Stimulation Of Mitochondrial Respiration Research Articles

Ketone body β-hydroxybutyrate (BHB) preserves mitochondrial bioenergetics

The ketogenic diet is an emerging therapeutic approach for refractory epilepsy, as well as certain rare and neurodegenerative disorders. The main ketone body, β-hydroxybutyrate (BHB), is the primary energy substrate endogenously produced in a ketogenic diet, however, mechanisms of its therapeutic actions remain unknown. Here, we studied the effects of BHB on mitochondrial energetics, both in non-stimulated conditions and during glutamate-mediated hyperexcitation. We found that glutamate-induced hyperexcitation stimulated mitochondrial respiration in cultured cortical neurons, and that this response was greater in cultures supplemented with BHB than with glucose. BHB enabled a stronger and more sustained maximal uncoupled respiration, indicating that BHB enables neurons to respond more efficiently to increased energy demands such as induced during hyperexcitation. We found that cytosolic Ca2+ was required for BHB-mediated enhancement of mitochondrial function, and that this enhancement was independent of the mitochondrial glutamate-aspartate carrier, Aralar/AGC1. Our results suggest that BHB exerts its protective effects against hyperexcitation by enhancing mitochondrial function through a Ca2+-dependent, but Aralar/AGC1-independent stimulation of mitochondrial respiration.

Ultrastructure of Myofascial Trigger Points of Skeletal Muscles of Rats after Photobiostimulation with Low-Intensity Red Light.

The effectiveness of low-intensity red light on myofascial trigger points in skeletal muscle of mature rats was evaluated by electron microscopy and high-resolution respirometry. The revealed changes in mitochondrial ultrastructure and activity of the respiratory chain enzymes indicate the development of hypoxia in the simulation area. Under the influence of low-intensity red light on myofascial trigger points, a decrease in the number of destructively altered muscle fibers and stimulation of mitochondrial respiration were found. These findings indicate intracellular regeneration and the stimulating effect of low-intensity red light on plastic processes.

Erythrocyte Respiratory Activity of the Mitochondrial Complex of the Black Sea Thornback Ray (Raja clavata L.) under the Influence of Certain Activators and Inhibitors In Vitro

Global warming implies the risk of a changing oxygen regime in the seas and oceans of our planet. The mitochondrial complex of nuclear erythrocytes of cartilaginous fish, as the energy basis of blood cells, has repeatedly encountered such climatic fluctuations throughout their evolutionary history. In this regard, the features of the adaptive strategy of the erythrocyte mitochondrial complex in the thornback ray (Raja clavata L.) are of interest from the evolutionary and ecological points of view. The rate of oxygen consumption in resuspended (Ht = 25–30%) erythrocytes taken from the Black Sea thornback ray in saline was studied by the polarographic method. A high “basal” rate of respiration in the erythrocytes of the thornback ray was shown, which ranged from 10.5 to 21.6 pmol O2 min−1·106 cells. The addition of substrates of the mitochondrial respiration activators glutamate, maleate, and succinate to the erythrocyte suspension caused a 2–6-fold increase in the respiratory activity of thornback ray erythrocytes. In cases where the rate of respiration of erythrocytes was high, protonophore–dinitrophenol caused an inhibition of the activity of mitochondrial respiration. At low respiration rates of erythrocytes, its effect was opposite and caused a stimulation of mitochondrial respiration. Oligomycin caused a significant inhibition of the respiratory activity of the red blood cell suspension of the thornback ray. This suppression of cell respiration was enhanced under conditions of exposure to the permeabilization of erythrocytes with digitonin. This can be recommended as one of the ways to block the respiratory activity of erythrocytes in cartilaginous fish. Another way of effectively blocking the respiration of the mitochondrial complex of the thornback ray’s erythrocytes was the effect of the blockers rotenone and sodium azide. The peculiarity of the mitochondria of the erythrocytes of the thornback ray was the absence of the complete inhibition of respiration by sodium azide (NaN3), which is characteristic of the mitochondria of other fish species. Our data on the activation of the “respiration” of erythrocytes in fish indicate that the potential capabilities of cold-blooded and warm-blooded vertebrates have rather similar characteristics. This may indicate the initial “laying” of the architecture of the inner membrane to support the energy potential of the mitochondria of the cell.

Antibiotic Pyrrolomycin as an Efficient Mitochondrial Uncoupler.

Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.

Tranexamic acid: Beyond antifibrinolysis.

Tranexamic acid (TXA) is a popular antifibrinolytic drug widely used in hemorrhagic trauma patients and cardiovascular, orthopedic, and gynecological surgical patients. TXA binds plasminogen and prevents its maturation to the fibrinolytic enzyme plasmin. A number of studies have demonstrated the broad life-saving effects of TXA in trauma, superior to those of other antifibrinolytic agents. Besides preventing fibrinolysis and blood loss, TXA has been reported to suppress posttraumatic inflammation and edema. Although the efficiency of TXA transcends simple inhibition of fibrinolysis, little is known about its mechanisms of action besides the suppression of plasmin maturation. Understanding the broader effects of TXA at the cell, organ, and organism levels are required to elucidate its potential mechanisms of action transcending antifibrinolytic activity. In this article, we provide a brief review of the current clinical use of TXA and then focus on the effects of TXA beyond antifibrinolytics such as its anti-inflammatory activity, protection of the endothelial and epithelial monolayers, stimulation of mitochondrial respiration, and suppression of melanogenesis.

Stimulation of Mitochondrial Respiration by Idebenone in Pro-inflammatory Microglia is Catalyzed by iNOS

Stimulation of Mitochondrial Respiration by Idebenone in Pro-inflammatory Microglia is Catalyzed by iNOS

Activity of Respiratory Pathways in Cultured Yam Cells under the Influence of Furostanol Glycosides

The role of furostanol glycosides (FGs) in the intensification of main metabolic processes in cultured yam (Dioscorea deltoidea Wall.) cells was shown. The effects of FGs were associated with changes in the complicated network of intracellular enzymatic reactions. This was evidenced by an increase in the activities of the enzymes from the glucose glycolytic oxidation pathway (hexokinase and fructose-1,6-difosphate aldolase) and the malate dehydrogenase complex (oxidoreductive—NADH/NAD-malate dehydrogenase and malic enzyme—decarboxylating NADP-malate dehydrogenase) as well as the stimulation of mitochondrial respiration accompanied by changes in activity of the alternative respiratory pathway. At the lag-phase of the growth cycle, the cell treatment with FGs was capable to guide glucose conversion through the glycolytic pathway. This was witnessed by a decrease in the activity of glucose-6-phosphate dehydrogenase and increase in that of hexokinase. Inhibition of NADPH oxidase afforded by diphenyliodonium chloride resulted in a decrease in activities of the key enzymes of glycolysis and the pentose phosphate cycle of glucose oxidation. Simultaneously, the aldolase activity increased and raised the content of its product glyceraldehyde 3-phosphate. The effect may explain the earlier-observed activation of the aldehyde-utilizing enzymes diminishing the initial POL level. The modulating action of FGs on activities of the enzymes of sugar glycolytic oxidation was shown. This was expressed as the activation of the tested enzymes at the lag- and exponential phases of the cellular growth and as the absence of the FG effect at the stationary phase. The short-term exposure of yam cells to FGs intensified the total mitochondrial respiration. An inhibitory analysis revealed the dominance of the cytochrome respiration pathway at the lag-phase, while its contribution became considerably lower at the later growth phases. FGs did not affect the rate of this respiration path but increased the potential activity of alternative oxidase (AO) during the lag- and exponential phases of the growth cycle. The FG-induced activation of AO, which may be intended to maintain a balance between carbohydrate metabolism and the rate of electron transport, is discussed.

ω-Hydroxypalmitic and α,ω-Hexadecanedioic Acids As Activators of Free Respiration and Inhibitors of H2O2 Generation in Liver Mitochondria

The effects of palmitic acid (PA) and the products of its ω-oxidation–ω-hydroxypalmitic acid (HPA) and α,ω-hexadecanedioic acid (HDA)—on respiration in the absence of ATP synthesis and on the H2O2 generation were studied on isolated liver mitochondria energized by oxidation of succinate. It was shown that HPA stimulates respiration and reduces the difference in electrical potentials (Δψ) in mitochondria to the same extent as is typical for PA and classical protonophore uncouplers. Under similar conditions, the stimulation of mitochondrial respiration by HDA is not accompanied by a decrease in Δψ and is suppressed by 10 μM cyclosporin A. In this regard, HDA is considered as a “decoupling” agent that switches the complexes of the mitochondrial respiratory chain to the idle mode. It was established that in the presence of 20 mM potassium chloride in the sucrose incubation medium, stimulation of respiration by HPA (in contrast to PA and HDA) is suppressed by 60% in the case of pre-incubation with 3 mM magnesium chloride. We found that under these conditions, HPA at a concentration of 25 μM and above induces mitochondrial swelling, which is completely suppressed by magnesium chloride. HPA as an inducer of mitochondrial swelling is less efficient in the absence of potassium chloride than in its presence. We assume that, in contrast to PA and HDA, HPA produces its activating influence on free respiration in mitochondria owing to a combination of its ionophore and protonophore effects. It was established that PA, HDA, and HPA in concentrations equally stimulating respiration of mitochondria in state 2 are equally efficient in inhibiting the H2O2 generation. We conclude that the effects of these fatty acids as inhibitors of H2O2 generation in isolated liver mitochondria are due to stimulation of respiration in various ways and are not associated with a decrease in Δψ.

The Role of Calcium in the Regulation of Mitochondrial Respiration under Various Substrate Combinations in the Heart and Kidney

It has been recognized that the efficiency of mitochondrial oxidative phosphorylation (OxPhos) and energy metabolism depends on the choice of respiratory substrates, which feed electrons to the mitochondrial electron transport chain. In addition, mitochondrial enzymes, such as pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and alpha‐ketoglutarate dehydrogenase (AKGDH), have been shown to be stimulated by calcium ions (Ca2+) in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on the efficiency of mitochondrial OxPhos and energy metabolism in in‐situ and in‐vivo states in the heart and kidney under different substrate combinations are not well‐understood. In this study, we investigated the effect of a variety of substrate combinations and Ca2+ alternations that optimize mitochondrial respiration and ATP synthesis. We hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is both substrate dependent and tissue specific. To study mitochondrial function under a specific substrate combination and Ca2+, cardiac and renal mitochondria were isolated from Sprague‐Dawley rats, and oxygen consumption rates (JO2) were measured in the presence and absence of ADP that corresponded to leak (state 2) and ADP‐stimulated (state 3) states, respectively. We first measured the time courses of JO2 at various ADP concentrations to find the saturated ADP‐stimulated JO2. This saturated ADP concentration was then used to measure the time‐courses of JO2 under different [Ca2+]. The substrate combinations used include pyruvate + malate (PM), glutamate + malate (GM), alpha‐ketoglutarate + malate (AM), palmitoyl‐L‐carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). Different substrates led to different levels of the saturated state 3 JO2. On average, it was 9 times higher in the heart compared to the kidney with the PM substrate. Also, the alterations in the respiratory substrates led to dramatically different respiratory rates/dynamics in the isolated mitochondria of the chosen organs. The results also demonstrated that Ca2+ significantly increased mitochondrial state 3 JO2 with GM and AM substrates compared to the other substrates in both organs (Table 1). Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increase in [Ca2+] had a stimulatory effect while large increases had inhibitory effects (Figure 1). These results suggest that energized isolated mitochondria in the presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, proper [Ca2+], and specific tissue source of mitochondria to achieve an optimal level of OxPhos and energy metabolism comparable to in‐vivo conditions.Support or Funding InformationThis work was supported by the NIH grants P01‐GM066730, P01‐HL116264, R01‐HL‐122662, and U01‐HL122199.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Deregulation and Mitochondrial Bioenergetics in GDAP1-Related CMT Disease.

The pathology of Charcot-Marie-Tooth (CMT), a disease arising from mutations in different genes, has been associated with an impairment of mitochondrial dynamics and axonal biology of mitochondria. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause several forms of CMT neuropathy, but the pathogenic mechanisms involved remain unclear. GDAP1 is an outer mitochondrial membrane protein highly expressed in neurons. It has been proposed to play a role in different aspects of mitochondrial physiology, including mitochondrial dynamics, oxidative stress processes, and mitochondrial transport along the axons. Disruption of the mitochondrial network in a neuroblastoma model of GDAP1-related CMT has been shown to decrease Ca2+ entry through the store-operated calcium entry (SOCE), which caused a failure in stimulation of mitochondrial respiration. In this review, we summarize the different functions proposed for GDAP1 and focus on the consequences for Ca2+ homeostasis and mitochondrial energy production linked to CMT disease caused by different GDAP1 mutations.

A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector

BackgroundLimited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C10) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). MethodsProton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. ResultsWe compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C4-mitoFluo). MitoFluo, but not C4-mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C4-mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C4-mitoFluo. MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. ConclusionsA long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. General significanceMitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.

β‐cell Deletion of Nr4a1 and Nr4a3 Nuclear Receptors Impedes Mitochondrial Respiration and Insulin Secretion

Insulin secretion from β‐cells is dependent on the stimulation of mitochondrial respiration in response to an increase in glucose supply. Various studies have clearly demonstrated that the Nr4a family of orphan nuclear receptors are essential for fuel utilization and mitochondrial function in liver, muscle and fat. We have previously demonstrated that overexpression of Nr4a1 and Nr4a3 are sufficient to induce proliferation of pancreatic β‐cells. We examined the impact of nuclear receptors Nr4a1 and Nr4a3 on pancreatic β‐cell mitochondrial function and proliferation using lentivirus mediated shRNA knock‐down of Nr4a1 and Nr4a3 in INS‐1 derived 832/13 rat insulinoma cells. β‐cells deficient in Nr4a1 and Nr4a3 have a 37% reduction in proliferation rates (p<0.05). The reduction in β‐cell proliferation with Nr4a1 and Nr4a3 deficiency was accompanied by a significant decrease in glucose stimulated insulin secretion rates (GSIS) (26% decrease, p<0.05). GSIS was determined in cells preincubated in secretion assay buffer (SAB) for 1.5 hours containing 2.5 mM glucose followed by incubation in SAB with 16.7 mM glucose. High resolution respirometry (Oroboros O2k) was used to evaluate mitochondrial respiratory function and capacity with substrate‐uncoupler‐inhibitor titration protocols for both intact and permeabilized β‐cells. Permeabilized cells allow for an in depth assessment of chemiosmotic coupling within the β‐cells while the intact protocol evaluates a more physiologically relevant condition. We have shown that deletions of the Nr4a nuclear receptors clearly impact cellular respiration, lowering the oxygen consumption rate in all respiratory states of intact β‐cells between 40–65% and permeabilized β‐cells by 23–50% compared to GFP (p<0.05). Our results indicate that knockdown of Nr4a1 and Nr4a3 results in decreased expression Idh3a, Idh3g, Sdhb, and Mdh2, which would impede TCA cycle progression, and is consistent with the reduction observed in O2 consumption, ATP production and ultimately glucose stimulated insulin secretion. These data demonstrate for the first time that the Nr4a orphan nuclear receptor family members Nr4a1 and Nr4a3 are critical for basal β‐cell mitochondrial function and insulin secretion.Support or Funding InformationBrigham Young University

Glutamine and succinate stabilize shelf-life color of frozen and thawed meat by stimulation of mitochondrial respiration

Glutamine and succinate stabilize shelf-life color of frozen and thawed meat by stimulation of mitochondrial respiration

Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

The myeloid translocation gene 16 (MTG16) co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1) heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1). Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs) in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.

Protection against neurodegeneration with low-dose methylene blue and near-infrared light.

Neurons are metabolically protected against degeneration using low-level methylene blue and near-infrared light interventions. Both of these novel interventions act by a cellular mechanism involving enhancement of the electron transport chain in mitochondria, which promotes energy metabolism and neuronal survival (Gonzalez-Lima et al., 2014). Methylene blue preferentially enters neuronal mitochondria after systemic administration, and at low-doses forms an electron cycling redox complex that donates electrons to the mitochondrial electron transport chain. Low-level near-infrared light applied transcranially delivers photons to cortical neurons that are accepted by cytochrome oxidase, which causes increased cell respiration and cerebral blood flow. Breakthrough in vivo studies with these interventions suggest that targeting mitochondrial respiration may be beneficial for protection against different types of neurodegenerative disorders. The purpose of this paper is to provide an update on the cellular mechanisms mediating the neuroprotective effects of low doses of methylene blue and near-infrared light, and to argue that the neurotherapeutic benefits of these two different interventions share the same cellular mechanism of action based on stimulation of mitochondrial respiration. Presented first is the explanation of the biochemical redox action of low-dose methylene as an electron cycler on the mitochondrial electron transport. Presented second is the explanation of the biophysical action of near-infrared light as a photon donor to cytochrome oxidase that also serves to stimulate mitochondrial electron transport. We finish with a comparison of these two interventions and how they share a common cellular mechanism with similar properties such as energy transfer, low-dose hormetic dose-responses, and enhanced capacity for oxidative metabolic energy production, which serve to protect nervous tissue from degeneration.

Heat Shock Protein 22 (Hsp22) Regulates Oxidative Phosphorylation upon Its Mitochondrial Translocation with the Inducible Nitric Oxide Synthase in Mammalian Heart

ObjectivesStress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 overexpression in vivo stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo significantly reduces respiration. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS.Methods and ResultsAdenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a N-terminal 20 amino acid putative mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, WT-Hsp22 accumulated in mitochondria by 2.5 fold (P<0.05) and increased oxygen consumption rates by 2-fold (P<0.01). This latter effect was abolished upon addition of the selective iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2.5-fold (P<0.01), and also increased iNOS mitochondrial localization by 4.5 fold vs β-gal (P<0.05). Upon comparable overexpression, the N20-Hsp22 mutant did not show significant mitochondrial translocation or stimulation of mitochondrial respiration. Moreover, although N20-Hsp22 did increase global iNOS expression by 4.6-fold, it did not promote iNOS mitochondrial translocation.ConclusionTranslocation of both Hsp22 and iNOS to the mitochondria is necessary for Hsp22-mediated stimulation of oxidative phosphorylation.

Low-level laser (light) therapy increases mitochondrial membrane potential and ATP synthesis in C2C12 myotubes with a peak response at 3-6 h.

Low-level laser (light) therapy has been used before exercise to increase muscle performance in both experimental animals and in humans. However, uncertainty exists concerning the optimum time to apply the light before exercise. The mechanism of action is thought to be stimulation of mitochondrial respiration in muscles, and to increase adenosine triphosphate (ATP) needed to perform exercise. The goal of this study was to investigate the time course of the increases in mitochondrial membrane potential (MMP) and ATP in myotubes formed from C2C12 mouse muscle cells and exposed to light-emitting diode therapy (LEDT). LEDT employed a cluster of LEDs with 20 red (630 ± 10 nm, 25 mW) and 20 near-infrared (850 ± 10 nm, 50 mW) delivering 28 mW cm(2) for 90 s (2.5 J cm(2)) with analysis at 5 min, 3 h, 6 h and 24 h post-LEDT. LEDT-6 h had the highest MMP, followed by LEDT-3 h, LEDT-24 h, LEDT-5 min and Control with significant differences. The same order (6 h > 3 h > 24 h > 5 min > Control) was found for ATP with significant differences. A good correlation was found (r = 0.89) between MMP and ATP. These data suggest an optimum time window of 3-6 h for LEDT stimulate muscle cells.

Abstract LB-58: Role of mitochondrial ROS in activation of glioma stem cells and promotion of cancer development

Abstract Glioblastoma stem cells (GSCs) play a major role in brain tumor development, and they can be induced to undergo apparent differentiation morphology by serum. It is generally assumed that induction of differentiation by serum in cancer stem cells would reduce their ability to form tumors. However, how serum triggers such change in GSC differentiation status and whether it really affects their ability to form tumor remain unclear. Here we show that serum exposure activates mitochondrial respiration and causes an increase in mitochondrial ROS and stress responses, leading to NFκB activation and down-regulation of stem cell markers including CD133, Olig2, SOX2 and Notch1. Chemical perturbation of mitochondrial electron transport chain causes ROS increase in serum-induced GSCs and further decreases stem cell markers, while antioxidants reduce ROS and prevent the loss of stem markers. Surprisingly, the serum-induced GSCs exhibit greater ability to form tumor in both orthotopic and subcutaneous xenograft models, which can be suppressed by antioxidants. Our study suggests that stimulation of mitochondrial respiration and ROS generation is critical in activation of GSCs and promoting tumor development in vivo. Although exposure of glioma stem cells to serum causes a decrease in stem cell markers and a loss of neurosphere morphology, the seemingly differentiated GSCs surprisingly exhibit greater ability to form tumors in vivo. A decrease in expression of certain stem cell markers such as CD133 and SOX2 may not necessarily indicate a decrease in tumorigenicity in vivo, and that redox signaling may play a major role in regulation of tumor formation from cancer stem cells. Citation Format: Feng Wang, Shuqiang Yuan, Gang Chen, Sangbae Kim, Jing Yang, Feng Li, Ju-Seog Lee, Xiao-Nan Li, Rui-hua Xu, Peng Huang. Role of mitochondrial ROS in activation of glioma stem cells and promotion of cancer development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2014-LB-58

Abstract 28: Increased Expression Of Mitochondrial Inducible Nitric Oxide Synthase By Heat Shock Protein Hsp22/h11 Kinase Promotes Oxidative Phosphorylation In Mammalian Heart

Aims: Stress-inducible heat shock protein 22 (Hsp22) confers protection against ischemia through induction of the inducible isoform of nitric oxide synthase (iNOS). Hsp22 over-expression in vivo significantly stimulates cardiac mitochondrial respiration, whereas Hsp22 deletion in vivo shows a reciprocal effect. It has also been shown in Drosophila that Hsp22 is expressed in the mitochondria that depends on its N-terminal domain. We hypothesized that Hsp22-mediated regulation of mitochondrial function is dependent upon its mitochondrial translocation together with iNOS. Methods and Results: Adenoviruses harboring either the full coding sequence of Hsp22 (Ad-WT-Hsp22) or a mutant lacking a 20 amino acid putative N-terminal mitochondrial localization sequence (Ad-N20-Hsp22) were generated, and infected in rat neonatal cardiomyocytes. Compared to β-Gal control, Ad-WT-Hsp22 accumulated in mitochondria by 2.5 fold (P&lt;0.05), reduced chelerythrine-induced apoptosis by 60% (P&lt;0.01), and increased oxygen consumption rate by 2-fold (P&lt;0.01). This latter effect was abolished upon addition of the specific iNOS inhibitor, 1400W. Ad-WT-Hsp22 significantly increased global iNOS expression by about 2-fold (P&lt;0.01), and also increased its mitochondrial localization by 2.5 fold vs β-gal (P&lt;0.05). Upon comparable over-expression, the Ad-N20-Hsp22 mutant did not show significant mitochondrial translocation, protection against apoptosis or stimulation of mitochondrial respiration. Although Ad-N20-Hsp22 did increase global iNOS expression by 6-fold it did not significantly promote iNOS mitochondrial translocation. Conclusion: Translocation of both Hsp22 and iNOS to the mitochondria is necessary for the stimulation of oxidative metabolism and protection against apoptosis.

Comparative Study of Changes in Energy Metabolism in Rat Cardiomyocytes in Postinfarction Cardiosclerosis and Diabetes Mellitus

Cardiomyocyte energy metabolism in experimental unfolding postinfarction cardiosclerosis and diabetes mellitus was studied. Postinfarction cardiosclerosis formed 6 weeks after coronary artery occlusion. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (60 mg/kg). The rate of oxygen consumption in postinfarction cardiosclerosis and diabetes increased by 3.4 and 4.2 times, respectively. Stimulation of mitochondrial respiration (ATP, palmitic acid) significantly increased oxygen consumption in animals with postinfarction cardiosclerosis and significantly reduced this process in diabetes. The content of LDH and SDH in the myocardium of animals with diabetes and postinfarction cardiosclerosis was significantly below the control. Hence, the development of postinfarction cardiosclerosis and diabetes mellitus were characterized by reduced generation of ATP in anaerobic and aerobic pathways and oxidative phosphorylation in cardiomyocytes.