Luke I Szweda

University of Kentucky, Lexington, Kentucky, United States

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Publications (114)572.74 Total impact

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    ABSTRACT: The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.
    Cell 04/2014; 157(3):565-79. · 31.96 Impact Factor
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    ABSTRACT: Obesity enhances the risk for the development of type 2 diabetes and cardiovascular disease. Loss in insulin sensitivity and diminished ability of muscle to take up and utilize glucose is a characteristic of type 2 diabetes. Paradoxically, regulatory mechanisms that promote utilization of fatty acids appear to initiate diet-induced insulin insensitivity. In this review, we discuss recent findings implicating increased mitochondrial production of the pro-oxidant, H2O2, due to enhanced utilization of fatty acids, as a signal to diminish reliance on glucose and its metabolites for energy. In the short term, the ability to preferentially utilize fatty acids may be beneficial, promoting a metabolic shift that ensures use of available fat by skeletal muscle and heart while preventing intracellular glucose accumulation and toxicity. However, with prolonged consumption of high dietary fat and ensuing obesity, the near exclusive dependence on fatty acid oxidation for production of energy by the mitochondria drives insulin resistance, diabetes, and cardiovascular disease.
    AJP Heart and Circulatory Physiology 06/2013; · 4.01 Impact Factor
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    ABSTRACT: ABSTRACT Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently binds amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated to be intimately associated with pathological lesions of Alzheimer disease (AD), suggesting oxidative stress is a major component in the disease. Here, we examined the HNE-crosslinking modifications by using an antibody specific for a lysine-lysine crosslink. Since in a prior study we noted no immunolabeling of neuritic plaques or neurofibrillary tangles but instead found strong labeling of axons, we focused this study on axons. Axonal labeling was examined in mouse sciatic nerve, and immunoblotting showed the crosslink was restricted to neurofilament heavy and medium subunit, which while altering migration, did not indicate larger NF aggregates, indicative of intermolecular crosslinks. Examination of mice at various ages showed the extent of modification remaining relatively constant through the lifespan. These findings demonstrate lipid-crosslinking peroxidation primarily involves lysine rich neurofilaments and is restricted to intramolecular crosslinks.
    Free Radical Research 04/2013; · 3.28 Impact Factor
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    ABSTRACT: α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione-lipoic acid adduct. Nevertheless, 1:1 lipoic acid-glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme's native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition providing further evidence that specific lipoic acid residues within the E2 complex are susceptible to glutathionylation. These findings offer chemical information necessary for the identification of mechanisms and physiological implications of KGDH glutathionylation.
    Free Radical Biology and Medicine 04/2013; · 5.27 Impact Factor
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    ABSTRACT: Alzheimer disease (AD) is an age-related neurodegenerative disease characterized by the presence of three pathological hallmarks: synapse loss, extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFTs). The major component of SP is amyloid β-peptide (Aβ), which has been shown to induce oxidative stress. The AD brain shows increased levels of lipid peroxidation products, including 4-hydroxy-2-nonenal (HNE). HNE can react covalently with Cys, His, or Lys residues on proteins, altering structure and function of the latter. In the present study we measured the levels of the HNE-modified lipoic acid in brain of subjects with AD and age-matched controls. Lipoic acid is a key co-factor for a number of proteins including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, key complexes for cellular energetics. We observed a significant decrease in the levels of HNE-lipoic acid in the AD brain compared to that of age-matched controls. To investigate this phenomenon further, the levels and activity of lipoamide dehydrogenase (LADH) were measured in AD and control brains. Additionally, LADH activities were measured after in-vitro HNE-treatment to mice brains. Both LADH levels and activities were found to be significantly reduced in AD brain compared to age-matched control. HNE-treatment also reduced the LADH activity in mice brain. These data are consistent with a two-hit hypothesis of AD: oxidative stress leads to lipid peroxidation that, in turn, causes oxidative dysfunction of key energy-related complexes in mitochondria, triggering neurodegeneration. This study is consonant with the notion that lipoic acid supplementation could be a potential treatment for the observed loss of cellular energetics in AD and potentiate the antioxidant defense system to prevent or delay the oxidative stress in and progression of this devastating dementing disorder.
    Redox biology. 01/2013; 1(1):80-5.
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    Clair Crewe, Michael Kinter, Luke I Szweda
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    ABSTRACT: Cardiac function depends on the ability to switch between fatty acid and glucose oxidation for energy production in response to changes in substrate availability and energetic stress. In obese and diabetic individuals, increased reliance on fatty acids and reduced metabolic flexibility are thought to contribute to the development of cardiovascular disease. Mechanisms by which cardiac mitochondria contribute to diet-induced metabolic inflexibility were investigated. Mice were fed a high fat or low fat diet for 1 d, 1 wk, and 20 wk. Cardiac mitochondria isolated from mice fed a high fat diet displayed a diminished ability to utilize the glycolytically derived substrate pyruvate. This response was rapid, occurring within the first day on the diet, and persisted for up to 20 wk. A selective increase in the expression of pyruvate dehydrogenase kinase 4 and inhibition of pyruvate dehydrogenase are responsible for the rapid suppression of pyruvate utilization. An important consequence is that pyruvate dehydrogenase is sensitized to inhibition when mitochondria respire in the presence of fatty acids. Additionally, increased expression of pyruvate dehydrogenase kinase 4 preceded any observed diet-induced reductions in the levels of glucose transporter type 4 and glycolytic enzymes and, as judged by Akt phosphorylation, insulin signaling. Importantly, diminished insulin signaling evident at 1 wk on the high fat diet did not occur in pyruvate dehydrogenase kinase 4 knockout mice. Dietary intervention leads to a rapid decline in pyruvate dehydrogenase kinase 4 levels and recovery of pyruvate dehydrogenase activity indicating an additional form of regulation. Finally, an overnight fast elicits a metabolic response similar to that induced by high dietary fat obscuring diet-induced metabolic changes. Thus, our data indicate that diet-induced inhibition of pyruvate dehydrogenase may be an initiating event in decreased oxidation of glucose and increased reliance of the heart on fatty acids for energy production.
    PLoS ONE 01/2013; 8(10):e77280. · 3.53 Impact Factor
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    ABSTRACT: Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific upregulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal c protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H(2)O(2) production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H(2)O(2) produced by increased fat metabolism. To determine if the catalase-specific response is a common feature of physiologic conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fed versus fasted mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24hr. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H(2)O(2)-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.
    Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
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    BMC proceedings 06/2012; 6(3).
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    ABSTRACT: The loading of macrophages with oxidized low density lipoprotein (LDL) is a key part of the initiation and progression of atherosclerosis. Oxidized LDL contains a wide ranging set of toxic species, yet the molecular events that allow macrophages to withstand loading with these toxic species are not completely characterized. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the cellular stress response. However, the specific parts of the Nrf2-dependent stress response are diverse, with both tissue- and treatment-dependent components. The goal of these experiments was to develop and use a quantitative proteomic approach to characterize the Nrf2-dependent response in macrophages to oxidized LDL. Cultured mouse macrophages, the J774 macrophage-like cell line, were treated with a combination of oxidized LDL, the Nrf2-stabilizing reagent tert- butylhydroquinone (tBHQ), and/or Nrf2 siRNA. Protein expression was determined using a quantitative proteomics assay based on selected reaction monitoring. The assay was multiplexed to monitor a set of 28 antioxidant and stress response proteins, 6 housekeeping proteins, and 1 non-endogenous standard protein. The results have two components. The first component is the validation of the multiplexed, quantitative proteomics assay. The assay is shown to be fundamentally quantitative, precise, and accurate. The second component is the characterization of the Nrf2-mediated stress response. Treatment with tBHQ and/or Nrf2 siRNA gave statistically significant changes in the expression of a subset of 11 proteins. Treatment with oxidized LDL gave statistically significant increases in the expression of 7 of those 11 proteins plus one additional protein. All of the oxLDL-mediated increases were attenuated by Nrf2 siRNA. These results reveal a specific, multifaceted response of the foam cells to the incoming toxic oxidized LDL.
    PLoS ONE 01/2012; 7(11):e50016. · 3.53 Impact Factor
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    ABSTRACT: Clinical and experimental evidence supports that chronic oxidative stress is a primary contributing factor to numerous retinal degenerative diseases, such as age-related macular degeneration (AMD). Eyes obtained postmortem from AMD patients have extensive free radical damage to the proteins, lipids, DNA, and mitochondria of their retinal pigment epithelial (RPE) cells. In addition, several mouse models of chronic oxidative stress develop many of the pathological hallmarks of AMD. However, the extent to which oxidative stress is an etiologic component versus its involvement in disease progression remains a major unanswered question. Further, whether the primary target of oxidative stress and damage is photoreceptors or RPE cells, or both, is still unclear. In this review, we discuss the major functions of RPE cells with an emphasis on the oxidative challenges these cells encounter and the endogenous antioxidant mechanisms employed to neutralize the deleterious effects that such stresses can elicit if left unchecked.
    International review of cell and molecular biology 01/2012; 298:135-77. · 4.97 Impact Factor
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    ABSTRACT: Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.
    Free Radical Biology and Medicine 11/2011; 52(3):699-704. · 5.27 Impact Factor
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    ABSTRACT: Intracellular proteins are degraded by a number of proteases, including the ubiquitin-proteasome pathway (UPP). Impairments in the UPP occur during the aging of a variety of tissues, although little is known in regards to age-related alterations to the UPP during the aging of adipose tissue. The UPP is known to be involved in regulating the differentiation of a variety of cell types, although the potential changes in the UPP during adipose differentiation have not been fully elucidated. How the UPP is altered in aging adipose tissue and adipocyte differentiation and the effects of proteasome inhibition on adipocyte homeostasis and differentiation are critical issues to elucidate experimentally. Adipogenesis continues throughout the life of adipose tissue, with continual differentiation of preadipocytes essential to maintaining tissue function during aging, and UPP alterations in mature adipocytes are likely to directly modulate adipose function during aging. In this study we demonstrate that aging induces alterations in the activity and expression of principal components of the UPP. Additionally, we show that multiple changes in the UPP occur during the differentiation of 3T3-L1 cells into adipocytes. In vitro data link observed UPP alterations to increased levels of oxidative stress and altered adipose biology relevant to both aging and differentiation. Taken together, these data demonstrate that changes in the UPP occur in response to adipose aging and adipogenesis and strongly suggest that proteasome inhibition is sufficient to decrease adipose differentiation, as well as increasing oxidative stress in mature adipocytes, both of which probably promote deleterious effects on adipose aging.
    Free Radical Biology and Medicine 08/2011; 51(9):1727-35. · 5.27 Impact Factor
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    ABSTRACT: α-Ketoglutarate dehydrogenase (KGDH), a key regulatory enzyme within the Krebs cycle, is sensitive to mitochondrial redox status. Treatment of mitochondria with H₂O₂ results in reversible inhibition of KGDH due to glutathionylation of the cofactor, lipoic acid. Upon consumption of H₂O₂, glutathione is removed by glutaredoxin restoring KGDH activity. Glutathionylation appears to be enzymatically catalysed or require a unique microenvironment. This may represent an antioxidant response, diminishing the flow of electrons to the respiratory chain and protecting sulphydryl residues from oxidative damage. KGDH is, however, also susceptible to oxidative damage. 4-Hydroxy-2-nonenal (HNE), a lipid peroxidation product, reacts with lipoic acid resulting in enzyme inactivation. Evidence indicates that HNE modified lipoic acid is cleaved from KGDH, potentially the first step of a repair process. KGDH is therefore a likely redox sensor, reversibly altering metabolism to reduce oxidative damage and, under severe oxidative stress, acting as a sentinel of mitochondrial viability.
    Free Radical Research 01/2011; 45(1):29-36. · 3.28 Impact Factor
  • Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2011; 51.
  • Alzheimer's and Dementia 07/2010; 6(4). · 17.47 Impact Factor
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    ABSTRACT: Biological Sciences SymposiaHydroxynonenal-Generated Crosslinking Fluorophore and Mitochondria-derived Lipoic Acid Accumulation in Alzheimer Disease Reveal a Dichotomy of Protein TurnoverArticle author querycastellani r [PubMed] [Google Scholar]moreira p [PubMed] [Google Scholar]aliev g [PubMed] [Google Scholar]shenk j [PubMed] [Google Scholar]siedlak s [PubMed] [Google Scholar]harris p [PubMed] [Google Scholar]sayre l [PubMed] [Google Scholar]szweda l [PubMed] [Google Scholar]zhu x [PubMed] [Google Scholar]smith m [PubMed] [Google Scholar]perry g [PubMed] [Google Scholar]szweda p [PubMed] [Google Scholar]RJ Castellania1, P Moreiraa2, G Alieva3, JC Shenka3, SL Siedlaka4, PLR Harrisa4, LM Sayrea4, LI Szwedaa5, X Zhua4, MA Smitha4, G Perrya3 and PA Szwedaa5a1 University of Maryland
    Microscopy and Microanalysis 06/2010; 16:1014 - 1015. · 2.50 Impact Factor
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    ABSTRACT: The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.
    Journal of Biological Chemistry 05/2010; 285(29):21943-50. · 4.65 Impact Factor
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    ABSTRACT: AIM: The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilonPKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. METHODS AND RESULTS: Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished deltaPKC translocation by 3.8-fold and increased epsilonPKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of deltaPKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilonPKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilonPKC during IPC restored deltaPKC levels at the mitochondria while decreasing epsilonPKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a deltaPKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. CONCLUSION: Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, deltaPKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilonPKC.
    Cardiovascular Research 01/2010; 85(2-85):385. · 5.81 Impact Factor
  • Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2010; 49.
  • Paul M Rindler, Luke I Szweda, Mike Kinter
    Free Radical Biology and Medicine - FREE RADICAL BIOL MED. 01/2010; 49.

Publication Stats

5k Citations
572.74 Total Impact Points


  • 2013
    • University of Kentucky
      Lexington, Kentucky, United States
  • 2010–2013
    • University of Oklahoma Health Sciences Center
      • Department of Biochemistry and Molecular Biology
      Oklahoma City, OK, United States
  • 2006–2012
    • Oklahoma Medical Research Foundation
      • Free Radical Biology and Aging Program
      Oklahoma City, Oklahoma, United States
  • 1996–2011
    • Case Western Reserve University
      • • Department of Pathology (University Hospitals Case Medical Center)
      • • Department of Physiology and Biophysics
      Cleveland, OH, United States
  • 2006–2009
    • Stanford University
      • Department of Chemical and Systems Biology
      Stanford, CA, United States
  • 2004
    • Stanford Medicine
      Stanford, California, United States
  • 2001–2002
    • Paris Diderot University
      Lutetia Parisorum, Île-de-France, France
  • 1999
    • University of Wisconsin, Madison
      • Department of Pathology and Laboratory Medicine
      Madison, MS, United States
  • 1992–1998
    • National Heart, Lung, and Blood Institute
      • Biochemistry and Biophysics Center
      Maryland, United States
  • 1994
    • National Institutes of Health
      • Chemical Biology Laboratory
      Bethesda, MD, United States