Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 µM low-dose range (LDR) of CCCP. Within the 20-50 µM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage.

Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice.

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28h; p=0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to ß-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.

Loss of myocardial protection against myocardial infarction in middle-aged transgenic mice overexpressing cardiac thioredoxin-1.

Thioredoxin-1 (Trx1) protects the heart from ischemia/reperfusion (I/R) injury. Given that the age at which the first episode of coronary disease takes place has considerably decreased, life at middle-aged (MA) emerges as a new field of study. The aim was determine whether infarct size, Trx1 expression and activity, Akt and GSK-3ß were altered in young (Y) and MA mice overexpressing cardiac Trx1, and in a dominant negative (DN-Trx1) mutant of Trx1. Langendorff-perfused hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (R). We used 3 and 12 month-old male of wild type (WT), Trx1, and DN-Trx1. Trx1 overexpression reduced infarct size in young mice (WT-Y: 46.8±4.1% vs. Trx1-Y: 27.6±3.5%, p < 0.05). Trx1 activity was reduced by 52.3±3.2% (p < 0.05) in Trx1-MA, accompanied by an increase in nitration by 17.5±0.9%, although Trx1 expression in transgenic mice was similar between young and middle-aged. The expression of p-Akt and p-GSK-3ß increased during reperfusion in Trx1-Y. DN-Trx1 mice showed neither reduction in infarct size nor Akt and GSK-3ß phosphorylation. Our data suggest that the lack of protection in Trx1 middle-aged mice even with normal Trx1 expression may be associated to decreased Trx1 activity, increased nitration and inhibition of p-Akt and p-GSK-3ß.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.

Diastolic function during hemorrhagic shock in rabbits.

Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.

Defective leptin-AMP-dependent kinase pathway induces nitric oxide release and contributes to mitochondrial dysfunction and obesity in ob/ob mice.

AIMS: Obesity arises on defective neuroendocrine pathways that increase energy intake and reduce mitochondrial metabolism. In the metabolic syndrome, mitochondrial dysfunction accomplishes defects in fatty acid oxidation and reciprocal increase in triglyceride content with insulin resistance and hyperglycemia. Mitochondrial inhibition is attributed to reduced biogenesis, excessive fission, and low adipokine-AMP-activated protein kinase (AMPK) level, but lateness of the respiratory chain contributes to perturbations. Considering that nitric oxide (NO) binds cytochrome oxidase and inhibits respiration, we explored NO as a direct effector of mitochondrial dysfunction in the leptin-deficient ob/ob mice. RESULTS: A remarkable three- to fourfold increase in neuronal nitric oxide synthase (nNOS) expression and activity was detected by western blot, citrulline assay, electronic and confocal microscopy, flow cytometry, and NO electrode sensor in mitochondria from ob/ob mice. High NO reduced oxygen uptake in ob/ob mitochondria by inhibition of complex IV and nitration of complex I. Low metabolic status restricted ß-oxidation in obese mitochondria and displaced acetyl-CoA to fat synthesis; instead, small interference RNA nNOS caused a phenotype change with fat reduction in ob/ob adipocytes. INNOVATION: We evidenced that leptin increases mitochondrial respiration and fat utilization by potentially inhibiting NO release. Accordingly, leptin administration to ob/ob mice prevented nNOS overexpression and mitochondrial dysfunction in vivo and rescued leptin-dependent effects by matrix NO reduction, whereas leptin-Ob-Rb disruption increased the formation of mitochondrial NO in control adipocytes. We demonstrated that in ob/ob, hypoleptinemia is associated with critically low mitochondrial p-AMPK and that, oppositely to p-Akt2, p-AMPK is a negative modulator of nNOS. CONCLUSION: Thereby, defective leptin-AMPK pathway links mitochondrial NO to obesity with complex I syndrome and dysfunctional mitochondria.

Mitochondrial nitric oxide synthase: a masterpiece of metabolic adaptation, cell growth, transformation, and death.

Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)-dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome.

BACKGROUND: In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO(2) and H(2)O or to drive glucose storage to glycogen. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4-8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (-25 to -60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-(14)C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37+/-3 vs 10+/-1 micromolO(2)/h.g tissue and 13+/-1 vs 7.2+/-1 micromol (3)H(2)O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half. CONCLUSIONS/SIGNIFICANCE: These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis.

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.

The biological significance of mtNOS modulation.

In the last years, nitric oxide synthases (NOS) have been localized in mitochondria. At this site, NO yield directly regulates the activity of cytochrome oxidase, O(2) uptake and the production of reactive oxygen species. Recent studies showed that translocated neuronal nitric oxide synthase (nNOS) is posttranslationally modified including phosphorylation at Ser 1412 (in mice) and myristoylation in an internal residue. Different studies confirm that modified nNOS alpha is the main modulable isoform in mitochondria. Modulation of mtNOS was observed in different situations, like adaptation to reduced O(2) availability and hypoxia, adaptation to low environmental temperature, and processes linked to life and death by effects on kinases and transcription factors. We present here evidence about the role of mtNOS in the analyzed conditions.

Hypothyroid phenotype is contributed by mitochondrial complex I inactivation due to translocated neuronal nitric-oxide synthase.

Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-alpha (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3',5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype.

Mitochondrial nitric oxide synthase drives redox signals for proliferation and quiescence in rat liver development.

Mitochondrial nitric oxide synthase (mtNOS) is a fine regulator of oxygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cycle progression. From this perspective, we examined liver mtNOS modulation and mitochondrial redox changes in developing rats from embryonic days 17-19 and postnatal day 2 (proliferating hepatocyte phenotype) through postnatal days 15-90 (quiescent phenotype). mtNOS expression and activity were almost undetectable in fetal liver, and progressively increased after birth by tenfold up to adult stage. NO-dependent mitochondrial hydrogen peroxide (H(2)O(2)) production and Mn-superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolic H(2)O(2) concentration ([H(2)O(2)](ss)) and cell fluorescence. mtNOS-dependent [H(2)O(2)](ss) was a good predictor of extracellular signal-regulated kinase (ERK)/p38 activity ratio, cyclin D1, and tissue proliferation. At low 10(-11)-10(-12) M [H(2)O(2)](ss), proliferating phenotypes had high cyclin D1 and phospho-ERK1/2 and low phospho-p38 mitogen-activated protein kinase, while at 10(-9) M [H(2)O(2)](ss), quiescent phenotypes had the opposite pattern. Accordingly, leading postnatal day 2-isolated hepatocytes to embryo or adult redox conditions with H(2)O(2) or NO-H(2)O(2) scavengers, or with ERK inhibitor U0126, p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio, cyclin D1 expression, and [(3)H] thymidine incorporation in the cells. Accordingly, p38 inhibitor SB202190 or N-acetyl-cysteine prevented H(2)O(2) inhibitory effects on proliferation. In conclusion, the results suggest that a synchronized increase of mtNOS and derived H(2)O(2) operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence.

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease.

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.

Sepsis: citoquinas, radicales libres y óxido nítrico / Sepsis: cytokine, free radicals and nitric oxide

La sepsis y sus complicaciones, el shock séptico y el síndrome de disfunción orgánica múltiple (MODS - multiple organ dysfunction syndrome) mantienen desde hace años el triste privilegio de ser las primeras causas de muerte en las salas de terapia intensiva y postquirúrgica; el aumento de su incidencia está en relación con el desarrollo de procedimientos más invasivos, los tratamientos inmunosupresores, la quimioterapia, la mayor edad de los enfermos, los síndromes de inmunodeficiencia y las floras hospitalarias multirresistentes. Se estima en 400.000 el número de pacientes afectados anualmente en los Estados Unidos y, a pesar de sofisticados y extremadamente caros procedimientos de sostén vital y de los antibióticos, la mortalidad no ha disminuido en los últimos años. Probablemente, esta detención en el progreso terapéutico, se deba a la extrema complejidad de los mecanismos patogénicos en juego y a lo incompleto de su conocimiento y comprensión. El problema de las infecciones graves y de la sepsis (del griego putrefacción), es antiguo y acompaña al hombre desde sus orígenes remotos, como ejemplo de lo cual, basta recordar la peste, la fiebre tifoidea, la gangrena, la peritonitis y las infecciones puerperales. En realidad se trata de un enfrentamiento ancestral entre bacterias y organismos superiores, en el que, desafortunadamente, a menudo triunfan las primeras (AU)

Sepsis: citoquinas, radicales libres y óxido nítrico / Sepsis: cytokine, free radicals and nitric oxide

La sepsis y sus complicaciones, el shock séptico y el síndrome de disfunción orgánica múltiple (MODS - multiple organ dysfunction syndrome) mantienen desde hace años el triste privilegio de ser las primeras causas de muerte en las salas de terapia intensiva y postquirúrgica; el aumento de su incidencia está en relación con el desarrollo de procedimientos más invasivos, los tratamientos inmunosupresores, la quimioterapia, la mayor edad de los enfermos, los síndromes de inmunodeficiencia y las floras hospitalarias multirresistentes. Se estima en 400.000 el número de pacientes afectados anualmente en los Estados Unidos y, a pesar de sofisticados y extremadamente caros procedimientos de sostén vital y de los antibióticos, la mortalidad no ha disminuido en los últimos años. Probablemente, esta detención en el progreso terapéutico, se deba a la extrema complejidad de los mecanismos patogénicos en juego y a lo incompleto de su conocimiento y comprensión. El problema de las infecciones graves y de la sepsis (del griego putrefacción), es antiguo y acompaña al hombre desde sus orígenes remotos, como ejemplo de lo cual, basta recordar la peste, la fiebre tifoidea, la gangrena, la peritonitis y las infecciones puerperales. En realidad se trata de un enfrentamiento ancestral entre bacterias y organismos superiores, en el que, desafortunadamente, a menudo triunfan las primeras