Little is known concerning the time-course and structural dynamics of reactivation of compensatory myogenesis in denervated muscle, its initiating cellular mechanisms, and the relationship between this process and the progression of postdenervation atrophy. The purpose of this study was to investigate the interrelations between temporal and spatial patterns of the myogenic response in denervated muscle and progressive atrophy of muscle fibers. Another objective was to study whether reactivation of myogenesis correlates with destabilization of the differentiated state and death of denervated muscle cells. It has remained unclear whether muscle fiber atrophy was the primary factor activating the myogenic response, what levels of cellular atrophy were associated with its activation, and whether the initiation and intensity of myogenesis depended on the local and individual heterogeneity of atrophic changes among fibers. For this reason, our objective was also to identify the levels of atrophic and degenerative changes in denervated muscle fibers that are correlated with activation of the myogenic response. We found that the reactivation of myogenesis in the tibialis anterior and extensor digitorum longus muscles of the rat starts between days 10-21 following nerve transection, before atrophy has attained advanced level, long before dead cells are found in the tissue. Formation of new muscle fibers reaches its maximum between 2 and 4 months following denervation and gradually decreases with progressive postdenervation atrophy. The myogenic response is biphasic and includes two distinct processes. The first process resembles the formation of secondary and tertiary generations of myotubes during normal muscle development and dominates during the first 2 months of denervation. During this period, activated satellite cells form new myotubes on live differentiated muscle fibers. Most of the daughter myotubes in 1-and 2-month denervated muscle develop on the surface of fast type parent muscle fibers, and some of the newly formed muscle fibers express slow myosin. Some fast type parent fibers are weakly or, more rarely, moderately immunopositive for embryonic isomyosin. This indicates that reactivation of myogenesis may also depend on the fiber type. The level of atrophy, destabilization of the differentiated myofiber phenotype, and degenerative changes of individual fibers in denervated muscle are very heterogeneous. The myogenic response of the first type is associated predominantly with fibers of average and higher than average levels of atrophy. Muscle cells that undergo a lesser degree of atrophy also form daughter fibers, although with a lower incidence. We did not find any correlation between the size of newly formed fibers and the level of atrophy of parent fibers. The topographical distribution of new myotubes both in the peripheral and central areas of the mid-belly equatorial sections at the early stages following nerve transection indicates that myogenesis of the first type represents a systemic reactio...

Cells of the precardiac mesoderm (stages 4-6) and dividing myocytes of early hearts (stages 10-15) were tagged with a replication-incompetent retrovirus (CXL) (Mikawa et al., 1991b) encoding bacterial P-galactosidase (0-gal). Two protocols were used to infect the cardiogenic cells.(1) Small blocks (-50 pm2) of anterolateral mesoderm were dissected from gastrula-stage embryos (stages 4-6) and incubated in liquid medium containing the retrovirus. After removal of CXL, the tissues were dispersed into single-cell suspensions and pressure injected into the precardiac areas of recipient embryos (stages 4-6). Such embryos were then incubated in vitro at 37°C for 2 days (New, 1968), and those embryos with beating hearts were fixed for X-gal histochemistry and paraffii serial sectioning. (2) CXL was pressure injected in ovo (embryonic stages 4-15) into cardiogenic tissues and the eggs subsequently returned to an incubator. At selected stages of development embryos or whole hearts were fixed, stained with X-gal, and serially sectioned after paraffin embedding. The first method showed that (1) cells of the precardiac mesoderm could be infected with the retrovirus, (2) the transplanted cells would differentiate into beating myocytes, and (3) 0-gal expression was sufficiently high to be detected histochemically. With the second procedure we could show that (1) P-gal-tagged cells formed colonies in the myocardium, (2) the labeled cells were exclusively myocytes, (3) the number of cells per colony increased with increasing age of embryonic development, (4) the size of colonies was larger in the left than the right ventricle, (5) many of the colonies were transmural, i.e., they extended from epicardial to endocardia1 layers of the myocardium and generally exhibited a cone or funnel-shape with the base of the cone nearest the epicardium, (6) the orientation of myocytes within each colony changed at different layers of the myocardium, and (7) the cones contained both Pgal+ and P-gal-myocytes. DNA labeling studies with [3H]thymidine indicated that cardiogenic cells divided every 16-18 hr during the first week of development and that the CXL-labeled cells divided indistinguishably from unlabeled myocytes.Based on these observations a model growth of the myocardium is presented.

This study, conducted on 25-month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long-term denervated muscles. Despite a ϳ97.6% loss in mean cross-sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing ϳ1.5 times more fibers than age-matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end-plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural-cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long-term denervated muscles, as compared with age-matched control muscles. In 25-month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long-term denervated muscle. Anat Rec 263: 139 -154, 2001.

Obscurin (approximately 800 kDa) in striated muscle closely surrounds sarcomeres at the level of the M-band and Z-disk where, we hypothesize, it participates in the assembly of the contractile apparatus and membrane systems required for Ca2+ homeostasis. In this study, we used small inhibitory RNA (siRNA) technology to reduce the levels of obscurin in primary cultures of skeletal myotubes to study its role in myofibrillogenesis and the organization of the sarcoplasmic reticulum (SR). siRNA-treated myotubes showed a specific and dramatic reduction in the approximately 800 kDa form of obscurin by reverse transcription-polymerase chain reaction, immunoblotting, and immunofluorescence. M-bands and A-bands, but not Z-disks or I-bands, were disrupted when the synthesis of obscurin was inhibited. Small ankyrin 1, an integral protein of the network SR that binds to obscurin, also failed to align around developing sarcomeres in treated myotubes. Myosin and myomesin levels were significantly reduced in treated myotubes but alpha-actinin was not, suggesting that down-regulation of obscurin destabilizes proteins of the M-band and A-band but not of the Z-disk. Our findings suggest that obscurin is required for the assembly of the M-band and A-band and for the regular alignment of the network SR around the contractile apparatus.

Obscurin/obscurin-MLCK is a giant sarcomere-associated protein with multiple isoforms whose interactions with titin and small ankyrin-1 suggest that it has an important role in myofibril assembly, structural support, and the sarcomeric alignment of the sarcoplasmic reticulum. In this study, we characterized the zebrafish orthologue of obscurin and examined its role in striated myofibril assembly. Zebrafish obscurin was expressed in the somites and central nervous system by 24 hours post-fertilization (hpf) and in the heart by 48 hpf. Depletion of obscurin using two independent morpholino antisense oligonucleotides resulted in diminished numbers and marked disarray of skeletal myofibrils, impaired lateral alignment of adjacent myofibrils, disorganization of the sarcoplasmic reticulum, somite segmentation defects, and abnormalities of cardiac structure and function. This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate and organize new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies.