Identification of fiber types in rat skeletal muscle based on the sensitivity of myofibrillar actomyosin ATPase to copper

Experiments are reported demonstrating that differential rates of inactivation of the histochemical staining for myofibrillar actomyosin ATPase in rat skeletal muscle fibers exist following inclusion of low concentrations of Cu2+ in the preincubation medium. This response of rat muscle occurs at near neutral (7.40), acid (4.60), and alkaline (10.30) pH. The response to Cu2+ appears to result from a binding of Cu2+ onto the myofibrillar complex, probably on myosin itself, as it can be reversed by soaking of the pretreated muscle sections in sodium cyanide or the Cu2+ chelator diethyldithiocarbamate. The pattern of modification of the staining pattern following pretreatment with Cu2+ is the mirror image of that produced by pretreatment with acid. The results demonstrate that the inclusion of Cu2+ in the preincubation media for the myofibrillar actomyosin ATPase can be a useful tool to differentiate fiber types. They also support the earlier conclusion that three distinct types of type II fibers can be identified in rat skeletal muscle based on the histochemical staining for myofibrillar actomyosin ATPase.     

Comparison of fiber types in skeletal muscles from ten animal species based on sensitivity of the myofibrillar actomyosin ATPase to acid or copper

Summary Comparisons were made of the histochemical characteristics of skeletal muscle from 10 animal species. The basic comparison was made from the staining patterns for the myofibrillar actomyosin ATPase produced by preincubation of fresh frozen cross-sections of muscle at alkaline pH (10.30) or acid pH (4.60) with those produced by preincubation in media containing Cu²⁺ at alkaline pH (10.30), near neutral pH (7.40), or acid pH (4.60). Muscle sections were also stained for reduced nicotinamide adenine dinucleotide tetrazolium reductase and alpha-glycerophosphate dehydrogenase to provide an indication of the relative oxidative and glycolytic capacity of the different fiber types. Type II fibers in mixed fibered muscles were either very sensitive, moderately sensitive, or relatively insensitive to inactivation of the myofibrillar actomyosin ATPase after acid preincubation. These fibers were identified as type IIA1, IIA2, and IIA3, respectively. The myofibrillar actomyosin ATPase of the type I fibers of these muscles, with the exception of those in mouse muscle, was activated by pretreatment with acid. A separation of animal species was possible based on the stability of the IIA1 fibers to inclusion of Cu²⁺ in the preincubation medium. For one group of animals (rat, mouse, monkey, man, dog, rabbit, and cow), a reciprocal relationship existed between lability to acid and stability to Cu²⁺ for type IIA1 and IIA3 fibers, respectively. For the second group of animals (horse, ass, and cat) there was a parallel relationship between lability or stability of the type IIA1 and IIA3 fibers to pretreatment with either acid or Cu²⁺ Visiting scholar from the Laboratory of Biomechanics and Physiology, College of General Education, Yamaguchi University, Yamaguchi 753, JapanSupported in part by Washington State Equine Research Program grant #105 3925 0042     

Comparison of fiber types in skeletal muscles from ten animal species based on sensitivity of the myofibrillar actomyosin ATPase to acid or copper

Comparisons were made of the histochemical characteristics of skeletal muscle from 10 animal species. The basic comparison was made from the staining patterns for the myofibrillar actomyosin ATPase produced by preincubation of fresh frozen cross-sections of muscle at alkaline pH (10.30) or acid pH (4.60) with those produced by preincubation in media containing Cu2+ at alkaline pH (10.30), near neutral pH (7.40), or acid pH (4.60). Muscle sections were also stained for reduced nicotinamide adenine dinucleotide tetrazolium reductase and alpha-glycerophosphate dehydrogenase to provide an indication of the relative oxidative and glycolytic capacity of the different fiber types. Type II fibers in mixed fibered muscles were either very sensitive, moderately sensitive, or relatively insensitive to inactivation of the myofibrillar actomyosin ATPase after acid preincubation. These fibers were identified as type IIA1, IIA2, and IIA3, respectively. The myofibrillar actomyosin ATPase of the type I fibers of these muscles, with the exception of those in mouse muscle, was activated by pretreatment with acid. A separation of animal species was possible based on the stability of the IIA1 fibers to inclusion of Cu2+ in the preincubation medium. For one group of animals (rat, mouse, monkey, man, dog, rabbit, and cow), a reciprocal relationship existed between lability to acid and stability to Cu2+ for type IIA1 and IIA3 fibers, respectively. For the second group of animals (horse, ass, and cat) there was a parallel relationship between lability or stability of the type IIA1 and IIA3 fibers to pretreatment with either acid or Cu2+.     

Differentiation of fiber types in skeletal muscle from the sequential inactivation of myofibrillar actomyosin ATPase during acid preincubation

A method is described for identifying fiber types of skeletal muscle from several mammalian species on the basis of the sequential inactivation of myofibrillar actomyosin ATPase during acid preincubation. When this method is used in combination with the standard alkaline preincubation at least 5 types of fibers can be identified. Of these, 2 are type I fibers with those of the slow twitch soleus muscle being different from those that exist in mixed muscles. The 3 subtypes of type II fibers exist independent of their metabolic properties. The need for careful standardization of histochemical methods for the visualization of myofibrillar actomyosin ATPase and the implication of the existence of different fiber types in apparently homogeneous muscle for the preparation of antibodies used for immunocytochemical methods of fiber identification are discussed.     

Influence of ionic composition,buffering agent,and pH on the histochemical demonstration of myofibrillar actomyosin ATPase

Summary The influence of the composition of the preincubation medium on the histochemical demonstration of myofibrillar actomyosin ATPase, including a variety of carboxylic acid and non-carboxylic acid buffering compounds and neutral salts, was studied. In inorganic salt-free systems the rate of the activation of type I fibers and inactivation of type II fibers was accelerated when the carboxylic acids had longer chain length or multiple carobxyl groups. Of these factors, the number of carboxyl groups was dominant with a 100 mM citrate buffer producing a sharp differentiation between fiber types. In contrast, the time course of the response was exceptionally long in an acetate buffer. The time course of the ATPase reaction was also modified by other buffers at pH 4.60. The most notable were an ascorbate — glycine buffer system which produced little or no deviation from the alkaline preincubation staining pattern after prolonged preincubation and a pyrophosphate system which produced a rapid change. Neutral salts in the preincubation medium accelerated the time course of the inactivation — activation process with the order for the halogen salts of K⁺ being F⁻<Cl⁻<Br⁻<I⁻, which is a progression by molecular weight. The only sequence for cations on the myofibrillar actomyosin ATPase was Li⁺< Na⁺<K⁺. The response to salts was concentration dependent. An interaction existed between buffering compound, type of salt, and pH. These experiments demonstrate that the histochemical differentiation of fiber types by the myofibrillar actomyosin ATPase reaction depends upon a modification of some component(s) of the myofibrillar complex that can be influenced by a number of factors.     

Effect of varying the preincubation and incubation temperature on the reaction pattern for myosin ATPase in rat skeletal muscle

The temperature of the preincubation and incubation medium influences the staining pattern and intensity of the reverse myofibrillar ATPase in rat skeletal muscle. The activity in some fibers was raised and in others depressed by varying the temperature of the preincubation and incubation medium between 4 and 37 degrees C. This indicates the presence of reverse ATPase isoenzymes with different temperature sensitivities.     

Evidence for myosin heterogeneity inDrosophila melanogaster

Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca²⁺ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles.The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.     

Dynamic exercise training in foxhounds. II. Analysis of skeletal muscle

The purpose of this study was to determine whether 8-12 wk of endurance training produces biochemical and histochemical adaptations in skeletal muscle in foxhounds. Analyses were performed on samples removed from gastrocnemius, triceps, and semitendinosus muscles of foxhounds before and after a treadmill running program. Biochemical analysis showed that training did not alter the activities of phosphofructokinase, beta-hydroxyacyl-CoA dehydrogenase, succinate dehydrogenase, or total phosphorylase. Histochemical analysis of myofibrillar actomyosin ATPase demonstrated three distinct classes of type II fibers and one type I fiber in the semitendinosus and triceps muscles and two type II and two type I fibers in the gastrocnemius muscle. Fiber type distribution and oxidative and glycolytic potentials, as indicated by nicotinamide adenine dinucleotide tetrazolium reductase or alpha-glycerophosphate dehydrogenase staining intensity, were unaltered by training. Similarly, capillary density, capillary-to-fiber ratios, and capillary area-to-fiber area ratios did not change with training. Thus, unlike humans and other mammals (i.e., rat), these foxhounds did not manifest biochemical or histochemical adaptations in skeletal muscle as the result of endurance training. This is consistent with the results of the study in which endurance training produced a 27% increase in maximal cardiac output and a 4% increase in maximal arteriovenous O2 extraction in foxhounds.     

Influence of exercise on cardiac and skeletal muscle myofibrillar proteins

The purpose of this study was to examine the Ca²⁺-Mg²⁺ myofibrillar ATPase and protein composition of cardiac and skeletal muscle following strenuous activity to voluntary exhaustion. Sprague-Dawley rats (200 g) were assigned to a control and exercised group, with the run group completing 25 m·min^–1 and 8% grade for 1 hour. Following activity, the myocardial Ca²⁺–Mg²⁺ myofibrillar ATPase activity -pCa relationship had undergone a rightward shift in the curve. Electrophoretic analysis revealed a change in the pattern of cardiac myofibrillar protein bands, particularly in the 38–42 Kdalton region. Enzymatic analysis of myofibrillar proteins from plantaris muscle, revealed no change in Ca²⁺ regulation following exercise. Electronmicrographic and electrophoretic analysis revealed extensively disrupted sarcomeric structure and a change in the ratio of several plantaris myofibrillar proteins. No difference was observed for myosin: Actin: tropomyosin ratios; however a dramatic reduction in 58 and 95 Kdalton proteins were evident. The results indicate that prolonged running is associated with similar responses in cardiac and skeletal muscle myofibrillar protein compositions. The abnormalities in myofibrillar ultrastructure may implicate force transmission failure as a factor in exercised-induced muscle damage and/or fatigue.     

Histochemical, enzymatic, and contractile properties of skeletal muscle fibers in the lizard Dipsosaurus dorsalis

Lizard skeletal muscle fiber types were investigated in the iliofibularis (IF) muscle of the desert iguana (Dipsosaurus dorsalis). Three fiber types were identified based on histochemical staining for myosin ATPase (mATPase), succinic dehydrogenase (SDH), and alphaglycerophosphate dehydrogenase (alphaGPDH) activity. The pale region of the IF contains exclusively fast-twitch-glycolytic (FG) fibers, which stain dark for mATPase and alphaGPDH, light SDH. The red region of the IF contains fast-twitch-oxidative-glycolytic (FOG) fibers, which stain dark for all three enzymes, and tonic fibers, which stain light for mATPase, dark for SDH, and moderate for alphaGPDH. Enzymatic activities of myofibrillar ATPase, citrate synthase, and alphaGPDH confirm these histochemical interpretations. Lizard FG and FOG fibers possess twitch contraction times and resistance to fatigue comparable to analogous fibers in mammals, but are one-half as oxidative and several times as glycolytic as analogous fibers in rats. Lizard tonic fibers demonstrate the acetylcholine sensitivity common to other vertebrate tonic fibers.     

A new histochemical method for magnesium actomyosin adenosine triphosphatase at physiological pH

A new lead-precipitation technique for demonstrating magnesium-activated actomyosin adenosine triphosphatase (ATPase) at physiological pH and electrolyte levels in fixed skeletal muscle sections is reported. This method is compared with standard acid- and alkali-denatured muscle stained for calcium myosin ATPase as well as calcium-formalin denatured and pyrophosphate-formalin denatured muscle also stained for calcium myosin ATPase. The technique was developed using hamster skeletal muscle; however, it has also been applied to human, rat, and cat muscle. The fiber-type staining intensities of the formalin-denatured magnesium actomyosin ATPase closely resemble those of the formalin-denatured calcium myosin ATPase in rodents, but intensities in Type 1 fibers are reversed relative to calcium myosin ATPase in human muscle. Cat muscle shows intermediate characteristics.     

The contractile proteins of smooth muscle. Properties and components of a Ca2+-sensitive actomyosin from chicken gizzard.

The preparation and characterization of a Ca²⁺-sensitive actomyosin from chicken gizzard is described. The pH curve of the Mg²⁺ ATPase activity of the actomyosin was dominated by the activity of the myosin component, and this gave rise to the acid and alkaline optima. Skeletal muscle myosin showed a similar curve. Both the activation of myosin ATPase by actin, and the Ca²⁺ sensitivity were confined to the neutral pH region. The subunit composition of the Ca²⁺-sensitive actomyosin was interesting in that no components corresponding to skeletal muscle troponin were obvious. It is suggested that the activity of gizzard actomyosin is regulated by a protein on the thin filaments with a subunit weight of ~130,000.     

A metachromatic dye-ATPase method for the simultaneous identification of skeletal muscle fiber types I, IIA, IIB and IIC.

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C, followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, IIA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.     

A Metachromatic Dye-Atpase Method for The Simultaneous Identification of Skeletal Muscle Fiber Types I, IIA, IIB and IIC

The histochemical demonstration of quantitative differences in myofibrillar ATPase activity at the selective pH optima of the various types of human skeletal muscle fibers is the most widely used technique for their differentiation. The basis of the reaction is the deposition of insoluble salts of inorganic phosphate cleaved from ATP by myofibrillar ATPase(s) followed by substitution of the phosphates with less soluble chromogenic salts. Doriguzzi and associates reported using metachromatic dyes to demonstrate quantitative differences in phosphate deposition among different fiber types. Following routine ATPase histochemistry and staining with either azure A or toluidine blue, fibers with low ATPase activity (and low phosphate content) were stained metachromatically while fibers with high ATPase activity (and high phosphate content) were orthochromatic with the intensity of color proportional to the content of insoluble phosphate. The metachromasia was readily lost after immoderate washing in aqueous solutions or routine dehydration in ethanol, with consequent diminished fiber type distinction. A critical modification of this technique is reported in which incubation of frozen sections of human skeletal muscle in ATP-containing medium is carried out at room temperature (22-24 C), rather than the usual 37 C., followed by a revised washing and dehydration protocol. With these modifications, the four human skeletal muscle fiber types (types I, HA, IIB, and IIC) can be identified rapidly and reliably in single sections, obviating the need for examination of serial sections. The tinctorial differentiation allows fiber type identification even in black and white photographs.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag1 fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag2 fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag1 fibers.     

Histochemical heterogeneity of fibers in the abdominal superficial flexor muscles of the Norway lobster, Nephrops norvegicus (L.).

The superficial flexor muscle in the abdomen of the Norway lobster Nephrops norvegicus (L.), comprises medial and lateral bundles with distinct fiber type composition. Fibers of the medial bundle have long sarcomeres (> 9 microns) and a thick fringe of subsarcolemmal mitochondria. In histochemical tests they have a low total myofibrillar ATPase activity, a pH-stable isoform of myosin ATPase, and a high level of oxidative enzyme activity. A few fibers of the lateral bundle also display these morphological and histochemical properties. However, the majority of lateral fibers have shorter sarcomeres (< 8 microns), no subsarcolemmal mitochondria, but a well-developed tubular system. They also have a higher total myofibrillar ATPase activity, a pH-labile isoform of myosin ATPase, and a low level of oxidative enzyme activity. The heterogeneous pattern of different fiber types in the lateral bundle of this muscle is similar but not identical in the different abdominal segments and in different individuals.     

Variable pH dependence of the myosin-ATPase in different muscles of the rat

Summary For the histochemical demonstration of the Myosin-ATPase (EC 3.6.1.3) the pH of both the preincubation and the incubation medium was varied in steps of 1 within a small range: 10.2 to 10.5 and 9.3 to 9.6, respectively. The optimum combinations of both pH values, defined as the ones providing most consistent contrast among the three major types of muscle fibers were determined in 9 different muscles of the rat. The spectrum of optimum combinations differs considerably from muscle to muscle. The reduction of the incubation pH by only 0.1 may drastically change the staining pattern. This probably reflects the unspecifity of the histochemical procedure as well as the plasticity of the ATPase systems. To cope with the lability of the myosin-ATPase the optimum pH values of both media should be determined for each muscle separately.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site

In order to determine whether polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca²⁺ + Mg²⁺-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca²⁺ + Mg²⁺-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca²⁺ + Mg²⁺-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca²⁺ + Mg²⁺-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca²⁺-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca²⁺. Its lack of cross-reactivity with proteins that do not bind Ca²⁺ suggests that it has specificity for antigenic determinants that make up the Ca²⁺-binding sites in several Ca²⁺-binding proteins including the Ca²⁺ + Mg²⁺-dependent ATPase.This paper is dedicated to the memory of Dr. David E. Green.