Ultrastructural and metabolic profiles on single muscle fibers of different types after hindlimb suspension in rats

The relationships between ultrastructural and metabolic profiles in different types of single muscle fiber after hindlimb suspension in rats were examined. Glycolytic (lactate dehydrogenase, LDH; phosphofructokinase, PFK) and oxidative (succinate dehydrogenase, SDH; malate dehydrogenase, MDH) enzyme activities in extensor digitorum longus (EDL) and soleus (SOL) muscles were measured. Relative mitochondrial and lipid droplet volumes were also measured in single muscle fiber of different types. Glycolytic enzyme activity in EDL muscle and oxidative enzyme activity in soleus muscle decreased following suspension for 2 weeks. LDH and PFK activities in fast-twitch (FG, fast-twitch glycolytic; FOG, fast-twitch oxidative glycolytic) fibers and oxidative enzymes in FOG and FG fibers decreased following suspension. Relative mitochondrial volume decreased significantly in all types (SO, slow-twitch oxidative; FOG, and FG) of fibers following suspension. The mitochondrial volume in SO fiber of the control group was significantly (p less than 0.01) higher than that of suspended group; however, SDH and MDH activities were not different between the control and suspended groups. The structural and metabolic changes following hindlimb suspension were influenced by different factors, respectively. Changes in ultrastructural and metabolic profiles in response to the hindlimb suspension differed according to the type of fibers.     

Different metabolic responses to exercise training programmes in single rat muscle fibres

Summary The aim of this report is to elucidate the effects of exercise training on metabolic properties of different muscle fibre types of the rat hindlimb. Single muscle fibres were dissected from soleus (SOL) or extensor digitorum longus (EDL) muscles of Wistar strain male rats trained on a treadmill for 16 weeks. Each fibre was typed histochemically (SO, slow-twitch oxidative; FOG, fast-twitch oxidative glycolytic; FG, fast-twitch glycolytic). Then glycolytic and oxidative enzymes (CK, LDH, PFK, PK, SDH, and MDH) activities were measured biochemically. Slow,-type fibres (SO) were hypertrophied following endurance training and fast-twitch fibres (FOG and FG) were hypertrophied following sprint training. In EDL muscles the distribution of the slow-type fibres was reduced following the sprint training. The activity of glycolytic enzymes increased significantly in the fast-type fibres (FOG and FG) following sprint training, while oxidative enzymes activities increased in both fast (FOG and FG) and slow (SO) muscle fibres following the endurance training. Neither glycolytic nor oxidative enzymes' activities always increased equally in all types of fibre following exercise training. Consequently, the metabolic profiles in each type of single muscle fibre were affected differently by different intensities of exercise training. These results suggest that the functional (enzymes activity) and structural (muscle fibre hypertrophy) changes of skeletal muscle fibre following exercise training appeared gradually, and would be controlled by different factors.     

Structure,innervation, and age-associated changes of mouse forearm muscles

In spite of a decline in muscle strength with age, the cause of the overall decrease in motor performance in aged mammals, including rodents, is incompletely understood. To add clarity, the gross organization, innervation, histochemical fiber types, and age-associated changes are described for mouse forearm muscles used in a variety of motor functions. The anterior (flexor) and posterior (extensor) forearm compartments have the same arrangement of muscles and gross pattern of innervation as the rat. Two primary histochemical fiber types, fast/oxidative/glycolytic (FOG) and fast/glycolytic (FG), with characteristic hitsochemical staining patterns were observed in all forearm muscles. Additionally, there was a small population of slow/oxidative (SO) fibers confined to the deep region of a single muscle, the flexor carpi ulnaris (FCU). Between 18 and 26 months the FCU muscle displayed fibers with morphological features distinct from earlier ages. Fibers displayed a greater variation in size, a loss of their uniform polygonal shape, and a dramatic increase in clumps of subsarcolemmal mitochondria, lysosomes, and lipofuscin granules. Many of the fibers had a distinctly atrophic, angular shape consistent with recent denervation. Morphometric analyses of the FCU's source of innervation, the ulnar nerve and one of its ventral roots (C8), were consistent with the denervation-like changes in the muscle fibers. Although, there was no net loss of myelinated axons between 4 and 26 months of age, there was a significant increase in the density of degenerating cells in both the ulnar nerve and ventral root C8. © 1993 Wiley-Liss, Inc.     

Histoenzymology and morphometry of the masticatory muscles of tufted capuchin monkey (Cebus apella Linnaeus, 1758)

Samples of the anterior and posterior regions of the masseter and temporal muscles and of the anterior belly of the digastric muscle of 4 adult male tufted capuchin monkeys (Cebus apella) were removed and stained with HE and submitted to the m-ATPase reaction (with alkaline and acid preincubation) and to the NADH-TR and SDH reactions. The results of the histoenzymologic reactions were similar, except for acid reversal which did not occur in fibers of the fast glycolytic (FG) type in the mandibular locomotor muscles. FG fibers had a larger area and were more frequent in all regions studied. No significant differences in frequency or area of each fiber type were detected, considering the anterior and posterior regions of the masseter and temporal muscles. The frequency of fibers of the fast oxidative glycolytic (FOG) and slow oxidative (SO) types and of FOG area differed significantly between the anterior belly of the digastric muscle and the mandibular locomotor muscle. The predominance of fast twitch (FG and FOG) fibers and the multipenniform and bipenniform internal architecture of the masseter and temporal muscles, respectively, are characteristics that permit the powerful bite typical of tufted capuchin monkeys.     

Differences in ultrastructural and metabolic profiles within the same type of fibres in various muscles of young and adult rats

Single fibres from tibialis anterior, extensor digitorum longus, gastrocnemius and soleus muscles in young (4–week–old) and adult (35–week–old) Wistar male rats were classified into three types on the basis of their enzyme–histochemical features: slow–twitch oxidative (SO), fast–twitch oxidative and glycolytic (FOG) and fast–twitch glycolytic (FG) fibres. Ultrastructural (volume density of mitochondria: V_mt and Z line width) and metabolic (phosphofructokinase: PFK and succinate dehydrogenase: SDH activities) profiles were measured. PFK activity in all types of fibres¹ was higher in adult rats, and the difference between the two age–groups (adult/young) was largest between FG, FOG and SO fibres respectively. SDH activity and V_mt were lower in adult rats in a similar way in all fibres. A significant positive correlation was observed between the V_mt and SDH activity in both age–groups. This positive correlation was very specific in fast–twitch and slow–twitch fibres. Changes in the V_mt did not relate directly to the changes in fibre cross–sectional area. The overall pattern indicates that glycolytic capacity of fast–twitch fibres in flexor muscles (TA and EDL) is higher than in extensor muscles (GC and SOL), and that oxidative capacity of all types of fibre in extensor muscles is higher than in flexor muscles. These profiles were changed by growth, and may be related to the specific differences in pattern of activity of each skeletal muscle, and may reflect differences in the recruitment order of different muscles.     

Effect of hypobaric hypoxia on rat soleus muscle fibers and their innervating motoneurons: a review

Mammalian skeletal muscle fibers are classified into three basic types based on their enzyme histochemical profiles: fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG), and slow-twitch oxidative (SO) types. The type-shift of fibers from FOG to SO in the slow soleus muscle of rats occurs during postnatal growth. Our previous studies have demonstrated that hypoxic exposure inhibits a growth-related type-shift of fibers from FOG to SO in the rat soleus muscle, irrespective of the duration and age at which the animals are exposed to hypoxia. Our previous studies have also revealed that a high percentage of FOG fibers in the soleus muscle of the hypoxia-adapted rats is found only under hypoxic conditions. Furthermore, we have found that these adaptations in fibers of the rat soleus muscle correspond well with those in motoneurons at the ventral horn of the spinal cord that innervate the muscle fibers.     

The time course of glycogen depletion in single fibers of chronically stimulated rabbit fast-twitch muscle

A time course study was conducted to investigate the possibility of a relationship between fiber degeneration and glycogen depletion in chronically nerve-stimulated extensor digitorum longus muscle of the rabbit. Muscles were stimulated 12 h daily at 10 Hz using alternating one-hour periods of stimulation and rest. When measured for the first time after 3 h (1 h stimulation, 1 h rest, 1 h stimulation), microphotometry revealed complete glycogen depletion of all fiber types (fast glycolytic, FG; fast oxidative glycolytic, FOG; slow oxidative, SO). Different responses were noted beginning at day 4. At this time point, all FOG and SO fibers recovered their glycogen stores with some of the FOG population attaining levels higher than the FOG fibers in the unstimulated, contralateral muscle. Approximately 28% of the FG fibers recovered to normal glycogen values, whereas 58% remained depleted and 14% displayed overshoting glycogen levels. Fifteen percent of all fibers were glycogen-depleted after 12 days of stimulation. At this time, classic fiber types could no longer be distinguished. Fiber degeneration, which was recognized by the invasion of nonmuscle cells, began after 6 days and was restricted to the glycogen-depleted fibers. By this time, there was also a significant increase in DNA content. Exhaustions of glycogen, the main fuel of the FG fibers, is believed to cause a collapse of energy-supply and ATP-driven ionic pumps. The latter could be the initial step of fiber deterioration.     

Early changes in fiber profile and capillary density in long-term stimulated muscles

Predominantly fast skeletal muscles of rabbits [tibialis anterior (TA), extensor digitorum longus (EDL)] were stimulated at a frequency naturally occurring in nerves to slow muscles (10 Hz continuously) for 8 h/day for 2--4 days. Such stimulation is known to convert all glycolytic fibers to oxidative and to increase capillary density. Our aim was to study early stages of conversion to investigate the factors responsible for the changes. Staining of quick-frozen sections for myosin ATPase, succinic dehydrogenase, and alkaline phosphatase was used to study the distribution of different fiber types and to measure fiber cross-sectional areas, capillaries per square millimeter, and capillary-to-fiber ratios in each fiber category. TA but not EDL showed conversion of fast glycolytic to fast oxidative fibers after 2 days, more after 4 days of stimulation. In both muscles, the largest fast glycolytic fibers were diminished in number after stimulation. There was significant increase in total capillaries per square millimeter after 4 days and some increase after 2 days of stimulation. The increase in capillaries per square millimeter exceeded the increase in the number of fibers per square millimeter, and since there was no change in mean fiber area, the increase is attributed to capillary growth. In EDL, there was an increase in the number of capillaries supplying both fast glycolytic and fast oxidative fibers, suggesting that capillary growth precedes fiber type conversion. In TA, the number of capillaries supplying fast oxidative fibers was increased but that to fast glycolytic fibers, was not. This is consistent with capillary growth simultaneous with or following fiber conversion. In both TA and EDL the number of capillaries perfused after contraction was higher in stimulated muscles, suggesting that increased capillary flow contributed to capillary growth.     

Differentiation of rat skeletal muscle fibres during development and ageing

The purpose of the present study was to determine at which point in the period from embryonic day 21 up to postnatal day (PD) 75, the different fibre types and subtypes are detectable in rat extensor digitorum longus, soleus and gastrocnemius muscles using immunohistochemical, enzyme histochemical and cytophotometrical methods. Moreover, fibre type-specific changes in metabolic profile and changes in fibre type population during postnatal development were analysed. Before birth, no clear differentiation of fibre types was found. At PD 1, slow and fast fibres were typed by antibodies against neonatal, slow and fast myosin heavy chains (MHCs). At PD 8, the different ATPase activities of slow and fast MHCs after alkaline preincubation were detected histochemically. At PD 21, differences in acid stability of ATPase activity of fast MHC isoforms revealed the fast subtypes IIA and IIB (including IIX). At this age, also differences in metabolic properties (oxidative and glycolytic enzyme activities) of fibres were detected for the first time by cytophotometry classifying the fibres into SO, FOG I, FOG II and FG. Before the age of 21 days, the fast fibres were metabolically undifferentiated. During further development and ageing, the population of FG fibres with high glycolytic activity increased at the expense of FOG fibres suggesting FOG to FG transformation. Cytophotometrical measurements revealed that the muscle fibres developed their highest contractile, oxidative and glycolytic activity at PD 21, the time of weaning. In this way, muscle fibres may be prepared for the higher demands for posture and mobility after leaving the nest.     

Levels of enzymes of energy metabolism are controlled by activity of cultured rat myotubes

We have investigated the effects of inhibiting the spontaneous activity of cultured rat myotubes on several representative enzymes of glycolytic and oxidative metabolism. The results presented demonstrate that contractile activity in the absence of nerves can regulate the amounts of these enzymes and indicate that muscle activity may partially control development of the metabolic types of muscle fibers. Control muscle cells have relatively high levels of glycolytic enzymes and low oxidative enzymes and metabolically most closely resemble fast glycolytic fibers. The divalent cation ionophore A23187 caused enzyme levels of the cultured cells to change towards those found in tonically contracting skeletal muscle fibers in vivo. The evidence presented suggests that calcium may mediate certain of the effects associated with muscle contraction on enzymes of energy metabolism.     

Histochemical changes of rat skeletal muscles induced by cold acclimation

After cold acclimation of rats the augmentation of succinic dehydrogenase activity in the fast-twitch oxidative glycolytic (FOG) and the slow-twitch oxidative (SO) fibers was observed in entire regions of the soleus, the extensor digitorum longus, the plantaris, the longissimus and the gastrocnemius muscles. Furthermore, a tendency to increased proportion of the FOG and the SO fibers was observed more prominently in superficial regions than in deep regions of a large muscle such as the gastrocnemius muscle.     

Comparison of physiological and histochemical properties of motor units after cross-reinnervation of antagonistic muscles in the cat hindlimb

1. Physiological and histochemical properties of the cat ankle extensor muscles, the lateral and medial gastrocnemius, and the soleus were studied after cross-reinnervation by flexor motoneurons. 2. Tibial and common peroneal nerves were cut and cross-united in the popliteal fossa of 2- to 6-mo-old cats. Eighteen to 24 mo later, single motor units were isolated by dissection and stimulation of ventral root filaments and classified into four types: fast-twitch, fatigable (FF), fast-twitch with intermediate fatigue resistance (FI), fast-twitch, fatigue-resistant (FR), and slow, fatigue-resistant (S). Muscle fibers were classified as fast glycolytic (FG), fast, oxidative glycolytic (FOG), and slow oxidative (SO) on the basis of histochemical staining. 3. Although motor-unit force was normally well correlated with the size of the innervating motor axon in the cross-reinnervated muscles, the force of different unit types overlapped considerably. The reinnervated motor units also showed a higher than normal degree of fatigability. 4. The range of muscle unit forces in cross-reinnervated triceps surae muscles was the same as in the normally innervated triceps surae muscles. This range is 2-3 times greater than the flexor muscles, which the common peroneal nerve normally supplies. The range of contraction speed of units in the cross-reinnervated extensor muscles was comparable to that in the flexor muscles, consistent with a motoneuron-specific determination of muscle speed (28). 5. SO and FOG muscle fibers were found in all reinnervated triceps surae muscles, but FG fibers were only found in reinnervated medial gastrocnemius (MG) and lateral gastrocnemius (LG) muscles, consistent with previous findings of the resistance of soleus muscles to complete conversion (10, 16, 20, 21). Type grouping of muscle fibers was characteristic of the reinnervated muscles. 6. Reinnervated SO muscle fibers were larger than the corresponding fibers in normally innervated muscles as were the estimated number of muscle fibers innervated by slow motor axons. Nonetheless, the force generated by the S motor units remained relatively smaller than FR and FF units. The relative contributions of the number, cross-sectional area and specific tension to the force generation of reinnervated motor units are discussed.     

Histochemical differentiation of fibers in the rat slow and fast twitch muscles.

Wistar male rats were sacrificed at 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days, and at 5 and 10 weeks of age. The histochemical differentiation of slow twitch soleus and fast twitch plantaris muscle fibers was examined from the enzyme activities of adenosine triphosphatase (slow- or fast-contracting), succinate dehydrogenase (high- or low-oxidative), and alpha-glycerophosphate dehydrogenase (high- or low-glycolytic). The soleus muscle fibers differentiated into slow-contracting (S) and fast-contracting (F) fibers at 7 days of age. In the plantaris muscle, differentiation into S and F fibers in the deep portion occurred earlier (9 days) than in the superficial portion (11 days). Thereafter, fiber type shifts between S and F were observed in both muscles. Differentiation into fast-contracting oxidative glycolytic (FOG), fast-contracting glycolytic (FG), and slow-contracting oxidative (SO) fibers occurred in both muscles at 15 and 17 days of age. After subdivision into the three fiber types, a type shift from FOG to FG was observed in both the deep and superficial portions of the plantaris muscle.     

Statin-induced muscle necrosis in the rat: distribution, development, and fibre selectivity

Simvastatin and cerivastatin have been used to investigate the development of statin-induced muscle necrosis in the rat. This was similar for both statins and was treatment-duration dependent, only occurring after 10 days had elapsed even if the dose was increased, and still occurring after this time when dosing was terminated earlier as a result of morbidity. It was then widespread and affected all areas of the muscular system. However, even when myotoxicity was severe, particular individual muscles and some types of fibres within affected muscles were spared consistently. Fibre typing of spared muscles and of acutely necrotic fibres within affected muscles indicated a differential fibre sensitivity to statin-induced muscle necrosis. The fibres showed a necrotic response to statin administration that matched their oxidative/glycolytic metabolic nature: Least sensitive --> I < - > IIA < - > IID < - > IIB <-- most sensitive. Type I and IIB fibres represent metabolic extremes of a continuum of metabolic properties through the fibre types with type I fibres most oxidative in metabolism and type IIB fibres most glycolytic. In addition, in some (nonnecrotic) glycolytic fibres from muscles showing early multifocal single fibre necrosis the only subcellular alterations present in isolation of any other changes were mitochondrial. These changes were characterised by an increased incidence of vacuolation and the formation of myelinoid vesicular bodies that accumulated in the subsarcolemmal areas. These findings suggest an important early involvement of mitochondria in selective glycolytic muscle fibre necrosis following inhibition of the enzyme HMG-CoA reductase.     

Specific mitochondrial responses to running training are induced in each type of rat single muscle fibers

The effects of training by running (sprint or endurance) on the mitochondrial volume, number, and succinate dehydrogenase (SDH) activity of different types of single muscle fibers, and on the elemental composition of soleus and extensor digitorum longus muscles were studied employing histochemical, electron microscopic, and electron probe-micro analysis (EPMA). Newly weaned male Wistar rats were trained on a motor-driven treadmill endurance exercise for 14 weeks. The relative mitochondrial volume per single muscle fiber of slow-twitch oxidative (SO) fiber was significantly increased following endurance training (p less than 0.01). There was no significant correlation between mitochondrial volume and number of SO fibers following endurance exercise training. Following sprint training, both mitochondrial volume and number of fast-twitch oxidative glycolytic (FOG) fibers increased significantly (p less than 0.01), with significant correlation (r = 0.69) between these parameters. SDH activity was higher in the order of SO, FOG, and fast-twitch glycolytic (FG) fibers following endurance training; however, the activity was higher in the order FOG, SO, and FG fibers following sprint training. The potassium concentration in cytoplasm of the soleus muscle showed a tendency to decrease following both types of training. These results suggest that the oxidative capacity of each type of muscle fiber does not always increase equally following training. Changes in mitochondrial number and volume in response to the two different types of training differed according to the type of fiber.     

Effects of age on the number and histochemical properties of muscle fibers and motoneurons in the rat extensor digitorum longus muscle

The number and histochemical properties of muscle fibers and motoneurons were studied in the extensor digitorum longus muscle in female albino rats at the ages of 10, 60 and 120 weeks. The number of fast twitch glycolytic (FG) fibers was decreased at 60 weeks, while that of fast twitch oxidative glycolytic (FOG) fibers was decreased at 120 weeks. The number and oxidative enzyme activity of motoneurons were decreased later at 120 weeks. Thus, it is suggested that the decrease in FG fibers at 60 weeks was due to selective muscle fiber atrophy or to degeneration of neuromuscular junctions, while on the other hand, the decrease in FOG fibers at 120 weeks was due mainly to both a decrease in motoneurons and a type shift of fibers from FOG to FG.     

Effects of lengthened immobilization on functional and histochemical properties of rabbit tibialis anterior muscle.

The imposition of long-term lengthened immobilization on rabbit tibialis anterior (TA) muscles resulted in rapid increases in slow oxidative (SO) fibre number. After 2 weeks, SO number had increased 2-fold and was 5 times greater after 6 weeks immobilization. There were also fibre-type-specific effects on SO, fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibre areas. Twitch strength was unchanged throughout immobilization whilst production of tetanic tension was impaired during the initial period but had returned to control levels by 6 weeks. Twitch contraction times and isometrically determined rates of rise and relaxation were largely unaffected by immobilization despite the marked increase in expression of slow myosin. The change in phenotypic expression of the lengthened TA was not attributable to chronically increased levels of activation since integrated EMG activity was unchanged from control values throughout the immobilization period. Thus it is suggested that a chronic increase in tension consequent on the lengthening procedure is a potent stimulus for fast-to-slow myosin transformation.     

Histochemical profiles of fibers in the rat tibialis anterior muscle during early postnatal development

The enzyme activities of intra- and extrafusal fibers in the tibialis anterior muscle of rats during postnatal development have been investigated. Muscle fibers 1 day after birth showed a uniform reaction for adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), and alpha-glycerophosphate dehydrogenase (alpha-GPD) activities. Fast-twitch (F) and slow-twitch (S) fibers with ATPase activity were found at 9 and 11 days. Thereafter, the type shift of muscle fibers from S to F was observed in the deep and middle portions. Fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and slow-twitch oxidative (SO) fibers with ATPase, SDH, and alpha-GPD activities were found at 15 (the superficial portion) and 17 days (the deep and middle portions). The histochemical differentiation of intrafusal muscle fibers (7 and 9 days) was found earlier than that of extrafusal muscle fibers.     

A histochemical analysis of identified compartments of cat lateral gastrocnemius muscle

The lateral gastrocnemius muscle of cats can be divided into four discrete subvolumes or compartments which are supplied by the primary branches of its muscle nerve. The histochemical profile of each compartment was determined from the reaction for myosin ATPase after acid preincubation. Fibers were classified as fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG), or slow-twitch oxidative (SO). Each compartment in each cat examined was found to contain a relatively uniform distribution of different types of fibers. The most proximal LG compartment contains mainly type FG fibers, with relatively few type SO fibers; the most distal compartment, while still predominated by type FG fibers, contains a significantly larger proportion of type SO fibers. The histochemical profile of the intermediate compartments indicates that they contain fibers which lie intermediate in composition between these two. These results are consistent with the notion that LG compartments consist of aggregations of motor units, arranged such that muscle fibers comprising a single motor unit are contained within a single compartment.