Mitochondrial changes in flight muscles of normal and flightless Drosophila melanogaster with age

Fine structural changes in mitochondrial morphology pertaining to size, number and growth were examined in flight muscles of normal and experimentally dewinged male Drosphila melanogaster ranging up to 26 days of age. In the normal winged flies, the number of mitochondria decreases during the first week of adult life whereas the size of individual mitochondrial profile increases significantly. Changes in mitochondrial size and number are due to the fusion of mitochondria. Fused mitochondria are extremely large in size and irregular in shape. In 26-day old normal flies, the number of mitochondria increases while the mitochondrial size is reduced indicating mitochondrial division. In comparison to the normal flies, dewinged flies exhibit a similar degree of mitochondrial fusion and growth during the first week of life. However, the extent of mitochondrial fission in 26-day old dewinged flies is greater than in the normal flies of this age. Structural mechanisms of mitochondrial fusion and fission are described. The objective of this study was to examine the relative effects of age and flight activity on the mitochondria.     

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.     

Physiological Cost of Physical Exercise and Mitochondrial Volume in Working Muscles of Subjects Exposed to Long-Term Hypokinesia: Effects of Local Resistance Exercise

The results of changes in the physiological cost of 30-min submaximal aerobic bicycle ergometric exercise and characteristics of the mitochondrial apparatus of m. vastus lateralis were assessed comparatively during 120-day (–6°) antiorthostatic hypokinesia either without prophylactic measures or with low-intensity resistance exercise training for 60 days using a Penguin exercise suit. Hypokinesia was accompanied by an increase in the working heart rate and lactate accumulation in the blood during the test exercise, as well as by a decrease in the myofibril size and the volume density of mitochondria in the m. vastus lateralis fibers. The patterns of dynamic changes in the lactate concentration in the blood during exercise training and in the volume density of central mitochondria were found to be similar. A correlation between the rate of lactate accumulation in the blood during the test exercise and the volume density of mitochondria in the working muscle appeared after long-term (60 days) exposure to hypokinesia. The use of the Penguin exercise suit in dynamic mode during prolonged (60-day) exposure to hypokinesia completely prevented the following effects: atrophy of slow-type fibers, a decrease in the volume density of central mitochondria, and an increase in the level of lactate accumulation in the blood under conditions of a standard submaximal aerobic exercise load. The correlation links between the oxidative potential of working muscle and the energy supply of muscular work are discussed.     

Mechanism of Chilling Injury in Sweet Potato: X. Change in Lipid-Protein Interaction in Mitochondria from Cold-stored Tissue

Seventy per cent of the phospholipid in mitochondria from sweet potato roots was removed by aqueous acetone treatment. The amount of phospholipid that could be rebound to these lipid-depleted mitochondria roughly corresponded to the amount of phospholipid in untreated mitochondria. The activities of NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, cytochrome oxidase, and succinoxidase in lipid-depleted mitochondria were restored by addition of mitochondrial phospholipid to about 60, 50, 15, and 35%, respectively, in comparison to untreated mitochondria. The capacity of lipid-depleted mitochondria from 14-day cold-stored tissue to bind mitochondrial phospholipid from healthy tissue was lower than that from healthy tissue. However, there was no large difference in activities of NADH-cytochrome c oxidoreductase and succinate-cytochrome c oxidoreductase between both phospholipid rebound lipid-depleted mitochondria from healthy and 14-day cold-stored tissues. On the other hand, activity of succinoxidase in phospholipid rebound lipid-depleted mitochondria from 14-day cold-stored tissue was decreased by about 50% of that from healthy tissue. Furthermore, the capacity of lipid-depleted mitochondria from 2-day cold-stored tissue to bind mitochondrial phospholipid from healthy tissue was higher than that from healthy tissue.     

Quantitative morphologic studies on the myocardium in early stages of ontogenesis and after application of various cardioactive agents

The authors have morphometrically studied the differentiation of the myocardium in dynamic phases of the embryonic and postnatal development in chickens and Syrian Hamsters. Moreover, they investigated the action of the beta-adrenalytic substances Practolol and Trimepranol on ultrastructure of the cardiac muscle in adult animals. The volume of mitochondria in myocardial cells in 6-day old chicken embryos amounts to 5.65% of the total cell volume, in 12-day old embryos 14.35%, in 18-day old embryos 19.60%, in 1-day old chickens 23.24% which is nearly as much as in adult animals. The volume of myofibrils in 6-day old embryos is about 3.2%, in 12-day old embryos about 7.4%, in 18-day old embryos about 16.4% and in 1-day old chickens about 21.2%. The differences between individual groups are statistically significant. The dynamics of differentiation of the myocardium in Syrian Hamsters was studied in 5 phases, namely in 14-day old embryos and in postnatal phases on the 2nd, 5th, 14th and 21st days after birth. Most cells in 14-day old embryos are rather immature. Participation of the volume of mitochondria, myofibrils, equipment of mitochondria with cristae etc. considerably increase in postnatal phases. These findings suggest that the heart of mammals is rather immature at birth and will differentiate mainly in the postnatal developmental phases. Many morphometric findings, as regards the action of beta-adrenalytic drugs on the ultrastructure of the myocardium in adult rabbits, point to the fact that application of these substances will give rise to degenerative alterations in approximately 10% of myocardial cells. Theoretical explantation of these mechanisms is being discussed.     

Structural changes in L-cell mitochondria exposed to cyanide

Using light and electron microscopy, as well as stereological analysis, a study was made of structural changes in mitochondria of cultured L-cells acted upon with 1 mM cyanide. After 23 hours of incubation, in cells cultured in the presence of cyanide the volume density of mitochondria, the volume of the average mitochondrion and the surface area of its membranes increased nearly twice as much. Concurrently, the number of mitochondria decrease also practically two-fold without any reliable changes in the surface area of membranes per unit of mitochondrion volume. A shorter (7 hours) incubation of L-cells in the presence of cyanide results in a decreased volume density of mitochondria in the cytoplasm, decreased volume of the average mitochondrion, without any significant changes in other above mentioned parameters. It is supposed that the changes in mitochondria under a prolonged cyanide treatment of cells are conditioned by the fusion of mitochondria as well as by a compensatory increase in the total surface of mitochondrial membranes.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

ULTRASTRUCTURAL BASES FOR METABOLICALLY LINKED MECHANICAL ACTIVITY IN MITOCHONDRIA : I. Reversible Ultrastructural Changes with Change in Metabolic Steady State in Isolated Liver Mitochondria

By means of a new "quick-sampling" method, micropellets of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 min in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volume of the matrix decreases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate between the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substrate and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changes in the manner of folding of the inner membrane and in the manner of packing of small granules in the matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : II. Ultrastructural Studies

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.     

Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica

The objective of this study was to elucidate the possible cause of increased oxidative stress observed in the adult housefly during aging. The hypothesis that increased production of oxygen radicals may be a cause of the increased oxidative stress was tested by comparison of 8-day and 15-day old flies, which represent the stage of full maturation and the beginning of the dying phase, respectively. Rates of both antimycin A-resistant respiration of isolated mitochondria and O2 generation at ubiquinone-cytochrome b site by submitochondrial particles increased during aging and were associated with life expectancy of flies. Flies destined to die earlier than their cohorts of the same age exhibited a relatively higher rate of O2- production. Age-related increase in O2- generation was not associated with corresponding changes in ubiquinone content of mitochondria.     

Electron microscopic and polarographic study of rat liver mitochondria during freezing and thawing in sucrose and saline media

Results are presented of studying structural and functional changes in rat liver mitochondria during freezing-thawing in sucrose and saline medium. It is found that maximal disturbances in the structural and functional state of mitochondria are observed in the saline medium.     

Ultrastructure of Candida utilis in continuous culture with various concentrations of the carbon source introduced by different technics

The object of the work was to study changes in the ultrastructure of Candida utilis cells induced by increasing concentrations of glucose during chemostat cultivation and continuous cultivation with glucose pulse feeding. The results indicate that the cell structure and metabolism change vectorially with an increase of glucose concentration regardless of how glucose was added. An increase of the input glucose concentration is followed by an increase in the periplasmic space, the content of glycogen, the length and diameter of mitochondria, and the size of the nucleus. However, in the case of glucose pulse feeding, the above changes in the cell structure occur at a considerably lower input concentration as compared to the chemostat culture. Under these conditions, microtubuli are assembled in the cytoplasm in response to the glucose stimulus.     

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism--a model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.     

Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed.     

Rapid pacing of embryoid bodies impairs mitochondrial ATP synthesis by a calcium-dependent mechanism—A model of in vitro differentiated cardiomyocytes to study molecular effects of tachycardia

Tachycardia may cause substantial molecular and ultrastructural alterations in cardiac tissue. The underlying pathophysiology has not been fully explored. The purpose of this study was (I) to validate a three-dimensional in vitro pacing model, (II) to examine the effect of rapid pacing on mitochondrial function in intact cells, and (III) to evaluate the involvement of L-type-channel-mediated calcium influx in alterations of mitochondria in cardiomyocytes during rapid pacing. In vitro differentiated cardiomyocytes from P19 cells that formed embryoid bodies were paced for 24 h with 0.6 and 2.0 Hz. Pacing at 2.0 Hz increased mRNA expression and phosphorylation of ERK1/2 and caused cellular hypertrophy, indicated by increased protein/DNA ratio, and oxidative stress measured as loss of cellular thiols. Rapid pacing additionally provoked structural alterations of mitochondria. All these changes are known to occur in vivo during atrial fibrillation. The structural alterations of mitochondria were accompanied by limitation of ATP production as evidenced by decreased endogenous respiration in combination with decreased ATP levels in intact cells. Inhibition of calcium inward current with verapamil protected against hypertrophic response and oxidative stress. Verapamil ameliorated morphological changes and dysfunction of mitochondria. In conclusion, rapid pacing-dependent changes in calcium inward current via L-type channels mediate both oxidative stress and mitochondrial dysfunction. The in vitro pacing model presented here reflects changes occurring during tachycardia and, thus, allows functional analyses of the signaling pathways involved.     

Structural dynamics of the mitochondrial compartment.

The metabolic activities of mitochondria have been extensively characterized. However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism. There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions. Segregation and recombination of mitochondrial genomes are also processes dependent upon the dynamic nature of the mitochondrial compartment. These regulatory and structural aspects of mitochondrial compartment dynamics will play an important role in the analysis of mitochondrial function and pathology.     

Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

Inhibition of mitochondrial-specific protein symthesis in human lymphocytes and platelets is dependent upon the stage of cellular differentiation

Small lymphocytes differentiate into functionally active blast cells in vitro upon stimulation with such mitogens as phytohemagglutinin and sodium periodate. If stimulated lymphocytes are subsequently treated with the nucleic acid intercalating dye ethidium bromide, electron-dense complexes containing nucleic acid are formed in mitochondria, protein synthesis in mitochondria is inhibited, and lymphoblast division ceases. Formation of complexes and the development of morphologically abnormal mitochondria provide ultrastructural evidence of mitochondrial protein inhibition and serve as markers for mitogen-responsive lymphocytes. The formation of these abnormalities in all mitochondria of treated megakaryocytes and 22% of mitochondria in platelets indicates that platelets contain functional nucleic acid and that the induced structural changes may be occurring in a less-differentiated (i.e., younger) subpopulation of circulating platelets.     

Fatty acid composition of rat liver mitochondrial phospholipids during ethanol inhalation

Male Sprague-Dawley rats were exposed to increasing concentrations (15-22 mg/l) of ethanol vapor over a 4-day period. Phospholipids were analyzed in liver mitochondria isolated from ethanol-treated and pair-weighted control animals. After a 2-day inhalation period, the proportion of monoenoic acids in total phospholipids increased, whereas that of arachidonic acid decreased. These changes were more striking in phosphatidylcholine (PC) than in phosphatidylethanolamine (PE). The decrease in 20:4 may be related to increased lipid peroxidation. After a 4-day inhalation period, quite different changes in phospholipid fatty acids were found. They consisted in a trend towards a more unsaturated system, the proportion of 20:4 being increased in PC and that of 22:6 in PE. This increase in polyunsaturated acids might be related to a direct ethanol effect on lipid structure and/or metabolism that would be linked to the high blood alcohol level present at this stage of ethanol intoxication.