Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations. Nuclear bag1 fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag2 fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with anti-neonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag1 fibers lacked staining for M-protein, whereas bag2 fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag1 fibers were less reactive and clear striations were not observed, in contrast to bag2 and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested. Taken together, the data show that in adult rat soleus, slow tonic and neonatal myosin heavy chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type IIA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle

Summary This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type HA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.     

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.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag1 fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag2 fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag1 fibers.     

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.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag1, bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag1 and bag2 fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag1, bag2 and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

Effect of growth on muscle capillarity and fiber type composition in rat diaphragm

The effect of growth on the capillarity and fiber type composition of the diaphragm, soleus and extensor digitorum longus (EDL) muscles of rats weighing between 55 and 330 g have been studied. Muscle samples obtained from the anesthetized rat were rapidly frozen and sliced transversely in a cryostat. The sections were stained histochemically by the SDH method and the myosin ATPase method after preincubation at pH 4.3 to typify fibers (FG, FOG and SO fibers). To visualize capillaries, the myosin ATPase method after preincubation at pH 4.0 was used. The percentage of FOG fibers decreased in all muscles with growth. While the FG and SO fibers increased in the diaphragm, SO fibers increased in the soleus, and FG fibers increased in the EDL. The capillary density showed a hyperbolic decrease with growth in all muscles, while the number of capillaries around each fiber increased in all muscles with growth. It is concluded that growth causes the changing properties of the motoneurons and the new capillary formation in the diaphragm muscle, as well as the soleus and EDL muscles.     

Immunohistochemical demonstration of embryonic myosin heavy chains in adult mammalian intrafusal fibers

Summary Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag₁ fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag₂ fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag₁ fibers.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in rat.

The histochemical ATPase activity and the myosin light chains of a rat fast muscle (extensor digitorum longus, EDL) and a rat slow muscle (soleus) during development have been investigated. Both muscles initially synthesize fast myosin light chains and show the intense histochemical ATPase activity characteristic of adult fast muscle fibers. After birth, the soleus begins to accumulate slow fibers with their characteristic low histochemical ATPase activity, and slow myosin light chains begin to appear. Sciatic neurectomy prevents the development of slow fibers and the synthesis of slow myosin light chains in the soleus, while the EDL is unaffected. Similarly, cordotomy of an adult rat results, in the soleus, in the appearance of fibers with more intense staining for ATPase and an increase in fast myosin light chains. The EDL is unchanged by cordotomy. As a result, we suggest that slow muscle development, but not fast muscle development, is dependent upon the functional activity of the nervous system.     

Aggregation of myonuclei and the spread of slow-tonic myosin immunoreactivity in developing muscle spindles.

The pattern of regional expression of a slow-tonic myosin heavy chain (MHC) isoform was studied in developing rat soleus intrafusal muscle fibers. Binding of the slow-tonic antibody (ATO) began at the equator of prenatal intrafusal fibers where sensory nerve endings are located, and spread into the polar regions of nuclear bag2 and bag1 fibers but not nuclear chain fibers during ontogeny. The onset of the ATO reactivity coincided with the appearance of equatorial clusters of myonuclei (nuclear bag formations) in bag1 and bag2 fibers. Moreover, the intensity of the ATO reaction was strongest in the region of equatorial myonuclei and decreased with increasing distance from the equator of bag1 and bag2 fibers at all stages of prenatal and postnatal development. The polar expansion of ATO reactivity continued throughout the postnatal development of bag1 fibers, but ceased shortly after birth in bag2 fiber coincident with innervation by motor axons. Thus, afferents that innervate the equator might induce the slow-tonic MHC isoform in bag2 and bag1 fibers by regulating the myosin gene expression by equatorial myonuclei, and efferents or twitch contractile activity might inhibit the spread of the slow-tonic MHC isoform into the poles of bag2 but not bag1 fibers. Absence of ATO binding in chain fibers suggests that chain myotubes may not be as susceptible to the effect of afferents as are myotubes that develop into bag2 and bag1 fibers. The different patterns of slow-tonic MHC expression in the three types of intrafusal fiber may therefore result from the interaction of three elements: sensory neurons, motor neurons, and intrafusal myotubes.     

Anti-myosin heavy chain monoclonal antibodies reveal two IIB (fast) fiber subtypes

Indirect immunofluorescence analysis of different rat skeletal muscles using anti-myosin heavy chain (MHC) monoclonal antibodies (MAb) revealed the presence of two immunologically distinct kinds of fibers within the IIB fibers, histochemically identified by myosin ATPase staining. Some IIB fibers (designated here as IIB1) were unreactive with one anti-fast MHC MAb, whereas they did react with another anti-fast MHC MAb; other IIB fibers (designated here as IIB2) reacted with both anti-fast MAbs. Neither of the two IIB fiber subtypes was significantly reactive with a neonatal MHC MAb. The number of each IIB fiber subtype was age-dependent, at least in the plantaris muscle. IIB1 fibers were observed only in the superficial portion of the plantaris and gastrocnemius muscle. The ratio of IIB1:IIB2 fibers was about the same throughout the extensor digitorum longus and extraocular muscles. Therefore, the two kinds of IIB fibers here observed have a different myosin heavy chain content. On the basis of their specific immunoreactivities, we suggest that IIB1 fibers contain the previously described MHCB. IIB2 fibers contain either a unique new MHC isoform or a mixture of at least two MHC, possibly composed of the MHCB and either the previously described MHCA or a new MHC isoform.     

The occurrence of "mixed" nuclear bag intrafusal fibers in the cat muscle spindle

A total of 147 muscle spindles was studied histochemically in serial transverse sections of 42 cat tenuissimus muscle specimens. Nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers were distinguished by the differential staining resulting from the reactions for myosin adenosine 5'-triphosphatase and nicotinamide adenine dinucleotide tetrazolium reductase. The majority of intrafusal fibers were of the same histochemical type at both fiber poles. However, seven muscle spindles contained one nuclear bag fiber each that presented as a bag1 in one pole and as a bag2 in the other pole. These "mixed" nuclear bag fibers were found in spindles that also contained at least one bag1 and one bag2 fiber of equivalent histochemical presentation in both fiber poles. The "mixed" bag fibers displayed differences of apparent fiber diameter and relative polar length between the two fiber poles. The motor innervation pattern, as revealed by staining for cholinesterase, was also dissimilar between the two poles of "mixed" bag fibers. The study indicates that the spindle equatorial region may in some instances serve as a boundary between two morphologically and histochemically different poles of the same intrafusal fiber.     

A comparative study of muscle spindles in slow and fast neonatal muscles of normal and dystrophic mice

Muscle spindles from the slow-twitch soleus and the fast-twitch extensor digitorum longus (EDL) muscles of genetically dystrophic mice of the dy2J/dy2J strain were compared with age-matched normal animals at neonatal ages of 1-3 weeks according to histochemical, quantitative, and ultrastructural parameters. Intrafusal fibers in both the soleus and EDL exhibited similar regional differences in myosin ATPase activity, and conformed to those noted previously in various adult species. In distal polar regions, all nuclear bag fibers resembled extrafusal fibers of the type 1 variety, whereas in capsular zones they could be divided into two subtypes. Nuclear chain fibers possessed a staining pattern similar to type 2 extrafusal fibers, and in contrast to the bag fibers they exhibited no regional variations. These features were consistently observed in both the normal and dystrophic muscles at all ages. Spindles varied only slightly in their number and distribution in the two types of muscle, and their location followed the neurovascular branching pattern in each. Irrespective of age or genotype, spindles in the soleus were more homogeneously dispersed, but those in the EDL were concentrated along the dorsal aspect of the muscle. No significant differences were noted in the total number of spindles between normal and dystrophic muscles. In addition, no dramatic differences were observed in the muscle spindle index for soleus and EDL. The first obvious disease-related changes were noted in extrafusal fibers of the soleus of 3-week-old mice, and spindles were often located close to these areas of fiber degeneration. Despite alterations in the surrounding tissue, however, spindles appeared morphologically unaltered in dystrophy. These observations indicate that intrafusal fibers of spindles in neonatal mice appear enzymatically and histologically unaffected in incipient stages of progressive muscular dystrophy.     

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.     

Diversity in expression of myosin heavy chain isoforms and M-band proteins in rat muscle spindles

Summary The composition of adult rat soleus muscle spindles, with respect to myosin heavy chain isoforms and M-band proteins, was studied by light-microscope immunohistochemistry. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal myosin isoforms as well as against myomesin, M-protein and the MM form of creatine kinase. Intrafusal fiber types were distinguished according to the pattern of ATPase activity following acid and alkaline preincubations.Nuclear bag₁ fibers were always strongly stained throughout with anti-slow tonic myosin, were positive for anti-slow twitch myosin towards and in the C-region but were unstained with anti-fast twitch and anti-neonatal myosins. The staining of nuclear bag₂ fibers was in general highly variable. However, they were most often strongly stained by anti-slow tonic myosin in the A-region and gradually lost this reactivity towards the poles, whereas a positive reaction with anti-slow twitch myosins was found along the whole fiber. Regional staining variability with antineonatal and anti-fast myosins was apparent, often with decreasing intensity towards the polar regions. Nuclear chain fibers showed strong transient reactivity with anti-slow tonic myosin in the equatorial region, did not react with anti-slow twitch and were always evenly stained by anti-fast twitch and anti-neonatal myosins. All three intrafusal fiber types were stained with anti-myomesin. Nuclear bag₁ fibers lacked staining for M-protein, whereas bag₂ fibers displayed intermediate staining, with regional variability, often increasing in reactivity towards the polar regions. Chain fibers were always strongly stained by anti-M-protein. The MM form of creatine kinase was present in all three fiber types, but bag₁ fibers were less reactive and clear striations were not observed, in contrast to bag₂ and chain fibers. Out of 38 cross sectioned spindles two were found to have an atypical fiber composition, (lack of chain fibers) and a rather diverse staining pattern for the different antibodies tested.Taken together, the data show that in adult rat solcus, slow tonic and neonatal myosin heavy, chain isoforms are only expressed in the muscle spindle fibers and that each intrafusal fiber type has a unique, although variable, composition of myosin heavy chain isoforms and M-band proteins. We propose that both motor and sensory innervation might be the determining factors regulating the variable expression of myosin heavy chain isoforms and M-band proteins in intrafusal fibers of rat muscle spindles.     

Variation and limitations in fiber enzymatic and size responses in hypertrophied muscle.

The present study was designed to determine whether the degree and kind of adaptation of a muscle fiber to a functional overload (FO) are determined by properties that are intrinsic to that fiber. The study also addresses the question of the capability of fibers to maintain a normal level of coordination of proteins per fiber as fiber volume changes dramatically. The plantaris muscle of six adult female cats was overloaded for 12 wk by bilateral synergist removal. Plantaris muscle fiber mean size doubled after FO, although some very small fibers that stained dark for adenosinetriphosphatase (ATPase) were observed in some of the FO muscles. There appeared to be no change in total succinate dehydrogenase activity per fiber. A reduction in succinate dehydrogenase activity per unit volume was observed in a substantial number of fibers, reflecting a disproportionate increase in fiber volume relative to mitochondrial volume. In contrast, total alpha-glycerophosphate dehydrogenase activity and actomyosin ATPase activity increased as fiber size increased, whereas there was no change in alpha-glycerophosphate dehydrogenase and ATPase activities per unit volume. Control and FO muscle fibers generally expressed either a fast or slow myosin heavy chain type, but in some cases FO muscle fibers expressed both fast and slow myosin heavy chains. The persistence of variability in fiber sizes and enzyme activities in fibers of overloaded muscles suggests a wide range in the adaptive potential of individual fibers to FO. These data indicate that a severalfold increase in cell size may occur without significant qualitative changes in the coordination of protein regulation associated with metabolic pathways and ATP utilization.     

Progressive resistance training reduces myosin heavy chain coexpression in single muscle fibers from older men.

The purpose of this study was to examine myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following 12 wk of progressive resistance training (PRT). A needle biopsy was taken from the vastus lateralis to determine fiber-type expression [ATPase (pH 4.54) and MHC/MLC] in seven healthy men (age = 74.0 +/- 1.8 yr). Subjects were also tested for 1-repetition maximum (1-RM), pre- and posttraining. The progressive knee extensor protocol consisted of three sets at 80% of 1-RM 3 days/wk for 12 wk. Freeze-dried, single muscle fibers were dissected for MHC and MLC analysis and then subjected to SDS-PAGE and silver staining, pre- and posttraining. MHC expression increased in the I (10.4%; P < 0.05) and decreased in I/IIa (9.0%; P < 0.05), I/IIa/x (0.9%; P < 0.05), and IIa/x (8.9%; P < 0.05) isoforms, with no change in the IIa and IIx isoforms, pre- vs. posttraining (total fibers = 3,059). The MLC(3f)-to-MLC(2) ratio did not change with the PRT in either the MHC I or MHC IIa isoforms (total fibers = 902), pre- to posttraining. ATPase fiber distribution did not significantly differ following training (I: 50. 4 +/- 6.7 vs. 51.9 +/- 7.9, IIa: 36.8 +/- 5.3 vs. 41.1 +/- 7.0, IIb: 12.8 +/- 5.6 vs. 7.0 +/- 4.0%; pre- vs. posttraining, respectively). 1-RM increased (51.9%; P < 0.05) from pre- to posttraining. The PRT provide a stimulus for alterations in MHC isoforms, which demonstrated a decrease in all hybrid isoforms and an increase in MHC I expression (not found in the ATPase results), unlike the MLC ratio (3:2), which was not altered with training.     

Metabolic and morphologic properties of single muscle fibers in the rat after spaceflight, Cosmos 1887

The adaptation of a slow (soleus, Sol) and a fast (medial gastrocnemius, MG) skeletal muscle to spaceflight was studied in five young male rats. The flight period was 12.5 days and the rats were killed approximately 48 h after returning to 1 g. Five other rats that were housed in cages similar to those used by the flight rats were maintained at 1 g for the same period of time to serve as ground-based controls. Fibers were classified as dark or light staining for myosin adenosine triphosphatase (ATPase). On the average, the fibers in the Sol of the flight rats atrophied twice as much as those in the MG. Further, the fibers located in the deep (close to the bone and having the highest percentage of light ATPase and high oxidative fibers in the muscle cross section) region of the MG atrophied more than the fibers located in the superficial (away from the bone and having the lowest percentage of light ATPase and high oxidative fibers in the muscle cross-section) region of the muscle. Based on quantitative histochemical assays of single muscle fibers, succinate dehydrogenase (SDH) activity per unit volume was unchanged in fibers of the Sol and MG. However, in the Sol, but not the MG, the total amount of SDH activity in a 10-microns-thick section of a fiber decreased significantly in response to spaceflight. Based on population distributions, it appears that the alpha-glycerophosphate dehydrogenase (GPD) activities were elevated in the dark ATPase fibers in the Sol, whereas the light fibers in the Sol and both fiber types in the MG did not appear to change. The ratio of GPD to SDH activities increased in the dark (but not light) fibers of the Sol and was unaffected in the MG. Immunohistochemical analyses indicate that approximately 40% of the fibers in the Sol of flight rats expressed a fast myosin heavy chain compared with 22% in control rats. Further, 31% of the fibers in the Sol of flight rats expressed both fast and slow myosin heavy chains compared with 8% in control rats. Immunohistochemical changes in the MG were minimal. These data suggest that the magnitude and direction of enzymatic activity and cell volume changes are dependent on the muscle, the region of the muscle, and the type of myosin expressed in the fibers. Further, the ability of fibers to maintain normal or even elevated activities per unit volume of some metabolic enzymes is remarkable considering the marked and rapid decrease in fiber volume.