RESUMO
T-2 toxin, an omnipresent environmental contaminant, poses a serious risk to the health of humans and animals due to its pronounced cardiotoxicity. This study aimed to elucidate the molecular mechanism of cardiac tissue damage by T-2 toxin. Twenty-four male Sprague-Dawley rats were orally administered T-2 toxin through gavage for 12 weeks at the dose of 0, 10, and 100 nanograms per gram body weight per day (ng/(g·day)), respectively. Morphological, pathological, and ultrastructural alterations in cardiac tissue were meticulously examined. Non-targeted metabolomics analysis was employed to analyze alterations in cardiac metabolites. The expression of the Sirt3/FoxO3α/MnSOD signaling pathway and the level of oxidative stress markers were detected. The results showed that exposure to T-2 toxin elicited myocardial tissue disorders, interstitial hemorrhage, capillary dilation, and fibrotic damage. Mitochondria were markedly impaired, including swelling, fusion, matrix degradation, and membrane damage. Metabonomics analysis unveiled that T-2 toxin could cause alterations in cardiac metabolic profiles as well as in the Sirt3/FoxO3α/MnSOD signaling pathway. T-2 toxin could inhibit the expressions of the signaling pathway and elevate the level of oxidative stress. In conclusion, the T-2 toxin probably induces cardiac fibrotic impairment by affecting amino acid and choline metabolism as well as up-regulating oxidative stress mediated by the Sirt3/FoxO3α/MnSOD signaling pathway. This study is expected to provide targets for preventing and treating T-2 toxin-induced cardiac fibrotic injury.
Assuntos
Proteína Forkhead Box O3 , Estresse Oxidativo , Ratos Sprague-Dawley , Transdução de Sinais , Superóxido Dismutase , Toxina T-2 , Animais , Toxina T-2/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Masculino , Proteína Forkhead Box O3/metabolismo , Superóxido Dismutase/metabolismo , Fibrose , Doenças Metabólicas/induzido quimicamente , Regulação para Cima/efeitos dos fármacos , Sirtuína 3/metabolismo , Miocárdio/patologia , Miocárdio/metabolismoRESUMO
Rationale: MG53's known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53's role in the development of type 2 diabetes mellitus. Objective: This study aims to address this controversy - whether MG53's myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. Study design: We determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. Conclusion: Overall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is ~10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans (~10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for type 2 diabetes mellitus and its complications.
Assuntos
Insulina , Fígado , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt , Receptor de Insulina , Animais , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Receptor de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fígado/metabolismo , Fígado/efeitos dos fármacos , Insulina/metabolismo , Insulina/farmacologia , Miocárdio/metabolismo , Células Hep G2 , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Masculino , Transdução de Sinais/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Citocinas/metabolismo , Proteínas de MembranaRESUMO
OBJECTIVE: Aim: The aim of the study was to investigate the activity of bioenergetic processes in rats under conditions of simultaneous exposure to malathion and carbon tetrachloride and after the use of enterosgel. PATIENTS AND METHODS: Materials and Methods: Experiments were conducted on rats. The rats were divided into nine groups.Malathion was administered daily (for 30 days) at a dose of 20 mg / kg body weight of the animal. Tetrachloromethane was administered twice (every other day) as a 50% oil solution at a dose of 1.0 ml / kg body weight. The intensity of energy supply processes was assessed by the activity of succinate dehydrogenase and cytochrome oxidase, impaired carbohydrate metabolism in terms of glucose and glycogen. RESULTS: Results: It was noted that succinate dehydrogenase activity in the liver decreased 2 times, in the myocardium - 1.6 times. On the thirty and seventh day of administration of toxicants after enterosorbent use, succinate dehydrogenase activity increased in the liver by 20%, cytochrome oxidase by 27%, in the myocardium - by 31% and 23%, respectively. The content of glucose in the serum after exposure to toxicants increased maximally (2.4 times) at the end of the study. In contrast, the glycogen content in the liver decreased by 48%, in the myocardium by 13%. The use of enterosgel resulted in a decrease in serum glucose. CONCLUSION: Conclusions: The use of enterosgel leads to the restoration of energy processes in the body of affected rats, which is confirmed by increased activity of mitochondrial enzymes, lowering glucose and increasing glycogen in the studied organs.
Assuntos
Tetracloreto de Carbono , Metabolismo Energético , Fígado , Malation , Succinato Desidrogenase , Animais , Ratos , Metabolismo Energético/efeitos dos fármacos , Succinato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/enzimologia , Masculino , Miocárdio/metabolismo , Ratos Wistar , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , InseticidasRESUMO
Background: Ischemic preconditioning-induced serum exosomes (IPC-exo) protected rat heart against myocardial ischemia/reperfusion injury. However, whether IPC-exo regulate replacement fibrosis after myocardial infarction (MI) and the underlying mechanisms remain unclear. MicroRNAs (miRs) are important cargos of exosomes and play an essential role in cardioprotection. We aim to investigate whether IPC-exo regulate post-MI replacement fibrosis by transferring cardioprotective miRs and its action mechanism. Methods: Exosomes obtained from serum of adult rats in control (Con-exo) and IPC groups were identified and analyzed, subsequently intracardially injected into MI rats following ligation. Their miRs profiles were identified using high-throughput miR sequencing to identify target miRs for bioinformatics analysis. Luciferase reporter assays confirmed target genes of selected miRs. IPC-exo transfected with selected miRs antagomir or NC were intracardially administered to MI rats post-ligation. Cardiac function and degree of replacement fibrosis were detected 4 weeks post-MI. Results: IPC-exo exerted cardioprotective effects against excessive replacement fibrosis. MiR sequencing and RT-qPCR identified miR-133a-3p as most significantly different between IPC-exo and Con-exo. MiR-133a-3p directly targeted latent transforming growth factor beta binding protein 1 (LTBP1) and protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA). KEGG analysis showed that transforming growth factor-ß (TGF-ß) was one of the most enriched signaling pathways with miR-133a-3p. Comparing to injection of IPC-exo transfected with miR-133a-3p antagomir NC, injecting IPC-exo transfected with miR-133a-3p antagomir abolished protective effects of IPC-exo on declining excessive replacement fibrosis and cardiac function enhancement, while increasing the messenger RNA and protein expression of LTBP1, PPP2CA, and TGF-ß1in MI rats. Conclusion: IPC-exo inhibit excessive replacement fibrosis and improve cardiac function post-MI by transferring miR-133a-3p, the mechanism is associated with directly targeting LTBP1 and PPP2CA, and indirectly regulating TGF-ß pathway in rats. Our finding provides potential therapeutic effect of IPC-induced exosomal miR-133a-3p for cardiac repair.
Assuntos
Exossomos , MicroRNAs , Infarto do Miocárdio , Proteína Fosfatase 2 , Animais , MicroRNAs/sangue , MicroRNAs/genética , Infarto do Miocárdio/sangue , Infarto do Miocárdio/terapia , Infarto do Miocárdio/genética , Exossomos/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fibrose , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/terapia , Miocárdio/metabolismo , Precondicionamento Isquêmico/métodos , Precondicionamento Isquêmico Miocárdico/métodosRESUMO
This study investigated the role of Poly (ADP-ribose) Polymerase (PARP) in myocardial ischemia-reperfusion injury (MIRI) in elderly mice. It involves 30 elderly male KM mice divided into three groups: Sham, MIRI, and DPQ, where the MIRI and DPQ groups undergo myocardial ischemia-reperfusion with the DPQ group also receiving DPQ for PARP-1 inhibition. Over three weeks, assessments include histological analysis of myocardial lesions, left ventricular ejection fraction (LVEF) measurements, and evaluations of serum cardiac enzymes and inflammatory markers. This approach aims to understand the protective effects of DPQ in MIRI, focusing on its impact on cardiac health and inflammation via the JAK2/STAT3 pathway. The findings suggest that PARP activation exacerbates cardiac dysfunction and inflammation in MIRI by possibly modulating the JAK2/STAT3 signaling pathway. Inhibition of PARP-1 with DPQ mitigates these effects, as indicated by reduced myocardial lesions and inflammatory infiltration, improved LVEF, and altered levels of inflammatory markers and signaling molecules. However, the differences in STAT3 and p-STAT3 protein expression between the DPQ and MIRI groups were not statistically significant, suggesting that while PARP inhibition affects many aspects of MIRI pathology, its impact on the JAK2/STAT3 pathway may not fully explain the observed benefits. This study contributes to our understanding of the complex mechanisms underlying myocardial ischemia-reperfusion injury, particularly in the context of aging. It highlights the potential of PARP inhibition as a therapeutic strategy to attenuate cardiac dysfunction and inflammation in MIRI, though further research is necessary to fully elucidate the underlying molecular pathways and to explore the clinical relevance of these findings in humans.
Assuntos
Janus Quinase 2 , Traumatismo por Reperfusão Miocárdica , Miocárdio , Fator de Transcrição STAT3 , Transdução de Sinais , Animais , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Masculino , Camundongos , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Miocárdio/patologia , Miocárdio/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Envelhecimento , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
Ferroptosis is an important pathological mechanism of chronic heart failure (CHF). This study aimed to investigate the protective mechanism of Astragaloside IV (AS-IV) on CHF rats by integrating bioinformatics and ferroptosis. CHF-related targets and ferroptosis-related targets were collected. After the intersection, the common targets were obtained. The PPI network of the common targets was constructed, and topological analysis of the network was carried out. The target with the highest topological parameter values was selected as the key target. The key target p53 was obtained through bioinformatics analysis, and its molecular docking model with AS-IV was obtained, as well as molecular dynamics simulation analysis. The rat models of CHF after myocardial infarction were established by ligation of left coronary artery and treated with AS-IV for 4 weeks. AS-IV treatment significantly improved cardiac function in CHF rats, improved cardiomyocyte morphology and myocardial fibrosis, reduced mitochondrial damage, decreased myocardial MDA and Fe2+ content, increased GSH content, inhibited the expression of p53 and p-p53, and up-regulated the expression of SLC7A11 and GPX4. In conclusion, AS-IV improved cardiac function in CHF rats, presumably by regulating p53/SLC7A11/GPX4 signaling pathway and inhibiting myocardial ferroptosis.
Assuntos
Biologia Computacional , Ferroptose , Insuficiência Cardíaca , Saponinas , Triterpenos , Animais , Ferroptose/efeitos dos fármacos , Triterpenos/farmacologia , Saponinas/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Ratos , Biologia Computacional/métodos , Masculino , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Simulação de Acoplamento Molecular , Doença Crônica , Modelos Animais de Doenças , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Simulação de Dinâmica Molecular , Miocárdio/metabolismo , Miocárdio/patologiaRESUMO
Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless, a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process, including hypoxic stress, fibrosis, and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions, leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology, demonstrating that the phased combination of TGFß inhibitor SB431542, Rho kinase inhibitor Y27632, and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction, providing a crucial platform for discovering therapeutic targets for ischemic heart disease.
Assuntos
Dioxóis , Fibrose , Infarto do Miocárdio , Piridinas , Engenharia Tecidual , Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/terapia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Camundongos , Humanos , Piridinas/farmacologia , Piridinas/uso terapêutico , Engenharia Tecidual/métodos , Dioxóis/farmacologia , Dioxóis/uso terapêutico , Miocárdio/patologia , Miocárdio/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Modelos Animais de Doenças , Transdução de Sinais , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Remodelação Ventricular/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Coração/fisiopatologia , Coração/efeitos dos fármacos , AmidasRESUMO
Chronic fibrotic tissue disrupts various organ functions. Despite significant advances in therapies, mortality and morbidity due to heart failure remain high, resulting in poor quality of life. Beyond the cardiomyocyte-centric view of heart failure, it is now accepted that alterations in the interstitial extracellular matrix (ECM) also play a major role in the development of heart failure. Here, we show that protein kinase N (PKN) is expressed in cardiac fibroblasts. Furthermore, PKN mediates the conversion of fibroblasts into myofibroblasts, which plays a central role in secreting large amounts of ECM proteins via p38 phosphorylation signaling. Fibroblast-specific deletion of PKN led to a reduction of myocardial fibrotic changes and cardiac dysfunction in mice models of ischemia-reperfusion or heart failure with preserved ejection fraction. Our results indicate that PKN is a therapeutic target for cardiac fibrosis in heart failure.
Assuntos
Fibroblastos , Fibrose , Insuficiência Cardíaca , Miocárdio , Miofibroblastos , Proteína Quinase C , Animais , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/genética , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Camundongos , Miocárdio/patologia , Miocárdio/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C/genética , Masculino , Humanos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Knockout , Matriz Extracelular/metabolismo , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
ABSTRACT: The role of intravenous immunoglobulin in protecting the diabetic heart from ischemia/reperfusion (I/R) injury is unclear. Hearts isolated from adult diabetic and nondiabetic Wistar rats (n = 8 per group) were treated with intravenous immunoglobulin (IVIG) either 2 hours before euthanasia, before ischemia, or at reperfusion. Hemodynamic data were acquired using the Isoheart software version 1.524-S. Ischemia/reperfusion (I/R) injury was evaluated by 2,3,5-triphenyltetrazolium chloride staining and troponin T levels. The levels of apoptosis markers, caspases-3/8, antioxidant enzymes, superoxide dismutase and catalase, glucose transporters, GLUT-1 and GLUT-4, phosphorylated ERK1/2, and phosphorylated eNOS were estimated by Western blotting. Proinflammatory and anti-inflammatory cytokine levels were evaluated using enzyme-linked immunosorbent assays. Intravenous immunoglobulin administration abolished the effects of I/R injury in hearts subjected to hyperglycemia when infused at reperfusion, before ischemia, or at reperfusion in 4-week diabetic rat hearts and only at reperfusion in 6-week diabetic rat hearts. IVIG infusion resulted in a significant (P < 0.05) recovery of cardiac hemodynamics and decreased infarct size. IVIG also reduced the levels of troponin T, apoptotic enzymes, and proinflammatory cytokines. IVIG significantly (P < 0.05) increased the levels of anti-inflammatory cytokines, antioxidant enzymes, GLUT-4, and phosphorylated eNOS. Intravenous immunoglobulin protected the hearts from I/R injury if infused at reperfusion in the presence of hyperglycemia, in 4- and 6-week diabetic rat hearts, and when infused before ischemia in 4-week diabetic rat hearts. IVIG exerts its cardioprotective effects associated with the upregulated phosphorylated eNOS/GLUT-4 pathway.
Assuntos
Diabetes Mellitus Experimental , Transportador de Glucose Tipo 4 , Traumatismo por Reperfusão Miocárdica , Óxido Nítrico Sintase Tipo III , Ratos Wistar , Transdução de Sinais , Animais , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Transportador de Glucose Tipo 4/metabolismo , Masculino , Imunoglobulinas Intravenosas/farmacologia , Apoptose/efeitos dos fármacos , Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/tratamento farmacológico , Ratos , Estresse Oxidativo/efeitos dos fármacos , Citocinas/metabolismo , Preparação de Coração Isolado , Mediadores da Inflamação/metabolismoRESUMO
Enhancement of Connexin43 (Cx43) and ferroptosis are respectively associated with the exacerbation of myocardial ischemia-reperfusion injury (MIRI) in diabetes. Myocardial vulnerability to ischemic insult has been shown to vary during early and later phases of diabetes in experimental settings. Whether or not Connexin43 (Cx43) and ferroptosis interplay during MIRI in diabetes is unknown. We, thus, aimed to investigate whether or not the content of myocardial Cx43 may be attributable to myocardial vulnerability to MIRI at different stages of diabetes and also to explore the potential interplay between Cx43 and ferroptosis in this pathology. Age-matched control and subgroups of Streptozotocin-induced diabetic mice were subjected to MIRI induced by 30 minutes coronary artery occlusion and 2 hours reperfusion respectively at 1, 2 and 5 weeks of diabetes. Rat cardiac H9C2 cells were exposed to high glucose (HG) for 48h in the absence or presence of Cx43 gene knockdown followed by hypoxia/reoxygenation (HR) respectively for 6 and 12 hours. Post-ischemic myocardial infarct size was reduced in 1 and 2 weeks DM mice concomitant with enhanced GPX4 and reduced cardiac Cx43 and ferroptosis as compared to control. By contrast, cardiac GPX4 was significantly reduced while Cx43 increased at DM 5 weeks (D5w) which was correspondent to significant increases in ferroptosis and myocardial infarction. Post-ischemic cardiac function was improved in 1 and 2 weeks but worsened in 5w DM mice as compared with non-diabetic control. GAP19 (Cx43 inhibitor) significantly attenuated ferroptosis and reduced myocardial infarction in D5w mice. Erastin (ferroptosis activator) reversed the cardioprotective effect of GAP19. In vitro, HR significantly reduced cell viability accompanied with reduced GPX4 but elevated Cx43 expression, MDA production and ferroptosis. Cx43 gene knockdown in H9C2 resulted in a significant increase in GPX4, reduction in MDA and ferroptosis, and subsequently reduced post-hypoxic cell viability. The beneficial effects of Cx43 gene knock-down was minified or eliminated by Erastin. It is concluded that Cx43 overexpression exacerbates MIRI under diabetic conditions via promoting ferroptosis, while its down-regulation at early state of diabetes is attributable to enhanced myocardial tolerance to MIRI.
Assuntos
Conexina 43 , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Ferroptose , Traumatismo por Reperfusão Miocárdica , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Animais , Ferroptose/genética , Conexina 43/metabolismo , Conexina 43/genética , Camundongos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Ratos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/genética , Masculino , Técnicas de Silenciamento de Genes , Humanos , Linhagem Celular , Miocárdio/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismoRESUMO
Sarcomere activation in striated muscle requires both thin filament-based and thick filament-based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments.
Assuntos
Cálcio , Miocárdio , Animais , Cálcio/metabolismo , Suínos , Miocárdio/metabolismo , Sarcômeros/metabolismo , Miosinas/metabolismo , Troponina/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologiaRESUMO
This article focuses on screening the major secreted proteins by the ischemia-challenged cardiac stromal fibroblasts (CF), the assessment of their expression status and functional role in the post-ischemic left ventricle (LV) and in the ischemia-challenged CF culture and to phenotype CF at single cell resolution based on the positivity of the identified mediators. The expression level of CRSP2, HSP27, IL-8, Cofilin-1, and HSP90 in the LV tissues following coronary artery bypass graft (CABG) and myocardial infarction (MI) and CF cells followed the screening profile derived from the MS/MS findings. The histology data unveiled ECM disorganization, inflammation and fibrosis reflecting the ischemic pathology. CRSP2, HSP27, and HSP90 were significantly upregulated in the LV-CABG tissues with a concomitant reduction ion LV-MI whereas Cofilin-1, IL8, Nrf2, and Troponin I were downregulated in LV-CABG and increased in LV-MI. Similar trends were exhibited by ischemic CF. Single cell transcriptomics revealed multiple sub-phenotypes of CF based on their respective upregulation of CRSP2, HSP27, IL-8, Cofilin-1, HSP90, Troponin I and Nrf2 unveiling pathological and pro-healing phenotypes. Further investigations regarding the underlying signaling mechanisms and validation of sub-populations would offer novel translational avenues for the management of cardiac diseases.
Assuntos
Fibroblastos , Infarto do Miocárdio , Análise de Célula Única , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Fibroblastos/metabolismo , Humanos , Células Estromais/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/genética , Cofilina 1/metabolismo , Cofilina 1/genética , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Transcriptoma , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genéticaRESUMO
Hyperhomocysteinemia (HHcy) is considered an independent risk factor of cardiovascular diseases. Among the proposed mechanisms underlying homocysteine toxicity are altered protein expression and induction of oxidative stress. In the present study, we explored protein abundance and parameters related to oxidative stress in heart homogenates of rats exposed to chronic mild HHcy. Using two-dimensional gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry 22 altered proteins (6 upregulated and 14 downregulated) were identified. For eight proteins the altered abundances were validated by Western blot analysis. Identified proteins are primarily involved in energy metabolism (mainly enzymes of glycolysis, pyruvate dehydrogenase complex, citric acid cycle, and ATP synthase), cardiac muscle contraction (alpha-actin and myosin light chains), stress response (heat-shock protein beta1 and alphaB-crystallin) and antioxidant defense (glutathione peroxidase 1). Diminished antioxidant defense was confirmed by decreases in total antioxidant capacity and GSH/GSSG ratio. Consistent with the decline in enzymatic and non-enzymatic antioxidant defense the protein oxidative modification, as determined by tyrosine nitration, was significantly increased. These findings suggest that both, altered protein expression and elevated oxidative stress contribute to cardiovascular injury caused by HHcy. Keywords: Homocysteine, Heart, Protein abundance, Antioxidant capacity, Nitrotyrosines.
Assuntos
Hiper-Homocisteinemia , Miocárdio , Estresse Oxidativo , Ratos Wistar , Animais , Hiper-Homocisteinemia/metabolismo , Masculino , Ratos , Miocárdio/metabolismoRESUMO
The effects of alpha-pinene (AP), a monoterpenoid, known for its antioxidant, anti-inflammatory, and anti-apoptotic properties, on methotrexate (MTX)-induced cardiac and hepatic damage were investigated in this study. Male Sprague-Dawley rats were divided into Control, Vehicle, AP, MTX, and AP+MTX groups (n=7). AP (50 mg/kg/day, 14 days) was applied subcutaneously in the AP and AP+MTX groups. MTX (20 mg/kg) was injected three days before sacrification. Serum CK-MB, troponin T, ALT, and AST levels, as well as cardiac and hepatic MDA, GSH, caspase-3, and p53 levels, were measured by ELISA. Histological changes in tissues were evaluated by scoring in terms of tissue damage and cellular degeneration parameters after hematoxylin-eosin staining. MTX caused significant increase in serum CK-MB, troponin T, ALT, and AST levels, hepatic and cardiac lipid peroxidation, GSH depletion, and caspase-3 level. However, tissue levels of p53 did not change significantly. MTX-induced histological deterioration was observed in both tissues. These MTX-induced changes were significantly reduced in the AP+MTX group. Present results show that MTX-induced cardiac and hepatic damage is prevented by AP pretreatment. This protection can be attributed to the antioxidant and anti-apoptotic properties of AP. Considering the importance of MTX in cancer treatment, AP appears to have highly promising potential as a cardioprotective and hepatoprotective agent in anti-tumoral therapy. Key words: MDA, GSH, Caspase-3, p53, Oxidative stress, Apoptosis.
Assuntos
Monoterpenos Bicíclicos , Metotrexato , Ratos Sprague-Dawley , Animais , Masculino , Metotrexato/toxicidade , Ratos , Monoterpenos Bicíclicos/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/metabolismo , Miocárdio/patologia , Miocárdio/metabolismo , Monoterpenos/farmacologia , Monoterpenos/uso terapêuticoRESUMO
Systemic amyloidosis involves the deposition of misfolded proteins in organs/tissues, leading to progressive organ dysfunction and failure. Congo red is the gold-standard chemical stain for visualizing amyloid deposits in tissue, showing birefringence under polarization microscopy. However, Congo red staining is tedious and costly to perform, and prone to false diagnoses due to variations in amyloid amount, staining quality and manual examination of tissue under a polarization microscope. We report virtual birefringence imaging and virtual Congo red staining of label-free human tissue to show that a single neural network can transform autofluorescence images of label-free tissue into brightfield and polarized microscopy images, matching their histochemically stained versions. Blind testing with quantitative metrics and pathologist evaluations on cardiac tissue showed that our virtually stained polarization and brightfield images highlight amyloid patterns in a consistent manner, mitigating challenges due to variations in chemical staining quality and manual imaging processes in the clinical workflow.
Assuntos
Amiloide , Aprendizado Profundo , Microscopia de Fluorescência , Coloração e Rotulagem , Humanos , Birrefringência , Amiloide/metabolismo , Microscopia de Fluorescência/métodos , Coloração e Rotulagem/métodos , Vermelho Congo , Microscopia de Polarização/métodos , Amiloidose/patologia , Amiloidose/metabolismo , Amiloidose/diagnóstico por imagem , Imagem Óptica/métodos , Placa Amiloide/patologia , Placa Amiloide/metabolismo , Placa Amiloide/diagnóstico por imagem , Miocárdio/patologia , Miocárdio/metabolismoRESUMO
Cancer cachexia manifests as whole body wasting, however, the precise mechanisms governing the alterations in skeletal muscle and cardiac anabolism have yet to be fully elucidated. In this study, we explored changes in anabolic processes in both skeletal and cardiac muscles in the Yoshida AH-130 ascites hepatoma model of cancer cachexia. AH-130 tumor-bearing rats experienced significant losses in body weight, skeletal muscle, and heart mass. Skeletal and cardiac muscle loss was associated with decreased ribosomal (r)RNA, and hypophosphorylation of the eukaryotic factor 4E binding protein 1. Endoplasmic reticulum stress was evident by higher activating transcription factor mRNA in skeletal muscle and growth arrest and DNA damage-inducible protein (GADD)34 mRNA in both skeletal and cardiac muscles. Tumors provoked an increase in tissue expression of interferon-γ in the heart, while an increase in interleukin-1ß mRNA was apparent in both skeletal and cardiac muscles. We conclude that compromised skeletal muscle and heart mass in the Yoshida AH-130 ascites hepatoma model involves a marked reduction translational capacity and efficiency. Furthermore, our observations suggest that endoplasmic reticulum stress and tissue production of pro-inflammatory factors may play a role in the development of skeletal and cardiac muscle wasting.
Assuntos
Caquexia , Músculo Esquelético , Miocárdio , Resposta a Proteínas não Dobradas , Animais , Caquexia/metabolismo , Caquexia/etiologia , Caquexia/patologia , Caquexia/genética , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ratos , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Wistar , Estresse do Retículo Endoplasmático , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genéticaRESUMO
This study aimed to investigate the regulatory mechanism of Linggui Zhugan Decoction(LGZGD)-medicated serum on the fibrosis of cardiac fibroblasts(CFs) and the protein expression of the Wnt/ß-catenin signaling pathway. Blank serum and LGZGD-medicated serum were prepared, and primary CFs were isolated and cultured using trypsin-collagenase digestion and differential adhesion method. Immunofluorescence labeling was used to identify primary CFs. Cells were divided into normal control group, model group, 20% blank serum group, and 5%, 10%, and 20% LGZGD-medicated serum groups. Except for the normal control group, all other groups were stimulated with hydrogen peroxide(H_2O_2) after pretreatment with 20% blank serum or 5%, 10%, 20% LGZGD-medicated serum for 12 hours to establish a model of fibrosis in primary CFs. Scratch healing assay was used to observe cell migration ability. ELISA was used to detect the content of collagen type â (Col â ) and type â ¢(Col â ¢). Western blot was used to detect the protein expression of α-smooth muscle actin(α-SMA), Wnt1, glycogen synthase kinase 3ß(GSK-3ß), phosphorylated GSK-3ß(p-GSK-3ß), ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-catenin and matrix metalloproteinase 9(MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and ß-catenin. CFs with Wnt1 overexpression were prepared and treated with H_2O_2. The following groups were set up: normal control group, model group, 20% LGZGD-medicated serum group, empty plasmid+20% LGZGD-medicated serum group, and Wnt1 overexpression+20% LGZGD-medicated serum group. ELISA was used to detect the content and ratio of Col â and Col â ¢. Western blot was used to detect the protein expression of α-SMA, Wnt1, GSK-3ß, p-GSK-3ß, ß-catenin, and nuclear ß-catenin. RT-qPCR was used to detect the gene expression of ß-catenin and MMP9. Immunofluorescence staining showed that CFs expressed Vimentin positively, appearing green, with blue nuclei and purity greater than 90%, which were identified as primary CFs. RESULTS:: showed that compared with the normal control group, CFs in the model group had enhanced healing rate, increased content of Col â and Col â ¢, increased ratio of Col â /Col â ¢, upregulated protein expression of α-SMA, Wnt1, p-GSK-3ß, ß-catenin, nuclear ß-catenin, decreased GSK-3ß expression, elevated mRNA expression of ß-catenin and MMP9, and enhanced fluorescence intensity and expression of ß-catenin and α-SMA. Compared with the model group, 5%, 10%, 20% LGZGD-medicated serum significantly inhibited cell migration ability, reduced the content of Col â and Col â ¢, decreased ratio of Col â /Col â ¢, downregulated protein expression of α-SMA, Wnt1, p-GSK-3ß, ß-catenin, nuclear ß-catenin, increased GSK-3ß expression, decreased mRNA expression of ß-catenin and MMP9, and reduced fluorescence intensity and expression of ß-catenin and α-SMA. Compared with the empty plasmid+20% LGZGD-medicated serum group, the effect of LGZGD-medicated serum was significantly reversed after overexpression of Wnt1. LGZGD can reduce excessive deposition of collagen fibers, inhibit excessive proliferation of fibroblasts, and improve the process of myocardial fibrosis. The improvement of myocardial fibrosis by LGZGD is related to the regulation of the Wnt/ß-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.
Assuntos
Medicamentos de Ervas Chinesas , Fibrose , Miocárdio , Ratos Sprague-Dawley , Via de Sinalização Wnt , beta Catenina , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/administração & dosagem , Ratos , beta Catenina/metabolismo , beta Catenina/genética , Miocárdio/metabolismo , Miocárdio/patologia , Masculino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células CultivadasRESUMO
This study aims to investigate the effects of Linggui Zhugan Decoction(LGZGD) on myocardial fibrosis(MF) and the Lats1/Yap signaling pathway in mice after myocardial infarction(MI), exploring its role and mechanism in inhibiting MF. The MI-induced ischemic mouse model was established by left anterior descending coronary artery ligation, followed by continuous intervention for six weeks. Doppler ultrasound imaging-system of small animals was used to detect left ventricular ejection fraction(LVEF), left ventricular fractional shortening(LVFS), left ventricular internal diameter at end-systole(LVIDs), and left ventricular internal diameter at end-diastole(LVIDd). Pathological changes in myocardial tissue were observed by HE and Masson staining. Serum levels of creatine kinase isoenzyme MB(CK-MB) and lactate dehydrogenase(LDH) were detected by using ELISA. Myocardial tissue mRNA levels of Lats1, Yap, and connective tissue growth factor(CTGF) were determined by RT-qPCR. Protein expression of alpha-smooth muscle actin(α-SMA), collagen â (Col â ), collagen â ¢(Col â ¢), tissue inhibitor of metal protease 1(TIMP1), matrix metallopeptidase 2(MMP2), Yap, p-Yap, and n-Yap was determined by Western blot. Compared with the sham group, the model group showed significantly decreased LVEF and LVFS levels, increased LVIDd and LVIDs levels(P<0.01), disordered arrangement of myocardial cells, partial fracture of myocardial fibers, and massive deposition of collagen fibers. Moreover, serum levels of CK-MB and LDH were significantly increased(P<0.01), while myocardial tissue mRNA levels of Lats1 were significantly decreased(P<0.01), and mRNA levels of Yap and CTGF were significantly increased(P<0.01). Protein expression of α-SMA, Col â , Col â ¢, MMP2, Yap, and n-Yap was significantly increased(P<0.01), while protein expression of Lats1, TIMP1, p-Yap, and the ratio of p-Yap/Yap were significantly decreased(P<0.01). Compared with the model group, after intervention with LGZGD(9.36 g·kg~(-1)), mice showed significantly increased LVEF and LVFS levels, decreased LVIDd and LVIDs levels(P<0.01), more orderly arrangement of myocardial cells, significantly reduced myocardial fiber fracture and collagen fiber deposition. Serum levels of CK-MB and LDH were significantly decreased(P<0.01), while myocardial tissue mRNA levels of Lats1 were significantly increased(P<0.01), and mRNA levels of Yap and CTGF were significantly decreased(P<0.01). Protein expression of α-SMA, Col â , Col â ¢, MMP2, Yap, and n-Yap was significantly decreased(P<0.01), while protein expression of Lats1, TIMP1, p-Yap, and the ratio of p-Yap/Yap were significantly increased(P<0.01). LGZGD can inhibit MF in mice after MI and improve mouse cardiac function, which is closely related to the activation of the Lats1/Yap signaling pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Medicamentos de Ervas Chinesas , Fibrose , Infarto do Miocárdio , Miocárdio , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Proteínas de Sinalização YAP , Animais , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Masculino , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
Myocarditis is a major cause of heart failure and death, particularly in young individuals. Current treatments are mainly symptomatic, but emerging therapies focus on targeting inflammation and fibrosis pathways. Natural bioactive compounds like flavonoids and phenolic acids show promising anti-inflammatory and antioxidant properties. Corticosteroids are frequently employed in the treatment of autoimmune myocarditis and appear to lower mortality rates compared to conventional therapies for heart failure. This study aims to explore the effects of Mangiferin on pro-inflammatory cytokine levels, nitro-oxidative stress markers, histopathological alterations, and cardiac function in experimental myosin-induced autoimmune myocarditis. The effects were compared to Prednisone, used as a reference anti-inflammatory compound, and Trolox, used as a reference antioxidant. The study involved 30 male Wistar-Bratislava rats, which were randomly divided into five groups: a negative control group (C-), a positive control group with induced myocarditis using a porcine myosin solution (C+), three groups with induced myocarditis receiving Mangiferin (M), Prednisone (P), or Trolox (T) as treatment. Cardiac function was evaluated using echocardiography. Biochemical measurements of nitro-oxidative stress and inflammatory markers were conducted. Finally, histopathological changes were assessed. At echocardiography, the evaluation of the untreated myocarditis group showed a trend toward decreased left ventricular ejection fraction (LVEF) but was not statistically significant, while all treated groups showed some improvement in LVEF and left ventricular fraction shortening (LVFS). Significant changes were seen in the Mangiferin group, with lower end-diastolic left ventricular posterior wall (LVPWd) by day 21 compared to the Trolox group (p < 0.001). In the first week of the experiment, levels of interleukins (IL)-1ß, IL-6, and tumour necrosis factor (TNF)-α were significantly higher in the myosin group compared to the negative control group (p < 0.001, p < 0.001, p < 0.01), indicating the progression of inflammation in this group. Treatment with Mangiferin, Prednisone, and Trolox caused a significant reduction in IL-1ß compared to the positive control group (p < 0.001). Notably, Mangiferin resulted in a superior reduction in IL-1ß compared to Prednisone (p < 0.05) and Trolox (p < 0.05). Furthermore, Mangiferin treatment led to a statistically significant increase in total oxidative capacity (TAC) (p < 0.001) and a significant reduction in nitric oxide (NOx) levels (p < 0.001) compared to the negative control group. Furthermore, when compared to the Prednisone-treated group, Mangiferin significantly reduced NOx levels (p < 0.001) and increased TAC levels (p < 0.001). Mangiferin treatment significantly lowered creatine kinase (CK) and aspartate aminotransferase (AST) levels on day 7 (p < 0.001 and p < 0.01, respectively) and reduced CK levels on day 21 (p < 0.01) compared to the untreated group. In the nontreated group, the histological findings at the end of the experiment were consistent with myocarditis. In the group treated with Mangiferin, only one case exhibited mild inflammatory infiltrates, represented by mononucleated leukocytes admixed with few neutrophils, with the severity graded as mild. Statistically significant correlations between the grades (0 vs. 1-2) and the study groups have been highlighted (p < 0.005). This study demonstrated Mangiferin's cardioprotective effects in autoimmune myocarditis, showing reduced oxidative stress and inflammation. Mangiferin appears promising as a treatment for acute myocarditis, but further research is needed to compare its efficacy with other treatments like Trolox and Prednisone.