Fiber types in canine muscles: myosin isoform expression and functional characterization

This study was aimed to achieve a definitive and unambiguous identification of fiber types in canine skeletal muscles and of myosin isoforms that are expressed therein. Correspondence of canine myosin isoforms with orthologs in other species as assessed by base sequence comparison was the basis for primer preparation and for expression analysis with RT-PCR. Expression was confirmed at protein level with histochemistry, immunohistochemistry, and SDS-PAGE combined together and showed that limb and trunk muscles of the dog express myosin heavy chain (MHC) type 1, 2A, and 2X isoforms and the so-called "type 2dog" fibers express the MHC-2X isoform. MHC-2A was found to be the most abundant isoform in the trunk and limb muscle. MHC-2X was expressed in most but not all muscles and more frequently in hybrid 2A-2X fibers than in pure 2X fibers. MHC-2B was restricted to specialized extraocular and laryngeal muscles, although 2B mRNA, but not 2B protein, was occasionally detected in the semimembranosus muscle. Isometric tension (P(o)) and maximum shortening velocity (V(o)) were measured in single fibers classified on the basis of their MHC isoform composition. Purified myosin isoforms were extracted from single muscle fibers and characterized by the speed (V(f)) of actin filament sliding on myosin in an in vitro motility assay. A close proportionality between V(o) and V(f) indicated that the diversity in V(o) was due to the different myosin isoform composition. V(o) increased progressively in the order 1/slow < 2A < 2X < 2B, thus confirming the identification of the myosin isoforms and providing their first functional characterization of canine muscle fibers.     

Fiber architecture of canine abdominal muscles.

During respiration, abdominal muscles experience loads, not only in the muscle-fiber direction but also transverse to the fibers. We wondered whether the abdominal muscles exhibit a fiber architecture that is similar to the diaphragm muscle, and, therefore, we chose two adjacent muscles: the internal oblique (IO), with about the same muscle length as the diaphragm, and the transverse abdominis (TA), which is twice as long as the diaphragm. First, we used acetylcholinesterase staining to examine the distribution of neuromuscular junctions on both surfaces of the TA and IO muscles in six dogs. A maximum of four irregular bands of neuromuscular junctions crossed the IO, and as many as six bands crossed the TA, which is consistent with a discontinuous fiber architecture. In six additional dogs, we examined fiber architecture of these muscles by microdissecting 103 fascicles from the IO and 139 from the TA. Each fascicle contained between 20 and 30 muscle fibers. The mean length of nonspanning fibers (NSF) ranged from 2.8 +/- 0.3 cm in the IO to 4.3 +/- 0.5 cm in the TA, and the mean length of spanning fibers ranged from 4.3 +/- 0.5 cm in the IO to 7.6 +/- 1.4 cm in the TA. NSF accounted for 89.6 +/- 1.5% of all fibers dissected from the IO and 99.1 +/- 0.2% of all fibers dissected from the TA. The percentage of NSF with both ends tapered was 6.2 +/- 1.0 and 41.0 +/- 2.3% for IO and TA, respectively. These data show that fiber architecture in either IO or TA is discontinuous, with much more short-tapered fibers in the TA than in the IO. When abdominal muscles are submaximally activated, as during both normal expiration and maximal expiratory efforts, muscle force could be transmitted to the cell membrane and to the extracellular intramuscular connective tissue by shear linkage, presumably via structural transmembrane proteins.     

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.     

Distribution of muscle fiber types and EMG activity in cat intercostal muscles

The electromyogram (EMG) activity and histochemical properties of intercostal muscles in the anesthetized cat were studied. The parasternal muscles were consistently active during inspiration. The external intercostals in the rostral spaces and the ventral portions of the midthoracic spaces were also recruited during inspiration. The remaining external intercostals were typically silent, regardless of the level of respiratory drive. The internal intercostal muscles located in the caudal spaces were occasionally recruited during expiration. There was a clear correlation between recruitment patterns of the intercostals and the histochemically defined fiber type properties of the muscles. Intercostal muscles that were routinely recruited during inspiration had a significantly higher proportion of slow-oxidative muscle fibers.     

Oxidative phosphorylation in mitochondria from different fiber types of chicken muscles

Isolated mitochondria from different types of muscle fibers from chickens 3 to 5 weeks were studied to evaluate the comparative oxidation of various substrates. Pectoralis (alphaW fibers), lateral adductor (betaR fibers), and medial adductor (alphaR fibers) were the muscles used. Oxygen consumption rates, RCR, and ADP/O ratios were measured to study mitochondrial function. Mitochondria from pectoralis muscle utilized pyruvate, succinate, L-glutamate, alpha-glycerophosphate, and beta-hydroxybutyrate. Mitochondria from the other two muscle types utilized all of those substrates except alpha-glycerophosphate. In each muscle type utilization of NADH was minimum and was not coupled with phosphorylation of ADP. Thus, in alphaW muscles oxidation of alpha-glycerophosphate may play an important role in transport of cytoplasmic NADH to the mitochondrial respiratory chain. In alphaR and betaR muscles "shuttle" systems other than alpha-glycerophosphate oxidation, e.g., beta-hydroxybutyrate, may perform that important role.     

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.     

Histochemical and biochemical characterization of two slow fiber types in decapod crustacean muscles

Myofibrillar proteins in muscles of the claws and abdomen of lobster, Homarus americanus, and the claws of fiddler crab, Uca pugnax, and land crab, Gecarcinus lateralis, have been analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fibers contained numerous isoforms of structural and regulatory proteins in assemblages correlated with fiber type. One fast (F) and two slow (S1 and S2) fibers were identified. All F fibers possessed two isoforms of paramyosin (P1 and P2), while all slow fibers, with the exception of Uca major claw, contained only the P2 variant. S1 and S2 fibers were distinguished by the distribution of a large isoform of troponin-T (T1; Mr = 55,000); S2 fibers in all three species contained T1 in addition to one or two smaller-molecular-weight variants usually associated with S1 fibers. In order to determine whether the slow fibers differed in histochemical properties, land crab claw closer muscle was cryosectioned and stained for myofibrillar ATPase and NADH diaphorase activities. Most S2 fibers had lower ATPase and higher NADH diaphorase activities than S1 fibers, which indicated that S2 fibers had a lower rate of contraction and were more fatigue-resistant than S1 fibers. It is proposed that the S1 and S2 fibers defined by biochemical and histochemical criteria are identical to the slow-twitch and tonic fibers, respectively characterized physiologically.     

Effect of stimulation of pulmonary C-fiber receptors on canine respiratory muscles

The effects of stimulation of pulmonary C-fiber receptors on the distribution of motor activity to upper airway, rib cage, and abdominal muscles were studied in anesthetized, tracheotomized, spontaneously breathing dogs. Stimulation of pulmonary C-fiber receptors by injection of capsaicin (3-20 micrograms/kg) into the right atrium resulted in complete cessation of electrical activity of the upper airway dilating muscles (UADM) and the inspiratory chest wall pumping muscles. The activity of abdominal muscles was also inhibited. The duration of electrical silence was longer for the diaphragm than for the UADM. Upper airway constricting muscles and expiratory intercostal muscles, including the triangularis sterni, remained tonically active during the apneic period. The responses of these muscles were qualitatively the same when the animals breathed 100% O2, 7% CO2 in O2, or 12% O2 in N2, and without or in the presence of an expiratory threshold load. Bilateral vagotomy abolished the inhibitory effects of capsaicin on UADM, chest wall, and abdominal muscle activity, suggesting that the vagus is the major afferent pathway for the reflex. The qualitative difference in the response of intercostal expiratory muscles and abdominal muscles suggests that these two groups of synergistic muscles may be independently regulated.     

Fiber types in rat laryngeal muscles and their transformations after denervation and reinnervation.

The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allotypes and that laryngeal muscle fibers are subject to neural regulation.     

Functional limitations to glucose uptake in muscles comprised of different fiber types

Skeletal muscle glucose uptake requires delivery of glucose to the sarcolemma, transport across the sarcolemma, and the irreversible phosphorylation of glucose by hexokinase (HK) inside the cell. Here, a novel method was used in the conscious rat to address the roles of these three steps in controlling the rate of glucose uptake in soleus, a muscle comprised of type I fibers, and two muscles comprised of type II fibers. Experiments were performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed, constant infusions of 3-O-methyl-[3H]glucose (3-O-MG) and [1-14C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration, which distributes based on the transsarcolemmal glucose gradient (TSGG), were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G](im) and [G](om), respectively) in muscle. Muscle glucose uptake was much lower in muscle comprised of type II fibers than in soleus under both basal and insulin-stimulated conditions. Under all conditions, the TSGG in type II muscle exceeded that in soleus, indicating that glucose transport plays a more important role to limit glucose uptake in type II muscle. Although hyperinsulinemia increased [G](im) in soleus, indicating that phosphorylation was a limiting factor, type II muscle was limited primarily by glucose delivery and glucose transport. In conclusion, the relative importance of glucose delivery, transport, and phosphorylation in controlling the rate of insulin-stimulated muscle glucose uptake varies between muscle fiber types, with glucose delivery and transport being the primary limiting factors in type II muscle.     

Capillarity and fiber types in locomotory muscles of wild common coots,Fulica atra

Six locomotory muscles of wild common coots, Fulica atra, were analyzed histochemically. Capillarity and fiber-type distributions were correlated to the functional implications and physiological needs of each muscle. Leg muscles exhibit three unevenly distributed fiber types, a pattern that reflects the great variety of terrestrial and aquatic locomotory performances that coots are able to develop. Aerobic zones are presumably recruited during steady swimming and diving, while regions with anaerobic characteristics may be used for bursts of activity such as sprint swimming or during take off, when coots run along the water's surface. Fiber types and capillarization in wing muscles have a marked oxidative trend. High wing beat frequencies, short and broad wings, and the long distance migrations that these birds perform indicate that the presence of high numbers of oxidative fibers and the well developed capillary supply are needed for enhanced oxygen uptake. The pectoralis muscle, except in its deep part, has exclusively fast oxidative fibers with a very high staining intensity for succinate dehydrogenase assay as compared to the same fiber type of other muscles. Its predominant role in flapping flight justifies these characteristics that are typical of fibers with high aerobic metabolism. The deep part of the pectoralis muscle presents a low proportion of an unusual slow anaerobic fiber type. These fibers could play a role during feeding dives when the bird presses the air out of the feathers by tightening the wings against the body. A linear relationship between capillary and fiber densities in all coot muscles studied reflects an adjustment between fiber diameter and vascularization in order to obtain the oxygen for mitochondrial supply. This strategy seems a suitable way to cope with the rigid aerobic constraints that flying and diving impose upon the coot's physiology. J. Morphol. 237:147–164, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation of muscle fiber types in aneurogenic brachial muscles of the chick embryo

Cross-reinnervation studies performed ex ovo with newly hatched chicks demonstrate that peripheral motor neurons control the phenotypic characteristics of avian muscles. The present experiments were designed to determine whether or not nerves play a similar role during the initial expression of muscle fiber types. Previous experiments indicated that differentiation of specific fiber types occurs during the first week of embryogenesis, temporally coincident with the penetration of nerves within muscle masses. These observations suggested that peripheral nerves may be associated with the initial differentiation of fiber types. To test this hypothesis directly, anterior limb buds of the chick embryo were rendered aneurogenic by deletion of the brachial segment of the neural tube. To ensure a completely aneurogenic environment for developing brachial muscles, surgery was performed at day 2 in ovo before the exit of ventral root fibers. Experimental and control embryos from Stage (St) 25 (4.5 d) through St 45 (19d) were analyzed histochemically by a silver-cholinesterase reaction to detect nerves and by the myosin ATPase reaction, following alkali and acid preincubation, to determine the fiber type composition of the muscles. In addition, the total volume of aneurogenic and control muscles was compared. Results demonstrate that the characteristic myosin ATPase profiles of individual aneurogenic and innervated (control) muscles were identical throughout the entire period analyzed. Therefore, we conclude that these enzymic profiles are endogenously expressed and are not under neuronal control during early embryogenesis. Furthermore, the entire sequence of events from the migration of myogenic cells to the anterior limb bud through the division of the primary muscle masses to form individual brachial muscles proceeded on schedule in the absence of nerves. Since the growth of aneurogenic muscles was impaired, we conclude that during embryogenesis peripheral motor nerves are necessary initially for the proper growth of muscles and ultimately, for their survival. They are not involved, however, with either the initial formation or initial differentiation of individual brachial muscles.     

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 fiber types in embryonic chick limb muscles innervated by foreign motoneurons

The differentiation of distinct myotube fiber types in chick limb muscle development is coincident with innervation. The role of motoneurons in influencing fiber type differentiation was analyzed by causing chick hind limb muscles to be innervated by inappropriate motoneurons and then examining experimental muscles for changes in the distribution of myosin ATPase fiber types. Motoneuron innervation of limb muscles was altered by performing either limb shifts, limb reversals, or large spinal cord reversals on early neural tube or limb bud stage chick embryos. The distribution of fiber types was then analyzed in muscles from stage 36 (E10) to stage 45 (E20) embryos after processing hind limb sections for myosin ATPase histochemistry. In the majority of experimental muscles examined (267/312), the distribution of myosin ATPase fiber types was unaltered. In the remaining experimental muscles (14%), alterations in the distribution of myosin ATPase fiber types occurred, indicating that in some cases, foreign innervation may alter the developmental program of differentiating myotubes. The results suggest that myotubes differentiate myosin ATPase staining characteristics according to an intrinsic program and that these differentiating myotubes are selectively innervated by motoneurons of the appropriate type under most conditions including normal development. Under exceptional circumstances of motoneuron-muscle fiber type mismatch, embryonic motoneurons can alter fiber type expression.     

Elementary steps of the cross-bridge cycle in fast-twitch fiber types from rabbit skeletal muscles

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca(2+)-activated at 20 degrees C and the effects of MgATP, phosphate (P, P(i)), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme: where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k(2), 198-526 s(-1); k(-2), 51-328 s(-1); k(4), 13.6-143 s(-1); k(-4), 13.6-81 s(-1)) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k(-2), k(4)) varied more among fiber types than their reversals (k(2), k(-4)). The equilibrium constants K(1) (MgATP affinity) and K(2) (=k(2)/k(-2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K(4) (=k(4)/k(-4), force generation) and K(5) (P(i) affinity) were larger in IIB than IIA and IID fibers. K(1) showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM(-1)), IID (4.9 mM(-1)), and IIB (0.84 mM(-1)). Similarly, the MgADP affinity (K(0)) was larger in type IID fibers than in type IIB fibers.     

Fiber types and some contractile properties of extensor digitorum longus and soleus muscles in different animal species

We studied the fiber types and contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles from young adult mice, rats and guinea pigs, and the correlation between these two parameters. Individual fibers in both muscles were classified as fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG) or slow-twitch oxidative (SO) fibers according to Peter et al., and type II B, II A, or I fibers according to Brooke & Kaiser. Contractile properties were measured in situ at 37 degrees C. The isometric twitch contraction time (CT) and one-half relaxation time (1/2 RT) tended to be shortened in proportion to the area occupied by type II fibers, and type II B fibers. However, the differences between CT and fiber types were not always uniform among the three species. The CT of the rat EDL, in spite of its higher proportion of type II B fibers about 10% was the same as that of the guinea-pig EDL. The SOL of the mouse, composed of about 50% type I (SO) fibers, had a CT about as short as that of the EDL. In the case of the classification by Peter et al., the relationship between the percentage of subgroups of fast-twitch fibers and the CT or 1/2 RT, but not the resistance to fatigue, was not obvious. The resistance to fatigue tended to be enhanced in proportion to the area occupied by FOG in the EDL and by SO (type I) in the SOL. These results suggest that the contractile properties of individual fibers identified histochemically are distinct among animal species, producing interspecies differences in fiber types along with different contractile properties. However, it may be possible to compare the difference between fiber types and CT or 1/2 RT in the classification based on the pH lability of myosin ATPase, and also the difference between fiber types and resistance to fatigue in the classification based on the oxidative enzyme.     

Effect of lung volume on the respiratory action of the canine pectoral muscles.

Lung volume influences the mechanical action of the primary inspiratory and expiratory muscles by affecting their precontraction length, alignment with the rib cage, and mechanical coupling to agonistic and antagonistic muscles. We have previously shown that the canine pectoral muscles exert an expiratory action on the rib cage when the forelimbs are at the torso's side and an inspiratory action when the forelimbs are held elevated. To determine the effect of lung volume on intrathoracic pressure changes produced by the canine pectoral muscles, we performed isolated bilateral supramaximal electrical stimulation of the deep pectoral and superficial pectoralis (descending and transverse heads) muscles in 15 adult supine anesthetized dogs during hyperventilation-induced apnea. Lung volume was altered by application of a negative or positive pressure (+/- 30 cmH2O) to the airway. In all animals, selective electrical stimulation of the descending, transverse, and deep pectoral muscles with the forelimbs held elevated produced negative intrathoracic pressure changes (i.e., an inspiratory action). Moreover, with the forelimbs elevated, increasing lung volume decreased both pectoral muscle fiber precontraction length and the negative intrathoracic pressure changes generated by contraction of each of these muscles. Conversely, with the forelimbs along the torso, increasing lung volume lengthened pectoral muscle precontraction length and augmented the positive intrathoracic pressure changes produced by muscle contraction (i.e., an expiratory action). These results indicate that lung volume significantly affects the length of the canine pectoral muscles and their mechanical actions on the rib cage.     

Thin filament diversity and physiological properties of fast and slow fiber types in astronaut leg muscles.

Slow type I fibers in soleus and fast white (IIa/IIx, IIx), fast red (IIa), and slow red (I) fibers in gastrocnemius were examined electron microscopically and physiologically from pre- and postflight biopsies of four astronauts from the 17-day, Life and Microgravity Sciences Spacelab Shuttle Transport System-78 mission. At 2.5-microm sarcomere length, thick filament density is approximately 1,012 filaments/microm(2) in all fiber types and unchanged by spaceflight. In preflight aldehyde-fixed biopsies, gastrocnemius fibers possess higher percentages (approximately 23%) of short thin filaments than soleus (9%). In type I fibers, spaceflight increases short, thin filament content from 9 to 24% in soleus and from 26 to 31% in gastrocnemius. Thick and thin filament spacing is wider at short sarcomere lengths. The Z-band lattice is also expanded, except for soleus type I fibers with presumably stiffer Z bands. Thin filament packing density correlates directly with specific tension for gastrocnemius fibers but not soleus. Thin filament density is inversely related to shortening velocity in all fibers. Thin filament structural variation contributes to the functional diversity of normal and spaceflight-unloaded muscles.