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In this in vitro study we induced ischemic injury on H9c2 rat cardiomyoblasts using the oxygen-glucose deprivation model (OGD). We monitored if the addition of healthy or mitochondria-depleted cells can save OGD treated cells from post-ischemic injury. We were able to significantly improve the surviving cell number of oxidatively damaged H9c2 cells by the addition of healthy cells to the culture. On the contrary, cells with disturbed mitochondria did not increase the number of surviving cells. High-resolution confocal time-lapse imaging also proved that mitochondria are drifting from cell-to-cell through tunneling membrane bridges, however, they do not get into the cytoplasm of the other cell. We conclude that addition of healthy cells to severly injured post-ischemic cardiomyoblasts can rescue them from death during the first 24h after reoxigenation. Grafted cells must maintain their mitochondria in an actively respiring state, and although cell contact is required for the mechanism, neither cell fusion nor organelle transfer occurs. This novel mechanism opens a new possiblity for cell-based cardiac repair in ischemic heart disease., (Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.) more...

Poly(ADP-ribose) polymerase (PARP) is an intracellular enzyme involved in DNA repair and in building poly-ADP-ribose polymers on nuclear proteins using NAD(+). While the majority of PARP resides in the nucleus, several studies indicated that PARP may also be located in the cytosol or in the mitochondrial matrix. In this study we found several poly-ADP-ribosylated proteins in isolated rat liver mitochondria following hydrogen peroxide (H(2)O(2)) or nitric oxide donor treatment. Protein poly-ADP-ribosylation was more intense in isolated mitochondria than in whole tissue homogenates and it was not associated with increased nuclear PARP activity. We identified five poly-ADP-ribose (PAR) positive mitochondrial bands by protein mass fingerprinting. All of the identified enzymes exhibited decreased activity or decreased levels following oxidative or nitrosative stress. One of the identified proteins is dihydrolipoamide dehydrogenase (DLDH), a component of the alpha-ketoglutarate dehydrogenase (KGDH) complex, which uses NAD(+) as a substrate. This raised the possibility that KGDH may have a PARP-like enzymatic activity. The intrinsic PARP activity of KGDH and DLDH was confirmed using a colorimetric PARP assay kit and by the incubation of the recombinant enzymes with H(2)O(2). The KGDH enzyme may, therefore, have a novel function as a PARP-like enzyme, which may play a role in regulating intramitochondrial NAD(+) and poly(ADP-ribose) homeostasis, with possible roles in physiology and pathophysiology. more...

New discoveries in the last decade significantly altered our view on mitochondria. They are no longer viewed as energy-making slaves but rather individual cells-within-the-cell. In particular, it has been suggested that many important cellular mechanisms involving specific enzymes and ion channels, such as nitric oxide synthase (NOS), ATP-dependent K+ (KATP) channels, and poly-(APD-ribose) polymerase (PARP), have a distinct, mitochondrial variant. Unfortunately, exploring these parallel systems in mitochondria have technical limitations and inappropriate methods often led to inconsistent results. For example, the intriguing possibility that mitochondria are significant sources of nitric oxide (NO) via a unique mitochondrial NOS variant has attracted intense interest among research groups because of the potential for NO to affect functioning of the electron transport chain. Nonetheless, conclusive evidence concerning the existence of mitochondrial NO synthesis is yet to be presented. This review summarizes the experimental evidence gathered over the last decade in this field and highlights new areas of research that reveal surprising dimensions of NO production and metabolism by mitochondria. more...

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS. more...

It is more than 10 years now that mitochondria are suspected to be sources of nitric oxide (NO). This hypothesis is intriguing since NO has multiple targets within the organelle and it is even suggested that mitochondria are the primary targets of NO in the cell. Most remarkably, nanomolar concentrations of NO can inhibit mitochondrial respiration, so even a small amount of NO in the mitochondrial matrix may regulate ATP synthesis. Therefore, the idea that mitochondria themselves are capable of NO production is an important concept in several physiological and pathological mechanisms. However, this field of research generates surprisingly few original papers and the published studies contain conflicting results. The reliability of the results is frequently questioned since they are seldom reproduced by independent investigators. Until 2003, all papers published in this field showed affirmative results but since then several studies directly challenged the existence of a mitochondrial nitric oxide synthase. The present review aims to summarize the most recent developments in mitochondrial NO production, highlights a few unsolved questions, and proposes new directions for future work in this research area. more...

Mitochondrial responses to ischemic stress play an important role in necrosis and apoptosis of brain cells. Recent studies using several different experimental preparations have shown that activation of ATP-sensitive potassium channels in mitochondria (mitoK(ATP) channels) is able to protect neurons and astroglia against injury and death. Thus, targeting of mitoK(ATP) channels appears to be a novel approach to neuroprotection. However, little is known about the mechanisms involved. The purpose of this review is to detail the current state of knowledge about this important, emerging area of investigation, and to provide suggestions for future studies. more...

Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism. more...

Activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) has been shown to protect against cell death following ischemia/reperfusion in the heart but not in brain. We examined whether mK(ATP) activation with diazoxide (DIZ) prevents neuronal cell death following hypoxia-ischemia (HI) in 7-day-old rat pups. Rat pups were subjected to HI (left carotid ligation; 8% O(2); 2.5 h), following administration of vehicle, 1.9 mg/kg DIZ, 3.8 mg/kg DIZ or DIZ plus 10 mg/kg 5-hydroxydecanoic acid (mK(ATP) antagonist). Total infarct volume was reduced from 99.8+/-2.7% in vehicle animals to 80.6+/-4.2% in 3.8 mg/kg DIZ treated animals (n=85, P<0.05). Western blotting showed K(ATP) subunits concentrated in mitochondria. Fluorescent studies indicated DIZ directly depolarized the mitochondria. In conclusion, selective opening of mK(ATP) prior to HI results in neuroprotection in immature rats. more...

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K(ATP) channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia. more...