Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

miR-1 as a Key Epigenetic Regulator in Early Differentiation of Cardiac Sinoatrial Region.

A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. In this study, miR-1 potential role in early cardiac chamber differentiation was analyzed through specific signaling pathways. For this, we performed in chick embryos functional experiments by means of miR-1 microinjections into the posterior cardiac precursors-of both primitive endocardial tubes-committed to sinoatrial region fates. Subsequently, embryos were subjected to whole mount in situ hybridization, immunohistochemistry and RT-qPCR analysis. As a relevant novelty, our results revealed that miR-1 increased Amhc1, Tbx5 and Gata4, while this microRNA diminished Mef2c and Cripto expressions during early differentiation of the cardiac sinoatrial region. Furthermore, we observed in this developmental context that miR-1 upregulated CrabpII and Rarß and downregulated CrabpI, which are three crucial factors in the retinoic acid signaling pathway. Interestingly, we also noticed that miR-1 directly interacted with Hdac4 and Calm1/Calmodulin, as well as with Erk2/Mapk1, which are three key factors actively involved in Mef2c regulation. Our study shows, for the first time, a key role of miR-1 as an epigenetic regulator in the early differentiation of the cardiac sinoatrial region through orchestrating opposite actions between retinoic acid and Mef2c, fundamental to properly assign cardiac cells to their respective heart chambers. A better understanding of those molecular mechanisms modulated by miR-1 will definitely help in fields applied to therapy and cardiac regeneration and repair.

Identification of atrial-enriched lncRNA Walras linked to cardiomyocyte cytoarchitecture and atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras, and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture.

Genomic and Non-Genomic Regulatory Mechanisms of the Cardiac Sodium Channel in Cardiac Arrhythmias.

Nav1.5 is the predominant cardiac sodium channel subtype, encoded by the SCN5A gene, which is involved in the initiation and conduction of action potentials throughout the heart. Along its biosynthesis process, Nav1.5 undergoes strict genomic and non-genomic regulatory and quality control steps that allow only newly synthesized channels to reach their final membrane destination and carry out their electrophysiological role. These regulatory pathways are ensured by distinct interacting proteins that accompany the nascent Nav1.5 protein along with different subcellular organelles. Defects on a large number of these pathways have a tremendous impact on Nav1.5 functionality and are thus intimately linked to cardiac arrhythmias. In the present review, we provide current state-of-the-art information on the molecular events that regulate SCN5A/Nav1.5 and the cardiac channelopathies associated with defects in these pathways.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment.

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Molecular Mechanisms of lncRNAs in the Dependent Regulation of Cancer and Their Potential Therapeutic Use.

Deep whole genome and transcriptome sequencing have highlighted the importance of an emerging class of non-coding RNA longer than 200 nucleotides (i.e., long non-coding RNAs (lncRNAs)) that are involved in multiple cellular processes such as cell differentiation, embryonic development, and tissue homeostasis. Cancer is a prime example derived from a loss of homeostasis, primarily caused by genetic alterations both in the genomic and epigenetic landscape, which results in deregulation of the gene networks. Deregulation of the expression of many lncRNAs in samples, tissues or patients has been pointed out as a molecular regulator in carcinogenesis, with them acting as oncogenes or tumor suppressor genes. Herein, we summarize the distinct molecular regulatory mechanisms described in literature in which lncRNAs modulate carcinogenesis, emphasizing epigenetic and genetic alterations in particular. Furthermore, we also reviewed the current strategies used to block lncRNA oncogenic functions and their usefulness as potential therapeutic targets in several carcinomas.

Post-Transcriptional Regulation of Molecular Determinants during Cardiogenesis.

Cardiovascular development is initiated soon after gastrulation as bilateral precardiac mesoderm is progressively symmetrically determined at both sides of the developing embryo. The precardiac mesoderm subsequently fused at the embryonic midline constituting an embryonic linear heart tube. As development progress, the embryonic heart displays the first sign of left-right asymmetric morphology by the invariably rightward looping of the initial heart tube and prospective embryonic ventricular and atrial chambers emerged. As cardiac development progresses, the atrial and ventricular chambers enlarged and distinct left and right compartments emerge as consequence of the formation of the interatrial and interventricular septa, respectively. The last steps of cardiac morphogenesis are represented by the completion of atrial and ventricular septation, resulting in the configuration of a double circuitry with distinct systemic and pulmonary chambers, each of them with distinct inlets and outlets connections. Over the last decade, our understanding of the contribution of multiple growth factor signaling cascades such as Tgf-beta, Bmp and Wnt signaling as well as of transcriptional regulators to cardiac morphogenesis have greatly enlarged. Recently, a novel layer of complexity has emerged with the discovery of non-coding RNAs, particularly microRNAs and lncRNAs. Herein, we provide a state-of-the-art review of the contribution of non-coding RNAs during cardiac development. microRNAs and lncRNAs have been reported to functional modulate all stages of cardiac morphogenesis, spanning from lateral plate mesoderm formation to outflow tract septation, by modulating major growth factor signaling pathways as well as those transcriptional regulators involved in cardiac development.

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.

LncRNA H19 Impairs Chemo and Radiotherapy in Tumorigenesis.

Various treatments based on drug administration and radiotherapy have been devoted to preventing, palliating, and defeating cancer, showing high efficiency against the progression of this disease. Recently, in this process, malignant cells have been found which are capable of triggering specific molecular mechanisms against current treatments, with negative consequences in the prognosis of the disease. It is therefore fundamental to understand the underlying mechanisms, including the genes-and their signaling pathway regulators-involved in the process, in order to fight tumor cells. Long non-coding RNAs, H19 in particular, have been revealed as powerful protective factors in various types of cancer. However, they have also evidenced their oncogenic role in multiple carcinomas, enhancing tumor cell proliferation, migration, and invasion. In this review, we analyze the role of lncRNA H19 impairing chemo and radiotherapy in tumorigenesis, including breast cancer, lung adenocarcinoma, glioma, and colorectal carcinoma.

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).

Non-coding RNAs and Atrial Fibrillation.

Atrial fibrillation is the most frequent type of cardiac arrhythmia in humans, with an estimate incidence of 1-2% in the general population, rising up to 8-10% in the elderly. Cardiovascular risk factors such as diabetes, obesity, hypertension and hyperthyroidism can increase the occurrence of AF. The onset of AF triggers additional AF episodes, leading to structural and electrical remodeling of the diseased heart. Understanding the molecular bases of atrial fibrillation have greatly advance over the last decade demonstrating a pivotal role of distinct ion channels in AF pathophysiology. A new scenario has opened on the understanding of the molecular mechanisms underlying AF, with the discovery of non-coding RNAs and their wide implication in multiple disease states, including cardiac arrhythmogenic pathologies. microRNAs are small non-coding RNAs of 22-24 nucleotides that are capable of regulating gene expression by interacting with the mRNA transcript 3'UTRs and promoting mRNA degradation and/or protein translation blockage. Long non-coding RNAs are a more diverse group of non-coding RNAs, providing transcriptional and post-transcriptional roles and subclassified according to their functional properties. In this chapter we summarized current state-of-the-art knowledge on the functional of microRNAs and long non-coding RNAs as well as their cross-talk regulatory mechanisms in atrial fibrillation.

Genetics and Epigenetics of Atrial Fibrillation.

Atrial fibrillation (AF) is known to be the most common supraventricular arrhythmia affecting up to 1% of the general population. Its prevalence exponentially increases with age and could reach up to 8% in the elderly population. The management of AF is a complex issue that is addressed by extensive ongoing basic and clinical research. AF centers around different types of disturbances, including ion channel dysfunction, Ca2+-handling abnormalities, and structural remodeling. Genome-wide association studies (GWAS) have uncovered over 100 genetic loci associated with AF. Most of these loci point to ion channels, distinct cardiac-enriched transcription factors, as well as to other regulatory genes. Recently, the discovery of post-transcriptional regulatory mechanisms, involving non-coding RNAs (especially microRNAs), DNA methylation, and histone modification, has allowed to decipher how a normal heart develops and which modifications are involved in reshaping the processes leading to arrhythmias. This review aims to provide a current state of the field regarding the identification and functional characterization of AF-related epigenetic regulatory networks.

Negative Fgf8-Bmp2 feed-back is regulated by miR-130 during early cardiac specification.

It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification.

Long-range regulatory interactions at the 4q25 atrial fibrillation risk locus involve PITX2c and ENPEP.

BACKGROUND: Recent genome-wide association studies have uncovered genomic loci that underlie an increased risk for atrial fibrillation, the major cardiac arrhythmia in humans. The most significant locus is located in a gene desert at 4q25, approximately 170 kilobases upstream of PITX2, which codes for a transcription factor involved in embryonic left-right asymmetry and cardiac development. However, how this genomic region functionally and structurally relates to PITX2 and atrial fibrillation is unknown. RESULTS: To characterise its function, we tested genomic fragments from 4q25 for transcriptional activity in a mouse atrial cardiomyocyte cell line and in transgenic mouse embryos, identifying a non-tissue-specific potentiator regulatory element. Chromosome conformation capture revealed that this region physically interacts with the promoter of the cardiac specific isoform of Pitx2. Surprisingly, this regulatory region also interacts with the promoter of the next neighbouring gene, Enpep, which we show to be expressed in regions of the developing mouse heart essential for cardiac electrical activity. CONCLUSIONS: Our data suggest that de-regulation of both PITX2 and ENPEP could contribute to an increased risk of atrial fibrillation in carriers of disease-associated variants, and show the challenges that we face in the functional analysis of genome-wide disease associations.

MiR-23b and miR-199a impair epithelial-to-mesenchymal transition during atrioventricular endocardial cushion formation.

BACKGROUND: Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial-mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf-beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp- and Tgf-beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages. RESULTS: qRT-PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR-23b, miR-199a, and miR-15a displayed increased expression during early AVC development whereas others such as miR-130a and miR-200a display decreased expression levels. Functional analyses of miR-23b, miR-199a, and miR-15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT-related genes that are predicted to be targeted by these microRNAs. CONCLUSIONS: Our data demonstrate that miR-23b and miR-199a over-expression can impair atrioventricular EMT.

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.

Absence of family history and phenotype-genotype correlation in pediatric Brugada syndrome: more burden to bear in clinical and genetic diagnosis.

Brugada syndrome (BrS) is an autosomal-dominant genetic cardiac disorder caused in 18-30 % of the cases by SCN5A gene mutations and manifested by an atypical right bundle block pattern with ST segment elevation and T wave inversion in the right precordial leads. The syndrome is usually detected after puberty. The identification of BrS in pediatric patients is thus a rare occurrence, and most of the reported cases are unmasked after febrile episodes. Usually, having a family history of sudden death represents the first reason to perform an ECG in febrile children. However, this practice makes the sporadic cases of cardiac disease and specially the asymptomatic ones excluded from this diagnosis. Here, we report a sporadic case of a 2-month-old male patient presented with vaccination-related fever and ventricular tachycardia associated with short breathing, palpitation and cold sweating. ECG changes were consistent with type 1 BrS. SCN5A gene analysis of the proband and his family revealed a set of mutations and polymorphisms differentially distributed among family members, however, without any clear genotype-phenotype correlation. Based on our findings, we think that genetic testing should be pursued as a routine practice in symptomatic and asymptomatic pediatric cases of BrS, with or without family history of sudden cardiac death. Similarly, our study suggests that pediatrician should be encouraged to perform an ECG profiling in suspicious febrile children and quickly manage fever since it is the most important factor unmasking BrS in children.

MEF2C Directly Interacts with Pre-miRNAs and Distinct RNPs to Post-Transcriptionally Regulate miR-23a-miR-27a-miR-24-2 microRNA Cluster Member Expression.

Transcriptional regulation constitutes a key step in gene expression regulation. Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS box family involved in the early development of several cell types, including muscle cells. Over the last decade, a novel layer of complexity modulating gene regulation has emerged as non-coding RNAs have been identified, impacting both transcriptional and post-transcriptional regulation. microRNAs represent the most studied and abundantly expressed subtype of small non-coding RNAs, and their functional roles have been widely documented. On the other hand, our knowledge of the transcriptional and post-transcriptional regulatory mechanisms that drive microRNA expression is still incipient. We recently demonstrated that MEF2C is able to transactivate the long, but not short, regulatory element upstream of the miR-23a-miR-27a-miR-24-2 transcriptional start site. However, MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the miR-23a-miR-27a-miR-24-2 mature cluster members without affecting pri-miRNA expression levels, thus supporting additional MEF2C-driven regulatory mechanisms. Within this study, we demonstrated a complex post-transcriptional regulatory mechanism directed by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members, distinctly involving different domains of the MEF2C transcription factor and the physical interaction with pre-miRNAs and Ksrp, HnRNPa3 and Ddx17 transcripts.

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.

Understanding Epicardial Cell Heterogeneity during Cardiogenesis and Heart Regeneration.

The outermost layer of the heart, the epicardium, is an essential cell population that contributes, through epithelial-to-mesenchymal transition (EMT), to the formation of different cell types and provides paracrine signals to the developing heart. Despite its quiescent state during adulthood, the adult epicardium reactivates and recapitulates many aspects of embryonic cardiogenesis in response to cardiac injury, thereby supporting cardiac tissue remodeling. Thus, the epicardium has been considered a crucial source of cell progenitors that offers an important contribution to cardiac development and injured hearts. Although several studies have provided evidence regarding cell fate determination in the epicardium, to date, it is unclear whether epicardium-derived cells (EPDCs) come from specific, and predetermined, epicardial cell subpopulations or if they are derived from a common progenitor. In recent years, different approaches have been used to study cell heterogeneity within the epicardial layer using different experimental models. However, the data generated are still insufficient with respect to revealing the complexity of this epithelial layer. In this review, we summarize the previous works documenting the cellular composition, molecular signatures, and diversity within the developing and adult epicardium.