Drosophila Erect wing (Ewg) controls mitochondrial fusion during muscle growth and maintenance by regulation of the Opa1-like gene

Mitochondrial biogenesis and morphological changes are associated with tissue-specific functional demand, but the factors and pathways that regulate these processes have not been completely identified. A lack of mitochondrial fusion has been implicated in various developmental and pathological defects. The spatiotemporal regulation of mitochondrial fusion in a tissue such as muscle is not well understood. Here, we show in Drosophila indirect flight muscles (IFMs) that the nuclear-encoded mitochondrial inner membrane fusion gene, Opa1-like, is regulated in a spatiotemporal fashion by the transcription factor/co-activator Erect wing (Ewg). In IFMs null for Ewg, mitochondria undergo mitophagy and/or autophagy accompanied by reduced mitochondrial functioning and muscle degeneration. By following the dynamics of mitochondrial growth and shape in IFMs, we found that mitochondria grow extensively and fuse during late pupal development to form the large tubular mitochondria. Our evidence shows that Ewg expression during early IFM development is sufficient to upregulate Opa1-like, which itself is a requisite for both late pupal mitochondrial fusion and muscle maintenance. Concomitantly, by knocking down Opa1-like during early muscle development, we show that it is important for mitochondrial fusion, muscle differentiation and muscle organization. However, knocking down Opa1-like, after the expression window of Ewg did not cause mitochondrial or muscle defects. This study identifies a mechanism by which mitochondrial fusion is regulated spatiotemporally by Ewg through Opa1-like during IFM differentiation and growth.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Lack of myostatin results in excessive muscle growth but impaired force generation

The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null (Mstn(-/-)) and compact (Berlin High Line, BEH(c/c)). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn(-/-) muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Muscle growth in Nile tilapia (Oreochromis niloticus): histochemical, ultrastructural and morphometric study

Muscle growth in Nile tilapia (Oreochromis niloticus) was studied focusing on histochemical, ultrastructural, and morphometric characteristics of muscle fibers. Based on body length (cm), we studied four groups: G1 = 1.36+/-0.09, G2 = 3.38+/-0.44, G3 = 8.90+/-1.47, and G4 = 28.30+/-3.29 (mean+/-S.D.). All groups showed intense reaction to NADH-TR in subdermal fibers and weak or no reaction in deep layer fibers. In G3 and G4, an intermediate layer was also observed with fibers presenting weak reaction; in G4, groups of fibers with intense reaction were observed in the subdermal region. The myosin ATPase (m-ATPase) activities were acid-stable and alkali-labile in subdermal fibers; most deep layer fibers were alkali-stable and acid-labile. Intermediate fibers were acid-labile and alkali-stable. Two fiber populations were observed near deep muscle layer: one large presenting weak acid- and alkali-stable and the other small alkali-stable.During growth, muscle fiber hypertrophy was more evident in intermediate and white fibers for G3 and G4. However, in these groups, the presence of fiber diameters less than or equal to21 mum suggested that there is still substantial fiber recruitment, confirmed by ultrastructural study, but hypertrophy is the main mechanism contributing to increase in muscular mass. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Short periods of fasting followed by refeeding change the expression of muscle growth-related genes in juvenile Nile tilapia (Oreochromis niloticus)

Muscle growth mechanisms are controlled by molecular pathways that can be affected by fasting and refeeding. In this study, we hypothesized that short period of fasting followed by refeeding would change the expression of muscle growth-related genes in juvenile Nile tilapia (Oreochromis niloticus). The aim of this study was to analyze the expression of MyoD, myogenin and myostatin and the muscle growth characteristics in the white muscle of juvenile Nile tilapia during short period of fasting followed by refeeding. Juvenile fish were divided into three groups: (FC) control, feeding continuously for 42. days, (F5) 5. days of fasting and 37. days of refeeding, and (F10) 10. days of fasting and 32. days of refeeding. At days 5 (D5), 10 (D10), 20 (D20) and 42 (D42), fish (n = 14 per group) were anesthetized and euthanized for morphological, morphometric and gene expression analyses. During the refeeding, fasted fish gained weight continuously and, at the end of the experiment (D42), F5 showed total compensatory mass gain. After 5 and 10. days of fasting, a significant increase in the muscle fiber frequency (class 20) occurred in F5 and F10 compared to FC that showed a high muscle fiber frequency in class 40. At D42, the muscle fiber frequency in class 20 was higher in F5. After 5. days of fasting, MyoD and myogenin gene expressions were lower and myostatin expression levels were higher in F5 and F10 compared to FC; at D42, MyoD, myogenin and myostatin gene expression was similar among all groups. In conclusion, this study showed that short periods of fasting promoted muscle fiber atrophy in the juvenile Nile tilapia and the refeeding caused compensatory mass gain and changed the expression of muscle growth-related genes that promote muscle growth. These fasting and refeeding protocols have proven useful for understanding the effects of alternative warm fish feeding strategies on muscle growth-related genes. © 2013.

Morphology and expression of genes related to skeletal muscle growth in juveniles of pirarucu (Arapaima gigas, Arapaimatidae, Teleostei)

Skeletal muscle growth in the pirarucu (Arapaima gigas) is highly interesting to fish farmers because it provides information about how the mechanism in muscle mass increase, characteristic of the species, is regulated. Pirarucu has specific muscle growth that highlights the species's significance and commercial value. Current research evaluates the morphology and the growth-related gene expression in the red and white skeletal muscles of the pirarucu. Muscle samples were collected from the lateral anterior region and frozen in liquid nitrogen. Histological sections were performed and stained by HE for morphological analysis. Red and white muscle samples were used to determine MyoD, myogenin, and myostatin genes expression by Real-time Polymerase Chain Reaction. Although MyoD and myogenin were not statistically different in the two types of muscles, myostatin was significantly higher in the white rather than in the red muscle. Results show the muscle growth characteristics of the species and may be helpful for improving aquaculture management programs.

Rearing temperature induces changes in muscle growth and gene expression in juvenile pacu (Piaractus mesopotamicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of incubation temperature on muscle morphology and growth in the pacu (Piaractus mesopotamicus)

This study describes the influence of incubation temperature during initial development phase on the morphology and muscle growth characteristics in the pacu (Piaractus mesopotamicus). Pacu eggs were incubated at 25, 27, and 29 degreesC until hatching. After day 5, fish from each temperature were transferred to 5001 tanks. At hatching and after 5, 25, and 60 days, muscle samples were collected, some were frozen in liquid nitrogen and others fixed in 4% paraformaldehyde or 2.5% glutaraldehyde. These samples were used for morphological, histochemical, immunohistochemical, and morphometric analysis. At hatching, we observed a superficial monolayer of small diameter fibers, lying just beneath the skin surrounding several round cells. From day 5, we observed two distinct populations of muscle fibers distributed in two layers: (1) red-in a superficial region with aerobic activity, and following acid preincubation, high mATPase activity, and 2) white-with anaerobic activity, and following alkaline preincubation, high mATPase activity. Twenty-five days after hatching, an intermediate layer and cell proliferating zones could be seen in the dorsal fin muscle region, with intermediate characteristics. Throughout the experimental period, there was an increase in muscle mass due to new fiber recruitment in the cell proliferating zones and between the more differentiated fibers in red, intermediate, and white muscles. This was more obvious from day 25, and at 29 degreesC than at 25 and 27 degreesC. Fiber hypertrophy occurred from hatching to 60 days and was more evident from 5 to 25 days. The number of proliferating nuclei (PCNA-labelling) increased from hatching to 60 days, and was more obvious in the 29 degreesC group at 60 days. Our results show that at incubation temperatures of 25, 27 and 29 degreesC, hypertrophy was predominantly from hatching to 25 days, after that muscle growth by hyperplastic mechanism increased. The interaction of muscle hypertrophic and hyperplastic growth processes in the 29 degreesC group produced the largest fish at the end of the experiment. (C) 2004 Elsevier B.V. All rights reserved.

Morphological and histochemical study of the myotomal muscle in pacu (Piaractus mesopotamicus Holmberg, 1887) during the initial growth phases

Morphological and histochemical methods were used to evaluate the myotomal muscle characteristics of pacu (Piaractus mesopotamicus) from hatching to 40 days old. During the larval period, the musculature consisted predominantly of white muscle. White and red muscle mass increased at 10, 20, 30 and 40 days after hatching. The larvae had round muscle fibers with a moderate degree of maturation and central nuclei. In subsequent phases, small and immature fibers were visible near larger and more differentiated fibers. Undifferentiated cells or presumptive myoblasts located in embryogenic zones were visible in the dorsal and ventral regions, and were more evident at 30 and 40 days. The red muscle fibers located in the subdermal region, had oxidative metabolism and slow contraction, whereas the more predominant white muscle fibers had glycolytic metabolism and fast contraction. Our findings indicate that during the initial phases, myotomal muscle growth in pacu occurs by both, muscle fiber hypertrophy and hyperplasia. The analysis of frequency of red and white muscle fibers shows that hyperplastic growth is intense in this period. As the growth rate in adult fish is related to the number of muscle fibers in young fish, extrinsic factors could change the muscle fiber phenotype and influence their ultimate size.

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0.0%: T2 =1.1%; T3 = 1.7%; T4 = 4.0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH-TR reaction and strong acid-stable mATPase activity, and a deep white one, most of the Muscle mass, formed by fibres with weak NADH-TR reaction and strong alkali-stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid-stable or acid-labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1-T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters <= 8 mu m was higher in the lysine treatments, and with diameters between 16 and 24 mu m, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 mu m and in T1-T4 were between 8 and 16 mu m. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy. (c) 2005 the Fisheries Society of the British

Differential MMP-2 and MMP-9 Activity and Collagen Distribution in Skeletal Muscle from pacu (Piaractus mesopotamicus) During Juvenile and Adult Growth Phases

Here, we evaluated collagen distribution and matrix metalloproteinases (MMPs) MMP-2 and MMP-9 activities in skeletal muscle of pacu (Piaractus mesopotamicus) during juvenile and adult growth phases. Muscle samples from juvenile and adult fishes were processed by histochemistry for collagen system fibers and for gelatin-zymography for MMP-2 and MMP-9 activities analysis. Picrosirius staining revealed a myosept, endomysium, and perimysium-like structures in both growth phases and muscle types, with increased areas of collagen fibers in adults, mainly in red muscle. Reticulin staining showed that reticular fibers in the endomysium-like structure were thinner and discontinuous in the red muscle fibers. The zymography revealed clear bands of the pro-MMP-9, active-MMP-9, intermediate-MMP-2, and active-MMP-2 forms in red and white muscle in both growth phases. MMP-2 activity was more intense in juvenile than adult muscle fibers. Comparing the red and white muscle types, MMP-2 activity was significantly higher in red muscle in adult phase only. The activity of MMP-9 forms was similar in juvenile red and white muscles and in the adult red muscle, without any activity in adult white muscle. In conclusion, our results show that, in pacu, the higher activities of MMP-2 and -9 are associated with the rapid muscle growth in juvenile age and in adult fish, these activities are related with a different red and white muscle physiology. This study may contribute to the understanding muscle growth mechanisms and may also contribute to analyse red and the white muscle parameters of firmness and softness, respectively, of the commercial product. Anat Rec, 292:387-395, 2009. (C) 2009 Wiley-Liss, Inc.

Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantitative expression of myogenic regulatory factors MyoD and myogenin in pacu (Piaractus mesopotamicus) skeletal muscle during growth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Muscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The influence of initial feeding on muscle development and growth in pacu Piaractus mesopotamicus larvae

The effects of different feeding schemes on pacu Piaractus mesopotamicus early development were evaluated with respect to growth, survival, muscle development, and differential gene expression of MyoD and myogenin. The pacu larvae (4 days post hatch-dph, 0.77 mg wet weight) were given six feeding treatments intentionally designed to cause variations in the larvae growth rate: (A) only artemia nauplii; (CD) only a commercial diet; (ED) only a semi-purified experimental diet; (ACD) and (AED) two treatments that involved weaning; and (S) starvation. Early weaning from artemia nauplii to the formulated diets (ACD and AED) affected growth and survival of the pacu larvae compared with the exclusive use of artemia (A). Starvation (S) and the commercial diet (CD) caused total mortality in pacu larvae at 18 dph. The experimental diet (ED) assured low fish survival and growth. The skeletal muscle morphology was not affected by the delay in somatic growth from early weaning onto the formulated diets. Three distinct muscle compartments were observed throughout the larval development in treatments A, ACD and AED: superficial, deep and intermediate, accompanied by muscle thickening. Severe undernourishment caused drastic differences in growth and in the morphology of the muscle fibers. Pacu larvae fed only formulated diets (CD and ED) showed muscle characteristics similar to the larvae in starvation (S) during the first 15 dph. At 27 and 35 dph, a slight increase in epaxial muscle mass was noted in larvae fed only the experimental diet (ED). At 35 dph, we observed a high frequency of fibers >= 40 mu m in the larvae that were weaned onto the formulated diets (ACD and AED), indicative of hypertrophy. In contrast, the larvae fed only artemia nauplii (A) displayed a larger number of fibers with diameters <= 20 mu m, which is indicative of hyperplasia. The expression of the MyoD and myogenin genes in pacu larvae at 35 dph was not affected by initial feeding (p>0.05). In conclusion, the formulated diets used impaired pacu larvae growth and survival; therefore, they were inadequate for pacu, at least at the times they were introduced. Artemia nauplii were the most adequate food source during first feeding of the pacu, and they produced bigger fish upon completion of the experiment. Moreover, the contribution of hyperplasia to the skeletal muscle growth appeared higher in fast- than in slow-growing pacu larvae. (C) 2011 Elsevier By. All rights reserved.