Reserve capacity for ATP consumption during isometric contraction in human skeletal muscle fibers.

Maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) and ATP consumption rate during maximum isometric activation (ATP(iso)) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 -- (ratio of ATP(iso) to V(max) ATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21--66 yr). A quantitative histochemical procedure was used to measure V(max) ATPase. In permeabilized fibers, ATP(iso) was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC(2X) and MHC(2A) compared with fibers singularly expressing MHC(2A) and MHC(slow) (39 vs. 52 and 56%, respectively). Tension cost (ratio of ATP(iso) to generated force) also varied with fiber type, being highest in fibers coexpressing MHC(2X) and MHC(2A). We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.     

Effect of denervation on ATP consumption rate of diaphragm muscle fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC(2X) isoform but not in fibers expressing MHC(slow) and MHC(2A). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP(iso)) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC(2X). In single-type-identified, permeabilized DIAm fibers, ATP(iso) was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V(o)) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k(TR)) following quick release and restretch] was also examined. Two weeks after DNV, ATP(iso) was significantly reduced in fibers expressing MHC(2X), but unaffected in fibers expressing MHC(slow) and MHC(2A). This effect of DNV on fibers expressing MHC(2X) persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V(o) and k(TR) were slowed in fibers expressing MHC(2X), consistent with the effect on ATP(iso). The difference between V(max) ATPase and ATP(iso) reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC(2X). DNV-induced reductions in ATP(iso) and V(max) ATPase of fibers expressing MHC(2X) reflect the underlying decrease in MHC content, while reduction in ATP(iso) also reflects a slowing of cross-bridge cycling rate.     

ATP consumption rate per cross bridge depends on myosin heavy chain isoform.

In the present study, we tested the hypothesis that intrinsic differences in ATP consumption rate per cross bridge exist across rat diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. During maximum Ca(2+) activation (pCa 4.0) of single, Triton X-permeabilized Dia(m) fibers, isometric ATP consumption rate was determined by using an NADH-linked fluorometric technique. The MHC concentration in single Dia(m) fibers was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. Isometric ATP consumption rate varied across Dia(m) fibers expressing different MHC isoforms, being highest in fibers expressing MHC(2X) (1.14 +/- 0.08 nmol. mm(-3). s(-1)) and/or MHC(2B) (1.33 +/- 0.08 nmol. mm(-3). s(-1)), followed by fibers expressing MHC(2A) (0.77 +/- 0.11 nmol. mm(-3). s(-1)) and MHC(Slow) (0.46 +/- 0.03 nmol. mm(-3). s(-1)). These differences in ATP consumption rate also persisted when it was normalized for MHC concentration in single Dia(m) fibers. Normalized ATP consumption rate for MHC concentration varied across Dia(m) fibers expressing different MHC isoforms, being highest in fibers expressing MHC(2X) (2.02 +/- 0.19 s(-1)) and/or MHC(2B) (2.64 +/- 0.15 s(-1)), followed by fibers expressing MHC(2A) (1.57 +/- 0.16 s(-1)) and MHC(Slow) (0.77 +/- 0.05 s(-1)). On the basis of these results, we conclude that there are intrinsic differences in ATP consumption rate per cross bridge in Dia(m) fibers expressing MHC isoforms.     

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia(m)) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P(o)) and maximum unloaded shortening velocity (V(o)). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC(neo) in the Dia(m) persisted, and emergence of MHC(2X) and MHC(2B) was delayed. By D-21 and D-28, relative expression of MHC(2A) and MHC(2B) was reduced in DNV compared with control (CTL) animals. Expression of MHC(neo) also reappeared in adult Dia(m) by 2-3 wk after DNV, and relative expression of MHC(2B) was reduced. At each age, P(o) was reduced and V(o) was slowed by DNV, compared with CTL. In CTL Dia(m), postnatal changes in P(o) and V(o) were associated with an increase in fast MHC isoform expression. In DNV Dia(m), no such association existed. We conclude that, in the Dia(m), DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.     

Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat diaphragm muscle fibers.

The present study examined Ca(2+) sensitivity of diaphragm muscle (Dia(m)) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Dia(m) fibers expressing the MHC(slow) isoform have greater Ca(2+) sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca(2+) sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Dia(m) fibers. The Ca(2+) concentration at which 50% maximal force was generated (pCa(50)) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa(50) for Dia(m) fibers expressing MHC(slow) was significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca(2+) sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Dia(m) fibers expressing MHC(slow) contained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca(2+) sensitivity in Dia(m) fibers.     

Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers.

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Dia(m)) fibers, 2) Hyp decreases the maximum specific force (F(max)) of Dia(m) fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and F(max) to a greater extent in fibers expressing MHC type 2X (MHC(2X)) and/or MHC type 2B (MHC(2B)). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC(2X) and/or MHC(2B). On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased F(max) across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (approximately 40 vs. approximately 20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC(2A) was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC(2X) and/or MHC(2B) in the Hyp Dia(m) than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC(2X) and/or MHC(2B) and that the reduction of F(max) with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.     

Maximum specific force depends on myosin heavy chain content in rat diaphragm muscle fibers.

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Dia(m)) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Dia(m) fibers. Maximum specific force was determined by activation of single Dia(m) fibers in the presence of a high-calcium solution (pCa, -log Ca(2+) concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.     

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.     

Diversity of fast myosin heavy chain expression during development of gastrocnemius, bicep brachii, and posterior latissimus dorsi muscles in normal and dystrophic chickens

The expression of fast myosin heavy chain (MHC) isoforms was examined in developing bicep brachii, lateral gastrocnemius, and posterior latissimus dorsi (PLD) muscles of inbred normal White Leghorn chickens (Line 03) and genetically related inbred dystrophic White Leghorn chickens (Line 433). Utilizing a highly characterized monoclonal antibody library we employed ELISA, Western blot, immunocytochemical, and MHC epitope mapping techniques to determine which MHCs were present in the fibers of these muscles at different stages of development. The developmental pattern of MHC expression in the normal bicep brachii was uniform with all fibers initially accumulating embryonic MHC similar to that of the pectoralis muscle. At hatching the neonatal isoform was expressed in all fibers; however, unlike in the pectoralis muscle the embryonic MHC isoform did not disappear. With increasing age the neonatal MHC was repressed leaving the embryonic MHC as the only detectable isoform present in the adult bicep brachii muscle. While initially expressing embryonic MHC in ovo, the post-hatch normal gastrocnemius expressed both embryonic and neonatal MHCs. However, unlike the bicep brachii muscle, this pattern of expression continued in the adult muscle. The adult normal gastrocnemius stained heterogeneously with anti-embryonic and anti-neonatal antibodies indicating that mature fibers could contain either isoform or both. Neither the bicep brachii muscle nor the lateral gastrocnemius muscle reacted with the adult specific antibody at any stage of development. In the developing posterior latissimus dorsi muscle (PLD), embryonic, neonatal, and adult isoforms sequentially appeared; however, expression of the embryonic isoform continued throughout development. In the adult PLD, both embryonic and adult MHCs were expressed, with most fibers expressing both isoforms. In dystrophic neonates and adults virtually all fibers of the bicep brachii, gastrocnemius, and PLD muscles were identical and contained embryonic and neonatal MHCs. These results corroborate previous observations that there are alternative programs of fast MHC expression to that found in the pectoralis muscle of the chicken (M.T. Crow and F.E. Stockdale, 1986, Dev. Biol. 118, 333-342), and that diversification into fibers containing specific MHCs fails to occur in the fast muscle fibers of the dystrophic chicken. These results are consistent with the hypothesis that avian muscular dystrophy is a developmental disorder that is associated with alterations in isoform switching during muscle maturation.     

Developmental effects on myonuclear domain size of rat diaphragm fibers.

During early postnatal development in rat diaphragm muscle (Diam), significant fiber growth and transitions in myosin heavy chain (MHC) isoform expression occur. Similar to other skeletal muscles, Diam fibers are multinucleated, and each myonucleus regulates the gene products within a finite volume: the myonuclear domain (MND). We hypothesized that postnatal changes in fiber cross-sectional area (CSA) are associated with increased number of myonuclei so that the MND size is maintained. The Diam was removed at postnatal days 14 (P-14) and 28 (P-28). MHC isoform expression was determined by SDS-PAGE. Fiber CSA, myonuclear number, and MND size were measured using confocal microscopy. By P-14, significant coexpression of MHC isoforms was present with no fiber displaying singular expression of MHCNeo. By P-28, singular expression was predominant. MND size was not different across fiber types at P-14. Significant fiber growth was evident by P-28 at all fiber types (fiber CSA increased by 61, 93, and 147% at fibers expressing MHCSlow, MHC2A, and MHC2X, respectively). The number of myonuclei per unit of fiber length was similar across fibers at P-14, but it was greater at fibers expressing MHC2X at P-28. The total number of myonuclei per fiber also increased between P-14 and P-28 at all fiber types. Accordingly, MND size increased significantly by P-28 at all fiber types, and it became larger at fibers expressing MHC2X compared with fibers expressing MHCSlow or MHC2A. These results suggest that MND size is not maintained during the considerable fiber growth associated with postnatal development of the Diam.     

Aging, muscle fiber type, and contractile function in sprint-trained athletes.

Biopsy samples were taken from the vastus lateralis of 18- to 84-yr-old male sprinters (n = 91). Fiber-type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content were identified using ATPase histochemistry and SDS-PAGE. Specific tension and maximum shortening velocity (V(o)) were determined in 144 single skinned fibers from younger (18-33 yr, n = 8) and older (53-77 yr, n = 9) runners. Force-time characteristics of the knee extensors were determined by using isometric contraction. The cross-sectional area of type I fibers was unchanged with age, whereas that of type II fibers was reduced (P < 0.001). With age there was an increased MHC I (P < 0.01) and reduced MHC IIx isoform content (P < 0.05) but no differences in MHC IIa. Specific tension of type I and IIa MHC fibers did not differ between younger and older subjects. V(o) of fibers expressing type I MHC was lower (P < 0.05) in older than in younger subjects, but there was no difference in V(o) of type IIa MHC fibers. An aging-related decline of maximal isometric force (P < 0.001) and normalized rate of force development (P < 0.05) of knee extensors was observed. Normalized rate of force development was positively associated with MHC II (P < 0.05). The sprint-trained athletes experienced the typical aging-related reduction in the size of fast fibers, a shift toward a slower MHC isoform profile, and a lower V(o) of type I MHC fibers, which played a role in the decline in explosive force production. However, the muscle characteristics were preserved at a high level in the oldest runners, underlining the favorable impact of sprint exercise on aging muscle.     

Phrenic motoneuron morphology during rapid diaphragm muscle growth.

In the adult rat, there is a general correspondence between the sizes of motoneurons, motor units, and muscle fibers that has particular functional importance in motor control. During early postnatal development, after the establishment of singular innervation, there is rapid growth of diaphragm muscle (Dia(m)) fibers. In the present study, the association between Dia(m) fiber growth and changes in phrenic motoneuron size (both somal and dendritic) was evaluated from postnatal day 21 (D21) to adulthood. Phrenic motoneurons were retrogradely labeled with fluorescent tetramethylrhodamine dextran (3,000 MW), and motoneuron somal volumes and surface areas were measured using three-dimensional confocal microscopy. In separate animals, phrenic motoneurons retrogradely labeled with choleratoxin B-fragment were visualized using immunocytochemistry, and dendritic arborization was analyzed by camera lucida. Between D21 and adulthood, Dia(m) fiber cross-sectional area increased by approximately 164% overall, with the growth of type II fibers being disproportionate to that of type I fibers. There was also substantial growth of phrenic motoneurons ( approximately 360% increase in total surface area), during this same period, that was primarily attributable to an expansion of dendritic surface area. Comparison of the distribution of phrenic motoneuron surface areas between D21 and adults suggests the establishment of a bimodal distribution that may have functional significance for motor unit recruitment in the adult rat.     

Alterations in diaphragm contractility after nandrolone administration: an analysis of potential mechanisms.

The aim of this study was to evaluate the potential mechanisms underlying the improved contractility of the diaphragm (Dia) in adult intact male hamsters after nandrolone (Nan) administration, given subcutaneously over 4 wk via a controlled-release capsule (initial dose: 4.5 mg. kg-1. day-1; with weight gain, final dose: 2.7 mg. kg-1. day-1). Control (Ctl) animals received blank capsules. Isometric contractile properties of the Dia were determined in vitro after 4 wk. The maximum velocity of unloaded shortening (Vo) was determined in vitro by means of the slack test. Dia fibers were classified histochemically on the basis of myofibrillar ATPase staining and fiber cross-sectional area (CSA), and the relative interstitial space was quantitated. Ca2+-activated myosin ATPase activity was determined by quantitative histochemistry in individual diaphragm fibers. Myosin heavy chain (MHC) isoforms were identified electrophoretically, and their proportions were determined by using scanning densitometry. Peak twitch and tetanic forces, as well as Vo, were significantly greater in Nan animals compared with Ctl. The proportion of type IIa Dia fibers was significantly increased in Nan animals. Nan increased the CSA of all fiber types (26-47%), whereas the relative interstitial space decreased. The relative contribution of fiber types to total costal Dia area was preserved between the groups. Proportions of MHC isoforms were similar between the groups. There was a tendency for increased expression of MHC2B with Nan. Ca2+-activated myosin ATPase activity was increased 35-39% in all fiber types in Nan animals. We conclude that, after Nan administration, the increase in Dia specific force results from the relatively greater Dia CSA occupied by hypertrophied muscle fibers, whereas the increased ATPase activity promotes a higher rate of cross-bridge turnover and thus increased Vo. We speculate that Nan in supraphysiological doses have the potential to offset or ameliorate conditions associated with enhanced proteolysis and disordered protein turnover.     

Plasticity in Skeletal, Cardiac, and Smooth Muscle: Selected Contribution: Mechanisms underlying increased force generation by rat diaphragm muscle fibers during development

It has beenfound that maximum specific force (F_max; force percross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change inF_max reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridgesin parallel) and/or a greater force per cross bridge in fibersexpressing fast MHC isoforms compared with slow and neonatal MHCisoforms (MHC_slow and MHC_neo, respectively).Single Triton 100-X-permeabilized fibers were activated at a pCa of4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibersexpressing MHC_slow and coexpressing MHC_neoproduced less force than fibers expressing fast MHC isoforms. Weconclude that lower force per cross bridge in fibers expressingMHC_slow and MHC_neo contributes to the lowerF_max seen in early postnatal development.

     

Emergence of the mature myosin phenotype in the rat diaphragm muscle

Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2X and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in approximately 27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day greater than or equal to 115, less than 4% of the myofibers expressed MHC2B). Fibers which contained MHC beta/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHC beta/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was greater than or equal to 115 days old.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

Enzyme‐ and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos)

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (α‐GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC‐I, ‐IIa, and ‐IIx are expressed in the muscles of adolescent bears. MHC‐I isoform was expressed in Type‐I fibers and coexpressed with presumably ‐IIa isoform, in Type‐IIC fibers. Surprisingly, two antibodies specific to rat MHC‐IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC‐IIb and all fast fiber staining with antibody that according to our experience recognizes MHC‐IIa and ‐IIx of rat. Furthermore, quite high‐oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC‐IIa and ‐IIx isoform expression in fast fibers of bear. However, in adult, full‐grown animal, only MHC‐I and MHC‐IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight‐bearing and locomotor demands of these mammals. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.     

Evidence for three fast myosin heavy chain isoforms in type II skeletal muscle fibers in the adult llama (Lama glama).

Skeletal muscle fiber types classified on the basis of their content of different myosin heavy chain (MHC) isoforms were analyzed in samples from hindlimb muscles of adult sedentary llamas (Lama glama) by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies, myofibrillar ATPase (mATPase) histochemistry, and quantitative histochemistry of fiber metabolic and size properties. The immunohistochemical technique allowed the separation of four pure (i.e., expressing a unique MHC isoform) muscle fiber types: one slow-twitch (Type I) and three fast-twitch (Type II) phenotypes. The same four major fiber types could be objectively discriminated with two serial sections stained for mATPase after acid (pH 4.5) and alkaline (pH 10.5) preincubations. The three fast-twitch fiber types were tentatively designated as IIA, IIX, and IIB on the basis of the homologies of their immunoreactivities, acid denaturation of their mATPase activity, size, and metabolic properties expressed at the cellular level with the corresponding isoforms of rat and horse muscles. Acid stability of their mATPase activity increased in the rank order IIA>IIX>IIB. The same was true for size and glycolytic capacity, whereas oxidative capacity decreased in the same rank order IIA>IIX>IIB. In addition to these four pure fibers (I, IIA, IIX, and IIB), four other fiber types with hybrid phenotypes containing two (I+IIA, IIAX, and IIXB) or three (IIAXB) MHCs were immunohistochemically delineated. These frequent phenotypes (40% of the semitendinosus muscle fiber composition) had overlapped mATPase staining intensities with their corresponding pure fiber types, so they could not be delineated by mATPase histochemistry. Expression of the three fast adult MHC isoforms was spatially regulated around islets of Type I fibers, with concentric circles of fibers expressing MHC-IIA, then MHC-IIX, and peripherally MHC-IIB. This study demonstrates that three adult fast Type II MHC isoproteins are expressed in skeletal muscle fibers of the llama. The general assumption that the very fast MHC-IIB isoform is expressed only in small mammals can be rejected.