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).     

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).     

Origin of intrafusal muscle fibers in the rat

Summary The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag₂ fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag₁ fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag₂ and bag₁ intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.     

Origin of intrafusal muscle fibers in the rat

The expression of several isoforms of myosin heavy chain (MHC) by intrafusal and extrafusal fibers of the rat soleus muscle at different stages of development was compared by immunocytochemistry. The first intrafusal myotube to form, the bag2 fiber, expressed a slow-twitch MHC isoform identical to that expressed by the primary extrafusal myotubes. The second intrafusal myotube to form, the bag1 fiber, expressed a fast-twitch MHC similar to that initially expressed by the secondary extrafusal myotubes. At subsequent stages of development, the equatorial and juxtaequatorial regions of bag2 and bag1 intrafusal myofibers began to express a slow-tonic myosin isoform not expressed by extrafusal fibers, and ceased to express some of the MHC isoforms present initially. Myotubes which eventually matured into chain fibers expressed initially both the slow-twitch and fast-twitch MHC isoforms similar to some secondary extrafusal myotubes. In contrast, adult chain fibers expressed the fast-twitch MHC isoform only. Hence intrafusal myotubes initially expressed no unique MHCs, but rather expressed MHCs similar to those expressed by extrafusal myotubes at the same chronological stage of muscle development. These observations suggest that both intrafusal and extrafusal fibers develop from common pools of bipotential myotubes. Differences in MHC expression observed between intrafusal and extrafusal fibers of rat muscle might then result from a morphogenetic effect of afferent innervation on intrafusal myotubes.     

Histochemistry of intrafusal muscle fibers outside the spindle capsule.

Regional differences in histochemical properties along the length of rat intrafusal muscle fibers were examined. Outside the muscle spindle capsule the nuclear bagfibers lose their hitherto characteristic ATPase activity and stain in a manner similar to that of extrafusal type I muscle fibers, perhaps in relationship to spindle skeletofusimotor innervation.     

Innervation of developing intrafusal muscle fibers in the rat

The chronology of development of spindle neural elements was examined by electron microscopy in fetal and neonatal rats. The three types of intrafusal muscle fiber of spindles from the soleus muscle acquired sensory and motor innervation in the same sequence as they formed--bag2, bag1, and chain. Both the primary and secondary afferents contacted developing spindles before day 20 of gestation. Sensory endings were present on myoblasts, myotubes, and myofibers in all intrafusal bundles regardless of age. The basic features of the sensory innervation--first-order branching of the parent axon, separation of the primary and secondary sensory regions, and location of both primary and secondary endings beneath the basal lamina of the intrafusal fibers--were all established by the fourth postnatal day. Cross-terminals, sensory terminals shared by more than one intrafusal fiber, were more numerous at all developmental stages than in mature spindles. No afferents to immature spindles were supernumerary, and no sensory axons appeared to retract from terminations on intrafusal fibers. The earliest motor axons contacted spindles on the 20th day of gestation or shortly afterward. More motor axons supplied the immature spindles, and a greater number of axon terminals were visible at immature intrafusal motor endings than in adult spindles; hence, retraction of supernumerary motor axons accompanies maturation of the fusimotor system analogous to that observed during the maturation of the skeletomotor system. Motor endings were observed only on the relatively mature myofibers; intrafusal myoblasts and myotubes lacked motor innervation in all age groups. This independence of the early stages of intrafusal fiber assembly from motor innervation may reflect a special inherent myogenic potential of intrafusal myotubes or may stem from the innervation of spindles by sensory axons.     

Distribution of SERCA isoforms in human intrafusal fibers

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a membrane protein that plays a crucial role in muscle relaxation by transporting cytosolic Ca²⁺ into the lumen of the sarco/endoplasmic reticulum. In this study, the presence of SERCA1 and SERCA2 was investigated in human intrafusal fibers by immunocytochemistry. Nuclear bag₁ fibers contained both SERCA1 and SERCA2 isoforms, with predominant staining seen with SERCA2 in the A and B regions. Most nuclear bag₂ fibers also contained SERCA1 and SERCA2 isoforms and their coexistence frequently occurred in the A region. SERCA1 was present whereas SERCA2 was generally absent in the nuclear chain fibers. The staining intensity seen with the SERCA1 monoclonal antibody varied in the order of chain>bag₁>bag₂. The expression of SERCA1 isoform was found to correlate with the presence of fast myosin heavy chain (MyHC) isoform in nuclear chain fibers, whereas for nuclear bag fibers there was no such apparent correlation between patterns of expression of SERCA and MyHC isoforms. The phenotype revealed for the human bag fibers was very sophisticated and adapted to attain a very wide range of contraction and relaxation velocities.     

Expression of type-specific MHC isoforms in rat intrafusal muscle fibers.

Myosin heavy chain (MHC) expression by intrafusal fibers was studied by immunocytochemistry to determine how closely it parallels MHC expression by extrafusal fibers in the soleus and tibialis anterior muscles of the rat. Among the MHC isoforms expressed in extrafusal fibers, only the slow-twitch MHC of Type 1 extrafusal fibers was expressed along much of the fibers. Monoclonal antibodies (MAb) specific for this MHC bound to the entire length of bag2 fibers and the extracapsular region of bag1 fibers. The fast-twitch MHC isoform strongly expressed by bag2 and chain fibers had an epitope not recognized by MAb to the MHC isoforms characteristic of developing muscle fibers or the three subtypes (2A, 2B, 2X) of Type 2 extrafusal fibers. Therefore, intrafusal fibers may express a fast-twitch MHC that is not expressed by extrafusal fibers. Unlike extrafusal fibers, all three intrafusal fiber types bound MAb generated against mammalian heart and chicken limb muscles. The similarity of the fast-twitch MHC of bag2 and chain fibers and the slow-tonic MHC of bag1 and bag2 fibers to the MHC isoforms expressed in avian extrafusal fibers suggests that phylogenetically primitive MHCs might persist in intrafusal fibers. Data are discussed relative to the origin and regional regulation of MHC isoforms in intrafusal and extrafusal fibers of rat hindlimb muscles.     

Pax7 shows higher satellite cell frequencies and concentrations within intrafusal fibers of muscle spindles

Intrafusal fibers within muscle spindles make up a small subpopulation of muscle fibers. These proprioceptive fibers differ from most extrafusal fibers because, even in maturity, their diameters remain small, and they retain expression of developmental myosins. Although both extrafusal and intrafusal fibers contain satellite cells (SCs), comparatively little is known about intrafusal SCs. Analyzing chicken fast-phasic posterior (PLD) and slow-tonic anterior (ALD) latissimus dorsi muscles, we show that SCs of both intrafusal and extrafusal fibers express Pax7. We further test the hypotheses that intrafusal fibers display parameters reflective of extrafusal immaturity. These hypotheses are that intrafusal fibers contain (a) higher SC frequencies (number of SC nuclei/all nuclei within basal lamina) and concentrations (closer together) and (b) smaller myonuclear domains than do adjacent extrafusal fibers. IHC techniques were applied to PLD and ALD muscles excised at 30 and 138 days posthatch. The hypotheses were validated, suggesting that intrafusal fibers have greater capacities for growth, regeneration, and repair than do adjacent extrafusal fibers. During maturation, extrafusal and intrafusal fibers show similar trends of decreasing SC frequencies and concentrations and increases in myonuclear domains. Thus, extrafusal and intrafusal fibers alike should exhibit reduced capacities for growth, regeneration, and repair during maturation.     

Histochemistry of rat intrafusal muscle fibers and their motor innervation.

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bag1 and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intra- and extracapsular regions were compared. Simple "diffuse" and more elaborate "plate" motor nerve terminals were demonstrated histochemically along the poles of bag1 and bag2 fibers by staining for cholinesterase. One motor terminal of the "plate" appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.     

Myosin light chain patterns in histochemically typed single fibers of the rat skeletal muscle.

1. This study was conducted to investigate the relationship between histochemical fiber types and myosin light chain patterns in rat single muscle fibers. 2. The hybrid of fast and slow light chains was observed in type I and II fibers of the soleus and type II fibers of the red portion of lateral head of the gastrocnemius muscles. 3. We also observed 7 types of light chain composition. Of the 7 types, 5 types were explainable by assuming the coexistence of isomyosins with either fast or slow light chains. However, the other 2 types could not be accounted for without hypothesizing the presence of isomyosins with promiscuous light chain distribution.     

Quantitative studies on the regeneration of myelinated nerve fibers. I. Variations in the number and the size of myelinated fibers in the nerves of normal rats]

The number and size of myelinated nerve fibers have been determined at standard levels in the nerve to medial head of right and left gastrocnemius muscles of 24 normal rats (11 males and 13 females). The mean values of all results were comparable on right and left sides. Thus, 271 +/- 5 myelinated nerve fibers were found in the right nerve and 272 +/- 4 in the left; their mean diameter were respectively 8.1 +/- 0.1 and 8.0 +/- 0.1 micron. There were 60.1% of large nerve fibers on the right side and 59,9% on the left, their mean diameters being 10.5 and 10.6 micron. Some variations occured in all these values, depending of the weight and sex of the animals. Nevertheless, the differences between both sides of a same rat were negligible and the histograms of both nerves could be superposed. Accordingly, in the operated animals, the contralateral nerve may be used as control.     

The topography of long nuclear chain intrafusal fibers in the cat muscle spindle

Summary Muscle spindles were studied histochemically in serial transverse sections of specimens of the cat tenuissimus muscle. The nuclear chain intrafusal muscles fibers were separated into three subtypes, called long, intermediate and typical. The long chain and intermediate chain fibers tended to assume a particular position within the axial bundle of intrafusal fibers. The fibers were usually located in that layer of chain fibers that was positioned farthest away from the bag₂ fiber. Furthermore, they were usually situated adjacent to the bag₁ fiber throughout much of the extent of the spindle pole. Some long chain and intermediate chain fibers had several fiber nuclei abreast at the equator rather than a single row of central nuclei, as in most nuclear chain fibers. The relative position of intrafusal fibers within the cat spindle may reflect their order of formation during development, with the fibers retaining, to a variable degree, their association with the bag₂ fiber which acted as template. Thus, the axial position of long chain and intermediate chain fibers suggests that they are among the first nuclear chain fibers to form. This may play a role in the known preferential innervation of these chain fibers by skeleto-fusimotor axons.