RESUMO
Extranuclear localization of long noncoding RNAs (lncRNAs) is poorly understood. Based on machine learning evaluations, we propose a lncRNA-mitochondrial interaction pathway where polynucleotide phosphorylase (PNPase), through domains that provide specificity for primary sequence and secondary structure, binds nuclear-encoded lncRNAs to facilitate mitochondrial import. Using FVB/NJ mouse and human cardiac tissues, RNA from isolated subcellular compartments (cytoplasmic and mitochondrial) and cross-linked immunoprecipitate (CLIP) with PNPase within the mitochondrion were sequenced on the Illumina HiSeq and MiSeq, respectively. lncRNA sequence and structure were evaluated through supervised [classification and regression trees (CART) and support vector machines (SVM)] machine learning algorithms. In HL-1 cells, quantitative PCR of PNPase CLIP knockout mutants (KH and S1) was performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. One hundred twelve (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most abundant. Most noncoding RNAs binding PNPase were lncRNAs, including Malat1. lncRNA fragments bound to PNPase compared against randomly generated sequences of similar length showed stratification with SVM and CART algorithms. The lncRNAs bound to PNPase were used to create a criterion for binding, with experimental validation revealing increased binding affinity of RNA designed to bind PNPase compared to control RNA. The binding of lncRNAs to PNPase was decreased through the knockout of RNA binding domains KH and S1. In conclusion, sequence and secondary structural features identified by machine learning enhance the likelihood of nuclear-encoded lncRNAs binding to PNPase and undergoing import into the mitochondrion.NEW & NOTEWORTHY Long noncoding RNAs (lncRNAs) are relatively novel RNAs with increasingly prominent roles in regulating genetic expression, mainly in the nucleus but more recently in regions such as the mitochondrion. This study explores how lncRNAs interact with polynucleotide phosphorylase (PNPase), a protein that regulates RNA import into the mitochondrion. Machine learning identified several RNA structural features that improved lncRNA binding to PNPase, which may be useful in targeting RNA therapeutics to the mitochondrion.
Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Humanos , Camundongos , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Ligação ProteicaRESUMO
Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.
Assuntos
Ácidos e Sais Biliares , Dieta Ocidental , Animais , Masculino , Camundongos , Acil Coenzima A/metabolismo , Ácidos e Sais Biliares/metabolismo , Ácido Quenodesoxicólico , Ácidos Graxos/metabolismo , Fígado/metabolismo , Oxirredução , Nudix HidrolasesRESUMO
Diabetes mellitus has been linked to an increase in mitochondrial microRNA-378a (miR-378a) content. Enhanced miR-378a content has been associated with a reduction in mitochondrial genome-encoded mt-ATP6 abundance, supporting the hypothesis that miR-378a inhibition may be a therapeutic option for maintaining ATP synthase functionality during diabetes mellitus. Evidence also suggests that long noncoding RNAs (lncRNAs), including lncRNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (Kcnq1ot1), participate in regulatory axes with microRNAs (miRs). Prediction analyses indicate that Kcnq1ot1 has the potential to bind miR-378a. This study aimed to determine if loss of miR-378a in a genetic mouse model could ameliorate cardiac dysfunction in type 2 diabetes mellitus (T2DM) and to ascertain whether Kcnq1ot1 interacts with miR-378a to impact ATP synthase functionality by preserving mt-ATP6 levels. MiR-378a was significantly higher in patients with T2DM and 25-wk-old Db/Db mouse mitochondria, whereas mt-ATP6 and Kcnq1ot1 levels were significantly reduced when compared with controls. Twenty-five-week-old miR-378a knockout Db/Db mice displayed preserved mt-ATP6 and ATP synthase protein content, ATP synthase activity, and preserved cardiac function, implicating miR-378a as a potential therapeutic target in T2DM. Assessments following overexpression of the 500-bp Kcnq1ot1 fragment in established mouse cardiomyocyte cell line (HL-1) cardiomyocytes overexpressing miR-378a revealed that Kcnq1ot1 may bind and significantly reduce miR-378a levels, and rescue mt-ATP6 and ATP synthase protein content. Together, these data suggest that Kcnq1ot1 and miR-378a may act as constituents in an axis that regulates mt-ATP6 content, and that manipulation of this axis may provide benefit to ATP synthase functionality in type 2 diabetic heart.
Assuntos
Diabetes Mellitus Tipo 2 , MicroRNAs , RNA Longo não Codificante , Trifosfato de Adenosina , Animais , Diabetes Mellitus Tipo 2/genética , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Air pollution is a complex mixture of particles and gases, yet current regulations are based on single toxicant levels failing to consider potential interactive outcomes of co-exposures. We examined transcriptomic changes after inhalation co-exposure to a particulate and a gaseous component of air pollution and hypothesized that co-exposure would induce significantly greater impairments to mitochondrial bioenergetics. A whole-body inhalation exposure to ultrafine carbon black (CB), and ozone (O3) was performed, and the impact of single and multiple exposures was studied at relevant deposition levels. C57BL/6 mice were exposed to CB (10 mg/m3) and/or O3 (2 ppm) for 3 h (either a single exposure or four independent exposures). RNA was isolated from lungs and mRNA sequencing performed using the Illumina HiSeq. Lung pathology was evaluated by histology and immunohistochemistry. Electron transport chain (ETC) activities, electron flow, hydrogen peroxide production, and ATP content were assessed. RESULTS: Compared to individual exposure groups, co-exposure induced significantly greater neutrophils and protein levels in broncho-alveolar lavage fluid as well as a significant increase in mRNA expression of oxidative stress and inflammation related genes. Similarly, a significant increase in hydrogen peroxide production was observed after co-exposure. After single and four exposures, co-exposure revealed a greater number of differentially expressed genes (2251 and 4072, respectively). Of these genes, 1188 (single exposure) and 2061 (four exposures) were uniquely differentially expressed, with 35 mitochondrial ETC mRNA transcripts significantly impacted after four exposures. Both O3 and co-exposure treatment significantly reduced ETC maximal activity for complexes I (- 39.3% and - 36.2%, respectively) and IV (- 55.1% and - 57.1%, respectively). Only co-exposure reduced ATP Synthase activity (- 35.7%) and total ATP content (30%). Further, the ability for ATP Synthase to function is limited by reduced electron flow (- 25%) and translation of subunits, such as ATP5F1, following co-exposure. CONCLUSIONS: CB and O3 co-exposure cause unique transcriptomic changes in the lungs that are characterized by functional deficits to mitochondrial bioenergetics. Alterations to ATP Synthase function and mitochondrial electron flow underly a pathological adaptation to lung injury induced by co-exposure.
Assuntos
Poluentes Atmosféricos , Ozônio , Poluentes Atmosféricos/toxicidade , Animais , Exposição por Inalação/efeitos adversos , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Ozônio/toxicidade , Fuligem/toxicidade , TranscriptomaRESUMO
Ambient air, occupational settings, and the use and distribution of consumer products all serve as conduits for toxicant exposure through inhalation. While the pulmonary system remains a primary target following inhalation exposure, cardiovascular implications are exceptionally culpable for increased morbidity and mortality. The epidemiological evidence for cardiovascular dysfunction resulting from acute or chronic inhalation exposure to particulate matter has been well documented, but the mechanisms driving the resulting disturbances remain elusive. In the current review, we aim to summarize the cellular and molecular mechanisms that are directly linked to cardiovascular health following exposure to a variety of inhaled toxicants. The purpose of this review is to provide a comprehensive overview of the biochemical changes in the cardiovascular system following particle inhalation exposure and to highlight potential biomarkers that exist across multiple exposure paradigms. We attempt to integrate these molecular signatures in an effort to provide direction for future investigations. This review also characterizes how molecular responses are modified in at-risk populations, specifically the impact of environmental exposure during critical windows of development. Maternal exposure to particulate matter during gestation can lead to fetal epigenetic reprogramming, resulting in long-term deficits to the cardiovascular system. In both direct and indirect (gestational) exposures, connecting the biochemical mechanisms with functional deficits outlines pathways that can be targeted for future therapeutic intervention. Ultimately, future investigations integrating "omics"-based approaches will better elucidate the mechanisms that are altered by xenobiotic inhalation exposure, identify biomarkers, and guide in clinical decision making.
Assuntos
Poluentes Atmosféricos/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Sistema Cardiovascular/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Material Particulado/efeitos adversos , Adaptação Fisiológica , Adolescente , Adulto , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/fisiopatologia , Reprogramação Celular , Epigênese Genética , Feminino , Humanos , Masculino , Exposição Materna/efeitos adversos , Camundongos , Pessoa de Meia-Idade , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Medição de Risco , Fatores de Risco , Adulto JovemRESUMO
Type 2 diabetes mellitus (T2DM) is a systemic disease characterized by hyperglycemia, hyperlipidemia, and organismic insulin resistance. This pathological shift in both circulating fuel levels and energy substrate utilization by central and peripheral tissues contributes to mitochondrial dysfunction across organ systems. The mitochondrion lies at the intersection of critical cellular pathways such as energy substrate metabolism, reactive oxygen species (ROS) generation, and apoptosis. It is the disequilibrium of these processes in T2DM that results in downstream deficits in vital functions, including hepatocyte metabolism, cardiac output, skeletal muscle contraction, ß-cell insulin production, and neuronal health. Although mitochondria are known to be susceptible to a variety of genetic and environmental insults, the accumulation of mitochondrial DNA (mtDNA) mutations and mtDNA copy number depletion is helping to explain the prevalence of mitochondrial-related diseases such as T2DM. Recent work has uncovered novel mitochondrial biology implicated in disease progressions such as mtDNA heteroplasmy, noncoding RNA (ncRNA), epigenetic modification of the mitochondrial genome, and epitranscriptomic regulation of the mtDNA-encoded mitochondrial transcriptome. The goal of this review is to highlight mitochondrial dysfunction observed throughout major organ systems in the context of T2DM and to present new ideas for future research directions based on novel experimental and technological innovations in mitochondrial biology. Finally, the field of mitochondria-targeted therapeutics is discussed, with an emphasis on novel therapeutic strategies to restore mitochondrial homeostasis in the setting of T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Tecido Adiposo/metabolismo , Encéfalo/metabolismo , DNA Mitocondrial , Endotélio Vascular/metabolismo , Epigênese Genética , Exercício Físico , Humanos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Musculares/metabolismo , Miocárdio/metabolismo , Sistema Nervoso Periférico/metabolismo , TranscriptomaRESUMO
BACKGROUND: Diabetes mellitus is a chronic disease that impacts an increasing percentage of people each year. Among its comorbidities, diabetics are two to four times more likely to develop cardiovascular diseases. While HbA1c remains the primary diagnostic for diabetics, its ability to predict long-term, health outcomes across diverse demographics, ethnic groups, and at a personalized level are limited. The purpose of this study was to provide a model for precision medicine through the implementation of machine-learning algorithms using multiple cardiac biomarkers as a means for predicting diabetes mellitus development. METHODS: Right atrial appendages from 50 patients, 30 non-diabetic and 20 type 2 diabetic, were procured from the WVU Ruby Memorial Hospital. Machine-learning was applied to physiological, biochemical, and sequencing data for each patient. Supervised learning implementing SHapley Additive exPlanations (SHAP) allowed binary (no diabetes or type 2 diabetes) and multiple classification (no diabetes, prediabetes, and type 2 diabetes) of the patient cohort with and without the inclusion of HbA1c levels. Findings were validated through Logistic Regression (LR), Linear Discriminant Analysis (LDA), Gaussian Naïve Bayes (NB), Support Vector Machine (SVM), and Classification and Regression Tree (CART) models with tenfold cross validation. RESULTS: Total nuclear methylation and hydroxymethylation were highly correlated to diabetic status, with nuclear methylation and mitochondrial electron transport chain (ETC) activities achieving superior testing accuracies in the predictive model (~ 84% testing, binary). Mitochondrial DNA SNPs found in the D-Loop region (SNP-73G, -16126C, and -16362C) were highly associated with diabetes mellitus. The CpG island of transcription factor A, mitochondrial (TFAM) revealed CpG24 (chr10:58385262, P = 0.003) and CpG29 (chr10:58385324, P = 0.001) as markers correlating with diabetic progression. When combining the most predictive factors from each set, total nuclear methylation and CpG24 methylation were the best diagnostic measures in both binary and multiple classification sets. CONCLUSIONS: Using machine-learning, we were able to identify novel as well as the most relevant biomarkers associated with type 2 diabetes mellitus by integrating physiological, biochemical, and sequencing datasets. Ultimately, this approach may be used as a guideline for future investigations into disease pathogenesis and novel biomarker discovery.
Assuntos
DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Cardiomiopatias Diabéticas/genética , Epigênese Genética , Genômica/métodos , Mitocôndrias Cardíacas/genética , Modelos Genéticos , Máquina de Vetores de Suporte , Integração de Sistemas , Ilhas de CpG , Metilação de DNA , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/etiologia , Progressão da Doença , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Hemoglobinas Glicadas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Medição de Risco , Fatores de RiscoRESUMO
BACKGROUND: Nano-titanium dioxide (nano-TiO2) is amongst the most widely utilized engineered nanomaterials (ENMs). However, little is known regarding the consequences maternal ENM inhalation exposure has on growing progeny during gestation. ENM inhalation exposure has been reported to decrease mitochondrial bioenergetics and cardiac function, though the mechanisms responsible are poorly understood. Reactive oxygen species (ROS) are increased as a result of ENM inhalation exposure, but it is unclear whether they impact fetal reprogramming. The purpose of this study was to determine whether maternal ENM inhalation exposure influences progeny cardiac development and epigenomic remodeling. RESULTS: Pregnant FVB dams were exposed to nano-TiO2 aerosols with a mass concentration of 12.09 ± 0.26 mg/m3 starting at gestational day five (GD 5), for 6 h over 6 non-consecutive days. Aerosol size distribution measurements indicated an aerodynamic count median diameter (CMD) of 156 nm with a geometric standard deviation (GSD) of 1.70. Echocardiographic imaging was used to assess cardiac function in maternal, fetal (GD 15), and young adult (11 weeks) animals. Electron transport chain (ETC) complex activities, mitochondrial size, complexity, and respiration were evaluated, along with 5-methylcytosine, Dnmt1 protein expression, and Hif1α activity. Cardiac functional analyses revealed a 43% increase in left ventricular mass and 25% decrease in cardiac output (fetal), with an 18% decrease in fractional shortening (young adult). In fetal pups, hydrogen peroxide (H2O2) levels were significantly increased (~ 10 fold) with a subsequent decrease in expression of the antioxidant enzyme, phospholipid hydroperoxide glutathione peroxidase (GPx4). ETC complex activity IV was decreased by 68 and 46% in fetal and young adult cardiac mitochondria, respectively. DNA methylation was significantly increased in fetal pups following exposure, along with increased Hif1α activity and Dnmt1 protein expression. Mitochondrial ultrastructure, including increased size, was observed at both fetal and young adult stages following maternal exposure. CONCLUSIONS: Maternal inhalation exposure to nano-TiO2 results in adverse effects on cardiac function that are associated with increased H2O2 levels and dysregulation of the Hif1α/Dnmt1 regulatory axis in fetal offspring. Our findings suggest a distinct interplay between ROS and epigenetic remodeling that leads to sustained cardiac contractile dysfunction in growing and young adult offspring following maternal ENM inhalation exposure.
Assuntos
Epigênese Genética/efeitos dos fármacos , Cardiopatias/induzido quimicamente , Exposição Materna/efeitos adversos , Nanopartículas/toxicidade , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismo , Titânio/toxicidade , Animais , Feminino , Coração Fetal/citologia , Coração Fetal/efeitos dos fármacos , Coração Fetal/metabolismo , Cardiopatias/embriologia , Cardiopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Nanopartículas/administração & dosagem , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Titânio/administração & dosagemRESUMO
>99% of the mitochondrial proteome is nuclear-encoded. The mitochondrion relies on a coordinated multi-complex process for nuclear genome-encoded mitochondrial protein import. Mitochondrial heat shock protein 70 (mtHsp70) is a key component of this process and a central constituent of the protein import motor. Type 2 diabetes mellitus (T2DM) disrupts mitochondrial proteomic signature which is associated with decreased protein import efficiency. The goal of this study was to manipulate the mitochondrial protein import process through targeted restoration of mtHsp70, in an effort to restore proteomic signature and mitochondrial function in the T2DM heart. A novel line of cardiac-specific mtHsp70 transgenic mice on the db/db background were generated and cardiac mitochondrial subpopulations were isolated with proteomic evaluation and mitochondrial function assessed. MicroRNA and epigenetic regulation of the mtHsp70 gene during T2DM were also evaluated. MtHsp70 overexpression restored cardiac function and nuclear-encoded mitochondrial protein import, contributing to a beneficial impact on proteome signature and enhanced mitochondrial function during T2DM. Further, transcriptional repression at the mtHsp70 genomic locus through increased localization of H3K27me3 during T2DM insult was observed. Our results suggest that restoration of a key protein import constituent, mtHsp70, provides therapeutic benefit through attenuation of mitochondrial and contractile dysfunction in T2DM.
Assuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas Mitocondriais/genética , Miocárdio/metabolismo , Animais , Diabetes Mellitus Tipo 2/patologia , Epigênese Genética , Humanos , Peroxidação de Lipídeos/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/genética , Miocárdio/patologia , Estresse Oxidativo/genética , Transporte Proteico/genética , Proteoma/genéticaRESUMO
This case report chronicles the diagnostic odyssey and resolution of a 27-year-old female with a complex neurodevelopmental disorder (NDD) using Whole Exome Sequencing (WES). The patient presented to a precision medicine clinic with multiple diagnoses including intellectual disability, autism spectrum disorder (ASD), obsessive-compulsive disorder (OCD), tics, seizures, and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Although this patient previously had chromosomal microarray and several single-gene tests, the underlying cause of this patient's symptoms remained elusive. WES revealed a pathogenic missense mutation in the HNRNPU gene, associated with HNRNPU-related neurodevelopmental disorder (HNRNPU-NDD) and developmental and epileptic encephalopathy-54 (DEE54, OMIM: # 617391). Following this diagnoses, other treating clinicians identified additional indications for genetic testing, however, as the WES data was readily available, the clinical team was able to re-analyze the WES data to address their inquiries without requiring additional tests. This emphasizes the pivotal role of WES in expediting diagnoses, reducing costs, and providing ongoing clinical utility throughout a patient's life. Accessible WES data in primary care settings can enhance patient care by informing future genetic inquiries, enhancing coordination of care, and facilitating precision medicine interventions, thereby mitigating the burden on families and the healthcare system.
RESUMO
Speckle tracking echocardiography (STE) has been utilized to evaluate independent spatial alterations in the diabetic heart, but the progressive manifestation of regional and segmental cardiac dysfunction in the type 2 diabetic (T2DM) heart remains understudied. Therefore, the objective of this study was to elucidate if machine learning could be utilized to reliably describe patterns of the progressive regional and segmental dysfunction that are associated with the development of cardiac contractile dysfunction in the T2DM heart. Non-invasive conventional echocardiography and STE datasets were utilized to segregate mice into two pre-determined groups, wild-type and Db/Db, at 5, 12, 20, and 25 weeks. A support vector machine model, which classifies data using a single line, or hyperplane, that best separates each class, and a ReliefF algorithm, which ranks features by how well each feature lends to the classification of data, were used to identify and rank cardiac regions, segments, and features by their ability to identify cardiac dysfunction. STE features more accurately segregated animals as diabetic or non-diabetic when compared with conventional echocardiography, and the ReliefF algorithm efficiently ranked STE features by their ability to identify cardiac dysfunction. The Septal region, and the AntSeptum segment, best identified cardiac dysfunction at 5, 20, and 25 weeks, with the AntSeptum also containing the greatest number of features which differed between diabetic and non-diabetic mice. Cardiac dysfunction manifests in a spatial and temporal fashion, and is defined by patterns of regional and segmental dysfunction in the T2DM heart which are identifiable using machine learning methodologies. Further, machine learning identified the Septal region and AntSeptum segment as locales of interest for therapeutic interventions aimed at ameliorating cardiac dysfunction in T2DM, suggesting that machine learning may provide a more thorough approach to managing contractile data with the intention of identifying experimental and therapeutic targets.
Assuntos
Diabetes Mellitus Tipo 2 , Cardiopatias , Disfunção Ventricular Esquerda , Camundongos , Animais , Diabetes Mellitus Tipo 2/complicações , Modelos Animais de Doenças , Ecocardiografia/métodos , Cardiopatias/complicaçõesRESUMO
N6-methyladenosine (m6A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it's role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO2) contributes to cardiac m6A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO2 with a target aerosol mass concentration of 12 mg/m3. At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m6A antibodies was performed followed by sequencing of immunoprecipitant (m6A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO2 exposed mice (Padj ≤ 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m6A methylation sites (Padj ≤ 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P ≤ 0.05). The lack of m6A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO2 inhalation exposure.
Assuntos
Adenosina/análogos & derivados , Genes Mitocondriais , Exposição por Inalação , Gravidez , Camundongos , Feminino , Animais , Cromatografia Líquida , Espectrometria de Massas em Tandem , Camundongos Endogâmicos , RNA , RNA MensageiroRESUMO
We used segmental strain analysis to evaluate whether intrinsic (diet-induced obesity [DIO]) and extrinsic (unpredictable chronic mild stress [UCMS]) stressors can alter deformational patterns of the left ventricle. Six-week-old male C57BL/6J mice were randomized into the lean or obese group (n = 24/group). Mice underwent 12 wk of DIO with a high-fat diet (HFD). At 18 wk, lean and obese mice were further randomized into UCMS and non-UCMS groups (UCMS, 7 h/d, 5 d/wk, for 8 wk). Echocardiography was performed at baseline (6 wk), post-HFD (18 wk) and post-UCMS (26 wk). Machine learning was applied to the DIO and UCMS groups. There was robust predictive accuracy (area under the receiver operating characteristic curve [AUC] = 0.921) when comparing obese with lean mice, with radial strain changes in the lateral (-64%, p ≤ 0.001) and anterior free (-53%, p < 0.001) walls being most informative. The ability to predict mice that underwent UCMS, irrespective of diet, was assessed (AUC = 0.886), revealing longitudinal strain rate of the anterior midwall and radial strain of the posterior septal wall as the top features. The wall segments indicate a predilection for changes in deformation patterns to the free wall (DIO) and septal wall (UCMS), indicating disease-specific alterations to the myocardium.
Assuntos
Ventrículos do Coração , Miocárdio , Animais , Dieta Hiperlipídica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , ObesidadeRESUMO
Breast cancer metastases are most commonly found in bone, an indication of poor prognosis. Pathway-based biomarkers identification may help elucidate the cellular signature of breast cancer metastasis in bone, further characterizing the etiology and promoting new therapeutic approaches. We extracted gene expression profiles from mouse macrophages from the GEO dataset, GSE152795 using the GEO2R webtool. The differentially expressed genes (DEGs) were filtered by log2 fold-change with threshold 1.5 (FDR < 0.05). STRING database and Enrichr were used for GO-term analysis, miRNA and TF analysis associated with DEGs. Autodock Vienna was exploited to investigate interaction of anti-cancer drugs, Actinomycin-D and Adriamycin. Sensitivity and specificity of DEGs was assessed using receiver operating characteristic (ROC) analyses. A total of 61 DEGs, included 27 down-regulated and 34 up-regulated, were found to be significant in breast cancer bone metastasis. Major DEGs were associated with lipid metabolism and immunological response of tumor tissue. Crucial DEGs, Bcl3, ADGRG7, FABP4, VCAN, and IRF4 were regulated by miRNAs, miR-497, miR-574, miR-138 and TFs, CCDN1, STAT6, IRF8. Docking analysis showed that these genes possessed strong binding with the drugs. ROC analysis demonstrated Bcl3 is specific to metastasis. DEGs Bcl3, ADGRG7, FABP4, IRF4, their regulating miRNAs and TFs have strong impact on proliferation and metastasis of breast cancer in bone tissues. In conclusion, present study revealed that DEGs are directly involved in of breast tumor metastasis in bone tissues. Identified genes, miRNAs, and TFs can be possible drug targets that may be used for the therapeutics. However, further experimental validation is necessary.
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Antineoplásicos , MicroRNAs , Neoplasias , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Dactinomicina/metabolismo , Doxorrubicina , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , MicroRNAs/genética , Neoplasias/genéticaRESUMO
Maternal engineered nanomaterial (ENM) exposure during gestation has been associated with negative long-term effects on cardiovascular health in progeny. Here, we evaluate an epitranscriptomic mechanism that contributes to these chronic ramifications and whether overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) can preserve cardiovascular function and bioenergetics in offspring following gestational nano-titanium dioxide (TiO2) inhalation exposure. Wild-type (WT) and mPHGPx (Tg) dams were exposed to nano-TiO2 aerosols with a mass concentration of 12.01 ± 0.50 mg/m3 starting from gestational day (GD) 5 for 360 mins/day for 6 nonconsecutive days over 8 days. Echocardiography was performed in pregnant dams, adult (11-week old) and fetal (GD 14) progeny. Mitochondrial function and global N6-methyladenosine (m6A) content were assessed in adult progeny. MPHGPx enzymatic function was further evaluated in adult progeny and m6A-RNA immunoprecipitation (RIP) was combined with RT-qPCR to evaluate m6A content in the 3'-UTR. Following gestational ENM exposure, global longitudinal strain (GLS) was 32% lower in WT adult offspring of WT dams, with preservation in WT offspring of Tg dams. MPHGPx activity was significantly reduced in WT offspring (29%) of WT ENM-exposed dams, but preserved in the progeny of Tg dams. M6A-RIP-qPCR for the SEC insertion sequence region of mPHGPx revealed hypermethylation in WT offspring from ENM-exposed WT dams, which was thwarted in the presence of the maternal transgene. Our findings implicate that m6A hypermethylation of mPHGPx may be culpable for diminished antioxidant capacity and resultant mitochondrial and cardiac deficits that persist into adulthood following gestational ENM inhalation exposure.
Assuntos
Nanoestruturas , Efeitos Tardios da Exposição Pré-Natal , Adulto , Antioxidantes , Feminino , Feto , Coração , Humanos , Exposição Materna , GravidezRESUMO
Nano-titanium dioxide (nano-TiO2), though one of the most utilized and produced engineered nanomaterials (ENMs), diminishes cardiovascular function through dysregulation of metabolism and mitochondrial bioenergetics following inhalation exposure. The molecular mechanisms governing this cardiac dysfunction remain largely unknown. The purpose of this study was to elucidate molecular mediators that connect nano-TiO2 exposure with impaired cardiac function. Specifically, we were interested in the role of microRNA (miRNA) expression in the resulting dysfunction. Not only are miRNA global regulators of gene expression, but also miRNA-based therapeutics provide a realistic treatment modality. Wild type and MiRNA-378a knockout mice were exposed to nano-TiO2 with an aerodynamic diameter of 182 ± 1.70 nm and a mass concentration of 11.09 mg/m3 for 4 h. Cardiac function, utilizing the Vevo 2100 Imaging System, electron transport chain complex activities, and mitochondrial respiration assessed cardiac and mitochondrial function. Immunoblotting and qPCR examined molecular targets of miRNA-378a. MiRNA-378a-3p expression was increased 48 h post inhalation exposure to nano-TiO2. Knockout of miRNA-378a preserved cardiac function following exposure as revealed by preserved E/A ratio and E/SR ratio. In knockout animals, complex I, III, and IV activities (â¼2- to 6-fold) and fatty acid respiration (â¼5-fold) were significantly increased. MiRNA-378a regulated proteins involved in mitochondrial fusion, transcription, and fatty acid metabolism. MiRNA-378a-3p acts as a negative regulator of mitochondrial metabolic and biogenesis pathways. MiRNA-378a knockout animals provide a protective effect against nano-TiO2 inhalation exposure by altering mitochondrial structure and function. This is the first study to manipulate a miRNA to attenuate the effects of ENM exposure.