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1.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070501

RESUMO

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Diafragma/metabolismo , Mitocôndrias/metabolismo , Insuficiência Respiratória/metabolismo , Timidina Quinase/deficiência , Timidina Quinase/genética , Actinas/metabolismo , Adulto , Autopsia , Encéfalo/metabolismo , Catalase/metabolismo , Diafragma/enzimologia , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Fosforilação Oxidativa , Peroxirredoxina VI/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteoma/genética , Proteoma/metabolismo , Insuficiência Respiratória/genética , Insuficiência Respiratória/mortalidade , Superóxido Dismutase/metabolismo , Timidina Quinase/metabolismo , Regulação para Cima
2.
Skelet Muscle ; 11(1): 11, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941271

RESUMO

BACKGROUND: Old age is associated with a significantly increased mortality in COVID-19 patients exposed to long-term controlled mechanical ventilation (CMV) and suggested to be due to the hyperinflammatory response associated with the viral infection. However, our understanding of age-related differences in the response to CMV in the absence of a viral infection remains insufficient. METHODS: Young (7-8 months) and old (28-32 months) F344 BN hybrid rats were exposed to the ICU condition for 5 days, i.e., complete immobilization, mechanical ventilation, and extensive monitoring. Transcriptomic (RNA-Seq) and proteomics (Proximity Extension Assay) analyses of the diaphragm and proteomics analysis of plasma were conducted to investigate the molecular differences between young and old rats exposed to the ICU condition. RESULTS: According to multi-omics analyses, significant differences were observed in the diaphragm between young and old rats in response to 5 days CMV and immobilization. In young rats, metabolic pathways were primarily downregulated in response to immobilization (post-synaptic blockade of neuromuscular transmission). In old rats, on the other hand, dramatic immune and inflammatory responses were observed, i.e., an upregulation of specific related pathways such as "IL-17 signaling pathway", along with a higher level of inflammatory factors and cytokine/chemokine in plasma. CONCLUSIONS: The dramatically increased mortality in old ICU patients with COVID-19-associated hyperinflammation and cytokine storm need not only reflect the viral infection but may also be associated with the ventilator induced diaphragm dysfunction (VIDD) and hyperinflammatory responses induced by long-term CMV per se. Although mechanical ventilation is a life-saving intervention in COVID-19 ICU patients, CMV should be cautiously used especially in old age and other means of respiratory support may be considered, such as negative pressure ventilation.


Assuntos
Diafragma/metabolismo , Mediadores da Inflamação/sangue , Proteoma , Respiração Artificial , Transcriptoma , Fatores Etários , Animais , Biomarcadores/sangue , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Projetos Piloto , Mapas de Interação de Proteínas , Proteômica , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Transdução de Sinais
3.
Molecules ; 26(8)2021 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918827

RESUMO

This study aims to assess the safety of the Opuntia dillenii (Ker-Gawl) haw. seed oil (ODSO) and its effect on the glucose absorption activity of the isolated rat hemidiaphragm. This oil's safety was studied by exploring its acute (doses 1, 3, 5, and 7 mL/kg) and subacute (doses 1 and 2 mL/kg) toxicities in albino mice and Wistar rats, respectively. The safety of the ODSO was also assessed by studying its effect on the HepG2 cell viability in vitro. The effect of ODSO, or combined with the insulin, on the glucose absorption activity of isolated rat hemidiaphragm was evaluated at the dose 1 g/L in vitro. The results demonstrated the safety of ODSO. Indeed, this study showed that this oil does not produce any mortality or signs of toxicity after the single-dose administration in mice. Additionally, the daily intake of the ODSO during four weeks does not induce a significant variation in the biochemical parameters and body weight of rats compared with the control group. Besides, the cell viability of HepG2 did not change in the presence of ODSO. On the other hand, the ODSO increased the glucose absorption activity of the isolated rat hemidiaphragm, and this activity was significantly enhanced when combined with insulin. This study confirms, on one side, the safety of this oil and its efficacy and, on the other side, encourages its potential use as a complement to treat diabetes.


Assuntos
Absorção Fisiológica , Diafragma/metabolismo , Glucose/metabolismo , Opuntia/química , Óleos Vegetais/farmacologia , Sementes/química , Testes de Toxicidade Aguda , Absorção Fisiológica/efeitos dos fármacos , Administração Oral , Animais , Bilirrubina/sangue , Biomarcadores/sangue , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diafragma/efeitos dos fármacos , Feminino , Células Hep G2 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Óleos Vegetais/administração & dosagem , Ratos Wistar
5.
Neuroscience ; 460: 31-42, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33548369

RESUMO

In vertebrates, muscle activity is dependent on acetylcholine (ACh) released from neuromuscular junctions (NMJs), and changes in cholinergic neurotransmission are linked to a variety of neuromuscular diseases, including congenital myasthenic syndromes (CMS). The storage and release of ACh depends on the activity of the Vesicular Acetylcholine Transporter (VAChT), a rate-limiting step for cholinergic neurotransmission whose loss of function mutations was shown to cause human congenital myasthenia. However, we know much less about increased VAChT activity, due to copy number variations, for example. Therefore, here we investigated the impact of increased VAChT expression and consequently ACh levels at the synaptic cleft of the diaphragm NMJs. We analyzed structure and function of nerve and muscles from a mouse model of cholinergic hyperfunction (ChAT-ChR2-EYFP) with increased expression of VAChT. Our results showed a significant increase of ACh released under evoked stimuli. However, we observed deleterious changes in synaptic vesicles cycle (impaired endocytosis and decrease in vesicles number), together with structural alterations of NMJs. Interestingly, ultrastructure analyses showed that synaptic vesicles from ChAT-ChR2-EYFP mice NMJs were larger, which might be related to increased ACh load. We also observed that these larger synaptic vesicles were less rounded in comparison with control. Finally, we showed that ChAT-ChR2-EYFP mice NMJs have compromised safety factor, possible due to the structural alterations we described. These findings reveal that physiological cholinergic activity is important to maintain the structure and function of the neuromuscular system and help to understand some of the neuromuscular adverse effects experienced by chronically increased NMJ neurotransmission, such as individuals treated with cholinesterase inhibitors.


Assuntos
Variações do Número de Cópias de DNA , Diafragma , Animais , Colinérgicos , Diafragma/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Transmissão Sináptica , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
8.
Redox Biol ; 38: 101802, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33279868

RESUMO

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfortunately, an unintended consequence of prolonged MV is the rapid development of diaphragmatic atrophy and contractile dysfunction, known as ventilator-induced diaphragm dysfunction (VIDD). Although the mechanism(s) responsible for VIDD are not fully understood, abundant evidence reveals that oxidative stress leading to the activation of the major proteolytic systems (i.e., autophagy, ubiquitin-proteasome, caspase, and calpain) plays a dominant role. Of the proteolytic systems involved in VIDD, calpain has received limited experimental attention due to the longstanding dogma that calpain plays a minor role in inactivity-induced muscle atrophy. Guided by preliminary experiments, we tested the hypothesis that activation of calpains play an essential role in MV-induced oxidative stress and the development of VIDD. This premise was rigorously tested by transgene overexpression of calpastatin, an endogenous inhibitor of calpains. Animals with/without transfection of the calpastatin gene in diaphragm muscle fibers were exposed to 12 h of MV. Results confirmed that overexpression of calpastatin barred MV-induced activation of calpain in diaphragm fibers. Importantly, deterrence of calpain activation protected the diaphragm against MV-induced oxidative stress, fiber atrophy, and contractile dysfunction. Moreover, prevention of calpain activation in the diaphragm forstalled MV-induced mitochondrial dysfunction and prevented MV-induced activation of caspase-3 along with the transcription of muscle specific E3 ligases. Collectively, these results support the hypothesis that calpain activation plays an essential role in the early development of VIDD. Further, these findings provide the first direct evidence that calpain plays an important function in inactivity-induced mitochondrial dysfunction and oxidative stress in skeletal muscle fibers.


Assuntos
Calpaína , Respiração Artificial , Animais , Calpaína/genética , Calpaína/metabolismo , Diafragma/metabolismo , Humanos , Mitocôndrias , Debilidade Muscular/etiologia , Debilidade Muscular/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/metabolismo
9.
Gene ; 770: 145356, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33333219

RESUMO

The diaphragm is the main inspiratory muscle, and the chronic phase post-myocardial infarction (MI) is characterized by diaphragm morphological, contractile, and metabolic abnormalities. However, the mechanisms of diaphragm weakness are not fully understood. In the current study, we aimed to identify the transcriptome changes associated with diaphragm abnormalities in the chronic stage MI. We ligated the left coronary artery to cause MI in rats and performed RNA-sequencing (RNA-Seq) in diaphragm samples 16 weeks post-surgery. The sham group underwent thoracotomy and pericardiotomy but no artery ligation. We identified 112 differentially expressed genes (DEGs) out of a total of 9664 genes. Myocardial infarction upregulated and downregulated 42 and 70 genes, respectively. Analysis of DEGs in the framework of skeletal muscle-specific biological networks suggest remodeling in the neuromuscular junction, extracellular matrix, sarcomere, cytoskeleton, and changes in metabolism and iron homeostasis. Overall, the data are consistent with pathological remodeling of the diaphragm and reveal potential biological targets to prevent diaphragm weakness in the chronic stage MI.


Assuntos
Diafragma/metabolismo , Proteínas Musculares/biossíntese , Infarto do Miocárdio/metabolismo , RNA-Seq , Transcriptoma , Animais , Diafragma/patologia , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Ratos , Ratos Sprague-Dawley
10.
Int J Mol Sci ; 21(19)2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-33007822

RESUMO

Oxidative damage to the diaphragm as a result of cervical spinal cord injury (SCI) promotes muscle atrophy and weakness. Respiratory insufficiency is the leading cause of morbidity and mortality in cervical spinal cord injury (SCI) patients, emphasizing the need for strategies to maintain diaphragm function. Hyperbaric oxygen (HBO) increases the amount of oxygen dissolved into the blood, elevating the delivery of oxygen to skeletal muscle and reactive oxygen species (ROS) generation. It is proposed that enhanced ROS production due to HBO treatment stimulates adaptations to diaphragm oxidative capacity, resulting in overall reductions in oxidative stress and inflammation. Therefore, we tested the hypothesis that exposure to HBO therapy acutely following SCI would reduce oxidative damage to the diaphragm muscle, preserving muscle fiber size and contractility. Our results demonstrated that lateral contusion injury at C3/4 results in a significant reduction in diaphragm muscle-specific force production and fiber cross-sectional area, which was associated with augmented mitochondrial hydrogen peroxide emission and a reduced mitochondrial respiratory control ratio. In contrast, rats that underwent SCI followed by HBO exposure consisting of 1 h of 100% oxygen at 3 atmospheres absolute (ATA) delivered for 10 consecutive days demonstrated an improvement in diaphragm-specific force production, and an attenuation of fiber atrophy, mitochondrial dysfunction and ROS production. These beneficial adaptations in the diaphragm were related to HBO-induced increases in antioxidant capacity and a reduction in atrogene expression. These findings suggest that HBO therapy may be an effective adjunctive therapy to promote respiratory health following cervical SCI.


Assuntos
Diafragma/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxigênio/metabolismo , Traumatismos da Medula Espinal/terapia , Animais , Diafragma/patologia , Modelos Animais de Doenças , Transporte de Elétrons/genética , Humanos , Oxigenação Hiperbárica , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia
11.
Int J Mol Sci ; 21(21)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105841

RESUMO

It is established that cancer cachexia causes limb muscle atrophy and is strongly associated with morbidity and mortality; less is known about how the development of cachexia impacts the diaphragm. The purpose of this study was to investigate cellular signaling mechanisms related to mitochondrial function, reactive oxygen species (ROS) production, and protein synthesis during the development of cancer cachexia. C57BL/J6 mice developed Lewis Lung Carcinoma for either 0 weeks (Control), 1 week, 2 weeks, 3 weeks, or 4 weeks. At designated time points, diaphragms were harvested and analyzed. Mitochondrial respiratory control ratio was ~50% lower in experimental groups, which was significant by 2 weeks of cancer development, with no difference in mitochondrial content markers COXIV or VDAC. Compared to the controls, ROS was 4-fold elevated in 2-week animals but then was not different at later time points. Only one antioxidant protein, GPX3, was altered by cancer development (~70% lower in experimental groups). Protein synthesis, measured by a fractional synthesis rate, appeared to become progressively lower with the cancer duration, but the mean difference was not significant. The development and progression of cancer cachexia induces marked alterations to mitochondrial function and ROS production in the diaphragm and may contribute to increased cachexia-associated morbidity and mortality.


Assuntos
Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Diafragma/fisiopatologia , Mitocôndrias Musculares/metabolismo , Animais , Antioxidantes/metabolismo , Caquexia/etiologia , Carcinoma Pulmonar de Lewis/fisiopatologia , Diafragma/metabolismo , Proteína Forkhead Box O3/metabolismo , Glutationa Peroxidase/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Musculares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
12.
J Appl Physiol (1985) ; 129(5): 1062-1074, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32909923

RESUMO

Obesity is associated with bioenergetic dysfunction of peripheral muscles; however, little is known regarding the impact of obesity on the diaphragm. We hypothesized that obesity would be associated with diaphragm dysfunction attributable to mitochondrial oxygen consumption and structural and ultrastructural changes. Wistar rat litters were culled to 3 pups to induce early postnatal overfeeding and consequent obesity. Control animals were obtained from unculled litters. From postnatal day 150, diaphragm ultrasound, computed tomography, high-resolution respirometry, immunohistochemical, biomolecular, and ultrastructural histological analyses were performed. The diaphragms of obese animals, compared with those of controls, presented changes in morphology as increased thickening fraction, diaphragm excursion, and diaphragm dome height, as well as increased mitochondrial respiratory capacity coupled to ATP synthesis and maximal respiratory capacity. Fatty acid synthase gene expression was also higher in obese animals, suggesting a source of energy for the respiratory chain. Myosin heavy chain-IIA was increased, indicating shift from glycolytic toward oxidative muscle fiber profile. Diaphragm tissue also exhibited ultrastructural changes, such as compact, round, and swollen mitochondria with fainter cristae and more lysosomal bodies. Dynamin-1 expression in the diaphragm was reduced in obese rats, suggesting decreased mitochondrial fission. Furthermore, gene expressions of peroxisome γ proliferator-activated receptor coactivator-1α and superoxide dismutase-2 were lower in obese animals than in controls, which may indicate a predisposition to oxidative injury. In conclusion, in the obesity model used herein, muscle fiber phenotype was altered in a manner likely associated with increased mitochondrial respiratory capability, suggesting respiratory adaptation to increased metabolic demand.NEW & NOTEWORTHY Obesity has been associated with peripheral muscle dysfunction; however, little is known about its impact on the diaphragm. In the current study, we found high oxygen consumption in diaphragm tissue and changes in muscle fiber phenotypes toward a more oxidative profile in experimental obesity.


Assuntos
Diafragma , Obesidade , Animais , Diafragma/metabolismo , Metabolismo Energético , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Ratos , Ratos Wistar
13.
Cells ; 9(7)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674346

RESUMO

Abundant evidence reveals that activation of the renin-angiotensin system promotes skeletal muscle atrophy in several conditions including congestive heart failure, chronic kidney disease, and prolonged mechanical ventilation. However, controversy exists about whether circulating angiotensin II (AngII) promotes skeletal muscle atrophy by direct or indirect effects; the centerpiece of this debate is the issue of whether skeletal muscle fibers express AngII type 1 receptors (AT1Rs). While some investigators assert that skeletal muscle expresses AT1Rs, others argue that skeletal muscle fibers do not contain AT1Rs. These discordant findings in the literature are likely the result of study design flaws and additional research using a rigorous experimental approach is required to resolve this issue. We tested the hypothesis that AT1Rs are expressed in both human and rat skeletal muscle fibers. Our premise was tested using a rigorous, multi-technique experimental design. First, we established both the location and abundance of AT1Rs on human and rat skeletal muscle fibers by means of an AngII ligand-binding assay. Second, using a new and highly selective AT1R antibody, we carried out Western blotting and determined the abundance of AT1R protein within isolated single muscle fibers from humans and rats. Finally, we confirmed the presence of AT1R mRNA in isolated single muscle fibers from rats. Our results support the hypothesis that AT1Rs are present in both human and rat skeletal muscle fibers. Moreover, our experiments provide the first evidence that AT1Rs are more abundant in fast, type II muscle fibers as compared with slow, type I fibers. Together, these discoveries provide the foundation for an improved understanding of the mechanism(s) responsible for AngII-induced skeletal muscle atrophy.


Assuntos
Músculo Esquelético/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Adolescente , Adulto , Angiotensina II/metabolismo , Animais , Diafragma/metabolismo , Feminino , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor Tipo 1 de Angiotensina/genética , Sarcolema/metabolismo , Adulto Jovem
14.
Exp Biol Med (Maywood) ; 245(14): 1280-1289, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32493122

RESUMO

IMPACT STATEMENT: Diabetes mellitus is a group of chronic metabolic disorders, which causes serious damage to a variety of organs, such as the retina, heart, and skeletal muscle. The diaphragm is an important skeletal muscle involved in respiration in mammals. Fibrosis of the diaphragm muscle affects its contractility, which in turn impairs respiratory function. Accumulating evidence suggests that exogenous hydrogen sulfide (H2S) exhibits anti-fibrotic activity in diabetes mellitus, but whether and how H2S exerts this anti-fibrotic effect in the diabetic diaphragm remains unclear. The current work for the first time reveals that exogenous H2S attenuates hyperglycemia-induced fibrosis of the diaphragm muscle and strengthens diaphragmatic biomechanical properties in diabetes mellitus, and the mechanism may involve the alleviation of collagen deposition by suppression of the nucleotide-binding oligomerization domain-like receptor protein (NLRP) 3 inflammasome-mediated inflammatory reaction. Therefore, H2S supplementation could be used as an efficient targeted therapy against the NLRP3 inflammasome in the diabetic diaphragm.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diafragma/efeitos dos fármacos , Fibrose/tratamento farmacológico , Sulfeto de Hidrogênio/farmacologia , Músculo Esquelético/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Colágeno/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/metabolismo , Diafragma/metabolismo , Fibrose/metabolismo , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Músculo Esquelético/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Estreptozocina/farmacologia
15.
Cell Physiol Biochem ; 54(4): 567-576, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32496722

RESUMO

BACKGROUND/AIMS: Cigarette smoking is a key factor in systemic inflammation and oxidative stress, and it has also been associated with the loss of muscle strength and an elevated risk of pulmonary diseases. Thus, this study aimed to analyze the effects of cigarette smoking on the diaphragm muscle structure of postmortem samples. METHODS: Immunohistochemical techniques were used for muscle remodeling (metalloproteinases 2 and 9), inflammation (cyclooxygenase-2), oxidative stress (8-hydroxy-2'-deoxyguanosine), and vascularization (vascular endothelial growth factor). Hematoxylin and eosin stain was used for histopathological analysis and Picrosirius stain was used to highlight the collagen fibers. RESULTS: Cigarette smokers had an increase of diaphragm muscle remodeling, oxidative stress, inflammation, and vascularization compared to non-smokers. CONCLUSION: Diaphragm muscle structure may be negatively affected by cigarette smoking.


Assuntos
Fumar Cigarros/efeitos adversos , Diafragma/metabolismo , Diafragma/patologia , Inflamação/patologia , Estresse Oxidativo/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autopsia , Estudos Transversais , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Fumantes , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Reprod Sci ; 27(8): 1627-1636, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32430714

RESUMO

Thoracic endometriosis (TE) syndrome is a clinical condition known as an extrapelvic form of endometriosis with the presence of functioning endometrial tissue involving lung parenchyma, pleura, chest wall, or diaphragm. In an effort to obtain an endometriosis ex vivo model, we established the spontaneously growing TH-EM1 cell line from endometriotic implants in lung parenchyma from a woman with TE. Maintained in long-term culture, the cells grew as large mesenchymal-like cells with a doubling time between 5 and 6 days. Treatment with medroxyprogesterone acetate (10-7 mol/L) inhibited the TH-EM1 cells growth and induced morphological changes to an epithelial-like cells. Strong expression of the nuclear estrogen receptors, progesterone receptors, and erytropoietin receptors were found in both the pulmonary implant and the TH-EM1 cells by immunohistochemical analysis. Consistent immunoreactivity of TH-EM1 cells for CD9, CD13, CD73, CD90, CD105, and CD157 was revealed by flow cytometry. Likewise, the embryonic markers, SRY-box 2 (SOX-2) and the Nanog molecules, were detected in 76% and 52% of the cells, while fetal hemoglobin and a-globin were detected in 76% and 65% of TH-EM1 cells, respectively. By RHG banding, normal metaphases were observed, while the microarray chromosomal analysis showed gains of DNA sequences located on the segments 8p23.1, 11p15.5, and 12p11.23. The described in vitro cellular model can serve as a useful tool to study the pathogenesis of endometriosis and to improve the knowledge of molecular mechanisms controlling the endometriotic cell dissemination potential.


Assuntos
Endometriose/genética , Endometriose/patologia , Endométrio/patologia , Células Estromais/patologia , Doenças Torácicas/metabolismo , Doenças Torácicas/patologia , Adulto , Técnicas de Cultura de Células/métodos , Proliferação de Células/fisiologia , Diafragma/metabolismo , Diafragma/patologia , Endometriose/metabolismo , Endométrio/metabolismo , Feminino , Humanos , Células Estromais/metabolismo , Doenças Torácicas/genética
17.
Am J Physiol Lung Cell Mol Physiol ; 319(2): L228-L238, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32460519

RESUMO

Clinical studies indicate that sepsis-induced diaphragm dysfunction is a major contributor to respiratory failure in mechanically ventilated patients. Currently there is no drug to treat this form of diaphragm weakness. Sepsis-induced muscle dysfunction is thought to be triggered by excessive mitochondrial free radical generation; we therefore hypothesized that therapies that target mitochondrial free radical production may prevent sepsis-induced diaphragm weakness. The present study determined whether MitoTEMPOL, a mitochondrially targeted free radical scavenger, could reduce sepsis-induced diaphragm dysfunction. Using an animal model of sepsis, we compared four groups of mice: 1) sham-operated controls, 2) animals with sepsis induced by cecal ligation puncture (CLP), 3) sham controls given MitoTEMPOL (10 mg·kg-1·day-1 ip), and 4) CLP animals given MitoTEMPOL. At 48 h after surgery, we measured diaphragm force generation, mitochondrial function, proteolytic enzyme activities, and myosin heavy chain (MHC) content. We also examined the effects of delayed administration of MitoTEMPOL (by 6 h) on CLP-induced diaphragm weakness. The effects of MitoTEMPOL on cytokine-mediated alterations on muscle cell superoxide generation and cell size in vitro were also assessed. Sepsis markedly reduced diaphragm force generation. Both immediate and delayed MitoTEMPOL administration prevented sepsis-induced diaphragm weakness. MitoTEMPOL reversed sepsis-mediated reductions in mitochondrial function, activation of proteolytic pathways, and decreases in MHC content. Cytokines increased muscle cell superoxide generation and decreased cell size, effects that were ablated by MitoTEMPOL. MitoTEMPOL and other compounds that target mitochondrial free radical generation may be useful therapies for sepsis-induced diaphragm weakness.


Assuntos
Antioxidantes/farmacologia , Diafragma/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Mitocôndrias/efeitos dos fármacos , Debilidade Muscular/etiologia , Debilidade Muscular/prevenção & controle , Sepse/complicações , Animais , Ceco/efeitos dos fármacos , Ceco/metabolismo , Citocinas/metabolismo , Diafragma/metabolismo , Modelos Animais de Doenças , Ligadura/métodos , Masculino , Camundongos , Mitocôndrias/metabolismo , Debilidade Muscular/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Proteólise/efeitos dos fármacos , Sepse/metabolismo
18.
Int J Mol Med ; 45(6): 1644-1660, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32186768

RESUMO

Decreased diaphragm function is a crucial factor leading to reduced ventilatory efficiency and worsening of quality of life in chronic obstructive pulmonary disease (COPD). Exercise training has been demonstrated to effectively improve the function of the diaphragm. However, the mechanism of this process has not been identified. The emergence of metabolomics has allowed the exploration of new ideas. The present study aimed to analyze the potential biomarkers of exercise­dependent enhancement of diaphragm function in COPD using metabolomics. Sprague Dawley rats were divided into three groups: COPD + exercise group (CEG); COPD model group (CMG); and control group (CG). The first two groups were exposed to cigarette smoke for 16 weeks to establish a COPD model. Then, the rats in the CEG underwent aerobic exercise training for 9 weeks. Following confirmation that exercise effectively improved the diaphragm function, a gas chromatography tandem time­of­flight mass spectrometry analysis system was used to detect the differential metabolites and associated pathways in the diaphragm muscles of the different groups. Following exercise intervention, the pulmonary function and diaphragm contractility of the CEG rats were significantly improved compared with those of the CMG rats. A total of 36 different metabolites were identified in the comparison between the CMG and the CG. Pathway enrichment analysis indicated that these different metabolites were involved in 17 pathways. A total of 29 different metabolites were identified in the comparison between the CMG and the CEG, which are involved in 14 pathways. Candidate biomarkers were selected, and the pathways analysis of these metabolites demonstrated that 2 types of metabolic pathways, the nicotinic acid and nicotinamide metabolism and arginine and proline metabolism pathways, were associated with exercise­induced pulmonary rehabilitation.


Assuntos
Biomarcadores/metabolismo , Diafragma/metabolismo , Metaboloma/fisiologia , Músculo Esquelético/metabolismo , Condicionamento Físico Animal/fisiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Modelos Animais de Doenças , Masculino , Metabolômica/métodos , Contração Muscular/fisiologia , Ratos , Ratos Sprague-Dawley
19.
Thyroid ; 30(7): 1079-1090, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32200709

RESUMO

Background: Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. This study aimed at evaluating the relationship between TH signaling, mitochondrial function, and the antioxidant defense system in the diaphragms of septic mice. Methods: Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms, and livers were collected. TH metabolism and responses were analyzed by measuring messenger RNA (mRNA) expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10, and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and real-time polymerase chain reaction (qPCR), and function with oxygraphy. The diaphragm antioxidative defense system was examined by qPCR, analyzing superoxide dismutase (SOD) 1 (Sod1), mitochondrial superoxide dismutase (SOD 2; Sod2), extracellular superoxide dismutase (SOD 3; Sod3), glutathione peroxidase 1 (Gpx1), and catalase (Cat) expression. The effect of TH replacement was tested by treating the mice with T4 and triiodothyronine (T3) (CLP+TH) after surgery. Results: CLP mice presented reduced total plasma T4 concentrations, downregulated Dio1, and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10, and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encoding PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression, and reduced maximal respiration. In addition, septic animals presented a three-fold increase in Ucp3 and G6pdh expression; downregulated Sod3, Gpx1, and Cat expression; and upregulated Sod2 expression, potentially due to elevated reactive oxygen species levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice. Conclusions: Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction, and oxidative stress defense.


Assuntos
Diafragma/metabolismo , Mitocôndrias/metabolismo , Sepse/metabolismo , Transdução de Sinais/fisiologia , Hormônios Tireóideos/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Fígado/metabolismo , Camundongos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo
20.
Clin Chim Acta ; 505: 92-97, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32070725

RESUMO

INTRODUCTION: Carnitine is essential for long-chain fatty acid oxidation in muscle and heart. Tissue stores are regulated by organic cation/Cn transporter plasmalemmal Octn2. We previously demonstrated low carnitine in quadriceps/gluteus and heart of adult mdx mice. METHODS: We studied protein and mRNA expression of Octn2, mitochondrial Octn1 and peroxisomal Octn3 in adult male C57BL/10ScSn-DMD mdx/J quadriceps, heart, and diaphragm compared to C57BL/10SnJ mice. RESULTS: We demonstrated reduction in mOctn2 expression on Western blot and similar expression of mOctn1 and mOctn3 in mdx quadriceps, heart and diaphragm. There was a significant upregulation of mOctn1 and mOctn2 mRNA by qRT-PCR in mdx quadriceps and of mOctn2 and mOctn3 mRNA in mdx heart. We showed upregulation of mdx mOctn1 and mOctn3 mRNA but no increase in protein expression. DISCUSSION: Dystrophin deficiency likely disrupts Octn2 expression decreasing muscle carnitine uptake thus contributing to membranotoxic long-chain acyl-CoAs with sarcolemmal and organellar membrane oxidative injury providing a treatment rationale for early L-carnitine in DMD.


Assuntos
Carnitina/química , Carnitina/uso terapêutico , Músculo Esquelético/química , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Miocárdio/química , Proteínas de Transporte de Cátions Orgânicos/biossíntese , Proteínas de Transporte de Cátions Orgânicos/genética , Membro 5 da Família 22 de Carreadores de Soluto/biossíntese , Membro 5 da Família 22 de Carreadores de Soluto/genética , Simportadores/biossíntese , Simportadores/genética , Animais , Carnitina/metabolismo , Diafragma/metabolismo , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
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