Myocardin regulates expression of contractile genes in smooth muscle cells and is required for closure of the ductus arteriosus in mice.
ABSTRACT: Myocardin (Myocd) is a potent transcriptional coactivator that has been implicated in cardiovascular development and adaptation of the cardiovascular system to hemodynamic stress. To determine the function of myocardin in the developing cardiovascular system, Myocd(F/F)/Wnt1-Cre(+) and Myocd(F/F)/Pax3-Cre(+) mice were generated in which the myocardin gene was selectively ablated in neural crest-derived SMCs populating the cardiac outflow tract and great arteries. Both Myocd(F/F)/Wnt1-Cre(+) and Myocd(F/F)/Pax3-Cre(+) mutant mice survived to birth, but died prior to postnatal day 3 from patent ductus arteriosus (PDA). Neural crest-derived SMCs populating the ductus arteriosus (DA) and great arteries exhibited a cell autonomous block in expression of myocardin-regulated genes encoding SMC-restricted contractile proteins. Moreover, Myocd-deficient vascular SMCs populating the DA exhibited ultrastructural features generally associated with the SMC synthetic, rather than contractile, phenotype. Consistent with these findings, ablation of the Myocd gene in primary aortic SMCs harvested from Myocd conditional mutant mice caused a dramatic decrease in SMC contractile protein expression. Taken together, these data demonstrate that myocardin regulates expression of genes required for the contractile phenotype in neural crest-derived SMCs and provide new insights into the molecular and genetic programs that may underlie PDA.
Project description:Myocardin is a muscle-restricted transcriptional coactivator that activates a serum response factor (SRF)-dependent gene program required for cardiogenesis and embryonic survival. To identify myocardin-dependent functions in smooth muscle cells (SMCs) during postnatal development, mice harboring a SMC-restricted conditional, inducible Myocd null mutation were generated and characterized. Tamoxifen-treated SMMHC-Cre(ERT2)/Myocd(F/F) conditional mutant mice die within 6 mo of Myocd gene deletion, exhibiting profound derangements in the structure of great arteries as well as the gastrointestinal and genitourinary tracts. Conditional mutant mice develop arterial aneurysms, dissection, and rupture, recapitulating pathology observed in heritable forms of thoracic aortic aneurysm and dissection (TAAD). SMCs populating arteries of Myocd conditional mutant mice modulate their phenotype by down-regulation of SMC contractile genes and up-regulation of extracellular matrix proteins. Surprisingly, this is accompanied by SMC autonomous activation of endoplasmic reticulum (ER) stress and autophagy, which over time progress to programmed cell death. Consistent with these observations, Myocd conditional mutant mice develop remarkable dilation of the stomach, small intestine, bladder, and ureters attributable to the loss of visceral SMCs disrupting the muscularis mucosa. Taken together, these data demonstrate that during postnatal development, myocardin plays a unique, and important, role required for maintenance and homeostasis of the vasculature, gastrointestinal, and genitourinary tracts. The loss of myocardin in SMCs triggers ER stress and autophagy, which transitions to apoptosis, revealing evolutionary conservation of myocardin function in SMCs and cardiomyocytes.
Project description:Integrin-linked kinase (ILK) is located at focal adhesions and links the extracellular matrix (ECM) to the actin cytoskeleton via ?1- and ?3-integrins. ILK plays a role in the activation of kinases including protein kinase B/Akt and glycogen synthase kinase 3? and regulates cell proliferation, motility, and survival.To determine the function of ILK in vascular smooth muscle cells (SMCs) in vivo.SM22Cre(+)Ilk(Fl/Fl) conditional mutant mice were generated in which the Ilk gene was selectively ablated in SMCs. SM22Cre(+)Ilk(Fl/Fl) conditional mutant mice survive to birth but die in the perinatal period exhibiting multiple vascular pathologies including aneurysmal dilatation of the aorta and patent ductus arteriosus (PDA). Defects in morphogenetic development of the aorta were observed as early as E12.5 in SM22Cre(+)Ilk(Fl/Fl) mutant embryos. By late gestation (E16.5 to 18.5), striking expansion of the thoracic aorta was observed in ILK mutant embryos. Histological analyses revealed that the structural organization of the arterial tunica media is severely disrupted with profound derangements in SMC morphology, cell-cell, and cell-matrix relationships, including disruption of the elastic lamellae. ILK deletion in primary aortic SMCs results in alterations of RhoA/cytoskeletal signaling transduced through aberrant localization of myocardin-related transcription factor (MRTF)-A repressing the transcription and expression of SMC genes, which are required for the maintenance of the contractile SMC phenotype.These data identify a molecular pathway linking ILK signaling to the contractile SMC gene program. Activation of this pathway is required for morphogenetic development of the aorta and ductus arteriosus during embryonic and postnatal survival.
Project description:Smooth muscle cells (SMCs) are remarkably plastic. Their reversible differentiation is required for growth and wound healing but also contributes to pathologies such as atherosclerosis and restenosis. Although key regulators of the SMC phenotype, including myocardin (MYOCD) and KLF4, have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported.Using human SMCs, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA-modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its product, 5-hydroxymethylcytosine (5-hmC), are enriched in contractile SMCs but reduced in dedifferentiated SMCs. TET2 knockdown inhibits expression of key procontractile genes, including MYOCD and SRF, with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, whereas TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regulates phenotypic modulation through opposing effects on chromatin accessibility at the promoters of procontractile versus dedifferentiation-associated genes. Notably, we find that TET2 binds and 5-hmC is enriched in CArG-rich regions of active SMC contractile promoters (MYOCD, SRF, and MYH11). Loss of TET2 and 5-hmC positively correlates with the degree of injury in murine models of vascular injury and human atherosclerotic disease. Importantly, localized TET2 knockdown exacerbates injury response, and local TET2 overexpression restores the 5-hmC epigenetic landscape and contractile gene expression and greatly attenuates intimal hyperplasia in vivo.We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation.
Project description:BACKGROUND:Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS:In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS:Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH.
Project description:BACKGROUND:Myocardin (Myocd) is a strong coactivator that binds the serum response factor (SRF) transcription factor over CArG elements embedded within smooth muscle cell (SMC) and cardiac muscle cyto-contractile genes. Here, we sought to ascertain whether Myocd-mediated gene expression confers a structural and physiological cardiac or SMC phenotype. METHODS AND RESULTS:Adenoviral-mediated expression of Myocd in the BC(3)H1 cell line induces cardiac and SMC genes while suppressing both skeletal muscle markers and cell growth. Immunofluorescence microscopy shows that SRF and a SMC-like cyto-contractile apparatus are elevated with Myocd overexpression. A short hairpin RNA to Srf impairs BC(3)H1 cyto-architecture; however, cotransduction with Myocd results in complete restoration of the cyto-architecture. Electron microscopic studies demonstrate a SMC ultrastructural phenotype with no evidence for cardiac sarcomerogenesis. Biochemical and time-lapsed videomicroscopy assays reveal clear evidence for Myocd-induced SMC-like contraction. CONCLUSIONS:Myocd is sufficient for the establishment of a SMC-like contractile phenotype.
Project description:Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise alpha and beta subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.
Project description:Little is known about vascular smooth muscle cell (SMC) phenotypic modulation in the cerebral circulation or pathogenesis of intracranial aneurysms. Tumor necrosis factor-alpha (TNF-?) has been associated with aneurysms, but potential mechanisms are unclear. Cultured rat cerebral SMCs overexpressing myocardin induced expression of key SMC contractile genes (SM-?-actin, SM-22?, smooth muscle myosin heavy chain), while dominant-negative cells suppressed expression. Tumor necrosis factor-alpha treatment inhibited this contractile phenotype and induced pro-inflammatory/matrix-remodeling genes (monocyte chemoattractant protein-1, matrix metalloproteinase-3, matrix metalloproteinase-9, vascular cell adhesion molecule-1, interleukin-1 beta). Tumor necrosis factor-alpha increased expression of KLF4, a known regulator of SMC differentiation. Kruppel-like transcription factor 4 (KLF4) small interfering RNA abrogated TNF-? activation of inflammatory genes and suppression of contractile genes. These mechanisms were confirmed in vivo after exposure of rat carotid arteries to TNF-? and early on in a model of cerebral aneurysm formation. Treatment with the synthesized TNF-? inhibitor 3,6-dithiothalidomide reversed pathologic vessel wall alterations after induced hypertension and hemodynamic stress. Chromatin immunoprecipitation assays in vivo and in vitro demonstrated that TNF-? promotes epigenetic changes through KLF4-dependent alterations in promoter regions of myocardin, SMCs, and inflammatory genes. In conclusion, TNF-? induces phenotypic modulation of cerebral SMCs through myocardin and KLF4-regulated pathways. These results demonstrate a novel role for TNF-? in promoting a pro-inflammatory/matrix-remodeling phenotype, which has important implications for the mechanisms behind intracranial aneurysm formation.
Project description:The ductus arteriosus is a specialized blood vessel containing highly differentiated and contractile vascular smooth muscle, derived largely from neural crest cells, that is essential for fetal life but typically closes after birth. Impaired development of the ductus arteriosus or disruption of signaling pathways that initiate postnatal closure can result in persistent patency of the ductus arteriosus, the third most common congenital heart defect. We found that Tfap2beta, a transcription factor associated with patent ductus arteriosus in humans, was uniquely expressed in mouse ductal smooth muscle. Endothelin-1 and the hypoxia-induced transcription factor, Hif2alpha were also highly enriched in ductal smooth muscle at embryonic day 13.5 and were dependent on Tfap2beta for their expression in this domain. Hif2alpha functioned as a negative regulator of Tfap2beta-induced transcription by disrupting protein-DNA interactions, suggesting a negative feedback loop regulating Tfap2beta activity. Our data indicate that Tfap2beta, Et-1, and Hif2alpha act in a transcriptional network during ductal smooth muscle development and that disruption of this pathway may contribute to patent ductus arteriosus by affecting the development of smooth muscle within the ductus arteriosus.
Project description:Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle ?-actin(+) and smooth muscle myosin heavy chain(+) SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.
Project description:<h4>Background</h4>Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/?-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.