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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1996

Muscle organizers in Drosophila: the role of persistent larval fibers in adult flight muscle development

In many organisms muscle formation depends on specialized cells that prefigure the pattern of the musculature and serve as templates for myoblast organization and fusion. These include muscle pioneers in insects and muscle organizing cells in leech. In Drosophila, muscle founder cells have been proposed to play a similar role in organizing larval muscle development during embryogenesis. During metamorphosis in Drosophila, following histolysis of most of the larval musculature, there is a second round of myogenesis that gives rise to the adult muscles. It is not known whether muscle founder cells organize the development of these muscles. However, in the thorax specific larval muscle fibers do not histolyze at the onset of metamorphosis, but instead serve as templates for the formation of a subset of adult muscles, the dorsal longitudinal flight muscles (DLMs). Because these persistent larval muscle fibers appear to be functioning in many respects like muscle founder cells, we investigated whether they were necessary for DLM development by using a microbeam laser to ablate them singly and in combination. We found that, in the absence of the larval muscle fibers, DLMs nonetheless develop. Our results show that the persistent larval muscle fibers are not required to initiate myoblast fusion, to determine DLM identity, to locate the DLMs in the thorax, or to specify the total DLM fiber volume. However, they are required to regulate the number of DLM fibers generated. Thus, while the persistent larval muscle fibers are not obligatory for DLM fiber formation and differentiation, they are necessary to ensure the development of the correct number of fibers.

Non-NASA Center↗
NASA NTRS1998

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Non-NASA Center↗
NASA NTRS1998

Nerve-muscle interactions during flight muscle development in Drosophila

During Drosophila pupal metamorphosis, the motoneurons and muscles differentiate synchronously, providing an opportunity for extensive intercellular regulation during synapse formation. We examined the existence of such interactions by developmentally delaying or permanently eliminating synaptic partners during the formation of indirect flight muscles. When we experimentally delayed muscle development, we found that although adult-specific primary motoneuron branching still occurred, the higher order (synaptic) branching was suspended until the delayed muscle fibers reached a favourable developmental state. In reciprocal experiments we found that denervation caused a decrease in the myoblast pool. Furthermore, the formation of certain muscle fibers (dorsoventral muscles) was specifically blocked. Exceptions were the adult muscles that use larval muscle fibers as myoblast fusion targets (dorsal longitudinal muscles). However, when these muscles were experimentally compelled to develop without their larval precursors, they showed an absolute dependence on the motoneurons for their formation. These data show that the size of the myoblast pool and early events in fiber formation depend on the presence of the nerve, and that, conversely, peripheral arbor development and synaptogenesis is closely synchronized with the developmental state of the muscle.

Non-NASA Center↗
NASA NTRS2003

Gravity Plays an Important Role in Muscle Development and the Differentiation of Contractile Protein Phenotype

Several muscles in the body exist mainly to work against gravity. Whether gravity is important in the development of these muscles is not known. By examining the basic proteins that compose muscle, questions about the role of gravity in muscle development can be answered. Myosin heavy chains (MHCs) are a family of proteins critically important for muscle contraction. Several types of MHCs exist (e.g., neonatal, slow, fast), and each type is produced by a particular gene. Neonatal MHCs are produced early in life. Slow MHCs are important in antigravity muscles, and fast MHCs are found in fast-twitch power muscles. The gene that is turned on or expressed will determine which MHC is produced. Early in development, antigravity skeletal muscles (muscles that work against gravity) normally produce a combination of the neonatal/embryonic MHCs. The expression of these primitive MHCs is repressed early in development; and the adult slow and fast MHC genes become fully expressed. We tested the hypothesis that weightbearing activity is critical for inducing the normal expression of the slow MHC gene typically expressed in adult antigravity muscles. Also, we hypothesized that thyroid hormone, but not opposition to gravity, is necessary for expressing the adult fast IIb MHC gene essential for high-intensity muscle performance. Groups of normal thyroid and thyroid-deficient neonatal rats were studied after their return from the 16-day Neurolab mission and compared to matched controls. The results suggest: (1) Weightlessness impaired body and limb skeletal muscle growth in both normal and thyroid-deficient animals. Antigravity muscles were impaired more than those used primarily for locomotion andor nonweightbearing activity. (2) Systemic and muscle expression of insulin-like growth factor-I (IGF-I), an important body and tissue growth factor, was depressed in flight animals. (3) Normal slow, type I MHC gene expression was markedly repressed in the normal thyroid flight group. (4) Fast IIb MHC gene expression was enhanced in fast-twitch muscles of normal thyroid animals exposed to spaceflight; however, thyroid deficiency markedly repressed expression of this gene independently of spaceflight. In summary, the absence of gravity, when imposed at critical stages of development, impaired body and skeletal muscle growth, as well as expression of the MHC gene family of motor proteins. This suggests that normal weightbearing activity is essential for establishing body and muscle growth in neonatal animals, and for expressing the motor gene essential for supporting antigravity functions.

Adams, Gregory A.↗
NASA NTRS1993

Skeletal muscle satellite cells cultured in simulated microgravity

Satellite cells are postnatal myoblasts responsible for providing additional nuclei to growing or regenerating muscle cells. Satellite cells retain the capacity to proliferate and differentiate in vitro and therefore provide a useful model to study postnatal muscle development. Most culture systems used to study postnatal muscle development are limited by the two-dimensional (2-D) confines of the culture dish. Limiting proliferation and differentiation of satellite cells in 2-D could potentially limit cell-cell contacts important for developing the level of organization in skeletal muscle obtained in vivo. Culturing satellite cells on microcarrier beads suspended in the High-Aspect-Ratio-Vessel (HARV) designed by NASA provides a low shear, three-dimensional (3-D) environment to study muscle development. Primary cultures established from anterior tibialis muscles of growing rats (approximately 200 gm) were used for all studies and were composed of greater than 75 % satellite cells. Different inoculation densities did not affect the proliferative potential of satellite cells in the HARV. Plating efficiency, proliferation, and glucose utilization were compared between 2-D flat culture and 3-D HARV culture. Plating efficiency (cells attached - cells plated x 100) was similar between the two culture systems. Proliferation was reduced in HARV cultures and this reduction was apparent for both satellite cells and non-satellite cells. Furthermore, reduction in proliferation within the HARV could not be attributed to reduced substrate availability since glucose levels in media from HARV and 2-D cell culture were similar. Morphologically, microcarrier beads within the HARVS were joined together by cells into three-dimensional aggregates composed of greater than 10 beads/aggregate. Aggregation of beads did not occur in the absence of cells. Myotubes were often seen on individual beads or spanning the surface of two beads. In summary, proliferation and differentiation of satellite cells on microcarrier beads within the HARV bioreactor results in a three dimensional level of organization that could provide a more suitable model to study postnatal muscle development.

Molnar, Greg↗
NASA NTRS2002

Influence of Unweighting on Insulin Signal Transduction in Muscle

Unweighting of the juvenile soleus muscle is characterized by an increased binding capacity for insulin relative to muscle mass due to sparing of the receptors during atrophy. Although carbohydrate metabolism and protein degradation in the unweighted muscle develop increased sensitivity to insulin in vivo, protein synthesis in vivo and system A amino acid transport in vitro do not appear to develop such an enhanced response. The long-term goal is to identify the precise nature of this apparent resistance in the insulin signal transduction pathway and to consider how reduced weight-bearing may elicit this effect, by evaluating specific components of the insulin signalling pathway. Because the insulin-signalling pathway has components in common with the signal transduction pathway for insulin-like growth factor (IGF-1) and potentially other growth factors, the study could have important implications in the role of weight-bearing function on muscle growth and development. Since the insulin signalling pathway diverges following activation of insulin receptor tyrosine kinase, the immediate specific aims will be to study the receptor tyrosine kinase (IRTK) and those branches, which lead to phosphorylation of insulin receptor substrate-1 (IRS-1) and of Shc protein. To achieve these broader objectives, we will test in situ, by intramuscular injection, the responses of glucose transport, system A amino acid transport and protein synthesis to insulin analogues for which the receptor has either a weaker or much stronger binding affinity compared to insulin. Studies will include: (1) estimation of the ED(sub 50) for each analogue for these three processes; (2) the effect of duration (one to four days) of unweighting on the response of each process to all analogues tested; (3) the effect of unweighting and the analogues on IRTK activity; and (4) the comparative effects of unweighting and analogue binding on the tyrosine phosphorylation of IRTK, IRS-1, and Shc protein.

Tischler, Marc E.↗
NASA NTRS1999

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Rosenthal, Nadia A.↗
NASA NTRS2000

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

NASA Discipline Musculoskeletal↗
NASA NTRS1993

Space research on organs and tissues

Studies in space on various physiological systems have and will continue to provide valuable information on how they adapt to reduced gravitational conditions, and how living in a 1 g (gravity) environment has guided their development. Muscle and bone are the most notable tissues that respond to unweighting caused by lack of gravity. The function of specific muscles and bones relates directly to mechanical loading, so that removal of 'normal forces' in space, or in bedridden patients, causes dramatic loss of tissue mass. The cardiovascular system is also markedly affected by reduced gravity. Adaptation includes decreased blood flow to the lower extremities, thus decreasing the heart output requirement. Return to 1 g is associated with a period of reconditioning due to the deconditioning that occurs in space. Changes in the cardiovascular system are also related to responses of the kidney and certain endocrine (hormone-producing) organs. Changes in respiratory function may also occur, suggesting an effect on the lungs, though this adaptation is poorly understood. The neurovestibular system, including the brain and organs of the inner ear, must adapt to the disorientation caused by lack of gravity. Preliminary findings have been reported for liver. Additionally, endocrine organs responsible for release of hormones such as insulin, growth hormone, glucocorticoids, and thyroid hormone may respond to spaceflight.

Tischler, Marc E.↗
NASA NTRS2000

Dependence of normal development of skeletal muscle in neonatal rats on load bearing

Antigravity function plays an important role in determining the morphological and physiological properties of the neuromuscular system. Inhibition of the normal development of the neuromuscular system is induced by hindlimb unloading during the neonatal period in rats. However, the role of gravitational loading on the development of skeletal muscle in rats is not well understood. It could be hypothesized that during the early postnatal period, i.e. when minimal weight-supporting activity occurs, the activity imposed by gravity would be of little consequence in directing the normal development of the skeletal musculature. We have addressed this issue by limiting the amount of postnatal weight-support activity of the hindlimbs of rats during the lactation period. We have focused on the development of three characteristics of the muscle fibers, i.e. size, myonuclear number and myosin heavy chain expression.

Non-NASA Center↗
NASA NTRS1999

Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

Baldwin, K. M.↗
NASA NTRS1997

Molecular events underlying skeletal muscle atrophy and the development of effective countermeasures

Skeletal muscle adapts to loading; atrophying when exposed to unloading on Earth or in spaceflight. Significant atrophy (decreases in muscle fiber cross-section of 11-24%) in humans has been noted after only 5 days in space. Since muscle strength is determined both by muscle cross-section and synchronization of motor unit recruitment, a loss in muscle size weakens astronauts, which would increase risks to their safety if an emergency required maximal muscle force. Numerous countermeasures have been tested to prevent atrophy. Resistant exercise together with growth hormone and IGF-I are effective countermeasures to unloading as most atrophy is prevented in animal models. The loss of muscle protein is due to an early decrease in protein synthesis rate and a later increase in protein degradation. The initial decrease in protein synthesis is a result of decreased protein translation, caused by a prolongation in the elongation rate. A decrease in HSP70 by a sight increase in ATP may be the factors prolonging elongation rate. Increases in the activities of proteolytic enzymes and in ubiquitin contribute to the increased protein degradation rate in unloaded muscle. Numerous mRNA concentrations have been shown to be altered in unloaded muscles. Decreases in mRNAs for contractile proteins usually occur after the initial fall in protein synthesis rates. Much additional research is needed to determine the mechanism by which muscle senses the absence of gravity with an adaptive atrophy. The development of effective countermeasures to unloading atrophy will require more research.

Non-NASA Center↗
NASA NTRS2002

Effects of microgravity on myogenic factor expressions during postnatal development of rat skeletal muscle

To clarify the role of gravity in the postnatal development of skeletal muscle, we exposed neonatal rats at 7 days of age to microgravity. After 16 days of spaceflight, tibialis anterior, plantaris, medial gastrocnemius, and soleus muscles were removed from the hindlimb musculature and examined for the expression of MyoD-family transcription factors such as MyoD, myogenin, and MRF4. For this purpose, we established a unique semiquantitative method, based on RT-PCR, using specific primers tagged with infrared fluorescence. The relative expression of MyoD in the tibialis anterior and plantaris muscles and that of myogenin in the plantaris and soleus muscles were significantly reduced (P < 0.001) in the flight animals. In contrast, MRF4 expression was not changed in any muscle. These results suggest that MyoD and myogenin, but not MRF4, are sensitive to gravity-related stimuli in some skeletal muscles during postnatal development.

Non-NASA Center↗
NASA NTRS1979

Development of a bedrest muscle stress apparatus

In attempting further to define the deleterious effects of spaceflight on the human body, measurement systems and techniques were devised to determine the loss of skeletal muscle strength and tone as a result of spaceflight exposure. In order to determine how the muscle degradation process progresses with time during nonuse, a system for measuring muscle stress during bedrest was developed. The Bedrest Muscle Stress Apparatus is configured to slip snugly over the foot board of a standard hospital bed. Data collected with this device correlated well with pre- and post-bedrest data collected with the original skeletal muscle stress apparatus.

Booher, C. R.↗
NASA NTRS1997

Development of Human Muscle Protein Measurement with MRI

It is known that micro-gravity has a strong influence on the human musculoskeletal system. A number of studies have shown that significant changes in skeletal muscles occur in both space flight and bedrest simulation. In our 5 week bedrest study, the cross-sectional area of soleus-gastrocnemius decreased about 12% while the cross-sectional area of anterior calf muscles decreased about 4%. Using volume measurements, these losses increased after 17 weeks to approximately 30% and 21% respectively. Significant muscle atrophy was also found on the SL-J crew members after only 8 days in space. It is important that these effects are fully understood so that countermeasures can be developed. The same knowledge might also be useful in preventing muscle atrophy related to other medical problems. A major problem with anatomical measurements of muscle during bed rest and microgravity is the influence of fluid shifts and water balance on the measurement of muscle volume, especially when the exposure duration is short and the atrophy is relatively small. Fluid shifts were documented in Skylab by visual observations of blood vessel distention, rapid changes in limb volume, center of mass measurements and subjective descriptions such as puffy faces and head fullness. It has been reported that the muscle water content of biopsied soleus muscles decreased following 8 hours of head down tilt bed rest. Three aspects of fluid shifts that can affect volume measurements are: first, the shift of fluid that occurs whenever there is a change from upright to a recumbent position and vice versa; second, the potential for fluid accumulation in the lower limbs resulting from muscle damage caused by overextending atrophied muscle or swelling caused by deconditioned precapillary sphincter muscles during reambulation; third, the net change of hydration level during and after bed rest or spaceflight. Because of these transitory fluid shifts, muscle protein is expected to represent muscle capacity better than does muscle volume. The purpose of this study is to test the feasibility of using MRI to quantify of muscle protein and water content changes in muscle.

Lin, Chen↗
NASA NTRS2001

Biologically inspired toys using artificial muscles

Recent developments in electroactive polymers, so-called artificial muscles, could one day be used to make bionics possible. Meanwhile, as this technology evolves novel mechanisms are expected to emerge that are biologically inspired.

bionics↗
NASA NTRS2001

Ontogenetic, gravity-dependent development of rat soleus muscle

We tested the hypothesis that rat soleus muscle fiber growth and changes in myosin phenotype during the postnatal, preweaning period would be largely independent of weight bearing. The hindlimbs of one group of pups were unloaded intermittently from postnatal day 4 to day 21: the pups were isolated from the dam for 5 h during unloading and returned for nursing for 1 h. Control pups were either maintained with the dam as normal or put on an alternating feeding schedule as described above. The enlargement of mass (approximately 3 times), increase in myonuclear number (approximately 1.6 times) and myonuclear domain (approximately 2.6 times), and transformation toward a slow fiber phenotype (from 56 to 70% fibers expressing type I myosin heavy chain) observed in controls were inhibited by hindlimb unloading. These properties were normalized to control levels or higher within 1 mo of reambulation beginning immediately after the unloading period. Therefore, chronic unloading essentially stopped the ontogenetic developmental processes of 1) net increase in DNA available for transcription, 2) increase in amount of cytoplasm sustained by that DNA pool, and 3) normal transition of myosin isoforms that occur in some fibers from birth to weaning. It is concluded that normal ontogenetic development of a postural muscle is highly dependent on the gravitational environment even during the early postnatal period, when full weight-bearing activity is not routine.

Non-NASA Center↗