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OBJECTIVES: To evaluate the study design and feasibility of drug administration and safety in a randomized clinical trial of recombinant human annexin A5 (SY-005), a constitutively expressed protein with anti-inflammatory, antiapoptotic, and anticoagulant properties, in patients with severe coronavirus disease 2019 (COVID-19). DESIGN: Double-blind, randomized clinical trial. SETTING: Two ICUs at an academic medical center. PATIENTS/SUBJECTS: Adults admitted to the ICU with a confirmed diagnosis of COVID-19 and requiring ventilatory or vasopressor support. INTERVENTIONS: SY-005, a recombinant human annexin A5, at 50 or 100 µg/kg IV every 12 hours for 7 days. MEASUREMENTS AND MAIN RESULTS: We enrolled 18 of the 55 eligible patients (33%) between April 21, 2021, and February 3, 2022. We administered 82% (196/238) of the anticipated doses of study medication and 86% (169/196) were given within 1 hour of the scheduled time. There were no drug-related serious adverse events. We captured 100% of the data that would be required for measuring clinical outcomes in a phase 2 or 3 trial. LIMITATIONS: The small sample size was a result of decreasing admissions of patients with COVID-19, which triggered a stopping rule for the trial. CONCLUSIONS: Although enrollment was low, administration of SY-005 to critically ill patients with COVID-19 every 12 hours for up to 7 days was feasible and safe. Further clinical trials of annexin A5 for the treatment of COVID-19 are warranted. Given reduction of severe COVID-19 disease, future studies should explore the safety and effectiveness of SY-005 use in non-COVID-related sepsis.
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Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.
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BACKGROUND: Maternal exercise lowers the incidence of congenital heart defects (CHDs) induced by pregestational diabetes. However, the molecular mechanisms underlying the beneficial effects of maternal exercise remain unclear. The present study aimed to identify circular RNA (circRNA), microRNA (miRNA) and mRNA networks that are regulated by maternal exercise in fetal hearts of pregestational diabetes. METHODS: Pregestational diabetes was induced in adult C57BL/6 female mice by streptozotocin. The expression profiles of circRNAs, miRNAs and mRNAs in E10.5 fetal hearts of offspring of control and diabetic mothers with or without exercise were analyzed using next generation sequencing. circRNA-miRNA-mRNA networks in fetal hearts were mapped and key candidate transcripts were verified by qPCR analysis. RESULTS: Pregestational diabetes dysregulated the expression of 206 circRNAs, 66 miRNAs and 391 mRNAs in fetal hearts. Maternal exercise differentially regulated 188 circRNAs, 57 miRNAs and 506 mRNAs in fetal hearts of offspring of pregestational diabetes. A total of 5 circRNAs, 12 miRNAs, and 28 mRNAs were incorporated into a final maternal exercise-associated regulatory network in fetal hearts of offspring of maternal diabetes. Notably, maternal exercise normalized the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs in fetal hearts of pregestational diabetes. CONCLUSION: Maternal exercise reverses the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs, and partially normalizes circRNA, miRNA, and mRNA expression profiles in fetal hearts of pregestational diabetes. These findings shed new light on the potential mechanisms of the beneficial effects of maternal exercise on the developing heart in diabetic pregnancies.
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Diabetes Gestacional , MicroRNAs , Humanos , Gravidez , Animais , Camundongos , Feminino , MicroRNAs/genética , RNA Circular/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Camundongos Endogâmicos C57BL , Diabetes Gestacional/genética , Coração Fetal/metabolismo , Redes Reguladoras de GenesRESUMO
Objective: Annexin A5 is a phosphatidylserine binding protein with anti-inflammatory, anticoagulant and anti-apoptotic properties. Preclinical studies have shown that annexin A5 inhibits pro-inflammatory responses and improves organ function and survival in rodent models of sepsis. This clinical trial aimed to evaluate the pharmacokinetic (PK) properties of the recombinant human annexin A5 (SY-005) in severe COVID-19. Methods: This was a pilot randomized, double-blind, placebo-controlled trial. Severe COVID-19 patients were randomly assigned to receive intravenous 50 µg/kg (low dose, n = 3), 100 µg/kg (high dose, n = 5) of SY-005 or placebo (n = 5) every 12 h for 7 days. Plasma SY-005 levels were assessed using enzyme-linked immunosorbent assay (ELISA) and the PK parameters were determined using non-compartmental analysis. Results: All patients treated with SY-005 had a normal baseline estimated glomerular filtration rate (eGFR, 104-125 mL/min/1.73 m2). Both low and high doses of SY-005 were cleared within 6 h after intravenous administration. Plasma maximum concentrations (Cmax), half-life, clearance and volume distribution of low and high doses of SY-005 were 402.4 and 848.9 ng/mL, 0.92 and 0.96 h, 7.52 and 15.19 L/h, and 9.98 and 20.79 L, respectively. Daily pre-dose circulating annexin A5 levels were not significantly different when SY-005 was administered at the low or the high dose 12-h intervals. There was no significant effect on activated partial thromboplastin time (aPTT) or INR (international normalized ratio of prothrombin time) during 7 days of SY-005 treatment. Conclusion: SY-005 doses of 50 and 100 µg/kg were detectable and subsequently cleared from the plasma in severe COVID-19 patients with normal baseline renal function. There was no significant plasma SY-005 accumulation 6 h after drug administration and coagulation was not altered during 7 days of treatment. Clinical trials Registration: This study was registered with ClinicalTrials.gov (NCT04748757, first posted on 10 February 2021).
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Introduction: Cannabis is increasingly being consumed by pregnant women for recreational purposes as well as for its antiemetic and anxiolytic effects despite limited studies on its safety during pregnancy. Importantly, phytocannabinoids found in cannabis can pass through the placenta and enter the fetal circulation. Recent reports suggest gestational cannabis use is associated with negative fetal outcomes, including fetal growth restriction and perinatal intensive care, however, the effects of delta-9-tetrahydrocannabinol (THC) on fetal heart development remains to be elucidated. Materials and Methods: We aimed to determine the outcomes of maternal THC exposure on fetal heart development in mice by administering 0, 5, or 10 mg/kg/day of THC orally to C57BL/6 dams starting at embryonic day (E)3.5. Offspring were collected at E12.5 for molecular analysis, at E17.5 to analyze cardiac morphology or at postnatal day (PND)21 to assess heart function. Results: Maternal THC exposure in E17.5 fetuses resulted in an array of cardiac abnormalities with an incidence of 44% and 55% in the 5 and 10 mg/kg treatment groups, respectively. Maternal THC exposure in offspring resulted in ventricular septal defect, higher semilunar valve volume relative to orifice ratio, and higher myocardial wall thickness. Notably, cell proliferation within the ventricular myocardium was increased, and expression of multiple cardiac transcription factors was downregulated in THC-exposed E12.5 fetuses. Furthermore, heart function was compromised with lower left ventricular ejection fraction, fractional shortening, and cardiac output in PND21 pups exposed to THC compared to controls. Discussion: The results show that maternal THC exposure during gestation induces myocardial hyperplasia and semilunar valve thickening in the fetal heart and postnatal cardiac dysfunction. Our study suggests that maternal cannabis consumption may induce abnormalities in the developing heart and cardiac dysfunction in postnatal life.
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The epicardium is a potential source of cardiac progenitors to support reparative angiogenesis after myocardial infarction (MI) through epithelial-to-mesenchymal transition (EMT). Primary cilia are recognized as hubs of cellular signaling, and their presence can alter downstream pathways to modulate EMT. The present study aimed to examine the effects of inhibiting intraflagellar transport protein-88 (Ift88), a protein vital to ciliary assembly, on epicardial EMT and cardiac remodeling post-MI. Epicardium derived cells (EPDCs) were cultured from E13.5 heart explants and treated with adenoviral vector encoding short-hairpin RNA against the mouse Ift88 (Ad-shIft88) to disassemble the primary cilium. Effects of Ad-shIft88 on epicardial EMT and cardiac remodeling were examined in mice post-MI. Our results show that Ad-shIft88 enhanced EMT of cultured EPDCs. In adult mice, intra-myocardial administration of Ad-shIft88 increased the number of Wilms tumor 1 (Wt1) positive cells in the epicardium and myocardium, promoted expression of genes associated with epicardial EMT, and enhanced capillary and arteriolar densities post-MI. Additionally, intra-myocardial Ad-shIft88 treatment attenuated cardiac hypertrophy and improved myocardial function three weeks post-MI. In conclusion, knockdown of Ift88 improves epicardial EMT, neovascularization and cardiac remodeling in the ischemic heart. Our study highlights the primary cilium as a potential therapeutic target post-MI.
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Infarto do Miocárdio , Remodelação Ventricular , Animais , Proteínas de Transporte/metabolismo , Transição Epitelial-Mesenquimal/genética , Camundongos , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Neovascularização Patológica/metabolismo , Pericárdio , RNA , Proteínas Supressoras de Tumor , Proteínas WT1/metabolismoRESUMO
Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose-dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial-to-mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.
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Cardiopatias Congênitas , Efeitos Tardios da Exposição Pré-Natal , Abandono do Hábito de Fumar , Animais , Feminino , Cardiopatias Congênitas/induzido quimicamente , Humanos , Masculino , Camundongos , Nicotina/efeitos adversos , Gravidez , Dispositivos para o Abandono do Uso de Tabaco/efeitos adversosRESUMO
BACKGROUND: Left ventricular noncompaction (LVNC) is a cardiomyopathy that can lead to arrhythmias, embolic events and heart failure. Despite our current knowledge of cardiac development, the mechanisms underlying noncompaction of the ventricular myocardium are still poorly understood. The small GTPase Rac1 acts as a crucial regulator of numerous developmental events. The present study aimed to investigate the cardiomyocyte specific role of Rac1 in embryonic heart development. METHODS AND RESULTS: The Nkx2.5-Cre transgenic mice were crossed with Rac1f/f mice to generate mice with a cardiomyocyte specific deletion of Rac1 (Rac1Nkx2.5) during heart development. Embryonic Rac1Nkx2.5 hearts at E12.5-E18.5 were collected for histological analysis. Overall, Rac1Nkx2.5 hearts displayed a bifid apex, along with hypertrabeculation and a thin compact myocardium. Rac1Nkx2.5 hearts also exhibited ventricular septal defects (VSDs) and double outlet right ventricle (DORV) or overriding aorta. Cardiomyocytes had a rounded morphology and were highly disorganized, and the myocardial expression of Scrib, a planar cell polarity protein, was reduced in Rac1Nkx2.5 hearts. In addition, cell proliferation rate was significantly decreased in the Rac1Nkx2.5 ventricular myocardium at E9.5. CONCLUSIONS: Rac1 deficiency in the myocardium impairs cardiomyocyte elongation and organization, and proliferative growth of the heart. A spectrum of CHDs arises in Rac1Nkx2.5 hearts, implicating Rac1 signaling in the ventricular myocardium as a crucial regulator of OFT alignment, along with compact myocardium growth and development.
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NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfß2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.
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Transição Epitelial-Mesenquimal/fisiologia , Cardiopatias Congênitas/patologia , Coração/embriologia , NADPH Oxidase 2/metabolismo , Animais , Apoptose , Proliferação de Células , Coxins Endocárdicos/embriologia , Coxins Endocárdicos/metabolismo , Coxins Endocárdicos/patologia , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Camundongos , NADPH Oxidase 2/deficiência , NADPH Oxidase 2/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Endothelial nitric oxide synthase (eNOS) and oxidative stress are critical to embryonic coronary artery development. Maternal diabetes increases oxidative stress and reduces eNOS activity in the fetal heart. Sapropterin (Kuvan®) is an orally active, synthetic form of tetrahydrobiopterin (BH4) and a co-factor for eNOS with antioxidant properties. The aim of the present study was to examine the effects of sapropterin on fetal coronary artery development during pregestational diabetes in mice. Diabetes was induced by streptozotocin to adult female C57BL/6 mice. Sapropterin (10â¯mg/kg/day) was orally administered to pregnant mice from E0.5 to E18.5. Fetal hearts were collected at E18.5 for coronary artery morphological analysis. Sapropterin treatment to diabetic dams reduced the incidence of coronary artery malformation in offspring from 50.0% to 20.6%. Decreases in coronary artery luminal diameter, volume and abundance in fetal hearts from diabetic mothers, were prevented by sapropterin treatment. Maternal diabetes reduced epicardial epithelial-to-mesenchymal transition (EMT) and expression of transcription and growth factors critical to coronary artery development including hypoxia-inducible factor 1a (Hif1a), Snail1, Slug, ß-catenin, retinaldehyde dehydrogenase 2 (Aldh1a2), basic fibroblast growth factor (bFGF) and vascular endothelial group factor receptor 2 (Vegfr2) in E12.5 hearts. Additionally, eNOS phosphorylation was lower while oxidative stress was higher in E12.5 hearts from maternal diabetes. Notably, these abnormalities were all restored to normal levels after sapropterin treatment. In conclusion, sapropterin treatment increases eNOS activity, lowers oxidative stress and reduces coronary artery malformation in offspring of pregestational diabetes. Sapropterin may have therapeutic potential in preventing coronary artery malformation in maternal diabetes.
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Antioxidantes/farmacologia , Biopterinas/análogos & derivados , Doença da Artéria Coronariana/tratamento farmacológico , Vasos Coronários/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/farmacologia , Administração Oral , Animais , Antioxidantes/administração & dosagem , Biopterinas/administração & dosagem , Biopterinas/farmacologia , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Relação Dose-Resposta a Droga , Feminino , Hipoglicemiantes/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , EstreptozocinaRESUMO
Women with pre-gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia-induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre-gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes-induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial-mesenchymal transition at E12.5 was significantly lower with pre-gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non-exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre-gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.
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Diabetes Mellitus Experimental/complicações , Diabetes Gestacional/patologia , Cardiopatias Congênitas/etiologia , Estresse Oxidativo , Condicionamento Físico Animal , Animais , Glicemia/metabolismo , Capilares/anormalidades , Proliferação de Células , Anomalias dos Vasos Coronários/patologia , Embrião de Mamíferos/patologia , Transição Epitelial-Mesenquimal , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Pericárdio/embriologia , Pericárdio/patologia , Fosforilação , GravidezRESUMO
Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.
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Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sequência de Aminoácidos , Sinalização do Cálcio , Cisteína/química , Cisteína/metabolismo , Motivos EF Hand , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Retículo Sarcoplasmático/metabolismo , Alinhamento de Sequência , Molécula 1 de Interação Estromal/química , TermodinâmicaRESUMO
Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+ depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.
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Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/farmacologia , Molécula 1 de Interação Estromal/metabolismo , Animais , Células Cultivadas , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neurônios/citologia , Neurônios/enzimologia , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genéticaRESUMO
Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up-regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose-tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF-Akt (protein kinase B)-mammalian target of rapamycin (mTOR)-NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose-induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF-Akt-mTOR-NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.
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Envelhecimento/patologia , Diabetes Mellitus Experimental/patologia , Diabetes Gestacional/patologia , Traumatismo por Reperfusão Miocárdica/patologia , NADPH Oxidase 2/metabolismo , Regulação para Cima , Animais , Animais Recém-Nascidos , Apoptose , Células Cultivadas , Feminino , Glucose/toxicidade , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Background Tetrahydrobiopterin is a cofactor of endothelial NO synthase ( eNOS ), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan), an orally active synthetic form of tetrahydrobiopterin on eNOS uncoupling and congenital heart defects ( CHD s) induced by pregestational diabetes mellitus in mice. Methods and Results Adult female mice were induced to pregestational diabetes mellitus by streptozotocin and bred with normal male mice to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHD s were identified by histological analysis. Cell proliferation, eNOS dimerization, and reactive oxygen species production were assessed in the fetal heart. Pregestational diabetes mellitus results in a spectrum of CHD s in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHD s from 59% to 27%, and major abnormalities, such as atrioventricular septal defect and double-outlet right ventricle, were absent in the sapropterin-treated group. Lineage tracing reveals that pregestational diabetes mellitus results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes mellitus were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered reactive oxygen species levels in the fetal heart. Conclusions Sapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation, and prevents the development of CHD s in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHD s in pregestational diabetes mellitus.
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Biopterinas/análogos & derivados , Cardiopatias Congênitas/prevenção & controle , Animais , Biopterinas/uso terapêutico , Diabetes Gestacional , Feminino , Cardiopatias Congênitas/etiologia , Camundongos , GravidezRESUMO
Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit Gq- and Gs- mediated GPCR signalling. We previously demonstrated that RGS2 can also inhibit protein translation and can thereby attenuate cell growth. This G protein-independent inhibitory effect has been mapped to a 37 amino acid domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq- and Gs- activating agents. In the current study, we investigated the potential cardioprotective role of RGS2eb by determining whether RGS2eb transgenic (RGS2eb TG) mice with cardiomyocyte specific overexpression of RGS2eb show resistance to the development of hypertrophy in comparison to wild-type (WT) controls. Using transverse aortic constriction (TAC) in a pressure-overload hypertrophy model, we demonstrated that cardiac hypertrophy was inhibited in RGS2eb TG mice compared to WT controls following four weeks of TAC. Expression of the hypertrophic markers atrial natriuretic peptide (ANP) and ß-myosin heavy chain (MHC-ß) was also reduced in RGS2eb TG compared to WT TAC animals. Furthermore, cardiac function in RGS2eb TG TAC mice was significantly improved compared to WT TAC mice. Notably, cardiomyocyte cell size was significantly decreased in TG compared to WT TAC mice. These results suggest that RGS2 may limit pathological cardiac hypertrophy at least in part via the function of its eIF2B-binding domain.
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Cardiomegalia/genética , Cardiomegalia/metabolismo , Expressão Gênica , Miócitos Cardíacos/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas RGS/genética , Transdução de Sinais , Animais , Biomarcadores , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Testes de Função Cardíaca , Hemodinâmica , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/genética , Proteínas RGS/químicaRESUMO
Sepsis is a systemic inflammatory response to infection with a high mortality but has no specific treatment despite decades of research. North American (NA) ginseng (Panax quinquefolius) is a popular natural health product with anti-oxidant and anti-inflammatory properties. The aim of the present study was to investigate the effects of NA ginseng on pro-inflammatory cytokine expression and cardiac function in endotoxemia, a model of sepsis. Mice were challenged with lipopolysaccharide (LPS) to induce endotoxemia. Myocardial expression of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory cytokine that causes cardiac dysfunction, was upregulated in mice with endotoxemia, which was accompanied by increases in NOX2 expression, superoxide generation and ERK1/2 phosphorylation. Notably, pretreatment with NA ginseng aqueous extract (50mg/kg/day, oral gavage) for 5days significantly inhibited NOX2 expression, superoxide generation, ERK1/2 phosphorylation and TNF-α expression in the heart during endotoxemia. Importantly, cardiac function and survival in endotoxemic mice were significantly improved. Additionally, pretreatment with ginseng extract inhibited superoxide generation, ERK1/2 phosphorylation and TNF-α expression induced by LPS in cultured cardiomyocytes. We conclude that NA ginseng inhibits myocardial NOX2-ERK1/2-TNF-α signaling pathway and improves cardiac function in endotoxemia, suggesting that NA ginseng may have the potential in the prevention of clinical sepsis.
Assuntos
Anti-Inflamatórios/farmacologia , Endotoxemia/tratamento farmacológico , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , NADPH Oxidase 2/metabolismo , Panax/química , Extratos Vegetais/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anti-Inflamatórios/isolamento & purificação , Células Cultivadas , Modelos Animais de Doenças , Endotoxemia/induzido quimicamente , Endotoxemia/enzimologia , Endotoxemia/fisiopatologia , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosforilação , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Superóxidos/metabolismo , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.
Assuntos
Coração/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Regeneração/fisiologia , Animais , Vasos Coronários/cirurgia , Modelos Animais de Doenças , Ecocardiografia , Feminino , Ligadura/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/diagnóstico por imagem , Reprodutibilidade dos TestesRESUMO
Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload-induced Ca(2+) release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1(f/f) and cardiac-specific Rac1 knockdown (Rac1(ckd) ) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1(f/f) mice. Remarkably, I/R-induced ventricular arrhythmia was significantly decreased in Rac1(ckd) compared to Rac1(f/f) mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R-induced ventricular arrhythmia. Ca(2+) imaging analysis showed that in response to a 6 mM external Ca(2+) concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca(2+) waves in Rac1(f/f) cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R-induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1(ckd) mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.
Assuntos
Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/prevenção & controle , Cálcio/metabolismo , Ventrículos do Coração/patologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Arritmias Cardíacas/patologia , Eletrocardiografia , Técnicas de Silenciamento de Genes , Frequência Cardíaca , Camundongos Endogâmicos C57BL , Reperfusão Miocárdica , Miócitos Cardíacos/metabolismo , Oxirredução , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Superóxidos/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
BACKGROUND: The small GTPase Rac1 regulates diverse cellular functions, including both apicobasal and planar cell polarity pathways; however, its role in cardiac outflow tract (OFT) development remains unknown. In the present study, we aimed to examine the role of Rac1 in the anterior second heart field (SHF) splanchnic mesoderm and subsequent OFT development during heart morphogenesis. METHODS AND RESULTS: Using the Cre/loxP system, mice with an anterior SHF-specific deletion of Rac1 (Rac1(SHF)) were generated. Embryos were collected at various developmental time points for immunostaining and histological analysis. Intrauterine echocardiography was also performed to assess aortic valve blood flow in embryos at embryonic day 18.5. The Rac1(SHF) splanchnic mesoderm exhibited disruptions in SHF progenitor cellular organization and proliferation. Consequently, this led to a spectrum of OFT defects along with aortic valve defects in Rac1(SHF) embryos. Mechanistically, it was found that the ability of the Rac1(SHF) OFT myocardial cells to migrate into the proximal OFT cushion was severely reduced. In addition, expression of the neural crest chemoattractant semaphorin 3c was decreased. Lineage tracing showed that anterior SHF contribution to the OFT myocardium and aortic valves was deficient in Rac1(SHF) hearts. Furthermore, functional analysis with intrauterine echocardiography at embryonic day 18.5 showed aortic valve regurgitation in Rac1(SHF) hearts, which was not seen in control hearts. CONCLUSIONS: Disruptions of Rac1 signaling in the anterior SHF results in aberrant progenitor cellular organization and defects in OFT development. Our data show Rac1 signaling to be a critical regulator of cardiac OFT formation during embryonic heart development.