Mitochondrial sensitivity to AZT

The possibility of tissue-specific effects regarding mitochondrial sensitivity to AZT was evaluated in this study. When mitochondria isolated from liver, kidney, skeletal and cardiac muscle were oxidizing glutamate, a dose-dependent inhibition by AZT of state 3 respiration was observed; using succinate as substrate the inhibition occurred only in skeletal and cardiac muscle mitochondria. The same results were obtained with FCCP-uncoupled mitochondria. NADH oxidase of intact and disrupted mitochondria, isolated from all four tissues was strongly inhibited. Succinate oxidase activity was inhibited by AZT only in intact mitochondria from skeletal and cardiac muscles, suggesting the involvement of succinate transport systems. Similarly, inhibition by the drug of the hydrolytic activity of H⁺-ATPase was observed only in mitochondria of these tissues. These effects taken together, indicate a tissue/carrier-specific inhibition in vitro, although its precise mechanism requires further research. © 1998 John Wiley & Sons, Ltd.     

The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein

We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide. The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria. A mitochondrial polypeptide with an apparent molecular weight of 21 000 was detected with a polyclonal antibody raised against an ORF221 fusion protein. In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria. The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.     

Developmental potential of parthenogenetic cells: role of genotype-specific modifiers.

The developmental potential of parthenogenetic cells derived from different mouse strains was investigated by examining their distribution in various tissues of adult aggregation chimeras. Using GPI-1 allozymes as marker, no striking differences were observed between chimeras whose parthenogenetic cells were derived from activated oocytes isolated from females of different genetic backgrounds, (C57BL/6 x CBA/J) F1, CFLP, 129, and SWR. In all the combinations tested, parthenogenetic cells were consistently absent from skeletal muscle, but there were varying contributions to most other tissues. These results suggest that the maternal duplication of chromosomes containing imprinted gene(s) responsible for the systematic elimination of parthenogenetic cells from skeletal muscle, are not subject to a pronounced influence of genotype-specific modifiers. However, the contribution of parthenogenetic cells to the brain does appear to be influenced by strain background, since a marked improvement in the survival of CFLP, 129 and perhaps SWR parthenogenetic cells in chimeric brains was observed compared with F2 cells.     

Mitochondrial DNA depletion and fatal infantile hepatic failure due to mutations in the mitochondrial polymerase γ (POLG) gene: a combined morphological/enzyme histochemical and immunocytochemical/biochemical and molecular genetic study

Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16‐months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome‐c‐oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox‐positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox‐activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB‐ and mtTFA‐antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non‐homogenous defect pattern.     

Enhanced Gene Transduction into Skeletal Muscle of Mice In Vivo with Pluronic Block Copolymers and Ultrasound Exposure

The pluronic block copolymers are able to enhance the ultrasound-induced gene delivery in vitro. In the present study, the effects of pluronics on the efficiency of gene transfer into skeletal muscle in vivo under sonoporation were investigated. Plasmid DNA encoding green fluorescent protein (GFP) in combination with three different pluronics, F127, L61, and P85, was injected into the tibialis anterior (TA) muscle of mice with and without adjunct ultrasound (1 MHz, 3 W/cm² 1 min, 20% duty cycle). Mice were killed 1 week after injection. The TA muscles were removed and snap frozen immediately in isopentane cooled by liquid nitrogen and sections of 7 μm thick were cut. Transfection efficiency was assessed by counting the number of GFP-positive fibers under fluorescence microscopy, and tissue damage by hematoxylin and eosin staining. The results suggested that all three pluronics significantly enhanced transgene expression in skeletal muscle (P < 0.01), especially the P85 showed significantly higher efficiency than the other two pluronics (P < 0.05). Ultrasound synergistically enhanced the gene delivery efficiency with P85 (P < 0.01), but was unable to do so with F127 and L61 groups. In short, P85 displays significantly synergistic effect with ultrasound for enhancing plasmid DNA transduction in skeletal muscle of mice in vivo.     

Proteins of Mitochondrial Synthesis

Proteins synthesized in vitro by mitochondria isolated from 48-h germinating seeds of Vigna sinensis (L.) Savi and incubated in the presence of ¹⁴C-labelled amino acids from Chlorella protein hydrolysate, have been found associated with nine products separable by acrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Cytoplasmic contribution to these products was practically eliminated by the use of cycloheximide. Most of the radioactivity was incorporated into proteins having molecular weights between 10,000 and 65,000 as determined by comparing their electrophoretic mobilities with those of standard, reference proteins.     

Palmityl-CoA synthetase activity in the muscle of dystrophic mice.

Palmityl-CoA synthetase activity (acid CoA ligase (AMP), E C 6.2.1.3.) was determined using the radioassay method. The rate of formation of palmityl-CoA under the optimal conditions established was 20 nmoles per mg protein per min for mitochondria and 5.8 nmoles for the 9000 × g supernatant. The activity of palmityl-CoA synthetase in mitochondria from skeletal muscle of dystrophic mice was not significantly different from that obtained in normal littermate controls, whereas the activity of this enzyme in the 9000 × g supernatant fraction from dystrophic muscle preparation was found to be significantly higher than for the corresponding controls. It is concluded that the previously observed decrease in palmitate-1-¹⁴C oxidation in dystrophic muscle mitochondria was not due to a defect in the activation of palmitic acid.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.     

A multicopy c-Myc transgene as a nuclear label: overgrowth of Myctg50 cells in allophenic mice.

To trace cell lineages and the origin and fate of cells in transplantation and embryo chimeras, a DNA/DNA in situ hybridization cell labelling system was developed, based on a 50-copy murine c-myc transgene on mouse chromosome 8. Elevated levels of cMyc mRNA were found in Myc*tg50 (Myc^tg50/0 and Myc^tg50/Myc^tg50) transgenic tissues, but adult transgenic NMRI mice were anatomically and histologically indistinguishable from control NMRI mice and did not develop tumours on a wild-type or nude (nu / nu) background. The hybridization label detected transgenic nuclei with an efficiency of ～80%. In muscle grafts, this transgene label was successfully applied to trace donor cells in a labelled host and to study the invasion of a graft by host cells. When the cMyc hybridization was used in allophenic mice of the control|acNMRI-Myc^tg50/? (nu /+ or +/+) type, an up to a three-fold excess of MYC*tg50 positive over control nuclei was found in all organs examined (ventricle, skeletal muscle, liver, small intestine). This overgrowth of MYC*tg50 cells is probably due to transgene expression. Four out of seven (C57BL/6×BALB/c) or (C57BL/6×NMRI)|acMYC*tg50 allophenic mice displayed anatomical abnormalities, e.g. an enlarged thymus and a tumour in the groin region. As these abnormalities were only observed in allophenic mice, they might be due to the imbalance of growth potential between MYC*tg50 transgenic and normal cells in the same individual.     

Tissue-specific expression atlas of murine mitochondrial tRNAs

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNA^Lys, tRNA^Glu, and tRNA^Gln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation.     

Mouse lines with photo‐activatable mitochondria to study mitochondrial dynamics

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines withphoto‐activatable mitochondria (PhAM). In the PhAM ^floxed line, a mitochondrially localized version of the photo‐convertible fluorescent protein Dendra2 (mito‐Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP‐flanked termination signal. Expression of Cre in PhAM ^floxed cells results in bright mito‐Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM ^floxed line expresses mito‐Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM ^excised), the expression of mito‐Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo‐conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states. © genesis 1–11, 2012. © 2012 Wiley Periodicals, Inc.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.     

Mitochondrial creatine kinase functional development in post-natal rat skeletal muscle. A combined polarographic/31P NMR study

Mitochondrial creatine kinase (Mi-CK) function in viable mitochondria from developing rat skeletal muscle was assessed both by polarographic measurements of creatine-induced respiration and 31P NMR spectroscopy measurements of phosphocreatine (PCr) synthesis. Creatine-induced respiration was observed in very young rats and increased by 50% to 35 days of age. PCr synthesis was present in 7 day old animals and increased by 300% reaching levels measured in 35 day and adult muscle. Unlike reports showing Mi-CK enzymatic activities but no mitochondrial function in several situations, a concomitant progression of enzymatic activity and mitochondrial function was evidenced during the developmental stages of skeletal muscle Mi-CK in altricious animals. These results correlated with the progressive pattern of muscle differentiation during development of motricity in such animals. The observation that Mi-CK is functional in skeletal muscle mitochondria very early after birth, strongly favors the notion that adaptations in skeletal muscle of Mi-CK knock-out mice occur early.     

Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2+

In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.     

Hyperactive Intracellular Calcium Signaling Associated with Localized Mitochondrial Defects in Skeletal Muscle of an Animal Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1^G93A mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca²⁺ release activity, which can include propagating Ca²⁺ waves. These Ca²⁺ waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca²⁺ uptake by Ru360 lead to cell-wide propagation of such Ca²⁺ release events. Our data reveal that mitochondria regulate Ca²⁺ signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.     

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na⁺K⁺ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Mitochondrial intermembrane inclusion bodies: The common denominator between human mitochondrial myopathies and creatine depletion due to impairment of cellular energetics

Mitochondrial inclusion bodies are often described in skeletal muscle of patients suffering diseases termed mitochondrial myopathies. A major component of these structures was discovered as being creatine kinase. Similar creatine kinase enriched inclusion bodies in the mitochondria of creatine depleted adult rat cardiomyocytes have been demonstrated. Structurally similar inclusion bodies are observed in mitochondria of ischemic and creatine depleted rat skeletal muscle. This paper describes the various methods for inducing mitochondrial inclusion bodies in rodent skeletal muscle, and compares their effects on muscle metabolism to the metabolic defects of mitochondrial myopathy muscle. We fed rats with a creatine analogue guanidino propionic acid and checked their soled for mitochondrial inclusion bodies, with the electron microscope. The activity of creatine kinase was analysed by measuring creatine stimulated oxidative phosphorylation in soleus skinned fibres using an oxygen electrode . The guanidino propionic acid-rat soleus mitochondria displayed no creatine stimulation, whereas control soleus did, even though the GPA soled had a five fold increase in creatine kinase protein per mitochondrial protein. The significance of these results in light of their relevance to human mitochondrial myopathies and the importance of altered muscle metabolism in the formation of these crystalline structures are discussed. (Mol Cell Biochem 174: 283–289, 1997)