Changes In Mitochondrial Properties Research Articles

Lineage-specific changes in mitochondrial properties during neural stem cell differentiation.

Neural stem cells (NSCs) reside in discrete regions of the adult mammalian brain where they can differentiate into neurons, astrocytes, and oligodendrocytes. Several studies suggest that mitochondria have a major role in regulating NSC fate. Here, we evaluated mitochondrial properties throughout NSC differentiation and in lineage-specific cells. For this, we used the neurosphere assay model to isolate, expand, and differentiate mouse subventricular zone postnatal NSCs. We found that the levels of proteins involved in mitochondrial fusion (Mitofusin [Mfn] 1 and Mfn 2) increased, whereas proteins involved in fission (dynamin-related protein 1 [DRP1]) decreased along differentiation. Importantly, changes in mitochondrial dynamics correlated with distinct patterns of mitochondrial morphology in each lineage. Particularly, we found that the number of branched and unbranched mitochondria increased during astroglial and neuronal differentiation, whereas the area occupied by mitochondrial structures significantly reduced with oligodendrocyte maturation. In addition, comparing the three lineages, neurons revealed to be the most energetically flexible, whereas astrocytes presented the highest ATP content. Our work identified putative mitochondrial targets to enhance lineage-directed differentiation of mouse subventricular zone-derived NSCs.

Abstract 12789: Time-Dependent Changes in Mitochondrial Properties and Function in Dogs During Electrically Maintained Atrial Fibrillation

Introduction: Atrial fibrillation (AF) causes atrial remodeling, leading to progressive resistance to therapy. Changes in cell metabolism likely contribute to remodeling; here, we studied the nature and time course of mitochondrial (MITO) changes during AF . Methods: Atrial cardiomyocytes were isolated from control dogs and dogs kept in AF for 24 hrs, 3 days, 1 and 3 wks by atrial tachypacing at 600 bpm (N=10/group). MITO were visualized with transmission electron microscopy. [Ca 2+ ] Mito (Rhod-2 AM), MITO ROS production (MitoSOX), membrane potential (αψ, JC-1), mPTP opening (TMRM) and FAD autofluorescence were measured via confocal microscope, NADH autofluorescence was determined under UV light. Protein expression was assayed by western blotting. Results: MITO elongation occurred following 24-hr and 3-day AF, while 3-week AF induced MITO fragmentation, rupture and shrinking (A). MITO fusion protein (MFN1) decreased from 3 days to 3 wks of AF and fission protein pDRP1ser-616 was increased at 1 and 3 wks of AF, potentially accounting for MITO fragmentation. [Ca 2+ ] Mito increased progressively with AF-time (B). MITO ROS production increased at 24 hr AF, and remained elevated from 3 days to 3 wks of AF (C). SOD2 was reduced after 3 days AF. NOX4 expression was increased at 3 wks AF. mPTP opening frequency and duration increased progressively during AF (D). AF increased JC-1 fluorescence, indicating depolarized αψ, beginning 24 hours after AF-onset (E). NADH increased by 15% at 24 hr AF, and then decreased progressively with AF-time (F). FAD was decreased during AF. Mitotempo (5-μM) reversed AF-induced atrial-cell I CaL downregulation (G) and reduced triggered activity (H). Conclusions: MITO are activated early during AF, but then show progressive changes likely due to ROS autotoxicity. MITO dysfunction and impaired bioenergetics may contribute to AF maintenance/ progress. MITO-targeted antioxidant intervention may have potential as a novel therapeutic approach for AF.

The crucial roles of mitochondria in supporting C4 photosynthesis.

C4 photosynthesis involves a series of biochemical and anatomical traits that significantly improve plant productivity under conditions that reduce the efficiency of C3 photosynthesis. We explore how evolution of the three classical biochemical types of C4 photosynthesis (NADP-ME, NAD-ME and PCK types) has affected the functions and properties of mitochondria. Mitochondria in C4 NAD-ME and PCK types play a direct role in decarboxylation of metabolites for C4 photosynthesis. Mitochondria in C4 PCK type also provide ATP for C4 metabolism, although this role for ATP provision is not seen in NAD-ME type. Such involvement has increased mitochondrial abundance/size and associated enzymatic capacity, led to changes in mitochondrial location and ultrastructure, and altered the role of mitochondria in cellular carbon metabolism in the NAD-ME and PCK types. By contrast, these changes in mitochondrial properties are absent in the C4 NADP-ME type and C3 leaves, where mitochondria play no direct role in photosynthesis. From an eco-physiological perspective, rates of leaf respiration in darkness vary considerably among C4 species but does not differ systematically among the three C4 types. This review outlines further mitochondrial research in key areas central to the engineering of the C4 pathway into C3 plants and to the understanding of variation in rates of C4 dark respiration.

HIF-1α/Actl6a/H3K9ac axis is critical for pluripotency and lineage differentiation of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are important models for analyzing cellular metabolism and individual development. As a hypoxia-inducible factor subunit, HIF-1α plays an important role in maintaining the pluripotency of PSCs under hypoxic conditions. However, the mechanisms underlying the self-renewal and pluripotency maintenance of human induced pluripotent stem cells (hiPSCs) via regulating HIF-1α largely remain elusive. In this study, we found that disrupting the expression of HIF-1α reduced self-renewal and pluripotency of hiPSCs. Additionally, HIF-1α-knockdown led to lower mitochondrial membrane potential (ΔΨm ) and higher reactive oxygen species production in hiPSCs. However, HIF-1α-overexpression increased ATP content in hiPSCs, while the role of HIF-1α-knockdown was opposite. The embryoid body (EB) and teratoma formation assays showed that HIF-1α-knockdown promoted endoderm differentiation and development in vitro and in vivo. In terms of the underlying molecular mechanisms, HIF-1α-knockdown inhibited the expression of Actl6a and histone H3K9ac acetylation (H3K9ac). Actl6a knockdown reduced the expression of H3K9ac and the pluripotency of hiPSCs, and also affected endoderm differentiation. These data suggest that hindering HIF-1α expression causes the changes in mitochondrial properties and metabolic disorders in hiPSCs. Furthermore, HIF-1α affects hiPSC pluripotency, and germ layer differentiation via Actl6a and histone acetylation.

Changes in mitochondrial properties may contribute to enhanced resistance to ischemia-reperfusion injury in the diabetic rat heart.

Diabetes mellitus, besides having deleterious effects, induces cardiac adaptation that may reduce the heart's susceptibility to ischemia-reperfusion (IR) injury. This study aimed to investigate whether changes in mitochondrial properties are involved in the mechanisms of increased resistance of the diabetic heart to IR. Adult male Wistar rats were made diabetic by a single dose of streptozotocin (65 mg·kg-1, i.p.), and on the day 8, Langendorff-perfused hearts were subjected to 30 min global ischemia and 40 min reperfusion. Baseline preischemic parameters in the diabetic hearts did not differ markedly from those in the nondiabetic controls, except for lower left ventricular developed pressure, higher mitochondrial membrane fluidity, and protein levels of manganese superoxide dismutase. On the other hand, diabetic hearts showed significantly better post-IR functional restoration and reduced arrhythmogenesis associated with lower reactive oxygen species production as compared with healthy controls. IR decreased membrane fluidity in both experimental groups; however, it led to a complete recovery of mitochondrial Mg2+-ATPase activity in diabetics in contrast to its reduction in nondiabetics. These findings indicate that the heart may become adapted to diabetes-induced alterations that might increase its tolerance to an ischemic insult. Preserved mitochondrial function might play a role in the mechanisms of the heart's resistance to IR injury in diabetics.

Mechanisms and costs of mitochondrial thermal acclimation in a eurythermal killifish (Fundulus heteroclitus)

Processes acting at the level of the mitochondria have been suggested to affect the thermal limits of organisms. To determine whether changes in mitochondrial properties could underlie shifts in thermal limits, we examined how mitochondrial properties are affected by thermal acclimation in the eurythermal killifish, Fundulus heteroclitus - a species with substantial plasticity in whole-organism thermal limits. We hypothesized that thermal acclimation would result in functional changes in the mitochondria that could result in trade-offs in function during acute thermal shifts. We measured the mitochondrial respiration rate (V̇O2 ) through multiple complexes of the electron transport system following thermal acclimation (to 5, 15, 33°C) and assessed maintenance of mitochondrial membrane potential (Δp) and rates of reactive oxygen species (ROS) production as an estimate of costs. Acclimation to 5°C resulted in a modest compensation of mitochondrial respiration at low temperatures, but these mitochondria were able to maintain Δp with acute exposure to high temperatures, and ROS production did not differ between acclimation groups, suggesting that these increases in mitochondrial capacity do not alter mitochondrial thermal sensitivity. Acclimation to 33°C suppressed mitochondrial respiration as a result of effects on NADH dehydrogenase (complex I). These high-temperature acclimated fish nonetheless maintained levels of Δp and ROS production similar to those of the other acclimation groups. This work demonstrates that killifish mitochondria can successfully acclimate to a wide range of temperatures without incurring major functional trade-offs during acute thermal shifts and that high-temperature acclimation results in a suppression of metabolism, consistent with patterns observed at the organismal level.

Genetic inactivation of mitochondria-targeted redox enzyme p66ShcA preserves neuronal viability and mitochondrial integrity in response to oxidative challenges

Mitochondria are essential to neuronal viability and function due to their roles in ATP production, intracellular calcium regulation, and activation of apoptotic pathways. Accordingly, mitochondrial dysfunction has been indicated in a wide variety of neurodegenerative diseases, including Alzheimer's disease (AD), Huntington's disease, amyotrophic lateral sclerosis, stroke, and multiple sclerosis (MS). Recent evidence points to the permeability transition pore (PTP) as a key player in mitochondrial dysfunction in these diseases, in which pathologic opening leads to mitochondrial swelling, rupture, release of cytochrome c, and neuronal death. Reactive oxygen species (ROS), which are inducers of PTP opening, have been prominently implicated in the progression of many of these neurodegenerative diseases. In this context, inactivation of a mitochondria-targeted redox enzyme p66ShcA (p66) has been recently shown to prevent the neuronal cell death leading to axonal severing in the murine model of MS, experimental autoimmune encephalomyelitis (EAE). To further characterize the response of neurons lacking p66, we assessed their reaction to treatment with stressors implicated in neurodegenerative pathways. Specifically, p66-knockout (p66-KO) and wild-type (WT) neurons were treated with hydrogen peroxide (H2O2) and nitric oxide (NO), and assessed for cell viability and changes in mitochondrial properties, including morphology and ROS production. The results showed that p66-KO neurons had greater survival following treatment with each stressor and generated less ROS when compared to WT neurons. Correspondingly, mitochondria in p66-KO neurons showed diminished morphological changes in response to these challenges. Overall, these findings highlight the importance of developing mitochondria-targeted therapeutics for neurodegenerative disorders, and emphasize p66, mitochondrial ROS, and the PTP as key targets for maintaining mitochondrial and neuronal integrity.

Effects of temperature and cadmium exposure on the mitochondria of oysters (Crassostrea virginica) exposed to hypoxia and subsequent reoxygenation

Intertidal bivalves are commonly exposed to multiple stressors including periodic hypoxia, temperature fluctuations and pollution, which can strongly affect energy metabolism. We used top-down control and elasticity analyses to determine the interactive effects of intermittent hypoxia, cadmium (Cd) exposure and acute temperature stress on mitochondria of the eastern oyster Crassostrea virginica. Oysters were acclimated at 20°C for 30 days in the absence or presence of 50 μg l(-1) Cd and then subjected to a long-term hypoxia (6 days at <0.5% O(2) in seawater) followed by normoxic recovery. Mitochondrial function was assessed at the acclimation temperature (20°C), or at elevated temperature (30°C) mimicking acute temperature stress in the intertidal zone. In the absence of Cd or temperature stress, mitochondria of oysters showed high resilience to transient hypoxia. In control oysters at 20°C, hypoxia/reoxygenation induced elevated flux capacity of all three studied mitochondrial subsystems (substrate oxidation, phosphorylation and proton leak) and resulted in a mild depolarization of resting mitochondria. Elevated proton conductance and enhanced capacity of phosphorylation and substrate oxidation subsystems may confer resistance to hypoxia/reoxygenation stress in oyster mitochondria by alleviating production of reactive oxygen species and maintaining high aerobic capacity and ATP synthesis rates during recovery. Exposure to environmental stressors such as Cd and elevated temperatures abolished the putative adaptive responses of the substrate oxidation and phosphorylation subsystems, and strongly enhanced proton leak in mitochondria of oysters subjected to hypoxia/reoxygenation stress. Our findings suggest that Cd exposure and acute temperature stress may lead to the loss of mitochondrial resistance to hypoxia and reoxygenation and thus potentially affect the ability of oysters to survive periodic oxygen deprivation in coastal and estuarine habitats.

Differentiation‐related changes in mitochondrial properties as indicators of stem cell competence

Several methods may be used to assess stem cell competence, including the expression of cell surface markers and telomerase activity. We hypothesized that mitochondrial characteristics might be an additional and reliable way to verify stem cell competence. In a multipotent, adult monkey stromal stem cell line, previously shown to differentiate into adipocytes, chondrocytes, and osteocytes, we found that several mitochondrial properties change with increasing passage number in culture. Cells from the earliest passage (P11) versus those from a later passage (P17) are characterized by: (a) a much higher percentage of cells (85% vs. 18%) with a perinuclear arrangement of mitochondria; (b) a much lower percentage of cells (1% vs. 57%) with an aggregated mitochondrial arrangement, in which mitochondria appear to coalesce into large clumps; (c) a much lower percentage of cells with lipid droplets (1% vs. 36%), suggesting less differentiation into adipocytes; (d) a 5.6-fold lower ATP content per cell (0.45 vs. 2.51 pmoles ATP/cell; and (e) a 10-fold higher rate of oxygen consumption (37.8 vs. 3.8 nmoles O2/min/10(3) cells), indicating a higher metabolic activity. Collectively, these data indicate that the perinuclear arrangement of mitochondria, accompanied by a low ATP/cell content and a high rate of oxygen consumption, may be valid indicators of stem cell differentiation competence, while departures from this profile indicate that cells are differentiating or perhaps becoming senescent. These results represent the first characterization of mitochondrial properties reported for a primate stem cell line.

Metabolic responses to low temperature in fish muscle.

For most fish, body temperature is very close to that of the habitat. The diversity of thermal habitats exploited by fish as well as their capacity to adapt to thermal change makes them excellent organisms in which to examine the evolutionary and phenotypic responses to temperature. An extensive literature links cold temperatures with enhanced oxidative capacities in fish tissues, particularly skeletal muscle. Closer examination of inter-species comparisons (i.e. the evolutionary perspective) indicates that the proportion of muscle fibres occupied by mitochondria increases at low temperatures, most clearly in moderately active demersal species. Isolated muscle mitochondria show no compensation of protein-specific rates of substrate oxidation during evolutionary adaptation to cold temperatures. During phenotypic cold acclimation, mitochondrial volume density increases in oxidative muscle of some species (striped bass Morone saxatilis, crucian carp Carassius carassius), but remains stable in others (rainbow trout Oncorhynchus mykiss). A role for the mitochondrial reticulum in distributing oxygen through the complex architecture of skeletal muscle fibres may explain mitochondrial proliferation. In rainbow trout, compensatory increases in the protein-specific rates of mitochondrial substrate oxidation maintain constant capacities except at winter extremes. Changes in mitochondrial properties (membrane phospholipids, enzymatic complement and cristae densities) can enhance the oxidative capacity of muscle in the absence of changes in mitochondrial volume density. Changes in the unsaturation of membrane phospholipids are a direct response to temperature and occur in isolated cells. This fundamental response maintains the dynamic phase behaviour of the membrane and adjusts the rates of membrane processes. However, these adjustments may have deleterious consequences. For fish living at low temperatures, the increased polyunsaturation of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of reactive oxygen species (ROS), increase proton leak and favour peroxidation of these membranes. Minimisation of mitochondrial oxidative capacities in organisms living at low temperatures would reduce such damage.

Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms.

A hydrophobic cleft formed by the BH1, BH2 and BH3 domains of Bcl-xL is responsible for interactions between Bcl-xL and BH3-containing death agonists. Mutants were constructed which did not bind to Bax but retained anti-apoptotic activity. Since Bcl-xL can form an ion channel in synthetic lipid membranes, the possibility that this property has a role in heterodimerization-independent cell survival was tested by replacing amino acids within the predicted channel-forming domain with the corresponding amino acids from Bax. The resulting chimera showed a reduced ability to adopt an open conductance state over a wide range of membrane potentials. Although this construct retained the ability to heterodimerize with Bax and to inhibit apoptosis, when a mutation was introduced that rendered the chimera incapable of heterodimerization, the resulting protein failed to prevent both apoptosis in mammalian cells and Bax-mediated growth defect in yeast. Similar to mammalian cells undergoing apoptosis, yeast cells expressing Bax exhibited changes in mitochondrial properties that were inhibited by Bcl-xL through heterodimerization-dependent and -independent mechanisms. These data suggest that Bcl-xL regulates cell survival by at least two distinct mechanisms; one is associated with heterodimerization and the other with the ability to form a sustained ion channel.

Changes in Mitochondrial Properties During Low Temperature Storage of Apples

ABSTRACTMitochondria were isolated from ‘Cortland’ and ‘Winesap’ apples stored at 90% RH and either 1° or 5°C. Respiratory control ratios of mitochondria isolated from ‘Nured Winesap’ apples stored at 5°C were consistently lower than from those stored at 1°C. Succinoxidase and Ca +2 ATPase activity was higher in mitochondria of ‘Cortland’ apples stored at 1°C. Large increases were observed in mitochondrial Ca +2 uptake after 4 wk storage of ‘Nured Winesap’ apples at 5°C and after 1 wk storage of ‘Cortland’ apples at 1°C. Although no symptoms of chilling injury were observed in the tissue of either cultivar, the mitochondrial response suggested chilling resistance in ‘Nured Winesap’ apples and chilling sensitivity in ‘Cortland’ apples.

Functional changes in mitochondrial properties as a result of their membrane cryodestruction: II. Influence of freezing and thawing on ATP complex activity of intact liver mitochondria

The influence of the freeze-thawing rates on ATP synthetase (ATPase) complex of intact liver mitochondria was investigated. It was shown that the increase in latent ATPase activity and decrease in ATP synthetase activity resulted from an influence on the inner mitochondrial membrane. An increase in freeze-thawing rates led to the preservation of ATP synthetase activity and ATP hydrolysis reduction. Kinetic parameter changes of the ATP synthetase reaction resulted from an insignificant nonspecific increase in the inner mitochondrial membrane permeability and changes in its electrochemical potential level.

Functional changes in mitochondrial properties as a result of their membrane cryodestruction: I. Influence of freezing and thawing on succinate-ferricyanide reductase of intact liver mitochondria

The interaction of the succinate dehydrogenase complex of rat liver mitochondria with an artificial electron acceptor (K 3Fe(CN) 6), impermeable to the mitochondrial membrane as an index of a cryoinjury is investigated. It is shown that the freeze-thawing stimulates succinate-ferricyanide reductase (SFCR) activity of intact mitochondria. The increase of the freezing and thawing rates leads to a decrease in the released SFCR activity. The released SFCR activity after low-temperature treatment is a consequence of a nonspecific change in membrane ferricyanide permeability. The released SFCR activity decreases as the freezing and thawing rates increase.