Histochemical and biochemical studies on the red and white muscle in rabbit

1. The total amount of triglyceride was 6.00 +/- 0.14 mg/g wet tissue in soleus, 1.50 +/- 0.52 in extensor digitorum longus and 1.83 +/- 0.88 in gastrocnemius muscle. 2. The amounts of triglycerides in the individual types were calculated to be very large, moderate and very small in type 1, 2A and 2B, respectively, when compared with histochemical studies. 3. Differences in fatty acid composition of triglycerides were seen between the soleus and extensor digitorum longus, and gastrocnemius showed intermediate values. 4. These results might be important corresponding to differences in energy metabolism in different fiber types.     

Histochemical profile of articularis humeri muscle in the horse

In this study the histochemical fiber type analysis of the articularis humeri muscle of the horse was performed. This muscle is composed of type I and type IIA fibers. A large number of spindles has been observed and the presence of these receptors could help to understand the role played by this muscle.     

Misclassification of hybrid fast fibers in resistance-trained human skeletal muscle using histochemical and immunohistochemical methods

ABSTRACT: Staron, RS, Herman, JR, and Schuenke, MD. Misclassification of hybrid fast fibers in resistance-trained human skeletal muscle using histochemical and immunohistochemical methods. J Strength Cond Res 26(10): 2616-2622, 2012-Sixteen healthy untrained women participated in a 6-week progressive resistance training program to compare 2 common methods of classifying fiber types. The women were a subset from a previous study and were randomly divided into 2 groups: traditional strength training (TS, n = 9) and non-exercising control (C, n = 7). The TS group performed 3 lower limb exercises (leg press, squat, and knee extension) using 6-10 repetitions maximum 2 days per week for the first week and 3 days per week for the remaining 5 weeks (17 total workouts). Pre- and posttraining vastus lateralis muscle biopsies were analyzed for fiber type composition using 2 popular methods: myosin adenosine triphosphatase (mATPase) histochemistry and myosin heavy chain (MHC) immunohistochemistry. Six fiber types (I, IC, IIC, IIA, IIAX, and IIX) were delineated using each method separately and in combination. Because of the subjective nature of each method (visual assessment of staining intensities), IIAX fibers expressing a small amount of MHCIIa were misclassified as type IIX using mATPase histochemistry, whereas those expressing a small amount of MHCIIx were misclassified as type IIA using MHC immunohistochemistry. As such, either method used separately resulted in an underestimation of the type IIAX fiber population. In addition, the use of mATPase histochemistry alone resulted in an overestimation of type IIX, whereas there was an overestimation of type IIA using MHC immunohistochemistry. These fiber typing errors were most evident after 6 weeks of resistance training when fibers were in transition from type IIX to IIA. These data suggest that the best approach to more accurately determine muscle fiber type composition (especially after training) is the combination of mATPase histochemical and MHC immunohistochemical methods.     

Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers

Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers can be exposed to various histochemical reactions and the staining patterns compared on the same slide to those of frozen muscle and skinned bundles. By this procedure, three fiber types were distinguished by both Ca2+-ATPase and SDH reactions. The fiber typings determined from both enzyme systems correlated well with each other. Although we were able to differentiate only between slow and fast fibers by SDS-PAGE, these results corroborated the histochemical classification. This procedure will clearly be useful in skinned single muscle fiber mechanics experiments performed to determine functional differences among fiber types.     

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers

Summary Combined histochemical and biochemical analyses were performed on single fibers of rabbit soleus muscle. Histochemically, four fiber types (I, IC, IIC, IIA) were defined. Of these, types I and IIA were separate, histochemically homogeneous groups. A heterogeneous C fiber population exhibited a continuum of staining intensities between types I and IIA. Microelectrophoretic analyses of specific, histochemically defined fibers revealed that type I fibers contained exclusively HCI, whereas type IIA fibers contained only HCIIa. The C fibers were characterized by the coexistence of both heavy chains in varying ratios, type HC with a predominance of HCI and type IIC with a predominance of HCIIa. A direct correlation existed between the myosin heavy chain composition and the histochemical mATPase staining and was especially evident in the C fiber population with its variable HCI/HCIIa ratio. This correlation did not apply to the myosin light chain complement.     

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers

Combined histochemical and biochemical analyses were performed on single fibers of rabbit soleus muscle. Histochemically, four fiber types (I, IC, IIC, IIA) were defined. Of these, types I and IIA were separate, histochemically homogeneous groups. A heterogeneous C fiber population exhibited a continuum of staining intensities between types I and IIA. Microelectrophoretic analyses of specific, histochemically defined fibers revealed that type I fibers contained exclusively HCI, whereas type IIA fibers contained only HCIIa. The C fibers were characterized by the coexistence of both heavy chains in varying ratios, type IC with a predominance of HCI and type IIC with a predominance of HCIIa. A direct correlation existed between the myosin heavy chain composition and the histochemical mATPase staining and was especially evident in the C fiber population with its variable HCI/HCIIa ratio. This correlation did not apply to the myosin light chain complement.     

miR-222 is involved in the regulation of genistein on skeletal muscle fiber type

In skeletal muscle, the composition of the fiber types has a profound impact on athletic performance, such as endurance or strength output. The proportions of muscle fiber types have also been associated with certain diseases, including dyskinesia, obesity and insulin resistance. Genistein, a natural estrogen, has been demonstrated to regulate fatty acid oxidation and insulin sensitivity in skeletal muscle. However, it is unknown whether genistein can regulate skeletal muscle fiber types. Furthermore, the mechanism of its effect on skeletal muscle energy metabolism is not entirely clear. In this study, in vivo and in vitro experiments were used to explore the effect of genistein on the muscle fiber-type transitions and muscle metabolism. The results indicated that genistein not only promotes skeletal muscle development but increases the expression of slow muscle fibers in mice as well. It was also demonstrated that genistein altered the ratios of fiber type and promoted mitochondrial biogenesis in C2C12 myoblasts. Interestingly, the expression of miR-222 was decreased by genistein, and it was demonstrated that this microRNA targets the PGC1α gene. In C2C12 myoblasts, miR-222 appears to regulate fiber type conversion and mitochondrial biogenesis. However, this function was significantly reduced following genistein treatment. These results suggest that miR-222 may be involved in the regulation of genistein on skeletal muscle fiber and muscle metabolism, and genistein may be used to improve muscle health.     

Lycopene increases the proportion of slow-twitch muscle fiber by AMPK signaling to improve muscle anti-fatigue ability

Lycopene has a wide range of biological functions, especially its antioxidant capacity. However, effects of lycopene on muscle fatigue resistant and muscle fiber type conversion are unknown. In this study, we found that lycopene significantly prolonged the swimming time to exhaustion in mice. We also showed that lycopene increased the proportion of slow-twitch muscle fiber by promoting muscle fiber type conversion from fast-twitch to slow-twitch in mice and in C2C12 myotubes. The AMP-activated protein kinase (AMPK) signaling was activated by lycopene. AMPK upstream and downstream regulators including nuclear respiratory factor 1, calcium calmodulin-dependent protein kinase kinase-β, sirtuin 1 and peroxisome proliferator activated receptor-γ coactivator-1ɑ were also increased by lycopene. AMPK inhibitor compound C markedly attenuated the lycopene-induced skeletal muscle fiber type conversion in C2C12 myotubes. Taken together, we provided the first evidence that lycopene increases the proportion of slow-twitch muscle fiber through AMPK signaling pathway to improve fatigue resistant of skeletal muscle.     

Ontogeny of the Pectoralis muscle in the little brown bat,Myotis lucifugus

The ontogeny of a primary flight muscle, the pectoralis, in the little brown bat (Myotis lucifugus: Vespertilionidae) was studied using histochemical, immunocytochemical, and electrophoretic techniques. In fetal and early neonatal (postnatal age 1–6 days) Myotis, histochemical techniques for myofibrillar ATPase (mATPase) and antibodies for slow and fast myosins demonstrated the presence of two fiber types, here called types I and IIa. These data correlated with multiple transitional myosin heavy chain isoforms and native myosin isoforms demonstrated with SDS-PAGE and 4% pyrophosphate PAGE. There was a decrease in the distribution and number of type I fibers with increasing postnatal age. At postnatal age 8–9 days, the adult phenotype was observed with regard to muscle fiber type (100% type IIa fibers) and myosin isoform profile (single adult MHC and native myosin isoforms). This “adult” fiber type profile and myosin isoform composition preceeded adult function by about 2 weeks. For example, little brown bats were incapable of sustained flight until approximately postnatal day 24, and myofiber size did not achieve adult size until approximately postnatal day 25. Although Myotis pectoralis is unique in being composed of 100% type IIa fibers, transitional fiber types and isoforms were present. These transitional forms had been observed previously in other mammals bearing mixed adult muscle fibers and which undergo transitional stages in muscle ontogeny. However, in Myotis pectoralis, this transition transpires relatively early in development. © 1994 Wiley-Liss, Inc.     

Histochemical and electronmicroscopical analysis of muscle fiber in myotomes of teleost fish (Noemacheilus barbatulus L.)

A quantitative histochemical study was carried out on axial musculature of Noemacheilus barbatulus L. On the basis of succinate dehydrogenase (SDH) and myofibrillar ATP-ase activity, 5 types of muscle fibers are described. When the SDH method was used, red, tonic, intermediate, and white muscle fibers were easily observed. However, histochemical reaction for myofibrillar ATP-ase activity, after alkaline preincubation (pH = 10.4), revealed another type of fiber zone laying between the intermediate and white muscle fiber regions and forming a transitional zone. Electron microscopic observation showed significant differences in sarcomere organization and thickness of myosin filaments of the various muscle fiber types.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

Summary The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar.Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

The occurrence of "mixed" nuclear bag intrafusal fibers in the cat muscle spindle

A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5'-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag1 in one pole and as a bag2 in the other pole. These "mixed" nuclear bag fibers were found in spindles that also contained at least one bag1 and one bag2 fiber of equivalent histochemical presentation in both fiber poles. The "mixed" bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of "mixed" bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag1 fiber, and another spindle lacked a bag2 fiber. Several incomplete spindles also lacked bag1 fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag1 fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

Histochemical patterns in normal and splaylegged piglet muscle fibers

The histochemical pattern of muscle fiber types of the longissimus dorsi and biceps femoris muscles was investigated in normal and splaylegged piglets at birth and seven days later. Only slight differences between the muscle fibers at birth were found using histochemical reactions for alkaline adenosine triphosphatase (ATPase), succinate dehydrogenase (SDH), phosphorylase (PH) activities, and for the periodic acid-Schiff (PAS) reaction. With the method for acid-preincubated ATPase activity, high activity was observed in Type I muscle fibers and low activity in Type II muscle fibers in animals of both groups investigated. However, a higher number of Type I fibers was found in muscles of normal piglets, suggesting a faster and more advanced process of transformation of Type II into Type I muscle fibers in unaffected animals. Thus the histochemical conversion appears to be retarded in muscles of splaylegged animals, which have a histochemical pattern similar to that of normal prenatal animals. Cholinesterase activity in motor endplates was well developed; its staining revealed smaller sized and irregularly arranged endplates in muscles of affected piglets. Fiber type differentiation in muscles of animals which recovered from splayleg becomes fully developed and comparable to normal piglets seven days after birth. The number of fibers which became converted from Type II to Type I was increased; the fiber types were differentiated with regard to the PAS reaction and to their ATPase, SDH and PH activities. Morphological features of motor endplates in muscles of normal and surviving splaylegged piglets are similar. Histochemical investigation of the fiber type differentiation thus suggests that full recovery occurs within the first week of postnatal life in muscles affected by pathological changes accompanying splayleg.     

Histochemical and myosin composition of vampire bat (Desmodus rotundus) pectoralis muscle targets a unique locomotory niche

The vampire bat pectoralis muscle contains at least four fiber types distributed in a nonhomogeneous pattern. One of these fiber types, here termed IIe, can be elucidated only by adenosine triphosphatase (ATPase) histochemistry combined with reactions against antifast and antislow myosin antibodies. The histochemical and immunohistochemical observations indicate a well-developed specialization of function within specific regions of the muscle. In parallel, analyses of native myosin isoforms and myosin heavy chain isoforms indicate two points. First, the histochemical “type IIe” fiber is predominant in cranial portions of the muscle, and myosin extracted from these regions exhibits a unique electrophoretic mobility not observed in the myosin isoforms of more traditional laboratory mammals. Second, the type I fibers are confined to the pectoralis abdominalis muscle and a small adjacent region of the caudal part of the pectoralis. This pattern of type I fiber distribution is considered a derived character state compared to muscle histochemical phenotype and isoform composition in the pectoralis muscles of other phyllostomids we have studied (Artibeus jamaicensis, Artibeus lituratus, Carollia perspicillata). We relate this to the unique locomotory needs of the common vampire bat, Desmodus rotundus. © 1993 Wiley-Liss, Inc.     

One-bag-fiber muscle spindles in tenuissimus muscles of the cat

Summary Over 150 complete and 139 incomplete single muscle spindles were examined in serial transverse sections of cat tenuissimus muscles in search for spindles lacking one of the two types of nuclear bag intrafusal fiber. Several histochemical reactions were used to type the intrafusal muscle fibers and assess the spindle motor and sensory innervation. One complete spindle lacked a bag₁ fiber, and another spindle lacked a bag₂ fiber. Several incomplete spindles also lacked bag₁ fibers. In addition, ten double tandem spindles contained one capsular unit each that lacked the bag₁ fiber, and one triple tandem spindle had two such capsules. All one-bag-fiber spindles had primary sensory innervation, but none had secondary sensory innervation. Their motor innervation was similar to that of the usual two-bag-fiber spindles in the number and disposition of intrafusal motor endings. It is unclear whether the one-bag fiber spindles, either single or tandem-linked, are products of an aberrant spindle development or represent a true anatomical and functional subcategory of the cat muscle spindle.     

A histochemical characterization of muscle fiber types in the avian M. stapedius

The muscle fiber types and sizes in the M. stapedius (middle ear muscle) of the domestic chicken, Gallus gallus were determined histochemically on the basis of their reactions to myofibrillar adenosine triphosphatase (mATPase), succinic dehydrogenase and NADH diaphorase. Only type II fibers were identified at pH 9.4 and 4.2. At pH 4.6 three levels of activity were seen: high, intermediate and low. With the staining techniques three subtypes of fibers for oxidative enzymes, Types II1 (highly glycolytic), II12 (intermediately glycolytic and lipolytic) and II123 (highly lipolytic) were identified. Fiber diameter was also measured for the different fiber types. The average fiber diameter was around 20 micron for each fiber type. Although similar in size, the fiber types were markedly different in their histochemical properties. These findings plus those of earlier physiological studies suggest that the M. stapedius of G. gallus is a fast twitch, muscle with fibers of similar diameter showing mainly fatigue resistance characteristics.     

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.     

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.     

Human skeletal muscle fiber type alteration with high-intensity intermittent training

The response of muscle fiber type proportions and fiber areas to 15 weeks of strenuous high-intensity intermittent training was investigated in twenty-four carefully ascertained sedentary (14 women and 10 men) and 10 control (4 women and 6 men) subjects. The supervised training program consisted mainly of series of supramaximal exercise lasting 15 s to 90 s on a cycle ergometer. Proportions of muscle fiber type and areas of the fibers were determined from a biopsy of the vastus lateralis before and after the training program. No significant change was observed for any of the histochemical characteristics in the control group. Training significantly increased the proportion of type I and decreased type IIb fibers, the proportion of type IIa remained unchanged. Areas of type I and IIb fibers increased significantly with training. These results suggest that high-intensity intermittent training in humans may alter the proportion of type I and the area of type I and IIb fibers and in consequence that fiber type composition in human vastus lateralis muscle is not determined solely by genetic factors.