Uniform myosin isoforms in the flight muscles for little brown bats, Myotis lucifugus

The present study used muscle histochemistry and polyacrylamide gel electrophoresis of native myosin and myosin heavy chains to establish a correlation, if any, between chiropteran histochemical fiber types and myosin isoform composition. Histochemical analysis of the primary flight muscle, the pectoralis profundus, documented the presence of a single histochemical fiber type, here termed Type II. Electrophoresis of native myosin isolated from pectoralis muscle yielded a single isoform that comigrated with the FM-3 isoform of rat diaphragm. Heavy chain analysis of the Myotis pectoralis demonstrated a single heavy chain with comparable electrophoretic mobility to rat IIa myosin heavy chain. These data demonstrate unique histochemical and biochemical homogeneity in the myosin composition of the pectoralis muscle of Myotis lucifugus. Thus this muscle is extremely specialized for flight at histochemical, morphologic, and molecular levels. These data contrast with the mixed myosin and histochemical fiber types found in other mammals, as well as in other muscles of Myotis lucifugus.     

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.     

Ontogeny of flight in the little brown bat,Myotis lucifugus: behavior,morphology, and muscle histochemistry

Summary Postnatal changes in wing morphology, flight ability, muscle morphology, and histochemistry were investigated in the little brown bat, Myotis lucifugus. The pectoralis major, acromiodeltoideus, and quadriceps femoris muscles were examined using stains for myofibrillar ATPase, succinate dehydrogenase (SDH), and mitochondrial -glycerophosphate dehydrogenase (-GPDH) enzyme reactions. Bats first exhibited spontaneous, drop-evoked flapping behavior at 10 days, short horizontal flight at 17 days, and sustained flight at 24 days of age. Wing loading decreased and aspect ratio increased during postnatal development, each reaching adult range before the onset of sustained flight. Histochemically, fibers from the three muscles were undifferentiated at birth and had lower oxidative and glycolytic capacities compared to other age groups. Cross-sectional areas of fibers from the pectoralis and acromiodeltoideus muscles increased significantly at an age when dropevoked flapping behavior was first observed, suggesting that the neuromuscular mechanism controlling flapping did not develop until this time. Throughout the postnatal growth period, pectoralis and acromiodeltoideus muscle mass and fiber cross-sectional area increased significantly. By day 17 the pectoralis muscle had become differentiated in glycolytic capacity, as indicated by the mosaic staining pattern for -GPDH. By contrast, the quadriceps fibers were relatively large at birth and slowly increased in size during the postnatal period. Fiber differentiation was evident at the time young bats began to fly, as indicated by a mosaic pattern of staining for myosin ATPase. These results indicate that flight muscles (pectoralis and acromiodeltoideus) are less well developed at birth and undergo rapid development just before the onset of flight. By contrast the quadriceps femoris muscle, which is required for postural control, is more developed at birth than the flight muscles and grows more slowly during subsequent development.     

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

Pectoralis muscle morphology in the little brown bat,Myotis lucifugus: A non-convergence with birds

Recent studies of muscle architecture demonstrate that many mammalian muscles are composed of short, interdigitating fibers. In addition, the avian pectoralis, a muscle capable of producing high frequency oscillations has been shown to possess a serially arranged pattern of muscle endplate in all sizes of birds studied. The pectoralis muscle of the little brown bat, Myotis lucifugus (Chiroptera: Vespertilionidae), is composed of fairly uniform fibers that span the length of the muscle and is characterized by a zone of motor endplates within the middle third of the muscle. The homogeneous fiber architecture of the bat pectoralis muscle is in contrast to the serial arrangement of endplates (and presumably muscle muscle fibers) in the avian pectoralis in species equivalent in size to Myotis. The short fiber organization and motor endplate pattern observed in most birds is thus not a requisite design for flying vertebrates. © 1994 Wiley-Liss, Inc.     

Aberrant post‐translational modifications compromise human myosin motor function in old age

Novel experimental methods, including a modified single fiber in vitro motility assay, X‐ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging‐related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force‐generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X‐ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age‐specific myosin post‐translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.     

Fiber type and myosin heavy chain compositions of adult pretarsal orbicularis oculi muscle

Summary Pretarsal orbicularis oculi muscle (POOM) is an important structure of eyelid movement in human. The aim of this study was to investigate fiber histomorphology and myosin heavy chain (MyHC) isoform composition of adult POOM, and to clarify their age-related changes. Eyelid specimens from 58 subjects (age range, 21 to 91 years) were collected during upper blepharoplasty procedures. Serial cross sections of POOM were ATPase-stained and examined under miscroscope. Quantitative measures of muscle fiber size and fiber type distribution were obtained in 35 subjects with adequate fiber cross sections. Relative MyHC isoform contents of POOM were retrieved by gel electrophoresis in all 58 subjects. Examination of the histochemical staining revealed an abundance of type II fiber ( >85%) in human POOM, with more type IIX than IIA fibers. Decreased mean area of all fibers and type IIA fibers were noted in the old group when compared to the young. As for MyHC analysis, the relative content of MyHC isoforms exhibited an order of IIX > IIA > I, and the relative MyHC IIA content showed a negative correlation with age. Comparing with previous studies of limb or masticatory muscles, adult POOM exhibits a unique fiber and MyHC composition, as well as a different aging pattern.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Summary Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.     

Histochemistry of flight muscles in the Jamaican fruit bat, Artibeus jamaicensis: implications for motor control

Two fast-twitch fiber types are histochemically identified in the primary flight muscles of Artibeus jamaicensis. These are classified as type IIa and IIb according to an acid-preincubation staining protocol for myosin ATPase. All fibers in the bat flight muscles exhibit relatively intense staining properties for NADH-TR, suggesting a high oxidative capacity. The glycolytic potential of all fibers is rather low, as assessed by stains for alpha-GPD. This two-type histochemical profile appears to parallel biphasic electromyographic patterns observed in these muscles and leads us to propose that flight muscle histochemistry and activation are mediated by a "two-gear" neuromuscular control system. In contrast, earlier studies on Tadarida brasiliensis demonstrate the existence of a "one-gear" neuromuscular control system, exemplified by the presence of one fiber type. These observations are discussed with respect to the natural history and flight styles of several species.     

NFATc1 and slow-to-fast transition of myosin heavy chain isoforms in gravitational unloading of the rat soleus

An attempt was made to determine whether or not the concentration of NFATc1 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1) in nuclear and cytoplasmic extracts is related to an increase in the concentration of fibers containing type IIa myosin heavy chains under modeled gravitational unloading of m. soleus. Experiments were carried out on Wistar rats using the Morey-Holton tail suspension model. It was found that the soleus contains three isoforms of NFATc1 (140, 110, and 86 kDa). Under unloading, the 140-kDa isoform is translocated into the nucleus, the concentration of the 110-kDa isoform in the cytoplasmic extract decreases, and the concentration of the 86-kDa isoform in the nuclear extract increases. Under gravitational unloading of the muscle, the concentration of fibers containing type IIa myosin heavy chains increases. The increase in the concentration of the 140-and 86-kDa NFATc1 isoforms in the nucleus is accompanied by a decrease in the fraction of muscle fibers containing type I myosin heavy chains and an increase in the fraction containing type IIa chains.     

Muscle Fiber Types in the Pectoralis of the White Pelican,a Soaring Bird

The pectoralis muscle (M. pectoralis) of many premier soaring birds contains a smaller, accessory, deep belly in addition to the much larger superficial belly found in all flying birds. Here we describe the muscle fiber types in both the superficial and deep bellies of the pectoralis of one such adept soaring species, the white pelican (Pelecanus erythrorhynchos).Histochemical techniques are used to demonstrate both nicotinamide adenine dinucleotide (reduced) and myofibrillar adenosine triphosphatase activities within the muscle fibers. Immunocytochemical methods employing several monoclonal antibodies, each directed against a different myosin heavy chain epitope of the chicken, are also used to characterize the fibers. While the superficial belly of the muscle consists entirely of fast-twitch oxidative-glycolytic fibers, the deep belly is composed exclusively of slow fibers. These slow fibers are labelled by two different antibodies specific for chicken slow myosin. We suggest that the fibers of the superficial belly are best suited to flapping flight, and that the fibers of the deep belly would be recruited only during soaring flight. Furthermore, we hypothesize that the deep belly found in the pectoralis of soaring species probably evolved from a deep neuromuscular compartment of the superficial belly.     

Novel myosin isoform in nuclear chain fibers of rat muscle spindles produced in response to endurance swimming.

With the use of myosin adenosinetriphosphatase (ATPase) and immunofluorescence staining methods, the adaptive responses of intrafusal and extrafusal fibers to endurance swimming were studied in frozen sections of rat soleus (SOL) and extensor digitorum longus (EDL) muscles. Glycogen depletion confirmed muscle fatigue at the end of a standardized bout of exercise. No significant age-dependent changes in myosin isoforms were detected in any fibers. The 12-wk training increased type I fibers by 10.9% in the SOL and type IIa fibers in the EDL by 16.6%. In trained muscle sections, both staining methods identified a permuted chain fiber, expressed the same as the myosin isoform in the bag2 fiber. However, no exercise-induced change of myosin isoform profile was found in the bag1 and bag2 fibers. Myosin ATPase (and immunofluorescence) staining showed the percentage of permuted chain fibers increased from 0 to 6.7% (5.6%) after 6 wk of training and to 19.2% (14.1%) after 12 wk of training and that it was still at 6.1% (4.2%) 10 wks after training. A novel myosin isoform may thus be expressed in nuclear chain fibers by repetitive recruitment of muscle spindles.     

Muscular design in the equine interosseus muscle

We studied the forelimb interosseus muscle in horses, Equus caballus, to determine the muscular properties inherent in its function. Some authors have speculated that the equine interosseus contains muscle fibers at birth only to undergo loss of these fibers through postnatal ontogeny. We describe the muscle fibers in eight interosseus specimens from adult horses. These fibers were studied histochemically using myosin ATPase studies and immunocytochemically using several antibodies directed against type I and type II myosin heavy chain antibodies. We determined that 95% of the fibers were type I, presumed slow-twitch fibers. All fibers exhibited normal morphological appearance in terms of fiber diameter and cross-sectional area, suggesting that the muscles are undergoing normal cycles of recruitment. SDS-PAGE studies of myosin heavy chain isoforms were consistent with these observations of primarily slow-twitch muscle. Fibers were determined to be approximately 800 microm long when studied using nitric acid digestion protocols. Short fiber length combined with high pinnation angles suggest that the interosseus muscle is able to generate large amounts of force but can produce little work (measured as pulling the distal tendon proximally). While the equine interosseus muscle has undergone a general reduction of muscle content during its evolution, it remains composed of a significant muscular component that likely contributes to forelimb stability and elastic storage of energy during locomotion.     

Relationship between myosin heavy chain IId isoform and fibre types in soleus muscle of the rat after hindlimb suspension

The relationship between the myosin heavy chain (HC) IId isoform and histochemically defined fibre types was investigated in the rat soleus muscle after hindlimb suspension. After 4 weeks of suspension, right and left muscles were removed and fibre type composition and total fibre number were examined by histochemical myosin adenosine triphosphatase staining sections. Myosin HC isoforms were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. After the suspension, there was a significant decrease in the percentage of type I fibres and a concomitant increase in that of type IIa fibres. However, the total number of fibres was not affected by suspension. The synthesis of HC IId isoform, which was not found in the control, and the decrease in the ratio of slow type myosin heavy chain isoform (HC I) were observed after suspension. These results would may suggest that the change of fibre type composition was caused by a shift from type I to IIa fibres after suspension. Furthermore, it could be suggested that the synthesis of HC IId isoform occurred during the stage of type shift from type I to IIa fibres.     

Quantifying the temporospatial expression of postnatal porcine skeletal myosin heavy chain genes.

Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.     

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.     

大鼠与家兔生后各年龄阶段跖肌纤维的比较

应用建立在肌球蛋白重链异构体基础上的标准肌动球蛋白ATP酶和琥珀酸脱氢酶组织化学方法,分析大鼠和家兔出生后发育各年龄阶段跖肌纤维型分布。在生后2周至24周龄的大鼠和家兔Ⅰ、ⅡX型肌纤维百分比例减少,而ⅡA、ⅡB型纤维则增加。进行大量单肌纤维的组织化学特征的比较和相关性探讨。结果显示动物平均体重与跖肌的平均湿重随生后发育逐渐增加,Ⅰ、ⅡX、ⅡA及ⅡB型纤维均在生后各年龄组的全部肌肉内被发现,但出生后2日龄组是个例外。在生后发育期间,雄性大鼠和家兔ⅡB型纤维的平均肌纤维型构成要大于雌性大鼠和家兔,而雄性大鼠和家兔Ⅰ、ⅡX、ⅡA型三种氧化组织化学分类的肌纤维型构成均小于雌性大鼠和家兔。大鼠Ⅰ、ⅡX、ⅡA和ⅡB型纤维的平均横切面积显然要比家兔的同类型肌纤维要小。在大鼠和家兔可见明显的性别差异。大鼠和家兔的ⅡX型纤维横切面积是最小的,Ⅰ、ⅡA型纤维呈中等大小,ⅡB型纤维最大。该重要的测试有助于我们深入研究啮齿类动物快肌纤维生理特征的适应。