ABSTRACT
Rationale: Autophagy dysregulation is known to be a mechanism of doxorubicin (DOX)-induced cardiotoxicity (DIC). Mitochondrial-Endoplasmic Reticulum Contacts (MERCs) are where autophagy initiates and autophagosomes form. However, the role of MERCs in autophagy dysregulation in DIC remains elusive. FUNDC1 is a tethering protein of MERCs. We aim to investigate the effect of DOX on MERCs in cardiomyocytes and explore whether it is involved in the dysregulated autophagy in DIC. Methods: We employed confocal microscopy and transmission electron microscopy to assess MERCs structure. Autophagic flux was analyzed using the mCherry-EGFP-LC3B fluorescence assay and western blotting for LC3BII. Mitophagy was studied through the mCherry-EGFP-FIS1 fluorescence assay and colocalization analysis between LC3B and mitochondria. A total dose of 18 mg/kg of doxorubicin was administrated in mice to construct a DIC model in vivo. Additionally, we used adeno-associated virus (AAV) to cardiac-specifically overexpress FUNDC1. Cardiac function and remodeling were evaluated by echocardiography and Masson's trichrome staining, respectively. Results: DOX blocked autophagic flux by inhibiting autophagosome biogenesis, which could be attributed to the downregulation of FUNDC1 and disruption of MERCs structures. FUNDC1 overexpression restored the blocked autophagosome biogenesis by maintaining MERCs structure and facilitating ATG5-ATG12/ATG16L1 complex formation without altering mitophagy. Furthermore, FUNDC1 alleviated DOX-induced oxidative stress and cardiomyocytes deaths in an autophagy-dependent manner. Notably, cardiac-specific overexpression of FUNDC1 protected DOX-treated mice against adverse cardiac remodeling and improved cardiac function. Conclusions: In summary, our study identified that FUNDC1-meditated MERCs exerted a cardioprotective effect against DIC by restoring the blocked autophagosome biogenesis. Importantly, this research reveals a novel role of FUNDC1 in enhancing macroautophagy via restoring MERCs structure and autophagosome biogenesis in the DIC model, beyond its previously known regulatory role as an mitophagy receptor.
Subject(s)
Autophagy , Cardiotoxicity , Doxorubicin , Endoplasmic Reticulum , Membrane Proteins , Mitochondrial Proteins , Myocytes, Cardiac , Animals , Doxorubicin/adverse effects , Doxorubicin/pharmacology , Mice , Autophagy/drug effects , Cardiotoxicity/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Membrane Proteins/metabolism , Membrane Proteins/genetics , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondria/metabolism , Mitochondria/drug effects , Mitophagy/drug effects , Male , Autophagosomes/metabolism , Autophagosomes/drug effects , Mice, Inbred C57BL , Disease Models, AnimalABSTRACT
Objective: To investigate the effect of Sanshentongmai (SSTM) mixture on the regulation of oxidative damage to rat cardiomyocytes (H9C2) through microRNA-146a and its mechanism. Methods: H9C2 were cultured in vitro, H2O2 was used as an oxidant to create an oxidative damage model in H9C2 cells. SSTM intervention was administered to the H9C2 cells. Then, the changes in H2O2-induced oxidative damage in H9C2 cells and the expression of microRNA-146a were observed to explore the protective effect of SSTM on H9C2 and its mechanism. H9C2 cells cultured i n vitro were divided into 3 groups, including a control group, a model group of H2O2-induced oxidative damage (referred to hereafter as the model group), and a group given H2O2 modeling plus SSTM intervention at 500 µg/L for 72 h (referred to hereafter as the treatment group). The cell viability was measured by CCK8 assay. In addition, the levels of N-terminal pro-brain natriuretic peptide (Nt-proBNP), nitric oxide (NO), high-sensitivity C-reactive protein (Hs-CRP), and angiotensin were determined by enzyme-linked immunosorbent assay (ELISA). The expression level of microRNA-146a was determined by real-time PCR (RT-PCR). Result: H9C2 cells were pretreated with SSTM at mass concentrations ranging from 200 to 1500 µg/L. Then, CCK8 assay was performed to measure cell viability and the findings showed that the improvement in cell proliferation reached its peak when the mass concentration of SSTM was 500 µg/L, which was subsequently used as the intervention concentration. ELISA was performed to measure the indicators related to heart failure, including Nt-proBNP, NO, Hs-CRP, and angiotensin â ¡. Compared with those of the control group, the expressions of Nt-proBNP and angiotensin â ¡ in the treatment group were up-regulated (P<0.05), while the expression of NO was down-regulated (P<0.05). There was no significant difference in the expression of Hs-CRP between the treatment group and the control group. These findings indicate that SSTM could effectively ameliorate oxidative damage in H9C2 rat cardiomyocytes. Finally, according to the RT-PCR findings for the expression of microRNA-146a in each group, H2O2 treatment at 15 µmol/L could significantly reduce the expression of microRNA-146a, and the expression of microRNA-146a in the treatment group was nearly doubled compared with that in the model group. There was no significant difference between the treatment group and the control group. Conclusion: SSTM can significantly resist the H2O2-induced oxidative damage of H9C2 cells and may play a myocardial protective role by upregulating microRNA-146a.
Subject(s)
Drugs, Chinese Herbal , Hydrogen Peroxide , MicroRNAs , Myocytes, Cardiac , Oxidative Stress , Up-Regulation , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/cytology , Animals , MicroRNAs/metabolism , MicroRNAs/genetics , Rats , Oxidative Stress/drug effects , Hydrogen Peroxide/toxicity , Drugs, Chinese Herbal/pharmacology , Up-Regulation/drug effects , Cell Survival/drug effects , Cell Line , Drug CombinationsABSTRACT
There is a known genetic susceptibility to anthracycline-induced cardiac dysfunction in childhood cancer survivors, but this has not been adequately shown in adolescent and young adult (AYA) patients. Our aim was to determine if the previously identified variants associated with cardiac dysfunction in childhood cancer patients affect AYA cancer patients similarly. Forty-five variants were selected for analysis in 253 AYAs previously treated with anthracyclines. We identified four variants that were associated with cardiac dysfunction: SLC10A2:rs7319981 (p = 0.017), SLC22A17:rs4982753 (p = 0.019), HAS3:rs2232228 (p = 0.023), and RARG:rs2229774 (p = 0.050). HAS3:rs2232228 and SLC10A2:rs7319981 displayed significant effects in our AYA cancer survivor population that were in the opposite direction than that reported in childhood cancer survivors. Genetic variants in the host genes were further analyzed for additional associations with cardiotoxicity in AYA cancer survivors. The host genes were then evaluated in a panel of induced pluripotent stem cell-derived cardiomyocytes to assess changes in levels of expression when treated with doxorubicin. Significant upregulation of HAS3 and SLC22A17 expression was observed (p < 0.05), with non-significant anthracycline-responsivity observed for RARG. Our study demonstrates that there is a genetic influence on cardiac dysfunction in AYA cancer patients, but there may be a difference in the role of genetics between childhood and AYA cancer survivors.
Subject(s)
Anthracyclines , Cancer Survivors , Cardiotoxicity , Genetic Predisposition to Disease , Humans , Adolescent , Anthracyclines/adverse effects , Young Adult , Male , Female , Cardiotoxicity/genetics , Adult , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Polymorphism, Single Nucleotide/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Heart Diseases/chemically induced , Heart Diseases/genetics , Antibiotics, Antineoplastic/adverse effects , Risk FactorsABSTRACT
Doxorubicin (DOX) is a highly effective chemotherapeutic agent whose clinical use is hindered by the onset of cardiotoxic effects, resulting in reduced ejection fraction within the first year from treatment initiation. Recently it has been demonstrated that DOX accumulates within mitochondria, leading to disruption of metabolic processes and energetic imbalance. We previously described that phosphoinositide 3-kinase γ (PI3Kγ) contributes to DOX-induced cardiotoxicity, causing autophagy inhibition and accumulation of damaged mitochondria. Here we intend to describe the maladaptive metabolic rewiring occurring in DOX-treated hearts and the contribution of PI3Kγ signalling to this process. Metabolomic analysis of DOX-treated WT hearts revealed an accumulation of TCA cycle metabolites due to a cycle slowdown, with reduced levels of pyruvate, unchanged abundance of lactate and increased Acetyl-CoA production. Moreover, the activity of glycolytic enzymes was upregulated, and fatty acid oxidation downregulated, after DOX, indicative of increased glucose oxidation. In agreement, oxygen consumption was increased in after pyruvate supplementation, with the formation of cytotoxic ROS rather than energy production. These metabolic changes were fully prevented in KD hearts. Interestingly, they failed to increase glucose oxidation in response to DOX even with autophagy inhibition, indicating that PI3Kγ likely controls the fuel preference after DOX through an autophagy-independent mechanism. In vitro experiments showed that inhibition of PI3Kγ inhibits pyruvate dehydrogenase (PDH), the key enzyme of Randle cycle regulating the switch from fatty acids to glucose usage, while decreasing DOX-induced mobilization of GLUT-4-carrying vesicles to the plasma membrane and limiting the ensuing glucose uptake. These results demonstrate that PI3Kγ promotes a maladaptive metabolic rewiring in DOX-treated hearts, through a two-pronged mechanism controlling PDH activation and GLUT-4-mediated glucose uptake.
Subject(s)
Cardiotoxicity , Doxorubicin , Energy Metabolism , Fatty Acids , Glucose , Oxidation-Reduction , Animals , Doxorubicin/toxicity , Glucose/metabolism , Fatty Acids/metabolism , Energy Metabolism/drug effects , Class Ib Phosphatidylinositol 3-Kinase/metabolism , Glycolysis/drug effects , Autophagy/drug effects , Male , Signal Transduction/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Citric Acid Cycle/drug effects , Mice, Inbred C57BL , Heart Diseases/chemically induced , Heart Diseases/metabolism , Heart Diseases/pathology , Heart Diseases/prevention & control , Heart Diseases/physiopathology , Mitochondria, Heart/metabolism , Mitochondria, Heart/drug effects , Mitochondria, Heart/pathology , Mitochondria, Heart/enzymology , Mice, Knockout , Disease Models, Animal , Reactive Oxygen Species/metabolism , Glucose Transporter Type 4/metabolism , Antibiotics, Antineoplastic/toxicity , Antibiotics, Antineoplastic/adverse effectsSubject(s)
Inflammasomes , Isoproterenol , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Rats , Inflammasomes/metabolism , Inflammasomes/antagonists & inhibitors , Inflammasomes/drug effects , Cell Line , Myoblasts, Cardiac/drug effects , Myoblasts, Cardiac/metabolism , Myoblasts, Cardiac/pathology , Cardiomegaly/chemically induced , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomegaly/prevention & control , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathologySubject(s)
Antioxidants , Oxidative Stress , Quercetin , Quercetin/pharmacology , Quercetin/analogs & derivatives , Oxidative Stress/drug effects , Animals , Acetylation , Antioxidants/pharmacology , Inflammation/metabolism , Inflammation/drug therapy , Male , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Anti-Inflammatory Agents/pharmacology , Rats , Palmitic Acid/toxicity , Palmitic Acid/pharmacologySubject(s)
Autophagy , Benzhydryl Compounds , Cellular Senescence , Glucosides , Imidazoles , Myocytes, Cardiac , Pyridazines , Sodium-Glucose Transporter 2 Inhibitors , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Myocytes, Cardiac/metabolism , Cellular Senescence/drug effects , Autophagy/drug effects , Benzhydryl Compounds/pharmacology , Animals , Imidazoles/pharmacology , Pyridazines/pharmacology , Glucosides/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Cells, Cultured , RatsSubject(s)
Doxorubicin , Macrophages , Doxorubicin/adverse effects , Animals , Macrophages/drug effects , Macrophages/metabolism , Antibiotics, Antineoplastic/adverse effects , Cardiotoxicity , Humans , Myocardium/pathology , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Myocytes, Cardiac/metabolismSubject(s)
Antibiotics, Antineoplastic , Cardiotoxicity , Doxorubicin , Myocytes, Cardiac , Humans , Doxorubicin/adverse effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Antibiotics, Antineoplastic/adverse effects , RNA Splicing/drug effects , Alternative Splicing/drug effectsSubject(s)
Action Potentials , Induced Pluripotent Stem Cells , Long QT Syndrome , Myocytes, Cardiac , Humans , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Long QT Syndrome/chemically induced , Long QT Syndrome/physiopathology , Action Potentials/drug effects , Phenotype , Heart Rate/drug effects , Pharmacogenomic VariantsSubject(s)
Machine Learning , Humans , Air Pollutants/adverse effects , Air Pollution/adverse effects , Risk Assessment , Time Factors , Particulate Matter/adverse effects , Environmental Monitoring , Environmental Exposure/adverse effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolismSubject(s)
Action Potentials , Induced Pluripotent Stem Cells , Long QT Syndrome , Myocytes, Cardiac , Humans , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Long QT Syndrome/physiopathology , Long QT Syndrome/metabolism , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/drug effects , Action Potentials/drug effects , Female , Estrogens/pharmacology , Estrogens/metabolism , Estradiol/pharmacology , PhenotypeABSTRACT
Cyclic nucleotide phosphodiesterases (PDEs) modulate neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple isoforms of PDEs with different enzymatic properties and subcellular locally regulate cyclic nucleotide levels and associated cellular functions. This organisation is severely disrupted during hypertrophy and heart failure (HF), which may contribute to disease progression. Clinically, PDE inhibition has been seen as a promising approach to compensate for the catecholamine desensitisation that accompanies heart failure. Although PDE3 inhibitors such as milrinone or enoximone can be used clinically to improve systolic function and relieve the symptoms of acute CHF, their chronic use has proved detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as potential new targets for the treatment of HF, each with a unique role in local cyclic nucleotide signalling pathways. In this review, we describe cAMP and cGMP signalling in cardiomyocytes and present the different families of PDEs expressed in the heart and their modifications in pathological cardiac hypertrophy and HF. We also review results from preclinical models and clinical data indicating the use of specific PDE inhibitors or activators that may have therapeutic potential in CI.
Title: Les phosphodiestérases des nucléotides cycliques - Cibles thérapeutiques dans l'hypertrophie et l'insuffisance cardiaques. Abstract: Les phosphodiestérases des nucléotides cycliques (PDE) modulent la régulation neuro-hormonale de la fonction cardiaque en dégradant l'AMPc et le GMPc. Dans les cardiomyocytes, de multiples isoformes de PDE, aux propriétés enzymatiques et aux localisations subcellulaires différentes, régulent localement les niveaux de nucléotides cycliques et les fonctions cellulaires associées. Cette organisation est fortement perturbée au cours de l'hypertrophie et de l'insuffisance cardiaque à fraction d'éjection réduite (IC), ce qui peut contribuer à la progression de la maladie. Sur le plan clinique, l'inhibition des PDE a été considérée comme une approche prometteuse pour compenser la désensibilisation aux catécholamines qui accompagne l'IC. Bien que des inhibiteurs de la PDE3, tels que la milrinone ou l'énoximone, puissent être utilisés cliniquement pour améliorer la fonction systolique et soulager les symptômes de l'IC aiguë, leur utilisation chronique s'est avérée préjudiciable. D'autres PDE, telles que les PDE1, PDE2, PDE4, PDE5, PDE9 et PDE10, sont apparues comme de nouvelles cibles potentielles pour le traitement de l'IC, chacune ayant un rôle unique dans les voies de signalisation locales des nucléotides cycliques. Dans cette revue, nous décrivons la signalisation de l'AMPc et du GMPc dans les cardiomyocytes et présentons les différentes familles de PDE exprimées dans le cÅur ainsi que leurs modifications dans l'hypertrophie cardiaque pathologique et dans l'IC. Nous évaluons également les résultats issus de modèles précliniques ainsi que les données cliniques indiquant l'utilisation d'inhibiteurs ou d'activateurs de PDE spécifiques qui pourraient avoir un potentiel thérapeutique dans l'IC.
Subject(s)
Cardiomegaly , Heart Failure , Phosphodiesterase Inhibitors , Humans , Cardiomegaly/drug therapy , Heart Failure/drug therapy , Animals , Phosphodiesterase Inhibitors/therapeutic use , Phosphodiesterase Inhibitors/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , 3',5'-Cyclic-AMP Phosphodiesterases/physiology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Molecular Targeted Therapy/methods , Cyclic GMP/metabolism , Cyclic GMP/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Cyclic AMP/metabolism , Cyclic AMP/physiology , Phosphoric Diester Hydrolases/metabolism , Phosphoric Diester Hydrolases/physiologyABSTRACT
Objective: The aim of this study was to explore the role and mechanism of ferroptosis in SiO 2-induced cardiac injury using a mouse model. Methods: Male C57BL/6 mice were intratracheally instilled with SiO 2 to create a silicosis model. Ferrostatin-1 (Fer-1) and deferoxamine (DFO) were used to suppress ferroptosis. Serum biomarkers, oxidative stress markers, histopathology, iron content, and the expression of ferroptosis-related proteins were assessed. Results: SiO 2 altered serum cardiac injury biomarkers, oxidative stress, iron accumulation, and ferroptosis markers in myocardial tissue. Fer-1 and DFO reduced lipid peroxidation and iron overload, and alleviated SiO 2-induced mitochondrial damage and myocardial injury. SiO 2 inhibited Nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant genes, while Fer-1 more potently reactivated Nrf2 compared to DFO. Conclusion: Iron overload-induced ferroptosis contributes to SiO 2-induced cardiac injury. Targeting ferroptosis by reducing iron accumulation or inhibiting lipid peroxidation protects against SiO 2 cardiotoxicity, potentially via modulation of the Nrf2 pathway.
Subject(s)
Disease Models, Animal , Ferroptosis , Iron Overload , Mice, Inbred C57BL , Myocytes, Cardiac , Silicon Dioxide , Silicosis , Animals , Ferroptosis/drug effects , Male , Mice , Iron Overload/metabolism , Silicon Dioxide/toxicity , Silicosis/metabolism , Silicosis/drug therapy , Silicosis/pathology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Deferoxamine/pharmacology , Phenylenediamines/pharmacology , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Oxidative Stress/drug effects , Iron/metabolism , Cyclohexylamines/pharmacologyABSTRACT
Cellular therapy is considered a better option for the treatment of degenerative disorders. Different cell types are being used for tissue regeneration. Despite extensive research in this field, several issues remain to be addressed concerning cell transplantation. One of these issues is the survival and homing of administered cells in the injured tissue, which depends on the ability of these cells to adhere. To enhance cell adherence and survival, Rap1 GTPase was activated in mesenchymal stem cells (MSCs) as well as in cardiomyocytes (CMs) by using 8-pCPT-2'-O-Me-cAMP, and the effect on gene expression dynamics was determined through quantitative reverse transcriptase-polymerase chain reaction analysis. Pharmacological activation of MSCs and CMs resulted in the upregulation of connexin-43 and cell adhesion genes, which increased the cell adhesion ability of MSCs and CMs, and increased the fusion of MSCs with neonatal CMs. Treating stem cells with a pharmacological agent that activates Rap1a before transplantation can enhance their fusion with CMs and increase cellular regeneration.
Subject(s)
Mesenchymal Stem Cells , Myocytes, Cardiac , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Animals , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/genetics , Cell Fusion , Cells, Cultured , Rats , Animals, Newborn , rap1 GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/geneticsABSTRACT
BACKGROUND: The anthracycline chemotherapeutic antibiotic doxorubicin (DOX) can induce cumulative cardiotoxicity and lead to cardiac dysfunction. Long non-coding RNAs (lncRNAs) can function as important regulators in DOX-induced myocardial injury. OBJECTIVE: This study aims to investigate the functional role and molecular mechanism of lncRNA OXCT1 antisense RNA 1 (OXCT1-AS1) in DOX-induced myocardial cell injury in vitro. METHODS: Human cardiomyocytes (AC16) were stimulated with DOX to induce a myocardial cell injury model. OXCT1-AS1, miR-874-3p, and BDH1 expression in AC16 cells were determined by RT-qPCR. AC16 cell viability was measured by XTT assay. Flow cytometry was employed to assess the apoptosis of AC16 cells. Western blotting was used to evaluate protein levels of apoptosis-related markers. Dual-luciferase reporter assay was conducted to verify the binding ability between miR-874-3p and OXCT1-AS1 and between miR-874-3p and BDH1. The value of p<0.05 indicated statistical significance. RESULTS: OXCT1-AS1 expression was decreased in DOX-treated AC16 cells. Overexpression of OXCT1-AS1 reversed the reduction of cell viability and promotion of cell apoptosis caused by DOX. OXCT1-AS1 is competitively bound to miR-874-3p to upregulate BDH1. BDH1 overexpression restored AC16 cell viability and suppressed cell apoptosis under DOX stimulation. Knocking down BDH1 reversed OXCT1-AS1-mediated attenuation of AC16 cell apoptosis under DOX treatment. CONCLUSION: LncRNA OXCT1-AS1 protects human myocardial cells AC16 from DOX-induced apoptosis via the miR-874-3p/BDH1 axis.
FUNDAMENTO: O antibiótico quimioterápico antraciclina doxorrubicina (DOX) pode induzir cardiotoxicidade cumulativa e levar à disfunção cardíaca. RNAs não codificantes longos (lncRNAs) podem funcionar como importantes reguladores na lesão miocárdica induzida por DOX. OBJETIVO: Este estudo tem como objetivo investigar o papel funcional e o mecanismo molecular do RNA antisense lncRNA OXCT1 1 (OXCT1-AS1) na lesão celular miocárdica induzida por DOX in vitro. MÉTODOS: Cardiomiócitos humanos (AC16) foram estimulados com DOX para induzir um modelo de lesão celular miocárdica. A expressão de OXCT1-AS1, miR-874-3p e BDH1 em células AC16 foi determinada por RT-qPCR. A viabilidade das células AC16 foi medida pelo ensaio XTT. A citometria de fluxo foi empregada para avaliar a apoptose de células AC16. Western blotting foi utilizado para avaliar os níveis proteicos de marcadores relacionados à apoptose. O ensaio repórter de luciferase dupla foi conduzido para verificar a capacidade de ligação entre miR-874-3p e OXCT1-AS1 e entre miR-874-3p e BDH1. O valor de p<0,05 indicou significância estatística. RESULTADOS: A expressão de OXCT1-AS1 foi diminuída em células AC16 tratadas com DOX. A superexpressão de OXCT1-AS1 reverteu a redução da viabilidade celular e a promoção da apoptose celular causada pela DOX. OXCT1-AS1 está ligado competitivamente ao miR-874-3p para regular positivamente o BDH1. A superexpressão de BDH1 restaurou a viabilidade das células AC16 e suprimiu a apoptose celular sob estimulação com DOX. A derrubada do BDH1 reverteu a atenuação da apoptose de células AC16 mediada por OXCT1-AS1 sob tratamento com DOX. CONCLUSÃO: LncRNA OXCT1-AS1 protege células miocárdicas humanas AC16 da apoptose induzida por DOX através do eixo miR-874-3p/BDH1.
Subject(s)
Apoptosis , Doxorubicin , MicroRNAs , Myocytes, Cardiac , RNA, Long Noncoding , Humans , Doxorubicin/pharmacology , RNA, Long Noncoding/genetics , Apoptosis/drug effects , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Antibiotics, Antineoplastic/pharmacology , Cell Survival/drug effects , Reproducibility of Results , Blotting, Western , Flow Cytometry , RNA, Competitive EndogenousABSTRACT
BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection. METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385. RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs. CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
Subject(s)
Antioxidants , Extracellular Vesicles , Induced Pluripotent Stem Cells , Myocytes, Cardiac , NF-E2-Related Factor 2 , Proto-Oncogene Proteins c-akt , Signal Transduction , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Extracellular Vesicles/metabolism , NF-E2-Related Factor 2/metabolism , Humans , Proto-Oncogene Proteins c-akt/metabolism , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Signal Transduction/drug effects , Antioxidants/pharmacology , Oxidative Stress/drug effects , Cell Hypoxia/drug effects , Apoptosis/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , AnimalsABSTRACT
BACKGROUND: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). METHODS AND RESULTS: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. CONCLUSIONS: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.