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1.
Circulation ; 149(15): 1205-1230, 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38189150

ABSTRACT

BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.


Subject(s)
Atrial Fibrillation , Atrial Remodeling , Heart Failure , Humans , Mice , Animals , Atrial Fibrillation/genetics , Gene Regulatory Networks , Calcium/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Heart Atria , Heart Failure/genetics , Genomics , GATA4 Transcription Factor/genetics
2.
JAMA Cardiol ; 7(5): 521-528, 2022 05 01.
Article in English | MEDLINE | ID: mdl-35385050

ABSTRACT

Importance: Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of atherosclerotic cardiovascular disease, and mouse experiments suggest that CHIP related to Tet2 loss of function in myeloid cells accelerates atherosclerosis via augmented interleukin (IL) 1ß signaling. Objective: To assess whether individuals with CHIP have greater cardiovascular event reduction in response to IL-1ß neutralization in the Canankinumab Anti-inflammatory Thrombosis Outcomes Trial (CANTOS). Design, Setting, and Participants: This randomized clinical trial took place from April 2011 to June 2017 at more than 1000 clinical sites in 39 countries. Targeted deep sequencing of genes previously associated with CHIP in a subset of trial participants using genomic DNA prepared from baseline peripheral blood samples were analyzed. All participants had prior myocardial infarction and elevated high-sensitivity C-reactive protein level above 0.20 mg/dL. Analysis took place between June 2017 and December 2021. Interventions: Canakinumab, an anti-IL-1ß antibody, given at doses of 50, 150, and 300 mg once every 3 months. Main Outcomes and Measures: Major adverse cardiovascular events (MACE). Results: A total of 338 patients (8.6%) were identified in this subset with evidence for clonal hematopoiesis. As expected, the incidence of CHIP increased with age; the mean (SD) age of patients with CHIP was 66.3 (9.2) years and 61.5 (9.6) years in patients without CHIP. Unlike other populations that were not preselected for elevated C-reactive protein, in the CANTOS population variants in TET2 were more common than DNMT3A (119 variants in 103 patients vs 86 variants in 85 patients). Placebo-treated patients with CHIP showed a nonsignificant increase in the rate of MACE compared with patients without CHIP using a Cox proportional hazard model (hazard ratio, 1.32 [95% CI, 0.86-2.04]; P = .21). Exploratory analyses of placebo-treated patients with a somatic variant in either TET2 or DNMT3A (n = 58) showed an equivocal risk for MACE (hazard ratio, 1.65 [95% CI, 0.97-2.80]; P = .06). Patients with CHIP due to somatic variants in TET2 also had reduced risk for MACE while taking canakinumab (hazard ratio, 0.38 [95% CI, 0.15-0.96]) with equivocal difference compared with others (P for interaction = .14). Conclusions and Relevance: These results are consistent with observations of increased risk for cardiovascular events in patients with CHIP and raise the possibility that those with TET2 variants may respond better to canakinumab than those without CHIP. Future studies are required to further substantiate this hypothesis. Trial Registration: ClinicalTrials.gov Identifier: NCT01327846.


Subject(s)
Antibodies, Monoclonal, Humanized , Atherosclerosis , Clonal Hematopoiesis , Dioxygenases , Antibodies, Monoclonal, Humanized/therapeutic use , Atherosclerosis/drug therapy , C-Reactive Protein/analysis , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Humans
4.
Cancer Res ; 80(24): 5597-5605, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33023946

ABSTRACT

In the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), inhibition of the IL1ß inflammatory pathway by canakinumab has been shown to significantly reduce lung cancer incidence and mortality. Here we performed molecular characterization of CANTOS patients who developed lung cancer during the study, including circulating tumor DNA (ctDNA) and soluble inflammatory biomarker analysis. Catalogue of Somatic Mutations in Cancer (COSMIC) database ctDNA mutations were detected in 65% (46/71) of the CANTOS patients with lung cancer, with 51% (36/71) having detectable ctDNA at the time point closest to lung cancer diagnosis and 43% (29/67) having detectable ctDNA at trial randomization. Mutations commonly found in lung cancer were observed with no evidence of enrichment in any mutation following canakinumab treatment. Median time to lung cancer diagnosis in patients with (n = 29) versus without (n = 38) detectable COSMIC ctDNA mutations at baseline was 407 days versus 837 days (P = 0.011). For serum inflammatory biomarker analysis, circulating levels of C-reactive protein (CRP), IL6, IL18, IL1 receptor antagonist, TNFα, leptin, adiponectin, fibrinogen, and plasminogen activator inhibitor-1 were determined. Patients with the highest level of baseline CRP or IL6, both downstream of IL1ß signaling, trended toward a shorter time to lung cancer diagnosis. Other inflammation markers outside of the IL1ß pathway at baseline did not trend with time to lung cancer diagnosis. These results provide further evidence for the importance of IL1ß-mediated protumor inflammation in lung cancer and suggest canakinumab's effect may be mediated in part by delaying disease progression of diverse molecular subtypes of lung cancer. SIGNIFICANCE: These findings suggest that targeting the IL1ß inflammatory pathway might be critical in reducing tumor-promoting inflammation and lung cancer incidence.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Antibodies, Monoclonal, Humanized/administration & dosage , Immunotherapy/methods , Interleukin-1beta/antagonists & inhibitors , Lung Neoplasms/epidemiology , Lung Neoplasms/prevention & control , Thrombosis/therapy , Biomarkers, Tumor/blood , C-Reactive Protein/analysis , Circulating Tumor DNA/blood , Circulating Tumor DNA/genetics , Dose-Response Relationship, Drug , Female , Humans , Incidence , Interleukin-1beta/blood , Longitudinal Studies , Lung Neoplasms/blood , Lung Neoplasms/mortality , Male , Mutation , Treatment Outcome
5.
Immunohorizons ; 3(7): 331-340, 2019 07 17.
Article in English | MEDLINE | ID: mdl-31356162

ABSTRACT

Ets1 is emerging as a key transcription factor that is required to prevent autoimmunity in mice and humans. Ets1 is expressed in both B and T cells, and mice lacking Ets1 are characterized by excess B and T cell activation, leading to enhanced formation of Ab-secreting cells and high titers of autoantibodies. In humans, genome-wide association studies have detected associations of single nucleotide polymorphisms in the human ETS1 gene with autoimmune diseases, including lupus. An increased fraction of CD4+ T cells from Ets1-/- mice have an activated effector-memory phenotype, and there are aberrations in differentiation that contribute to the autoimmune phenotype. In vitro studies of B cells suggest that Ets1 may have B cell-intrinsic effects as well. To confirm B cell-intrinsic roles for Ets1, we crossed CD19-Cre mice to mice with a floxed allele of Ets1. Mice with a B cell-specific deletion of Ets1 show increases in B cell activation, numbers of Ab-secreting cells, and levels of autoantibodies, despite the fact that T cells are normal. However, when compared with conventional Ets1 knockout mice, mice with B cell-specific loss of Ets1 have a significantly milder phenotype. These results demonstrate that Ets1 is required in B cells to prevent autoimmune responses but that loss of Ets1 activity in other cell types is required for maximal autoimmune phenotypes.


Subject(s)
Autoimmunity/immunology , B-Lymphocytes/immunology , Lymphocyte Activation , Proto-Oncogene Protein c-ets-1/metabolism , Alleles , Animals , Antigen-Antibody Complex/metabolism , Autoantibodies/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/genetics , Gene Knockout Techniques , Immunoglobulin G/blood , Immunoglobulin M/blood , Kidney/immunology , Kidney/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Proto-Oncogene Protein c-ets-1/genetics
6.
Ann Rheum Dis ; 76(4): 773-778, 2017 04.
Article in English | MEDLINE | ID: mdl-28153829

ABSTRACT

OBJECTIVES: Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc. METHODS: The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-ß-receptor I. RESULTS: Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis. CONCLUSIONS: These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.


Subject(s)
Aminopyridines/therapeutic use , Membrane Proteins/antagonists & inhibitors , Piperazines/therapeutic use , Scleroderma, Localized/prevention & control , Scleroderma, Systemic/prevention & control , Skin/pathology , Wnt Signaling Pathway/drug effects , Acyltransferases , Aminopyridines/pharmacology , Animals , Bleomycin , Disease Models, Animal , Disease Progression , Female , Fibrosis , Graft vs Host Disease/complications , Mice, Inbred BALB C , Piperazines/pharmacology , Protein Serine-Threonine Kinases/genetics , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/prevention & control , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Scleroderma, Localized/etiology , Scleroderma, Localized/metabolism , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Skin/metabolism , Transforming Growth Factor beta/metabolism
7.
J Am Coll Cardiol ; 68(16): 1769-1780, 2016 10 18.
Article in English | MEDLINE | ID: mdl-27737744

ABSTRACT

BACKGROUND: Evidence suggests that interleukin (IL)-1ß is important in the pathogenesis of atherosclerosis and its complications and that inhibiting IL-1ß may favorably affect vascular disease progression. OBJECTIVES: The goal of this study was to evaluate the effects of IL-1ß inhibition with canakinumab versus placebo on arterial structure and function, determined by magnetic resonance imaging. METHODS: Patients (N = 189) with atherosclerotic disease and either type 2 diabetes mellitus or impaired glucose tolerance were randomized to receive placebo (n = 94) or canakinumab 150 mg monthly (n = 95) for 12 months. They underwent magnetic resonance imaging of the carotid arteries and aorta. RESULTS: There were no statistically significant differences between canakinumab compared with placebo in the primary efficacy and safety endpoints. There was no statistically significant change in mean carotid wall area and no effect on aortic distensibility, measured at 3 separate anatomic sites. The change in mean carotid artery wall area was -3.37 mm2 after 12 months with canakinumab versus placebo. High-sensitivity C-reactive protein was significantly reduced by canakinumab compared with placebo at 3 months (geometric mean ratio [GMR]: 0.568; 95% confidence interval [CI]: 0.436 to 0.740; p < 0.0001) and 12 months (GMR: 0.56; 95% CI: 0.414 to 0.758; p = 0.0002). Lipoprotein(a) levels were reduced by canakinumab compared with placebo (-4.30 mg/dl [range: -8.5 to -0.55 mg/dl]; p = 0.025] at 12 months), but triglyceride levels increased (GMR: 1.20; 95% CI: 1.046 to 1.380; p = 0.01). In these patients with type 2 diabetes mellitus or impaired glucose tolerance, canakinumab had no effect compared with placebo on any of the measures assessed by using a standard oral glucose tolerance test. CONCLUSIONS: There were no statistically significant effects of canakinumab on measures of vascular structure or function. Canakinumab reduced markers of inflammation (high-sensitivity C-reactive protein and interleukin-6), and there were modest increases in levels of total cholesterol and triglycerides. (Safety & Effectiveness on Vascular Structure and Function of ACZ885 in Atherosclerosis and Either T2DM or IGT Patients; NCT00995930).


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Arteries/drug effects , Atherosclerosis/complications , Atherosclerosis/drug therapy , Diabetes Mellitus, Type 2/complications , Glucose Intolerance/complications , Interleukin-1beta/antagonists & inhibitors , Antibodies, Monoclonal, Humanized , Double-Blind Method , Female , Humans , Male , Middle Aged
8.
J Exp Med ; 213(5): 687-96, 2016 05 02.
Article in English | MEDLINE | ID: mdl-27069114

ABSTRACT

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that play a protective role in the response to helminth infection, but they also contribute to allergic lung inflammation. Here, we report that the deletion of the ETS1 transcription factor in lymphoid cells resulted in a loss of ILC2s in the bone marrow and lymph nodes and that ETS1 promotes the fitness of the common progenitor of all ILCs. ETS1-deficient ILC2 progenitors failed to up-regulate messenger RNA for the E protein transcription factor inhibitor ID2, a critical factor for ILCs, and these cells were unable to expand in cytokine-driven in vitro cultures. In vivo, ETS1 was required for the IL-33-induced accumulation of lung ILC2s and for the production of the T helper type 2 cytokines IL-5 and IL-13. IL-25 also failed to elicit an expansion of inflammatory ILC2s when these cells lacked ETS1. Our data reveal ETS1 as a critical regulator of ILC2 expansion and cytokine production and implicate ETS1 in the regulation of Id2 at the inception of ILC2 development.


Subject(s)
Cytokines/immunology , Immunity, Innate , Inhibitor of Differentiation Protein 2/immunology , Lymphocytes/immunology , Proto-Oncogene Protein c-ets-1/immunology , Animals , Cytokines/genetics , Inhibitor of Differentiation Protein 2/genetics , Mice , Mice, Knockout , Proto-Oncogene Protein c-ets-1/genetics
9.
Dev Biol ; 395(1): 50-61, 2014 Nov 01.
Article in English | MEDLINE | ID: mdl-25196150

ABSTRACT

FOG-2 is a multi-zinc finger protein that binds the transcriptional activator GATA4 and modulates GATA4-mediated regulation of target genes during heart development. Our previous work has demonstrated that the Nucleosome Remodeling and Deacetylase (NuRD) complex physically interacts with FOG-2 and is necessary for FOG-2 mediated repression of GATA4 activity in vitro. However, the relevance of this interaction for FOG-2 function in vivo has remained unclear. In this report, we demonstrate the importance of FOG-2/NuRD interaction through the generation and characterization of mice homozygous for a mutation in FOG-2 that disrupts NuRD binding (FOG-2(R3K5A)). These mice exhibit a perinatal lethality and have multiple cardiac malformations, including ventricular and atrial septal defects and a thin ventricular myocardium. To investigate the etiology of the thin myocardium, we measured the rate of cardiomyocyte proliferation in wild-type and FOG-2(R3K5A) developing hearts. We found cardiomyocyte proliferation was reduced by 31±8% in FOG-2(R3K5A) mice. Gene expression analysis indicated that the cell cycle inhibitor Cdkn1a (p21(cip1)) is up-regulated 2.0±0.2-fold in FOG-2(R3K5A) hearts. In addition, we demonstrate that FOG-2 can directly repress the activity of the Cdkn1a gene promoter, suggesting a model by which FOG-2/NuRD promotes ventricular wall thickening by repression of this cell cycle inhibitor. Consistent with this notion, the genetic ablation of Cdkn1a in FOG-2(R3K5A) mice leads to an improvement in left ventricular function and a partial rescue of left ventricular wall thickness. Taken together, our results define a novel mechanism in which FOG-2/NuRD interaction is required for cardiomyocyte proliferation by directly down-regulating the cell cycle inhibitor Cdkn1a during heart development.


Subject(s)
Cell Proliferation , DNA-Binding Proteins/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Myocytes, Cardiac/metabolism , Transcription Factors/metabolism , Animals , Animals, Newborn , Blotting, Western , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/physiopathology , Cell Survival/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA-Binding Proteins/genetics , Echocardiography , GATA4 Transcription Factor/genetics , GATA4 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Heart/embryology , Heart/physiology , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mice, 129 Strain , Mice, Knockout , Mice, Transgenic , Mutation , Myocardium/cytology , Myocardium/metabolism , Myocytes, Cardiac/cytology , Oligonucleotide Array Sequence Analysis , Protein Binding/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics
10.
Am J Respir Cell Mol Biol ; 46(4): 524-31, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22108299

ABSTRACT

The mechanistic links between exposure to airborne particulate matter (PM) pollution and the associated increases in cardiovascular morbidity and mortality, particularly in people with congestive heart failure (CHF), have not been identified. To advance understanding of this issue, genetically engineered mice (CREB(A133)) exhibiting severe dilated cardiomyopathic changes were exposed to ambient PM collected in Baltimore. CREB(A133) mice, which display aberrant cardiac physiology and anatomy reminiscent of human CHF, displayed evidence of basal autonomic aberrancies (compared with wild-type mice) with PM exposure via aspiration, producing significantly reduced heart rate variability, respiratory dysynchrony, and increased ventricular arrhythmias. Carotid body afferent nerve responses to hypoxia and hyperoxia-induced respiratory depression were pronounced in PM-challenged CREB(A133) mice, and denervation of the carotid bodies significantly reduced PM-mediated cardiac arrhythmias. Genome-wide expression analyses of CREB(A133) left ventricular tissues demonstrated prominent Na(+) and K(+) channel pathway gene dysregulation. Subsequent PM challenge increased tyrosine phosphorylation and nitration of the voltage-gated type V cardiac muscle α-subunit of the Na(+) channel encoded by SCN5A. Ranolazine, a Na(+) channel modulator that reduces late cardiac Na(+) channel currents, attenuated PM-mediated cardiac arrhythmias and shortened PM-elongated QT intervals in vivo. These observations provide mechanistic insights into the epidemiologic findings in susceptibility of human CHF populations to PM exposure. Our results suggest a multiorgan pathobiology inherent to the CHF phenotype that is exaggerated by PM exposure via heightened carotid body sensitivity and cardiac Na(+) channel dysfunction.


Subject(s)
Air Pollutants/toxicity , Arrhythmias, Cardiac/chemically induced , Carotid Body/drug effects , Particulate Matter/toxicity , Sodium Channels/metabolism , Animals , Baltimore , Bradycardia/chemically induced , Carotid Body/physiopathology , Cyclic AMP Response Element-Binding Protein/genetics , Disease Models, Animal , Gene Expression Regulation , Heart Failure/etiology , Heart Ventricles/drug effects , Mice , Mice, Inbred Strains , Mice, Mutant Strains , NAV1.5 Voltage-Gated Sodium Channel , Sodium Channels/genetics
11.
Birth Defects Res A Clin Mol Teratol ; 91(6): 586-9, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21538819

ABSTRACT

BACKGROUND: Developmental abnormalities of the abdominal aorta are exceedingly rare, and the molecular mechanisms underlying these defects are unknown. CASE: We present computed angiographic findings of a 64-year-old female with long-standing hypertension having an abdominal double aorta accompanied by a double inferior vena cava. The abdominal aorta bifurcated into two lumens just caudal to the diaphragm at the level of the 12th thoracic vertebra. The dorsal abdominal aortic trunk supplied several lumbar arteries, the inferior mesenteric artery, and both iliac arteries. The ventral abdominal aortic trunk supplied the celiac artery, both renal arteries, and the superior mesenteric artery. CONCLUSION: We propose that a defect in the development of the aortic vascular smooth muscle is a possible mechanism for this rare anomaly based on our current understanding of the formation of the aorta during early embryonic development.


Subject(s)
Aorta, Abdominal/abnormalities , Body Patterning , Vena Cava, Inferior/abnormalities , Angiography , Constriction, Pathologic , Female , Humans , Hypertension/drug therapy , Middle Aged , Renal Artery/abnormalities , Tomography, X-Ray Computed
12.
Am J Physiol Heart Circ Physiol ; 299(6): H2056-68, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20935148

ABSTRACT

Cardiac failure is associated with diminished activation of the transcription factor cyclic nucleotide regulatory element binding-protein (CREB), and heart-specific expression of a phosphorylation-deficient CREB mutant in transgenic mice [dominant negative CREB (dnCREB) mice] recapitulates the contractile phenotypes of cardiac failure (Fentzke RC, Korcarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant-negative CREB transcription factor in the heart. J Clin Invest 101: 2415-2426, 1998). In the present study, we demonstrated significantly elevated mortality and contractile dysfunction in female compared with male dnCREB mice. Female dnCREB mice demonstrated a 21-wk survival of only 17% compared with 67% in males (P < 0.05) and exclusively manifest decreased cardiac peroxisome proliferator-activated receptor-γ coactivator-1α and estrogen-related receptor-α content, suggesting sex-related effects on cardiac mitochondrial function. Hearts from 4-wk-old dnCREB mice of both sexes demonstrated diminished mitochondrial respiratory capacity compared with nontransgenic controls. However, by 12 wk of age, there was a significant decrease in mitochondrial density (citrate synthase activity) and deterioration of mitochondrial structure, as demonstrated by transmission electron microscopy, in female dnCREB mice, which were not found in male transgenic littermates. Subsarcolemmal mitochondria isolated from hearts of female, but not male, dnCREB mice demonstrated increased ROS accompanied by decreases in the expression/activity of the mitochondrial antioxidants MnSOD and glutathione peroxidase. These results demonstrate that heart-specific dnCREB expression results in mitochondrial respiratory dysfunction in both sexes; however, increased oxidant burden, reduced antioxidant expression, and disrupted mitochondrial structure are exacerbated by the female sex, preceding and contributing to the greater contractile morbidity and mortality. These results provide further support for the role of the CREB transcription factor in regulating mitochondrial integrity and identify a critical pathway that may contribute to sex differences in heart failure.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Heart Failure/metabolism , Mitochondria, Heart/metabolism , Mitochondrial Diseases/metabolism , Myocardium/metabolism , Age Factors , Animals , Apoptosis , Cell Respiration , Citrate (si)-Synthase/metabolism , Cyclic AMP Response Element-Binding Protein/genetics , Female , Genes, Dominant , Glutathione Peroxidase/metabolism , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Ion Channels/metabolism , Male , Mice , Mice, Inbred ICR , Mice, Transgenic , Mitochondria, Heart/ultrastructure , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Mitochondrial Diseases/physiopathology , Mitochondrial Proteins/metabolism , Myocardial Contraction , Myocardium/ultrastructure , Oxidative Stress , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation , Reactive Oxygen Species/metabolism , Receptors, Estrogen/metabolism , Sex Factors , Superoxide Dismutase/metabolism , Time Factors , Trans-Activators/metabolism , Transcription Factors , Uncoupling Protein 3 , Up-Regulation , Glutathione Peroxidase GPX1 , ERRalpha Estrogen-Related Receptor
14.
Circulation ; 121(24): 2661-71, 2010 Jun 22.
Article in English | MEDLINE | ID: mdl-20529999

ABSTRACT

BACKGROUND: Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H(2)O(2), is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this are unknown. METHODS AND RESULTS: SOD2 expression in PASMCs is decreased in PAH patients and FHRs with PAH. FHR PASMCs have higher proliferation and lower apoptosis rates than Sprague-Dawley rat PASMCs. Moreover, FHR PASMCs have hyperpolarized mitochondria, low H(2)O(2) production, and reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing mitochondria, decreasing H(2)O(2), and inhibiting caspase activity. Conversely, SOD2 overexpression in FHR PASMCs or therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PAs versus aortic SMCs and is reversed by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, restoring both SOD2 expression and the ratio of proliferation to apoptosis. Expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs. CONCLUSIONS: Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.


Subject(s)
Cell Proliferation , Epigenesis, Genetic/genetics , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Mitochondria, Muscle/enzymology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Adenoviridae/genetics , Animals , Apoptosis , Biomimetics , Disease Models, Animal , Epigenesis, Genetic/physiology , Humans , Hydrogen Peroxide/metabolism , Hypertension, Pulmonary/drug therapy , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Phenotype , Rats , Rats, Mutant Strains , Rats, Sprague-Dawley
15.
J Cell Sci ; 123(Pt 14): 2413-22, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20571050

ABSTRACT

Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.


Subject(s)
Membrane Proteins/biosynthesis , Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/metabolism , NFATC Transcription Factors/metabolism , RNA, Messenger/analysis , Animals , Calcium Signaling/drug effects , Calcium Signaling/genetics , Cardiotoxins/pharmacology , Cells, Cultured , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Knockout , Models, Animal , Muscle Proteins/genetics , Muscle, Skeletal/drug effects , Muscle, Skeletal/growth & development , Muscle, Skeletal/injuries , Muscle, Skeletal/pathology , Muscular Dystrophy, Duchenne/genetics , NFATC Transcription Factors/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Regeneration
16.
Development ; 137(9): 1543-51, 2010 May.
Article in English | MEDLINE | ID: mdl-20356956

ABSTRACT

Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1(-/-) mice have a nearly complete perinatal lethality. Histologic examination of Ets1(-/-) embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1(-/-) mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.


Subject(s)
Cell Differentiation/physiology , Cell Movement/physiology , Heart/embryology , Neural Crest/cytology , Proto-Oncogene Protein c-ets-1/metabolism , Aggrecans/metabolism , Animals , Blotting, Western , Butadienes/pharmacology , Cartilage/embryology , Cartilage/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Movement/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique , Heart/drug effects , Heart Defects, Congenital/genetics , In Situ Hybridization , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Neural Crest/embryology , Nitriles/pharmacology , Proto-Oncogene Protein c-ets-1/genetics , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor/metabolism , SOXD Transcription Factors/metabolism
17.
Circ Res ; 106(5): 818-32, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-20299672

ABSTRACT

The establishment of the coronary circulation is critical for the development of the embryonic heart. Over the last several years, there has been tremendous progress in elucidating the pathways that control coronary development. Interestingly, many of the pathways that regulate the development of the coronary vasculature are distinct from those governing vasculogenesis in the rest of the embryo. It is becoming increasingly clear that coronary development depends on a complex communication between the epicardium, the subepicardial mesenchyme, and the myocardium mediated in part by secreted growth factors. This communication coordinates the growth of the myocardium with the formation of the coronary vasculature. This review summarizes our present understanding of the role of these growth factors in the regulation of coronary development. Continued progress in this field holds the potential to lead to novel therapeutics for the treatment of patients with coronary artery disease.


Subject(s)
Coronary Vessels/metabolism , Myocardium/metabolism , Neovascularization, Physiologic , Pericardium/metabolism , Signal Transduction , Animals , Coronary Vessels/embryology , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Pericardium/embryology
18.
EMBO J ; 29(2): 457-68, 2010 Jan 20.
Article in English | MEDLINE | ID: mdl-20010697

ABSTRACT

The transcriptional co-factor Friend of GATA1 (FOG-1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N-terminus. To test the importance of FOG-1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG-1/NuRD interaction (FOG-1(R3K5A)). Homozygous FOG-1(R3K5A) mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG-1(R3K5A/R3K5A) megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG-1/NuRD interaction is required for the earlier described 'GATA Switch'. In addition, ablation of FOG-1/NuRD interaction led to inappropriate expression of mast cell and eosinophil-specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG-1(R3K5A/R3K5A) megakaryocytes, suggesting that FOG-1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG-1/NuRD interaction for the re-enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.


Subject(s)
Hematopoiesis , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cells, Cultured , Chromatin/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , Erythropoiesis , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Gene Expression Regulation , Granulocytes/cytology , Megakaryocytes/cytology , Megakaryocytes/metabolism , Mice , Mutation , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic
19.
Am J Physiol Gastrointest Liver Physiol ; 297(6): G1214-22, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19779014

ABSTRACT

GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH(2)-terminally truncated "short GATA5" comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated.


Subject(s)
Alternative Splicing , GATA5 Transcription Factor/genetics , Promoter Regions, Genetic , RNA, Messenger/metabolism , 3' Untranslated Regions , 5' Flanking Region , Animals , Atrial Natriuretic Factor/genetics , Atrial Natriuretic Factor/metabolism , Blotting, Northern , Dogs , Epithelial Cells/metabolism , Exons , GATA5 Transcription Factor/metabolism , Gastric Mucosa/metabolism , Genes, Reporter , Introns , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Smooth Muscle/metabolism , NIH 3T3 Cells , Protein Isoforms , Random Amplified Polymorphic DNA Technique , Respiratory Mucosa/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Initiation Site , Transcriptional Activation , Transfection
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