Publications by authors named "John M Hollander"

53 Publications

Mitochondrial membranes modify mutant huntingtin aggregation.

Biochim Biophys Acta Biomembr 2021 Jun 2;1863(10):183663. Epub 2021 Jun 2.

The C. Eugene Bennett Department of Chemistry, West Virginia University, 217 Clark Hall, Morgantown, WV 26506, United States; Rockefeller Neurosciences Institutes, West Virginia University, 1 Medical Center Dr., P.O. Box 9303, Morgantown, WV 26505, United States; Department of Neuroscience, West Virginia University, 1 Medical Center Dr., P.O. Box 9303, Morgantown, WV 26505, United States. Electronic address:

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract near the N-terminus of the huntingtin (htt) protein. Expanded polyQ tracts are prone to aggregate into oligomers and insoluble fibrils. Mutant htt (mhtt) localizes to variety of organelles, including mitochondria. Specifically, mitochondrial defects, morphological alteration, and dysfunction are observed in HD. Mitochondrial lipids, cardiolipin (CL) in particular, are essential in mitochondria function and have the potential to directly interact with htt, altering its aggregation. Here, the impact of mitochondrial membranes on htt aggregation was investigated using a combination of mitochondrial membrane mimics and tissue-derived mitochondrial-enriched fractions. The impact of exposure of outer and inner mitochondrial membrane mimics (OMM and IMM respectively) to mhtt was explored. OMM and IMM reduced mhtt fibrillization, with IMM having a larger effect. The role of CL in mhtt aggregation was investigated using a simple PC system with varying molar ratios of CL. Lower molar ratios of CL (<5%) promoted fibrillization; however, increased CL content retarded fibrillization. As revealed by in situ AFM, mhtt aggregation and associated membrane morphological changes at the surface of OMM mimics was markedly different compared to IMM mimics. While globular deposits of mhtt with few fibrillar aggregates were observed on OMM, plateau-like domains were observed on IMM. A similar impact on htt aggregation was observed with exposure to purified mitochondrial-enriched fractions. Collectively, these observations suggest mitochondrial membranes heavily influence htt aggregation with implication for HD.
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http://dx.doi.org/10.1016/j.bbamem.2021.183663DOI Listing
June 2021

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

Pharm Res 2021 May 12;38(5):803-817. Epub 2021 May 12.

Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, 1 Medical Center Drive, Morgantown, West Virginia, 26506, USA.

Purpose: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics.

Method: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke.

Results: NL-1 decreased hydrogen peroxide production with an IC of 5.95 μM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%.

Conclusion: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.
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http://dx.doi.org/10.1007/s11095-021-03046-4DOI Listing
May 2021

Enhanced antioxidant capacity prevents epitranscriptomic and cardiac alterations in adult offspring gestationally-exposed to ENM.

Nanotoxicology 2021 May 8:1-20. Epub 2021 May 8.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, WV, USA.

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 (TiO) inhalation exposure. Wild-type (WT) and mPHGPx (Tg) dams were exposed to nano-TiO aerosols with a mass concentration of 12.01 ± 0.50 mg/m 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 N-methyladenosine (mA) content were assessed in adult progeny. MPHGPx enzymatic function was further evaluated in adult progeny and mA-RNA immunoprecipitation (RIP) was combined with RT-qPCR to evaluate mA 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. MA-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 mA 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.
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http://dx.doi.org/10.1080/17435390.2021.1921299DOI Listing
May 2021

Cardiovascular adaptations to particle inhalation exposure: molecular mechanisms of the toxicology.

Am J Physiol Heart Circ Physiol 2020 08 19;319(2):H282-H305. Epub 2020 Jun 19.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia.

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.
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http://dx.doi.org/10.1152/ajpheart.00026.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473925PMC
August 2020

MiR-34a Interacts with Cytochrome c and Shapes Stroke Outcomes.

Sci Rep 2020 02 24;10(1):3233. Epub 2020 Feb 24.

Neuroscience, Center for Basic and Translational Stroke Research; West Virginia University, Morgantown, West Virginia, 26506, USA.

Blood-brain barrier (BBB) dysfunction occurs in cerebrovascular diseases and neurodegenerative disorders such as stroke. Opening of the BBB during a stroke has a negative impact on acute outcomes. We have recently demonstrated that miR-34a regulates the BBB by targeting cytochrome c (CYC) in vitro. To investigate the role of miR-34a in a stroke, we purified primary cerebrovascular endothelial cells (pCECs) from mouse brains following 1 h transient middle cerebral artery occlusion (tMCAO) and measured real-time PCR to detect miR-34a levels. We demonstrate that the miR-34a levels are elevated in pCECs from tMCAO mice at the time point of BBB opening following 1 h tMCAO and reperfusion. Interestingly, knockout of miR-34a significantly reduces BBB permeability, alleviates disruption of tight junctions, and improves stroke outcomes compared to wild-type (WT) controls. CYC is decreased in the ischemic hemispheres and pCECs from WT but not in miR-34a mice following stroke reperfusion. We further confirmed CYC is a target of miR-34a by a dural luciferase reporter gene assay in vitro. Our study provides the first description of miR-34a affecting stroke outcomes and may lead to discovery of new mechanisms and treatments for cerebrovascular and neurodegenerative diseases such as stroke.
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http://dx.doi.org/10.1038/s41598-020-59997-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040038PMC
February 2020

Pyrvinium Pamoate Use in a B cell Acute Lymphoblastic Leukemia Model of the Bone Tumor Microenvironment.

Pharm Res 2020 Jan 27;37(3):43. Epub 2020 Jan 27.

Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine, Morgantown, West Virginia, USA.

Purpose: Pyrvinium pamoate (PP) is an anthelmintic drug that has been found to have anti-cancer activity in several cancer types. In the present study, we evaluated PP for potential anti-leukemic activity in B cell acute lymphoblastic leukemia (ALL) cell lines, in an effort to evaluate the repurposing potential of this drug in leukemia.

Methods: ALL cells were treated with PP at various concentrations to determine its effect on cell proliferation. Metabolic function was tested by evaluating Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR). Lastly, 3D spheroids were grown, and PP was reformulated into nanoparticles to evaluate distribution effectiveness.

Results: PP was found to inhibit ALL proliferation, with varied selectivity to different ALL cell subtypes. We also found that PP's cell death activity was specific for leukemic cells, as primary normal immune cells were resistant to PP-mediated cell death. Metabolic studies indicated that PP, in part, inhibits mitochondrial oxidative phosphorylation. To increase the targeting of PP to a hypoxic bone tumor microenvironment (BTME) niche, we successfully encapsulated PP in a nanoparticle drug delivery system and demonstrated that it retained its anti-leukemic activity in a hemosphere assay.

Conclusion: We have demonstrated that PP is a novel therapeutic lead compound that counteracts the respiratory reprogramming found in refractory ALL cells and can be effectively formulated into a nanoparticle delivery system to target the BTME.
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http://dx.doi.org/10.1007/s11095-020-2767-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021357PMC
January 2020

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

Part Fibre Toxicol 2019 06 18;16(1):24. Epub 2019 Jun 18.

Division of Exercise Physiology, West Virginia University School of Medicine, PO Box 9227, 1 Medical Center Drive, Morgantown, WV, 26506, USA.

Background: Nano-titanium dioxide (nano-TiO) 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-TiO aerosols with a mass concentration of 12.09 ± 0.26 mg/m 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 (HO) 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-TiO results in adverse effects on cardiac function that are associated with increased HO 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.
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http://dx.doi.org/10.1186/s12989-019-0310-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582485PMC
June 2019

The role of SIRT1 in skeletal muscle function and repair of older mice.

J Cachexia Sarcopenia Muscle 2019 08 14;10(4):929-949. Epub 2019 Jun 14.

Laboratory of Muscle Biology and Sarcopenia, West Virginia University School of Medicine, Morgantown, USA.

Background: Sirtuin 1 (SIRT1) is a NAD+ sensitive deacetylase that has been linked to longevity and has been suggested to confer beneficial effects that counter aging-associated deterioration. Muscle repair is dependent upon satellite cell function, which is reported to be reduced with aging; however, it is not known if this is linked to an aging-suppression of SIRT1. This study tested the hypothesis that Sirtuin 1 (SIRT1) overexpression would increase the extent of muscle repair and muscle function in older mice.

Methods: We examined satellite cell dependent repair in tibialis anterior, gastrocnemius, and soleus muscles of 13 young wild-type mice (20-30 weeks) and 49 older (80+ weeks) mice that were controls (n = 13), overexpressed SIRT1 in skeletal muscle (n = 14), and had a skeletal muscle SIRT1 knockout (n = 12) or a satellite cell SIRT1 knockout (n = 10). Acute muscle injury was induced by injection of cardiotoxin (CTX), and phosphate-buffered saline was used as a vector control. Plantarflexor muscle force and fatigue were evaluated before or 21 days after CTX injection. Satellite cell proliferation and mitochondrial function were also evaluated in undamaged muscles.

Results: Maximal muscle force was significantly lower in control muscles of older satellite cell knockout SIRT1 mice compared to young adult wild-type (YWT) mice (P < 0.001). Mean contraction force at 40 Hz stimulation was significantly greater after recovery from CTX injury in older mice that overexpressed muscle SIRT1 than age-matched SIRT1 knockout mice (P < 0.05). SIRT1 muscle knockout models (P < 0.05) had greater levels of p53 (P < 0.05 MKO, P < 0.001 OE) in CTX-damaged tissues as compared to YWT CTX mice. SIRT1 overexpression with co-expression of p53 was associated with increased fatigue resistance and increased force potentiation during repeated contractions as compared to wild-type or SIRT1 knockout models (P < 0.001). Muscle structure and mitochondrial function were not different between the groups, but proliferation of satellite cells was significantly greater in older mice with SIRT1 muscle knockout (P < 0.05), but not older SIRT1 satellite cell knockout models, in vitro, although this effect was attenuated in vivo after 21 days of recovery.

Conclusions: The data suggest skeletal muscle structure, function, and recovery after CTX-induced injury are not significantly influenced by gain or loss of SIRT1 abundance alone in skeletal muscle; however, muscle function is impaired by ablation of SIRT1 in satellite cells. SIRT1 appears to interact with p53 to improve muscle fatigue resistance after repair from muscle injury.
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http://dx.doi.org/10.1002/jcsm.12437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711423PMC
August 2019

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

Cardiovasc Diabetol 2019 06 11;18(1):78. Epub 2019 Jun 11.

Division of Exercise Physiology, West Virginia University School of Medicine, PO Box 9227, 1 Medical Center Drive, Morgantown, WV, 26505, USA.

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.
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http://dx.doi.org/10.1186/s12933-019-0879-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560734PMC
June 2019

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

Nanotoxicology 2019 06 1;13(5):644-663. Epub 2019 Feb 1.

a Division of Exercise Physiology , West Virginia University School of Medicine , Morgantown , WV , USA.

Nano-titanium dioxide (nano-TiO), 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-TiO 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-TiO with an aerodynamic diameter of 182 ± 1.70 nm and a mass concentration of 11.09 mg/m 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-TiO. 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-TiO inhalation exposure by altering mitochondrial structure and function. This is the first study to manipulate a miRNA to attenuate the effects of ENM exposure.
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http://dx.doi.org/10.1080/17435390.2019.1570372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629495PMC
June 2019

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

Am J Physiol Endocrinol Metab 2019 02 2;316(2):E268-E285. Epub 2019 Jan 2.

Division of Exercise Physiology, West Virginia University School of Medicine , Morgantown, West Virginia.

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, β-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.
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http://dx.doi.org/10.1152/ajpendo.00314.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397358PMC
February 2019

Crystal structure of the mitochondrial protein mitoNEET bound to a benze-sulfonide ligand.

Commun Chem 2019 3;2. Epub 2019 Jul 3.

Department of Biochemistry, School of Medicine West Virginia University, Morgantown, WV 26506, USA.

MitoNEET (gene ) is a mitochondrial outer membrane [2Fe-2S] protein and is a potential drug target in several metabolic diseases. Previous studies have demonstrated that mitoNEET functions as a redox-active and pH-sensing protein that regulates mitochondrial metabolism, although the structural basis of the potential drug binding site(s) remains elusive. Here we report the crystal structure of the soluble domain of human mitoNEET with a sulfonamide ligand, furosemide. Exploration of the high-resolution crystal structure is used to design mitoNEET binding molecules in a pilot study of molecular probes for use in future development of mitochondrial targeted therapies for a wide variety of metabolic diseases, including obesity, diabetes and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
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http://dx.doi.org/10.1038/s42004-019-0172-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205193PMC
July 2019

Mitochondrial proteome disruption in the diabetic heart through targeted epigenetic regulation at the mitochondrial heat shock protein 70 (mtHsp70) nuclear locus.

J Mol Cell Cardiol 2018 06 4;119:104-115. Epub 2018 May 4.

Division of Exercise Physiology, Mitochondrial, Metabolism and Bioenergetics Working Group, West Virginia University School of Medicine, Morgantown, WV 26505, United States. Electronic address:

>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.
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http://dx.doi.org/10.1016/j.yjmcc.2018.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987221PMC
June 2018

Intermediary metabolism and fatty acid oxidation: novel targets of electron transport chain-driven injury during ischemia and reperfusion.

Am J Physiol Heart Circ Physiol 2018 04 29;314(4):H787-H795. Epub 2017 Dec 29.

Division of Cardiology, Department of Medicine, Virginia Commonwealth University , Richmond, Virginia.

Cardiac ischemia-reperfusion (I/R) damages the electron transport chain (ETC), causing mitochondrial and cardiomyocyte injury. Reversible blockade of the ETC at complex I during ischemia protects the ETC and decreases cardiac injury. In the present study, we used an unbiased proteomic approach to analyze the extent of ETC-driven mitochondrial injury during I/R. Isolated-perfused mouse (C57BL/6) hearts underwent 25-min global ischemia (37°C) and 30-min reperfusion. In treated hearts, amobarbital (2 mM) was given for 1 min before ischemia to rapidly and reversibly block the ETC at complex I. Mitochondria were isolated at the end of reperfusion and subjected to unbiased proteomic analysis using tryptic digestion followed by liquid chromatography-mass spectrometry with isotope tags for relative and absolute quantification. Amobarbital treatment decreased cardiac injury and protected respiration. I/R decreased the content ( P < 0.05) of multiple mitochondrial matrix enzymes involved in intermediary metabolism compared with the time control. The contents of several enzymes in fatty acid oxidation were decreased compared with the time control. Blockade of ETC during ischemia largely prevented the decreases. Thus, after I/R, not only the ETC but also multiple pathways of intermediary metabolism sustain damage initiated by the ETC. If these damaged mitochondria persist in the myocyte, they remain a potent stimulus for ongoing injury and the transition to cardiomyopathy during prolonged reperfusion. Modulation of ETC function during early reperfusion is a key strategy to preserve mitochondrial metabolism and to decrease persistent mitochondria-driven injury during longer periods of reperfusion that predispose to ventricular dysfunction and heart failure. NEW & NOTEWORTHY Ischemia-reperfusion (I/R) damages mitochondria, which could be protected by reversible blockade of the electron transport chain (ETC). Unbiased proteomics with isotope tags for relative and absolute quantification analyzed mitochondrial damage during I/R and found that multiple enzymes in the tricarboxylic acid cycle, fatty acid oxidation, and ETC decreased, which could be prevented by ETC blockade. Strategic ETC modulation can reduce mitochondrial damage and cardiac injury.
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http://dx.doi.org/10.1152/ajpheart.00531.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966772PMC
April 2018

Reactive oxygen species damage drives cardiac and mitochondrial dysfunction following acute nano-titanium dioxide inhalation exposure.

Nanotoxicology 2018 02 15;12(1):32-48. Epub 2017 Dec 15.

a Division of Exercise Physiology , West Virginia University School of Medicine , Morgantown , WV , USA.

Nanotechnology offers innovation in products from cosmetics to drug delivery, leading to increased engineered nanomaterial (ENM) exposure. Unfortunately, health impacts of ENM are not fully realized. Titanium dioxide (TiO) is among the most widely produced ENM due to its use in numerous applications. Extrapulmonary effects following pulmonary exposure have been identified and may involve reactive oxygen species (ROS). The goal of this study was to determine the extent of ROS involvement on cardiac function and the mitochondrion following nano-TiO exposure. To address this question, we utilized a transgenic mouse model with overexpression of a novel mitochondrially-targeted antioxidant enzyme (phospholipid hydroperoxide glutathione peroxidase; mPHGPx) which provides protection against oxidative stress to lipid membranes. MPHGPx mice and littermate controls were exposed to nano-TiO aerosols (Evonik, P25) to provide a calculated pulmonary deposition of 11 µg/mouse. Twenty-four hours following exposure, we observed diastolic dysfunction as evidenced by E/A ratios greater than 2 and increased radial strain during diastole in wild-type mice (p < 0.05 for both), indicative of restrictive filling. Overexpression of mPHGPx mitigated the contractile deficits resulting from nano-TiO exposure. To investigate the cellular mechanisms associated with the observed cardiac dysfunction, we focused our attention on the mitochondrion. We observed a significant increase in ROS production (p < 0.05) and decreased mitochondrial respiratory function (p < 0.05) following nano-TiO exposure which were attenuated in mPHGPx transgenic mice. In summary, nano-TiO inhalation exposure is associated with cardiac diastolic dysfunction and mitochondrial functional alterations, which can be mitigated by the overexpression of mPHGPx, suggesting ROS contribution in the development of contractile and bioenergetic dysfunction.
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http://dx.doi.org/10.1080/17435390.2017.1416202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777890PMC
February 2018

Regulating microRNA expression: at the heart of diabetes mellitus and the mitochondrion.

Am J Physiol Heart Circ Physiol 2018 02 6;314(2):H293-H310. Epub 2017 Oct 6.

Division of Exercise Physiology, West Virginia University School of Medicine , Morgantown, West Virginia.

Type 2 diabetes mellitus is a major risk factor for cardiovascular disease and mortality. Uncontrolled type 2 diabetes mellitus results in a systemic milieu of increased circulating glucose and fatty acids. The development of insulin resistance in cardiac tissue decreases cellular glucose import and enhances mitochondrial fatty acid uptake. While triacylglycerol and cytotoxic lipid species begin to accumulate in the cardiomyocyte, the energy substrate utilization ratio of free fatty acids to glucose changes to almost entirely free fatty acids. Accumulating evidence suggests a role of miRNA in mediating this metabolic transition. Energy substrate metabolism, apoptosis, and the production and response to excess reactive oxygen species are regulated by miRNA expression. The current momentum for understanding the dynamics of miRNA expression is limited by a lack of understanding of how miRNA expression is controlled. While miRNAs are important regulators in both normal and pathological states, an additional layer of complexity is added when regulation of miRNA regulators is considered. miRNA expression is known to be regulated through a number of mechanisms, which include, but are not limited to, epigenetics, exosomal transport, processing, and posttranscriptional sequestration. The purpose of this review is to outline how mitochondrial processes are regulated by miRNAs in the diabetic heart. Furthermore, we will highlight the regulatory mechanisms, such as epigenetics, exosomal transport, miRNA processing, and posttranslational sequestration, that participate as regulators of miRNA expression. Additionally, current and future treatment strategies targeting dysfunctional mitochondrial processes in the diseased myocardium, as well as emerging miRNA-based therapies, will be summarized.
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http://dx.doi.org/10.1152/ajpheart.00520.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867655PMC
February 2018

Exploring the mitochondrial microRNA import pathway through Polynucleotide Phosphorylase (PNPase).

J Mol Cell Cardiol 2017 09 11;110:15-25. Epub 2017 Jul 11.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, WV 26506, United States; Mitochondria, Metabolism & Bioenergetics Working Group, West Virginia University School of Medicine, Morgantown, WV 26506, United States. Electronic address:

Cardiovascular disease is the primary cause of mortality for individuals with type 2 diabetes mellitus. During the diabetic condition, cardiovascular dysfunction can be partially attributed to molecular changes in the tissue, including alterations in microRNA (miRNA) interactions. MiRNAs have been reported in the mitochondrion and their presence may influence cellular bioenergetics, creating decrements in functional capacity. In this study, we examined the roles of Argonaute 2 (Ago2), a protein associated with cytosolic and mitochondrial miRNAs, and Polynucleotide Phosphorylase (PNPase), a protein found in the inner membrane space of the mitochondrion, to determine their role in mitochondrial miRNA import. In cardiac tissue from human and mouse models of type 2 diabetes mellitus, Ago2 protein levels were unchanged while PNPase protein expression levels were increased; also, there was an increase in the association between both proteins in the diabetic state. MiRNA-378 was found to be significantly increased in db/db mice, leading to decrements in ATP6 levels and ATP synthase activity, which was also exhibited when overexpressing PNPase in HL-1 cardiomyocytes and in HL-1 cells with stable miRNA-378 overexpression (HL-1-378). To assess potential therapeutic interventions, flow cytometry evaluated the capacity for targeting miRNA-378 species in mitochondria through antimiR treatment, revealing miRNA-378 level-dependent inhibition. Our study establishes PNPase as a contributor to mitochondrial miRNA import through the transport of miRNA-378, which may regulate bioenergetics during type 2 diabetes mellitus. Further, our data provide evidence that manipulation of PNPase levels may enhance the delivery of antimiR therapeutics to mitochondria in physiological and pathological conditions.
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http://dx.doi.org/10.1016/j.yjmcc.2017.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854179PMC
September 2017

Mitochondrial miRNAs in diabetes: just the tip of the iceberg.

Can J Physiol Pharmacol 2017 Oct 3;95(10):1156-1162. Epub 2017 May 3.

a Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA.

Over the last 2 decades, mi(cro)RNAs have emerged as one of the key regulators of metabolic homeostasis. Most of the studies have highlighted that, in the cytoplasm, miRNAs directly bind to the 3'-UTR (untranslated region) of a mRNA. Conventional RNA-induced silencing complex (RISC) formation results in the post-transcriptional inhibition. This process is known to contribute to the development of metabolic diseases, including diabetes mellitus. Recent advancements with small RNA detection technologies have enabled us to identify miRNAs in the mitochondrial compartment of the cells. We have termed these miRNAs, which translocate into the mitochondria as mitochondrial miRNA, MitomiR. It has been demonstrated that MitomiRs can regulate gene expression, with some evidence even suggesting that, after translocation, MitomiRs can bind to the 3'-end of a mitochondrial gene, altering its regulation. Our main focus in this review is to highlight the potential role of MitomiR in the pathogenesis of metabolic disorders such as diabetes mellitus.
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http://dx.doi.org/10.1139/cjpp-2016-0580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854153PMC
October 2017

Excess coenzyme A reduces skeletal muscle performance and strength in mice overexpressing human PANK2.

Mol Genet Metab 2017 04 3;120(4):350-362. Epub 2017 Feb 3.

Department of Biochemistry, West Virginia University, Morgantown, WV 26506, USA. Electronic address:

Coenzyme A (CoA) is a cofactor that is central to energy metabolism and CoA synthesis is controlled by the enzyme pantothenate kinase (PanK). A transgenic mouse strain expressing human PANK2 was derived to determine the physiological impact of PANK overexpression and elevated CoA levels. The Tg(PANK2) mice expressed high levels of the transgene in skeletal muscle and heart; however, CoA was substantially elevated only in skeletal muscle, possibly associated with the comparatively low endogenous levels of acetyl-CoA, a potent feedback inhibitor of PANK2. Tg(PANK2) mice were smaller, had less skeletal muscle mass and displayed significantly impaired exercise tolerance and grip strength. Skeletal myofibers were characterized by centralized nuclei and aberrant mitochondria. Both the content of fully assembled complex I of the electron transport chain and ATP levels were reduced, while markers of oxidative stress were elevated in Tg(PANK2) skeletal muscle. These abnormalities were not detected in the Tg(PANK2) heart muscle, with the exception of spotty loss of cristae organization in the mitochondria. The data demonstrate that excessively high CoA may be detrimental to skeletal muscle function.
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http://dx.doi.org/10.1016/j.ymgme.2017.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382100PMC
April 2017

Maternal-engineered nanomaterial exposure disrupts progeny cardiac function and bioenergetics.

Am J Physiol Heart Circ Physiol 2017 Mar 23;312(3):H446-H458. Epub 2016 Dec 23.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia;

Nanomaterial production is expanding as new industrial and consumer applications are introduced. Nevertheless, the impacts of exposure to these compounds are not fully realized. The present study was designed to determine whether gestational nano-sized titanium dioxide exposure impacts cardiac and metabolic function of developing progeny. Pregnant Sprague-Dawley rats were exposed to nano-aerosols (~10 mg/m, 130- to 150-nm count median aerodynamic diameter) for 7-8 nonconsecutive days, beginning at gestational Physiological and bioenergetic effects on heart function and cardiomyocytes across three time points, fetal (gestational ), neonatal (4-10 days), and young adult (6-12 wk), were evaluated. Functional analysis utilizing echocardiography, speckle-tracking based strain, and cardiomyocyte contractility, coupled with mitochondrial energetics, revealed effects of nano-exposure. Maternal exposed progeny demonstrated a decrease in E- and A-wave velocities, with a 15% higher E-to-A ratio than controls. Myocytes isolated from exposed animals exhibited ~30% decrease in total contractility, departure velocity, and area of contraction. Bioenergetic analysis revealed a significant increase in proton leak across all ages, accompanied by decreases in metabolic function, including basal respiration, maximal respiration, and spare capacity. Finally, electron transport chain complex I and IV activities were negatively impacted in the exposed group, which may be linked to a metabolic shift. Molecular data suggest that an increase in fatty acid metabolism, uncoupling, and cellular stress proteins may be associated with functional deficits of the heart. In conclusion, gestational nano-exposure significantly impairs the functional capabilities of the heart through cardiomyocyte impairment, which is associated with mitochondrial dysfunction. Cardiac function is evaluated, for the first time, in progeny following maternal nanomaterial inhalation. The findings indicate that exposure to nano-sized titanium dioxide (nano-TiO) during gestation negatively impacts cardiac function and mitochondrial respiration and bioenergetics. We conclude that maternal nano-TiO inhalation contributes to adverse cardiovascular health effects, lasting into adulthood.
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http://dx.doi.org/10.1152/ajpheart.00634.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402018PMC
March 2017

Role of microRNA in metabolic shift during heart failure.

Am J Physiol Heart Circ Physiol 2017 Jan 14;312(1):H33-H45. Epub 2016 Oct 14.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia; and Mitochondria, Metabolism, and Bioenergentics Working Group, Morgantown, West Virginia

Heart failure (HF) is an end point resulting from a number of disease states. The prognosis for HF patients is poor with survival rates precipitously low. Energy metabolism is centrally linked to the development of HF, and it involves the proteomic remodeling of numerous pathways, many of which are targeted to the mitochondrion. microRNAs (miRNA) are noncoding RNAs that influence posttranscriptional gene regulation. miRNA have garnered considerable attention for their ability to orchestrate changes to the transcriptome, and ultimately the proteome, during HF. Recently, interest in the role played by miRNA in the regulation of energy metabolism at the mitochondrion has emerged. Cardiac proteome remodeling during HF includes axes impacting hypertrophy, oxidative stress, calcium homeostasis, and metabolic fuel transition. Although it is established that the pathological environment of hypoxia and hemodynamic stress significantly contribute to the HF phenotype, it remains unclear as to the mechanistic underpinnings driving proteome remodeling. The aim of this review is to present evidence highlighting the role played by miRNA in these processes as a means for linking pathological stimuli with proteomic alteration. The differential expression of proteins of substrate transport, glycolysis, β-oxidation, ketone metabolism, the citric acid cycle (CAC), and the electron transport chain (ETC) are paralleled by the differential expression of miRNA species that modulate these processes. Identification of miRNAs that translocate to cardiomyocyte mitochondria (miR-181c, miR-378) influencing the expression of the mitochondrial genome-encoded transcripts as well as suggested import modulators are discussed. Current insights, applications, and challenges of miRNA-based therapeutics are also described.
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http://dx.doi.org/10.1152/ajpheart.00341.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283914PMC
January 2017

Early detection of cardiac dysfunction in the type 1 diabetic heart using speckle-tracking based strain imaging.

J Mol Cell Cardiol 2016 Jan 3;90:74-83. Epub 2015 Dec 3.

Department of Exercise Physiology, Center for Cardiovascular and Respiratory Sciences, School of Medicine, West Virginia University, Morgantown, WV, 26505, United States. Electronic address:

Enhanced sensitivity in echocardiographic analyses may allow for early detection of changes in cardiac function beyond the detection limits of conventional echocardiographic analyses, particularly in a small animal model. The goal of this study was to compare conventional echocardiographic measurements and speckle-tracking based strain imaging analyses in a small animal model of type 1 diabetes mellitus. Conventional analyses revealed differences in ejection fraction, fractional shortening, cardiac output, and stroke volume in diabetic animals relative to controls at 6-weeks post-diabetic onset. In contrast, when assessing short- and long-axis speckle-tracking based strain analyses, diabetic mice showed changes in average systolic radial strain, radial strain rate, radial displacement, and radial velocity, as well as decreased circumferential and longitudinal strain rate, as early as 1-week post-diabetic onset and persisting throughout the diabetic study. Further, we performed regional analyses for the LV and found that the free wall region was affected in both the short- and long-axis when assessing radial dimension parameters. These changes began 1-week post-diabetic onset and remained throughout the progression of the disease. These findings demonstrate the use of speckle-tracking based strain as an approach to elucidate cardiac dysfunction from a global perspective, identifying left ventricular cardiac regions affected during the progression of type 1 diabetes mellitus earlier than contractile changes detected by conventional echocardiographic measurements.
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http://dx.doi.org/10.1016/j.yjmcc.2015.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725063PMC
January 2016

Cardiac and mitochondrial dysfunction following acute pulmonary exposure to mountaintop removal mining particulate matter.

Am J Physiol Heart Circ Physiol 2015 Dec 23;309(12):H2017-30. Epub 2015 Oct 23.

West Virginia University School of Medicine, Division of Exercise Physiology, Morgantown, West Virginia; Center for Cardiovascular and Respiratory Sciences, Morgantown, West Virginia;

Throughout the United States, air pollution correlates with adverse health outcomes, and cardiovascular disease incidence is commonly increased following environmental exposure. In areas surrounding active mountaintop removal mines (MTM), a further increase in cardiovascular morbidity is observed and may be attributed in part to particulate matter (PM) released from the mine. The mitochondrion has been shown to be central in the etiology of many cardiovascular diseases, yet its roles in PM-related cardiovascular effects are not realized. In this study, we sought to elucidate the cardiac processes that are disrupted following exposure to mountaintop removal mining particulate matter (PM MTM). To address this question, we exposed male Sprague-Dawley rats to PM MTM, collected within one mile of an active MTM site, using intratracheal instillation. Twenty-four hours following exposure, we evaluated cardiac function, apoptotic indices, and mitochondrial function. PM MTM exposure elicited a significant decrease in ejection fraction and fractional shortening compared with controls. Investigation into the cellular impacts of PM MTM exposure identified a significant increase in mitochondrial-induced apoptotic signaling, as reflected by an increase in TUNEL-positive nuclei and increased caspase-3 and -9 activities. Finally, a significant increase in mitochondrial transition pore opening leading to decreased mitochondrial function was identified following exposure. In conclusion, our data suggest that pulmonary exposure to PM MTM increases cardiac mitochondrial-associated apoptotic signaling and decreases mitochondrial function concomitant with decreased cardiac function. These results suggest that increased cardiovascular disease incidence in populations surrounding MTM mines may be associated with increased cardiac cell apoptotic signaling and decreased mitochondrial function.
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http://dx.doi.org/10.1152/ajpheart.00353.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698426PMC
December 2015

IL-15Rα deficiency in skeletal muscle alters respiratory function and the proteome of mitochondrial subpopulations independent of changes to the mitochondrial genome.

Mitochondrion 2015 Nov 14;25:87-97. Epub 2015 Oct 14.

Division of Exercise Physiology, United States; Center for Cardiovascular and Respiratory Sciences, United States; Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV, United States. Electronic address:

Interleukin-15 receptor alpha knockout (IL15RαKO) mice exhibit a greater skeletal muscle mitochondrial density with an altered mitochondrial morphology. However, the mechanism and functional impact of these changes have not been determined. In this study, we characterized the functional, proteomic, and genomic alterations in mitochondrial subpopulations isolated from the skeletal muscles of IL15RαKO mice and B6129 background control mice. State 3 respiration was greater in interfibrillar mitochondria and whole muscle ATP levels were greater in IL15RαKO mice supporting the increases in respiration rate. However, the state 3/state 4 ratio was lower, suggesting some degree of respiratory uncoupling. Proteomic analyses identified several markers independently in mitochondrial subpopulations that are associated with these functional alterations. Next Generation Sequencing of mtDNA revealed a high degree of similarity between the mitochondrial genomes of IL15RαKO mice and controls in terms of copy number, consensus coding and the presence of minor alleles, suggesting that the functional and proteomic alterations we observed occurred independent of alterations to the mitochondrial genome. These data provide additional evidence to implicate IL-15Rα as a regulator of skeletal muscle phenotypes through effects on the mitochondrion, and suggest these effects are driven by alterations to the mitochondrial proteome.
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http://dx.doi.org/10.1016/j.mito.2015.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5317373PMC
November 2015

Translational Regulation of the Mitochondrial Genome Following Redistribution of Mitochondrial MicroRNA in the Diabetic Heart.

Circ Cardiovasc Genet 2015 Dec 16;8(6):785-802. Epub 2015 Sep 16.

From the Department of Human Performances, Division of Exercise Physiology (R.J., D.T., C.E.N., D.L.S., J.C.S., T.L.C., W.A.B., S.E.L., J.M.H.), Center for Cardiovascular and Respiratory Sciences (R.J., D.T., C.E.N., D.L.S., T.L.C., W.A.B., S.E.L., J.M.H.), Department of Microbiology, Immunology and Cell Biology (I.M.), and Mary Babb Randolph Cancer Center (I.M.), West Virginia University School of Medicine, Morgantown.

Background: Cardiomyocytes are rich in mitochondria which are situated in spatially distinct subcellular regions, including those under the plasma membrane, subsarcolemmal mitochondria, and those between the myofibrils, interfibrillar mitochondria. We previously observed subpopulation-specific differences in mitochondrial proteomes following diabetic insult. The objective of this study was to determine whether mitochondrial genome-encoded proteins are regulated by microRNAs inside the mitochondrion and whether subcellular spatial location or diabetes mellitus influences the dynamics.

Methods And Results: Using microarray technology coupled with cross-linking immunoprecipitation and next generation sequencing, we identified a pool of mitochondrial microRNAs, termed mitomiRs, that are redistributed in spatially distinct mitochondrial subpopulations in an inverse manner following diabetic insult. Redistributed mitomiRs displayed distinct interactions with the mitochondrial genome requiring specific stoichiometric associations with RNA-induced silencing complex constituents argonaute-2 (Ago2) and fragile X mental retardation-related protein 1 (FXR1) for translational regulation. In the presence of Ago2 and FXR1, redistribution of mitomiR-378 to the interfibrillar mitochondria following diabetic insult led to downregulation of mitochondrially encoded F0 component ATP6. Next generation sequencing analyses identified specific transcriptome and mitomiR sequences associated with ATP6 regulation. Overexpression of mitomiR-378 in HL-1 cells resulted in its accumulation in the mitochondrion and downregulation of functional ATP6 protein, whereas antagomir blockade restored functional ATP6 protein and cardiac pump function.

Conclusions: We propose mitomiRs can translationally regulate mitochondrially encoded proteins in spatially distinct mitochondrial subpopulations during diabetes mellitus. The results reveal the requirement of RNA-induced silencing complex constituents in the mitochondrion for functional mitomiR translational regulation and provide a connecting link between diabetic insult and ATP synthase function.
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http://dx.doi.org/10.1161/CIRCGENETICS.115.001067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681669PMC
December 2015

Microvascular and mitochondrial dysfunction in the female F1 generation after gestational TiO2 nanoparticle exposure.

Nanotoxicology 2015 4;9(8):941-51. Epub 2015 Sep 4.

a Center for Cardiovascular and Respiratory Sciences .

Due to the ongoing evolution of nanotechnology, there is a growing need to assess the toxicological outcomes in under-studied populations in order to properly consider the potential of engineered nanomaterials (ENM) and fully enhance their safety. Recently, we and others have explored the vascular consequences associated with gestational nanomaterial exposure, reporting microvascular dysfunction within the uterine circulation of pregnant dams and the tail artery of fetal pups. It has been proposed (via work derived by the Barker Hypothesis) that mitochondrial dysfunction and subsequent oxidative stress mechanisms as a possible link between a hostile gestational environment and adult disease. Therefore, in this study, we exposed pregnant Sprague-Dawley rats to nanosized titanium dioxide aerosols after implantation (gestational day 6). Pups were delivered, and the progeny grew into adulthood. Microvascular reactivity, mitochondrial respiration and hydrogen peroxide production of the coronary and uterine circulations of the female offspring were evaluated. While there were no significant differences within the maternal or litter characteristics, endothelium-dependent dilation and active mechanotransduction in both coronary and uterine arterioles were significantly impaired. In addition, there was a significant reduction in maximal mitochondrial respiration (state 3) in the left ventricle and uterus. These studies demonstrate microvascular dysfunction and coincide with mitochondrial inefficiencies in both the cardiac and uterine tissues, which may represent initial evidence that prenatal ENM exposure produces microvascular impairments that persist throughout multiple developmental stages.
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http://dx.doi.org/10.3109/17435390.2014.984251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736545PMC
April 2017

Transgenic overexpression of mitofilin attenuates diabetes mellitus-associated cardiac and mitochondria dysfunction.

J Mol Cell Cardiol 2015 Feb 22;79:212-23. Epub 2014 Nov 22.

West Virginia University School of Medicine, Division of Exercise Physiology, Center for Cardiovascular and Respiratory Sciences, Morgantown, WV 26506, USA. Electronic address:

Mitofilin, also known as heart muscle protein, is an inner mitochondrial membrane structural protein that plays a central role in maintaining cristae morphology and structure. It is a critical component of the mitochondrial contact site and cristae organizing system (MICOS) complex which is important for mitochondrial architecture and cristae morphology. Our laboratory has previously reported alterations in mitochondrial morphology and proteomic make-up during type 1 diabetes mellitus, with mitofilin being significantly down-regulated in interfibrillar mitochondria (IFM). The goal of this study was to investigate whether overexpression of mitofilin can limit mitochondrial disruption associated with the diabetic heart through restoration of mitochondrial morphology and function. A transgenic mouse line overexpressing mitofilin was generated and mice injected intraperitoneally with streptozotocin using a multi low-dose approach. Five weeks following diabetes mellitus onset, cardiac contractile function was assessed. Restoration of ejection fraction and fractional shortening was observed in mitofilin diabetic mice as compared to wild-type controls (P<0.05 for both). Decrements observed in electron transport chain (ETC) complex I, III, IV and V activities, state 3 respiration, lipid peroxidation as well as mitochondria membrane potential in type 1 diabetic IFM were restored in mitofilin diabetic mice (P<0.05 for all). Qualitative analyses of electron micrographs revealed restoration of mitochondrial cristae structure in mitofilin diabetic mice as compared to wild-type controls. Furthermore, measurement of mitochondrial internal complexity using flow cytometry displayed significant reduction in internal complexity in diabetic IFM which was restored in mitofilin diabetic IFM (P<0.05). Taken together these results suggest that transgenic overexpression of mitofilin preserves mitochondrial structure, leading to restoration of mitochondrial function and attenuation of cardiac contractile dysfunction in the diabetic heart.
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http://dx.doi.org/10.1016/j.yjmcc.2014.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302057PMC
February 2015

Aging alters contractile properties and fiber morphology in pigeon skeletal muscle.

J Comp Physiol B 2014 Dec 23;184(8):1031-9. Epub 2014 Aug 23.

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, WV, 26506, USA,

In this study, we tested the hypothesis that skeletal muscle from pigeons would display age-related alterations in isometric force and contractile parameters as well as a shift of the single muscle fiber cross-sectional area (CSA) distribution toward smaller fiber sizes. Maximal force output, twitch contraction durations and the force-frequency relationship were determined in tensor propatagialis pars biceps muscle from young 3-year-old pigeons, middle-aged 18-year-old pigeons, and aged 30-year-old pigeons. The fiber CSA distribution was determined by planimetry from muscle sections stained with hematoxylin and eosin. Maximal force output of twitch and tetanic contractions was greatest in muscles from young pigeons, while the time to peak force of twitch contractions was longest in muscles from aged pigeons. There were no changes in the force-frequency relationship between the age groups. Interestingly, the fiber CSA distribution in aged muscles revealed a greater number of larger sized muscle fibers, which was verified visually in histological images. Middle-aged and aged muscles also displayed a greater amount of slow myosin containing muscle fibers. These data demonstrate that muscles from middle-aged and aged pigeons are susceptible to alterations in contractile properties that are consistent with aging, including lower force production and longer contraction durations. These functional changes were supported by the appearance of slow myosin containing muscle fibers in muscles from middle-aged and aged pigeons. Therefore, the pigeon may represent an appropriate animal model for the study of aging-related alterations in skeletal muscle function and structure.
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http://dx.doi.org/10.1007/s00360-014-0857-5DOI Listing
December 2014

Functional deficiencies of subsarcolemmal mitochondria in the type 2 diabetic human heart.

Am J Physiol Heart Circ Physiol 2014 Jul 28;307(1):H54-65. Epub 2014 Apr 28.

Division of Exercise Physiology and Center for Cardiovascular and Respiratory Sciences, West Virginia University School of Medicine, Morgantown, West Virginia;

The mitochondrion has been implicated in the development of diabetic cardiomyopathy. Examination of cardiac mitochondria is complicated by the existence of spatially distinct subpopulations including subsarcolemmal (SSM) and interfibrillar (IFM). Dysfunction to cardiac SSM has been reported in murine models of type 2 diabetes mellitus; however, subpopulation-based mitochondrial analyses have not been explored in type 2 diabetic human heart. The goal of this study was to determine the impact of type 2 diabetes mellitus on cardiac mitochondrial function in the human patient. Mitochondrial subpopulations from atrial appendages of patients with and without type 2 diabetes were examined. Complex I- and fatty acid-mediated mitochondrial respiration rates were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with no change in IFM. Electron transport chain (ETC) complexes I and IV activities were decreased in diabetic SSM compared with nondiabetic (P ≤ 0.05 for both), with a concomitant decline in their levels (P ≤ 0.05 for both). Regression analyses comparing comorbidities determined that diabetes mellitus was the primary factor accounting for mitochondrial dysfunction. Linear spline models examining correlative risk for mitochondrial dysfunction indicated that patients with diabetes display the same degree of state 3 and electron transport chain complex I dysfunction in SSM regardless of the extent of glycated hemoglobin (HbA1c) and hyperglycemia. Overall, the results suggest that independent of other pathologies, mitochondrial dysfunction is present in cardiac SSM of patients with type 2 diabetes and the degree of dysfunction is consistent regardless of the extent of elevated HbA1c or blood glucose levels.
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http://dx.doi.org/10.1152/ajpheart.00845.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080178PMC
July 2014

Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies.

Am J Physiol Heart Circ Physiol 2014 Jul;307(1):H1-14

Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle.
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http://dx.doi.org/10.1152/ajpheart.00747.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080170PMC
July 2014