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Skeletal muscles are the largest tissues in our body and the physiological function of muscle is essential to every aspect of life. The regulation of development, homeostasis, and metabolism is critical for the proper functioning of skeletal muscle. Consequently, understanding the processes involved in the regulation of myogenesis is of great interest. Non-coding RNAs especially microRNAs (miRNAs) are important regulators of gene expression and function. MiRNAs are small (~22 nucleotides long) noncoding RNAs known to negatively regulate target gene expression post-transcriptionally and are abundantly expressed in skeletal muscle. Gain- and loss-of function studies have revealed important roles of this class of small molecules in muscle biology and disease. In this review, we summarize the latest research that explores the role of miRNAs in skeletal muscle development, gene expression, and function as well as in muscle disorders like sarcopenia and Duchenne muscular dystrophy (DMD). Continuing with the theme of the current review series, we also briefly discuss the role of miRNAs in cancer cachexia.

Osteonecrosis resulting from heavy ethanol consumption is one of the major causes of nontraumatic osteonecrosis of the femoral head (ONFH). The underlying pathological and molecular mechanisms remain elusive. In this study, we performed deep RNA sequencing from femoral heads of patients diagnosed with late-stage alcohol-induced ONFH (AIONFH), other types of ONFH and traumatic injury (bone fracture). Genome-wide gene expression analyses identified 690 differentially expressed mRNAs in AIONFH. Gene annotation and pathway analyses revealed significant dysregulated genes involved in hemostasis, angiogenesis and bone remodeling processes from the late-stage AIONFH. Notably, ADH1B, which codes for one of the major alcohol dehydrogenases, is significantly upregulated in AIONFH samples. Further, we found that the ADH1B protein was primarily expressed in smooth muscle cells of the blood vessels, stromal cells and adipocytes of the femoral heads of AIONFH patients; but was absent in other ONFH samples. Our analyses also revealed unique long non-coding RNA (lncRNA) expression profiles and identified novel lncRNAs in AIONFH. In addition, we observed a close co-expression correlation between lncRNAs and mRNAs in AIONFH suggesting that cis-gene regulation represents a major mechanism of action of human femoral lncRNAs. Further, the expression signature of lncRNAs, but not mRNAs, distinguishes AIONFH from other types of ONFH. Taken together, our studies uncovered novel molecular signatures associated with late-stage AIONFH in which the dysregulation of several key signaling pathways within the femoral head may be involved in AIONFH. Subsequently, lncRNAs may serve as potential biomarkers for diagnosis and therapeutic treatment of AIONFH. Further studies are needed to confirm that ADH1B is specifically upregulated in AIONFH and not generally upregulated in patients who consume alcohol excessively.

This review aims to summarize and discuss the function and molecular mechanism of miRNA and lncRNA in the heart, focusing on ischemic and non-ischemic cardiomyopathy. Extensive studies in the past decades have identified numerous protein-coding genes that are highly expressed in the heart, playing essential roles in the regulation of cardiac gene expression, heart development, and function. Furthermore, mutations in many of these genes have been identified and are linked to cardiovascular disease. Intriguingly, it is now recognized that majority of our genome is “non-coding,” which produces a large amount of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Emerging evidence has indicated that these classes of non-coding RNAs participate in most (if not all) aspects of cardiac gene expression, cardiomyocyte proliferation, differentiation, and cardiac remodeling in response to stress. Recent findings have demonstrated important functions for non-coding RNA in ischemic and non-ischemic cardiomyopathy. It is expected that non-coding RNAs will become promising therapeutic targets for cardiovascular diseases.

Cardiomyopathy is associated with altered expression of genes encoding contractile proteins. Here we show that Trbp (Tarbp2), an RNA-binding protein, is required for normal heart function. Cardiac-specific inactivation in mice of Trbp (Trbp(cKO)) caused progressive cardiomyopathy and lethal heart failure. Loss of Trbp function resulted in upregulation of Sox6, repression of genes encoding normal cardiac slow-twitch myofiber proteins and pathologically increased expression of genes encoding skeletal fast-twitch myofiber proteins. Remarkably, knockdown of Sox6 fully rescued the Trbp-mutant phenotype, whereas mice overexpressing Sox6 phenocopied Trbp(cKO) mice. Trbp inactivation was mechanistically linked to Sox6 upregulation through altered processing of miR-208a, which is a direct inhibitor of Sox6. Transgenic overexpression of Mir208a sufficiently repressed Sox6, restored the balance in gene expression for fast- and slow-twitch myofiber proteins, and rescued cardiac function in Trbp(cKO) mice. Together, our studies identify a new Trbp-mediated microRNA-processing mechanism in the regulation of a linear genetic cascade essential for normal heart function.

Phosphorus (P) is a limiting macronutrient for diatom growth and productivity in the ocean. Much effort has been devoted to the physiological response of marine diatoms to ambient P change, however, the whole-genome molecular mechanisms are poorly understood. Here, we utilized RNA-Seq to compare the global gene expression patterns of a marine diatom Skeletonema costatum grown in inorganic P-replete, P-deficient, and inorganic- and organic-P resupplied conditions. In total 34,942 unique genes were assembled and 20.8% of them altered significantly in abundance under different P conditions. Genes encoding key enzymes/proteins involved in P utilization, nucleotide metabolism, photosynthesis, glycolysis, and cell cycle regulation were significantly up-regulated in P-deficient cells. Genes participating in circadian rhythm regulation, such as circadian clock associated 1, were also up-regulated in P-deficient cells. The response of S. costatum to ambient P deficiency shows several similarities to the well-described responses of other marine diatom species, but also has its unique features. S. costatum has evolved the ability to re-program its circadian clock and intracellular biological processes in response to ambient P deficiency. This study provides new insights into the adaptive mechanisms to ambient P deficiency in marine diatoms.

Long noncoding RNAs (LncRNAs) are RNA transcripts longer than 200 nucleotides that lack protein-coding potential. Although thousands of lncRNAs have been identified, only a few have been linked to cardiac gene expression and function. In this study, we identified, from genome-scale RNA-seq data, 12 candidate lncRNAs associated with cardiac hypertrophy (CH-lncRNAs). The expression of these lncRNAs was altered in mouse models of cardiac hypertrophy induced by transverse aortic constriction (TAC)- or CnA transgene. To determine the function of these lncRNAs, we developed an adeno-associated virus serotype 9 (AAV9)-based functional screening in postnatal mice. An AAV9:cTNT vector, in which the cardiac troponin T (cTNT) promoter was used to direct cardiac-specific expression of target genes, was utilized to overexpress or knockdown candidate lncRNAs in mouse hearts. Postnatal day 1 wild type or CnA transgenic pups were injected with AAV9 viruses and cardiac function was measured one and two months later. Thus far, we have tested 15 candidate lncRNAs for both gain- and loss-of-function studies. Among them, two lncRNAs were demonstrated regulating hypertrophy growth when knocked down. Finally, we identified the human homologues of CH-lncRNA through analyzing the conservation of the promoter regions of lncRNA genes. We showed that the expression of these human CH-lncRNA was dysregulated in human diseased hearts, suggesting the functional conservation of these lncRNAs in cardiac disease. Our study therefore demonstrated that lncRNAs are important regulator of cardiac hypertrophy and disease.

Global inactivation of Trbp, a regulator of miRNA pathways, resulted in developmental defects and postnatal lethality in mice. Recently, we showed that cardiac-specific deletion of Trbp caused heart failure. However, its functional role(s) in skeletal muscle has not been characterized. Using a conditional knockout model, we generated mice lacking Trbp in the skeletal muscle. Unexpectedly, skeletal muscle specific Trbp mutant mice appear to be phenotypically normal under normal physiological conditions. However, these mice exhibited impaired muscle regeneration and increased fibrosis in response to cardiotoxin-induced muscle injury, suggesting that Trbp is required for muscle repair. Using cultured myoblast cells we further showed that inhibition of Trbp repressed myoblast differentiation in vitro. The impaired myogenesis is associated with reduced expression of muscle-specific miRNAs, miR-1a and miR-133a. Together, our study demonstrated that Trbp participates in the regulation of muscle differentiation and regeneration.

MicroRNAs (miRNAs) are a class of small noncoding RNAs highly conserved across species. miRNAs regulate gene expression posttranscriptionally by base pairing to complementary sequences mainly in the 3'-untranslated region of their target mRNAs to induce mRNA cleavage and translational repression. Thousands of miRNAs have been identified in human and their function has been linked to the regulation of both physiological and pathological processes. The skeletal muscle is the largest human organ responsible for locomotion, posture, and body metabolism. Several conditions such as aging, immobilization, exercise, and diet are associated with alterations in skeletal muscle structure and function. The genetic and molecular pathways that regulate muscle development, function, and regeneration as well as muscular disease have been well established in past decades. In recent years, numerous studies have underlined the importance of miRNAs in the control of skeletal muscle development and function, through its effects on several biological pathways critical for skeletal muscle homeostasis. Furthermore, it has become clear that alteration of the expression of many miRNAs or genetic mutations of miRNA genes is associated with changes on myogenesis and on progression of several skeletal muscle diseases. The present review provides an overview of the current studies and recent progress in elucidating the complex role exerted by miRNAs on skeletal muscle physiology and pathology. © 2016 American Physiological Society. Compr Physiol 6:1279-1294, 2016.

MicroRNAs (miRNAs) negatively regulate gene expression at the post- transcriptional level, primarily by base-pairing with the 3′-untranslated region (3′-UTR) of their target mRNAs. Many miRNAs are expressed in a tissue/organ-specific manner and are associated with an increasing number of cell proliferation, differentiation, and tissue development events. Cardiac muscle expresses distinct genes encoding structural proteins and a subset of signal molecules that control tissue specification and differentiation. The transcriptional regulation of cardiomyocyte development has been well established, yet only until recently has it been uncovered that miRNAs participate in the regulatory networks. A subset of miRNAs are either specifically or highly expressed in cardiac muscle, providing an opportunity to understand how gene expression is controlled by miRNAs at the post-transcriptional level in this muscle type. miR-1, miR-133, miR-206, and miR-208 have been found to be muscle-specific, and thus have been called myomiRs. The discovery of myomiRs as a previously unrecognized component in the regulation of gene expression adds an entirely new layer of complexity to our understanding of cardiac muscle development. Investigating myomiRs will not only reveal novel molecular mechanisms of the miRNA-mediated regulatory network in cardiomyocyte development, but also raise new opportunities for therapeutic intervention for cardiovascular disease. WIREs Syst Biol Med 2011 3 183–190 DOI: 10.1002/wsbm.111 For further resources related to this article, please visit the WIREs website

MicroRNAs (miRNAs) are a class of small non-coding RNAs that are endogenously transcribed and processed into approximately 21- to approximately 23-nucleotide products. They are believed to function predominantly as sequence-targeted modifiers of gene expression through inhibition of post-transcriptional processes, including messenger RNA degradation and translational repression. Rapid expansion of functional studies of miRNAs in recent years has established a new paradigm in which miRNAs 'fine-tune' gene expression in complex biologic networks. In this review, we summarize the role of miRNA in heart development and cardiac pathogenesis, and discuss the implication of miRNAs as innovative therapeutic approaches for cardiovascular diseases.

We previously reported the importance of the serum response factor (SRF) cofactor myocardin in controlling muscle gene expression as well as the fundamental role for the inflammatory transcription factor NF-κB in governing cellular fate. Inactivation of myocardin has been implicated in malignant tumor growth. However, the underlying mechanism of myocardin regulation of cellular growth remains unclear. Here we show that NF-κB(p65) represses myocardin activation of cardiac and smooth muscle genes in a CArG-box-dependent manner. Consistent with their functional interaction, p65 directly interacts with myocardin and inhibits the formation of the myocardin/SRF/CArG ternary complex in vitro and in vivo . Conversely, myocardin decreases p65-mediated target gene activation by interfering with p65 DNA binding and abrogates LPS-induced TNF-α expression. Importantly, myocardin inhibits cellular proliferation by interfering with NF-κB-dependent cell-cycle regulation. Cumulatively, these findings identify a function for myocardin as an SRF-independent transcriptional repressor and cell-cycle regulator and provide a molecular mechanism by which interaction between NF-κB and myocardin plays a central role in modulating cellular proliferation and differentiation.

Aims Ischemic cardiomyopathy (ICM) resulting from myocardial infarction is a major cause of heart failure (HF). Recently, thousands of long non-coding RNAs (lncRNAs) have been discovered and implicated in a variety of biological processes. However, the role of most lncRNAs in HF remains largely unknown. The aim of this study is to test the hypothesis that the expression and function of lncRNAs are differentially regulated in diseased hearts. Methods and results In this study, we performed RNA deep sequencing of protein-coding and non-coding RNAs from cardiac samples of patients with ICM ( n = 15) and controls ( n = 15). Genome-wide transcriptome analysis confirmed that many protein-coding genes previously known to be involved in HF were altered in ICM hearts. Among the 145 differentially expressed lncRNAs identified in ICM hearts, we found a set of 35 lncRNAs that display strong positive expression correlation. Expression correlation coefficient analyses of differentially expressed lncRNAs and protein-coding genes revealed a strong association between lncRNAs and extracellular matrix (ECM) protein-coding genes. We overexpressed or knocked down selected lncRNAs in cardiac fibroblasts and our results suggest that lncRNAs are important regulators of fibrosis and the expression of ECM synthesis genes. Moreover, we show that lncRNAs participate in the TGF-β pathway to modulate the expression of ECM genes and myofibroblast differentiation. Conclusion Our studies demonstrate that the expression of many lncRNAs is dynamically regulated in ICM. lncRNAs regulate the expression and function of ECM and cardiac fibrosis during the development of ICM. Our results further indicate that lncRNAs may represent novel regulators of heart function and cardiac disorders, including ICM.

Differentiation of smooth muscle cells is accompanied by the transcriptional activation of an array of muscle-specific genes controlled by serum response factor (SRF). Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. Here, we show that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of smooth muscle genes. The promyogenic activity of myocardin is enhanced by p300, a histone acetyltransferase that associates with the transcription activation domain of myocardin. Conversely, class II histone deacetylases interact with a domain of myocardin distinct from the p300-binding domain and suppress smooth muscle gene activation by myocardin. These findings point to myocardin as a nexus for positive and negative regulation of smooth muscle gene expression by changes in chromatin acetylation.

Smooth muscle cells switch between differentiated and proliferative phenotypes in response to extracellular cues1, but the transcriptional mechanisms that confer such phenotypic plasticity remain unclear. Serum response factor (SRF) activates genes involved in smooth muscle differentiation and proliferation by recruiting muscle-restricted cofactors, such as the transcriptional coactivator myocardin, and ternary complex factors (TCFs) of the ETS-domain family, respectively2,3,4,5,6,7,8,9. Here we show that growth signals repress smooth muscle genes by triggering the displacement of myocardin from SRF by Elk-1, a TCF that acts as a myogenic repressor. The opposing influences of myocardin and Elk-1 on smooth muscle gene expression are mediated by structurally related SRF-binding motifs that compete for a common docking site on SRF. A mutant smooth muscle promoter, retaining responsiveness to myocardin and SRF but defective in TCF binding, directs ectopic transcription in the embryonic heart, demonstrating a role for TCFs in suppression of smooth muscle gene expression in vivo. We conclude that growth and developmental signals modulate smooth muscle gene expression by regulating the association of SRF with antagonistic cofactors.

Formation of the vascular system requires differentiation and patterning of endothelial and smooth muscle cells (SMCs). Although much attention has focused on development of the vascular endothelial network, the mechanisms that control vascular SMC development are largely unknown. Myocardin is a smooth and cardiac muscle-specific transcriptional coactivator of serum response factor, a ubiquitous transcription factor implicated in smooth muscle gene expression. When expressed ectopically in nonmuscle cells, myocardin can induce smooth muscle differentiation by its association with serum response factor. Here we report that mouse embryos homozygous for a myocardin loss-of-function mutation die by embryonic day 10.5 and show no evidence of vascular SMC differentiation. Myocardin is the only transcription factor known to be necessary and sufficient for vascular SMC differentiation.

Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF–myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.

Introduction: miR-208a, a small non-coding RNA expressed only in the heart, has a profound influence on cardiac gene expression. It has been previously demonstrated that genetic deletion of miR-208a does not affect viability or induce gross morphological heart defects, suggesting it is not required for normal cardiac growth and function (van Rooij et al., 2007; Callis et al., 2009). However, recent data from our lab indicate that miR-208a knock-out (KO) mice (developed in the laboratory of Da-Zhi Wang and subsequently maintained by inbreeding) display gross morphological cardiac abnormalities and dramatically reduced survival. Therefore, this investigation sought out to determine the mechanism(s) responsible for the maladaptive growth. Methods: Wild-type (WT) and miR-208a KO littermate mice (3-5 mon old) were used for this investigation. Cardiac function was assessed via echocardiography, cardiac fibrosis by collagen fiber staining, and gene expression by real-time PCR and Western blotting. We also measured basal Ca2+ transients and unloaded cell shortening in isolated mouse ventricular myocytes. Results: We observed KO mice have significantly lower survival rates, developed marked cardiac hypertrophy, fibrosis and have significantly reduced cardiac function. Compared to WT controls, cardiomyocytes from KO mice exhibited a significantly lower percent of cell shortening, slower rates of relaxation and contraction, lower amplitude, and slower kinetics (tau) of Ca2+ transients. Additionally, there was a reduced phosphorylation of phospholamban (Ser16) in miR-208 KO mice indicating a lower Ca2+ affinity of the SR Ca2+-ATPase. Conclusions: These data provide evidence that the observed reduction in cardiac function in miR-208a KO mice is likely due, in part, to alterations in cellular Ca2+ fluxes. To our knowledge, this is the first study to demonstrate genetic deletion of miR-208a induces a maladaptive phenotype at baseline. Our findings indicate the importance of considering variables such as genetic/environmental constraints when perturbing the expression of the miR-208a in the heart.

Cardiac hypertrophy is one of the primary responses of the heart to pathophysiological stress. However, the mechanism of the transition from compensative hypertrophic growth to cardiac dilation is poor understood. Recently, we identified a cardiac-specific expressed gene CIP. The expression of CIP is unchanged in hypertrophic heart but significantly down-regulated in dilated hearts, suggesting CIP may play an important role in the transition from cardiac hypertrophy to dilated cardiomyopathy. We generated CIP knockout mice and found that CIP is dispensable for cardiac development. Interestingly, CIP-null mutant mice developed severe cardiac dilation 4 weeks after TAC (transverse aortic constriction) surgery, while control mice were still at the stage of compensative hypertrophic growth. Echocardiography and histological examinations showed that mutant hearts had enlarged chamber with thinner ventricle wall and decreased cardiac performance compared to controls. The expression of marker genes of cardiac disease, BNP and Myh7, was elevated. Consistently, deletion of CIP in Myh6-CnA transgenic mice result in premature death, displaying severe left ventricle dilation. Conversely, cardiac-specific CIP overexpression inhibited pressure overload-induced cardiac hypertrophy. CIP transgenic mice exhibit decreased ventricle weight/body weight ratio, decreased cardiomyocyte cross-section area and repressed expression of hypertrophic related marker genes. CIP overexpression also protected the heart from developing cardiac dilation and preserved the cardiac function after prolonged pressure overload. We performed unbiased microarray assay to document the transcriptome in CIP knockout and control mice which were subjected to pressure overload (TAC). The analysis of Gene Ontology term indicated the Negative Regulation of Apoptosis was down-regulated while the Collagen/Extracellular Structure Organization was up-regulated in CIP-null hearts under TAC condition. In summary, our studies established CIP as a key regulator of the transition from cardiac hypertrophy to dilated cardiomyopathy. The protective effect of CIP in cardiac remodeling indicates that CIP could become a therapeutic target for cardiac diseases.

Rationale : Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. Objective : We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. Methods and Results : We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. Conclusions : Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.

MicroRNAs (miRNAs) are a class of small ∼22 nucleotide noncoding RNAs which regulate gene expression at the posttranscriptional level by either destabilizing and consequently degrading their targeted mRNAs or by repressing their translation. Emerging evidence has demonstrated that miRNAs are essential for normal mammalian development, homeostasis, and many other functions. In addition, deleterious changes in miRNA expression were associated with human diseases. Several muscle-specific miRNAs, including miR-1, miR-133, miR-206, and miR-208, have been shown to be important for normal myo-blast differentiation, proliferation, and muscle remodeling in response to stress. They have also been implicated in various cardiac and skeletal muscular diseases. miRNA-based gene therapies hold great potential for the treatment of cardiac and skeletal muscle diseases. Herein, we describe methods commonly applied to study the biological role of miRNAs, as well as techniques utilized to manipulate miRNA expression and to investigate their target regulation.

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin–glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.

MicroRNAs (miRNAs) are a class of small ~22 nt noncoding RNAs. miRNAs regulate gene expression at the posttranscriptional levels by destabilization and degradation of the target mRNA or by translational repression. Numerous studies have demonstrated that miRNAs are essential for normal mammalian development and organ function. Deleterious changes in miRNA expression play an important role in human diseases. We and others have previously reported several muscle-specific miRNAs, including miR-1/206, miR-133, and miR-208. These muscle-specific miRNAs are essential for normal myoblast differentiation and proliferation, and they have also been implicated in various cardiac and skeletal muscular diseases. miRNA-based gene therapies hold great potential for the treatment of cardiac and skeletal muscle disease(s). Herein, we introduce the methods commonly applied to study the biological role of miRNAs, as well as the techniques utilized to manipulate miRNA expression.

Myocardial differentiation is associated with the activation and expression of an array of cardiac specific genes. However, the transcriptional networks that control cardiac gene expression are not completely understood. Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of Serum Response Factor (SRF) and is able to potently activate cardiac and smooth muscle gene expression during development. We hypothesize that myocardin discriminates between cardiac and smooth muscle specific genes by associating with distinct co-factors. Here, we show that myocardin directly interacts with Tbx5, a member of the T-box family of transcription factors involved in the Holt-Oram syndrome. Tbx5 synergizes with myocardin to activate expression of the cardiac specific genes atrial natriuretic factor (ANF) and alpha myosin heavy chain (α-MHC), but not that of smooth muscle specific genes SM22 or smooth muscle myosin heavy chain (SM-MHC). We found that this synergistic activation of shared target genes is dependent on the binding sites for Tbx5, T-box factor-Binding Elements (TBEs). Myocardin and Tbx5 physically interact and their interaction domains were mapped to the basic domain and the coil domain of myocardin and Tbx5, respectively. Our analysis demonstrates that the Tbx5G80R mutation, which leads to the Holt-Oram syndrome in humans, failed to synergize with myocardin to activate cardiac gene expression. These data uncover a key role for Tbx5 and myocardin in establishing the transcriptional foundation for cardiac gene activation and suggest that the interaction of myocardin and Tbx5 maybe involved in cardiac development and diseases.

Background MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs of ~22 nucleotides in length. miRNAs regulate gene expression post-transcriptionally, primarily by associating with the 3' untranslated region (UTR) of their regulatory target mRNAs. Recent work has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis. Many miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related disorders. We have previously reported that the expression of muscle-specific miR-1 and miR-133 is induced during skeletal muscle differentiation and miR-1 and miR-133 play central regulatory roles in myoblast proliferation and differentiation in vitro. Methods In this study, we measured the expression of miRNAs in the skeletal muscle of mdx mice, an animal model for human muscular dystrophy. We also generated transgenic mice to overexpress miR-133a in skeletal muscle. Results We examined the expression of miRNAs in the skeletal muscle of mdx mice. We found that the expression of muscle miRNAs, including miR-1a, miR-133a and miR-206, was up-regulated in the skeletal muscle of mdx mice. In order to further investigate the function of miR-133a in skeletal muscle in vivo, we have created several independent transgenic founder lines. Surprisingly, skeletal muscle development and function appear to be unaffected in miR-133a transgenic mice. Conclusions Our results indicate that miR-133a is dispensable for the normal development and function of skeletal muscle.

microRNAs (miRNAs) are 21–23-nucleotide non-coding RNAs. It has become more and more evident that this class of small RNAs plays critical roles in the regulation of gene expression at the post-transcriptional level. MEF2A is a member of the MEF2 (myogenic enhancer factor 2) family of transcription factors. Prior report showed that the 3′-untranslated region (3′-UTR) of the Mef2A gene mediated its repression; however, the molecular mechanism underlying this intriguing observation was unknown. Here, we report that MEF2A is repressed by miRNAs. We identify miR-155 as one of the primary miRNAs that significantly represses the expression of MEF2A. We show that knockdown of the Mef2A gene by siRNA impairs myoblast differentiation. Similarly, overexpression of miR-155 leads to the repression of endogenous MEF2A expression and the inhibition of myoblast differentiation. Most importantly, reintroduction of MEF2A in miR-155 overexpressed myoblasts was able to partially rescue the miR-155-induced myoblast differentiation defect. Our data therefore establish miR-155 as an important regulator of MEF2A expression and uncover its function in muscle gene expression and myogenic differentiation.

MicroRNAs are recently discovered group of small noncoding RNAs (ribonucleic acids) which have been demonstrated to repress the expression of protein-coding genes in multiple biological processes. Rapid expansion of studies has begun to reveal roles for miRNAs in a wide range of biological processes, including cell proliferation, differentiation and apoptosis as well as in human diseases, such as cancer. miRNAs are expressed in the developing and adult heart, and dysregulated miRNA expression has been correlated with cardiovascular disorders. Genetic studies have identified distinct roles for specific miRNAs during cardiovascular development and cardiomyopathy. In addition, miRNAs have been shown to be necessary for normal development and physiological homeostasis. The focus of this review is to provide an overview of the current state of the molecular mechanisms and processes that miRNAs modulate within the multifaceted processes of cardiovascular development and progression of cardiovascular disease. Key Concepts: miRNAs are noncoding small RNAs that regulate gene expression posttranscriptionally. miRNAs are abundantly expressed in most organisms. A single miRNA may potentially repress hundreds of target genes. miRNA expression is altered in cardiovascular diseases. miRNAs modulate gene expression during normal cardiovascular development and in pathophysiological conditions. Gain- and lost-of-function studies indicate that miRNAs are key regulators of cardiovascular system homeostasis in responding to stress. Keywords: microRNA; gene expression; heart development; myocardium; cardiac remodelling; vascular smooth muscle cell; endothelium; vasculogenesis; angiogenesis; cardiovascular disease

microRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. miRNAs are known to mediate myriad cell processes, including proliferation, differentiation, and apoptosis. With more than 600 miRNAs identified in humans, it is generally believed that many miRNAs function through simultaneously inhibiting multiple regulatory mRNA targets, suggesting that miRNAs participate in regulating the expression of many, if not all, genes. While many miRNAs are expressed ubiquitously, some are expressed in a tissue specific manner. The muscle specific miR-1, miR-133 and miR-206 are perhaps the most studied and best-characterized miRNAs to date. Many studies demonstrate that these miRNAs are necessary for proper skeletal and cardiac muscle development and function, and have a profound influence on multiple myopathies, such as hypertrophy, dystrophy, and conduction defects.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

microRNAs (miRNAs) are a class of highly conserved small non-coding RNAs that negatively regulate gene expression post-transcriptionally. The emerging field of miRNA biology has begun to unravel roles for these regulatory molecules in a range of biological functions, including cardiac and skeletal muscle development, as well as in muscle-related disease processes. In this paper, we review the role of miRNAs in muscle biology. Recent genetic studies have demonstrated that miRNAs are required for both proper muscle development and function, with crucial roles for miRNAs being identified in regulating muscle cell proliferation and differentiation. Furthermore, dysregulated expression of miRNAs has been correlated to certain muscle-related diseases, including cardiac hypertrophy, cardiac arrhythmias, and muscular dystrophy.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that negatively regulate gene expression by inhibiting translation or promoting de-gradation of targeted messenger RNAs (mRNAs). More than one-third of human protein-coding mRNAs are predicted as being regulated by miRNAs, which have been implicated in processes as diverse as cancer and muscle biology. Now, recent studies demonstrate that the expression profiles of many miRNAs change during cardiac hypertrophy. Most importantly, both gain- and loss-of-function studies have established a clear functional correlation between miRNAs and cardiac hypertrophy. This previously unrecognized relationship sheds new light onto the regulatory mechanisms underlying the heart’s response to pathological stress and suggests the potential of miRNAs as therapeutic targets for cardiovascular disease.

Myocardin belongs to the SAP domain family of transcription factors and is expressed specifically in cardiac and smooth muscle during embryogenesis and in adulthood. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. However, the in vivo function of myocardin during cardiogenesis is not completely understood. Here we clone myocardin from chick embryonic hearts and show that myocardin protein sequences are highly conserved cross species. Detailed studies of chick myocardin expression reveal that myocardin is expressed in cardiac and smooth muscle lineage during early embryogenesis, similar to that found in mouse. Interestingly, the expression of myocardin in the heart was found enriched in the outflow tract and the sinoatrial segments shortly after the formation of linear heart tube. Such expression pattern is also maintained in later developing embryos, suggesting that myocardin may play a unique role in the formation of those cardiac modules. Similar to its mouse counterpart, chick myocardin is able to activate cardiac and smooth muscle promoter reporter genes and induce smooth muscle gene expression in nonmuscle cells. Ectopic overexpression of myocardin enlarged the embryonic chick heart. Conversely, repression of the endogenous chick myocardin using antisense oligonucleotides or a dominant negative mutant form of myocardin inhibited cardiogenesis. Together, our data place myocardin as one of the earliest cardiac marker genes for cardiogenesis and support the idea that myocardin plays an essential role in cardiac gene expression and cardiogenesis.

MicroRNAs (miRNAs) are a class of highly conserved, small non-coding RNAs that regulate gene expression post-transcriptionally. The emerging field of miRNA biology has begun to reveal roles for these regulatory molecules in a wide range of biological processes. Dysregulated miRNA expression has been correlated to diseased hearts in human patients, whereas inhibiting the maturation of miRNAs conditionally in murine hearts has revealed that miRNAs are essential for cardiac development and function. Moreover, genetic studies have identified distinct roles for specific miRNAs during cardiogenesis, cardiac hypertrophy and electrical conduction. These previously unrecognized relationships shed new light on the regulatory mechanisms underlying heart development and pathology and suggest the potential importance of miRNAs as diagnostic markers and therapeutic targets for cardiovascular disease.

MicroRNAs (miRNAs) are a recently discovered class of small non-coding RNAs, which are approximately 22 nucleotides in length. miRNAs negatively regulate gene expression by translational repression and target mRNA degradation. It has become clear that miRNAs are involved in many biological processes, including development, differentiation, proliferation, and apoptosis. Interestingly, many miRNAs are expressed in a tissue-specific manner and several miRNAs are specifically expressed in cardiac and skeletal muscles. In this review, we focus on those miRNAs that have been shown to be involved in muscle development. Compelling evidences have demonstrated that muscle miRNAs play an important role in the regulation of muscle proliferation and differentiation processes. However, it appears that miRNAs are not essential for early myogenesis and muscle specification. Importantly, dysregulation of miRNAs has been linked to muscle-related diseases, such as cardiac hypertrophy. A mutation resulting in a gain-of-function miRNA target site in the myostatin gene leads to down regulation of the targeted protein in Texel sheep. miRNAs therefore are a new class of regulators of muscle biology and they might become novel therapeutic targets in muscle-related human diseases.

Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 22 nucleotides that post-transcriptionally regulate gene expression. Increasing evidence points to the potential role of miRNAs in various biological processes. Here we show that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). Our results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, our studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development.

The interactions between serum response factor (SRF) and CArG elements are critical for smooth muscle cell (SMC) marker gene transcription. However, the mechanisms whereby SRF, which is expressed ubiquitously, contributes to SMC-specific transcription are unknown. Myocardin was recently cloned as a coactivator of SRF in the heart, but its role in regulating CArG-dependent expression of SMC differentiation marker genes has not been clearly elucidated. In this study, we examined the expression and the function of myocardin in SMCs. In adult mice, myocardin mRNA was expressed in multiple smooth muscle (SM) tissues including the aorta, bladder, stomach, intestine, and colon, as well as the heart. Myocardin was also expressed in cultured rat aortic SMCs and A404 SMC precursor cells. Of particular interest, expression of myocardin was induced during differentiation of A404 cells, although it was not expressed in parental P19 cells from which A404 cells were derived. Cotransfection studies in SMCs revealed that myocardin induced the activity of multiple SMC marker gene promoters including SM α-actin, SM-myosin heavy chain, and SM22α by 9- to 60-fold in a CArG-dependent manner, whereas myocardin short interfering RNA markedly decreased activity of these promoters. Moreover, adenovirus-mediated overexpression of a dominant-negative form of myocardin significantly suppressed expression of endogenous SMC marker genes, whereas adenovirus-mediated overexpression of wild-type myocardin increased expression. Taken together, results provide compelling evidence that myocardin plays a key role as a transcriptional coactivator of SMC marker genes through CArG-dependent mechanisms.

Members of the MEF2 family of transcription factors are upregulated during skeletal muscle differentiation and cooperate with the MyoD family of myogenic basic helix-loop-helix (bHLH) transcription factors to control the expression of muscle-specific genes. To determine the mechanisms that regulate MEF2 gene expression during skeletal muscle development, we analyzed the mouse Mef2c gene for cis-regulatory elements that direct expression in the skeletal muscle lineage in vivo. We describe a skeletal muscle-specific control region for Mef2c that is sufficient to direct lacZ reporter gene expression in a pattern that recapitulates that of the endogenous Mef2c gene in skeletal muscle during pre- and postnatal development. This control region is a direct target for the binding of myogenic bHLH and MEF2 proteins. Mutagenesis of the Mef2c control region shows that a binding site for myogenic bHLH proteins is essential for expression at all stages of skeletal muscle development, whereas an adjacent MEF2 binding site is required for maintenance but not for initiation of Mef2c transcription. Our findings reveal the existence of a regulatory circuit between these two classes of transcription factors that induces, amplifies and maintains their expression during skeletal muscle development.

Ankylosing spondylitis (AS) is an inflammatory rheumatic disease and strongly associated with human leucocyte antigen (HLA)-B27.1 A genome-wide scan for AS revealed the linkage locus on many non-major histocompatibility complex (MHC) loci of chromosomes.2,–,5 Among all non-MHC regions, we examined an 11cM region 16q23.1-24.1, with two announced positive STR markers: D16S515 and D16S516. Among the 101 genes in this region, proteasome 26S subunit non-ATPase 7 (PSMD7) was chosen as the only candidate gene because of its importance in the ubiquitin proteasome pathway (UPP). The UPP genes have been much studied in AS studies. Two MHC UPP genes: LMP2 and LMP7 were found to be associated with AS in Caucasian and Chinese populations.6,–,8 Two recent reports found that protein or gene expression levels of some UPP subunits were higher …

In order to comprehensively screen genetic variants leading to differential expression of the important human ABCB1 gene in the primary drug-metabolizing organ, ABCB1 mRNA expression levels were measured in 73 normal liver tissue samples from Chinese subjects. A set of Tag SNPs. were genotyped. In addition, imputation was performed within a 500 kb region around the ABCB1 gene using the reference panels of 1,000 Genome project and HapMap III. Bayesian regression was used to assess the strength of associations by compute Bayes Factors for imputed SNPs. Through imputation and linkage disequilibrium analysis, the imputed loci rs28373093, rs1002205, rs1029421, rs2285647, and rs10235835, may represent independent and strong association signals. rs28373093, a polymorphism 1.5 kb upstream from the ABCB1 transcription start site, has the strongest association. 2677 G>A/T and 3435C>T confer a clear gene-dosage effect on ABCB1 mRNA expression. The systematic characterization of gene-wide common quantitative trait loci associated with ABCB1 mRNA expression in normal liver tissues would provide the candidate markers to ABCB1-relevant clinical phenotypes in Chinese population.