Giancarlo Solaini

Hypoxia and mitochondrial oxidative metabolism

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondrias central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O2] sensors, convey signals to HIF-1 directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F1F0 ATPase in the hypoxia-induced mitochondrial autophagy.

Biochemical dysfunction in heart mitochondria exposed to ischaemia and reperfusion.

Heart tissue is remarkably sensitive to oxygen deprivation. Although heart cells, like those of most tissues, rapidly adapt to anoxic conditions, relatively short periods of ischaemia and subsequent reperfusion lead to extensive tissue death during cardiac infarction. Heart tissue is not readily regenerated, and permanent heart damage is the result. Although mitochondria maintain normal heart function by providing virtually all of the hearts ATP, they are also implicated in the development of ischaemic damage. While mitochondria do provide some mechanisms that protect against ischaemic damage (such as an endogenous inhibitor of the F1Fo-ATPase and antioxidant enzymes), they also possess a range of elements that exacerbate it, including ROS (reactive oxygen species) generators, the mitochondrial permeability transition pore, and their ability to release apoptotic factors. This review considers the process of ischaemic damage from a mitochondrial viewpoint. It considers ischaemic changes in the inner membrane complexes I-V, and how this might affect formation of ROS and high-energy phosphate production/degradation. We discuss the contribution of various mitochondrial cation channels to ionic imbalances which seem to be a major cause of reperfusion injury. The different roles of the H+, Ca2+ and the various K+ channel transporters are considered, particularly the K+(ATP) (ATP-dependent K+) channels. A possible role for the mitochondrial permeability transition pore in ischaemic damage is assessed. Finally, we summarize the metabolic and pharmacological interventions that have been used to alleviate the effects of ischaemic injury, highlighting the value of these or related interventions in possible therapeutics.

Oxidative phosphorylation in cancer cells.

Evidence suggests that mitochondrial metabolism may play a key role in controlling cancer cells life and proliferation. Recent evidence also indicates how the altered contribution of these organelles to metabolism and the resistance of cancer mitochondria against apoptosis-associated permeabilization are closely related. The hallmarks of cancer growth, increased glycolysis and lactate production in tumours, have raised attention due to recent observations suggesting a wide spectrum of oxidative phosphorylation deficit and decreased availability of ATP associated with malignancies and tumour cell expansion. More specifically, alteration in signal transduction pathways directly affects mitochondrial proteins playing critical roles in controlling the membrane potential as UCP2 and components of both MPTP and oxphos complexes, or in controlling cells life and death as the Bcl-2 proteins family. Moreover, since mitochondrial bioenergetics and dynamics, are also involved in processes of cells life and death, proper regulation of these mitochondrial functions is crucial for tumours to grow. Therefore a better understanding of the key pathophysiological differences between mitochondria in cancer cells and in their non-cancer surrounding tissue is crucial to the finding of tools interfering with these peculiar tumour mitochondrial functions and will disclose novel approaches for the prevention and treatment of malignant diseases. Here, we review the peculiarity of tumour mitochondrial bioenergetics and the mode it is linked to the cell metabolism, providing a short overview of the evidence accumulated so far, but highlighting the more recent advances.

Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its rho(0) counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated.

Protective effect of endogenous coenzyme Q on both lipid peroxidation and respiratory chain inactivation induced by an adriamycin-iron complex.

Mitochondria from beef heart have been partially depleted of coenzyme Q by pentane extraction. It has been found that lipid peroxidation induced by an adriamycin-iron complex proceeds at a higher rate in this preparation than in coenzyme Q reincorporated mitochondria. Moreover in coenzyme Q depleted mitochondria both NADH and succinate oxidase activities result more affected. These observations indicate that endogenous coenzyme Q can effectively protect mitochondria from membrane lipid oxidative damage induced by adriamycin-iron and can reduce the inactivation of NADH and succinate oxidases.

Increased state 4 mitochondrial respiration and swelling in early post‐ischemic reperfusion of rat heart

Isolated rat hearts were exposed to 30 min ischemia or to 30 min ischemia followed by 2, 5 or 40 min reperfusion and mitochondria were isolated at these different time points. ADP‐stimulated, succinate‐dependent respiration rate (state 3) was not significantly changed at the different time points examined. In contrast, state 4 (non‐ADP‐stimulated) respiration rate was significantly increased after 30 min ischemia, and it increased further during the first post‐ischemic reperfusion period. Mitochondrial swelling, as evaluated under conditions of the major controlled ion channels (i.e. permeability transition pore and ATP‐dependent mitochondrial K+ channel) closed, significantly increased in parallel. It is suggested that the inner mitochondrial membrane permeability is increased under exposure of the heart to ischemia and early reperfusion, and that the phenomenon is reversible upon subsequent long periods of reperfusion.

Glucose plays a main role in human fibroblasts adaptation to hypoxia

Hypoxia induces severe changes in cell biology, particularly affecting energy production pathways. Although the theme has been widely investigated, particularly in transformed cells, studies of the mitochondrial bioenergetics of normal cells exposed to both prolonged hypoxic periods and low or null glucose concentration have been scarcely addressed. To evaluate the mitochondrial changes of cells exposed to the latter conditions, we set experiments in which the contribution of the mitochondrial energy production was maximized at the expenses of low glycolysis. Human fibroblasts were exposed to 1% oxygen tension, a hypoxic condition experimentally established for these cells, and grown in glucose deficient media. At variance with fibroblasts grown in 5-25 mM glucose, fibroblasts grown in glucose deficiency adapted to hypoxia by reducing only slightly the mitochondrial mass and preserving a well structured network as it occurs in normoxia. Moreover, the oxidative phosphorylation (OXPHOS) rate of the mitochondria was enhanced, due to increased OXPHOS complexes level. The master transcriptional modulator induced by hypoxia HIF-1α and BNIP3, a factor activating mitochondrial autophagy, were expressed both in the presence and in the absence of glucose, but to a lower level in the latter condition. Similarly, the microtubule-associated protein light chain 3 active fragment (LC3-II), a typical marker of autophagy, was found less expressed in glucose-free medium than in the presence of glucose. Therefore, our data show for the first time that glucose availability significantly affects the hypoxia-induced HIF-1/BNIP3 response, and in particular glucose absence results in enhancing the OXPHOS rate.

Cytochrome b of fish mitochondria is strongly resistant to funiculosin, a powerful inhibitor of respiration

We report here some unusual properties of ubiquinol: cytochrome c reductase of eel and other fish mitochondria. The turnover rate of the reductase is clearly higher than in mammalian mitochondria and the binding constant for ubiquinone seems to be larger than in other vertebrates. Additionally, the reductase activity of fish mitochondria is resistant to some powerful inhibitors that bind to cytochrome b, in particular to funiculosin. After sequencing most of the gene of eel cytochrome b and comparing the deduced amino acid sequence with that of other fish and animals, we hypothesize that the decreased binding of funiculosin could be due to a few amino acid replacements in the third and fourth transmembrane helix of the protein. In particular, the presence of methionine instead of alanine at position 125 seems to be largely responsible for the strong resistance to funiculosin and also to the partial resistance to myxothiazol in all fish mitochondria. Correlations between some residue substitutions in cytochrome b and the different effects of funiculosin in different species are also considered.

Effects of niridazole and 5-nitroimidazoles on heart mitochondrial respiration

2.3 micromoles/mg protein of MFNI induced a 60% decrease in the heart mitochondrial ADP-stimulated oxygen uptake using glutamate-malate as substrate. The same amount of niridazole, ipronidazole, DA 3851 and ornidazole led to falls of less than 20% in the oxygen uptake, whilst metronidazole was ineffective. State 3 and state 3 mu (uncoupled) respiration were affected to the same extent. Oxygen-uptake using succinate as substrate was not inhibited indicating that the action was exerted at the NADH oxidation level. The relationship between electroreduction potentials of the test compounds and inhibition of respiration has been studied.