Yoshiko Nakae

Early Onset of Lipofuscin Accumulation in Dystrophin-Deficient Skeletal Muscles of DMD Patients and mdx Mice

Lipofuscin, the so-called ageing pigment, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals and redox-active metal ions. Usually it accumulates in post-mitotic, long-lived cells such as neurons and cardiac muscle cells. In contrast, it is rarely seen in either normal or diseased skeletal muscle fibres. In this paper, we report that lipofuscin accumulates at an early age in both human and murine dystrophic muscles. Autofluorescent lipofuscin granules were localized, using confocal laser scanning microscopy and electron microscopy, in dystrophin-deficient skeletal muscles of X chromosome-linked young Duchenne muscular dystrophy (DMD) patients and of mdx mice at various ages after birth. Age-matched normal controls were studied similarly. Autofluorescent lipofuscin granules were observed in dystrophic biceps brachii muscles of 2–7-year-old DMD patients where degeneration and regeneration of myofibres are active, but they were rarely seen in age-matched normal controls. In normal mice, lipofuscin first appears in diaphragm muscles nearly 20 weeks after birth but in mdx muscles it occurs much earlier, 4 weeks after birth, when the primary degeneration of dystrophin-deficient myofibres is at a peak. Lipofuscin accumulation increases with age in both mdx and normal controls and is always higher in dystrophic muscles than in age-matched normal controls. At the electron microscopical level, it was confirmed that the localisation of autofluorescent granules observed by light microscopy in dystrophin-deficient skeletal muscles coincided with lipofuscin granules in myofibres and myosatellite cells, and in macrophages accumulating around myofibres and in interstitial connective tissue. Our results agree with previous biochemical and histochemical data implying increased oxidative damages in DMD and mdx muscles. They indicate that dystrophin-deficient myofibres are either more susceptible to oxidative stress, or are subjected to higher intra- or extracellular oxidative stress than normal controls, or both.

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

Abstract The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions.

Kinetic behaviour of succinate dehydrogenase of three fibre types in skeletal muscle. I: Effects of temperature and a competitive inhibitor

SummaryThe kinetic behaviour of succinate dehydrogenase [EC 1.3.99.1] in three fibre types of rat gastrocnemius was examined by a quantitative histochemical method without disruption of the cellular structure. 2-(2-Benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-tetrazolium chloride (BPST) and phenazine methosulphate were used as electron acceptors. On measurement of the absorbance value at 530 nm of BPST formazan, produced by the succinate dehydrogenase reaction in sections, it was found that the staining intensity of succinate dehydrogenase was linearly proportional to both the incubation time and the thickness of the slice therefore, the initial velocity of the staining could be calculated. By Michaelis-Menten (1913) treatment of the dependence of the initial velocity on the substrate concentration in the absence and the presence of a competitive inhibitor, malonate, the Km andVmax values for succinate and the Ki value for malonate were obtained. The Km and Ki values of the three fibre types were similar. The ratio of theVmax values of type A, B and C fibres was 1.0∶2.0∶3.3. The temperature dependence of the kinetic parameters was very similar in the three fibre types. These findings confirm that the differences in the staining intensity of the three fibre types reflect differences in the amounts, but not the properties, of succinate dehydrogenase.

The diverse Michaelis constants and maximum velocities of lactate dehydrogenase in situ in various types of cell.

SummaryThe kinetics of lactate dehydrogenase in mouse cardiac muscle fibres, skeletal muscle fibres, gastric parietal cells, parotid gland ductal and acinar cells, oocytes and mouse and human hepatocytes were studied as a function of substrate concentration in sections of unfixed mouse and human tissues incubated at 37°C on lactate agarose gel films. The absorbances of the final reaction products deposited in single cells of various types were measured continuously as a function of incubation time using an image analysis system. The initial velocities (vi) of the dehydrogenase were calculated from two equations deduced previously by us, vi = a1∘A (equation 1) and vi = v + a2∘A (equation 2), where v and ∘A are, respectively, the gradient (steady-state velocity) and intercept of the linear regression line of absorbance on time for incubation times between 1 and 3 min, and a1 and a2 are constants characteristic for each cell type.Hanes plots using vi, calculated from equation 2 gave more consistent estimates of the Michaelis constant (Km) and the maximum reaction velocity (Vmax) than those employing either steady-state velocity measurements or vi calculated from equation 1. The Km thus found for mouse skeletal muscle fibres (10.4–12.5 mM) and hepatocytes (14.3–16.7 mM) agreed well with values determined previously in biochemical assays. However, the Km for cardiac muscle fibres (13.4 mM) was higher. The Km of the enzyme in gastric parietal cells, parotid gland cells and oocytes was in the range 7.6–9.7 mM. The Vmax were more diverse, ranging from 29 μmoles hydrogen equivalents/cm3 cytoplasm/min units in mouse parotid gland acinar cells, 59–68 units in skeletal and cardiac muscle fibres, 62–65 units in gastric parietal cells and oocytes, and 102–110 units in hepatocytes. The diversity found for Km and Vmax in different cell types confirms the value of the quantitative histochemical approach in revealing the heterogeneity of cellular metabolism in situ.

Initial reaction kinetics of succinate dehydrogenase in mouse liver studied with a real-time image analyser system

SummaryThe initial reaction kinetics of succinate dehydrogenase in situ were investigated in sections of mouse unfixed liver using an ARGUS-100 image analyser system. The sections were incubated on substrate-containing agarose gel films. Images of a section, illuminated with monochromatic light (584 nm), were captured with the image analyser in real time at intervals of 10 s during the incubation. The absorbances of selected hepatocytes in the successive images were determined as a function of time. In every cell, the absorbance increased non-linearly after the first minute of incubation. The initial velocity of the dehydrogenase was calculated from the linear activities during the first 20 s of incubation. Hanes plots of the initial velocities and succinate concentration yielded the following mean kinetic constants. For periportal hepatocytes, the apparentKm=1.2±0.8 mM andVmax=29±2 μmol hydrogen equivalents formed/cm3 hepatocyte cytoplasm per min. For pericentral hepatocytes,Km=1.4±1.0 mM andVmax=21±2 μmol hydrogen equivalents/cm3 per min. TheKm values are very similar to those determined previously from biochemical assays. These results, and the observed dependence of the initial velocity on the enzyme concentration, suggest that the technique reported here is valid for the histochemical assay of succinate dehydrogenase.

The initial reaction velocities of lactate dehydrogenase in various cell types

SummaryThe initial reaction velocities (vv) of lactate dehydrogenase in hepatocytes, cardiac muscle fibres, skeletal (gastrocnemius) muscle fibres, gastric parietal cells, ductal epithelial and acinar cells of the parotid gland, and oocytes were determined, by computer-assisted image analysis, in unfixed sections of these tissues incubated at 37°C on substrate-containing agarose gel films. They were found to fit the equations vi = a1∘A (equation 1) and vi − v = a2∘A (equation 2) reported previously for mouse hepatocytes (Nakae & Stoward, 1993a, b), where v and ∘A are, respectively, the gradients (or steady-state velocities) and the intercepts on the absorbance axis of the linear regression lines of the absorbance (A) of the finalreaction product on incubation times between 1 and 3 min, and a1 and a2 are constants. Both equations 1 and 2 fitted the observed vi closely for mouse (a1 = 2.7, a2 = 2.2) and human (a1 = 3.0, a2 = 1.9) hepatocytes. However, equation 2 fitted the observed vi better than equation 1 for mouse cardiac muscle fibres (a2 = 1.5), skeletal muscle fibres (a2 = 1.2), gastric parietal cells (a2 = 1.7), acinar (a2 = 1.4) and striated ductal (a2 = 2.2) epithelial cells of the parotid gland, and oocytes (a2 = 1.6). The values of vi calculated from the two equations agreed with the observed vi to within about 11%. They ranged from 105 μmole hydrogen equivalents/cm3 cell/min units in hepatocytes to 24 units in parotid acinar cells, but for other cell types they were between 46 and 61 units. These are all considerably higher than values reported previously.

Kinetic Parameters of Lactate Dehydrogenase in Liver and Gastrocnemius Determined by Three Quantitative Histochemical Methods

We determined the Michaelis constant (K m) and maximal velocity (V max) of lactate dehydrogenase (LDH) in periportal hepatocytes and skeletal muscle fibers by three different histochemical assay methods. Unfixed sections of mouse liver and gastrocnemius were incubated at 37C either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media with and without 18% polyvinyl alcohol (PVA) as a tissue protectant. The absorbances of the formazan final reaction products were continuously measured at 584 nm in the cytoplasm of individual cells as a function of incubation time, using an image analysis system. The kinetic parameters of purified rabbit skeletal muscle LDH incorporated into polyacrylamide gel sections were similarly determined. The intrinsic initial velocities (v i) of LDH, corrected for “nothing dehydrogenase,” were determined as described in the previous article. The K m and V max were calculated from Hanes plots of s/vi on l-lactate concentration (s). The K m values obtained with three assay methods were similar and in the range of 21.1–21.9 mM for pure LDH, 8.62–13.5 mM for LDH in mouse periportal hepatocytes, and 13.3–17.9 mM for LDH in mouse skeletal muscle fibers. The V max values determined on agarose gel substrate films and in aqueous assay media without PVA were in good agreeement but were 53–65% lower when 18% PVA was included in the medium. These results indicate that catalytic center activity k cat of LDH is retarded by the high viscosity of PVA media because PVA hardly inhibited the enzyme. The K m values of LDH determined histochemically in skeletal muscle fibers and periportal hepatocytes were respectively three to five times and two to three times higher than those determined biochemically. These differences may be due to interactions of LDH with intracellular components.

Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

We studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. The kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate (l-lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation v i - v = a°A, where v i is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and °A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed (v i - v) against °A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12–43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

Combined quantitative cytophotometric method for staining indolyl and sulfhydryl groups and protein.

Sulfenylation with 2-nitrophenylsulfenyl chloride (NPS-Cl), which is specific for tryptophyl and cysteinyl residues in protein, was applied to quantitative histochemistry. By measurement of the absorbance values at 370 nm of sections stained with NPS-Cl, Beer-Lamberts law was found to hold for NPS staining. Treatment of NPS-stained sections with 2-mercaptoethanol (ME) (NPS-ME staining) resulted in sulfenylation of tryptophyl residues only. For determination of the amounts of tryptophyl and cysteinyl residues per unit of protein, protein staining with Coomassie Brilliant Blue (CB) was combined with NPS and NPS-ME staining. CB and NPS-CBB staining also followed Beer-Lamberts law. By measuring the absorbance values at 370 and 650 nm of doubly stained sections, the relative contents of tryptophyl and cysteinyl residues in various tissue proteins were calculated. This method will be useful for the investigation of changes in both protein amount and composition.

Histochemical modification of the active site of succinate dehydrogenase withN-acetylimidazole

SummaryThe kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius withN-acetylimidazole was studied by a newly modified histochemical technique.1.Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely.2.Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme.3.The value of the inhibition constant (Ki=34µM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with theKi value (33µM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.