Mitochondrial Sequencing Identifies Long Non-Coding RNA Features that Promote Binding to PNPase.

Extranuclear localization of long non-coding 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 crosslinked 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, qPCR of PNPase CLIP knockout mutants (KH and S1) were performed. In vitro fluorescence assays assessed PNPase RNA binding capacity and verified with PNPase CLIP. 112 (mouse) and 1,548 (human) lncRNAs were identified in the mitochondrion with Malat1 being the most highly expressed. Most non-coding 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. Binding of lncRNAs to PNPase was decreased through 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 to bind to PNPase and undergo import into the mitochondrion.

Manipulation of the miR-378a/mt-ATP6 regulatory axis rescues ATP synthase in the diabetic heart and offers a novel role for lncRNA Kcnq1ot1.

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.

Machine-learning to stratify diabetic patients using novel cardiac biomarkers and integrative genomics.

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.

ROS promote epigenetic remodeling and cardiac dysfunction in offspring following maternal engineered nanomaterial (ENM) exposure.

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.

miRNA-378a as a key regulator of cardiovascular health following engineered nanomaterial inhalation exposure.

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 (∼2- to 6-fold) and fatty acid respiration (∼5-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.

Mitochondrial dysfunction in type 2 diabetes mellitus: an organ-based analysis.

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.