Hsp-27 expression under these conditions. Wound healing assays showed increased Brn-3b in Dox-treated migratory cells that also express Hsp-27. Interestingly, Hsp-27 phosphorylation and cellular localisation are also significantly altered at different times following Dox treatment. Thus, phospho-Hsp-27 (p-Hsp27) protein displayed widespread distribution after 24 hrs of Dox treatment but was restricted to the nucleus after 5 days. However, in drug-resistant cells (grown in Dox for > 1 month), p-Hsp-27 was excluded from nuclei and most of the cytoplasm and appeared to be associated with the cell membrane. Studies to determine how this protein promotes survival and migration in breast cancer cells showed that the protective effects were conferred by unphosphorylated Hsp-27 protein. Thus, complex and dynamic mechanisms underlie effects of Hsp-27 protein in breast cancer cells following treatment with chemotherapeutic drugs such as Dox, and this may contribute to invasiveness and drug resistance following chemotherapy.

Introduction In cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known. Methods Bioinformatics analysis was used to identify the regulatory promoter region and map transcription start site as well as transcription factor binding sites. Polymerase chain reaction (PCR) cloning was used to generate promoter constructs for reporter assays. Chromatin immunoprecipitation and site-directed mutagenesis were used to confirm the transcription start site and autoregulation. MCF-7 and Cos-7 breast cancer cells were used. Cells grown in culture were transfected with Brn-3b promoter and treated with growth factors or estradiol to test for effects on promoter activity. Quantitative reverse transcriptase PCR assays and immunoblotting were used to confirm changes in gene and protein expression. Results We cloned the Brn-3b promoter, mapped the transcription start site and showed stimulation by estradiol and growth factors, nerve growth factor and epidermal growth factor, which are implicated in breast cancer initiation and/or progression. The effects of growth factors are mediated through the mitogen-activated protein kinase pathway, whereas hormone effects act via oestrogen receptor α (ERα). Brn-3b also autoregulates its expression and cooperates with ERα to further enhance levels. Conclusions Key regulators of growth in cancer cells, for example, oestrogens and growth factors, can stimulate Brn-3b expression, and autoregulation also contributes to increasing Brn-3b in breast cancers. Since increasing Brn-3b profoundly enhances growth in these cells, understanding how Brn-3b is increased in breast cancers will help to identify strategies for reducing its expression and thus its effects on target genes, thereby reversing its effects in breast cancer cells.

Early hypertrophic responses help the heart to maintain cardiac output and compensate for increased demand or workload. However, prolonged stresses trigger death of non-dividing cardiomyocytes and accompanying fibrosis, which underlies progression to heart failure. However, the molecular mechanisms associated with transition from compensatory to pathological changes in the heart are not fully understood. Here we demonstrate that the hypertrophic agonist, Angiotensin II (AngII) stimulates the expression of transcription factor (TF) POU4F2/Brn-3b (referred to as Brn-3b) in intact mouse hearts and in neonatal rat ventricular cardiomyocyte (NRVC) or embryonic rat heart-derived cell line H9c2. AngII stimulates Brn-3b expression via MAPK/ERK1 signalling pathway whereas known hypertrophic mediator, calcineurin acts via NFAT binding sites present in the gene promoter. Induction of Brn-3b by AngII correlates with expression of hypertrophic markers, atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MHC) suggesting that increased Brn-3b may be part of the hypertrophic response in these cells. Under these conditions, Brn-3b simulates known target gene, cyclin D1, which is required for hypertrophic responses in cardiomyocytes. However, AngII also stimulates the growth inhibitory p53 protein, which together with pro-apoptotic Bax is associated with apoptotic cell death that characterises progression from pathological hypertrophy to heart failure. Brn-3b cooperates with p53 to enhance Bax expression so may drive apoptosis in cardiomyocytes under these conditions. Thus, stimulation of Brn-3b by hypertrophic agonist, AngII may result in distinct effects on cell fate depending on whether p53 is co-expressed.

BackgroundSTARS (STriated muscle Activator of Rho Signaling) is a sarcomeric protein expressed early in cardiac development that acts as an acute stress sensor for pathological remodeling. However the role of STARS in cardiac development and function is incompletely understood. Here, we investigated the role of STARS in heart development and function in the zebrafish model and in vitro.Methodology and principal findingsExpression of zebrafish STARS (zSTARS) first occurs in the somites by the 16 somite stage [17 hours post fertilization (hpf)]. zSTARS is expressed in both chambers of the heart by 48 hpf, and also in the developing brain, jaw structures and pectoral fins. Morpholino-induced knockdown of zSTARS alters atrial and ventricular dimensions and decreases ventricular fractional shortening (measured by high-speed video microscopy), with pericardial edema and decreased or absent circulation [abnormal cardiac phenotypes in 126/164 (77%) of morpholino-injected embryos vs. 0/152 (0%) of control morpholino embryos]. Co-injection of zsrf (serum response factor) mRNA rescues the cardiac phenotype of zSTARS knockdown, resulting in improved fractional shortening and ventricular end-diastolic dimensions. Ectopic over-expression of STARS in vitro activates the STARS proximal promoter, which contains a conserved SRF site. Chromatin immunoprecipitation demonstrates that SRF binds to this site in vivo and the SRF inhibitor CCG-1423 completely blocks STARS proximal reporter activity in H9c2 cells.Conclusions/significanceThis study demonstrates for the first time that STARS deficiency severely disrupts cardiac development and function in vivo and revealed a novel STARS-SRF feed-forward autoregulatory loop that could play an essential role in STARS regulation and cardiac function.

Cardiovascular diseases and in particular heart failure are major causes of morbidity and mortality in the Western world. Recently, the notion of promoting cardiac regeneration as a means to replace lost cardiomyocytes in the damaged heart has engendered considerable research interest. These studies envisage the utilization of both endogenous and exogenous cellular populations, which undergo highly specialized cell fate transitions to promote cardiomyocyte replenishment. Such transitions are under the control of regenerative gene regulatory networks, which are enacted by the integrated execution of specific transcriptional programs. In this context, it is emerging that the non-coding portion of the genome is dynamically transcribed generating thousands of regulatory small and long non-coding RNAs, which are central orchestrators of these networks. In this review, we discuss more particularly the biological roles of two classes of regulatory non-coding RNAs, i.e. microRNAs and long non-coding RNAs, with a particular emphasis on their known and putative roles in cardiac homeostasis and regeneration. Indeed, manipulating non-coding RNA-mediated regulatory networks could provide keys to unlock the dormant potential of the mammalian heart to regenerate. This should ultimately improve the effectiveness of current regenerative strategies and discover new avenues for repair. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Heart failure is a worldwide epidemic and represents a major cause of morbidity and mortality. Current clinical therapies for heart disease prolong survival by protecting the viable muscle, but they are unable to replenish lost cardiomyocytes to restore function. Over the last decade, the notion of promoting cardiac regeneration has engendered considerable research interest. New strategies envisage the transfer of stem cells into the damaged myocardium, the mobilization of cardiac precursor cells, the promotion of cardiomyocyte proliferation in situ and direct reprogramming of non-cardiac cells into electromechanically coupled cardiomyocytes. The molecular and cellular mechanisms underpinning these different regenerative avenues are under the control of integrated transcriptional programs, which are ultimately dependent on epigenomic reprogramming and reorganization of the genome nuclear architecture. Today, it is becoming evident that regulatory noncoding RNAs play fundamental roles in all these aspects of gene regulatory network activity. In particular, thousands of long noncoding RNAs are dynamically expressed across the entire genome during lineage-specific commitment, specialization, and differentiation, as well as during the response to environmental cues. Here, we review this emerging landscape, focusing particularly on a unique class of lncRNA emerging from enhancer sequences, the enhancer-associated lncRNAs, in the context of cardiac regeneration. We propose that characterizing and manipulating these enhancer-associated transcripts could provide a novel approach to awaken the dormant regenerative potential of the adult mammalian heart. Ultimately, this could lead to targeted noncoding RNA-based enhancer therapies to improve effectiveness of current regenerative strategies and provide new avenues for repair.

Background Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative in silico analysis coupled with an experimental promoter characterisation to delineate these mechanisms. Results Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in ms1 mRNA. An in silico comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with ms1 promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the ms1 promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, α and β). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs α and β correlates with the temporal induction in ms1 mRNA. Conclusion These findings suggest that the tissue specific and differentiation dependent up-regulation in ms1 mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the ms1 5' upstream sequence. We believe, through its activation of ms1, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.