Publications by authors named "Thomas H Sanderson"

33 Publications

Rapid Treatment with Intramuscular Magnesium Sulfate During Cardiopulmonary Resuscitation Does Not Provide Neuroprotection Following Cardiac Arrest.

Mol Neurobiol 2022 Jan 14. Epub 2022 Jan 14.

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.

Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective compound which protects neurons from ischemic injury by reducing neuronal calcium overload via NMDA receptor modulation and preventing calcium-induced mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO during CPR has the potential to target these mechanisms within an early therapeutic window. We hypothesize that IM MgSO administrated during CPR could achieve therapeutic serum magnesium levels within 15 min after ROSC and improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Male Long Evans rats were subjected to 8-min asphyxial cardiac arrest and block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO IM at the onset of CPR. Serum magnesium concentrations increased rapidly with IM delivery during CPR, achieving twofold to fourfold increase by 15 min after ROSC in all magnesium dose groups. Rats subjected to cardiac arrest or sham surgery were block randomized to treatment groups for assessment of neurological outcomes. We found that IM MgSO during CPR had no effect on ROSC rate (p > 0.05). IM MgSO treatment had no statistically significant effect on 10-day survival with good neurologic function or hippocampal CA1 pyramidal neuron survival compared to placebo treatment. In conclusion, a single dose IM MgSO during CPR achieves up to fourfold baseline serum magnesium levels within 15 min after ROSC; however, this treatment strategy did not improve survival, recovery of neurologic function, or neuron survival. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated.
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http://dx.doi.org/10.1007/s12035-021-02645-xDOI Listing
January 2022

Dose optimization of early high-dose valproic acid for neuroprotection in a swine cardiac arrest model.

Resusc Plus 2020 Mar-Jun;1-2:100007. Epub 2020 Jun 1.

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

Aim: High-dose valproic acid (VPA) improves the survival and neurologic outcomes after asphyxial cardiac arrest (CA) in rats. We characterized the pharmacokinetics, pharmacodynamics, and safety of high-dose VPA in a swine CA model to advance clinical translation.

Methods: After 8 ​min of untreated ventricular fibrillation CA, 20 male Yorkshire swine were resuscitated until return of spontaneous circulation (ROSC). They were block randomized to receive placebo, 75 ​mg/kg, 150 ​mg/kg, or 300 ​mg/kg VPA as 90-min intravenous infusion (n ​= ​5/group) beginning at ROSC. Animals were monitored for 2 additional hours then euthanized. Experimental operators were blinded to treatments.

Results: The mean(SD) total CA duration was 14.8(1.2) minutes. 300 ​mg/kg VPA animals required more adrenaline to maintain mean arterial pressure ≥80 ​mmHg and had worse lactic acidosis. There was a strong linear correlation between plasma free VPA C and brain total VPA (r ​= ​0.9494; p ​< ​0.0001). VPA induced dose-dependent increases in pan- and site-specific histone H3 and H4 acetylation in the brain. Plasma free VPA C is a better predictor than peripheral blood mononuclear cell histone acetylation for brain H3 and H4 acetylation (r ​= ​0.7189 for H3K27ac, r ​= ​0.7189 for pan-H3ac, and r ​= ​0.7554 for pan-H4ac; p ​< ​0.0001).

Conclusions: Up to 150 ​mg/kg VPA can be safely tolerated as 90-min intravenous infusion in a swine CA model. High-dose VPA induced dose-dependent increases in brain histone H3 and H4 acetylation, which can be predicted by plasma free VPA C as the pharmacodynamics biomarker for VPA target engagement after CA.
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http://dx.doi.org/10.1016/j.resplu.2020.100007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244526PMC
June 2020

Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons.

Cell Death Dis 2021 05 12;12(5):475. Epub 2021 May 12.

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.

Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.
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http://dx.doi.org/10.1038/s41419-021-03752-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115279PMC
May 2021

Inhibiting Mitochondrial Cytochrome Oxidase Downregulates Gene Transcription After Traumatic Brain Injury in .

Front Physiol 2021 15;12:628777. Epub 2021 Mar 15.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, United States.

Traumatic brain injuries (TBIs) caused by a sudden impact to the head alter behavior and impair physical and cognitive function. Besides the severity, type and area of the brain affected, the outcome of TBI is also influenced by the patient's biological sex. Previous studies reporting mitochondrial dysfunction mainly focused on exponential reactive oxygen species (ROS) generation, increased mitochondrial membrane potential, and altered mitochondrial dynamics as a key player in the outcome to brain injury. In this study, we evaluated the effect of a near-infrared (NIR) light exposure on gene expression in a TBI model. NIR interacts with cytochrome oxidase (COX) of the electron transport chain to reduce mitochondrial membrane potential hyperpolarization, attenuate ROS generation, and apoptosis. We subjected male and female flies to TBI using a high-impact trauma (HIT) device and subsequently exposed the isolated fly brains to a COX-inhibitory wavelength of 750 nm for 2 hours (hr). Genome-wide 3'-mRNA-sequencing of fly brains revealed that injured females exhibit greater changes in transcription compared to males at 1, 2, and 4 hours (hr) after TBI. Inhibiting COX by exposure to NIR downregulates gene expression in injured females but has minimal effect in injured males. Our results suggest that mitochondrial COX modulation with NIR alters gene expression in following TBI and the response to injury and NIR exposure varies by biological sex.
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http://dx.doi.org/10.3389/fphys.2021.628777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005633PMC
March 2021

Machine learning-based classification of mitochondrial morphology in primary neurons and brain.

Sci Rep 2021 03 4;11(1):5133. Epub 2021 Mar 4.

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.

The mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.
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http://dx.doi.org/10.1038/s41598-021-84528-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933342PMC
March 2021

Aerosol generation during chest compression and defibrillation in a swine cardiac arrest model.

Resuscitation 2021 02 15;159:28-34. Epub 2020 Dec 15.

Department of Emergency Medicine, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI, 48109, USA; Michigan Center for Integrative Research in Critical Care, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI, 48109, USA. Electronic address:

Aim: It remains unclear whether cardiac arrest (CA) resuscitation generates aerosols that can transmit respiratory pathogens. We hypothesize that chest compression and defibrillation generate aerosols that could contain the SARS-CoV-2 virus in a swine CA model.

Methods: To simulate witnessed CA with bystander-initiated cardiopulmonary resuscitation, 3 female non-intubated swine underwent 4 min of ventricular fibrillation without chest compression or defibrillation (no-flow) followed by ten 2-min cycles of mechanical chest compression and defibrillation without ventilation. The diameter (0.3-10 μm) and quantity of aerosols generated during 45-s intervals of no-flow and chest compression before and after defibrillation were analyzed by a particle analyzer. Aerosols generated from the coughs of 4 healthy human subjects were also compared to aerosols generated by swine.

Results: There was no significant difference between the total aerosols generated during chest compression before defibrillation compared to no-flow. In contrast, chest compression after defibrillation generated significantly more aerosols than chest compression before defibrillation or no-flow (72.4 ± 41.6 × 10 vs 12.3 ± 8.3 × 10 vs 10.5 ± 11.2 × 10; p < 0.05), with a shift in particle size toward larger aerosols. Two consecutive human coughs generated 54.7 ± 33.9 × 10 aerosols with a size distribution smaller than post-defibrillation chest compression.

Conclusions: Chest compressions alone did not cause significant aerosol generation in this swine model. However, increased aerosol generation was detected during chest compression immediately following defibrillation. Additional research is needed to elucidate the clinical significance and mechanisms by which aerosol generation during chest compression is modified by defibrillation.
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http://dx.doi.org/10.1016/j.resuscitation.2020.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833865PMC
February 2021

Cytochrome c oxidase-modulatory near-infrared light penetration into the human brain: Implications for the noninvasive treatment of ischemia/reperfusion injury.

IUBMB Life 2021 03 9;73(3):554-567. Epub 2020 Nov 9.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA.

Near-infrared light (IRL) has been evaluated as a therapeutic for a variety of pathological conditions, including ischemia/reperfusion injury of the brain, which can be caused by an ischemic stroke or cardiac arrest. Strategies have focused on modulating the activity of mitochondrial electron transport chain (ETC) enzyme cytochrome c oxidase (COX), which has copper centers that broadly absorb IRL between 700 and 1,000 nm. We have recently identified specific COX-inhibitory IRL wavelengths that are profoundly neuroprotective in rodent models of brain ischemia/reperfusion through the following mechanism: COX inhibition by IRL limits mitochondrial membrane potential hyperpolarization during reperfusion, which otherwise causes reactive oxygen species (ROS) production and cell death. Prior to clinical application of IRL on humans, IRL penetration must be tested, which may be wavelength dependent. In the present study, four fresh (unfixed) cadavers and isolated cadaver tissues were used to examine the transmission of infrared light through human biological tissues. We conclude that the transmission of 750 and 940 nm IRL through 4 cm of cadaver head supports the viability of IRL to treat human brain ischemia/reperfusion injury and is similar for skin with different skin pigmentation. We discuss experimental difficulties of working with fresh cadavers and strategies to overcome them as a guide for future studies.
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http://dx.doi.org/10.1002/iub.2405DOI Listing
March 2021

Endoplasmic reticulum-associated degradation regulates mitochondrial dynamics in brown adipocytes.

Science 2020 04 19;368(6486):54-60. Epub 2020 Mar 19.

Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48105, USA.

The endoplasmic reticulum (ER) engages mitochondria at specialized ER domains known as mitochondria-associated membranes (MAMs). Here, we used three-dimensional high-resolution imaging to investigate the formation of pleomorphic "megamitochondria" with altered MAMs in brown adipocytes lacking the Sel1L-Hrd1 protein complex of ER-associated protein degradation (ERAD). Mice with ERAD deficiency in brown adipocytes were cold sensitive and exhibited mitochondrial dysfunction. ERAD deficiency affected ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover of the MAM protein, sigma receptor 1 (SigmaR1). Thus, our study provides molecular insights into ER-mitochondrial cross-talk and expands our understanding of the physiological importance of Sel1L-Hrd1 ERAD.
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http://dx.doi.org/10.1126/science.aay2494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409365PMC
April 2020

Cytochrome c phosphorylation: Control of mitochondrial electron transport chain flux and apoptosis.

Int J Biochem Cell Biol 2020 04 2;121:105704. Epub 2020 Feb 2.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA; Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, MI 48201, USA. Electronic address:

Cytochrome c (Cytc)is a cellular life and death decision molecule that regulates cellular energy supply and apoptosis through tissue specific post-translational modifications. Cytc is an electron carrier in the mitochondrial electron transport chain (ETC) and thus central for aerobic energy production. Under conditions of cellular stress, Cytc release from the mitochondria is a committing step for apoptosis, leading to apoptosome formation, caspase activation, and cell death. Recently, Cytc was shown to be a target of cellular signaling pathways that regulate the functions of Cytc by tissue-specific phosphorylations. So far five phosphorylation sites of Cytc have been mapped and functionally characterized, Tyr97, Tyr48, Thr28, Ser47, and Thr58. All five phosphorylations partially inhibit respiration, which we propose results in optimal intermediate mitochondrial membrane potentials and low ROS production under normal conditions. Four of the phosphorylations result in inhibition of the apoptotic functions of Cytc, suggesting a cytoprotective role for phosphorylated Cytc. Interestingly, these phosphorylations are lost during stress conditions such as ischemia. This results in maximal ETC flux during reperfusion, mitochondrial membrane potential hyperpolarization, excessive ROS generation, and apoptosis. We here present a new model proposing that the electron transfer from Cytc to cytochrome c oxidase is the rate-limiting step of the ETC, which is regulated via post-translational modifications of Cytc. This regulation may be dysfunctional in disease conditions such as ischemia-reperfusion injury and neurodegenerative disorders through increased ROS, or cancer, where post-translational modifications on Cytc may provide a mechanism to evade apoptosis.
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http://dx.doi.org/10.1016/j.biocel.2020.105704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044036PMC
April 2020

Mitochondrial Quality Control: Role in Cardiac Models of Lethal Ischemia-Reperfusion Injury.

Cells 2020 01 15;9(1). Epub 2020 Jan 15.

Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.

The current standard of care for acute myocardial infarction or 'heart attack' is timely restoration of blood flow to the ischemic region of the heart. While reperfusion is essential for the salvage of ischemic myocardium, re-introduction of blood flow paradoxically (rather than rescues) a population of previously ischemic cardiomyocytes-a phenomenon referred to as 'lethal myocardial ischemia-reperfusion (IR) injury'. There is long-standing and exhaustive evidence that mitochondria are at the nexus of lethal IR injury. However, during the past decade, the paradigm of mitochondria as mediators of IR-induced cardiomyocyte death has been expanded to include the highly orchestrated process of mitochondrial quality control. Our aims in this review are to: (1) briefly summarize the current understanding of the pathogenesis of IR injury, and (2) incorporating landmark data from a broad spectrum of models (including immortalized cells, primary cardiomyocytes and intact hearts), provide a critical discussion of the emerging concept that mitochondrial dynamics and mitophagy (the components of mitochondrial quality control) may contribute to the pathogenesis of cardiomyocyte death in the setting of ischemia-reperfusion.
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http://dx.doi.org/10.3390/cells9010214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7016592PMC
January 2020

Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.

FASEB J 2019 12 28;33(12):13503-13514. Epub 2019 Sep 28.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA.

Cytochrome (Cyt) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cyt in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects using phosphorylated Cyt purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cyt at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cyt, making it a good model system. Both S47-phosphorylated and phosphomimetic Cyt showed a lower oxygen consumption rate in reaction with isolated Cyt oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cyt showed lower caspase-3 activity. Furthermore, phosphomimetic Cyt had decreased cardiolipin peroxidase activity and is more stable in the presence of HO. Our data suggest that S47 phosphorylation of Cyt is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.
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http://dx.doi.org/10.1096/fj.201901120RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894086PMC
December 2019

Use of resuscitative balloon occlusion of the aorta in a swine model of prolonged cardiac arrest.

Resuscitation 2019 07 20;140:106-112. Epub 2019 May 20.

University of Michigan, Department of Emergency Medicine, United States; University of Michigan, Department of Biomedical Engineering, United States; University of Michigan, Department of Michigan Center for Integrative Research in Critical Care, United States. Electronic address:

Aim: We examined the use of a Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) catheter during cardiopulmonary resuscitation (CPR) after cardiac arrest (CA) to assess its effect on haemodynamics such as coronary perfusion pressure (CPP), common carotid artery blood flow (CCA-flow) and end-tidal CO (PetCO) which are associated with increased return of spontaneous circulation (ROSC).

Methods: Six male swine were instrumented to measure CPP, CCA-Flow, and PetCO. A 7Fr REBOA was advanced into zone-1 of the aorta through the femoral artery. Ventricular fibrillation was induced and untreated for 8 min. CPR (manual then mechanical) was initiated for 24 min. Continuous infusion of adrenaline (epinephrine) was started at minute-4 of CPR. The REBOA balloon was inflated at minute-16 for 3 min and then deflated/inflated every 3 min for 3 cycles. Animals were defibrillated up to 6 times after the final cycle. Animals achieving ROSC were monitored for 25 min.

Results: Data showed significant differences between balloon deflation and inflation periods for CPP, CCA-Flow, and PetCO (p < 0.0001) with an average difference (SD) of 13.7 (2.28) mmHg, 15.5 (14.12) mL min and -4 (2.76) mmHg respectively. Three animals achieved ROSC and had significantly higher mean CPP (54 vs. 18 mmHg), CCA-Flow (262 vs. 135 mL min) and PetCO (16 vs. 8 mmHg) (p < 0.0001) throughout inflation periods than No-ROSC animals. Aortic histology did not reveal any significant changes produced by balloon inflation.

Conclusion: REBOA significantly increased CPP and CCA-Flow in this model of prolonged CA. These increases may contribute to the ability to achieve ROSC.
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http://dx.doi.org/10.1016/j.resuscitation.2019.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157798PMC
July 2019

Non-invasive treatment with near-infrared light: A novel mechanisms-based strategy that evokes sustained reduction in brain injury after stroke.

J Cereb Blood Flow Metab 2020 04 21;40(4):833-844. Epub 2019 May 21.

Department of Emergency Medicine, University of Michigan, Ann Arbor, MI, USA.

Ischemic stroke is a debilitating disease that causes significant brain injury. While restoration of blood flow is critical to salvage the ischemic brain, reperfusion can exacerbate damage by inducing generation of reactive oxygen species (ROS). Recent studies by our group found that non-invasive mitochondrial modulation with near-infrared (NIR) light limits ROS generation following global brain ischemia. NIR interacts with cytochrome oxidase (COX) to transiently reduce COX activity, attenuate mitochondrial membrane potential hyperpolarization, and thus reduce ROS production. We evaluated a specific combination of COX-inhibitory NIR (750 nm and 950 nm) in a rat stroke model with longitudinal analysis of brain injury using magnetic resonance imaging. Treatment with NIR for 2 h resulted in a 21% reduction in brain injury at 24 h of reperfusion measured by diffusion-weighted imaging (DWI) and a 25% reduction in infarct volume measured by T2-weighted imaging (T2WI) at 7 and 14 days of reperfusion, respectively. Additionally, extended treatment reduced brain injury in the acute phase of brain injury, and 7 and 14 days of reperfusion, demonstrating a >50% reduction in infarction. Our data suggest that mitochondrial modulation with NIR attenuates ischemia-reperfusion injury and evokes a sustained reduction in infarct volume following ischemic stroke.
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http://dx.doi.org/10.1177/0271678X19845149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168789PMC
April 2020

Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis.

FASEB J 2019 02 17;33(2):1540-1553. Epub 2018 Sep 17.

Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Wayne State University, Detroit, Michigan, USA.

Cytochrome c (Cyt c) plays a vital role in the mitochondrial electron transport chain (ETC). In addition, it is a key regulator of apoptosis. Cyt c has multiple other functions including ROS production and scavenging, cardiolipin peroxidation, and mitochondrial protein import. Cyt c is tightly regulated by allosteric mechanisms, tissue-specific isoforms, and post-translational modifications (PTMs). Distinct residues of Cyt c are modified by PTMs, primarily phosphorylations, in a highly tissue-specific manner. These modifications downregulate mitochondrial ETC flux and adjust the mitochondrial membrane potential (ΔΨ), to minimize reactive oxygen species (ROS) production under normal conditions. In pathologic and acute stress conditions, such as ischemia-reperfusion, phosphorylations are lost, leading to maximum ETC flux, ΔΨ hyperpolarization, excessive ROS generation, and the release of Cyt c. It is also the dephosphorylated form of the protein that leads to maximum caspase activation. We discuss the complex regulation of Cyt c and propose that it is a central regulatory step of the mammalian ETC that can be rate limiting in normal conditions. This regulation is important because it maintains optimal intermediate ΔΨ, limiting ROS generation. We examine the role of Cyt c PTMs, including phosphorylation, acetylation, methylation, nitration, nitrosylation, and sulfoxidation and consider their potential biological significance by evaluating their stoichiometry.-Kalpage, H. A., Bazylianska, V., Recanati, M. A., Fite, A., Liu, J., Wan, J., Mantena, N., Malek, M. H., Podgorski, I., Heath, E. I., Vaishnav, A., Edwards, B. F., Grossman, L. I., Sanderson, T. H., Lee, I., Hüttemann, M. Tissue-specific regulation of cytochrome c by post-translational modifications: respiration, the mitochondrial membrane potential, ROS, and apoptosis.
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http://dx.doi.org/10.1096/fj.201801417RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338631PMC
February 2019

Publisher Correction: Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury.

Sci Rep 2018 Apr 25;8(1):6729. Epub 2018 Apr 25.

Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI, 48201, USA.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-25184-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916910PMC
April 2018

Inhibitory modulation of cytochrome c oxidase activity with specific near-infrared light wavelengths attenuates brain ischemia/reperfusion injury.

Sci Rep 2018 02 22;8(1):3481. Epub 2018 Feb 22.

Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI, 48201, USA.

The interaction of light with biological tissue has been successfully utilized for multiple therapeutic purposes. Previous studies have suggested that near infrared light (NIR) enhances the activity of mitochondria by increasing cytochrome c oxidase (COX) activity, which we confirmed for 810 nm NIR. In contrast, scanning the NIR spectrum between 700 nm and 1000 nm revealed two NIR wavelengths (750 nm and 950 nm) that reduced the activity of isolated COX. COX-inhibitory wavelengths reduced mitochondrial respiration, reduced the mitochondrial membrane potential (ΔΨ), attenuated mitochondrial superoxide production, and attenuated neuronal death following oxygen glucose deprivation, whereas NIR that activates COX provided no benefit. We evaluated COX-inhibitory NIR as a potential therapy for cerebral reperfusion injury using a rat model of global brain ischemia. Untreated animals demonstrated an 86% loss of neurons in the CA1 hippocampus post-reperfusion whereas inhibitory NIR groups were robustly protected, with neuronal loss ranging from 11% to 35%. Moreover, neurologic function, assessed by radial arm maze performance, was preserved at control levels in rats treated with a combination of both COX-inhibitory NIR wavelengths. Taken together, our data suggest that COX-inhibitory NIR may be a viable non-pharmacologic and noninvasive therapy for the treatment of cerebral reperfusion injury.
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http://dx.doi.org/10.1038/s41598-018-21869-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823933PMC
February 2018

Mitochondrial Quality Control and Disease: Insights into Ischemia-Reperfusion Injury.

Mol Neurobiol 2018 03 11;55(3):2547-2564. Epub 2017 Apr 11.

Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI, 48201, USA.

Mitochondria are key regulators of cell fate during disease. They control cell survival via the production of ATP that fuels cellular processes and, conversely, cell death via the induction of apoptosis through release of pro-apoptotic factors such as cytochrome C. Therefore, it is essential to have stringent quality control mechanisms to ensure a healthy mitochondrial network. Quality control mechanisms are largely regulated by mitochondrial dynamics and mitophagy. The processes of mitochondrial fission (division) and fusion allow for damaged mitochondria to be segregated and facilitate the equilibration of mitochondrial components such as DNA, proteins, and metabolites. The process of mitophagy are responsible for the degradation and recycling of damaged mitochondria. These mitochondrial quality control mechanisms have been well studied in chronic and acute pathologies such as Parkinson's disease, Alzheimer's disease, stroke, and acute myocardial infarction, but less is known about how these two processes interact and contribute to specific pathophysiologic states. To date, evidence for the role of mitochondrial quality control in acute and chronic disease is divergent and suggests that mitochondrial quality control processes can serve both survival and death functions depending on the disease state. This review aims to provide a synopsis of the molecular mechanisms involved in mitochondrial quality control, to summarize our current understanding of the complex role that mitochondrial quality control plays in the progression of acute vs chronic diseases and, finally, to speculate on the possibility that targeted manipulation of mitochondrial quality control mechanisms may be exploited for the rationale design of novel therapeutic interventions.
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http://dx.doi.org/10.1007/s12035-017-0503-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5636654PMC
March 2018

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

J Biol Chem 2017 Jan 7;292(1):64-79. Epub 2016 Oct 7.

From the Center for Molecular Medicine and Genetics and

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ hyperpolarization, a known cause of ROS and trigger of apoptosis.
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http://dx.doi.org/10.1074/jbc.M116.744664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217700PMC
January 2017

Mitochondrial dynamics following global cerebral ischemia.

Mol Cell Neurosci 2016 10 25;76:68-75. Epub 2016 Aug 25.

Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, MI 48201, United States; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201, United States. Electronic address:

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056829PMC
http://dx.doi.org/10.1016/j.mcn.2016.08.010DOI Listing
October 2016

Unfolding the unfolded protein response: unique insights into brain ischemia.

Int J Mol Sci 2015 Mar 30;16(4):7133-42. Epub 2015 Mar 30.

Cardiovascular Research Institute and Departments of Emergency Medicine and Physiology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

The endoplasmic reticulum (ER) is responsible for processing of proteins that are destined to be secreted, enclosed in a vesicle, or incorporated in the plasma membrane. Nascent peptides that enter the ER undergo a series of highly regulated processing steps to reach maturation as they transit the ER. Alterations in the intracellular environment that induce ER stress are thought to interrupt these processing steps, and result in unfolding of proteins in the ER. Accumulation of unfolded proteins concurrently activates three transmembrane stress sensors, IRE1, ATF6 and PERK, and is referred to as the Unfolded Protein Response (UPR). Our understanding of the mechanisms of UPR induction has been assembled primarily from experiments inducing ER stress with chemical and genetic manipulations. However, physiological stress often induces activation of ER stress sensors in a distinct manner from the canonical UPR. The unique activation profiles in vivo have prompted us to examine the mechanism of UPR activation in neurons following cerebral ischemia.
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http://dx.doi.org/10.3390/ijms16047133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425008PMC
March 2015

Release of mitochondrial Opa1 following oxidative stress in HT22 cells.

Mol Cell Neurosci 2015 Jan 8;64:116-22. Epub 2015 Jan 8.

Department of Emergency Medicine, Wayne State University School of Medicine, 550 E. Canfield, Detroit, MI, USA; Cardiovascular Research Institute, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI, USA; Department of Physiology, Wayne State University School of Medicine, 550 E. Canfield, Detroit, MI, USA. Electronic address:

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.
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http://dx.doi.org/10.1016/j.mcn.2014.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429534PMC
January 2015

Clinical benefits of remote ischemic preconditioning: new insights...and new questions.

Circ Res 2014 Feb;114(5):748-50

From the Cardiovascular Research Institute (K.P., T.H.S., M.H.), Center for Molecular Medicine and Genetics (K.P., M.H.), Department of Physiology (K.P., T.H.S.), and Department of Emergency Medicine (K.P., T.H.S.), Wayne State University School of Medicine, Detroit, MI.

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http://dx.doi.org/10.1161/CIRCRESAHA.114.303331DOI Listing
February 2014

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

PLoS One 2013 5;8(11):e78627. Epub 2013 Nov 5.

Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078627PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818486PMC
August 2014

2-vessel occlusion/hypotension: a rat model of global brain ischemia.

J Vis Exp 2013 Jun 22(76). Epub 2013 Jun 22.

Department of Emergency Medicine, Wayne State University School of Medicine.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia (1). Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion (2). The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) (2), as first presented in its current form in Sanderson, et al. (2008) (3), which produces reproducible injury to selectively vulnerable brain regions (3-6). The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time. In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.
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http://dx.doi.org/10.3791/50173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728756PMC
June 2013

Mitochondrial dynamics: an emerging paradigm in ischemia-reperfusion injury.

Curr Pharm Des 2013 ;19(39):6848-57

Cardiovascular Research Institute, Wayne State University School of Medicine, Elliman Building, Room 1107, 421 E Canfield, Detroit, MI 48201 USA.

Cardiomyocytes and neurons are highly susceptible to ischemia-reperfusion injury; accordingly, considerable effort has been devoted to elucidating the cellular mechanisms responsible for ischemia-reperfusion-induced cell death and developing novel strategies to minimize ischemia-reperfusion injury. Maintenance of mitochondrial integrity is, without question, a critical determinant of cell fate. However, there is emerging evidence of a novel and intriguing extension to this paradigm: mitochondrial dynamics (that is, changes in mitochondrial morphology achieved by fission and fusion) may play an important but as-yet poorly understood role as a determinant of cell viability. Focusing on heart and brain, our aims in this review are to provide a synopsis of the molecular mechanisms of fission and fusion, summarize our current understanding of the complex relationships between mitochondrial dynamics and the pathogenesis of ischemia-reperfusion injury, and speculate on the possibility that targeted manipulation of mitochondrial dynamics may be exploited for the design of novel therapeutic strategies for cardio- and neuroprotection.
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http://dx.doi.org/10.2174/138161281939131127110701DOI Listing
July 2014

Molecular mechanisms of ischemia-reperfusion injury in brain: pivotal role of the mitochondrial membrane potential in reactive oxygen species generation.

Mol Neurobiol 2013 Feb 26;47(1):9-23. Epub 2012 Sep 26.

Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Stroke and circulatory arrest cause interferences in blood flow to the brain that result in considerable tissue damage. The primary method to reduce or prevent neurologic damage to patients suffering from brain ischemia is prompt restoration of blood flow to the ischemic tissue. However, paradoxically, restoration of blood flow causes additional damage and exacerbates neurocognitive deficits among patients who suffer a brain ischemic event. Mitochondria play a critical role in reperfusion injury by producing excessive reactive oxygen species (ROS) thereby damaging cellular components, and initiating cell death. In this review, we summarize our current understanding of the mechanisms of mitochondrial ROS generation during reperfusion, and specifically, the role the mitochondrial membrane potential plays in the pathology of cerebral ischemia/reperfusion. Additionally, we propose a temporal model of ROS generation in which posttranslational modifications of key oxidative phosphorylation (OxPhos) proteins caused by ischemia induce a hyperactive state upon reintroduction of oxygen. Hyperactive OxPhos generates high mitochondrial membrane potentials, a condition known to generate excessive ROS. Such a state would lead to a "burst" of ROS upon reperfusion, thereby causing structural and functional damage to the mitochondria and inducing cell death signaling that eventually culminate in tissue damage. Finally, we propose that strategies aimed at modulating this maladaptive hyperpolarization of the mitochondrial membrane potential may be a novel therapeutic intervention and present specific studies demonstrating the cytoprotective effect of this treatment modality.
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http://dx.doi.org/10.1007/s12035-012-8344-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725766PMC
February 2013

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology.

FASEB J 2012 Sep 22;26(9):3916-30. Epub 2012 Jun 22.

Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.
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http://dx.doi.org/10.1096/fj.11-203273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425824PMC
September 2012

Phosphorylation of mammalian cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease.

Adv Exp Med Biol 2012 ;748:237-64

Wayne State University School of Medicine, Detroit, MI, USA.

The mitochondrial oxidative phosphorylation (OxPhos) system not only generates the vast majority of cellular energy, but is also involved in the generation of reactive oxygen species (ROS), and apoptosis. Cytochrome c (Cytc) and cytochrome c oxidase (COX) represent the terminal step of the electron transport chain (ETC), the proposed rate-limiting reaction in mammals. Cytc and COX show unique regulatory features including allosteric regulation, isoform expression, and regulation through cell signaling pathways. This chapter focuses on the latter and discusses all mapped phosphorylation sites based on the crystal structures of COX and Cytc. Several signaling pathways have been identified that target COX including protein kinase A and C, receptor tyrosine kinase, and inflammatory signaling. In addition, four phosphorylation sites have been mapped on Cytc with potentially large implications due to its multiple functions including apoptosis, a pathway that is overactive in stressed cells but inactive in cancer. The role of COX and Cytc phosphorylation is reviewed in a human disease context, including cancer, inflammation, sepsis, asthma, and ischemia/reperfusion injury as seen in myocardial infarction and ischemic stroke.
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http://dx.doi.org/10.1007/978-1-4614-3573-0_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3727645PMC
September 2012

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation.

Biochim Biophys Acta 2012 Apr 13;1817(4):598-609. Epub 2011 Jul 13.

Wayne State University School of Medicine, Detroit, MI 48201, USA.

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.
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http://dx.doi.org/10.1016/j.bbabio.2011.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3229836PMC
April 2012

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis.

Mitochondrion 2011 May 4;11(3):369-81. Epub 2011 Feb 4.

Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201, USA.

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.
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http://dx.doi.org/10.1016/j.mito.2011.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075374PMC
May 2011
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