A cardiac microRNA governs systemic energy homeostasis by regulation of MED13.

Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.

Oxidative regulation of TDP-43 self-association by a ß-to-α conformational switch.

An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.

Transiently structured head domains control intermediate filament assembly.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, ß-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for ß-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease-causing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, ß-strand-enriched self-interactions may broadly influence cell morphology.

Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments.

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1-I/C IFs functions by forming labile cross-ß interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology.

Myomaker is essential for muscle regeneration.

Regeneration of injured adult skeletal muscle involves fusion of activated satellite cells to form new myofibers. Myomaker is a muscle-specific membrane protein required for fusion of embryonic myoblasts, but its potential involvement in adult muscle regeneration has not been explored. We show that myogenic basic helix-loop-helix (bHLH) transcription factors induce myomaker expression in satellite cells during acute and chronic muscle regeneration. Moreover, genetic deletion of myomaker in adult satellite cells completely abolishes muscle regeneration, resulting in severe muscle destruction after injury. Myomaker is the only muscle-specific protein known to be absolutely essential for fusion of embryonic and adult myoblasts.

Myomaker is a membrane activator of myoblast fusion and muscle formation.

Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury.

Vascular injury triggers dedifferentiation and cytoskeletal remodeling of smooth muscle cells (SMCs), culminating in vessel occlusion. Serum response factor (SRF) and its coactivator, myocardin, play a central role in the control of smooth muscle phenotypes by regulating the expression of cytoskeletal genes. We show that SRF and myocardin regulate a cardiovascular-specific microRNA (miRNA) cluster encoding miR-143 and miR-145. To assess the functions of these miRNAs in vivo, we systematically deleted them singly and in combination in mice. Mice lacking both miR-143 and miR-145 are viable and do not display overt abnormalities in smooth muscle differentiation, although they show a significant reduction in blood pressure due to reduced vascular tone. Remarkably, however, neointima formation in response to vascular injury is profoundly impeded in mice lacking these miRNAs, due to disarray of actin stress fibers and diminished migratory activity of SMCs. These abnormalities reflect the regulation of a cadre of modulators of SRF activity and actin dynamics by miR-143 and miR-145. Thus, miR-143 and miR-145 act as integral components of the regulatory network whereby SRF controls cytoskeletal remodeling and phenotypic switching of SMCs during vascular disease.

Activation of MRTF-A-dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing.

Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF-ß1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A-dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity.

Hippo pathway effector Yap promotes cardiac regeneration.

The adult mammalian heart has limited potential for regeneration. Thus, after injury, cardiomyocytes are permanently lost, and contractility is diminished. In contrast, the neonatal heart can regenerate owing to sustained cardiomyocyte proliferation. Identification of critical regulators of cardiomyocyte proliferation and quiescence represents an important step toward potential regenerative therapies. Yes-associated protein (Yap), a transcriptional cofactor in the Hippo signaling pathway, promotes proliferation of embryonic cardiomyocytes by activating the insulin-like growth factor and Wnt signaling pathways. Here we report that mice bearing mutant alleles of Yap and its paralog WW domain containing transcription regulator 1 (Taz) exhibit gene dosage-dependent cardiac phenotypes, suggesting redundant roles of these Hippo pathway effectors in establishing proper myocyte number and maintaining cardiac function. Cardiac-specific deletion of Yap impedes neonatal heart regeneration, resulting in a default fibrotic response. Conversely, forced expression of a constitutively active form of Yap in the adult heart stimulates cardiac regeneration and improves contractility after myocardial infarction. The regenerative activity of Yap is correlated with its activation of embryonic and proliferative gene programs in cardiomyocytes. These findings identify Yap as an important regulator of cardiac regeneration and provide an experimental entry point to enhance this process.

Regulation of PI3-kinase/Akt signaling by muscle-enriched microRNA-486.

microRNAs (miRNAs) play key roles in modulating a variety of cellular processes through repression of mRNA targets. In a screen for miRNAs regulated by myocardin-related transcription factor-A (MRTF-A), a coactivator of serum response factor (SRF), we discovered a muscle-enriched miRNA, miR-486, controlled by an alternative promoter within intron 40 of the Ankyrin-1 gene. Transcription of miR-486 is directly controlled by SRF and MRTF-A, as well as by MyoD. Among the most strongly predicted targets of miR-486 are phosphatase and tensin homolog (PTEN) and Foxo1a, which negatively affect phosphoinositide-3-kinase (PI3K)/Akt signaling. Accordingly, PTEN and Foxo1a protein levels are reduced by miR-486 overexpression, which, in turn, enhances PI3K/Akt signaling. Similarly, we show that MRTF-A promotes PI3K/Akt signaling by up-regulating miR-486 expression. Conversely, inhibition of miR-486 expression enhances the expression of PTEN and Foxo1a and dampens signaling through the PI3K/Akt-signaling pathway. Our findings implicate miR-486 as a downstream mediator of the actions of SRF/MRTF-A and MyoD in muscle cells and as a potential modulator of PI3K/Akt signaling.

Oxidative regulation of TDP-43 self-association by a ß-to-α conformational switch.

An evolutionarily conserved region of the TDP-43 low complexity domain twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the low complexity domain. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent, and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of low complexity domains. Significance Statement: The TDP-43 protein is a constituent of RNA granules involved in regulated translation. TDP-43 contains a C-terminal domain of 150 amino acids of low sequence complexity conspicuously decorated with ten methionine residues. An evolutionarily conserved region (ECR) of 20 residues within this domain can adopt either of two forms of labile secondary structure. Under normal conditions wherein methionine residues are reduced, the ECR forms a labile cross-ß structure that enables RNA granule condensation. Upon methionine oxidation, the ECR undergoes a conformational switch to become an α-helix incompatible with self-association and granule integrity. Oxidation of the TDP-43 low complexity domain is hypothesized to occur proximal to mitochondria, thus facilitating dissolution of RNA granules and activation of localized translation.

MicroRNA-218 regulates vascular patterning by modulation of Slit-Robo signaling.

RATIONALE: Establishment of a functional vasculature requires the interconnection and remodeling of nascent blood vessels. Precise regulation of factors that influence endothelial cell migration and function is essential for these stereotypical vascular patterning events. The secreted Slit ligands and their Robo receptors constitute a critical signaling pathway controlling the directed migration of both neurons and vascular endothelial cells during embryonic development, but the mechanisms of their regulation are incompletely understood. OBJECTIVE: To identify microRNAs regulating aspects of the Slit-Robo pathway and vascular patterning. METHODS AND RESULTS: Here, we provide evidence that microRNA (miR)-218, which is encoded by an intron of the Slit genes, inhibits the expression of Robo1 and Robo2 and multiple components of the heparan sulfate biosynthetic pathway. Using in vitro and in vivo approaches, we demonstrate that miR-218 directly represses the expression of Robo1, Robo2, and glucuronyl C5-epimerase (GLCE), and that an intact miR-218-Slit-Robo regulatory network is essential for normal vascularization of the retina. Knockdown of miR-218 results in aberrant regulation of this signaling axis, abnormal endothelial cell migration, and reduced complexity of the retinal vasculature. CONCLUSIONS: Our findings link Slit gene expression to the posttranscriptional regulation of Robo receptors and heparan sulfate biosynthetic enzymes, allowing for precise control over vascular guidance cues influencing the organization of blood vessels during development.

Myocyte enhancer factor 2 and class II histone deacetylases control a gender-specific pathway of cardioprotection mediated by the estrogen receptor.

RATIONALE: Gender differences in cardiovascular disease have long been recognized and attributed to beneficial cardiovascular actions of estrogen. Class II histone deacetylases (HDACs) act as key modulators of heart disease by repressing the activity of the myocyte enhancer factor (MEF)2 transcription factor, which promotes pathological cardiac remodeling in response to stress. Although it is proposed that HDACs additionally influence nuclear receptor signaling, the effect of class II HDACs on gender differences in cardiovascular disease remains unstudied. OBJECTIVE: We aimed to examine the effect of class II HDACs on post-myocardial infarction remodeling in male and female mice. METHODS AND RESULTS: Here we show that the absence of HDAC5 or -9 in female mice protects against maladaptive remodeling following myocardial infarction, during which there is an upregulation of estrogen-responsive genes in the heart. This genetic reprogramming coincides with a pronounced increase in expression of the estrogen receptor (ER)alpha gene, which we show to be a direct MEF2 target gene. ERalpha also directly interacts with class II HDACs. Cardioprotection resulting from the absence of HDAC5 or -9 in female mice can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of the ER target gene vascular endothelial growth factor-a. CONCLUSIONS: Our results reveal a novel gender-specific pathway of cardioprotection mediated by ERalpha and its regulation by MEF2 and class II HDACs.

Myocardin-related transcription factor-a controls myofibroblast activation and fibrosis in response to myocardial infarction.

RATIONALE: Myocardial infarction (MI) results in loss of cardiac myocytes in the ischemic zone of the heart, followed by fibrosis and scar formation, which diminish cardiac contractility and impede angiogenesis and repair. Myofibroblasts, a specialized cell type that switches from a fibroblast-like state to a contractile, smooth muscle-like state, are believed to be primarily responsible for fibrosis of the injured heart and other tissues, although the transcriptional mediators of fibrosis and myofibroblast activation remain poorly defined. Myocardin-related transcription factors (MRTFs) are serum response factor (SRF) cofactors that promote a smooth muscle phenotype and are emerging as components of stress-responsive signaling. OBJECTIVE: We aimed to examine the effect of MRTF-A on cardiac remodeling and fibrosis. METHODS AND RESULTS: Here, we show that MRTF-A controls the expression of a fibrotic gene program that includes genes involved in extracellular matrix production and smooth muscle cell differentiation in the heart. In MRTF-A-null mice, fibrosis and scar formation following MI or angiotensin II treatment are dramatically diminished compared with wild-type littermates. This protective effect of MRTF-A deletion is associated with a reduction in expression of fibrosis-associated genes, including collagen 1a2, a direct transcriptional target of SRF/MRTF-A. CONCLUSIONS: We conclude that MRTF-A regulates myofibroblast activation and fibrosis in response to the renin-angiotensin system and post-MI remodeling.

Mutations linked to neurological disease enhance self-association of low-complexity protein sequences.

Protein domains of low sequence complexity do not fold into stable, three-dimensional structures. Nevertheless, proteins with these sequences assist in many aspects of cell organization, including assembly of nuclear and cytoplasmic structures not surrounded by membranes. The dynamic nature of these cellular assemblies is caused by the ability of low-complexity domains (LCDs) to transiently self-associate through labile, cross-ß structures. Mechanistic studies useful for the study of LCD self-association have evolved over the past decade in the form of simple assays of phase separation. Here, we have used such assays to demonstrate that the interactions responsible for LCD self-association can be dictated by labile protein structures poised close to equilibrium between the folded and unfolded states. Furthermore, missense mutations causing Charcot-Marie-Tooth disease, frontotemporal dementia, and Alzheimer's disease manifest their pathophysiology in vitro and in cultured cell systems by enhancing the stability of otherwise labile molecular structures formed upon LCD self-association.

Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis.

Acute myocardial infarction (MI) due to coronary artery occlusion is accompanied by a pathological remodeling response that includes hypertrophic cardiac growth and fibrosis, which impair cardiac contractility. Previously, we showed that cardiac hypertrophy and heart failure are accompanied by characteristic changes in the expression of a collection of specific microRNAs (miRNAs), which act as negative regulators of gene expression. Here, we show that MI in mice and humans also results in the dysregulation of specific miRNAs, which are similar to but distinct from those involved in hypertrophy and heart failure. Among the MI-regulated miRNAs are members of the miR-29 family, which are down-regulated in the region of the heart adjacent to the infarct. The miR-29 family targets a cadre of mRNAs that encode proteins involved in fibrosis, including multiple collagens, fibrillins, and elastin. Thus, down-regulation of miR-29 would be predicted to derepress the expression of these mRNAs and enhance the fibrotic response. Indeed, down-regulation of miR-29 with anti-miRs in vitro and in vivo induces the expression of collagens, whereas over-expression of miR-29 in fibroblasts reduces collagen expression. We conclude that miR-29 acts as a regulator of cardiac fibrosis and represents a potential therapeutic target for tissue fibrosis in general.

An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133.

The muscle-specific microRNAs, miR-1 and miR-133, play important roles in muscle growth and differentiation. Here, we show that the MEF2 transcription factor, an essential regulator of muscle development, directly activates transcription of a bicistronic primary transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-specific enhancer located between the miR-1-2 and 133a-1 coding regions. This MEF2-dependent enhancer is activated in the linear heart tube during mouse embryogenesis and thereafter controls transcription throughout the atrial and ventricular chambers of the heart. MEF2 together with MyoD also regulates the miR-1-2/-133a-1 intragenic enhancer in the somite myotomes and in all skeletal muscle fibers during embryogenesis and adulthood. A similar muscle-specific intragenic enhancer controls transcription of the miR-1-1/-133a-2 locus. These findings reveal a common architecture of regulatory elements associated with the miR-1/-133 genes and underscore the central role of MEF2 as a regulator of the transcriptional and posttranscriptional pathways that control cardiac and skeletal muscle development.

PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells.

Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.

Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size.

The Hippo signaling pathway regulates growth of the heart and other tissues. Hippo pathway kinases influence the activity of various targets, including the transcriptional coactivator Yap, but the specific role of Yap in heart growth has not been investigated. We show that Yap is necessary and sufficient for embryonic cardiac growth in mice. Deletion of Yap in the embryonic mouse heart impeded cardiomyocyte proliferation, causing myocardial hypoplasia and lethality at embryonic stage 10.5. Conversely, forced expression of a constitutively active form of Yap in the embryonic heart increased cardiomyocyte number and heart size. Yap activated the insulin-like growth factor (IGF) signaling pathway in cardiomyocytes, resulting in inactivation of glycogen synthase kinase 3ß, which led to increased abundance of ß-catenin, a positive regulator of cardiac growth. Our results point to Yap as a critical downstream effector of the Hippo pathway in the control of cardiomyocyte proliferation and a nexus for coupling the IGF, Wnt, and Hippo signaling pathways with the developmental program for heart growth.

A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance.

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.