Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Mild Hyperbaric Oxygen Improves Decreased Oxidative Capacity of Spinal Motoneurons Innervating the Soleus Muscle of Rats with Type 2 Diabetes

Rats with type 2 diabetes exhibit decreased oxidative capacity, such as reduced oxidative enzyme activity, low-intensity staining for oxidative enzymes in fibers, and no high-oxidative type IIA fibers, in the skeletal muscle, especially in the soleus muscle. In contrast, there are no data available concerning the oxidative capacity of spinal motoneurons innervating skeletal muscle of rats with type 2 diabetes. This study examined the oxidative capacity of motoneurons innervating the soleus muscle of non-obese rats with type 2 diabetes. In addition, this study examined the effects of mild hyperbaric oxygen at 1.25 atmospheres absolute with 36 % oxygen for 10 weeks on the oxidative capacity of motoneurons innervating the soleus muscle because mild hyperbaric oxygen improves the decreased oxidative capacity of the soleus muscle in non-obese rats with type 2 diabetes. Spinal motoneurons innervating the soleus muscle were identified using nuclear yellow, a retrograde fluorescent neuronal tracer. Thereafter, the cell body sizes and succinate dehydrogenase activity of identified motoneurons were analyzed. Decreased succinate dehydrogenase activity of small-sized alpha motoneurons innervating the soleus muscle was observed in rats with type 2 diabetes. The decreased succinate dehydrogenase activity of these motoneurons was improved by mild hyperbaric oxygen. Therefore, we concluded that rats with type 2 diabetes have decreased oxidative capacity in motoneurons innervating the soleus muscle and this decreased oxidative capacity is improved by mild hyperbaric oxygen.     

Tissue effects of iron deficiency in the rat

Measurements of succinate dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase activities, iron, cytochrome c and myoglobin, were made on various hind-leg muscles, fast-twitch red and white muscle and heart and liver of male Wistar rats fed an iron-deficient diet on weaning. Rats fed the same diet and given 20 mg iron intraperitoneally as iron-dextran (Imferon) served as controls. For iron-repletion studies anemic rats (hemoglobin less than 7 g/dl) were given a single injection of 10 mg iron (Imferon) and the time course of change in the above parameters was followed up to 22 days after injection. The iron concentration of most iron-deficient muscles dropped to approx. 35% of control, the heart to 60% and liver to 13%. On repletion, the iron concentration of all tissues increase significantly by 4 days. While the levels of cytochrome c and myoglobin approximated the iron levels in muscle, they did not change significantly in the heart. Succinate dehydrogenase activity dropped profoundly in muscle, to 10-30% of control; on repletion, the activity increased significantly. Mitochondrial glycerol-3-phosphate dehydrogenase activity showed only small changes in iron-deficient tissues.     

Locomotor muscle profile of a deep (Kogia breviceps) versus shallow (Tursiops truncatus) diving cetacean

When a marine mammal dives, breathing and locomotion are mechanically uncoupled, and its locomotor muscle must power swimming when oxygen is limited. The morphology of that muscle provides insight into both its oxygen storage capacity and its rate of oxygen consumption. This study investigated the m. longissimus dorsi, an epaxial swimming muscle, in the long duration, deep‐diving pygmy sperm whale (Kogia breviceps) and the short duration, shallow‐diving Atlantic bottlenose dolphin (Tursiops truncatus). Muscle myoglobin content, fiber type profile (based upon myosin ATPase and succinate dehydrogenase assays), and fiber size were measured for five adult specimens of each species. In addition, a photometric analysis of sections stained for succinate dehydrogenase was used to create an index of mitochondrial density. The m. longissimus dorsi of K. breviceps displayed significantly a) higher myoglobin content, b) larger proportion of Type I (slow oxidative) fibers by area, c) larger mean fiber diameters, and d) lower indices of mitochondrial density than that of T. truncatus. Thus, this primary swimming muscle of K. breviceps has greater oxygen storage capacity, reduced ATP demand, and likely a reduced rate of oxygen consumption relative to that of T. truncatus. The locomotor muscle of K. breviceps appears able to ration its high onboard oxygen stores, a feature that may allow this species to conduct relatively long duration, deep dives aerobically. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

A Method for Myoglobin in Cryostat Sections of Muscle by Precipitation with Sulfosalicylic Acid

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixation prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

A method for myoglobin in cryostat sections of muscle by precipitation with sulfosalicylic acid.

Transverse cryostat sections of skeletal muscle were fixed in a solution containing 1.5% glutaraldehyde and 1.5% sulfosalicylic acid and stained in a solution containing equal volumes of 3% hydrogen peroxide and 50% ethanol saturated with o-tolidine. Myoglobin in the sarcoplasm of muscle fibers was precipitated and stained blue. Applicability of this method to cryostat sections, without glutaraldehyde fixations prior to freezing, allowed the myoglobin content of individual muscle fibers to be correlated with other histochemical characteristics of the same fibers seen in serial sections. In the dark red bovine sternomandibularis muscle, fibers with weak adenosine triphosphatase (ATPase) and strong succinate dehydrogenase (SDH) activity always exhibited strong myoglobin staining. An equal degree of staining was found in many fibers with strong ATPase and intermediate to strong SDH activity. Fibers with strong ATPase and weak SDH activity were less strongly stained than the preceding types.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.     

A successive histochemical staining for succinate dehydrogenase and "reversed"-ATPase in a single section for the skeletal muscle fibre typing

A procedure is described which simplifies the classification of skeletal muscle fibres in that it allows a simultaneous evaluation of both the oxidative capacity and the intensity of "reversed" ATPase of the fibres, and thus enables to distinguish three fibre types - SO, FOG and FG - in one tissue section. After preincubation at pH 4.1-4.2 the cryostat section is incubated for succinate dehydrogenase (SDH) and subsequently for "reversed"-ATPase. This is followed by the fixation with neutral buffered formaldehyde. The results of typing of chicken, minipig and rabbit fibres in a single muscle section stained with this technique are identical to those obtained with the usual method based on a comparison of serial sections of which one is stained for SDH activity the other for "reversed"-ATPase activity.     

Effects of genetic selection and voluntary activity on the medial gastrocnemius muscle in house mice.

In a previous study, we found that in house mice both genetic selection (10 generations of artificial selection for high voluntary activity on running wheels) and access to running wheels (7-8 weeks) elicited a modest increase in maximal oxygen consumption. Based on these results, we hypothesized that genetic selection would affect the changes in endurance and oxidative capacity of the medial gastrocnemius (MG) muscle induced by wheel access (training response). Wheel access increased the isotonic endurance of the MG in both genetically selected and random-bred (control) mice. However, this exercise-induced improvement in isotonic endurance of the MG was similar between genetically selected and control mice. Wheel access also increased the succinate dehydrogenase activity of MG muscle fibers in both selected and control lines. However, this exercise-induced increase in succinate dehydrogenase activity was comparable between genetically selected and control animals. Taken together, these results indicate that the modest increase in maximal oxygen consumption associated with genetic selection is not reflected by the training-induced changes in oxidative capacity and endurance of MG muscle fibers.     

Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle

Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced.     

Metabolic adaptations in skeletal muscle of streptozotocin-diabetic rats following exercise training

Compensatory metabolic adaptations induced in streptozotocin-diabetic rat skeletal muscle by submaximal endurance training have been investigated. The gastrocnemius muscles of sedentary streptozotocin-diabetic rats were found to have a lower than normal myoglobin content, succinate dehydrogenase activity, and capacity to oxidize pyruvate and palmitate-1-[¹⁴C]. The values of these parameters were significantly increased in the diabetic skeletal muscle by the training program, obtaining levels similar to those of normal sedentary animals.     

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections

A component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono- and diformazans are formed, and these exhibit over-lapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono- and diformazans in histochemical reactions.     

Topographic estimations by component spectroanalysis of two formazans of nitroblue tetrazolium in tissue sections

Summary A component-spectroanalysis technique was used to study the multicolor properties of histochemically stained tissue sections. We developed a method that makes it possible to obtain separately both the spectral patterns and spatial distributions of different color components in tissue sections. To illustrate the application of this technique, we examined the extinction spectrum of reduced nitroblue tetrazolium (NBT), which is used for the detection of dehydrogenase activity. Upon the reduction of NBT, mono-and diformazans are formed, and these exhibit overlapping extinction spectra. When succinate dehydrogenase (SDH) activity in rat liver lobules was examined using NBT, monoformazan was found to be present at higher concentrations than diformazan and to have a uniform distribution, whereas the concentration of diformazan increased with a steep gradient between the center and periphery of lobules. In rat skeletal muscle fibers, diformazan was present at higher concentrations than monoformazan. The level of SDH activity was topographically represented by the hydrogen concentration calculated from the concentrations of the two formazans. This method is effective for separating multiple components such as mono-and diformazans in histochemical reactions.     

Adaptations in metabolic capacity of rat soleus after paralysis.

To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., alpha-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.     

Targeting nucleotide-requiring enzymes: implications for diazoxide-induced cardioprotection

Modulation of mitochondrial respiratory chain, dehydrogenase, and nucleotide-metabolizing enzyme activities is fundamental to cellular protection. Here, we demonstrate that the potassium channel opener diazoxide, within its cardioprotective concentration range, modulated the activity of flavin adenine dinucleotide-dependent succinate dehydrogenase with an IC50 of 32 microM and reduced the rate of succinate-supported generation of reactive oxygen species (ROS) in heart mitochondria. 5-Hydroxydecanoic fatty acid circumvented diazoxide-inhibited succinate dehydrogenase-driven electron flow, indicating a metabolism-dependent supply of redox equivalents to the respiratory chain. In perfused rat hearts, diazoxide diminished the generation of malondialdehyde, a marker of oxidative stress, which, however, increased on diazoxide washout. This effect of diazoxide mimicked ischemic preconditioning and was associated with reduced oxidative damage on ischemia-reperfusion. Diazoxide reduced cellular and mitochondrial ATPase activities, along with nucleotide degradation, contributing to preservation of myocardial ATP levels during ischemia. Thus, by targeting nucleotide-requiring enzymes, particularly mitochondrial succinate dehydrogenase and cellular ATPases, diazoxide reduces ROS generation and nucleotide degradation, resulting in preservation of myocardial energetics under stress.     

Regional metabolic differences in the rat diaphragm

This study characterized the biochemical properties of the rat diaphragm by measuring the activities of selected citric acid cycle and glycolytic enzymes. The diaphragm was removed from 10 female Sprague-Dawley rats (180 days old) and dissected into five discrete anatomic regions: crural (region 1), left posterior costal (region 2), left anterior costal (region 3), right anterior costal (region 4), and right posterior costal (region 5). Sections were assayed for total protein concentration and the activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH). The SDH activity in the crural region was approximately 18% lower (P less than 0.05) than that in any costal region. Furthermore, protein concentration was significantly lower (P less than 0.05) in the crural region compared with all costal regions. In contrast, costal regions 2-5 did not significantly differ from each other in protein concentration or SDH activity. LDH activity did not differ significantly (P greater than 0.05) between regions. Finally, the LDH-to-SDH activity ratio was significantly higher (P less than 0.05) in the crural diaphragm compared with all costal regions. We conclude that the crural region of the rat diaphragm is significantly lower in oxidative capacity than all the costal regions. Investigators who use a rodent model to study diaphragmatic function and plasticity should consider the oxidative heterogeneity of the diaphragm when designing experiments.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

Succinate dehydrogenase activity in various parts of the rat heart in systole and diastole

After fixation of the rat heart at the state of systole and diastole, succinate dehydrogenase (SDH) activity has been revealed. In distributions of the muscle fibers according to their optic density, statistic characteristics connected with the first four moments have been taken into consideration. SDH activity is different in five cardiac parts. Individual changeability is more pronounced in the all cardiac parts at diastole. In comparison to the internal one at systole, it is lower in the all parts, and at diastole--in the atria only. The ratio of the internal and individual changeability is in such a state, that it should be taken into account in all statistical calculations, connected with SDH activity determination in cardiomyocytes.     

Effects of denervation on the activities of some tricarboxylic acid-cycle-associated dehydrogenases and adenine-metabolizing enzymes in rat diaphragm muscle

1. The activity of several tricarboxylic acid-cycle-associated dehydrogenases, adenine-metabolizing enzymes and glutathione reductase and the content of myoglobin were measured in rat diaphragm muscle after unilateral nerve section. 2. Consistent with morphological disintegration of the mitochondria there was a rapid diminution in activity of NAD- and NADP-linked isocitrate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase. 3. Creatine phosphokinase and adenylate kinase, by contrast, showed little change in activity; adenylate deaminase and glutathione reductase activities increased during the hypertrophic phase. The concentration of myoglobin at first declined, then increased again. 4. The distribution of enzymes between the left and right hemidiaphragms was found not to be uniform. 5. Activities of adenine-metabolizing enzymes in the diaphragm were as great as in white muscle. It is suggested that their reputedly lower activities in red muscle properly refer to muscle containing a high proportion of intermediate fibres, which is not the case with diaphragm. 6. The possible causes of the transient hypertrophy after nerve section are discussed.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.