Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study

The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.     

Complex three-dimensional patterns of myosin isoform expression: differences between and within specific extraocular muscles

Because complex structural differences in adult extraocular muscles may have physiological and pathophysiological significance, the three-dimensional pattern of myosin heavy chain (MHC) isoform expression within the orbital and global layers of the muscle bellies compared with the distal tendon ends was quantitatively assessed. Three of the six extraocular muscles of adult rabbits were examined for immunohistologic expression of all fast, fast IIA/X, slow, neonatal and developmental MHC isoforms. The percentages of myofibers positive for each of these 5 myosin isoforms were determined in the orbital and global layers. There were relatively similar patterns of fast and slow MHC expression in the orbital and global layers of each of the three muscles examined. There were high levels of developmental MHC in the orbital layers, but significantly fewer developmental MHC positive myofibers in the global layer. The most variable expression was found with the neonatal MHC. There were significant differences between the longitudinal expression of the various isoforms in the middle of each muscle compared with the tendon end. In the orbital layer of all three muscles examined, the large numbers of fibers positive for fast MHC in the middle of the muscle dramatically decreased at the tendon end, with a concomitant increase in expression of slow myosin. There was a greater number of developmental MHC-positive myofibers at the tendon end than in the middle of the muscle in all three muscles examined. In the global layer, the IIA/X-positive myofibers comprised only half of the total number of fast-positive myofibers whereas in the orbital layer they comprised all or almost all of the fast positive myofibers. The configuration of the extraocular muscles is more complex than might be indicated by previous studies. The lateral rectus muscle had the most individual pattern of MHC expression when compared with the inferior rectus and inferior oblique muscles. Together with dramatic cross-sectional MHC fiber type differences between the orbital and global layers of the muscles, there are pronounced longitudinal differences in the proportions of myofibers expressing these five MHC isoforms in the middle region of the muscles and those in the distal tendon ends. This longitudinal progression appears to occur both within single myofibers, as well as within the series of myofibers that comprise the length of the muscle. We also confirm that the number of myofibers is reduced at the tendonous end while the cross-sectional area of each of the remaining myofibers is proportionally increased with regard to those in the muscle belly. Future studies may yet require two additional schemes for anatomic classification of the named extraocular muscles. One will be based on immunohistochemical features of their constituent myofibers as a supplement to classifications based on their electron microscopic appearance, innervation patterns or relative position with regard to the globe and orbit. Another will be based on the proportional length and longitudinal position of individual myofibers within an individual extraocular muscle.     

Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells.

1. Theree-day-old cultures of myotomal muscle, obtained from embryos of Xenopus laevis, were stained with fluorescent conjugates of alpha-bungarotoxin and maintained in native toxin in order to ensure that ACh receptors subsequently inserted into the sarcolemma would not be stained. Neural tube cells were then added to the cultures. 2. When cultures were exmained 1-3 days later fluorescent stain was found to be associated with sites of nerve-muscle contact. In some cases the stain along the path of contact extended for greater distances than the patches of stain seen on non-contacted muscle cells. 3. The development of new areas of fluorescent stain at sites of nerve-muscle contact was confirmed by making successive observations on the same muscle cells over a period of a day. 4. Similar experiments on muscle cells not contacted by nerve revealed the formation of new receptor patches, usually in areas of cell growth. 5. The majority of fluorescent pathes on non-contacted muscle cells did not undergo changes in size or shape over the course of 1-2 days. However some examples of enlargement, shrinkage and disappearance were observed. 6. On the basis of these findings it is concluded that ACh receptors aggregate within the sarcolemma, spontaneously as well as in response to innervation. In the latter case extrajunctional receptors accumulate at the site of nerve contact thereby contributing to the development of high receptor density in the subneural muscle membrane. This process of receptors redistribution occurs in the absence of synaptic or contractile activity. 7. Possible mechanisms involved in the redistribution of ACh receptors are discussed in relation to those which appear to modulate ligand-induced changes in the distribution of lectin and immunoglobulin receptors.     

Myofiber apoptosis occurs in the inflammation and regeneration phase following eccentric contractions in rats

Eccentric contractions (ECC) induce myofibrillar collapse, edema, and inflammation in muscle cells. Although apoptosis of myonuclei following ECC is activated during the inflammatory phase, the apoptosis response of the regenerative phase remains to be elucidated. The aim of the present study was to determine the inflammatory and regenerative phase of the apoptosis responses induced by ECC. In anesthetized rats, the tibialis anterior muscles were subjected to ECC repeated 40 times, evoked by surface electric stimulation (100 Hz, 10 V) with mechanical muscle stretch. Apoptosis was examined in the control group and in groups 1, 3, 7, and 14 days after ECC (each group, n = 4–6). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive myonuclei were assessed by further labeling with dystrophin staining and DAPI. The expression of proteins related to apoptosis (Bcl-2 and Bax) was examined by Western blot assay. At 1 and 3 days, focal edema and necrotic myofibers invaded by mononuclear phagocytes were present, whereas regenerated myofibers with central nuclei were detected at 7 and 14 days. The occurrence of TUNEL-positive myonuclei increased significantly at 7 (7.0 ± 1.5%) and 14 days (5.6 ± 0.6%) compared with control (0.9 ± 0.5%). Further we found that myonuclear apoptosis was restricted to the subsarcolemmal space at 7 and 14 days and markedly absent from the central nucleus. The Bax/Bcl-2 ratio was significantly higher at 3 (4.5 ± 0.9) and 7 days (3.4 ± 0.5) after ECC. In conclusion, myofiber apoptotic responses following ECC are present not only in the inflammatory phase but also persist during the regenerative phase.     

Degenerative and regenerative features of myofibers differ among skeletal muscles in a murine model of muscular dystrophy

Skeletal muscle myofibers constantly undergo degeneration and regeneration. Histopathological features of 6 skeletal muscles (cranial tibial [CT], gastrocnemius, quadriceps femoris, triceps brachii [TB], lumbar longissimus muscles, and costal part of the diaphragm [CPD]) were compared using C57BL/10ScSn-Dmd ^mdx (mdx) mice, a model for muscular dystrophy versus control, C57BL/10 mice. Body weight and skeletal muscle mass were lower in mdx mice than the control at 4 weeks of age; these results were similar at 6–30 weeks. Additionally, muscular lesions were observed in all examined skeletal muscles in mdx mice after 4 weeks, but none were noted in the controls. Immunohistochemical staining revealed numerous paired box 7-positive satellite cells surrounding the embryonic myosin heavy chain-positive regenerating myofibers, while the number of the former and staining intensity of the latter decreased as myofiber regeneration progressed. Persistent muscular lesions were observed in skeletal muscles of mdx mice between 4 and 14 weeks of age, and normal myofibers decreased with age. Number of muscular lesions was lowest in CPD at all ages examined, while the ratio of normal myofibers was lowest in TB at 6 weeks. In CT, TB, and CPD, Iba1-positive macrophages, the main inflammatory cells in skeletal muscle lesions, showed a significant positive correlation with the appearance of regenerating myofibers. Additionally, B220-positive B-cells showed positive and negative correlation with regenerating and regenerated myofibers, respectively. Our data suggest that degenerative and regenerative features of myofibers differ among skeletal muscles and that inflammatory cells are strongly associated with regenerative features of myofibers in mdx mice.     

l-dopa-induced reversal in striatal glutamate following partial depletion of nigrostriatal dopamine with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

We have reported that 1 month following acute (20mg/kg x 4) or subchronic (30 mg/kg/day x 7d) administration of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, there is an increase or decrease, respectively, in the extracellular level of striatal glutamate as determined by in vivo microdialysis [Robinson S, Freeman P, Moore C, Touchon JC, Krentz L, Meshul CK (2003) Acute and subchronic MPTP administration differentially affects striatal glutamate synaptic function. Exp Neurol 180:73-86]. The goal of this study was to determine the effects of treatment with l-dopa (15 mg/kg) for 21 days on striatal glutamate starting on day 8 after the first dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was administered to mice. Following acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the increase in extracellular striatal glutamate due to lesion of the nigrostriatal pathway was completely reversed to a level below that found in the vehicle-treated group after l-dopa treatment. Subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment resulted in a decrease in striatal extracellular glutamate that was reversed to the level close to that observed in the vehicle-treated group. There was no change in the density of nerve terminal glutamate immunolabeling associated with the synaptic vesicle pool, suggesting that the alterations in extracellular glutamate most likely originated from the calcium-independent pool. There was a similar decrease in the relative density of tyrosine hydroxylase immunolabeling, a marker for dopamine terminals, within the dorsolateral striatum in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups that had been administered l-dopa. There was a decrease in the relative density of immunolabeling within the dorsolateral striatum for the glutamate transporter, GLT-1, following acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment in the groups administered either vehicle or l-dopa. There was no change in GLT-1 immunolabeling following subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The results demonstrate that the reversal in the extracellular level of striatal glutamate following l-dopa treatment in both the acute and subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated groups is not due to changes in either striatal dopamine nerve terminals or in the density of the glutamate transporter, GLT-1.     

Post-injury stretch promotes recovery in a rat model of muscle damage induced by lengthening contractions

We investigated the cellular mechanisms and therapeutic effect of post-injury stretch on the recovery process from muscle injury induced by lengthening contractions (LC). One day after LC, a single 15-min bout of muscle stretch was applied at an intensity of 3 mNm. The maximal isometric torque was measured before and at 2–21 days after LC. The myofiber size was analyzed at 21 days after LC. Developmental myosin heavy chain-immunoreactive (dMHC-ir) cells, a marker of regenerating myofibers, were observed in the early recovery stage (2–5 days after LC). We observed that LC-induced injury markedly decreased isometric torque and myofiber size, which recovered faster in rats that underwent stretch than in rats that did not. Regenerating myofiber with dMHC-ir cells was observed earlier in rats that underwent stretch. These results indicate that post-injury stretch may facilitate the regeneration and early formation of new myofibers, thereby promoting structural and functional recovery from LC-induced muscle injury.     

Dynamic contrast-enhanced MRI of muscle perfusion combined with MR angiography of collateral artery growth in a femoral artery ligation model

To assess the use of MRI for evaluating changes in muscle blood flow and number of collateral arteries, serial dynamic contrast-enhanced MRI (DCE-MRI) was combined with high-spatial-resolution contrast-enhanced MR angiography (MRA) in a peripheral ischemia model.The combined MRI (DCE-MRI and MRA) protocol was performed serially in 15 male rabbits at 2 h (day 0(+)), 7 days, and 21 days after femoral artery ligation. In the anterior tibial and soleus muscle, changes in resting muscle blood flow determined as the endothelial transfer coefficient (K(trans)) and arterial inflow delay from DCE-MRI and changes in the number of sub-millimeter sized collateral arteries as scored with MRA were measured. Directly after ligation, K(trans) in the anterior tibial muscle was reduced to 23% of that in the control limb, then recovered to 81% on day 7, and to 85 % on day 21. K(trans) in the soleus muscle recovered from a reduction to 63% on day 0(+), to 85% on day 7, and to 90% on day 21. The number of collaterals around the ligated femoral artery increased from 1.1 on day 0(+) to 4.2 on day 7, and 6.0 on day 21 in the ligated limb only. Combined DCE-MRI and MRA allows non-invasive serial monitoring of changes in muscle blood flow and growth of sub-millimeter sized collateral arteries in a rabbit femoral artery ligation model.     

Modification of the phenotypic expression of murine dystrophy: a morphological study

The extensor digitorum longus muscles of 4-6-week-old normal mice (129 ReJ) and dystrophic mice (129 ReJ dy/dy) were orthotopically transplanted. Grafted muscles were examined 1, 3, 7, 14, 20, 50, and 100 days post-transplantation. The myofibers of both types of grafts underwent a similar time course of necrosis and regeneration. Other than during the initial necrotic response, no evidence of necrotic myofibers was found in either type of grafted muscle. At 100 days post-transplantation, the grafted normal and dystrophic muscles were essentially similar, except that the dystrophic graft was of smaller size. Based on a comparison of the number of myofibers found at the 100-day grafts' widest girths [631 +/- 59 SEM, for normal grafts (Bourke and Ontell, 1984); 631 +/- 74 SEM, for dystrophic grafts], it is suggested that the regenerative capability of traumatized 4-6-week-old dystrophic muscle is similar to that of traumatized normal muscle. At 100 days post-transplantation, the grafted dystrophic muscle appeared "healthier" than untraumatized muscle from age-matched dystrophic mice, having less variation in myofiber diameter, better fascicular organization, and less connective tissue. The transplantation system demonstrates the possibility of modifying the expression of genetic programming of myopathic disorders using environmental manipulation.     

The morphological variability of neuromuscular junctions in the rat extraocular muscles: a scanning electron microscopical study

Six morphologically distinct types of neuromuscular junctions were identified by scanning electron microscopy in the rat extraocular muscles: two diffuse and four focal types. The diffuse junctions, spreading out extensively over the muscle fiber surface, were characterized by two types of varicose swellings (or terminal varicosities) of nerve endings. One type consisted of several ramifying nerve endings and shallow postsynaptic depressions with poorly-developed junctional folds. The other type consisted of a single axon and formed many synaptic contacts along the long axis of a muscle fiber. Round synaptic depressions facing the varicosities contained several junctional folds. The focal junctions, confined to an oval area on the muscle fiber surface, were characterized by the complexity and variability of their subneural apparatuses. Four different types of apparatuses, i.e., focal junctions, were found: 1) an apparatus consisting of labyrinthine gutters with numerous slit-like junctional folds, 2) apparatuses consisting of a large number (more than 20) of cup-like depressions with either a small number of pit-like junctional folds or 3) numerous slit-like ones, and 4) an apparatus consisting of a small number (about 10) of cup-like depressions with a few junctional folds. The findings indicate that the rat extraocular muscles contain six different types of muscle fibers.     

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.     

Haematological and acute-phase responses associated with delayed-onset muscle soreness in humans

Delayed-onset muscle soreness following unaccustomed or eccentric exercise is associated with inflammation, tissue necrosis and the release of muscle enzymes (Newham et al. 1983). We have investigated the time course of changes in circulating leucocytes and serum levels of some acute phase reactants, serum creatine kinase activity (CK) and muscle pain after a 40-min bout of bench-stepping exercise in eight healthy untrained subjects. Leg muscle soreness was greatest 2 days after the exercise bout. Peak serum CK values [mean (SD) 540 (502) IU·l^–1] occurred 1–7 days post-exercise. Serum C-reactive protein (CRP) was unchanged from pre-exercise levels [7.8 (3.4) mg·l^–1] immediately post-exercise [7.9 (2.3) mg·l^–1] but rose to a peak of 17.0 (3.9) mg·l^–1 1 day post-exercise, thereafter declining to basal levels. Serum levels of iron and zinc fell below pre-exercise levels for 1–3 days post-exercise. Serum albumin, IgG and IgM fell below pre-exercise levels from 1 day post-exercise, reaching minimal values (about 80% of basal levels) at 7 days post-exercise. The exercise did not appear to significantly affect serum levels of alpha-1-antitrypsin and alpha-1-acid glycoprotein. Two and three days after the exercise bout the circulating numbers of total leucocytes, neutrophils, monocytes and basophils fell 15–20% below pre-exercise levels, whereas lymphocytes, eosinophils and platelets were unchanged. The results indicate that a rapid acute phase inflammatory response is initiated within 1 day of a bout of exercise that induces delayed-onset muscle soreness, and that any later tissue necrosis that may occur is not accompanied by further marked changes in acute-phase reactants such as CRP.     

Promotion of muscle regeneration in the toad (Bufo viridis) gastrocnemius muscle by low-energy laser irradiation

The effect of low-energy laser (He-Ne) irradiation on the process of skeletal muscle regeneration after cold injury to the gastrocnemius muscle of the toad (Bufo viridis) was studied using quantitative histological and morphometric methods. The injured zones in the experimental toads were subjected to five direct He-Ne laser (632.8 nm wavelength) irradiations (6.0 mW for 2.3 min) every alternate day starting on the fourth day postinjury. Muscles that were injured as above, and subjected to redlight irradiation, served as a control group. Morphometric analysis was performed on histological sections of injured areas at 9, 14, and 30 days postinjury. At 9 days postinjury, mononucleated cells populated 69.3% ± 16.8% of the total area of injury. Thereafter, their volume fraction (percent of total injured zone) decreased gradually but more rapidly in the laserirradiated muscle than in the control. The volume fraction of the myotubes in the laser-irradiated muscles at 9 days of muscle regeneration was significantly higher (7.0% ± 2.2%) than in the control muscle (1.2% ± 0.4%). Young myofibers in the laser-irradiated muscles populated 15.5% ± 7.9% and 65.0% ± 9.5% of the injured area at 9 and 14 days of muscle regeneration, respectively, while in control muscles these structures were not evident at 9 days and made up only 5.3% ± 2.9% of the traumatized area at 14 days postinjury. The volume fraction of the young myofibers further increased by 30 days of muscle regeneration making up 75.7% ± 13.2% of the traumatized area, while in the laser-irradiated muscles most of the injured zone was filled with mature muscle fibers. It is concluded that He-Ne laser irradiation during the regeneration process markedly promotes muscle maturation in the injured zone following cold injury to the toad gastrocnemius muscle. © 1993 Wiley-Liss, Inc.     

Branched myofibers in long-term whole muscle transplants: a quantitative study

Orthotopic transplants of whole extensor digitorum longus muscles were performed on six 4-6-week-old 129 ReJ mice. One hundred days posttransplantation, the animals were killed and the regenerated muscles were processed for electron microscopy. The grafts contained polygonal-shaped myofibers with persistent central nuclei, organized into discrete muscle fascicles. No central area of fatty infiltration or fibrosis was observed. The mean number of myofibers in a regenerating transplanted muscle, as determined from an ultrathin section taken from the graft's widest girth, was 631 (SEM = +/- 59), a reduction of approximately 32% from that found in age-matched control muscle (Ontell et al., 1983). By following the myofibers in spaced, serial ultrathin sections along their length, it was found that the branched, regenerating myofibers found in immature grafts of normal muscle (Ontell et al., 1982) persisted in stabilized, long-term transplanted muscle. The frequency of branching was determined by following each fiber found at the widest girths of four of the grafts in spaced, serial ultrathin sections (15-micron intervals) for approximately 2% of the total length of the grafts. Over this distance, 6.6% of the fibers were involved in the branching phenomenon. The persistence of branched fibers in long-term grafts and the frequency with which the branching phenomenon was found to occur may have physiological consequences and should be investigated.     

超声对大鼠精囊腺超微结构及血清睾酮含量影响的研究

用超声处理大鼠阴囊区１０ｍｉｎ（１．７５Ｗ／ｃｍ２）。透射电镜观察处理后１ｈ至６０ｄ不同时间精囊腺粘膜和肌层的超微结构改变，并对电镜形态计量学所得的有关主细胞大分泌颗粒参数和血清睾酮放射免疫测定值作了统计学分析，结果表明主细胞、平滑肌细胞和毛细血管内皮细胞对超声敏感。处理后１ｈ，主细胞、平滑肌细胞和毛细血管内皮细胞已呈超微结构改变。３～１５ｄ退变显著加重，３０ｄ后呈恢复趋势，６０ｄ已接近正常。主细胞的主要改变是线粒体肿胀，高尔基复合体囊泡及粗面内质网池扩大、崩解，髓样结构与脂滴增多，以及核的退变等。处理后３ｄ、１５ｄ，主细胞大分泌颗粒（包括有芯及无芯）面数密度和体密度有明显减小，６０ｄ已恢复正常。大分泌颗粒平均直径变化不明显。处理后７ｄ、１５ｄ，血清睾酮显著降低，３０ｄ后恢复正常。本文联系超声抗生育作用，讨论了其对精囊腺影响的意义。     

Collagen synthesis by cultured arterial smooth muscle cells during spontaneous phenotypic modulation

Quantitative and qualitative changes in collagen synthetic activity by rabbit arterial smooth muscle cells were monitored during spontaneous phenotypic modulation from days 2-15 of culture. The cultured smooth muscle cells transformed into a synthetic phenotype, reaching a maximum of 94.6% on day 4, and then gradually returned to a contractile phenotype accounting for 59.3% on day 15 of culture. The maximum collagen synthesis was found on day 7 when the cells were in early quiescent phase and showed a 91.7% synthetic phenotype. With an increasing proportion of cells in a contractile state, total collagen synthesis per cell decreased in parallel with the reduction in total protein synthesis. Synthesis of type I collagen was predominant, and the proportion of type I + III collagen was over 85% during the entire period of culture. Synthetic activity of type IV collagen, however, was relatively increased, and reached 3.8 +/- 0.4% at day 15 in comparison with 0.8 +/- 0.1% in the late logarithmic growth phase on day 4. This significant increment of type IV collagen in vitro seems to be correlated with the phenotypic modulation of cultured smooth muscle cells into a contractile phenotype.     

Myonuclear degeneration in LMNA null mice

Lamins A/C, the major constituent of the nuclear lamina, confer mechanical stability to nuclei. We examined the myonuclei of LMNA null mice at the myotendinous junctions (MTJ), the site of longitudinal force transmission from contractile proteins to extracellular proteins. The right soleus and rectus femoris muscles of five null mutants aged 5-7 weeks and two wild-type animals aged 5 weeks and 6 months were examined by electron microscopy. The myofibers merging into the tendons were assessed for nuclear disintegration and cytoplasmic degeneration. The myofibers of the wild-type rectus femoris and soleus muscles revealed 19-27 nuclei/50 myofibers and 5-8/20, respectively, with no signs of degeneration. The rectus femoris muscle fibers of the null mice contained 75-117 myonuclei/50 myofibers, the soleus muscle, 13-36 nuclei/20 myofibers. Eleven to twenty-one per 50 myonuclei of the rectus femoris myonuclei showed chromatin clumping, nuclear fragmentation, nuclear inclusions and invaginations, and intranuclear filaments. The values were 12-19/50 for the soleus myonuclei. Moreover, 5-12/50 rectus femoris myofibers and 5-14/20, of the soleus myofibers showed cytoplasmic degeneration. None of these changes was found distal to the MTJ. These results favor the notion that myonuclei lacking a functional lamina are susceptible to mechanical stress in vivo. These alterations may contribute to the development of early joint contractures, a feature of ADEDMD.     

Myosatellite cells,growth, and regeneration in murine dystrophic muscle: A quantitative study

Patterns of growth and regeneration in 2-, 4-, 8-, and 17-week-old murine dystrophic (129 ReJ dy/dy) extensor digitorum longus muscles have been determined. Necrosis and myofiber loss, hypertrophy, and regeneration result in a reduced population of myofibers whose diameter distribution is more extensive than that found in the extensor digitorum longus muscles of age-matched normal mice. At the onset of dystrophic symptoms (2 weeks postnatal), the ratio of myosatellite cell nuclei to the total sublaminal nuclear population (myonuclei + myosatellite cells) is similar to that found in 2-week-old control muscles. The frequency of finding myosatellite cells decreases with age in both control and dystrophic muscles. Myosatellite cells account for 11%, 6%, 5%, and 3% of the total sublaminal nuclear population in control muscle and 12%, 8%, 6%, and 5% of the total sublaminal nuclear population in dystrophic muscle at 2, 4, 8, and 17 weeks, respectively. No preferential association of myosatellite cells with myofibers of a particular diameter is found in control muscle or in the two youngest dystrophic groups. At 8 and 17 weeks, myosatellite cells are less frequently encountered on small-diameter, regenerating myofibers of dystrophic muscle, and they are preferentially associated with large diameter, hypertrophied myofibers. The labeling index of myosatellite cells decreases with age in both normal and dystrophic muscle. At all ages the myosatellite cell labeling index is higher in dystrophic muscle (23%, 7%, 5%, and 2% at 2, 4, 8, and 17 weeks, respectively) than in normal muscle (5%, < 1% at 2 and 4 weeks, respectively), with no labeled myosatellite cells being found in 8- and 17-week-old normal muscles. It is suggested that the magnitude of the regenerative response of dystrophic murine muscle decreases with age and that this factor may be responsible for the inability of the regenerative response of dystrophic muscle to keep pace with the rapid muscle deterioration.