Benjamin W. C. Rosser

Pattern of Pax7 Expression During Myogenesis in the Posthatch Chicken Establishes a Model for Satellite Cell Differentiation and Renewal

The paired‐box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7‐expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity. Developmental Dynamics 231:489–502, 2004.

Pax7 Reveals a Greater Frequency and Concentration of Satellite Cells at the Ends of Growing Skeletal Muscle Fibers

The main sites of longitudinal growth in skeletal muscle are the ends of the fibers. This study tests the hypothesis that satellite cells (SCs) are at a greater frequency (#SC nuclei/all nuclei within basal laminae) and concentration (closer together) within growing fiber ends of posthatch chicken pectoralis. SCs were localized by their Pax7 expression, and fiber ends were identified by their retention of neonatal myosin heavy chains and small cross-sectional profiles. Whereas SC frequency decreased from about 20% at 9 days posthatch to <5% at 115 days, fiber ends retained a frequency of ∼16%. Calculated mean area of sarcolemma per SC revealed higher concentrations of SCs at fiber ends. There was also a strong inverse correlation between SC frequency and fiber profile cross-sectional size throughout development. This study suggests that SCs at fiber ends play a key role in the longitudinal growth of muscle fibers, and that fiber profile size may impact SC distribution.

Evolutionary Significance of Myosin Heavy Chain Heterogeneity in Birds

This article reviews the complexity, expression, genetics, regulation, function, and evolution of the avian myosin heavy chain (MyHC). The majority of pertinent studies thus far published have focussed on domestic chicken and, to a much lesser extent, Japanese quail. Where possible, information available about wild species has also been incorporated into this review. While studies of additional species might modify current interpretations, existing data suggest that some fundamental properties of myosin proteins and genes in birds are unique among higher vertebrates. We compare the characteristics of myosins in birds to those of mammals, and discuss potential molecular mechanisms and evolutionary forces that may explain how avian MyHCs acquired these properties. Microsc. Res. Tech. 50:473–491, 2000.

Metabolic capacity of individual muscle fibers from different anatomic locations.

We studied muscle fibers by quantitative biochemistry to determine whether metabolic capacity varied among fibers of a given type as a function of their anatomic location. Muscles were selected from both contiguous and diverse anatomic regions within the rats studied. The individual fibers, classified into myosin ATPase fiber types by histochemical means, were assessed for fiber diameters and analyzed for the activities of enzymes representing major energy pathways: malate dehydrogenase (MDH, oxidative), lactate dehydrogenase (LDH, glycolytic), and adenylokinase (AK, high-energy phosphate metabolism). We found that neither the average activities of each of the three enzymes nor the fiber diameters varied in Type I or Type IIa fibers selected from superficial to deep portions of the triceps surae of the hindlimb. However, the IIb fibers in the deep region of this muscle group had significantly greater oxidative capacity, less glycolytic capacity, and smaller diameters than the superficially situated IIb fibers. Type IIa fibers in lateral gastrocnemius, extensor digitorum longus, psoas, diaphragm, biceps brachii, superficial masseter, and superior rectus muscles were highly variable in both diameter and enzyme profiles, with a correlation between MDH activity and fiber diameter. Therefore, our results show that both intermuscular and intramuscular metabolic variations exist in muscle fibers of a given type.

Metabolic capacity of muscle fibers from high-altitude natives.

We evaluate the effects of chronic hypoxia on the metabolic phenotype of the muscle fiber types of humans. The subjects were three Quechua natives residing in the Peruvian Andes at an altitude greater than 3300 m, and three lowlanders from below 700 m. Biopsy specimens were obtained from the vastus lateralis muscles of volunteers. Muscle fibers were identified histochemically as type 1 (oxidative), 2a (oxidativeglycolytic) or 2b (glycolytic). The relative contribution of each fiber type to the total cross-sectional area of each biopsy sample was determined. In individual fibers, the activities of malate dehydrogenase (MDH, citric acid cycle), lactate dehydrogenase (LDH, glycolysis) and adenylokinase (high-energy phosphate) were quantified. The cross-sectional area of the muscle occupied by each fiber type is comparable between Quechuas and lowlanders. Type 1 fibers are the only fiber type to demonstrate statistically significant (P ≤ 50.05) differences in enzyme activities between Quechuas and lowlanders. MDH activity is, on average, 19.6% less (P ≤ 0.0001) and LDH activity 28.1% more (P ≤ 0.0001) in the type 1 fibers of the Quechuas. Chronic hypoxia appears to produce a shift from oxidative to glycolytic metabolism in those fibers which are typically the, most aerobic in human muscle.

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.

Retention of Pax3 Expression in Satellite Cells of Muscle Spindles

Intrafusal fibers within muscle spindles retain features characteristic of immaturity, unlike the larger and more numerous extrafusal fibers constituting the bulk of skeletal muscle. Satellite cells (SCs), myogenic progenitors, are detected on the surfaces of both intrafusal and extrafusal fibers, but little is known of spindle SCs. We have recently demonstrated that, like their extrafusal counterparts, SCs in muscle spindles of posthatch chickens express paired box transcription factor 7 (Pax7) protein. During vertebrate embryogenesis, myogenic progenitors express both Pax7 and Pax3 proteins. In postnatal mice, Pax3 appears in rare SC subsets, whereas Pax7 is expressed by all SCs within extrafusal fibers. Here we test the hypothesis that Pax3 protein maintains localized expression within SCs of muscle spindles. Immunohistochemical techniques were used to identify SCs by their Pax7 expression within anterior latissimus dorsi muscle excised from posthatch chickens of various ages. A greater percentage of SCs express Pax3 within intrafusal than extrafusal fibers at each age, and the proportion of SCs expressing Pax3 declines with aging. This is the first study to localize Pax3 expression in posthatch avian muscle and within SCs of muscle spindles. We suggest that Pax3-positive SCs are involved in fiber maintenance.

Pax7 Shows Higher Satellite Cell Frequencies and Concentrations within Intrafusal Fibers of Muscle Spindles

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

On the histochemical characterization and distribution of fast and slow muscle fibers in certain avian skeletal muscles

Histochemical techniques based upon the pH sensitivity of myofibrillar adenosine triphosphatase are used to differentiate avian skeletal muscle fibers as either fast or slow. Pars thoracica m. pectoralis (PM) of several avian species, Pars cranialis m. latissimi dorsi (LDCR) of the Japanese quail, and M. tensor propatagialis (TP) of the domestic pigeon are examined. Fast fibers predominate in the PM, and slow fibers in the LDCR. The TP shows marked internal variation in the distribution of muscle fibers. The occurrence of fast and slow muscle fibers, both intra- and inter-muscularly, is correlated with their functional adaptations.

Ultrastructural and cytological changes in the muscle fibers of the pectoralis of the giant Canada goose (Branta canadensis maxima) in disuse atrophy during molt

SummaryAdult male Branta canadensis maxima were collected from a nonmigratory feral population during their premolt, molt and postmolt phases. Lean dry weight of the pectoralis muscle decreased significantly (p≤0.0001) during molt, as a result of disuse atrophy. Histochemical analysis revealed that the region of the pectoralis muscle sampled consisted of Red (fast-twitch oxidative-glycolytic) and White (fast-twitch glycolytic) muscle fiber types, in an approximate ratio of 9 to 1. There was no significant (p= 0.1238) difference in the relative percentages of the two fiber types during the three periods of study. There was, however, a significant decrease in mean cross-sectional area of both Red (p≤0.0194) and White (p≤0.0001) fibers during molt. Red and White fiber areas were strongly correlated with each other during molt (r2=0.76, p=0.0010) and postmolt (r2=0.70, p=0.0052), but not during premolt (r2=0.02, p=0.7626). The latter finding may be related to fiber-type specific hypertrophy in premolt breeding males. Analysis of ultrastructure revealed that there was a significant (p=0.0003) decrease in the mean myofibrillar crosssectional area, and a significant increase in both the density (p=0.0227) and total number (p=0.0058) of myofibrils within the muscle fibers of the molting birds. These results indicate that the myofibrils split longitudinally during moltassociated disuse atrophy. A significant (p=0.0375) reduction in the amount of non-myofibrillar material (mitochondria) was also observed in the periphery of the muscle fibers of the molting birds. The changes observed during disuse atrophy are neither as pathological nor as extreme as those induced by experimental models of avian muscle atrophy.