C. Vijayasarathy

Retrograde Ca2+ signaling in C2C12 skeletal myocytes in response to mitochondrial genetic and metabolic stress: a novel mode of inter‐organelle crosstalk

We have investigated the mechanism of mitochondrial–nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m‐chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψm) and electron transport‐coupled ATP synthesis, which was also accompanied by an elevated steady‐state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor‐1 (RyR‐1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+‐responsive factors calineurin, calcineurin‐dependent NFATc (cytosolic counterpart of activated T‐cell‐specific nuclear factor) and c‐Jun N‐terminal kinase (JNK)‐dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF‐κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady‐state [Ca2+]i were normalized in genetically reverted cells which contain near‐normal mtDNA levels. We propose that the mitochondria‐to‐nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Mitochondria-to-nucleus stress signaling induces phenotypic changes, tumor progression and cell invasion.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial‐specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic‐free Ca2+ [Ca2+]c. In the present study we show that the mitochondria‐to‐nucleus stress signaling induces invasive phenotypes in otherwise non‐invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor‐specific markers cathepsin L and transforming growth factor β (TGFβ) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4‐ to 6‐fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca2+‐dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with ∼70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

Preferential effects of nicotine and 4-(N-methyl- N-nitrosamino)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase a4-4 induction and increased oxidative stress in the rat brain

We have investigated the in vivo effects of the tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on antioxidant defense systems in the mitochondrial, microsomal, and cytosolic compartments of rat brain, lung, and liver. Nicotine induced maximum oxidative stress in brain mitochondria, as seen from a 1.9-fold (P < 0.001) increase in thiobarbituric acid-reactive substance (TBARS) and a 2-fold (P < 0.001) increase in glutathione S-transferase (GST) A4-4 (also referred to as rGST 8-8) activities. These changes were accompanied by a 25-40% increase in reactive oxygen species and a 20-30% decrease in alcohol dehydrogenase activities. The 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-induced oxidative damage was apparent in the microsomal fraction of brain, lung, and liver, and it also increased 4-hydroxynonenal specific GST A4-4 activity in the brain and lung mitochondrial matrix fraction. The levels of microsomal thiobarbituric acid reactive substance, cytochrome P4502E1 activity, and reactive oxygen species were also increased significantly (P < 0.001) in all tissues. Both of these toxins induced the level of GST A4-4 mRNA in the brain, while they caused a marked reduction in the liver GST A4-4 mRNA pool. Additionally, the brain mitochondrial matrix showed a markedly higher level of 4-hydroxynonenal specific GST activity and mGST A4-4 antibody-reactive protein than did the cytosolic fraction. In conclusion, the present study provides evidence for the occurrence of GST A4-4 enzyme activity in mammalian mitochondria, in addition to demonstrating that both mitochondria and microsomes are intracellular targets for nicotine- and NNK-induced organ toxicity.

Competitive and noncompetitive inhibition of myocardial cytochrome C oxidase in sepsis

Sepsis is the most common cause of death in intensive care units worldwide. The basic pathophysiologic defect in sepsis, causing functional abnormalities in many organ systems, remains elusive. One potential cause is disruption of oxidative phosphorylation in mitochondria. Here, we report that oxidation of cytochrome c by myocardial cytochrome c oxidase, the terminal oxidase in the electron transport chain, is competitively inhibited early in experimental sepsis (cecal ligation with single or double 23-gauge puncture) in mice. In severe sepsis (cecal ligation and double puncture, 75% mortality at 48 h), inhibition becomes noncompetitive by 48 h. The development of noncompetitive inhibition is associated with a decrease in heme a,a3 content, which is the key active site in the functional subunit (I) and catalyzes the reduction of molecular oxygen. In addition, there are persistently decreased steady-state levels of subunit I mRNA and protein after cecal ligation and double puncture. Both loss of heme and loss of subunit I could explain the observed irreversible inhibition of cytochrome c oxidase. Noncompetitive inhibition of cytochrome c oxidase may interrupt oxidative phosphorylation, leading to sepsis-associated cardiac depression. Importantly, this abnormality may underlie sepsis-associated dysfunction in other organ systems.

Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments

The composition and activity of cytochrome c oxidase (COX) was studied in mitochondria from rat liver, brain, kidney and heart and also in different compartments of the bovine heart to see whether any correlation exists between known oxidative capacity and COX activity. Immunoblot analysis showed that the levels of ubiquitously expressed subunits IV and Vb are about 8-12-fold lower in liver mitochondria as compared to the heart, kidney and brain. The heart enzyme with higher abundance of COX IV and Vb showed lower turnover number (495) while the liver enzyme with lower abundance of these subunits exhibited higher turnover number of 750. In support of the immunoblot results, immunohistochemical analysis of heart and kidney tissue sections showed an intense staining with the COX Vb antibody as compared to the liver sections. COX Vb antibody stained certain tubular regions of the kidney more intensely than the other regions suggesting region specific variation in the subunit level. Bovine heart compartments showed variation in subunit levels and also differed in the kinetic parameters of COX. The right atrium contained relatively more Vb protein, while the left ventricle contained higher level of subunit VIa. COX from both the ventricles showed high Km for cytochrome c (23-37 microM) as compared to the atrial COX (Km 8-15 microM). These results suggest a correlation between tissue specific oxidative capacity/work load and changes in subunit composition and associated changes in the activity of COX complex. More important, our results suggest variations based on the oxidative load of cell types within a tissue.

Physiological Role of the N-terminal Processed P4501A1 Targeted to Mitochondria in Erythromycin Metabolism and Reversal of Erythromycin-mediated Inhibition of Mitochondrial Protein Synthesis

Recently, we showed that the major species of β-naphthoflavone-inducible rat liver mitochondrial P450MT2 consists of N-terminal truncated microsomal P4501A1 (+33/1A1) and that the truncated enzyme exhibits different substrate specificity as compared with intact P4501A1. The results of the present study show that P450MT2 targeted to COS cell mitochondria by transient transfection of P4501A1 cDNA is localized inside the mitochondrial inner membrane in a membrane-extrinsic orientation. Co-expression with wild type P4501A1 and adrenodoxin (Adx) cDNAs resulted in 5–7-fold higher erythromycin N-demethylation (ERND) in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells. Erythromycin, a potent inhibitor of bacterial and mitochondrial protein synthesis, caused 8–12-fold higher accumulation of CYP1A1 mRNA, preferential accumulation of P450MT2, and 5–6-fold higher ERND activity in the mitochondrial compartment of rat C6 glioma cells. Consistent with the increased mitochondrial ERND activity, co-expression with P4501A1 and Adx in COS cells rendered complete protection against erythromycin-mediated mitochondrial translation inhibition. Mutations that specifically affect the mitochondrial targeting of P4501A1 also abolished protection against mitochondrial translation inhibition. These results for the first time suggest a physiological function for the xenobiotic inducible cytochrome P4501A1 against drug-mediated mitochondrial toxicity.

Canine mitochondrial myopathy associated with reduced mitochondrial mRNA and altered cytochrome c oxidase activities in fibroblasts and skeletal muscle.

Skeletal muscle and fibroblast biopsies obtained from a normal dog and an old English sheep dog with exertional myopathy and lactic acidosis were examined for mitochondrial enzyme activities and mitochondrially coded mRNAs. The fibroblast cultures of the affected dog showed reduced cytochrome c oxidase (COX) I+II mRNA content (25% of control) and COX enzyme activities (23% of control). The skeletal muscle of the affected dog was similarly affected and showed not only decreased COX I+II mRNA content, but also decreased ATPase6 mRNA level. Apart from COX enzyme activity (62% of control), the oligomycin sensitive ATPase and NADH-Ferricyanide reductase activities were also reduced in the skeletal muscle of the affected dog (12-20% of control). These results suggest that a mitochondrial dysfunction may be the causative factor of the exertional metabolic myopathy with lactic acidosis in this affected old English sheep dog. These animals may serve as an excellent model for mitochondrial myopathies.

FUNCTIONAL CHARACTERIZATION OF A HUMAN DNASE-LIKE PROTEIN ENCODED BY A GENE POSITIONED IN XQ28

Xib, a gene recently reported to reside on the q28 region of the human X chromosome [Pergolizzi et al. (1996) Gene 168, 267-270], contains an open reading frame homologous to those of the DNase I family enzymes. The full open reading frame of this gene has been fused to the E. coli gene of the maltose binding protein and expressed in bacteria as a chimeric protein. The partially purified chimeric protein is enzymatically active. It introduces single and double stranded breaks into supercoiled DNA, at 30 degrees C in the absence of divalent cations and at a pH optimum of 5.2. To our knowledge this enzyme represents the first cloned human endonuclease with characteristics similar to those of acidic DNase II.

Inhibition of mitochondrial translation by calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide

The possible role of calmodulin in mitochondrial functions was investigated in Ehrlich ascites tumor cell and mouse liver mitochondria employing sulfonamide compounds as calmodulin indicators. N-[6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), the most potent of the sulfonamide compounds, inhibited mitochondrial protein synthesis and oxidative phosphorylation. The inhibitors had no significant effect on mitochondrial cytochrome c oxidase, oligomycin-sensitive ATPase and NADH dehydrogenase activities. Depletion of endogenous ATP pool seemed to be the main mechanism of inhibition of mitochondrial translation by sulfonamides. The results also show that mitochondria from hepatic tissues are relatively less sensitive to sulfonamide drugs as compared to the Ehrlich ascites tumor cell mitochondria. Results of Ca2+ autoradiography revealed 2-3-fold higher levels of calmodulin-like Ca2+ binding protein in extracts from Ehrlich ascites tumor cell mitoplasts as compared to mitoplasts from mouse liver. These results suggest cell and tissue specific variations in Ca(2+)-dependent processes in the mitochondrial compartment.