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Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. We have previously demonstrated through bioinformatics that constitutive adenylyl cyclase activation in TGAC8 mice is associated with the activation of inflammation-related signaling pathways. However, the immune response associated with chronic myocardial stress in the TGAC8 mouse remains unexplored. Here we demonstrate that chronic activation of adenylyl cyclase in cardiomyocytes of TGAC8 mice results in activation of cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. All these changes precede the appearance of cardiac fibrosis. We provide evidence indicating that RelA activation in cardiomyocytes with chronic activation of adenylyl cyclase is mediated by calcium-protein Kinase A (PKA) signaling. Using a model of chronic cardiomyocyte stress and accelerated aging, we highlight a novel, calcium/PKA/RelA-dependent connection between cardiomyocyte stress, myocardial inflammation, and systemic inflammation. These findings suggest that RelA-mediated signaling in cardiomyocytes might be an adaptive response to stress that, when chronically activated, ultimately contributes to both cardiac and systemic aging.
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Adenilil Ciclases , Envelhecimento , Inflamação , Miócitos Cardíacos , Transdução de Sinais , Fator de Transcrição RelA , Animais , Miócitos Cardíacos/metabolismo , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Camundongos , Transdução de Sinais/fisiologia , Envelhecimento/fisiologia , Envelhecimento/metabolismo , Fator de Transcrição RelA/metabolismo , Inflamação/metabolismo , Modelos Animais de Doenças , Adaptação Fisiológica , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
AIMS: High glucose levels and Glucose-6-Phosphate Dehydrogenase deficiency (G6PDd) have both tissue inflammatory effects. Here we determined whether G6PDd accelerates arterial aging (information linked stiffening) in diabetes. METHODS: Plasma glucose, interleukin 6 (IL6), and arterial stiffness (indexed as carotid-femoral Pulse Wave Velocity, PWV) and red blood cell G6PD activity were assessed in a large (4448) Sardinian population. RESULTS: Although high plasma glucose in diabetics, did not differ by G6DP status (178.2 ± 55.1 vs 169.0 ± 50.1 mg/dl) in G6DPd versus non-G6PDd subjects, respectively, IL6, and PWV (adjusted for age and glucose) were significantly increased in G6PDd vs non-G6PDd subjects (PWV, 8.0 ± 0.4 vs 7.2 ± 0.2 m/sec) and (IL6, 6.9 ± 5.0 vs 4.2 ± 3.0 pg/ml). In non-diabetics, neither fasting plasma glucose, nor IL6, nor PWV were impacted by G6PDd. CONCLUSION: G6PDd in diabetics is associated with increased inflammatory markers and accelerated arterial aging.
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Diabetes Mellitus , Deficiência de Glucosefosfato Desidrogenase , Rigidez Vascular , Humanos , Envelhecimento , Glicemia , Diabetes Mellitus/epidemiologia , Deficiência de Glucosefosfato Desidrogenase/epidemiologia , Interleucina-6 , Análise de Onda de PulsoRESUMO
Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMC from old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMC from the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMC with silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.
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Glicosídeos Cardíacos , Hipertensão , Músculo Liso Vascular , Animais , Masculino , Ratos , Envelhecimento/genética , Fator Natriurético Atrial , Células Cultivadas , Colágeno Tipo I , GMP Cíclico , Fibrose , Ratos Sprague-Dawley , Cloreto de Sódio na DietaRESUMO
Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.
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Aterosclerose , Placa Aterosclerótica , Camundongos , Animais , Placa Aterosclerótica/genética , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Senescência Celular/genética , Músculo Liso Vascular/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismoRESUMO
BACKGROUND: Photobiomodulation (PBM) therapy, a form of low-dose light therapy, has been noted to be effective in several age-associated chronic diseases such as hypertension and atherosclerosis. Here, we examined the effects of PBM therapy on age-associated cardiovascular changes in a mouse model of accelerated cardiac aging. METHODS: Fourteen months old Adenylyl cyclase type VIII (AC8) overexpressing transgenic mice (n = 8) and their wild-type (WT) littermates (n = 8) were treated with daily exposure to Near-Infrared Light (850 nm) at 25 mW/cm2 for 2 min each weekday for a total dose of 1 Einstein (4.5 p.J/cm2 or fluence 3 J/cm2 ) and compared to untreated controls over an 8-month period. PBM therapy was administered for 3.5 months (Early Treatment period), paused, due to Covid-19 restrictions for the following 3 months, and restarted again for 1.5 months. Serial echocardiography and gait analyses were performed at monthly intervals, and serum TGF-ß1 levels were assessed following sacrifice. RESULTS: During the Early Treatment period PBM treatments: reduced the age-associated increases in left ventricular (LV) mass in both genotypes (p = 0.0003), reduced the LV end-diastolic volume (EDV) in AC8 (p = 0.04); and reduced the left atrial dimension in both genotypes (p = 0.02). PBM treatments substantially increased the LV ejection fraction (p = 0.03), reduced the aortic wall stiffness (p = 0.001), and improved gait symmetry, an index of neuro-muscular coordination (p = 0.005). The effects of PBM treatments, measured following the pause, persisted. Total TGF-ß1 levels were significantly increased in circulation (serum) in AC8 following PBM treatments (p = 0.01). We observed a striking increase in cumulative survival in PBM-treated AC8 mice (100%; p = 0.01) compared to untreated AC8 mice (43%). CONCLUSION: PBM treatment mitigated age-associated cardiovascular remodeling and reduced cardiac function, improved neuromuscular coordination, and increased longevity in an experimental animal model. These responses correlate with increased TGF-ß1 in circulation. Future mechanistic and dose optimization studies are necessary to assess these anti-aging effects of PBM, and validation in future controlled human studies is required for effective clinical translation.
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COVID-19 , Terapia com Luz de Baixa Intensidade , Humanos , Camundongos , Animais , Lactente , Fator de Crescimento Transformador beta1 , Terapia com Luz de Baixa Intensidade/métodos , Envelhecimento , CoraçãoRESUMO
The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26 months) and adult (3-4 months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.
Assuntos
Fibrilação Atrial , Animais , Camundongos , 1-Metil-3-Isobutilxantina/farmacologia , Regulação para Baixo , NAD , AMP Cíclico , Trifosfato de Adenosina/farmacologiaRESUMO
The spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase-A signaling. The mean AP-cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine binds to adenosine receptors (A1, A3) that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gßγ signaling that activates IKAch,Ado. Adenosine also inhibits adenylyl cyclase activity via Gαi signaling, impacting cAMP-mediated protein kinase-A-dependent protein phosphorylation. We hypothesize that in addition to IKAch,Ado activation, adenosine impacts also Ca2+ via Gαi signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM adenosine substantially increased both the mean APCL (on average by 43%, n = 10) and AP beat-to-beat variability from 5.1 ± 1.7% to 7.2 ± 2.0% (n = 10) measured via membrane potential and 5.0 ± 2.2% to 10.6 ± 5.9% (n = 40) measured via Ca2+ (assessed as the coefficient of variability = SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic local Ca2+releases (LCRs) (Ca2+-clock effect): as LCR size distributions shifted to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of adenosine "drifted" upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher adenosine concentrations (>100 µM): small stochastic LCRs failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of adenosine to activate Gßγ and IKACh,Ado and to activate Gαi, suppressing adenylyl cyclase activity, both contribute to the adenosine-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.
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Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein-protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.
Assuntos
Proteínas 14-3-3 , Proteômica , Camundongos , Animais , Proteínas 14-3-3/metabolismo , Mitocôndrias/metabolismo , Coração , ImunoprecipitaçãoRESUMO
Background Age-associated aortic remodeling includes a marked increase in intimal medial thickness (IMT), associated with signs of inflammation. Although aortic wall milk fat globule-epidermal growth factor VIII (MFG-E8) increases with age, and is associated with aortic inflammation, it is not known whether MFG-E8 is required for the age-associated increase in aortic IMT. Here, we tested whether MFG-E8 is required for the age-associated increase in aortic IMT. Methods and Results To determine the role of MFG-E8 in the age-associated increase of IMT, we compared aortic remodeling in adult (20-week) and aged (96-week) MFG-E8 (-/-) knockout and age matched wild-type (WT) littermate mice. The average aortic IMT increased with age in the WT from 50±10 to 70±20 µm (P<0.0001) but did not significantly increase with age in MFG-E8 knockout mice. Because angiotensin II signaling is implicated as a driver of age-associated increase in IMT, we infused 30-week-old MFG-E8 knockout and age-matched littermate WT mice with angiotensin II or saline via osmotic mini-pumps to determine whether MFG-E8 is required for angiotensin II-induced aortic remodeling. (1) In WT mice, angiotensin II infusion substantially increased IMT, elastic lamina degradation, collagen deposition, and the proliferation of vascular smooth muscle cells; in contrast, these effects were significantly reduced in MFG-E8 KO mice; (2) On a molecular level, angiotensin II treatment significantly increased the activation and expression of matrix metalloproteinase type 2, transforming growth factor beta 1, and its downstream signaling molecule phosphorylated mother against decapentaplegic homolog 2, and collagen type I production in WT mice; however, in the MFG-E8 knockout mice, these molecular effects were significantly reduced; and (3) in WT mice, angiotensin II increased levels of aortic inflammatory markers phosphorylated nuclear factor-kappa beta p65, monocyte chemoattractant protein 1, tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 molecular expression, while in contrast, these inflammatory markers did not change in knockout mice. Conclusions Thus, MFG-E8 is required for both age-associated proinflammatory aortic remodeling and also for the angiotensin II-dependent induction in younger mice of an aortic inflammatory phenotype observed in advanced age. Targeting MFG-E8 would be a novel molecular approach to curb adverse arterial remodeling.
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Angiotensina II , Fator de Crescimento Epidérmico , Angiotensina II/farmacologia , Animais , Glicolipídeos , Glicoproteínas , Inflamação/metabolismo , Gotículas Lipídicas , Camundongos , Camundongos Knockout , Proteínas do Leite/genética , Proteínas do Leite/metabolismoRESUMO
Background Lower ankle-brachial index (ABI) values within the 0.90 to 1.40 range are associated with poorer mitochondrial oxidative capacity of thigh muscles in cross-sectional analyses. Whether ABI decline is associated with greater declines in thigh muscle oxidative capacity with aging is unknown. Method and Results We analyzed data from 228 participants (100 men) of the BLSA (Baltimore Longitudinal Study of Aging), aged 39 to 97 years, with an ABI between 0.9 and 1.40 at baseline and at follow-up (mean follow-up period of 2.8 years). We examined mitochondrial oxidative capacity of the left thigh muscle, by measuring the postexercise phosphocreatine recovery rate constant (kPCr) from phosphorus-31 magnetic resonance spectroscopy. Greater kPCr indicated higher mitochondrial oxidative capacity. Although kPCr was available on the left leg only, ABI was measured in both legs. Longitudinal rates of change (Change) of left and right ABI and kPCr of the left thigh muscle were estimated using linear mixed effects models, and their association was analyzed by standardized multiple linear regressions. In multivariate analysis including sex, age, baseline kPCr, both left and right baseline ABI, and ABI change in both legs, (kPCr)Change was directly associated with ipsilateral (left) (ABI)Change (standardized [STD]-ß=0.14; P=0.0168) but not with contralateral (right) (ABI)Change (P=0.22). Adjusting for traditional cardiovascular risk factors, this association remained significant (STD-ß=0.18; P=0.0051). (kPCr)Change was steeper in White race participants (STD-ß=0.16; P=0.0122) and body mass index (STD-ß=0.13; P=0.0479). There was no significant association with current smoking status (P=0.63), fasting glucose (P=0.28), heart rate (P=0.67), mean blood pressure (P=0.78), and low-density lipoprotein (P=0.75), high-density lipoprotein (P=0.82), or triglycerides (P=0.15). Conclusions In people without peripheral arterial disease, greater decline in ABI over time, but not baseline ABI, was associated with faster decline in thigh mitochondrial oxidative capacity in the ipsilateral leg. Further studies are needed to examine whether early interventions that improve lower extremity muscle perfusion can improve and prevent the decline of muscle energetics.
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Doença Arterial Periférica , Infecções Sexualmente Transmissíveis , Adulto , Idoso , Idoso de 80 Anos ou mais , Índice Tornozelo-Braço , Estudos Transversais , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/diagnóstico , Fatores de RiscoRESUMO
Background: Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? Methods: We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Results: Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Conclusion: Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.
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Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.
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Relógios Biológicos , Fosfoproteínas Fosfatases/metabolismo , Nó Sinoatrial/citologia , Potenciais de Ação/efeitos dos fármacos , Animais , Relógios Biológicos/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Simulação por Computador , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ventrículos do Coração/citologia , Toxinas Marinhas/farmacologia , Modelos Biológicos , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , CoelhosRESUMO
The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).
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Relógios Biológicos/fisiologia , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Nó Sinoatrial/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , AMP Cíclico/metabolismo , HumanosRESUMO
Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy. Additionally, disulfiram treatment reversed established diet-induced obesity and metabolic dysfunctions in middle-aged mice. Reductions in feeding efficiency and increases in energy expenditure were associated with body weight regulation in response to long-term disulfiram treatment. Loss of fat tissue and an increase in liver fenestrations were also observed in rats on disulfiram. Given the potent anti-obesogenic effects in rodents, repurposing disulfiram in the clinic could represent a new strategy to treat obesity and its metabolic comorbidities.
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Fármacos Antiobesidade/farmacologia , Peso Corporal/efeitos dos fármacos , Dissulfiram/farmacologia , Obesidade/tratamento farmacológico , Animais , Dieta/efeitos adversos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-ß1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-ß dependent, underlie its effects.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/induzido quimicamente , Bufanolídeos/farmacologia , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Hipertensão/complicações , Cloreto de Sódio , Remodelação Vascular/efeitos dos fármacos , Rigidez Vascular/efeitos dos fármacos , Animais , Aorta/metabolismo , Aorta/patologia , Aorta/fisiopatologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Pressão Sanguínea , Colágeno/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Fibrose , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Proteína Proto-Oncogênica c-fli-1/metabolismo , Ratos Wistar , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/sangue , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Spontaneous firing of sinoatrial (SA) node cells (SANCs) is regulated by cyclic adenosine monophosphate (cAMP)-mediated, protein kinase A (PKA)-dependent (cAMP/PKA) local subsarcolemmal Ca2+ releases (LCRs) from ryanodine receptors (RyR). The LCRs occur during diastolic depolarization (DD) and activate an inward Na+/Ca2+ exchange current that accelerates the DD rate prompting the next action potential (AP). Basal phosphodiesterases (PDEs) activation degrades cAMP, reduces basal cAMP/PKA-dependent phosphorylation, and suppresses normal spontaneous firing of SANCs. The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by 3-isobutyl-1-methylxanthine (IBMX) increases spontaneous firing of SANC by â¼50%. Though inhibition of single PDE1-PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by â¼50%. Here, we describe the expression and distribution of different PDE subtypes within rabbit SANCs, several specific targets (L-type Ca2+ channels and phospholamban) regulated by basal concurrent PDE3 + PDE4 activation, and critical importance of RyR Ca2+ releases for PDE-dependent regulation of spontaneous SANC firing. Colocalization of PDE3 and PDE4 beneath sarcolemma or in striated patterns inside SANCs strongly suggests that PDE-dependent regulation of cAMP/PKA signaling might be executed at the local level; this idea, however, requires further verification.
RESUMO
OBJECTIVE: Cytokines release by adipocytes could interact with TSH secretion. We evaluated the relationship between adipocytokines and TSH. We further tested for association of cytokines and thyroid autoimmunity. METHODS: We conducted a cross-sectional study in a community-based sample including 5385 individuals (2964 female) with TSH within the reference range. Subjects who reported taking thyroid medications or drugs that alter thyroid function were excluded. TSH, FT4, adiponectin, leptin, antibody against thyroperoxidase and against thyroglobulin were measured. Linear and logistic regression models were used to test for association. RESULTS: Females had higher adiponectin and leptin level and increased frequency of thyroid antibodies. In multiple regression analysis TSH was directly associated with leptin (ßâ¯=â¯0.003, pâ¯=â¯0.001) and the presence of circulating antibody against thyroperoxidase (ßâ¯=â¯0.315, pâ¯<â¯0.001), but negatively associated with age (ßâ¯=â¯-0.012, pâ¯<â¯0.001) and FT4 (ßâ¯=â¯-0.359, pâ¯<â¯0.001). Adiponectin, the presence of antibody against thyroglobulin and smoking habit were not associated with TSH levels (pâ¯=â¯0.223, pâ¯=â¯0.174 and pâ¯=â¯0.788, respectively). Logistic regression analysis revealed that higher adiponectin levels were associated with thyroid autoimmunity. CONCLUSIONS: Leptin is positively associated with TSH levels in euthyroid individuals, suggesting an effect of the adipokine on TSH secretion. Our results support the hypothesis that the leptin and pituitary-thyroid axis might interact in the context of energy homeostasis. The effect of adiponectin on thyroid autoimmunity will require more studies.
Assuntos
Adipocinas/metabolismo , Autoimunidade/fisiologia , Glândula Tireoide/metabolismo , Adipócitos/metabolismo , Adulto , Estudos Transversais , Citocinas/metabolismo , Feminino , Humanos , Leptina/metabolismo , Masculino , Pessoa de Meia-Idade , Tireoglobulina/metabolismo , Tireotropina/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismoRESUMO
BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.
Assuntos
Anticorpos/uso terapêutico , Bufanolídeos/imunologia , Placenta/metabolismo , Pré-Eclâmpsia/tratamento farmacológico , Artérias Umbilicais/metabolismo , Adulto , Animais , Anticorpos/imunologia , Pressão Sanguínea , Bufanolídeos/sangue , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Eritrócitos/enzimologia , Feminino , Fibrose , Idade Gestacional , Humanos , Imunoterapia , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Pré-Eclâmpsia/imunologia , Pré-Eclâmpsia/patologia , Gravidez , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Transativadores , Artérias Umbilicais/patologiaRESUMO
The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+-cAMP-protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. ß-Adrenergic receptor (ß-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating "arrested" human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When ß-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by ß-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.