Correlation of electrophysiological,histochemical, and mechanical properties in fibres of the coxa rotator muscle of the locust,Locusta migratoria

Summary The fibre composition of the anterior coxa rotator muscle of the locust middle leg (M92) was examined. The muscle is composed of 90–100 fibres. Muscle fibres were characterized with regard to innervation pattern, electrophysiological properties, and morphological parameters. Activity and isoenzyme composition of myofibrillar ATPase, succinic acid dehydrogenase (SDH) activity and glycogen content were examined employing histochemical techniques. Shortening velocity and the dependence of tension on intracellular Ca²⁺ were determined in skinned fibre experiments. A close match was observed between the innervation pattern of the muscle fibres and their histochemical and physiological properties. The combination of all parameters examined allowed an accurate classification of the muscle fibres into three types. Within a given type, broad variability of some properties was observed (SDH activity, Ca²⁺ sensitivity) while others assumed distinct values (innervation pattern, shortening velocity). The comprehensive characterization of muscle fibre properties permits a functional interpretation of fibre heterogeneity with regard to muscle performance. Fibres with the same innervation pattern may be recruited specifically, according to their electric properties and Ca²⁺ sensitivities. The resulting specific recruitment of fibres with different mechanical responses should allow a subtle control of muscular force, with regard to force amplitude, temporal characteristics of contraction, and metabolic cost.Abbreviations CI₁ common inhibitory neurone one - ejp ijp excitatory, inhibitory junctional potential - EGTA ethylene glycol-bis[-aminoethyl ether] N,N,N,N-tetraacetic acid - mATPase myofibrillar adenosinetriphosphatase - MOPSO 3-[N-morpholino]-2-hydroxypropanesulfonic acid - M92 anterior rotator muscle of the coxa - n Hill coefficient - pCa₅₀ pCa corresponding to half-maximal tension - P₀ maximal isometric tension - SDH succinic acid dehydrogenase - V _max maximal shortening velocity     

Further histochemical properties of rabbit skeletal muscle fibres

Summary The histochemical activities of succinic dehydrogenase (SDH), creatine kinase (CK), sarcoplasmic reticular ATPase (SR-ATPase) and myosin ATPase were studied in serial sections of rabbit adductor muscle. Three fibre types were distinguished depending upon the distribution of the enzyme activities. The type II white fibres posessing minimal SDH showed high myosin ATPase, SR-ATPase and ATPase dependent CK activities. Red oxidative fibres showing high SDH fell into two distinct groups: One category had mainly a peripheral localization of SDH and showed an enzymatic profile identical to that of type II white fibres. The second category of red fibres displayed both a homogeneous distribution of small diformazan granules throughout the fibre as well as a sub-sarcolemmal collection when tested for SDH activity but possessed very low amounts of reaction product of the various enzymes of the energetic metabolism studied. Since it is well established that the myosin ATPase of a fibre correlates with its contraction time, the present histochemical investigation provides further support for this concept by demonstrating the presence of high SR-ATPase and ATPase dependent CK activities in all white and red fibres rich in myosin ATPase.     

A histochemical analysis of mammalian oxidative skeletal muscle fibres using the enzymes of energetic metabolism

Summary Some histochemical parameters of the three main fibre types of rat vastus lateralis muscle were studied. Succinic dehydrogenase (SDH), creatine kinase (CK), sarcotubular ATPase (SR-ATPase) and mitochondrial ATPase activities were demonstrated in serial sections. The three fibre types, recognised by the distribution pattern of SDH activity, all show high CK activity. However, red Type I oxidative fibres when examined for ATPase and ATPase dependent CK activity, show distinct heterogeneity revealing sub-populations within the same homogeneous fibre type. Three distinct patterns were recognised in the red Type I fibres depending on the distribution of the final reaction product. The present histochemical evidence confirms the fact that subdivision of mammalian skeletal muscle into three fibre types is only approximate and probably more than three types exist.     

Histoenzymatic characterization of sub-types of type I fibres in fast muscles of rats.

The histochemical activities of succinic dehydrogenase (SDH), myofibrillar Adenosine triphosphatase (ATPase) and alpha glycerophosphate dehydrogenase were studied in serial sections of rat vastus lateralis (red) (RVL), gastrocnemius and diaphragm muscles. Three main fibre-types were distinguished. The "Type I" fibres of RVL and gastrocnemius muscles fell into two distinct groups: one category--"Type IA" showed very low ATPase activity. The second category of "Type IB" fibres displayed moderate ATPase reaction. The "Type IA" fibres were divisible into two sub-groups when tested for SDH reaction. "Type IA1" fibres possessed a homogenous distribution of diformazan granules throughout the fibre: "Type IA2" fibres displayed characteristic "moth-eaten" pattern of diformazan localization. The diaphragm muscle did not show either "Type IB" or "Type IA2" varieties. The great majority of TypeI fibres were sub-type IA1 in the three fast muscles studied. It is also demonstrated here that an inherent heterogeneity exists between Type I filores of diaphragm and leg muscles in regard to alpha-GPD localization. This histochemical data emphasizes the fact that subdivision of TypeI striated muscle fibres of mammalian animals into two sub-types is only approximate and that a further subcategorization is possible.     

Temperature induced variation in the distribution of different types of muscle fibre in the goldfish (Carassius auratus)

Summary Goldfish (Carassius auratus L.) were acclimated to environmental temperatures of 3 °C, 18 °C and 31 °C for a period of three months. Cytochemical techniques were used to study the metabolism and myofibrillar ATPase activities of individual muscle fibres. Fish muscle is composed of three basic fibre types each with distinct contractile and metabolic characteristics. Cold acclimation resulted in a shift to a more aerobic type of metabolism, particularly in the red and pink fibres. In addition, environmental temperature was found to affect the size and relative distribution of the different fibre types in the myotome. The total number of pink and red fibres increased significantly with cold acclimation. Mechanisms of environmentally-induced adaptation of muscle fibre phenotype are discussed.In addition to changes in the metabolism and distribution of muscle-fibre types, biochemical studies have provided evidence for different kinetic forms of Mg²⁺Ca²⁺ myofibrillar ATPase at different environmental temperatures. Activities of myofibrillar ATPase assayed at 31 °C were 2–3 times higher in fish acclimated to the higher environmental temperature. Activation enthalpy (H ) of the ATPase was also signficantly reduced in the cold adapted enzyme. Reduction of H in the cold acclimated ATPase is thought to reduce the temperature sensitivity of the activation process thus partly compensating for the reduced cell temperature.     

Intrafusal fibre types in rat limb muscle spindles

Summary Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali-and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length.Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

Studies on the swimming musculature of the rainbow trout I. Fibre types

A histochemical study has been made on the myotomal musculature of the rainbow trout Salmo gairdneri Richardson. Four main types of fibre can be distinguished on the basis of differences in fibre size, lipid content and succinic dehydrogenase and myofibrillar adenosine triphosphatase activity (ATPase activity). Early histological studies have concluded that in addition to a superficial strip of muscle small diameter 'red' fibres occur throughout the trout myotome. These small diameter fibres occurring in the bulk of the myotome are shown to differ from the superficial fibres with respect to their oxidative metabolism and myofibrillar ATPase. The relevance of this finding to studies on the energetics and swimming efficiency of this species are discussed.     

Intrafusal fibre types in rat limb muscle spindles: morphological and histochemical characteristics.

Morphological, histochemical and ultrastructural characteristics of intrafusal fibre types were studied in rat muscle spindles. The existence of three intrafusal fibre types, namely the typical bag, the intermediate bag and the chain fibres was confirmed. Intrafusal fibres differ in diameter, length and number of nuclei in the equatorial zone. Histochemically, typical bag fibres exhibit both alkali- and acid-stable ATPase activity and low SDH activity. Intermediate bag fibres possess low alkali-stable ATPase activity; after acid-preincubation, however, they have low activity only in the juxtaequatorial region, whereas in the polar zones they exhibit high acid-stable ATPase activity. The SDH activity varies from moderate to high. The chain fibres exhibit high alkali-stable and low acid-stable ATPase and high SDH activity in the extensor digitorum longus muscle, whereas in the soleus muscle the acid-stable ATPase activity varies from a low one to a high one, either among individual chain fibres in one spindle, and/or repeatedly along the fibre length. Since there are regional differences in morphological characteristics and in staining properties of intrafusal fibres, a reliable identification of intrafusal fibre types can only be achieved by an analysis of serial sections.     

A histoenzymatic study of rat intrafusal muscle fibres

Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K₂-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K₂-EDTA preincubation. Following either acetic acid solution or cold and room temperature K₂-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K₂-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

The claw closer muscle of two estuarine crab species,Cyrtograpsus angulatus and Neohelice granulata (Grapsoidea,Varunidae): histochemical fibre type composition

Longo, M.V. and Díaz, A.O. (2011). The claw closer muscle of two estuarine crab species, Cyrtograpsus angulatus and Neohelice granulata (Grapsoidea, Varunidae): histochemical fibre type composition. —Acta Zoologica (Stockholm) 00 : 1–7. This study permitted the characterization of four types of muscle fibres in the claw closer muscles of Cyrtograpsus angulatus and Neohelice granulata. Succinic dehydrogenase (SDH) for mitochondria, periodic acid Schiff (PAS) for glycogen, Sudan Black B for lipids and myosin‐adenosine triphosphatase (m‐ATPase) preincubated at alkaline and acid pHs were used for that purpose. The mean fibre diameters, the relative areas and frequencies of each muscle fibre type were calculated. Types I and IV would be considered ‘extreme’ groups with type I fibres large, weak and acid/alkaline‐labile m‐ATPase, weak SDH, PAS and Sudan, and type IV fibres small, very strong and acid/alkaline‐resistant m‐ATPase, strong SDH and PAS, and moderate Sudan. Types II and III would belong to a predominant ‘intermediate’ group. Type IV fibres were scarce in C. angulatus but represented 25% of the total fibre population in N. granulata. In C. angulatus, the relative area occupied by type I fibres was bigger than its relative proportion, whereas in N. granulata, types I and II had similar patterns. Concluding, variations in fibre type composition in the claw closer muscles of C. angulatus and N. granulata would be linked to different habitats and feeding behaviours.     

Calcium-stimulated myofibrillar ATPase activity correlates with shortening velocity of muscle fibres in Xenopus laevis

Summary The iliofibularis muscle ofXenopus laevis is reported to contain five types of fibres which have different force—velocity relationships. Ten fibres of each type were selected on the basis of succinate dehydrogenase activity, cross-sectional area and location in the muscle, in order to assess the validity of the fibre type classification.Maximum calcium-stimulated myofibrillar ATPase activity (V _max) and apparent Michaelis constant (K _m) for ATP were determined for these 50 fibres from serial sections. The values obtained varied according to the type of fibre. Type 1 had the highest and type 5 the lowest values forK _m andV _max.In a separate experiment, single freeze-dried fibres were used to determine the relationship between their ATP content and apparentK _m for ATP. There was a tendency for high ATP concentrations in fibres with highK _m values.When myofibrillar ATPase activity was related to the maximum velocity of shortening of the five fibre types, a significant correlation was found. It is concluded that calcium-stimulated myofibrillar ATPase histochemistry allows an estimate of the maximum shortening velocity of muscle fibres fromXenopus laevis.     

Morphological and histochemical characterization of the pectoral fin muscle of the stripped weakfish,Cynoscion guatucupa

The fibres of superficial and deep abductor muscles of the pectoral fins of the stripped weakfish, Cynoscion guatucupa have been studied using histochemical techniques: succinic dehydrogenase (SDH) for mitochondria, periodic acid–Schiff (PAS) for glycogen, myosin‐adenosintriphosphatase (mATPase) to identify different fibre types based on the contraction speed and modified ATPase to identify capillaries. The fibre diameters were measured, and the capillaries of the main fibre types – red, pink and white— were counted. The two muscles showed both macroscopically and microscopically two well‐differentiated zones with predominant white fibres. The area of insertion of muscles into the fin rays had red, pink and white fibres. The origin zone of the muscle into the bone was composed by white fibres only. Both zones of white muscle evidenced a mosaic of small, medium and large polygonal white fibres. Red, pink and white muscles showed a wide histochemical diversity of fibre subtypes. The area per peripheral capillary increased from the red to the white muscles. Due to the predominance of white fibres, the pectoral fins of C. guatucupa were mainly involved in rapid movements to stop/discontinue and stabilize the body during swimming.     

Characterization of swimming muscle in the Atlantic mackerel, Scomber scombrus L.

Both red and white muscle were removed from juvenile and adult Atlantic mackerel, Scomber scombrus L., for histochemical characterization of the muscle fibre types. Staining of white muscle for myosin ATPase, SDH, NADH diaphorase, GPDH and LDH revealed that these fibres are homogeneous. Red muscle was shown to be heterogeneous, of at least two fibre types recognizable on the basis of myosin ATPase staining with preincubation at a pH of 9·8. These two red types are dispersed throughout the red muscle and are present in both juveniles and adults. Red muscle is located both deep within the myotomes and as a superficial layer of muscle fibres. A third group of muscle fibres, intermediate in nature, was distinguished at the apex of the red muscle 'triangle,' between the epaxial and hypaxial muscle, using NADH diaphorase and myosin ATPase stains. This paper discusses the possibility that functionally different muscle fibres occur in the red swimming muscle of the Atlantic mackerel.     

Histochemical properties of the biventer cervicis muscle of the chick: a relationship between multiple innervation and slow-tonic fibre types

Summary Chick biventer cervicis muscle fibres have been studied histochemically. Fast-twitch, focally innervated () fibres represent 70–80% of the total fibres in this muscle. Two histochemical profiles of slow-tonic multi-innervated () fibres have been observed from embryonic life to the adult (three-months) stage. These two slow-tonic types differ in the activity of their histochemically demonstrated myofibrillar ATPase after either acid or alkaline preincubation, and after formalin fixation. Both slow-tonic fibre types have a high oxidative metabolism and are PAS-negative. They are referred as to ₁ and ₂R fibre types (slow-tonic oxidative) in an expansion of Ashmore's nomenclature, and compared to avian slow-tonic sub-types that have been described in recent reports. ₁ and ₂ fibre types exhibit a similar pattern of innervation. Possible explanations of the origin of histochemical heterogeneity in multiple innervated fibres are discussed.     

Histochemical sub-types of three fibre-types of avian skeletal muscle

Summary The soleus muscle of adult chicken was studied histochemically. Succinic dehydrogenase (SDH) and myofibrillar ATPase reaction, with or without preincubation in K₂-EDTA salt, were compared in serial frozen sections. Based upon the distributions of the above reactions, the three major fibre types distinguished were Type I red, Type II red and Type II white. On the basis of non preincubated ATPase reaction alone two sub-types of type I red fibres could be distinguished. However, following preincubation in a Cold solution of K₂-EDTA, Type II red fibres fell into two sub-types and Type II white fibres fell into three sub-types. Amalgamating the two already existing classifications, a more elaborate classification is presented for characterizing these different sub-types. The presence of two different or a spectrum of staining variations in a seemingly homogeneous population of muscle fibres in a given fibre-type emphasizes the possible correlation between this histochemical data and the heterogeneity of contraction times of the different motor units.     

Transitional stages in the histochemical development of muscle fibres during post-natal growth

Synopsis Serial frozen sections of longissimus dorsi muscles from seven pigs at different live weights (13 to 127 kg) were reacted for ATPase by the calcium method at an alkaline pH and for NADH oxidative activity. One hundred muscle fibres from each animal were identified individually in serial sections and their staining intensity was measured with a microscope photometer at 600 nm. For each section, staining intensity of fibres (% tranmission) was measured and converted to the nearest one-tenth unit of the range from the darkest to the lightest staining fibres. Frequency of occurrence of fibre types was plotted on a 10×10 grid using the range co-ordinates for NADH oxidative activity (vertical) and ATPase activity (horizontal). The commonly recognized histochemical fibre types in this muscle appeared as crowded areas in the grid but, in many cases, these areas were part of a continuous L shaped distribution. In fibres having an ATPase staining intensity of 1.0 and 0.9 units of the range, a continuous but skewed distribution with regard to NADH oxidative activity was detected. In fibres with NADH oxidative activity of 0.6 to 1.0 units of the range, a continuous but irregular distribution with regard to ATPase activity was detected. Within this range, there was some evidence of a growth-related shift towards weaker ATPase activity.     

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.     

Structural diversity in muscle fibres of chicken breast

Summary Chicken breast muscle is usually considered to be a relatively homogeneous white muscle and has therefore been widely used for studies of muscle proteins. In a previous study, however, we have found different M-region structures in different fibres from this muscle. Because of this result, we have now carried out a combined histochemical and ultrastructural survey of this muscle. In particular, we have made use of large transverse cryo-sections that include most of the muscle cross-section.Although the white region is fairly homogeneous in fibre content according to normal histochemical criteria (mAT-Pase), we have found that there is a gradation of fibre structure across the muscle. The bulk of the muscle stains conventionally for Type-II fibres according to mATPase tests (the white part) but, in the small red part of the muscle, there are also Type-I fibres together with the Type-II fibres. Superimposed on this division into Type-I and Type-II fibres are variations in fibre size, oxidative and glycolytic staining properties, and variations of Z-band width and M-band structure; there is no strict correlation among any of these parameters. The apparently uniform staining across most of the muscle when tested for myofibrillar ATPase may be a misleading indicator of fibre properties.     

Quantitative analysis of histochemical and immunohistochemical reactions in skeletal muscle fibres ofRana andXenopus

Summary Intensities of histochemical and immunohistochemical reactions in muscle fibres ofRana andXenopus have been estimated microphotometrically, and the data from serial sections statistically analysed. Quantitative validities of reactions and measurements have also been assessed against independent published evidence. It is concluded that NADH—tetrazolium reductase overestimates tonic-fibre aerobic capacities and the actomyosin ATPase reaction overestimates their contraction speeds. However, it appears that succinate dehydrogenase, despite being a near-equilibrium enzyme of particulate distribution, indicates the relative aerobic capacities of fibres with acceptable accuracy when lightly reacted. Capacities for aerobic and anaerobic metabolism are positively correlated over all types of fibre (r typically 0.6 for 200 fibres), perhaps as an adaptation to environmental hypoxia.Multivariate clusters (indicating fibre types) have been sought, using Ward's method with optimizing procedures (iterative relocation and multivariate-normal modelling). Cluster analysis confirms the subjective identifications of two slow/tonic types inXenopus (labelled T5 and S4) but of only one (T5) inRana. Division of the fast family twitch fibres into three types (F1–F3) in both genera, with metabolic capacity related inversely to apparent shortening velocity, is highly supportable by objective criteria. However, statistically significant subdivisions also present themselves.Rana F2 andXenopus F1 clusters can be bisected according to metabolic capacity; andXenopus F2 fibres fall into three subtypes reflecting different isomyosin contents. In the different types of twitch fibre, ratios of myofibrillar ATP consumption rate to aerobic capacity increase up to 30-fold with contraction speed, but anaerobic/aerobic ratios do so only 5-fold.     

Relationship among fibre type, myosin ATPase activity and contractile properties

Summary At least two types of skeletal muscle myosin have been described which differ in ATPase activity and stability in alkaline or acidic media. Differences in ATPase characteristics distinguish Type I and Type II fibres histochemically. In this study, ATPase activity of myosin from muscles of several species with known histochemical and contractile properties has been determined to test the hypothesis that (1) myosin ATPase activity, (2) histochemical determination of fibre types and (3) maximum shortening velocity, all provide equivalent estimates of contractile properties in muscles of mixed fibre types. Maximum shortening velocity appears to be proportional to ATPase activity as expected from previous reports by Barany. However, both myosin ATPase and the maximum shortening velocity exhibit curvilinear relationships to the fraction of cross-sectional area occupied by Type II fibres. Therefore, we reject the hypothesis and conclude that histochemically determined myofibrillar ATPase does not accurately reflect the intrinsic ATPase activity or shortening velocity in muscles of mixed fibre types. Our data are consistent with the presence of more than two myosin isozymes or with a mixture of isozymes within single muscle fibres.