Kirstin Winkler

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50–100 μg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon — tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given. (Mol Cell Biochem 184: 81–100, 1998)

Impairment of mitochondrial function in skeletal muscle of patients with amyotrophic lateral sclerosis

In skeletal muscle homogenates of 14 patients with sporadic amyotrophic lateral sclerosis, an approximately twofold lower specific activity of NADH:CoQ oxidoreductase in comparison to an age matched control group (n=28) was detected. This finding was confirmed by a detailed analysis of mitochondrial oxidative phosphorylation in skeletal muscle using saponin-permeabilized muscle fibers. (i) A significantly lowered maximal glutamate+malate and pyruvate+malate supported respiration of saponin-permeabilized fibers was detected in the patients group. (ii) Titrations with the specific inhibitor of NADH:CoQ oxidoreductase amytal revealed a higher sensitivity of respiration to this inhibitor indicating an elevated flux control coefficient of this enzyme. (iii) Applying functional imaging of mitochondria using ratios of NAD(P)H and flavoprotein autofluorescence images of saponin-permeabilized fibers we detected the presence of partially respiratory chain inhibited mitochondria on the single fiber level. A secondary defect of mitochondrial function due to the neurogenic changes in muscle seems to be unlikely since no mitochondrial abnormalities were detectable in biopsies of patients with spinal muscular atrophy. These results support the viewpoint that an impairment of mitochondria may be of pathophysiological significance in the etiology of amyotrophic lateral sclerosis.

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.

Visualization of defective mitochondrial function in skeletal muscle fibers of patients with sporadic amyotrophic lateral sclerosis

The mitochondrial function in skeletal muscle was investigated in skeletal muscle biopsies of 26 patients with sporadic amyotrophic lateral sclerosis (ALS) and compared with investigations of 28 age-matched control muscle samples and biopsies of 6 patients with spinal muscular atrophy (SMA) and two patients with Tay-Sachs disease. In comparison to the control, SMA and Tay-Sachs biopsies, we observed in the ALS samples a significant about two-fold lower activity of complex I of mitochondrial respiratory chain. To visualise the distribution of the mitochondrial defect in skeletal muscle fibers we applied confocal laser-scanning microscopy and video fluorescence microscopy of NAD(P)H and fluorescent flavoproteins. The redox change of mitochondrial NAD(P)H and flavoproteins on addition of mitochondrial substrates, ADP, or cyanide were determined by measurement of fluorescence intensities with dual-photon UV-excitation and single-photon blue excitation. In skeletal muscle fibers of ALS patients with abnormalities of mitochondrial DNA (multiple deletions, n=1, or lower mtDNA levels, n=14) we observed a heterogeneous distribution of the mitochondrial defects among individual fibers and even within single fibers. In some patients (n=3) a mitochondrial defect was also detectable in cultivated skin fibroblasts. These findings support the viewpoint that the observed impairment of mitochondrial function in muscle of certain ALS patients is caused by an intrinsic mitochondrial defect which may be of pathophysiological significance in the etiology of this neurodegenerative disease.

Application of inhibitor titrations for the detection of oxidative phosphorylation defects in saponin-skinned muscle fibers of patients with mitochondrial diseases.

Inhibitor titrations were applied to characterize functional changes in mitochondrial energy metabolism in the skeletal muscle of patients with mitochondrial diseases. For this we titrated the maximal mitochondrial respiration rate of saponin-skinned muscle fibers isolated from the skeletal muscle biopsy with the specific inhibitors of mitochondrial oxidative phosphorylation complexes I, IV and V-rotenone, azide and oligomycin. For three patients with deletions of mitochondrial DNA and one patient with a complex I deficiency the titrations revealed at rather normal respiration activities of saponin-skinned fibers significant differences to healthy controls: (i) The inhibitor titration curves of the affected enzyme were much steeper and (ii) for almost complete inhibition of respiration a smaller amount of the inhibitor is necessary. The detailed analysis of the titration curves within the framework of metabolic control theory indicated elevated flux control coefficients of the respective complex of respiratory chain. On the other hand, for one patient with a mitochondrial DNA depletion syndrome, decreased respiration activities of skinned fibers but no redistribution of flux control was observed. We conclude, therefore, that application of inhibitor titrations and the quantitative description of the titration curve can be a valuable approach to elucidate functional defects of mitochondrial oxidative phosphorylation.

Loss of heteroplasmy in the displacement loop of brain mitochondrial DNA in astrocytic tumors.

The aim of this study was the determination of D‐loop heteroplasmy in astrocytic brain tumors. DNA fragments corresponding to the hypervariable region 2 of the mitochondrial displacement loop (D‐loop) from 12 astrocytic tumors and 2 corresponding brain samples were cloned into a plasmid vector. Heteroduplex analysis revealed high sequence variability in the brain samples and a subfraction of grade 2 and grade 3 tumors. Furthermore, the results were suggestive of a very low degree of heteroplasmy in all glioblastomas. This was confirmed by direct sequencing of 223 cloned DNA samples from nine individuals. Heteroplasmy was caused in most cases by a well‐known length polymorphism of a homopolymeric c‐tract. Heteroplasmy of the two reference brain samples was lost in the corresponding tumors. Genes Chromosomes Cancer 26:80–83, 1999.

Detection of Respiratory Chain Defects in Cultivated Skin Fibroblasts and Skeletal Muscle of Patients with Parkinson's Disease

Parkinson’s disease (PD) is associated with a degeneration of dopaminergic neurons. The cause of this neuronal death is still unknown. Since 1989,1,2 the hypothesis of mitochondrial respiratory chain dysfunction as a possible basis for the neuronal death has been intensively debated. It is expected that the proposed mitochondrial defect as a possible pathogenic factor for PD should include mitochondria in other cell populations than neurons. Reported data obtained from different extracerebral tissues are controversial.3,4 Therefore, the direct proof of a suspected generalized mitochondrial dysfunction in PD is still missing. However, in muscle homogenates of patients with mitochondrial myopathies the mitochondrial defect is not always detectable with conventional enzyme assays for the determination of respiratory chain enzyme complexes I + III (NADH:cytochrome c reductase), II + III (succinate:cytochrome c reductase), and IV (cytochrome c oxidase).5 Therefore, we assessed the applicability of different in situ methods for the detection of putative mitochondrial respiratory chain defects in extracerebral tissues of PD patients. The functional behavior of mitochondria in digitonin-permeabilized skin fibroblasts and in saponinpermeabilized skeletal muscle fibers of PD patients was studied applying respiration experiments with different substrates using metabolic control analysis. Furthermore, we determined the redox state of the mitochondrial NAD system measuring the laser-excited autofluorescence of NAD(P)H and fluorescent flavoproteins in permeabilized skeletal muscle fibers.6 Fifteen patients with PD (4 female and 11 male, age range 37 to 78 years, mean 58 years) had a skeletal muscle and skin biopsy. The patients had had symptomatic PD for 2 to 13 years. The median UPDRS was 20 (range 4 to 56). All patients had the akinetic-rigid type of the disease. Skeletal muscle samples from diagnostic biopsies of 32 patients with discrete myopathic EMG abnormalities but no biopsy evidence for a manifest myopathy (17 male, 15 female, age range 29 to 72 years) were used as control, and all patients gave written informed consent. The study was approved by the Ethics Committee of Magdeburg University. Bundles of fibers from fresh M. vastus lateralis were used for preparation of saponin-permeabilized fibers.

[12] Functional imaging of mitochondrial redox state

Publisher Summary The use of intrinsic fluorophores allows evaluation of the function of mitochondrial oxidative phosphorylation without having the phototoxic side effects of the various fluorescent mitochondrial dyes. To detect the changes of the mitochondrial redox state in response to alterations of oxidative phosphorylation (OxPhos) at the single cell level, the fluorescence changes of the α-lipoamide dehydrogenase flavin moiety by confocal microscopy is investigated. Functional imaging of the mitochondrial redox state can be performed by microscopic detection of the flavoprotein fluorescence of the mitochondrially localized α-lipoamide dehydrogenase, which is in tight redox equilibrium with the mitochondrial NAD system. Direct detection of the NAD(P)H fluorescence signal for mitochondrial NAD redox state determinations can be performed in digitonin-permeabilized cells or saponin-permeabilized muscle fibers at considerably higher sensitivity. The procedure is applicable for cultured cells having low mitochondrial content. For the determination of putative heterogeneities of the mitochondrial redox state in permeabilized muscle fibers, the application of Fp/NAD(P)H ratio imaging is advantageous, because it offers an increased sensitivity with respect to metabolic alterations of the fluorescence signals. In sum, the monitoring of intrinsic fluorophores allows one to obtain insights into the mitochondrial function of single living cells, avoiding the problems of phototoxicity and photobleaching.

Laser-excited fluorescence studies of mitochondria) function in saponin-skinned skeletal muscle fibers of patients with chronic progressive external ophthalmoplegia

The functional behavior of mitochondria in skeletal muscle of patients with chronic progressive external ophthalmoplegia was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. Variations in the mitochondrial content and the presence of partially respiratory chain-inhibited mitochondria can be detected using this novel method.

mtDNA depletion and impairment of mitochondrial function in a case of a multisystem disorder including severe myopathy

The ratio of mtDNA and a nuclear reference gene was estimated by Southern blotting in the skeletal muscle DNA of a 3-year-old girl who suffered from congenital brain damage, focal epilepsy, hepatomegaly, malabsorption syndrome and severe myopathy. The signal ratio of mtDNA versus 18S rDNA was 22% of the mean value obtained from controls. No major deletions or insertions were found and the MERRF, MELAS and NARP mutations were ruled out. Mitochondrial DNA-encoded enzyme activities and mitochondrial respiration were reduced. The analysis of the NAD(P)H and flavoprotein redox states of intact fibres revealed the presence of mitochondrial dysfunction. In tissue sections a moderate elevation of type I and type II fibre diameter variation was detected, aberrant NADH- and succinate dehydrogenase staining and some ragged red fibres. This suggested that a mitochondrial disorder caused by a decrease in the amount of intact wild-type mtDNA was responsible for the severe myopathy.