Browsing by Author "Brown, David A."

Now showing 1 - 12 of 12
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    The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats
    Allen, Mitchell E.; Pennington, Edward Ross; Perry, Justin B.; Dadoo, Sahil; Makrecka-Kuka, Marina; Dambrova, Maija; Moukdar, Fatiha; Patel, Hetal D.; Han, Xianlin; Kidd, Grahame K.; Benson, Emily K.; Raisch, Tristan B.; Poelzing, Steven; Brown, David A.; Shaikh, Saame Raza (2020-07-17)
    Allen and Pennington et al. show that the cardiolipin-binding peptide elamipretide mitigates disease-induced fragmentation of cristae networks following cardiac ischemia reperfusion in rats. This study suggests that elamipretide targets mitochondrial membranes to sustain cristae networks, improving their bioenergetic function. Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.
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    Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog
    Bandara, Aloka B.; Drake, Joshua C.; Brown, David A. (2021-06-12)
    Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.
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    Detecting Transient Changes in Gait Using Fractal Scaling of Gait Variability in Conjunction with Gaussian Continuous Wavelet Transform
    Jaskowak, Daniel Joseph (Virginia Tech, 2019-01-31)
    Accelerometer data can be analyzed using a variety of methods which are effective in the clinical setting. Time-series analysis is used to analyze spatiotemporal variables in various populations. More recently, investigators have focused on gait complexity and the structure of spatiotemporal variations during walking and running. This study evaluated the use of time-series analyses to determine gait parameters during running. Subjects were college-age female soccer players. Accelerometer data were collected using GPS-embedded trunk-mounted accelerometers. Customized Matlab® programs were developed that included Gaussian continuous wavelet transform (CWT) to determine spatiotemporal characteristics, detrended fluctuation analysis (DFA) to examine gait complexity and autocorrelation analyses (ACF) to assess gait regularity. Reliability was examined using repeated running efforts and intraclass correlation. Proof of concept was determined by examining differences in each variable between various running speeds. Applicability was established by examining gait before and after fatiguing activity. The results showed most variables had excellent reliability. Test-retest R2 values for these variables ranged from 0.8 to 1.0. Low reliability was seen in bilateral comparisons of gait symmetry. Increases in running speed resulted in expected changes in spatiotemporal and acceleration variables. Fatiguing exercise had minimal effects on spatiotemporal variables but resulted in noticeable declines in complexity. This investigation shows that GPS-embedded trunk-mounted accelerometers can be effectively used to assess running gait. CWT and DFA yield reliable measures of spatiotemporal characteristics of gait and gait complexity. The effects of running speed and fatigue on these variables provides proof of concepts and applicability for this analytical approach.
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    Elevated perfusate [Na+] increases contractile dysfunction during ischemia and reperfusion
    King, D. Ryan; Padget, Rachel L.; Perry, Justin B.; Hoeker, Gregory S.; Smyth, James W.; Brown, David A.; Poelzing, Steven (2020-10-14)
    Recent studies revealed that relatively small changes in perfusate sodium ([Na+](o)) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+](o) modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+](o) that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia-reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia-reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+](o) decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+](o) at baseline and reduced the time to peak ischemic contracture. Ischemia-reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+](o) further depressed mechanical function. In summary, elevating [Na+](o) by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+](o), with cardiac ischemia may require further investigation.
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    High Resolution Respirometry of Heart Mitochondria in Healthy and Stressed States
    Wilson, Zachary T.; Perry, Justin B.; Brown, David A. (Virginia Tech, 2018-08)
    Heart disease remains the leading cause of death globally, claiming the lives of nearly 10 million people in 2016. Current standard-of-care therapies for heart disease patients reduce energy demands on the heart but do not treat underlying deficits in cellular energy production. Cardiac mitochondria are primarily responsible for the production of energy in the heart, and targeting dysfunctional mitochondria represents a promising solution to improving the prognosis of heart disease patients. Increased production of reactive oxygen species in heart disease damages mitochondrial function, ultimately decreasing cardiac energy supply. Isolated mitochondria exposed to hydrogen peroxide serves as a heart disease model where the reactive oxygen species damage the respiratory chain, a series of complexes responsible for the actual production of energy. N-acetylcysteine (NAC) is a drug precursor to glutathione, an endogenous antioxidant which reduces reactive oxygen species. In this project, isolated mitochondria are treated with hydrogen peroxide with or without NAC. If NAC is capable of rescuing the respiratory rate, that would suggest that NAC restores mitochondrial energy production in pathological states. If successful, these data would be the first step in determining if incorporation of NAC into heart disease treatment plans could begin to better treat the number of one cause of morbidity/mortality on the planet.
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    Mitochondrial function as a therapeutic target in heart failure
    Brown, David A.; Perry, Justin B.; Allen, Mitchell E.; Sabbah, Hani N.; Stauffer, Brian L.; Shaikh, Saame Raza; Cleland, John G. F.; Colucci, Wilson S.; Butler, Javed; Voors, Adriaan A.; Anker, Stefan D.; Pitt, Bertram; Pieske, Burkert; Filippatos, Gerasimos; Greene, Stephen J.; Gheorghiade, Mihai (2017-04)
    Heart failure is a pressing worldwide public-health problem with millions of patients having worsening heart failure. Despite all the available therapies, the condition carries a very poor prognosis. Existing therapies provide symptomatic and clinical benefit, but do not fully address molecular abnormalities that occur in cardiomyocytes. This shortcoming is particularly important given that most patients with heart failure have viable dysfunctional myocardium, in which an improvement or normalization of function might be possible. Although the pathophysiology of heart failure is complex, mitochondrial dysfunction seems to be an important target for therapy to improve cardiac function directly. Mitochondrial abnormalities include impaired mitochondrial electron transport chain activity, increased formation of reactive oxygen species, shifted metabolic substrate utilization, aberrant mitochondrial dynamics, and altered ion homeostasis. In this Consensus Statement, insights into the mechanisms of mitochondrial dysfunction in heart failure are presented, along with an overview of emerging treatments with the potential to improve the function of the failing heart by targeting mitochondria.
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    Mitochondrial Structure-Function in health and disease
    Allen, Mitchell Edison (Virginia Tech, 2019-04-25)
    Mitochondrial structure and function are inextricably linked ("structure-function"), with decrements in structure-function evident across diseases. Barriers to new therapies include a complete understanding of the underlying molecular culprits, as well as effective mitochondria-targeted therapies that mitigate injury. In these works, we investigate the role of cristae-shaping factors like cardiolipin in health and disease. In a series of studies, we tested the effects of the cell-permeable tetrapeptides, elamipretide and a postulated peptide, (arginine-tyrosine-lysine-phenylalanine; "RYKF"), on the recovery of mitochondrial structure-function after injury. Elamipretide is a clinical-stage compound currently under investigation for genetic and age-related mitochondrial diseases, yet the mechanism of action is not completely understood. We used a combination of physiological models, mitochondrial imaging, and biomimetic membrane studies to test the hypothesis that elamipretide and RYKF-cardiolipin interactions improved mitochondrial structure-function. Post-ischemic treatment with elamipretide sustained mitochondrial function across electron transport chain complexes. Endogenous RYKF expression similarly improved mitochondrial respiration after peroxide and hypoxia nutrient deprivation injuries. Using two parallel electron microscopy paradigms, we show elamipretide and RYKF treatment led to maintenance of mitochondrial ultrastructure and notably, improved cristae interconnectedness. Finally, we utilized a novel biomimetic membrane system to model the pathological mitochondrial membrane and found that elamipretide and RYKF both improved biophysical pressure-area relationships through a mechanism that appears to involve aggregating cardiolipin. Our data indicate that targeting pathophysiological mitochondrial membranes with cationic, lipophilic peptides can improve bioenergetics by sustaining cristae networks and support interdependent relationships between mitochondrial structure and function.
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    Novel approaches to treat mitochondrial complex-I mediated defects in disease
    Perry, Justin Bradley (Virginia Tech, 2019-04-25)
    Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.
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    Progression-Mediated Changes in Mitochondrial Morphology Promotes Adaptation to Hypoxic Peritoneal Conditions in Serous Ovarian Cancer
    Grieco, Joseph P.; Allen, Mitchell E.; Perry, Justin B.; Wang, Yao; Song, Yipei; Rohani, Ali; Compton, Stephanie L. E.; Smyth, James W.; Swami, Nathan S.; Brown, David A.; Schmelz, Eva M. (2021-01-13)
    Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.
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    Pulmonary Exposure to Magnéli Phase Titanium Suboxides Results in Significant Macrophage Abnormalities and Decreased Lung Function
    McDaniel, Dylan K.; Ringel-Scaia, Veronica M.; Morrison, Holly A.; Coutermarsh-Ott, Sheryl; Council-Troche, McAlister; Angle, Jonathan W.; Perry, Justin B.; Davis, Grace; Leng, Weinan; Minarchick, Valerie; Yang, Yi; Chen, Bo; Reece, Sky W.; Brown, David A.; Cecere, Thomas E.; Brown, Jared M.; Gowdy, Kymberly M.; Hochella, Michael F. Jr.; Allen, Irving C. (Frontiers, 2019-11-28)
    Coal is one of the most abundant and economic sources for global energy production. However, the burning of coal is widely recognized as a significant contributor to atmospheric particulate matter linked to deleterious respiratory impacts. Recently, we have discovered that burning coal generates large quantities of otherwise rare Magnéli phase titanium suboxides from TiO2 minerals naturally present in coal. These nanoscale Magnéli phases are biologically active without photostimulation and toxic to airway epithelial cells in vitro and to zebrafish in vivo. Here, we sought to determine the clinical and physiological impact of pulmonary exposure to Magnéli phases using mice as mammalian model organisms. Mice were exposed to the most frequently found Magnéli phases, Ti6O11, at 100 parts per million (ppm) via intratracheal administration. Local and systemic titanium concentrations, lung pathology, and changes in airway mechanics were assessed. Additional mechanistic studies were conducted with primary bone marrow derived macrophages. Our results indicate that macrophages are the cell type most impacted by exposure to these nanoscale particles. Following phagocytosis, macrophages fail to properly eliminate Magnéli phases, resulting in increased oxidative stress, mitochondrial dysfunction, and ultimately apoptosis. In the lungs, these nanoparticles become concentrated in macrophages, resulting in a feedback loop of reactive oxygen species production, cell death, and the initiation of gene expression profiles consistent with lung injury within 6 weeks of exposure. Chronic exposure and accumulation of Magnéli phases ultimately results in significantly reduced lung function impacting airway resistance, compliance, and elastance. Together, these studies demonstrate that Magnéli phases are toxic in the mammalian airway and are likely a significant nanoscale environmental pollutant, especially in geographic regions where coal combustion is a major contributor to atmospheric particulate matter.
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    Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus
    De Vries, Maaike C.; Brown, David A.; Allen, Mitchell E.; Bindoff, Laurence; Gorman, Grainne S.; Karaa, Amel; Keshavan, Nandaki; Lamperti, Costanza; McFarland, Robert; Ng, Yi Shiau; O'Callaghan, Mar; Pitceathly, Robert D. S.; Rahman, Shamima; Russel, Frans G. M.; Varhaug, Kristin N.; Schirris, Tom J. J.; Mancuso, Michelangelo (2020-07)
    Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.
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    Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice
    Hamm, Shelby E.; Fathalikhani, Daniel D.; Bukovec, Katherine E.; Addington, Adele K.; Zhang, Haiyan; Perry, Justin B.; McMillan, Ryan P.; Lawlor, Michael W.; Prom, Mariah J.; Vanden Avond, Mark A.; Kumar, Suresh N.; Coleman, Kirsten E.; Dupont, J.B.; Mack, David L.; Brown, David A.; Morris, Carl A.; Gonzalez, J. Patrick; Grange, Robert W. (Cell Press, 2021-02-25)
    We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9- CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wildtype (WT) mice were assigned to WTR (run) and WT (no run) groups.WTRandmdxRGTperformed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGTanimals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.