Comparative histochemical study of prosimian primate hindlimb muscles. II. Populations of fiber types

The populations of fiber types in hindlimb muscles of the tree shrew (Tupaia glis), lesser bushbaby (Galago senegalensis), and the slow loris (Nycticebus coucang) were described and an attempt was made to correlate populations of fiber types and locomotor patterns. Muscle fibers were assigned to one of the following groups: fast-twitch glycolytic (FG), fast-twitch oxidative-glycolytic (FOG), and slow-twitch oxidase (SO). Histochemical techniques for the demonstration of alkaline- and acid-stable ATPase, succinic dehydrogenase, and mitochondrial alpha-glycerophosphate dehydrogenase were used in the classification of muscle fibers. Results indicated that the FG fiber type is the predominant fiber type in muscles used for jumping, the FOG fiber type is predominant in muscles used for running, and the SO fiber type occurs in high percentages in postural muscles. The SO fiber was also the most common fiber in muscles of the slow loris-a species that exhibits a slow, deliberate, sustained locomotor pattern. Intramuscular regional variations in populations were seen in some larger muscles of the tree shrew, but not in the lesser bushbaby and slow loris. Our results did not support the contentions of others that analogous muscles in different species have similar populations of fiber types.     

Muscle fiber types in tetrapods. A comparative histochemical and morphometric study

A comparative histochemical and morphometric study in two groups of homologous muscles from different tetrapods (rat, pigeon, lizard and frog) was performed. On the basis of their fiber diameters and oxidative enzyme activities, an initial correlation between fiber types of all animals is observed, although in the lizard and frog muscles, another fiber type does exists that could not be demonstrated in higher vertebrates. When more than one histochemical techniques are used for the identification of each tetrapod fiber types, the lack of correlation between them becomes obvious. Thus, different animals groups, each showing a characteristic muscle metabolic pattern, could be distinguished.     

Muscle fiber types and size in trained and untrained muscles of elite athletes

Tissue samples were obtained from vastus lateralis and deltoid muscles of physical education students (n = 12), Greco-Roman wrestlers (n = 8), flat-water kayakers (n = 9), middle- and long-distance runners (n = 9), and olympic weight and power lifters (n = 7). Histochemical stainings for myofibrillar adenosinetriphosphatase and NADH-tetrazolium reductase were applied to assess the relative distribution of fast-twitch and slow-twitch (ST) muscle fiber types and fiber size. The %ST was not different in the vastus (mean SD 48 +/- 14) and deltoid (56 +/- 13) muscles. The %ST was higher (P less than 0.001), however, in the deltoid compared with vastus muscle of kayakers. This pattern was reversed in runners (P less than 0.001). The %ST of the vastus was higher (P less than 0.001) in runners than in any of the other groups. The %ST of the deltoid muscle was higher in kayakers than in students, runners (P less than 0.001), and lifters (P less than 0.05). The mean fiber area and the area of ST fibers were greater (P less than 0.01) in the vastus than the deltoid muscle. Our data show a difference in fiber type distribution between the trained and nontrained muscles of endurance athletes. This pattern may reflect the adaptive response to long-term endurance training.     

Differentiation of muscle fiber types in the chicken hindlimb

The differentiation of myotubes into fiber types was studied by examining the ATPase staining characteristics of chicken embryo thigh muscles. Two distinct fiber types, designated type IEMB and IIEMB, could be distinguished as early as stage 29. Paralysis of the embryo with d-tubocurarine prevented the differentiation of type IEMB but not type IIEMB characteristics. The two embryonic fiber types differed from each other, and mature type I and II fibers, in the acid and alkali labilities of their ATPases. Myotubes which were type IEMB at stage 29 matured into type I fibers, whereas those which were type IIEMB predominantly but not exclusively developed into type II fibers. The process of maturation involved sequential changes in the staining characteristics of the myotubes. Thus, the ultimate fiber type of a myotube can be detected long before it expresses its mature characteristics.     

Development of muscle fiber types in the prenatal rat hindlimb

Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).     

Morphofunctional and histochemical characteristics of the hindlimb muscles of the Rana temporaria in ontogeny

Mixed muscles of adult frogs respond to the increase in external potassium and to Ach by polyphasic contracture which is due to asynchronous activity of various groups of muscle fibers (fast phasic, intermediate and tonic ones). In the developing in vivo hindlimb muscles, the predominance of phasic contractile response and relatively weak tonic one were noted. In contrast to definitive muscles, in which maximum potassium and acetylcholine contractures are identical, growing muscles produce weak contractile reaction to Ach. Ach sensitivity of the developing muscles (as revealed by the contracture) is lower than in the definitive ones. Histochemical (studies on the lipid content and the activity of succinate dehydrogenase) and morphometric (the ratio of muscle fibers of different types at different stages of development, comparison of their diameters, relative size of tonic bundle, etc.) studies indicate that the development of morphological substrate for tonic contractions (tonic and intermediate muscle fibers) takes place at a lower rate as compared to the development of the substrate for phasic contractions. However, histochemically tonic fibers may be revealed already at the stage of myotubes.     

A histochemical study of the muscles of Drosophila melanogaster.

The thoracic muscles of Drosophila melanogaster can be classified into two classes, the fibrillar and the tubular muscles, on morphological grounds. Histochemical techniques were used to characterize these two classes of muscle according to their content of various enzymes (alpha-glycerophosphate, NAD-dependent isocitrate, malate and succinate dehydrogenases, fumarase, acid phosphatase, adenosine triphosphatase and acetylcholinesterase) and of glycogen. These investigations showed that the two muslces types are histochemically very different and, further, that the morphologically similar tubular muscles are heterogeneous with respect to their enzyme content. In particular, the tergal depressor of the trochanter of the second leg, the largest of the tubular muslces, has considerably less of all the enzymes studied, with the exception of acetylcholinesterase, than all the other tubular muscles examined. The histochemical techniqes were also used to follow the changes in enzyme levels that occur during development of the indirect flight muscle fibres. All the enzymes that are present in adult flight muslces showed an increase in staining intensity throughout muscle development. Some minor differences were observed in the time of appearance and rate of increase of intensity of the different enzymes.     

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.     

Myosin types and fiber types in cardiac muscle. I. Ventricular myocardium

Antisera against bovine atrial myosin were raised in rabbits, purified by affinity chromatography, and absorbed with insolubilized ventricular myosin. Specific anti-bovine atrial myosin (anti-bAm) antibodies reacted selectively with atrial myosin heavy chains, as determined by enzyme immunoassay combined with SDS-gel electrophoresis. In direct and indirect immunofluorescence assay, anti-bAm was found to stain all atrial muscle fibers and a minor proportion of ventricular muscle fibers in the right ventricle of the bovine heart. In contrast, almost all muscle fibers in the left ventricle were unreactive. Purkinje fibers showed variable reactivity. In the rabbit heart, all atrial muscle fibers were stained by anti-bAm, whereas ventricular fibers showed a variable response in both the right and left ventricle, with a tendency for reactive fibers to be more numerous in the right ventricle and in subepicardial regions. Diversification of fiber types with respect to anti-bAm reactivity was found to occur during late stages of postnatal development in the rabbit heart and to be influenced by thyroid hormone. All ventricular muscle fibers became strongly reactive after thyroxine treatment, whereas they became unreactive or poorly reactive after propylthiouracil treatment. These findings are consistent with the existence of different ventricular isomyosins whose relative proportions can vary according to the thyroid state. Variations in ventricular isomyosin composition can account for the changes in myosin Ca2+-activated ATPase activity previously observed in cardiac muscle from hyper- and hypothyroid animals and may be responsible for the changes in the velocity of contraction of ventricular myocardium that occur under these conditions. The differential distribution of ventricular isomyosins in the normal heart suggests that fiber types with different contractile properties may coexist in the ventricular myocardium.     

Distribution of fiber types in locomotory muscles of dogs

The distribution of Type I and Type II fibers, as determined from histochemical estimation of myofibrillar ATPase activity, was studied within and among the locomotory muscles of the forelimb, trunk, and hindlimb of three mongrel dogs. All Type II fibers had high oxidative capacities as estimated from the histochemical assay for reduced nicotinamide adenine dinucleotide tetrazolium reductase, so they were not further divided into subpopulations. Furthermore, Type I and Type II fibers had similar oxidative potentials as indicated by both histochemistry and biochemistry. Type I fiber populations ranged between 14% and 100% in the muscles sampled. The highest percentages of Type I fibers were found in deep muscles of physiological extensor groups in the arm and thigh that serve to resist gravity (antigravity muscles) when the dog is in the quadrupedal standing position. More superficial muscles in these same groups had fewer Type I fibers. The patterns of Type I fiber distribution among muscles in the antigravity groups of the forearm and leg were the opposite of those in the arm and thigh, with the more superficial muscles of the distal limb segments having more Type I fibers than the deeper muscles. In all limb segments, muscle groups that do not serve to resist gravity did not show as much intermuscular variation. Type I fiber populations in these muscles did not exceed 50%. A stratification of fiber types also existed within muscles, both in extensor and flexor groups, with the deeper portions of the muscles having more Type I fibers than the more superficial portions.     

Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb

The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.     

Histochemical determination of muscle fiber types in locomotor muscles of anuran amphibians.

1. A histochemical study using myosin ATPase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase reactions and a morphometric analysis with image analyser, was carried out in sartorius and gastrocnemius muscles of two anuran species, Rana perezi and Bufo calamita, that show different locomotor activities. 2. Four types of muscle fiber were found. There were interspecific variations in their proportions, with a predominance of oxidative muscle fibers in Bufo calamita. 3. These results agree with those obtained previously for the metabolic profile of several tissues from both species and point to a clear metabolic basis for the differences in locomotor activities between these two species.     

Distribution of three types of muscle fiber in the skeletal muscles of Rana temporaria frogs (according to histochemical findings)]

On 15 skeletal muscles with different functional profile (flexors and extensors, trunk muscles and muscles of the head) and different embryonic origin (somatic and visceral), it has been shown that muscles of R. temporaria are mixed. Muscle fibers are divided into three groups which differ in the content of lipids, the activity of succinic dehydrogenase and localization of myofibrils on a cross section. On the basis of the data obtained and those in literature, these fiber groups are qualified as phasic, tonic and transitional (slow phasic) ones. Both phasic and tonic fiber groups are uniform, whereas transitional fibers form a broad specturm. Most of the muscles studied contain all the three types of fibers. In muscles of the extremities (especially in flexors), tonic fibers together with transitional ones usually form more or less compact bundles. The highest content of tonic and transitional fibers was found in muscles which do not directly participate in locomotion. If muscles contain only two types of fibers (phasic and transitional ones), these fibers form a mosaic structure.     

Fiber types and fiber diameters in canine respiratory muscles

In the present study, we measured fiber types and fiber diameters in canine respiratory muscles and examined regional variation within the diaphragm. Samples of eight diaphragm regions, internal intercostals, external intercostals, transversus abdominis, and triceps brachii were removed from eight adult mongrel dogs, frozen, and histochemically processed for standard fiber type and fiber diameter determinations. The respiratory muscles were composed of types I and IIa fibers; no IIb fibers were identified. Fiber composition differed between muscles (P less than 0.0001). Normal type I percent (+/- SE) were: diaphragm 46 +/- 2, external intercostal 85 +/- 6, internal intercostals 48 +/- 3, transversus abdominis 53 +/- 1, and triceps 33 +/- 7. The diaphragm also contained a type I subtype [6 +/- 1% (SE)] previously thought only to occur in developing muscle. Fiber composition varied between diaphragm regions (P less than 0.01). Most notably, left medial crus contained 64% type I fibers. Fiber size also varied systematically among muscles (P less than 0.025) and diaphragm regions (P less than 0.0005). External intercostal fiber diameter was largest (47-50 microns) and diaphragm was smallest (34 microns). Within diaphragm, crural fibers were larger than costal (P less than 0.05). We conclude that there are systematic differences in fiber composition and fiber diameter of the canine respiratory muscles.     

Genomic data reject the hypothesis of a prosimian primate clade

The phylogenetic position of tarsiers within the primates has been a controversial subject for over a century. Despite numerous morphological and molecular studies, there has been weak support for grouping tarsiers with either strepsirrhine primates in a prosimian clade or with anthropoids in a haplorrhine clade. Here, we take advantage of the recently released whole genome assembly of the Philippine tarsier, Tarsius syrichta, in order to infer the phylogenetic relationship of Tarsius within the order Primates. We also present estimates of divergence times within the primates. Using a 1.26 million base pair multiple sequence alignment derived from 1078 orthologous genes, we provide overwhelming statistical support for the presence of a haplorrhine clade. We also present divergence date estimates using local relaxed molecular clock methods. The estimated time of the most recent common ancestor of extant Primates ranged from 64.9 Ma to 72.6 Ma, and haplorrhines were estimated to have a most recent common ancestor between 58.9 Ma and 68.6 Ma. Examination of rates of nucleotide substitution in the three major extant primate clades show that anthropoids have a slower substitution rate than either strepsirrhines or tarsiers. Our results provide the framework on which primate morphological, reproductive, and genomic features can be reconstructed in the broader context of mammalian phylogeny.