RESUMO
RATIONALE: ß1ARs (ß1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular ß1AR in cardiac contractility remains to be elucidated. OBJECTIVE: Test localization and function of intracellular ß1AR on cardiac contractility. METHODS AND RESULTS: Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of ß1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant ßAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant ßAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility. CONCLUSIONS: Functional ß1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular ß1ARs requires catecholamine transport via OCT3.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Frequência Cardíaca , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/genética , Fosforilação , Coelhos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 2/genética , Retículo Sarcoplasmático/metabolismo , Transdução de SinaisRESUMO
We have recently identified a pool of intracellular ß1 adrenergic receptors (ß1ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular ß1AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized ß1AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation-contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local ß1AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-ß1AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-ß1AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.
Assuntos
Corticosterona , Proteínas Quinases Dependentes de AMP Cíclico , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Animais , Cálcio/metabolismo , Catecolaminas/metabolismo , Catecolaminas/farmacologia , Cátions/metabolismo , Cátions/farmacologia , Corticosterona/metabolismo , Corticosterona/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Camundongos , Monoaminoxidase/metabolismo , Monoaminoxidase/farmacologia , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Fosforilação , Receptores Adrenérgicos beta 1/genética , Receptores Adrenérgicos beta 1/metabolismo , Retículo SarcoplasmáticoRESUMO
Spatiotemporal regulation of subcellular protein kinase A (PKA) activity for precise substrate phosphorylation is essential for cellular responses to hormonal stimulation. Ryanodine receptor 2 (RyR2) and (sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) represent two critical targets of ß adrenoceptor (ßAR) signaling on the sarcoplasmic reticulum membrane for cardiac excitation and contraction coupling. Using novel biosensors, we show that cardiac ß1AR signals to both RyR2 and SERCA2a nanodomains in cardiomyocytes from mice, rats, and rabbits, whereas the ß2AR signaling is restricted from these nanodomains. Phosphodiesterase 4 (PDE4) and PDE3 control the baseline PKA activity and prevent ß2AR signaling from reaching the RyR2 and SERCA2a nanodomains. Moreover, blocking inhibitory G protein allows ß2AR signaling to the RyR2 but not the SERCA2a nanodomains. This study provides evidence for the differential roles of inhibitory G protein and PDEs in controlling the adrenergic subtype signaling at the RyR2 and SERCA2a nanodomains in cardiomyocytes. Video abstract.
Assuntos
Sinalização do Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Proteínas Quinases Dependentes de AMP Cíclico , Proteínas de Ligação ao GTP , Camundongos , Fosforilação , Coelhos , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
Autophagy and apoptosis, as major modes of cell death, play critical roles in cellular homeostasis. Our previous study demonstrated that the cross-talk between autophagy and apoptosis regulated cadmium-induced testicular injury and self-recovery, influencing male fertility. However, the underlying mechanism remains blurry. Herein, our subfertility rat model indicated that cadmium-induced autophagy and apoptosis were ameliorated by the activation of SIRT3 and blunted by the inhibition of SIRT3 in rat testis. Further, generating SIRT3 overexpression and knockdown models in TM3 mouse Leydig cells, we found that melatonin (SIRT3 activator) and overexpression of SIRT3 rescued cadmium-induced autophagy and apoptosis in TM3 cells. Knockdown of SIRT3 induced autophagy and apoptosis, which failed to be reversed by melatonin in TM3 cells. Taken together, SIRT3 functions as a pivotal protective factor in testicular Leydig cells injury, and melatonin regulates the cross-talk between autophagy and apoptosis by SIRT3, ameliorating cadmium-induced testicular injury.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Células Intersticiais do Testículo/metabolismo , Melatonina/metabolismo , Sirtuína 3/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Células Cultivadas , Técnicas de Silenciamento de Genes , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Melatonina/farmacologia , Camundongos , Ratos Sprague-Dawley , Sirtuína 3/genética , Sirtuínas/metabolismo , Testículo/citologia , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Pre-mRNA splicing plays an important role in muscle function and diseases. The RNA binding motif 20 (RBM20) is a splicing factor that is predominantly expressed in muscle tissues and primarily regulates pre-mRNA splicing of Ttn, encoding a giant muscle protein titin that is responsible for muscle function and diseases. RBM20-mediated Ttn splicing has been mostly studied in heart muscle, but not in skeletal muscle. In this study, we investigated splicing specificity in different muscle types in Rbm20 knockout rats and hormonal effects on RBM20-mediated splicing both in cellulo and in vivo studies. The results revealed that RBM20 is differentially expressed across muscles and RBM20-mediated splicing is muscle-type specific. In the presence of RBM20, Ttn splicing responds to hormones in a muscle-type dependent manner, while in the absence of RBM20, Ttn splicing is not affected by hormones. In differentiated and undifferentiated C2C12 cells, RBM20-mediated splicing in response to hormonal effects is mainly through genomic signaling pathway. The knowledge gained from this study may help further understand muscle-specific gene splicing in response to hormone stimuli in different muscle types.
Assuntos
Conectina/genética , Músculo Esquelético/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Precursores de RNA/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Animais , Antitireóideos/farmacologia , Linhagem Celular , Conectina/metabolismo , Cruzamentos Genéticos , Feminino , Humanos , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Especificidade de Órgãos , Propiltiouracila/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Estreptozocina/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
Obesity is a major public health problem worldwide. In the United States, one-third of women of reproductive age are obese. Human studies show that maternal obesity (MO) predisposes offspring to cardiovascular disease. However, the underlying mechanisms remain unclear. Given the similarities between pregnancy in sheep and humans, we studied sheep to examine the impact of MO on fetal cardiomyocyte contractility at term. We observed that MO impaired cardiomyocyte contractility by reducing peak shortening and shortening/relengthening velocity, prolonging time to relengthening. MO disrupted Ca2+ homeostasis in fetal cardiomyocytes, increasing intracellular Ca2+ and inducing cellular Ca2+ insensitivity. The Ca2+-release channel was impaired, but Ca2+ uptake was unaffected by MO. The upstream kinases that phosphorylate the Ca2+-release channel-ryanodine receptor-2, PKA, and calmodulin-dependent protein kinase II-were activated in MO fetuses. Contractile dysfunction was associated with an increased ratio of myosin heavy chain (MHC)-ß to MHC-α and upregulated cardiac troponin (cTn)-T and tropomyosin, as well as cTn-I phosphorylation. In summary, this is the first characterization of the effects of MO on fetal cardiomyocyte contractility. Our findings indicate that MO impairs fetal cardiomyocyte contractility through altered intracellular Ca2+ handling, overloading fetal cardiomyocyte intracellular Ca2+ and aberrant myofilament protein composition. These mechanisms may contribute to developmental programming by MO of offspring cardiac function and predisposition to later life cardiovascular disease in the offspring.-Wang, Q., Zhu, C., Sun, M., Maimaiti, R., Ford, S. P., Nathanielsz, P. W., Ren, J., Guo, W. Maternal obesity impairs fetal cardiomyocyte contractile function in sheep.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Feto/patologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/patologia , Obesidade/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Feminino , Feto/metabolismo , Humanos , Miócitos Cardíacos/metabolismo , Fosforilação , Gravidez , OvinosRESUMO
Background: Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells lose cell-cell contacts and acquire expression of mesenchymal components and manifest a migratory phenotype. Recent studies indicated that EMT is involved in the development of keloids. Therefore, this study aims to investigate the mechanisms of the effects of metformin in hypoxia-induced EMT in keloid fibroblasts (KFs). Methods: KFs were cultured in a hypoxia incubator to induce EMT and were treated with or without metformin. Cell viability was evaluated by a cell counting kit 8 (CCK-8), and cell migration was measured by the transwell assay. The expression levels of HIF-1α, E-cadherin, vimentin, phosphorylated p70s6k (p-p70s6k) and pyruvate kinase M2 (PKM2) were evaluated by western blotting. Results: Hypoxia promoted EMT in KFs. Metformin significantly inhibited the expression of HIF-1α and partially abolished hypoxia-induced EMT. PKM2 is involved in hypoxia-induced EMT of KFs and metformin decreased the expression of p-p70s6k and PKM2. Conclusions: Metformin abolishes hypoxia-induced EMT in KFs by inhibiting the HIF-1α/PKM2 signaling pathway. Our study provides a novel mechanistic insight into potential use of metformin for treatment of keloids.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Queloide/tratamento farmacológico , Metformina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adulto , Proteínas de Transporte/metabolismo , Hipóxia Celular , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Orelha , Feminino , Fibroblastos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Queloide/patologia , Proteínas de Membrana/metabolismo , Metformina/uso terapêutico , Cultura Primária de Células , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Pele/citologia , Pele/patologia , Hormônios Tireóideos/metabolismo , Adulto Jovem , Proteínas de Ligação a Hormônio da TireoideRESUMO
RNA binding motif 20 (RBM20) is a key regulator of pre-mRNA splicing of titin and other genes that are associated with cardiac diseases. Hormones, like insulin, triiodothyronine (T3), and angiotensin II (Ang II), can regulate gene-splicing through RBM20, but the detailed mechanism remains unclear. This study was aimed at investigating the signaling mechanism by which hormones regulate pre-mRNA splicing through RBM20. We first examined the role of RBM20 in Z-, I-, and M-band titin splicing at different ages in wild type (WT) and RBM20 knockout (KO) rats using RT-PCR; we found that RBM20 is the predominant regulator of I-band titin splicing at all ages. Then we treated rats with propylthiouracil (PTU), T3, streptozotocin (STZ), and Ang II and evaluated the impact of these hormones on the splicing of titin, LIM domain binding 3 (Ldb3), calcium/calmodulin-dependent protein kinase II gamma (Camk2g), and triadin (Trdn). We determined the activation of mitogen-activated protein kinase (MAPK) signaling in primary cardiomyocytes treated with insulin, T3, and Ang II using western blotting; MAPK signaling was activated and RBM20 expression increased after treatment. Two downstream transcriptional factors c-jun and ETS Transcription Factor (ELK1) can bind the promoter of RBM20. A dual-luciferase activity assay revealed that Ang II, but not insulin and T3, can trigger ELK1 and thus promote transcription of RBM20. This study revealed that Ang II can trigger ELK1 through activation of MAPK signaling by enhancing RBM20 expression which regulates pre-mRNA splicing. Our study provides a potential therapeutic target for the treatment of cardiac diseases in RBM20-mediated pre-mRNA splicing.
Assuntos
Angiotensina II/farmacologia , Sistema de Sinalização das MAP Quinases , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Titin (TTN) has multifunctional roles in sarcomere assembly, mechanosignaling transduction, and muscle stiffness. TTN splicing generates variable protein sizes with different functions. Therefore, understanding TTN splicing is important to develop a novel treatment for TTN-based diseases. The I-band TTN splicing regulated by RNA binding motif 20 (RBM20) has been extensively studied. However, the Z- and M-band splicing and regulation remain poorly understood. Herein, we aimed to define the Z- and M-band splicing in striated muscles and determined whether RBM20 regulates the Z- and M-band splicing. We discovered four new Z-band TTN splicing variants, and one of them dominates in mouse, rat, sheep, and human hearts. But only one form can be detected in frog and chicken hearts. In skeletal muscles, three new Z repeats (Zr) were detected, and Zr4 to 6 exclusion dominates in the fast muscles, whereas Zr4 skipping dominates in the slow muscle. No developmental changes were detected in the Z-band. In the M-band, two new variants were discovered with alternative 3' splice site in exon363 (Mex5) and alternative 5' splice site in intron 362. However, only the sheep heart expresses two new variants rather than other species. Skeletal muscles express three M-band variants with altered ratios of Mex5 inclusion to Mex5 exclusion. Finally, we revealed that RBM20 does not regulate the Z- and M-band splicing in the heart, but does in skeletal muscles. Taken together, we characterized the Z- and M-band splicing and provided the first evidence of the role of RBM20 in the Z- and M-band TTN splicing.
Assuntos
Conectina/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Conectina/genética , Humanos , Camundongos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sarcômeros/metabolismo , Ovinos/genética , Ovinos/metabolismoAssuntos
Insuficiência Cardíaca/metabolismo , Monoaminoxidase/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de SinaisRESUMO
Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed.
Assuntos
Processamento Alternativo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sarcômeros/metabolismo , Animais , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Humanos , Miocárdio/patologia , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Sarcômeros/genética , Sarcômeros/patologiaRESUMO
Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.
Assuntos
Processamento Alternativo , Conectina/biossíntese , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Precursores de RNA/biossíntese , Proteínas de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Conectina/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Precursores de RNA/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Mutantes , Serina-Treonina Quinases TOR/genéticaRESUMO
BACKGROUND: Chronic sympathetic stimulation drives desensitization and downregulation of ß1 adrenergic receptor (ß1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ß1AR signaling in the heart. METHODS AND RESULTS: We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine ß1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular ß1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced ß1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the ß1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the ß1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced ß1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening. CONCLUSIONS: This study reveals distinct mechanisms for the downregulation of subcellular ß1AR signaling in the heart under chronic adrenergic stimulation.
Assuntos
Canais de Cálcio Tipo L , Proteínas Quinases Dependentes de AMP Cíclico , Regulação para Baixo , Isoproterenol , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Receptores Adrenérgicos beta 1 , Canal de Liberação de Cálcio do Receptor de Rianodina , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transdução de Sinais , Animais , Receptores Adrenérgicos beta 1/metabolismo , Masculino , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Isoproterenol/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Modelos Animais de Doenças , Camundongos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/fisiopatologia , Cardiomiopatias/metabolismo , Cardiomiopatias/induzido quimicamente , Transferência Ressonante de Energia de FluorescênciaRESUMO
The coronavirus disease 2019 (COVID-19) has merged as a global health threat since its outbreak in December 2019. Despite widespread recognition, there has been a paucity of studies focusing on the T cell receptor (TCR) bias in adaptive immunity induced by SARS-CoV-2. This research conducted a comparative analysis of the TCR immune repertoire to identify notable αß TCR bias sequences associated with the SARS-CoV-2 virus antigen. The present study encompassed 73 symptomatic COVID-19 patients, categorized as moderate/mild or severe/critical, along with 9 healthy controls. Our findings revealed specific TCR chains prominently utilized by moderate and severe patients, identified as TRAV30-J34-TRBV3-1-J2-7 and TRAV12-3-J6-TRBV28-J1-1, respectively. Additionally, our research explored critical TCR preferences in the bronchoalveolar lavage fluid (BALF) of COVID-19 patients at various disease stages. Indeed, monitoring the dynamics of immune repertoire changes in COVID-19 patients could serve as a crucial biomarker for predicting disease progression and recovery. Furthermore, the study explored TCR bias in both peripheral blood mononuclear cells (PBMCs) and BALF. The most common αß VJ pair observed in BALF was TRAV12-3-J18-TRBV7-6-J2-7. In addition, a comparative analysis with the VDJdb database indicated that the HLA-A*02:01 allele exhibited the widest distribution and highest frequency in COVID-19 patients across different periods. This comprehensive examination provided a global characterization of the TCR immune repertoire in COVID-19 patients, contributing significantly to our understanding of TCR bias induced by SARS-CoV-2.
Assuntos
COVID-19 , Receptores de Antígenos de Linfócitos T alfa-beta , SARS-CoV-2 , Humanos , COVID-19/imunologia , SARS-CoV-2/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Adulto , Líquido da Lavagem Broncoalveolar/imunologia , Idoso , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Imunidade Adaptativa/imunologia , Índice de Gravidade de DoençaRESUMO
Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac ß2 adrenoreceptor (ß2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac ß2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of ß2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.
RESUMO
5-Demethylnobiletin is the active ingredient in citrus polymethoxyflavones that could inhibit the proliferation of several tumor cells. However, the anti-tumor effect of 5-Demethylnobiletin on glioblastoma and the underlying molecular mechanisms are remains unknown. In our study, 5-Demethylnobiletin markedly inhibited the viability, migration and invasion of glioblastoma U87-MG, A172 and U251 cells. Further research revealed that 5-Demethylnobiletin induces cell cycle arrest at the G0/G1 phase in glioblastoma cells by downregulating Cyclin D1 and CDK6 expression levels. Furthermore, 5-Demethylnobiletin significantly induced glioblastoma cells apoptosis by upregulating the protein levels of Bax and downregulating the protein level of Bcl-2, subsequently increasing the expression of cleaved caspase-3 and cleaved caspase-9. Mechanically, 5-Demethylnobiletin trigged G0/G1 phase arrest and apoptosis by inhibiting the ERK1/2, AKT and STAT3 signaling pathway. Furthermore, 5-Demethylnobiletin inhibition of U87-MG cell growth was reproducible in vivo model. Therefore, 5-Demethylnobiletin is a promising bioactive agent that might be used as glioblastoma treatment drug.
RESUMO
The power system is evolving from a single energy system to an integrated energy system. In order to further improve the power generation and consumption balance capacity of the park integrated energy system (PIES), the park integrated energy system is gradually transitioning from the single park energy system operation mode to the multipark energy system operation mode. The design of multipark integrated energy system (MPIES) collaborative control strategy will become an important part to improve the power generation and consumption balance ability of the integrated energy system. In order to fully tap the regulation capacity of each PIES, we propose a coordinated control strategy for the integrated energy system in multiple parks considering the flexible substitution interval of multiple types of energy. Firstly, we analyze the influence of the types of regulation resources and the regulation incentive mechanism of the PIES on the regulation flexible range of the PIES. Then, based on the Markov decision process, a distributed cluster regulation model of MPIES considering regulation demand and regulation flexible interval is established. Finally, using multilayer deep Q networks (MLDQN), the distributed cluster regulation optimization algorithm of MPIES is given. The simulation results show that the proposed method can coordinate the regulation ability of each park integrated energy system in the MPIES, give full play to the large-scale advantage of the interconnection of the park integrated energy system, and improve the overall stability of the multipark integrated energy system.
Assuntos
Algoritmos , Simulação por ComputadorRESUMO
In recent years, with the development of smart grid, the power systems and other energy systems are gradually forming integrated energy systems. The electric-thermal integrated energy system is a mature integrated energy system at present. The electric-thermal integrated energy system uses modern communication technology to realize the comprehensive regulation of electric energy and thermal energy, which greatly improves the efficiency of energy use. However, this also greatly increases the risk of malicious tampering with the energy dispatch system. In this paper, we study the regulation of electric-thermal integrated energy systems considering false data injection attacks. First, we establish a compromised model of an electric-thermal integrated energy system considering false data injection attacks. Then, we designed vulnerable variable observers for different tampering scenarios to observe the tampered variables. Finally, considering the relationship between the observed data and the measured data, we design a tampering behavior detection method based on relation network. The simulation results verify the effectiveness of the detection method proposed in this paper.
RESUMO
RNA binding motif 20 (RBM20) cardiomyopathy has been detected in approximately 3% of populations afflicted with dilated cardiomyopathy (DCM). It is well conceived that RBM20 cardiomyopathy is provoked by titin isoform switching in combination with resting Ca2+ leaking. In this study, we characterized the cardiac function in Rbm20 knockout (KO) rats at 3-, 6-, 9-, and 12-months of age and examined the effect of the ryanodine receptor stabilizer S107 on resting intracellular levels and cardiomyocyte contractile properties. Our results revealed that even though Rbm20 depletion promoted expression of larger titin isoform and reduced myocardial stiffness in young rats (3 months of age), the established DCM phenotype required more time to embellish. S107 restored elevated intracellular Ca2+ to normal levels and ameliorated cardiomyocyte contractile properties in isolated cardiomyocytes from 6-month-old Rbm20 KO rats. However, S107 failed to preserve cardiac homeostasis in Rbm20 KO rats at 12 months of age, unexpectedly, likely due to the existence of multiple pathogenic mechanisms. Taken together, our data suggest the therapeutic promises of S107 in the management of RBM20 cardiomyopathy.