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BACKGROUND: Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. METHODS: We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. RESULTS: lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 (nuclear factor of activated T cells/myocyte enhancer factor-2) pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1 (KRAB-associated protein-1). lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. CONCLUSIONS: These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.
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Cardiomegalia , Ratones Noqueados , ARN Largo no Codificante , Animales , Humanos , Masculino , Ratones , Cardiomegalia/metabolismo , Cardiomegalia/genética , Cardiomegalia/prevención & control , Cardiomegalia/patología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/prevención & control , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Remodelación VentricularRESUMEN
BACKGROUND: Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins. METHODS: We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. RESULTS: We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. CONCLUSIONS: Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.
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Aterosclerosis , Placa Aterosclerótica , Animales , Ratones , Placa Aterosclerótica/metabolismo , Macrófagos/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Colesterol/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismoRESUMEN
BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.
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Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Humanos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Doxiciclina , Células Cultivadas , Células Madre Pluripotentes Inducidas/metabolismo , ARN Interferente Pequeño/metabolismo , Biosíntesis de Proteínas , Proliferación Celular , Regeneración , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismoRESUMEN
BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.
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Aneurisma de la Aorta Abdominal , ARN Largo no Codificante , Animales , Humanos , Ratones , Aneurisma de la Aorta Abdominal/metabolismo , Proliferación Celular , Células Cultivadas , Inflamación/genética , Inflamación/metabolismo , Luciferasas/metabolismo , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.
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Células Endoteliales/citología , Células Endoteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción MEF2/metabolismo , Neovascularización Fisiológica/genética , Animales , Células Cultivadas , Embrión no Mamífero , Células Endoteliales/enzimología , Elementos de Facilitación Genéticos/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Neovascularización Patológica/genética , Dominios y Motivos de Interacción de Proteínas , Retina/embriología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
Heart failure is a leading cause of fatality in Duchenne muscular dystrophy (DMD) patients. Previously, we discovered that cardiac and skeletal-muscle-enriched CIP proteins play important roles in cardiac function. Here, we report that CIP, a striated muscle-specific protein, participates in the regulation of dystrophic cardiomyopathy. Using a mouse model of human DMD, we found that deletion of CIP leads to dilated cardiomyopathy and heart failure in young, non-syndromic mdx mice. Conversely, transgenic overexpression of CIP reduces pathological dystrophic cardiomyopathy in old, syndromic mdx mice. Genome-wide transcriptome analyses reveal that molecular pathways involving fibrogenesis and oxidative stress are affected in CIP-mediated dystrophic cardiomyopathy. Mechanistically, we found that CIP interacts with dystrophin and calcineurin (CnA) to suppress the CnA-Nuclear Factor of Activated T cells (NFAT) pathway, which results in decreased expression of Nox4, a key component of the oxidative stress pathway. Overexpression of Nox4 accelerates the development of dystrophic cardiomyopathy in mdx mice. Our study indicates CIP is a modifier of dystrophic cardiomyopathy and a potential therapeutic target for this devastating disease.
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Cardiomiopatías , Cardiomiopatía Dilatada , Distrofia Muscular de Duchenne , Animales , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatía Dilatada/genética , Proteínas Co-Represoras , Distrofina/metabolismo , Corazón , Humanos , Ratones , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/patología , Proteínas NuclearesRESUMEN
BACKGROUND/AIMS: An obesogenic diet (high fat and sugar, low fiber) is associated with an increased risk for metabolic and cardiovascular disorders. Previous studies have demonstrated that epigenetic changes can modify gene transcription and protein function, playing a key role in the development of several diseases. The methyltransferase Set7 methylates histone and non-histone proteins, influencing diverse biological and pathological processes. However, the functional role of Set7 in obesity-associated metabolic and cardiovascular complications is unknown. METHODS: Wild type and Set7 knockout female mice were fed a normal diet or an obesogenic diet for 12 weeks. Body weight gain and glucose tolerance were measured. The 3T3-L1 cells were used to determine the role of Set7 in white adipogenic differentiation. Cardiac morphology and function were evaluated by histology and echocardiography. An ex vivo Langendorff perfusion system was used to model cardiac ischemia/reperfusion (I/R). RESULTS: Here, we report that Set7 protein levels were enhanced in the heart and perigonadal adipose tissue (PAT) of female mice fed an obesogenic diet. Significantly, loss of Set7 prevented obesogenic diet-induced glucose intolerance in female mice although it did not affect the obesogenic diet-induced increase in body weight gain and adiposity in these animals, nor did Set7 inhibition change white adipogenic differentiation in vitro. In addition, loss of Set7 prevented the compromised cardiac functional recovery following ischemia and reperfusion (I/R) injury in obesogenic diet-fed female mice; however, deletion of Set7 did not influence obesogenic diet-induced cardiac hypertrophy nor the hemodynamic and echocardiographic parameters. CONCLUSION: These data indicate that Set7 plays a key role in obesogenic diet-induced glucose intolerance and compromised myocardial functional recovery after I/R in obese female mice.
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Intolerancia a la Glucosa , Animales , Dieta Alta en Grasa/efectos adversos , Femenino , Isquemia , Ratones , Ratones Noqueados , Ratones Obesos , Obesidad/metabolismo , Reperfusión/efectos adversosRESUMEN
Cardiovascular diseases are the main cause of death worldwide. Recent studies have revealed the influence of histone-modifying enzymes in cardiac remodeling and heart dysfunction. The Set7 methyltransferase regulates the expression of several genes through the methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in cardiac remodeling and heart dysfunction remains unknown. To address this question, wild-type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol or saline. WT mice injected with isoproterenol displayed a decrease in Set7 activity in the heart. In addition, WT and Set7 KO mice injected with isoproterenol exhibited cardiac hypertrophy. Interestingly, Set7 deletion exacerbated cardiac hypertrophy in response to isoproterenol but attenuated myocardial fibrosis. Echocardiograms revealed that WT mice injected with isoproterenol had lowered ejection fractions and fractional shortening, and increased E'-wave deceleration time and E/A ratio compared with their controls. Conversely, Set7 KO mice did not show alteration in these parameters in response to isoproterenol. However, prolonged exposure to isoproterenol induced cardiac dysfunction both in WT and Set7 KO mice. Both isoproterenol and Set7 deletion changed the transcriptional profile of the heart. Moreover, Set7 deletion increased the expression of Pgc1α and mitochondrial DNA content in the heart, and reduced the expression of cellular senescence and inflammation markers in response to isoproterenol. Taken together, our data suggest that Set7 deletion attenuates isoproterenol-induced myocardial fibrosis and delays heart dysfunction, suggesting that Set7 plays an important role in cardiac remodeling and dysfunction in response to stress.
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Cardiomiopatías , Remodelación Ventricular , Ratones , Masculino , Animales , Isoproterenol/efectos adversos , Isoproterenol/metabolismo , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Cardiomegalia/metabolismo , Ratones Noqueados , Cardiomiopatías/inducido químicamente , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/genética , Fibrosis , Miocitos Cardíacos/metabolismo , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.
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Aterosclerosis/metabolismo , Endotelio Vascular/metabolismo , Factores de Transcripción MEF2/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Endotelio Vascular/patología , Femenino , Regulación de la Expresión Génica , Homeostasis , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/deficiencia , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción MEF2/deficiencia , Factores de Transcripción MEF2/genética , Masculino , Ratones , Ratones Noqueados , Receptores Notch/genética , Transducción de Señal , Transactivadores/metabolismoRESUMEN
Differentiated vascular smooth muscle cells (VSMCs) are crucial in maintaining vascular homeostasis. While the coding transcriptome of the differentiated VSMC phenotype has been defined, we know little about its noncoding signature. Herein, we identified a Myocardin-induced muscle specific long noncoding RNA (lncRNA) (Mymsl) downregulated upon VSMC phenotypic modulation. We demonstrated an essential role of a proximal consensus CArG element in response to MYOCD/SRF in vitro. To validate the in vivo role of this CArG element, we generated CArG mutant mice via CRISPR-Cas9 genome editing. While the CArG mutation had no impact on the expression of surrounding genes, it abolished Mymsl expression in SMCs, but not skeletal and cardiac muscle. Chromatin immunoprecipitation assays (ChIPs) showed decreased SRF binding to CArG region in mutants whereas the enrichment of H3K79Me2 remained the same. RNA-seq analysis showed a downregulation of matrix genes in aortas from Mymsl knockout mice, which was further validated in injured carotid arteries. Our study defined the transcriptional control of a novel lncRNA in SMCs via a single transcription factor binding site, which may offer a new strategy for generating SMC-specific knockout mouse models. We also provided in vivo evidence supporting the potential importance of Mymsl in vascular pathophysiology.
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Vasos Sanguíneos/metabolismo , Miocitos del Músculo Liso/metabolismo , ARN Largo no Codificante/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética , Animales , Aorta/metabolismo , Diferenciación Celular , Regulación hacia Abajo , Matriz Extracelular/metabolismo , Edición Génica , Genoma , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación/genética , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Sistemas de Lectura Abierta/genética , Fenotipo , ARN Largo no Codificante/genética , Factor de Respuesta Sérica/metabolismo , Transactivadores/metabolismoRESUMEN
BACKGROUND/AIMS: Obesity is a risk factor associated with cardiometabolic complications. Recently, we reported that miRNA-22 deletion attenuated high-fat diet-induced adiposity and prevented dyslipidemia without affecting cardiac hypertrophy in male mice. In this study, we examined the impact of miRNA-22 in obesogenic diet-induced cardiovascular and metabolic disorders in females. METHODS: Wild type (WT) and miRNA-22 knockout (miRNA-22 KO) females were fed a control or an obesogenic diet. Body weight gain, adiposity, glucose tolerance, insulin tolerance, and plasma levels of total cholesterol and triglycerides were measured. Cardiac and white adipose tissue remodeling was assessed by histological analyses. Echocardiography was used to evaluate cardiac function and morphology. RNA-sequencing analysis was employed to characterize mRNA expression profiles in female hearts. RESULTS: Loss of miRNA-22 attenuated body weight gain, adiposity, and prevented obesogenic diet-induced insulin resistance and dyslipidemia in females. WT obese females developed cardiac hypertrophy. Interestingly, miRNA-22 KO females displayed cardiac hypertrophy without left ventricular dysfunction and myocardial fibrosis. Both miRNA-22 deletion and obesogenic diet changed mRNA expression profiles in female hearts. Enrichment analysis revealed that genes associated with regulation of the force of heart contraction, protein folding and fatty acid oxidation were enriched in hearts of WT obese females. In addition, genes related to thyroid hormone responses, heart growth and PI3K signaling were enriched in hearts of miRNA-22 KO females. Interestingly, miRNA-22 KO obese females exhibited reduced mRNA levels of Yap1, Egfr and Tgfbr1 compared to their respective controls. CONCLUSION: This study reveals that miRNA-22 deletion induces cardiac hypertrophy in females without affecting myocardial function. In addition, our findings suggest miRNA-22 as a potential therapeutic target to treat obesity-related metabolic disorders in females.
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Cardiomegalia , Dieta Alta en Grasa/efectos adversos , Eliminación de Gen , Enfermedades Metabólicas , MicroARNs/genética , Miocardio , Obesidad , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Femenino , Enfermedades Metabólicas/inducido químicamente , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Miocardio/metabolismo , Miocardio/patología , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
Tetraspanins (TSPANs) comprise a large family of 4-transmembrane domain proteins. The importance of TSPANs in vascular smooth muscle cells (VSMCs) is unexplored. Given that TGF-ß1 and myocardin (MYOCD) are potent activators for VSMC differentiation, we screened for TGF-ß1 and MYOCD/serum response factor (SRF)-regulated TSPANs in VSMC by using RNA-seq analyses and RNA-arrays. TSPAN2 was found to be the only TSPAN family gene induced by TGF-ß1 and MYOCD, and reduced by SRF deficiency in VSMCs. We also found that TSPAN2 is highly expressed in smooth muscle-enriched tissues and down-regulated in in vitro models of VSMC phenotypic modulation. TSPAN2 expression is attenuated in mouse carotid arteries after ligation injury and in failed human arteriovenous fistula samples after occlusion by dedifferentiated neointimal VSMC. In vitro functional studies showed that TSPAN2 suppresses VSMC proliferation and migration. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that TSPAN2 is regulated by 2 parallel pathways, MYOCD/SRF and TGF-ß1/SMAD, via distinct binding elements within the proximal promoter. Thus, we identified the first VSMC-enriched and MYOCD/SRF and TGF-ß1/SMAD-dependent TSPAN family member, whose expression is intimately associated with VSMC differentiation and negatively correlated with vascular disease. Our results suggest that TSPAN2 may play important roles in vascular disease.-Zhao, J., Wu, W., Zhang, W., Lu, Y. W., Tou, E., Ye, J., Gao, P., Jourd'heuil, D., Singer, H. A., Wu, M., Long, X. Selective expression of TSPAN2 in vascular smooth muscle is independently regulated by TGF-ß1/SMAD and myocardin/serum response factor.
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Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Factor de Respuesta Sérica/metabolismo , Proteínas Smad/metabolismo , Tetraspaninas/metabolismo , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Diferenciación Celular , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Miocitos del Músculo Liso/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Factor de Respuesta Sérica/genética , Proteínas Smad/genética , Tetraspaninas/genética , Transactivadores/genética , Transcriptoma , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
OBJECTIVE: Laminar flow activates myocyte enhancer factor 2 (MEF2) transcription factors in vitro to induce expression of atheroprotective genes in the endothelium. Here we sought to establish the role of Mef2c in the vascular endothelium in vivo. APPROACH AND RESULTS: To study endothelial Mef2c, we generated endothelial-specific deletion of Mef2c using Tie2-Cre or Cdh5-Cre-ERT2 and examined aortas and carotid arteries by en face immunofluorescence. We observed enhanced actin stress fiber formation in the Mef2c-deleted thoracic aortic endothelium (laminar flow region), similar to those observed in normal aortic inner curvature (disturbed flow region). Furthermore, Mef2c deletion resulted in the de novo formation of subendothelial intimal cells expressing markers of differentiated smooth muscle in the thoracic aortas and carotids. Lineage tracing showed that these cells were not of endothelial origin. To define early events in intimal development, we induced endothelial deletion of Mef2c and examined aortas at 4 and 12 weeks postinduction. The number of intimal cell clusters increased from 4 to 12 weeks, but the number of cells within a cluster peaked at 2 cells in both cases, suggesting ongoing migration but minimal proliferation. Moreover, we identified cells extending from the media through fenestrations in the internal elastic lamina into the intima, indicating transfenestral smooth muscle migration. Similar transfenestral migration was observed in wild-type carotid arteries ligated to induce neointimal formation. CONCLUSIONS: These results indicate that endothelial Mef2c regulates the endothelial actin cytoskeleton and inhibits smooth muscle cell migration into the intima.
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Traumatismos de las Arterias Carótidas/metabolismo , Movimiento Celular , Células Endoteliales/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Comunicación Paracrina , Túnica Íntima/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/fisiopatología , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Genotipo , Hemodinámica , Humanos , Factores de Transcripción MEF2/deficiencia , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones Noqueados , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , Neointima , Fenotipo , Interferencia de ARN , Flujo Sanguíneo Regional , Transducción de Señal , Factores de Tiempo , Transfección , Túnica Íntima/patología , Túnica Íntima/fisiopatologíaRESUMEN
PURPOSE OF REVIEW: 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. RECENT FINDINGS: 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.
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Cardiomiopatías/genética , MicroARNs/genética , ARN Largo no Codificante/genética , Remodelación Ventricular , Animales , Cardiomiopatías/metabolismo , Humanos , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismoAsunto(s)
Proliferación Celular/fisiología , Perfilación de la Expresión Génica/métodos , Infarto del Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fosfohidrolasa PTEN/deficiencia , Animales , Ratones , Ratones Noqueados , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Fosfohidrolasa PTEN/genéticaRESUMEN
One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long noncoding RNAs (lncRNAs), could provide new, more targeted therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome-associated lncRNA and show that its expression was induced in the heart upon pathological cardiac hypertrophy and that its deletion in mice exacerbated stress-induced cardiac hypertrophy and augmented protein translation. In contrast, overexpression of CARDINAL attenuated cardiac hypertrophy in vivo and in vitro and suppressed hypertrophy-induced protein translation. Mechanistically, CARDINAL interacted with developmentally regulated GTP-binding protein 1 (DRG1) and blocked its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 was downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.
Asunto(s)
Cardiomegalia , Biosíntesis de Proteínas , ARN Largo no Codificante , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Ratones , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Humanos , Ratones Noqueados , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
Sox17 is a critical regulator of arterial identity during early embryonic vascular development. However, its role in adult endothelial cells (ECs) are not fully understood. Sox17 is highly expressed in arterial ECs but not in venous ECs throughout embryonic development to adulthood suggesting that it may play a functional role in adult arteries. Here, we investigated Sox17 mediated phenotypical changes in adult ECs. To precisely control the temporal expression level of Sox17, we designed a tetracycline-inducible lentiviral gene expression system to express Sox17 selectively in cultured venous ECs. We confirmed that Sox17-induced ECs exhibit a gene profile favoring arterial and tip cell identity. Furthermore, in comparison to control ECs, Sox17-activated ECs under shear leads to greater expression of arterial markers and suppression of venous identity. These data suggest that Sox17 enables greater hemodynamic adaptability of ECs in response to fluid shear stress. Here, we also demonstrate key morphogenic behaviors of Sox17-mediated ECs. In both vasculogenic and angiogenic 3D fibrin gel studies, Sox17-mediated ECs prefer to form cohesive vessels with one another while interfering the vessel formation of the control ECs. Sox17-mediated ECs elicit hyper-sprouting behavior in the presence of pericytes but not fibroblasts, suggesting Sox17 mediated sprouting frequency is dependent on supporting cell type. Using a microfluidic chip, we also show that Sox17-mediated ECs maintain thinner diameter vessels that do not widen under interstitial flow like the control ECs. Taken together, these data showed that Sox17 mediated EC gene expression and phenotypical changes are highly modulated in the context of biomechanical stimuli, suggesting Sox17 plays a role in regulating the arterial ECs adaptability under arterial hemodynamics as well as tip cells behavior during angiogenesis and vasculogenesis. The results from this study may be valuable in improving vein graft adaptation to arterial hemodynamics and bioengineering microvasculature for tissue engineering applications.
Asunto(s)
Arterias , Células Endoteliales , Diferenciación Celular , Células Cultivadas , Células Endoteliales/metabolismo , Hemodinámica , Factores de Transcripción SOXFRESUMEN
In this study, we sought to determine the role of tRNA-derived fragments in the regulation of gene expression during skeletal muscle cell proliferation and differentiation. We employed cell culture to examine the function of mt-Ty 5' tiRNAs. Northern blotting, RT-PCR as well as RNA-Seq, were performed to determine the effects of mt-Ty 5' tiRNA loss and gain on gene expression. Standard and transmission electron microscopy (TEM) were used to characterize cell and sub-cellular structures. mt-Ty 5'tiRNAs were found to be enriched in mouse skeletal muscle, showing increased levels in later developmental stages. Gapmer-mediated inhibition of tiRNAs in skeletal muscle C2C12 myoblasts resulted in decreased cell proliferation and myogenic differentiation; consistent with this observation, RNA-Seq, transcriptome analyses, and RT-PCR revealed that skeletal muscle cell differentiation and cell proliferation pathways were also downregulated. Conversely, overexpression of mt-Ty 5'tiRNAs in C2C12 cells led to a reversal of these transcriptional trends. These data reveal that mt-Ty 5'tiRNAs are enriched in skeletal muscle and play an important role in myoblast proliferation and differentiation. Our study also highlights the potential for the development of tiRNAs as novel therapeutic targets for muscle-related diseases.
Asunto(s)
Mioblastos Esqueléticos , Ratones , Animales , Línea Celular , Diferenciación Celular , Músculo Esquelético/fisiología , Proliferación CelularRESUMEN
Alanyl-transfer RNA synthetase 2 (AARS2) is a nuclear encoded mitochondrial tRNA synthetase that is responsible for charging of tRNA-Ala with alanine during mitochondrial translation. Homozygous or compound heterozygous mutations in the Aars2 gene, including those affecting its splicing, are linked to infantile cardiomyopathy in humans. However, how Aars2 regulates heart development, and the underlying molecular mechanism of heart disease remains unknown. Here, we found that poly(rC) binding protein 1 (PCBP1) interacts with the Aars2 transcript to mediate its alternative splicing and is critical for the expression and function of Aars2. Cardiomyocyte-specific deletion of Pcbp1 in mice resulted in defects in heart development that are reminiscent of human congenital cardiac defects, including noncompaction cardiomyopathy and a disruption of the cardiomyocyte maturation trajectory. Loss of Pcbp1 led to an aberrant alternative splicing and a premature termination of Aars2 in cardiomyocytes. Additionally, Aars2 mutant mice with exon-16 skipping recapitulated heart developmental defects observed in Pcbp1 mutant mice. Mechanistically, we found dysregulated gene and protein expression of the oxidative phosphorylation pathway in both Pcbp1 and Aars2 mutant hearts; these date provide further evidence that the infantile hypertrophic cardiomyopathy associated with the disorder oxidative phosphorylation defect type 8 (COXPD8) is mediated by Aars2. Our study therefore identifies Pcbp1 and Aars2 as critical regulators of heart development and provides important molecular insights into the role of disruptions in metabolism on congenital heart defects.