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.     

Myofibrillar ATPase histochemistry of rat skeletal muscles: a "two-dimensional" quantitative approach

In histochemical investigations of skeletal muscle, the fibers are commonly classified into three types according to their staining for myofibrillar ATPase (mATPase). In serial sections of skeletal muscles from normal Wistar rats, we compared two common staining methods for mATPase: (a) an ac-ATPase technique, with pre-incubation at pH 4.7, and (b) a fixed alk-ATPase technique, using treatment with 5% paraformaldehyde followed by pre-incubation at pH 10.4. In addition, the same fibers were stained in subsequent serial sections for succinate dehydrogenase (SDH) activity. Staining intensities were objectively evaluated by microphotometric measurements of optical density. Combining both mATPase methods in consecutive serial sections ("two-dimensional approach") led to the identification of four distinct clusters of fibers: Types I, IIA, and two subgroups of Type IIB, as separated by their staining densities for fixed alk-ATPase (IIBd dark, IIBm moderate). The mean intensity of SDH staining per fiber type, as measured in the central core of the fibers, was ranked such that IIA greater than I greater than IIBd greater than IIBm. The analyzed muscles (tibialis anterior, biceps brachii) were markedly heterogeneous with respect to the topographic distribution of different fiber types. In comparison to other muscle portions, the regions containing Type I fibers ("red" portions) showed a higher IIBd vs IIBm ratio and more intense SDH staining for either subtype of the IIB fibers. The IIBd fibers probably correspond to the Type 2X fibers of Schiaffino et al.     

The radial distribution of succinate dehydrogenase activity in porcine muscle fibres

Summary A microscope photometer with a computer-controlled scanning stage was used to map the distribution of succinate dehydrogenase (SDH) activity in transverse sections of skeletal muscle fibres of pigs from 9 to 29 weeks of age. Absorbance due to SDH activity was measured in successive concentric zones that converged on the central axis of the muscle fibre. In all fibres, there was less SDH activity in the axis of the fibre than in the periphery of the fibre. In fibres with strong adenosine triphosphatase (ATPase) activity and weak overall SDH activity, the radial gradient of SDH activity remained constant as fibres grew in cross-sectional area. However, in fibres with weak ATPase and strong SDH and in fibres with strong ATPase and strong SDH, the radial gradient of SDH activity increased as fibres grew larger. Changes in radial gradients were due to both decreased SDH activity in the axis and to increased SDH activity in the periphery. In all three fibre types, the overall SDH activity per fibre increased with age.     

Mosaic lectin and enzyme staining patterns in rat skeletal muscle.

We compared the localizations of lectin binding and activity for myosin ATPase and succinic dehydrogenase in sections of the gracilis, soleus, and masseter muscles from 10- and 60-day-old rats. In the 60-day-old rats, incubation of the muscle sections with the lectins ConA, GS-II, HPA, and jacalin gave rise to a mosaic staining pattern, especially in the gracilis muscle, in which the same fibers were strongly stained for ConA, GS-II, and HPA, whereas the staining with jacalin in these fibers was weak, and vice versa. There was no correspondence in the staining patterns for the enzymes and the lectins. In the masseter muscle only GS-II gave rise to distinct differences in the staining intensity between muscle fibers. In 10-day-old rats all fibers in the muscles were moderately stained with ConA, HPA, and jacalin, whereas a chessboard staining pattern could be observed after incubation with GS-II. In an extract of hindleg muscle from 60-day-old rats there was strong affinity for ConA and HPA and weak affinity for GS-II and jacalin, as shown by dot-blotting. After electrophoresis and blotting to nitrocellulose membranes, three muscle protein bands with apparent molecular weights of 100,000, 90,000, and 43,000 showed affinity for ConA, HPA, and GS-II, whereas no bands were jacalin positive. The complex lectin staining pattern in skeletal muscle might be related to development, specialization, and function of the muscles.     

Analysis of fiber types in the pigeon's metapatagialis muscle. I. Histochemistry, end plates and ultrastructure

The pigeon's metapatagialis muscle has a pale slip and two pink slips containing slow and fast fibers respectively. The fast fibers in the pink slip are of three types: large white fibers, small red fibers with a fibrillar pattern throughout, and small red fibers with a fibrillar pattern only in the L-band. The large fibers are intermediate in ATPase activity, low in SDH activity, the small fibers are high in ATPase and SDH activity. The slow fibers are intermediate in size and SDH staining, and low in ATPase activity. Cholinesterase staining for end plates corroborate the fast and slow identities. These results reinforce the idea that gross color should not be used in classifying a muscle's contractile or morphological parameters.     

Oxidative capacity and capillary density of diaphragm motor units

Motor units in the cat diaphragm (DIA) were isolated in situ by microdissection and stimulation of C5 ventral root filaments. Motor units were classified based on their isometric contractile force responses and fatigue indexes (FI). The muscle fibers belonging to individual units (i.e., the muscle unit) were identified using the glycogen-depletion method. Fibers were classified as type I or II based on histochemical staining for myofibrillar adenosine triphosphatase (ATPase) after alkaline preincubation. The rate of succinate dehydrogenase (SDH) activity of each fiber was determined using a microphotometric procedure. The location of capillaries was determined from muscle cross sections stained for ATPase after acid (pH = 4.2) preincubation. The capillarity of muscle unit fibers was determined by counting the number of capillaries surrounding fibers and by calculating the number of capillaries per fiber area. A significant correlation was found between the fatigue resistance of DIA units and the mean SDH activity of muscle unit fibers. A significant correlation was also observed between DIA unit fatigue resistance and both indexes of muscle unit fiber capillarity. The mean SDH activity and mean capillary density of muscle unit fibers were also correlated. We conclude that DIA motor unit fatigue resistance depends, at least in part, on the oxidative capacity and capillary density of muscle unit fibers.     

Localization of Myoglobin in Striated Muscle of the Domestic Pig; Benzidine and NADH2-TR Reactions

Tissue blocks 1 cm³ from longissimus (white) and trapezius (red) muscles of adult pigs were fixed in phosphate-buffered 2.5% glutaraldehyde, pH 7.4, for 4 hr at about 25 C; washed 4 hr in running tap water, and immersed in 30% w/v sucrose solution for 16 hr or more. After freezing in liquid N₂, cryostat sections were cut and floated into saturated aqueous benzidine containing 0.15% H₂O₂ at 25 C for 30 min. Stained sections were washed in distilled water and mounted on slides with glycerol jelly. Three distinguishable gradiations of color intensity were found: strong, intermediate, and negative. The trapezius had a greater number of myoglobin-positive fibers than the longissimus muscle. Myoglobin-positive and myoglobin-negative staining occurred in red and white fibers, respectively; intermediates were apparently more closely related to the red than to the white fibers. The NADH₂TR reaction showed the same sites as did the benzidine reaction.     

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.     

Effect of growth on muscle capillarity and fiber type composition in rat diaphragm

The effect of growth on the capillarity and fiber type composition of the diaphragm, soleus and extensor digitorum longus (EDL) muscles of rats weighing between 55 and 330 g have been studied. Muscle samples obtained from the anesthetized rat were rapidly frozen and sliced transversely in a cryostat. The sections were stained histochemically by the SDH method and the myosin ATPase method after preincubation at pH 4.3 to typify fibers (FG, FOG and SO fibers). To visualize capillaries, the myosin ATPase method after preincubation at pH 4.0 was used. The percentage of FOG fibers decreased in all muscles with growth. While the FG and SO fibers increased in the diaphragm, SO fibers increased in the soleus, and FG fibers increased in the EDL. The capillary density showed a hyperbolic decrease with growth in all muscles, while the number of capillaries around each fiber increased in all muscles with growth. It is concluded that growth causes the changing properties of the motoneurons and the new capillary formation in the diaphragm muscle, as well as the soleus and EDL muscles.     

Periodic weight support effects on rat soleus fibers after hindlimb suspension

The morphological and histochemical properties of the rat soleus were studied after 1 wk of hindlimb suspension, one model that removes the weight-bearing function of the hindlimbs. To examine the effectiveness of weight support activity in maintaining soleus mass, fiber size, and succinate dehydrogenase (SDH) activity, the hindlimbs of adult male Sprague-Dawley rats were suspended (HS) and half of these rats were walked on a treadmill for 40 min/day (10 min every 6 h) at 5 m/min and a 19 degree grade (HS-WS). Significant reductions in soleus mass and fiber size were found after 1 wk of HS. Weight support activity decreased the atrophic response by approximately 50%. In the alkaline myofibrillar adenosine triphosphatase (ATPase) dark-staining fibers, SDH activity was higher in the HS than control rats, whereas it was similar to control in the HS-WS rats. Total SDH activity (SDH activity X cross-sectional area) in fibers staining lightly for ATPase in HS and HS-WS rats was lower than in control rats, whereas in the darkly stained ATPase fibers it was similar among the three groups. No changes were observed in fiber type percentages after 1 wk of HS or HS-WS. The results suggest that short-duration, daily weight support activity can ameliorate, but not prevent, soleus atrophy induced by HS. Furthermore, fiber cross-sectional area is more responsive to periodic weight support in dark than light ATPase fibers. These results also demonstrate that muscle fiber atrophy need not be associated with a loss in SDH activity.     

Classification of muscle fiber types based on succinic dehydrogenase and myofibrillar ATPase reactions in normal and randomly reinnervated rat skeletal muscles.

In the normal and randomly reinnervated plantaris muscle of rat staining for succinic dehydrogenase (SDH) activity differentiates three fiber types (A, B and C), staining for myofibrillar adenosine triphosphatase (ATPase) differentiates three fiber types (alpha, beta and alpha beta). Here we present our finding type A corresponds to alpha beta fibers, B to beta or alpha beta, C to alpha or alpha beta. In normal soleus muscle both classifications were found to be compatible and B fibers correspond to beta and C to alpha fibers. An exception is the small percent of alpha beta fibers which correspond to B type. In randomly reinnervated soleus muscle changes in ATPase activity are not followed by changes in SDH staining and B fibers correspond to alpha, beta or alpha beta types.     

Size and metabolic properties of single muscle fibers in rat soleus after hindlimb suspension

The effects of 28 days of hindlimb suspension (HS) and HS plus 10 daily forceful lengthening contractions on rat soleus muscle fibers were studied. Compared with age-matched controls (CON), soleus wet weights of suspended rats were significantly decreased (approximately 49%). In HS rats, the light adenosinetriphosphatase (ATPase) fibers (staining lightly for myosin ATPase, pH = 8.8) atrophied more than the dark ATPase fibers (staining darkly for myosin ATPase, pH = 8.8). Single-fiber alpha-glycerophosphate dehydrogenase (GPD) and succinate dehydrogenase (SDH) activities and the proportion of dark ATPase fibers were higher in HS than CON rats. Daily forceful lengthening contractions did not prevent the suspension-induced changes. These results considered in conjunction with a collaborative study on the mechanical properties of HS rats (Roy et al., accompanying paper) suggest a shift in the contractile potential of the muscle following HS without a deficit in SDH, a metabolic property commonly associated with resistance to fatigue. The results support the view that soleus muscle fibers can change from a slow-twitch oxidative to a fast-twitch oxidative-glycolytic profile, but rarely to a fast-twitch glycolytic one, and that SDH and GPD activity per volume of tissue can be maintained or increased even when there are severe losses of contractile proteins.     

Localization of sarcoplasmic reticulum proteins in rat skeletal muscle by immunofluorescence

Ca++-Mg++-dependent ATPase and calsequestrin, the major intrinsic and extrinsic proteins, respectively, of the sarcoplasmic reticulum, were localized in cryostat sections of adult rat skeletal muscle by immunofluorescent staining and phase-contrast microscopy. Relatively high concentrations of both the ATPase and calsequestrin were found in fast-twitch myofibers while a very low concentration of the ATPase and a moderate concentration of calsequestrin were found in slow-twitch myofibers. These findings are consistent with previous biochemical studies of the isolated sarcoplasmic reticulum of slow-twitch and fast-twitch mammalian muscles. The distribution of the ATPase in muscle fibers is distinctly different from that of calsequestrin. While calsequestrin is present only near the interface between the I- and A-band regions of the sarcomere, the ATPase is found throughout the I-band region as well as in the center of the A-band region. In comparing these results with in situ ultrastructural studies of the distribution of sarcoplasmic reticulum in fast-twitch muscle, it appears that the ATPase is rather uniformly distributed throughout the sarcoplasmic reticulum while calsequestrin is almost exclusively confined to those regions of the membrane system which correspond to terminal cisternae. Fluorescent staining with these antisera was not observed in vascular smooth muscle cells present in the cryostat sections of the mammalian skeletal muscle used in this study.     

Diaphragm capillarity and oxidative capacity during postnatal development.

In the cat diaphragm, fiber capillarity, cross-sectional area, and succinate dehydrogenase (SDH) activity were measured across the first 6 wk of postnatal development. Fibers were classified as type I, IIa, IIb, or IIc on the basis of staining for myofibrillar adenosinetriphosphatase (ATPase). Capillaries were identified in sections stained for ATPase at pH 4.2. Fiber cross-sectional areas and SDH activities were quantified using an image-processing system. During postnatal development, the proportions of type I fibers increased while type II fibers decreased. At birth, all type II fibers were IIc. From the 1st to the 2nd postnatal wk, the proportion of type IIc fibers decreased while the numbers of IIa and IIb increased. Thereafter the proportion of type IIb fibers continued to increase while the number of IIa steadily declined. At birth, capillarity, cross-sectional areas, and SDH activities of type I and II fibers were low compared with other postnatal age groups. Fiber cross-sectional areas increased progressively with age. The number of capillaries surrounding type I and II fibers increased markedly by the 2nd wk and then continued to increase at a slower rate. The number of capillaries per fiber area reached a peak by the 2nd wk and then declined as fiber cross-sectional area increased. Postnatal changes in capillarity depended on fiber type, being greatest in IIb. SDH activities of type I and II fibers were initially low during the first 2 postnatal wk and then peaked by the 3rd wk. After the 6th wk, fiber SDH activities decreased to adult values. Among the type II fibers, IIb showed the greatest change in SDH activity during early postnatal development.     

Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes

Myoglobin plays various roles in oxygen supply to muscle mitochondria. It is difficult, and in some cases impossible, to study the relationship between the myoglobin concentration and the oxidative capacity of individual muscle cells because myoglobin has to be fixed in situ whereas determination of oxidative capacity, for example, succinate dehydrogenase activity, requires unfixed cryostat sections. We have investigated whether a vapour-fixation technique allows the use of serial sections to study the relationship between myoglobin and succinate dehydrogenase activity. The technique is used to study a rat soleus muscle, two human skeletal muscle biopsies and biopsies of two patients with chronic heart failure, and in a control and hypertrophied rat heart. Staining intensities were quantified by microdensitometry. The absorbance values were calibrated using sections cut from gelatine blocks containing known amounts of myoglobin. The results show that it is possible to use serial sections for the determination of the myoglobin concentration and succinate dehydrogenase activity, and indicate that myoglobin can lead to a substantial reduction (18–60%) of the extracellular oxygen tension required to prevent an anoxic core in muscle cells.     

Influence of spaceflight on rat skeletal muscle

The size, succinate dehydrogenase (SDH) and alpha-glycerolphosphate dehydrogenase (GPD) activities, and alkaline myofibrillar adenosinetriphosphatase (ATPase) staining properties were determined from quantitative histochemical analyses of single fibers from five hindlimb muscles of six male rats exposed to a 7-day National Aeronautics and Space Administration spaceflight mission (SL-3). These same properties were determined in a group of ground-based control rats housed under simulated environmental conditions. The wet weight of each of the flight muscles was significantly reduced relative to control. However, the loss of mass varied from 36% in the soleus to 15% in the extensor digitorum longus. The cross-sectional areas of fibers in the flight muscles also were reduced, except for the dark ATPase fibers in the medial gastrocnemius. The greatest relative fiber atrophy occurred in the muscles with the highest proportion of light ATPase fibers. An increase in the percentage of dark ATPase fibers also was observed in flight muscles with a predominance of light ATPase fibers. Also, there was an increase in the biochemically determined myofibrillar ATPase activity of tissue sections of the flight soleus. No changes in histochemical or biochemical measures of ATPase activity were observed in the flight extensor digitorum longus. In general, the SDH activity of flight muscles was maintained, whereas GPD activity either was maintained or increased. Based on a metabolic profile of ATPase, SDH, and GPD, there was an increase in the proportion of fast oxidative-glycolytic fibers in some muscles.     

Histochemical definition of muscle fibre types in the trunk musculature of a teleost fish (cod,Gadus morhua,L.)

Summary Cryostat sections incubated for myofibrillar ATPase, SDH, LDH, and -GPDH as well as p-phenylene-diamine stained semithin sections were used to define muscle fibre types in the trunk musculature of the cod (Gadus morhua, L.).Three zones (superficial, intermediate, deep) containing different muscle fibre types are present within both epaxial and hypaxial parts of each myomere subjacent to the lateral line.Atypical relations concerning myofibrillar ATPase activity probably reflects instability of myosin during storage of frozen tissue. The histochemical reaction does not distinguish between myofibrillar and mitochondrial ATPase in cod muscle.Based on ATPase and SDH activities, seven different histochemical profiles of muscle fibres can be identified in trunk musculature of this teleost fish. Attempts to homologize these fibre types with those in cyclostomes or those in higher animals proved futile. The higher number of histochemically defined muscle fibre types in cod might be explained by developmental processes and an admixture of immature fibres throughout life.     

Differential sensitivity to androgens within a sexually dimorphic muscle of male frogs (Xenopus laevis)

Male frogs use their forelimb flexor muscles to clasp females during the mating behavior known as amplexus. We investigated the effects of testosterone on a principal forelimb flexor, the flexor carpi radialis muscle (FCR), using morphological and histochemical techniques. Male Xenopus laevis were surgically manipulated to produce high or low levels of circulating testosterone for an 8-week period. After this treatment, measurement of fibers in muscle cross-sections revealed that average fiber size was positively correlated with testosterone level. This effect was not the same for all muscle fibers, however. Fibers in the shoulder region were more sensitive to testosterone than fibers in other regions of the muscle. Histochemical staining of cross-sections showed that the patterns of staining for myosin ATPase or succinic dehydrogenase (SDH) were not influenced by testosterone levels, but total SDH activity was increased by testosterone treatment. When sensitivity to testosterone was correlated with ATPase activity, fibers with high ATPase activity were found to be more sensitive to testosterone than fibers with low activity, regardless of position within the muscle. Most fibers with high ATPase activity were located in the shoulder region of the muscle. These fibers are innervated by different motor axons than are fibers in the elbow region of the muscle, and contractions of shoulder (but not elbow) region fibers, elicited by stimulation of motor axons, are slowed by testosterone treatment (Regnier and Herrera, 1993, J. Physiol. 461:565–581). © 1993 John Wiley & Sons, Inc.     

Histochemical fiber typing and staining intensity in cat and rat muscles.

In the gastrocnemius muscle of cat and rat, staining for oxidative enzymes differentiated three fiber types (A,B,C) and staining for adenosine triphosphate at pH 9.4 differentiated two fiber types (I, II) with a reliability of 90% and 98%, respectively. In cat 96% and in rat 90% of the fibers were typed identically after staining for nicotinamide adenine dinucleotidelinked lactic dehydrogenase (LDH) and succinic dehydrogenase (SDH). When differentiated by staining for LDH, A and B fibers were of type I. IN RAT, 80-90% OF ALL FIBERS WERE OF TYPE 22, COMPPRISING A, B and C fibers. Type I fibers stained for LDH intensely as did C fibers of type II, but stained intermediately for SDH. The degree of staining was measured by photometry. When fibers were stained for LDH, histograms of density showed three peaks corresponding to A, B and C fibers in cat, but only two peaks corresponding to A and C fibers in rat, In cat and rat, the densities of A, B and C fibers belonged to different populations. In soleus muscle of cat and rat stained for LDH, menadione-linked alpha-glycerophosphate dehydrogenase and adenosine triphosphatase at pH 9.4, the degree of staining differed from thatin any type of fiber in gastrocnemius muscle     

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

Hindlimb suspension (HS) results in whole muscle atrophic and metabolic changes that vary in magnitude in different hindlimb muscles. The present study was designed to investigate these effects in single fibers. Fiber type and size and the activities of two metabolic marker enzymes were determined in a deep (close to the bone) and a superficial (away from the bone) region of the medial gastrocnemius (MG) and the tibialis anterior (TA) of control (CON) and 28-day HS adult female rats. Fibers were classified as dark or light adenosinetriphosphatase (ATPase) based on their qualitative staining reaction for myosin ATPase following alkaline preincubation. Fiber area and succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities were determined in tissue sections by use of an image analysis system. After 28 days of HS, the mean body weights of the CON and HS were similar. MG atrophied 28%, whereas TA weight was maintained in the HS. Both dark and light ATPase fibers in the deep region of the MG had smaller cross-sectional areas following HS, with the atrophic response being approximately twice as great in the light ATPase fibers. No significant changes in fiber type composition in either muscle or in fiber sizes in the superficial region of the MG or in either region of the TA were observed. Mean SDH activities of both fiber types were significantly lower in the MG and TA following HS. In contrast, mean GPD activities were either increased or maintained in light and dark ATPase fibers of both muscles in HS. Changes in SDH and GPD activity could not be directly linked to changes in fiber cross-sectional area. In summary, these data suggest an independence of the mechanisms determining muscle fiber size and metabolic adaptations associated with HS.