ABSTRACT
Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.
Subject(s)
Diabetes Mellitus, Type 2/complications , Diabetic Neuropathies/physiopathology , Heart Rate/physiology , RGS Proteins/metabolism , Sinoatrial Node/metabolism , Animals , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Diabetes Mellitus, Type 2/genetics , Diabetic Neuropathies/etiology , Disease Models, Animal , Female , Heart Rate/drug effects , Humans , Male , Mice , Mice, Transgenic , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Parasympathetic Nervous System , RGS Proteins/antagonists & inhibitors , Sinoatrial Node/cytology , Sinoatrial Node/drug effects , Sinoatrial Node/innervationABSTRACT
Sinoatrial node (SAN) disease mechanisms are poorly understood, and therapeutic options are limited. Natriuretic peptide(s) (NP) are cardioprotective hormones whose effects can be mediated partly by the NP receptor C (NPR-C). We investigated the role of NPR-C in angiotensin II (Ang II)-mediated SAN disease in mice. Ang II caused SAN disease due to impaired electrical activity in SAN myocytes and increased SAN fibrosis. Strikingly, Ang II treatment in NPR-C-/- mice worsened SAN disease, whereas co-treatment of wild-type mice with Ang II and a selective NPR-C agonist (cANF) prevented SAN dysfunction. NPR-C may represent a new target to protect against the development of Ang II-induced SAN disease.
ABSTRACT
Natriuretic peptides (NPs) play essential roles in the regulation of cardiovascular function. NP effects are mediated by receptors known as NPR-A, NPR-B or NPR-C. NPs have potent effects on regulation of heart rate (HR) by the autonomic nervous system (ANS), but the role of NPR-C in these effects has not been investigated. Accordingly, we have used telemetric ECG recordings in awake, freely moving wildtype and NPR-C knockout (NPR-C-/-) mice and performed heart rate variability (HRV) analysis to assess alterations in sympatho-vagal balance on the heart following loss of NPR-C. Our novel data demonstrate that NPR-C-/- mice are characterized by elevations in HR, reductions in circadian changes in HR and enhanced occurrence of sinus pauses, indicating increased arrhythmogenesis and a loss of HRV. Time domain and frequency domain analyses further demonstrate that HRV is reduced in NPR-C-/- mice in association with a reduction in parasympathetic activity. Importantly, the low frequency to high frequency ratio was increased in NPR-C-/- mice indicating that sympathetic activity is also enhanced. These changes in autonomic regulation were confirmed using atropine and propranolol to antagonize the ANS. These findings illustrate that loss of NPR-C reduces HRV due to perturbations in the regulation of the heart by the ANS.
Subject(s)
Autonomic Nervous System/physiology , Heart Rate , Receptors, Atrial Natriuretic Factor/deficiency , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Autonomic Nervous System/physiopathology , Gene Knockout Techniques , Mice , Receptors, Atrial Natriuretic Factor/geneticsABSTRACT
Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca2+ overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca2+ dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca2+ concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca2+ currents were similar in myocytes from sham and OVX mice but Ca2+ transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2µM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300µM IBMX), or an AC activator (10µM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca2+ homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50µM). Interestingly, selective inhibition of PDE4 increased Ca2+ current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca2+ release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca2+ dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP/metabolism , Excitation Contraction Coupling , Myocardium/metabolism , Ovariectomy , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Adenylyl Cyclases/metabolism , Animals , Calcium Signaling/drug effects , Cell Size/drug effects , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Excitation Contraction Coupling/drug effects , Female , Isoquinolines/pharmacology , Mice, Inbred C57BL , Models, Biological , Myocardial Contraction/drug effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Organ Size/drug effects , Protein Kinase Inhibitors/pharmacology , Rolipram/pharmacology , Sarcoplasmic Reticulum/metabolism , Sulfonamides/pharmacologyABSTRACT
Atrial fibrillation (AF) is prevalent in aging populations; however not all individuals age at the same rate. Instead, individuals of the same chronological age can vary in health status from fit to frail. Our objective was to determine the impacts of age and frailty on atrial function and arrhythmogenesis in mice using a frailty index (FI). Aged mice were more frail and demonstrated longer lasting AF compared to young mice. Consistent with this, aged mice showed longer P wave duration and PR intervals; however, both parameters showed substantial variability suggesting differences in health status among mice of similar chronological age. In agreement with this, P wave duration and PR interval were highly correlated with FI score. High resolution optical mapping of the atria demonstrated reduced conduction velocity and action potential duration in aged hearts that were also graded by FI score. Furthermore, aged mice had increased interstitial fibrosis along with changes in regulators of extracellular matrix remodelling, which also correlated with frailty. These experiments demonstrate that aging results in changes in atrial structure and function that create a substrate for atrial arrhythmias. Importantly, these changes were heterogeneous due to differences in health status, which could be identified using an FI.
Subject(s)
Aging/genetics , Atrial Fibrillation/genetics , Frailty/genetics , Heart Atria/metabolism , Myocytes, Cardiac/metabolism , Action Potentials/physiology , Animals , Atrial Fibrillation/diagnostic imaging , Atrial Fibrillation/metabolism , Atrial Fibrillation/pathology , Biomarkers/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Collagen Type III/genetics , Collagen Type III/metabolism , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Fibrosis , Frailty/diagnostic imaging , Frailty/metabolism , Frailty/pathology , Gene Expression , Heart Atria/diagnostic imaging , Heart Atria/pathology , Heart Function Tests , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/pathology , Severity of Illness Index , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Voltage-Sensitive Dye ImagingABSTRACT
KEY POINTS: Sinoatrial node (SAN) function declines with age; however, not all individuals age at the same rate and health status can vary from fit to frail. Frailty was quantified in young and aged mice using a non-invasive frailty index so that the impacts of age and frailty on heart rate and SAN function could be assessed. SAN function was impaired in aged mice due to alterations in electrical conduction, changes in SAN action potential morphology and fibrosis in the SAN. Changes in SAN function, electrical conduction, action potential morphology and fibrosis were correlated with, and graded by, frailty. This study shows that mice of the same chronological age have quantifiable differences in health status that impact heart rate and SAN function and that these differences in health status can be identified using our frailty index. ABSTRACT: Sinoatrial node (SAN) dysfunction increases with age, although not all older adults are affected in the same way. This is because people age at different rates and individuals of the same chronological age vary in health status from very fit to very frail. Our objective was to determine the impacts of age and frailty on heart rate (HR) and SAN function using a new model of frailty in ageing mice. Frailty, which was quantified in young and aged mice using a frailty index (FI), was greater in aged vs. young mice. Intracardiac electrophysiology demonstrated that HR was reduced whereas SAN recovery time (SNRT) was prolonged in aged mice; however, both parameters showed heteroscedasticity suggesting differences in health status among mice of similar chronological age. Consistent with this, HR and corrected SNRT were correlated with, and graded by, FI score. Optical mapping of the SAN demonstrated that conduction velocity (CV) was reduced in aged hearts in association with reductions in diastolic depolarization (DD) slope and action potential (AP) duration. In agreement with in vivo results, SAN CV, DD slope and AP durations all correlated with FI score. Finally, SAN dysfunction in aged mice was associated with increased interstitial fibrosis and alterations in expression of matrix metalloproteinases, which also correlated with frailty. These findings demonstrate that age-related SAN dysfunction occurs in association with electrical and structural remodelling and that frailty is a critical determinant of health status of similarly aged animals that correlates with changes in HR and SAN function.
Subject(s)
Aging/physiology , Sinoatrial Node/physiology , Action Potentials , Animals , Fibrosis , Heart Rate , Male , Mice, Inbred C57BL , Sinoatrial Node/pathologyABSTRACT
BACKGROUND: Atrial natriuretic peptide (ANP) is a hormone with numerous beneficial cardiovascular effects. Recently, a mutation in the ANP gene, which results in the generation of a mutant form of ANP (mANP), was identified and shown to cause atrial fibrillation in people. The mechanism(s) through which mANP causes atrial fibrillation is unknown. Our objective was to compare the effects of wild-type ANP and mANP on atrial electrophysiology in mice and humans. METHODS AND RESULTS: Action potentials (APs), L-type Ca(2+) currents (ICa,L), and Na(+) current were recorded in atrial myocytes from wild-type or natriuretic peptide receptor C knockout (NPR-C(-/-)) mice. In mice, ANP and mANP (10-100 nmol/L) had opposing effects on atrial myocyte AP morphology and ICa,L. ANP increased AP upstroke velocity (Vmax), AP duration, and ICa,L similarly in wild-type and NPR-C(-/-) myocytes. In contrast, mANP decreased Vmax, AP duration, and ICa,L, and these effects were completely absent in NPR-C(-/-) myocytes. ANP and mANP also had opposing effects on ICa,L in human atrial myocytes. In contrast, neither ANP nor mANP had any effect on Na(+) current in mouse atrial myocytes. Optical mapping studies in mice demonstrate that ANP sped electric conduction in the atria, whereas mANP did the opposite and slowed atrial conduction. Atrial pacing in the presence of mANP induced arrhythmias in 62.5% of hearts, whereas treatment with ANP completely prevented the occurrence of arrhythmias. CONCLUSIONS: These findings provide mechanistic insight into how mANP causes atrial fibrillation and demonstrate that wild-type ANP is antiarrhythmic.
Subject(s)
Action Potentials/drug effects , Atrial Fibrillation/drug therapy , Atrial Fibrillation/physiopathology , Atrial Natriuretic Factor/pharmacology , Electrophysiological Phenomena/drug effects , Heart Atria/drug effects , Myocytes, Cardiac/drug effects , Action Potentials/physiology , Animals , Cardiac Pacing, Artificial , Heart Atria/physiopathology , Humans , Mice , Mice, KnockoutABSTRACT
Cardiovascular autonomic neuropathy (CAN) is a serious complication of diabetes mellitus that impairs autonomic regulation of heart rate (HR). This has been attributed to damage to the nerves that modulate spontaneous pacemaker activity in the sinoatrial node (SAN). Our objective was to test the hypothesis that impaired parasympathetic regulation of HR in diabetes is due to reduced responsiveness of the SAN to parasympathetic agonists. We used the Akita mouse model of type 1 diabetes to study the effects of the parasympathetic agonist carbachol (CCh) on SAN function using intracardiac programmed stimulation, high resolution optical mapping and patch-clamping of SAN myocytes. CCh decreased HR by 30% and increased corrected SAN recovery time (cSNRT) by 123% in wildtype mice. In contrast, CCh only decreased HR by 12%, and only increased cSNRT by 37% in Akita mice. These alterations were due to smaller effects of CCh on SAN electrical conduction and spontaneous action potential firing in isolated SAN myocytes. Voltage clamp experiments demonstrate that the acetylcholine-activated K(+) current (IKACh) is reduced in Akita SAN myocytes due to enhanced desensitization and faster deactivation kinetics. These IKACh alterations were normalized by treating Akita SAN myocytes with PI(3,4,5)P3 or an inhibitor of regulator of G-protein signaling 4 (RGS4). There was no difference in the effects of CCh on the hyperpolarization-activated current (If) between wildtype and Akita mice. Our study demonstrates that Akita diabetic mice demonstrate impaired parasympathetic regulation of HR and SAN function due to reduced responses of the SAN to parasympathetic agonists. Our experiments demonstrate a key role for insulin-dependent phosphoinositide 3-kinase (PI3K) signaling in the parasympathetic dysfunction seen in the SAN in diabetes.
Subject(s)
Parasympathetic Nervous System/physiopathology , Sinoatrial Node/innervation , Acetylcholine/pharmacology , Action Potentials/drug effects , Animals , Carbachol/pharmacology , Cardiotonic Agents/pharmacology , Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/physiopathology , Disease Models, Animal , Heart/drug effects , Heart/physiopathology , Insulin/administration & dosage , Insulin/pharmacology , Mice , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , RGS Proteins/antagonists & inhibitors , RGS Proteins/metabolism , Sinoatrial Node/drug effectsABSTRACT
Natriuretic peptides (NPs) are critical regulators of the cardiovascular system that are currently viewed as possible therapeutic targets for the treatment of heart disease. Recent work demonstrates potent NP effects on cardiac electrophysiology, including in the sinoatrial node (SAN) and atria. NPs elicit their effects via three NP receptors (NPR-A, NPR-B and NPR-C). Among these receptors, NPR-C is poorly understood. Accordingly, the goal of this study was to determine the effects of NPR-C ablation on cardiac structure and arrhythmogenesis. Cardiac structure and function were assessed in wild-type (NPR-C(+/+)) and NPR-C knockout (NPR-C(-/-)) mice using echocardiography, intracardiac programmed stimulation, patch clamping, high-resolution optical mapping, quantitative polymerase chain reaction and histology. These studies demonstrate that NPR-C(-/-) mice display SAN dysfunction, as indicated by a prolongation (30%) of corrected SAN recovery time, as well as an increased susceptibility to atrial fibrillation (6% in NPR-C(+/+) vs. 47% in NPR-C(-/-)). There were no differences in SAN or atrial action potential morphology in NPR-C(-/-) mice; however, increased atrial arrhythmogenesis in NPR-C(-/-) mice was associated with reductions in SAN (20%) and atrial (15%) conduction velocity, as well as increases in expression and deposition of collagen in the atrial myocardium. No differences were seen in ventricular arrhythmogenesis or fibrosis in NPR-C(-/-) mice. This study demonstrates that loss of NPR-C results in SAN dysfunction and increased susceptibility to atrial arrhythmias in association with structural remodelling and fibrosis in the atrial myocardium. These findings indicate a critical protective role for NPR-C in the heart.
Subject(s)
Atrial Fibrillation/genetics , Atrial Fibrillation/metabolism , Receptors, Atrial Natriuretic Factor/genetics , Sinoatrial Node/metabolism , Action Potentials , Animals , Cells, Cultured , Collagen/metabolism , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Receptors, Atrial Natriuretic Factor/metabolism , Sinoatrial Node/physiopathologyABSTRACT
Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1(TG)) mice, and the onset of ductal hyperplasia was accelerated in AEBP1(TG) mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1(TG) bone marrow cells into non-transgenic (AEBP1(NT)) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1(TG) mammary macrophages and epithelium. Shh expression was induced in AEBP1(TG) macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1(TG) mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1(TG) peritoneal macrophages. The conditioned AEBP1(TG) macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1(TG) macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.
Subject(s)
Carboxypeptidases/genetics , Carboxypeptidases/physiology , Hedgehog Proteins/metabolism , Mammary Glands, Animal/metabolism , Repressor Proteins/genetics , Repressor Proteins/physiology , Animals , Bone Marrow Transplantation , Cell Line , Coculture Techniques , Culture Media, Conditioned/pharmacology , Hyperplasia , Inflammation , Macrophages/metabolism , Mammary Glands, Animal/pathology , Mice , Mice, Transgenic , NF-kappa B/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Adipocyte enhancer binding protein 1 (AEBP1) is a multifunctional protein that negatively regulates the tumor suppressor PTEN and IκBα, the inhibitor of NF-κB, through protein-protein interaction, thereby promoting cell survival and inflammation. Mice homozygous for a disrupted AEBP1 gene developed to term but showed defects in growth after birth. AEBP1(-/-) females display lactation defect, which results in the death of 100% of the litters nursed by AEBP1(-/-) dams. Mammary gland development during pregnancy appears normal in AEBP1(-/-) dams; however these mice exhibit expansion of the luminal space and the appearance of large cytoplasmic lipid droplets (CLDs) in the mammary epithelial cells at late pregnancy and parturition, which is a clear sign of failed secretory activation, and accumulation of milk proteins in the mammary gland, presumably reflecting milk stasis following failed secretory activation. Eventually, AEBP1(-/-) mammary gland rapidly undergoes involution at postpartum. Stromal restoration of AEBP1 expression by transplanting wild-type bone marrow (BM) cells is sufficient to rescue the mammary gland defect. Our studies suggest that AEBP1 is critical in the maintenance of normal tissue architecture and function of the mammary gland tissue and controls stromal-epithelial crosstalk in mammary gland development.
Subject(s)
Carboxypeptidases/physiology , Lactation/physiology , Mammary Glands, Animal/metabolism , Milk/chemistry , Repressor Proteins/physiology , Animals , Blotting, Western , Bone Marrow Transplantation , Epithelial Cells/cytology , Female , Mammary Glands, Animal/growth & development , Mice , Mice, Knockout , PregnancyABSTRACT
Atherogenesis is a long-term process that involves inflammatory response coupled with metabolic dysfunction. Foam cell formation and macrophage inflammatory response are two key events in atherogenesis. Adipocyte enhancer-binding protein 1 (AEBP1) has been shown to impede macrophage cholesterol efflux, promoting foam cell formation, via peroxisome proliferator-activated receptor (PPAR)-γ1 and liver X receptor α (LXRα) downregulation. Moreover, AEBP1 has been shown to promote macrophage inflammatory responsiveness by inducing nuclear factor (NF)-κB activity via IκBα downregulation. Lipopolysaccharide (LPS)-induced suppression of pivotal macrophage cholesterol efflux mediators, leading to foam cell formation, has been shown to be mediated by AEBP1. Herein, we showed that AEBP1-transgenic mice (AEBP1(TG)) with macrophage-specific AEBP1 overexpression exhibit hyperlipidemia and develop atherosclerotic lesions in their proximal aortas. Consistently, ablation of AEBP1 results in significant attenuation of atherosclerosis (males: 3.2-fold, P = 0.001 [en face]), 2.7-fold, P = 0.0004 [aortic roots]; females: 2.1-fold, P = 0.0026 [en face], 1.7-fold, P = 0.0126 [aortic roots]) in the AEBP1(-/-)/low-density lipoprotein receptor (LDLR )(-/-) double-knockout (KO) mice. Bone marrow (BM) transplantation experiments further revealed that LDLR (-/-) mice reconstituted with AEBP1(-/-)/LDLR (-/-) BM cells (LDLR (-/-)/KO-BM chimera) display significant reduction of atherosclerosis lesions (en face: 2.0-fold, P = 0.0268; aortic roots: 1.7-fold, P = 0.05) compared with control mice reconstituted with AEBP1(+/+)/LDLR (-/-) BM cells (LDLR (-/-)/WT-BM chimera). Furthermore, transplantation of AEBP1(TG) BM cells with the normal apolipoprotein E (ApoE) gene into ApoE (-/-) mice (ApoE (-/-)/TG-BM chimera) leads to significant development of atherosclerosis (males: 2.5-fold, P = 0.0001 [en face], 4.7-fold, P = 0.0001 [aortic roots]; females: 1.8-fold, P = 0.0001 [en face], 3.0-fold, P = 0.0001 [aortic roots]) despite the restoration of ApoE expression. Macrophages from ApoE (-/-)/TG-BM chimeric mice express reduced levels of PPARγ1, LXRα, ATP-binding cassette A1 (ABCA1) and ATP-binding cassette G1 (ABCG1) and increased levels of the inflammatory mediators interleukin (IL)-6 and tumor necrosis factor (TNF)-α compared with macrophages of control chimeric mice (ApoE (-/-)/NT-BM ) that received AEBP1 nontransgenic (AEBP1(NT) ) BM cells. Our in vivo experimental data strongly suggest that macrophage AEBP1 plays critical regulatory roles in atherogenesis, and it may serve as a potential therapeutic target for the prevention or treatment of atherosclerosis.
Subject(s)
Apolipoproteins E/metabolism , Atherosclerosis/metabolism , Carboxypeptidases/metabolism , Receptors, LDL/metabolism , Repressor Proteins/metabolism , Animals , Aorta/metabolism , Aorta/pathology , Apolipoproteins E/genetics , Atherosclerosis/etiology , Atherosclerosis/genetics , Carboxypeptidases/genetics , Cholesterol/metabolism , Diet, Atherogenic/adverse effects , Dietary Fats/adverse effects , Female , Hyperlipidemias/etiology , Hyperlipidemias/genetics , Hyperlipidemias/metabolism , Immunohistochemistry , Liver X Receptors , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Orphan Nuclear Receptors/metabolism , PPAR gamma/metabolism , Receptors, LDL/genetics , Repressor Proteins/genetics , Sex FactorsABSTRACT
During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.
Subject(s)
Erythropoiesis , Protein Serine-Threonine Kinases/physiology , Protein-Tyrosine Kinases/physiology , Alleles , Anemia/metabolism , Animals , Antigens, CD/metabolism , Bone Marrow Transplantation , Cell Line , Fluorouracil/pharmacology , Humans , K562 Cells , Mice , Mice, Knockout , Mice, Transgenic , Receptors, Transferrin/metabolism , TransgenesABSTRACT
EPO functions primarily as an erythroblast survival factor, and its antiapoptotic actions have been proposed to involve predominantly PI3-kinase and BCL-X pathways. Presently, the nature of EPO-regulated survival genes has been investigated through transcriptome analyses of highly responsive, primary bone marrow erythroblasts. Two proapoptotic factors, Bim and FoxO3a, were rapidly repressed not only via the wild-type EPOR, but also by PY-deficient knocked-in EPOR alleles. In parallel, Pim1 and Pim3 kinases and Irs2 were induced. For this survival gene set, induction failed via a PY-null EPOR-HM allele, but was restored upon reconstitution of a PY343 STAT5-binding site within a related EPOR-H allele. Notably, EPOR-HM supports erythropoiesis at steady state but not during anemia, while EPOR-H exhibits near wild-type EPOR activities. EPOR-H and the wild-type EPOR (but not EPOR-HM) also markedly stimulated the expression of Trb3 pseudokinase, and intracellular serpin, Serpina-3G. For SERPINA-3G and TRB3, ectopic expression in EPO-dependent progenitors furthermore significantly inhibited apoptosis due to cytokine withdrawal. BCL-XL and BCL2 also were studied, but in highly responsive Kit(pos)CD71(high)Ter119(neg) erythroblasts, neither was EPO modulated. EPOR survival circuits therefore include the repression of Bim plus FoxO3a, and EPOR/PY343/STAT5-dependent stimulation of Pim1, Pim3, Irs2 plus Serpina-3G, and Trb3 as new antiapoptotic effectors.
Subject(s)
Apoptosis/physiology , Erythroblasts/physiology , Gene Expression Regulation/physiology , Receptors, Erythropoietin/physiology , Animals , Cell Survival , Flow Cytometry , Gene Expression , Gene Expression Profiling , Mice , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Erythropoietin (EPO's) actions on erythroblasts are ascribed largely to survival effects. Certain studies, however, point to EPO-regulated proliferation. To investigate this problem in a primary system, Kit(pos)CD71(high) erythroblasts were prepared from murine bone marrow, and were first used in the array-based discovery of EPO-modulated cell-cycle regulators. Five cell-cycle progression factors were rapidly up-modulated: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1), Ngfi-A binding protein 2 (Nab2), and cyclin D2. In contrast, inhibitory cyclin G2, p27/Cdkn1b, and B-cell leukemia/lymphoma 6 (Bcl6) were sharply down-modulated. For CYCLIN G2, ectopic expression also proved to selectively attenuate EPO-dependent UT7epo cell-cycle progression at S-phase. As analyzed in primary erythroblasts expressing minimal EPO receptor alleles, EPO repression of cyclin G2 and Bcl6, and induction of cyclin D2, were determined to depend on PY343 (and Stat5) signals. Furthermore, erythroblasts expressing a on PY-null EPOR-HM allele were abnormally distributed in G0/G1. During differentiation divisions, EPOR-HM Ter119(pos) erythroblasts conversely accumulated in S-phase and faltered in an apparent EPO-directed transition to G0/G1. EPO/EPOR signals therefore control the expression of select cell-cycle regulatory genes that are proposed to modulate stage-specific decisions for erythroblast cell-cycle progression.
Subject(s)
Bone Marrow/metabolism , Cell Cycle Proteins/metabolism , Cell Division , Erythroblasts/cytology , Erythroblasts/metabolism , Erythropoietin/pharmacology , Alleles , Animals , Cell Cycle Proteins/genetics , Cells, Cultured , Gene Expression Regulation , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolismABSTRACT
Epo's erythropoietic capacity is ascribed largely to its antiapoptotic actions. In part via gene profiling of bone marrow erythroblasts, Epo is now shown to selectively down-modulate the adhesion/migration factors chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4) and to up-modulate growth differentiation factor-3 (Gdf3), oncostatin-M (OncoM), and podocalyxin like-1 (PODXL). For PODXL, Epo dose-dependent expression of this CD34-related sialomucin was discovered in Kit(+)CD71(high) proerythroblasts and was sustained at subsequent Kit(-)CD71(high) and Ter119(+) stages. In vivo, Epo markedly induced PODXL expression in these progenitors and in marrow-resident reticulocytes. This was further associated with a rapid release of PODXL(+) reticulocytes to blood. As studied in erythroblasts expressing minimal Epo receptor (EpoR) alleles, efficient PODXL induction proved dependence on an EpoR-PY343 Stat5 binding site. Moreover, in mice expressing an EpoR-HM F343 allele, compromised Epo-induced PODXL expression correlated with abnormal anucleated red cell representation in marrow. By modulating this select set of cell-surface adhesion molecules and chemokines, Epo is proposed to mobilize erythroblasts from a hypothesized stromal niche and possibly promote reticulocyte egress to blood.
Subject(s)
Erythroblasts/physiology , Erythropoietin/pharmacology , Receptors, Erythropoietin/genetics , Sialoglycoproteins/genetics , Animals , Apoptosis/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/physiology , Cell Nucleus/physiology , Erythroblasts/drug effects , Flow Cytometry , Gene Expression Profiling , Mice , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/drug effects , Sialomucins/drug effects , Sialomucins/geneticsABSTRACT
Anemia due to chronic disease or chemotherapy often is ameliorated by erythropoietin (Epo). Present studies reveal that, unlike steady-state erythropoiesis, erythropoiesis during anemia depends sharply on an Epo receptor-phosphotyrosine-343-Stat5 signaling axis. In mice expressing a phosphotyrosine-null (PY-null) Epo receptor allele (EpoR-HM), severe and persistent anemia was induced by hemolysis or 5-fluorouracil. In short-term transplantation experiments, donor EpoR-HM bone marrow cells also failed to efficiently repopulate the erythroid compartment. In each context, stress erythropoiesis was rescued to WT levels upon the selective restoration of an EpoR PY343 Stat5-binding site (EpoR-H allele). As studied using a unique primary culture system, EpoR-HM erythroblasts exhibited marked stage-specific losses in Epo-dependent growth and survival. EpoR-H PY343 signals restored efficient erythroblast expansion, and the selective Epo induction of the Stat5 target genes proviral integration site-1 (Pim-1) and oncostatin-M. Bcl2-like 1 (Bcl-x), in contrast, was not significantly induced via WT-EpoR, EpoR-HM, or EpoR-H alleles. In Kit+ CD71+ erythroblasts, EpoR-PY343 signals furthermore enhanced SCF growth effects, and SCF modulation of Pim-1 kinase and oncostatin-M expression. In maturing Kit- CD71+ erythroblasts, oncostatin-M exerted antiapoptotic effects that likewise depended on EpoR PY343-mediated events. Stress erythropoiesis, therefore, requires stage-specific EpoR-PY343-Stat5 signals, some of which selectively bolster SCF and oncostatin-M action.
Subject(s)
Cell Differentiation/physiology , Erythropoiesis/physiology , Phosphotyrosine/metabolism , Receptors, Erythropoietin/physiology , STAT5 Transcription Factor/physiology , Signal Transduction/physiology , Anemia/blood , Anemia/chemically induced , Anemia/genetics , Animals , Binding Sites/genetics , Bone Marrow Cells/cytology , Cell Differentiation/genetics , Cell Survival/genetics , Cell Survival/physiology , Cells, Cultured , Erythroblasts/cytology , Erythropoiesis/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Phenylhydrazines/administration & dosage , Phosphotyrosine/genetics , Phosphotyrosine/physiology , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Erythropoietin/biosynthesis , Receptors, Erythropoietin/genetics , Signal Transduction/geneticsABSTRACT
Committed erythroid progenitor cells require exposure to erythropoietin (Epo) for their survival and for their quantitatively regulated transition to red blood cells. With regard to Epo signal transduction mechanisms, much has been learned from analyses in cell line models, fetal liver or spleen-derived primary erythroblasts and human CD34pos progenitor cells from cord blood or mobilized bone marrow. Presently, we have developed an ex vivo system that efficiently supports the expansion and development of murine adult bone-marrow-derived erythroid progenitor cells. This system is outlined together with its demonstrated utility in studying (for the first time) the signaling capacities of two knocked-in phosphotyrosine-deficient Epo receptor alleles (EpoR-H and EpoR-HM). Ways in which these studies advance an understanding of core Epo signal transduction events are outlined. Also introduced are two new putative negative regulators of Epo-dependent erythropoiesis, DYRK3 and DAPK2 kinases.