Myonuclear domain size varies along the lengths of maturing skeletal muscle fibers

In a skeletal muscle fiber, each myonucleus is responsible for gene expression in its surrounding cytoplasm. The region of cytoplasm associated with an individual myonucleus is termed myonuclear domain. However, little is known about domain size variation within individual muscle fibers. This study tests the hypothesis that myonuclear domains expressing neonatal myosin within end regions of maturing fibers will be smaller than domains elsewhere in the fibers. The model used is chicken pectoralis, where we have previously shown that during development repression of neonatal myosin radiates from the central region towards the fiber ends. Samples excised from birds aged nine through to 115 days after hatching were sectioned transversely. Using computer image analysis and immunocytochemistry, fiber profiles were classified as neonatal, transforming or adult. Each profile was also located in an adjacent dystrophin-labelled section, where myonuclei were visualized using haematoxylin and bisbenzamide. Variation in myonuclear length with age was not found to be significant (p = 0.925). Myonuclei were counted, and formulae used to calculate mean myonuclear domain size for each profile type. Myonuclear number/mm fiber was calculated to be adult (mean = 108.57 myonuclei/mm), transforming (65.82) and neonatal (25.23). Transforming profiles had significantly (p=0.027) more myonuclei/mm than neonatal, as did adult (p=0.005). Volume of cytoplasm/myonucleus was adult (mean = 16,132 microm3/myonucleus), transforming (12,899) and neonatal (8,130). Transforming and adult profiles had significantly (p<0.001) larger myonuclear domains than did neonatal profiles. Transforming and adult profiles did not differ in either myonuclei/mm (p=0.302) or volume of cytoplasm/myonucleus (p=0.413). This study demonstrates smaller domains at the terminal tips of maturing muscle fibers.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.     

Is functional hypertrophy and specific force coupled with the addition of myonuclei at the single muscle fiber level?

Muscle force is typically proportional to muscle size, resulting in constant force normalized to muscle fiber cross-sectional area (specific force). Mice overexpressing insulin-like growth factor-1 (IGF-1) exhibit a proportional gain in muscle force and size, but not the myostatin-deficient mice. In an attempt to explore the role of the cytoplasmic volume supported by individual myonuclei [myonuclear domain (MND) size] on functional capacity of skeletal muscle, we have investigated specific force in relation to MND and the content of the molecular motor protein, myosin, at the single muscle fiber level from myostatin-knockout (Mstn(-/-)) and IGF-1-overexpressing (mIgf1(+/+)) mice. We hypothesize that the addition of extra myonuclei is a prerequisite for maintenance of specific force during muscle hypertrophy. A novel algorithm was used to measure individual MNDs in 3 dimensions along the length of single muscle fibers from the fast-twitch extensor digitorum longus and the slow-twitch soleus muscle. A significant effect of the size of individual MNDs in hypertrophic muscle fibers on both specific force and myosin content was observed. This effect was muscle cell type specific and suggested there is a critical volume individual myonuclei can support efficiently. The large MNDs found in fast muscles of Mstn(-/-) mice were correlated with the decrement in specific force and myosin content in Mstn(-/-) muscles. Thus, myostatin inhibition may not be able to maintain the appropriate MND for optimal function.     

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (～51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.     

Effects of aging and gender on the spatial organization of nuclei in single human skeletal muscle cells

The skeletal muscle fibre is a syncitium where each myonucleus regulates the gene products in a finite volume of the cytoplasm, i.e., the myonuclear domain (MND). We analysed aging‐ and gender‐related effects on myonuclei organization and the MND size in single muscle fibres from six young (21–31 years) and nine old men (72–96 years), and from six young (24–32 years) and nine old women (65–96 years), using a novel image analysis algorithm applied to confocal images. Muscle fibres were classified according to myosin heavy chain (MyHC) isoform expression. Our image analysis algorithm was effective in determining the spatial organization of myonuclei and the distribution of individual MNDs along the single fibre segments. Significant linear relations were observed between MND size and fibre size, irrespective age, gender and MyHC isoform expression. The spatial organization of individual myonuclei, calculated as the distribution of nearest neighbour distances in 3D, and MND size were affected in old age, but changes were dependent on MyHC isoform expression. In type I muscle fibres, average NN‐values were lower and showed an increased variability in old age, reflecting an aggregation of myonuclei in old age. Average MND size did not change in old age, but there was an increased MND size variability. In type IIa fibres, average NN‐values and MND sizes were lower in old age, reflecting the smaller size of these muscle fibres in old age. It is suggested that these changes have a significant impact on protein synthesis and degradation during the aging process.     

Immunohistochemical studies on regulation of alternative splicing of fast skeletal muscle troponin T: non-uniform distribution of the exon x3 epitope in a single muscle fiber

Troponin T (TnT) isoforms of chicken fast skeletal muscle are classified into two types, breast-muscle-type (B-type) and leg-muscle-type (L-type) isoforms. These isoforms are produced from a single gene by differential alternative splicing of pre-mRNA. We investigated immunohistochemically the distribution of B-type TnT isoforms in chicken leg muscle (musculus biceps femoris), using anti-exon x3 that was raised against a synthetic peptide corresponding to exon x3 and recognized B-type, but not the L-type, TnT isoforms. Mosaic patterns of immunostaining showing locally different expression of B-type TnT isoforms in a single fiber were observed among fibers, and the non-uniform distribution of the isoforms was also detected in sectioned fibers and myofibrils from the muscle. The results indicated that regulation of pre-mRNA splicing of fast skeletal muscle TnT was different not only among the muscle fibers but also within a single fiber, suggesting that heterogeneous myonuclei in regulation of alternative splicings occur in a single muscle fiber.     

Distribution and innervation of short, interdigitated muscle fibers in parallel-fibered muscles of the cat hindlimb

The cat hindlimb contains several long, biarticular strap muscles composed of parallel muscle fascicles that attach to short tendons. Three of these muscles--sartorius, tenuissimus, and semitendinosus--were studied by dissecting individual gold-stained fibers and determining the surface distribution of acetylcholinesterase-stained end-plate zones. In each muscle, fascicles were composed of muscle fibers that ran only part of the fascicle length and tapered to end as fine strands that interdigitated with other tapering fibers within the muscle mass. Most muscle fibers measured 2-3 cm in length. Fascicles of muscle fibers were crossed by short transverse bands of endplates (1 mm wide by 1-5 mm long) that were spaced at fairly regular intervals from the origin to the insertion of the muscle. The endplate pattern suggested that the fiber fascicles were organized into multiple longitudinal strips. In the sartorius, the temporospatial distribution of electromyographic (EMG) activity evoked by stimulating fine, longitudinal branches of the parent nerve confirmed that each strip was selectively innervated by a small subset of the motor axons. These axons appeared to distribute their endings throughout the entire length of the fascicles, providing for synchronous activation of their in-series fibers.     

No change in myonuclear number during muscle unloading and reloading

Muscle fibers are the cells in the body with the largest volume, and they have multiple nuclei serving different domains of cytoplasm. A large body of previous literature has suggested that atrophy induced by hindlimb suspension leads to a loss of "excessive" myonuclei by apoptosis. We demonstrate here that atrophy induced by hindlimb suspension does not lead to loss of myonuclei despite a strong increase in apoptotic activity of other types of nuclei within the muscle tissue. Thus hindlimb suspension turns out to be similar to other atrophy models such as denervation, nerve impulse block, and antagonist ablation. We discuss how the different outcome of various studies can be attributed to difficulties in separating myonuclei from other nuclei, and to systematic differences in passive properties between normal and unloaded muscles. During reload, after hindlimb suspension, a radial regrowth is observed, which has been believed to be accompanied by recruitment of new myonuclei from satellite cells. The lack of nuclear loss during unloading, however, puts these findings into question. We observed that reload led to an increase in cross sectional area of 59%, and fiber size was completely restored to the presuspension levels. Despite this notable growth there was no increase in the number of myonuclei. Thus radial regrowth seems to differ from de novo hypertrophy in that nuclei are only added during the latter. We speculate that the number of myonuclei might reflect the largest size the muscle fibers have had in its previous history.     

Role of gravitational loading in the development of rat soleus muscle fibers

Effects of hindlimb unloading during the first 3 months after birth on the development of soleus muscle fibers were studied in rats. The mean absolute weigh and cross-sectional area of whole soleus muscle in the unloaded rats were -1/3 and 1/4 of those in the controls, respectively. But the unloading did not affect the lengths of muscle, at 90 degrees of ankle joint angle, and of muscle fibers sampled from tendon to tendon, and the total sarcomere number. Since the total number of fibers in soleus was not affected either, the inhibited increase of muscle mass following unloading was mainly due to the smaller CSA of individual fibers. Numbers of both myonuclei and satellite cells were significantly less in unloaded than control rats. The % distribution of fibers expressing pure type I myosin heavy chain was significantly less in unloaded than controls (-23 %). Further, muscle fibers with multiple innervation were noted in the unloaded rats. It is suggested that the development and/or differentiation of soleus muscle fibers are closely associated with gravitational loading and that the growth-associated increase in fiber number may be genetically programmed.     

Cytomorphometry of skeletal muscle: The influence of age and testosterone on the rat M. levator ani

Summary The levator ani muscle of the rat was examined by correlated light and electron microscopic morphometry. Corrections were made for shrinkage, compression, and differences in stretching. Age, castration, and subsequent testosterone treatment do not affect the fiber number, the filament lattice, and the size of the filaments and myonuclei. The fibers in intact growing males increase in width and length. The number of myonuclei rises, although relatively slower than the amount of contractile material.Castration, performed at six weeks, partially suppresses fiber growth. The increase of mean fiber width is more strongly inhibited than that of fiber length. Myonuclear multiplication is almost completely arrested in castrates, and the amount of contractile material per myonucleus is lower than in intact males of equal age.Testosterone, administered at about two months following orchidectomy, highly accelerates the transversal fiber growth, but fiber length is not significantly influenced. Between the fourth and seventh day of treatment a marked increase in myonuclear number occurs.Analysis of the frequency distribution of the individual fiber widths, which is logarithmic-normal in intact males, revealed that the hormonal influence on the net result of protein anabolism and catabolism markedly differs in the various fibers of a single muscle.With the technical assistance of Tineke J. Hoogenboezem.     

Dynamic Transition States of ErbB1 Phosphorylation Predicted by Spatial Stochastic Modeling

ErbB1 overexpression is strongly linked to carcinogenesis, motivating better understanding of erbB1 dimerization and activation. Recent single-particle-tracking data have provided improved measures of dimer lifetimes and strong evidence that transient receptor coconfinement promotes repeated interactions between erbB1 monomers. Here, spatial stochastic simulations explore the potential impact of these parameters on erbB1 phosphorylation kinetics. This rule-based mathematical model incorporates structural evidence for conformational flux of the erbB1 extracellular domains, as well as asymmetrical orientation of erbB1 cytoplasmic kinase domains during dimerization. The asymmetric dimer model considers the theoretical consequences of restricted transactivation of erbB1 receptors within a dimer, where the N-lobe of one monomer docks with the C-lobe of the second monomer and triggers its catalytic activity. The dynamic nature of the erbB1 phosphorylation state is shown by monitoring activation states of individual monomers as they diffuse, bind, and rebind after ligand addition. The model reveals the complex interplay between interacting liganded and nonliganded species and the influence of their distribution and abundance within features of the membrane landscape.     

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.     

A novel mode of polymerization of alpha1-proteinase inhibitor

Patients homozygous for the Z mutant form of alpha1-proteinase inhibitor (alpha1-PI) have an increased risk for the development of liver disease because of the accumulation in hepatocytes of inclusion bodies containing linear polymers of mutant alpha1-PI. The most widely accepted model of polymerization proposes that a linear, head-to-tail polymer forms by sequential insertion of the reactive center loop (RCL) of one alpha1-PI monomer between the central strands of the A beta-sheet of an adjacent monomer. This model derives primarily from two observations: peptides that are homologous with the RCL insert into the A beta-sheet of alpha1-PI monomer and this insertion prevents alpha1-PI polymerization. Normal alpha1-PI monomer does not spontaneously polymerize; however, here we show that the disulfide-linked dimer of normal alpha1-PI spontaneously forms linear polymers in buffer. The monomers within this dimer are joined head-to-head. Thus, the arrangement of monomers in these polymers must be different from that predicted by the loop-A sheet model. Therefore, we propose a new model for alpha1-PI polymer. In addition, polymerization of disulfide-linked dimer is not inhibited by the presence of the peptide even though dimer appears to interact with the peptide. Thus, RCL insertion into A beta-sheets may not occur during polymerization of this dimer.     

Effects of inactivity on fiber size and myonuclear number in rat soleus muscle.

The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size, and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a midthoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared with control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus atrophy occurred earlier than the shift in myosin heavy chain (MHC) profile. The number of myonuclei was approximately 30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei per millimeter in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus myonuclei were eliminated from fibers containing only type I MHC. Because the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.     

Distribution of myogenic progenitor cells and myonuclei is altered in women with vs. those without chronically painful trapezius muscle

It is hypothesized that repeated recruitment of low-threshold motor units is an underlying cause of chronic pain in trapezius myalgia. This study investigated the distribution of satellite cells (SCs), myonuclei, and macrophages in muscle biopsies from the trapezius muscle of 42 women performing repetitive manual work, diagnosed with trapezius myalgia (MYA; 44 ± 8 yr; mean ± SD) and 20 matched healthy controls (CON; 45 ± 9 yr). Our hypothesis was that muscle of MYA, in particular type I fibers, would demonstrate higher numbers of SCs, myonuclei, and macrophages compared with CON. SCs were identified on muscle cross sections by combined immunohistochemical staining for Pax7, type I myosin, and laminin, allowing the number of SCs associated with type I and II fibers to be determined. We observed a pattern of SC distribution in MYA previously only reported for individuals above 70 yr of age. Compared with CON, MYA demonstrated 19% more SCs per fiber associated with type I fibers (MYA 0.098 ± 0.039 vs. CON 0.079 ± 0.031; P < 0.05) and 40% fewer SCs associated with type II fibers (MYA 0.047 ± 0.017 vs. CON 0.066 ± 0.035; P < 0.05). The finding of similar numbers of macrophages between the two groups was not in line with our hypothesis and suggests that the elevated SC content of MYA was not due to heightened inflammatory cell contents, but rather to provide new myonuclei. The findings of greater numbers of SCs in type I fibers of muscle subjected to repeated low-intensity work support our hypothesis and provide new insight into stimuli capable of regulating SC content.     

Organelle positioning in muscles requires cooperation between two KASH proteins and microtubules

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(ΔKASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.     

Muscle tissue regeneration of the lymph hearts in adult Rana temporaria frogs. A study by light and electron microscopy autoradiography methods

The regeneration response of adult frog lymph heart muscle tissue was studied from 2 to 3 weeks after mechanical injury. High resolution autoradiographic studies showed that regenerative necrotic zones have many actively proliferating mononuclear cells deprived of cytoplasmic myofilaments. Some of them have numerous free ribosomes, so they might be identified as myoblasts. On the 13th day after injury newly-formed myotubes with chains of myonuclei and pictures of active sarcomerogenesis were observed. On the other hand, the surviving muscle fibers of the perinecrotic zone were rich in myonuclei at their growing ends. In the vicinity of nuclei, accumulation of a mass of non-differentiated cytoplasm rich in free ribosomes and polysomes, rough endoplasmic reticulum, Golgi apparatus, and centrioles are seen. Tritiated thymidine pulse-labeling showed that only rare myonuclei of the perinecrotic zone muscle fibers were labeled, whereas numerous non-differentiated cells of granulation tissue and myosatellites incorporated thymidine. The number of labeled myonuclei markedly increased 96 hours after 3HTdr administration. These data evidence that the myoblastic mechanism is predominant in the regeneration of adult frog lymph heart muscle tissue. It is necessary to emphasize that during the lymph heart muscle tissue reparative myogenesis some of the perinecrotic myonuclei are able to synthesize DNA and to divide mitotically, which distinguishes this type of muscle from skeletal muscle tissue of vertebrates.     

Influence of corticosteroids on myonuclear domain size in the rat diaphragm muscle.

Skeletal muscle fibers are multinucleated. Each myonucleus regulates gene products and protein expression in only a restricted portion of the muscle fiber, the myonuclear domain (MND). In the rat diaphragm muscle (DIAm), corticosteroid (CoS) treatment causes atrophy of fibers containing myosin heavy chain (MHC): MHC2X and/or MHC2B. We hypothesized that DIAm fiber MND size is maintained during CoS-induced atrophy. Adult male rats received methylprednisolone for 11 days at 1 (CoS-Low, n = 8) or 8 mg x kg(-1) x day(-1) (CoS-High, n = 8). Age-matched (CTL-AgeM, n = 8), sham-operated (SHAM-AgeM, n = 8), and weight-matched (CTL-WtM, n = 8) animals served as controls. In single DIAm fibers, cross-sectional area (CSA), MND size, and MHC expression were determined. Fiber CSA and MND size were similar in CTL-AgeM and SHAM-AgeM groups. Only fibers containing MHCslow or MHC2A displayed smaller CSA in CTL-WtM than in CTL-AgeM and SHAM-AgeM groups, and MND size was reduced in all fibers. Thus fibers containing MHCslow and MHC2A maintain the number of myonuclei, whereas MHC2X or MHC2B fibers show loss of myonuclei during normal muscle growth. Both CoS groups displayed smaller CSA and MND size than CTL-AgeM and SHAM-AgeM groups. However, compared with CTL-WtM DIAm fibers, only fibers containing MHC2X or MHC2B displayed reduced CSA and MND size after CoS treatment. Thus little, if any, loss of myonuclei was associated with CoS-induced atrophy of MHC2X or MHC2B DIAm fibers. In summary, MND size does not appear to be regulated during CoS-induced DIAm atrophy.     

Skeletal muscle morphology in power-lifters with and without anabolic steroids

The morphological appearance of the vastus lateralis (VL) muscle from high-level power-lifters on long-term anabolic steroid supplementation (PAS) and power-lifters never taking anabolic steroids (P) was compared. The effects of long- and short-term supplementation were compared. Enzyme-immunohistochemical investigations were performed to assess muscle fiber type composition, fiber area, number of myonuclei per fiber, internal myonuclei, myonuclear domains and proportion of satellite cells. The PAS group had larger type I, IIA, IIAB and IIC fiber areas (p<0.05). The number of myonuclei/fiber and the proportion of central nuclei were significantly higher in the PAS group (p<0.05). Similar results were seen in the trapezius muscle (T) but additionally, in T the proportion of fibers expressing developmental myosin isoforms was higher in the PAS group compared to the P group. Further, in VL, the PAS group had significantly larger nuclear domains in fibers containing ≥5 myonuclei. The results of AS on VL morphology in this study were similar to previously reported short-term effects of AS on VL. The initial effects from AS appear to be maintained for several years.