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Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.

Adaptation, Physiological↗

A subclass of bHLH proteins required for cardiac morphogenesis.

Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors. Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone had no effect on embryonic development, whereas together they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development.

Actins↗

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process.

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.

Amiloride↗

Effect of hypobaric hypoxia on fiber type composition of the soleus muscle in the developing rat.

The fiber type composition of the soleus muscle was investigated in male Sprague-Dawley rats exposed to hypobaric hypoxia of 460 mm Hg from 5 to 12 weeks of age. The muscle fibers were classified as fast-twitch oxidative (FO) and slow-twitch (S) on the basis of adenosine triphosphatase (ATPase) and succinate dehydrogenase (SDH) reactions. Intermediate fibers (INT) with intermediate ATPase and high SDH reaction intensities were also examined. A type shift of muscle fibers from FO to INT and S was found in the control group during development. After exposure to hypoxia, the hypoxia group had a significantly greater percentage of FO fibers than the age-matched control group. There was no significant change in the total number of fibers in the muscle during development and after exposure to hypoxia. These results indicate that the increased percentage of FO fibers found in the developing rat under hypoxic conditions is due to a hypoxia-induced inhibition of the type shift of muscle fibers from FO to S during development.

Animals↗

Transient disruption of nerve-muscle interaction shortly after birth permanently alters the development of the rat soleus muscle.

Transient paralysis of the rat soleus muscle shortly after birth leads to a permanent loss of motoneurones as revealed by retrograde labelling. Here we show that this loss of motoneurones is reflected in a reduction in the number of motor units. Soleus muscles in normal adult rats were found to have 27 (+/-0.6 S.E.M., n = 9) motor units. However, in muscles which had been treated with alpha-bungarotoxin (BTX) at birth and 3 days of age, causing paralysis lasting for 6-8 days, only 15(+/-0.6 S.E.M., n = 5) motor units remain. The effects of paralysis on the ability of the adult soleus muscle to develop force was also tested. Following treatment with a single BTX implant at birth, causing paralysis for 2-3 days, soleus muscles develop less tension (73.7% +/- 4.5 S.E.M., n = 8) and weigh less (88.2% +/- 3.8 S.E.M., n = 13) than their unoperated controls. This loss of muscle force is caused by a loss of muscle fibres, which in muscles that had been paralysed at birth was 81.4% (+/-4.1 S.E.M., n = 5) of control. Prolonging the duration of paralysis led to a greater reduction in force production, weight and the number of muscle fibres. Those muscles which had been paralysed at birth also took longer to relax during single twitch contractions. In addition, whereas normal soleus muscles contain around 20% of muscle fibres that do not react with antibodies to slow myosin HC, in soleus muscles paralysed at birth, 100% of the fibres reacted with this antibody. This study shows that disruption of neuromuscular interaction for a brief period after birth leads to a loss of motoneurones and a permanent impairment of muscle function.

Animals↗

Lysine-rich rice enhanced muscle growth and development in young rats.

Rice is the staple food for half of the world's population but is low in lysine content. We previously developed transgenic lysine-rich rice with enhanced free lysine content in rice seeds and demonstrated that it could improve skeletal growth and development in rats. However, the effects of lysine-rich rice on muscle remain to be studied. We hypothesized that lysine-rich rice was able to improve muscle growth in weaning rats via its anabolic effects on muscle metabolism. Male weaning Sprague-Dawley rats received lysine-rich rice (HFL) diet, wild-type rice (WT) diet, or wild-type rice with various doses of lysine supplementation (WT + Lys) diet (+ 0%, + 10%, + 20%, and + 40% lysine) for 70 days. Muscle strength and quality were analyzed by biomechanical test and muscle fiber typing of the extensor digitorum longus (EDL) muscles. Molecular mechanisms of lysine on muscle growth were also explored by rat serum biochemistry and cell culture systems. Results indicated that the HFL diet improved rats' muscle growth, strength, and physiological cross-sectional area (CSA) over the WT diet group. The CSAs of fast-twitch muscle fibers (Type IIb and IIx) were also increased. In addition, the HFL increased serum insulin-like growth factor 1 (IGF-1) and decreased serum myostatin (MSTN) concentrations. The cell culture model showed that lysine deficiency reduced IGF-1 expression and inhibited myoblast differentiation associated with muscle growth. Our findings showed that lysine-rich rice improved muscle growth and development in weaning rats. Higher dietary lysine possibly inhibited MSTN and activated of IGF-1 signaling pathway for muscle growth and development.

Animals↗

Evaluation of physical dimension changes as nondestructive measurements for monitoring rigor mortis development in broiler muscles.

Studies were conducted to develop a non-destructive method for monitoring the rate of rigor mortis development in poultry and to evaluate the effectiveness of electrical stimulation (ES). In the first study, 36 male broilers in each of two trials were processed at 7 wk of age. After being bled, half of the birds received electrical stimulation (400 to 450 V, 400 to 450 mA, for seven pulses of 2 s on and 1 s off), and the other half were designated as controls. At 0.25 and 1.5 h postmortem (PM), carcasses were evaluated for the angles of the shoulder, elbow, and wing tip and the distance between the elbows. Breast fillets were harvested at 1.5 h PM (after chilling) from all carcasses. Fillet samples were excised and frozen for later measurement of pH and R-value, and the remainder of each fillet was held on ice until 24 h postmortem. Shear value and pH means were significantly lower, but R-value means were higher (P < 0.05) for the ES fillets compared to the controls, suggesting acceleration of rigor mortis by ES. The physical dimensions of the shoulder and elbow changed (P < 0.05) during rigor mortis development and with ES. These results indicate that physical measurements of the wings maybe useful as a nondestructive indicator of rigor development and for monitoring the effectiveness of ES. In the second study, 60 male broilers in each of two trials were processed at 7 wk of age. At 0.25, 1.5, 3.0, and 6.0 h PM, carcasses were evaluated for the distance between the elbows. At each time point, breast fillets were harvested from each carcass. Fillet samples were excised and frozen for later measurement of pH and sacromere length, whereas the remainder of each fillet was held on ice until 24 h PM. Shear value and pH means (P < 0.05) decreased, whereas sarcomere length means (P < 0.05) increased over time, indicating rigor mortis development. Elbow distance decreased (P < 0.05) with rigor development and was correlated (P < 0.01) with shear value (r = 0.2581), sarcomere length (r = -0.3079), and pH (r = 0.6303). These results suggest that elbow distance could be used in conjunction with other detection methods for optically automating measurement of rigor mortis development in broiler carcasses.

Animals↗

Initial steps of myogenesis in somites are independent of influence from axial structures.

Formation of paraxial muscles in vertebrate embryos depends upon interactions between early somites and the neural tube and notochord. Removal of both axial structures results in a complete loss of epaxial myotomal muscle, whereas hypaxial and limb muscles develop normally. We report that chicken embryos, after surgical removal of the neural tube at the level of the unsegmented paraxial mesoderm, start to develop myotomal cells that express transcripts for the muscle-specific regulators MyoD and myogenin. These cells also make desmin, indicating that the initial steps of axial skeletal muscle formation can occur in the absence of the neural tube. However, a few days following the extirpation, the expression of MyoD and myogenin transcripts gradually disappears, and becomes almost undetectable after 4 days. From these observations we conclude that the neural tube is not required for the generation of the skeletal muscle cell lineage, but may support the survival or maitenance of further differentiation of the myotomal cell compartment. Notochord transplanted medially or laterally to the unsegmented paraxial mesoderm leads to a ventralization of axial structures but does not entirely prevent the early appearance of myoblasts expressing MyoD transcripts. However, the additional notochord inhibits subsequent development and maturation of myotomes. Taken together, our data suggest that neural tube promotes, and notochord inhibits, the process of myogenesis in axial muscles at a developmental step following the initial expression of myogenic bHLH regulators.

Animals↗

Early maturation of force production in pig tracheal smooth muscle during fetal development.

The contractility of airway smooth muscle is fully established at late term at birth but its responsiveness during fetal life has not been defined. In this study, the contractile force of airway smooth muscle to acetylcholine (ACh), K+ depolarizing solution, and electrical field stimulation (EFS) was measured in tracheas from small fetal pigs. Contraction to either agonist and to EFS was detectable in fetuses of as low as 9 g body weight, which corresponds to approximately 36 days of gestation. Isometric force increased progressively with age, reaching 4.1 +/- 0.4 mN for K+ and 5.8 +/- 0.5 mN for ACh (10(-4) M) at 600 g fetal weight (90 days). However, when normalized for cross sectional area of smooth muscle, the stress was essentially the same from 17- to 600-g fetuses. (K+: 17 g = 74.4 +/- 10.6 mN/mm2, 600 g = 89.3 +/- 13.0 mN/mm2; ACh [10(-4) M]: 17 g = 76.3 +/- 16.0 mN/mm2, 600 g = 127.0 +/- 13.0 mN/mm2). The sensitivities of the various groups to ACh were not significantly different (e.g., EC50: 30 g = 4.0 +/- 0.2 x 10(-6) M, 600 g = 3.7 +/- 1.1 x 10(-6) M). EFS produced frequency-dependent contractile responses in all groups. With increasing fetal size, there was a corresponding increase in force. When this force was normalized to a maximum ACh response (10(-4) M), there was no significant difference between groups of fetuses. Histologic examination showed the major tissue components of the trachea were present in fetuses above 7 g. Immunocytochemistry detected myosin, caldesmon, and filamin in the smooth muscle from fetuses of 7 g and above, showing that contractile and actin-binding proteins were present from a very early age. It is concluded that smooth muscle contractile function is well developed very early in fetal life in pigs.

Acetylcholine↗

Thy-1-like immunoreactivity in developing chicken skeletal muscle: identification of a cross-reactive slow-fiber specific molecule that is not Thy-1.

We investigated the Thy-1-like immunoreactivity during the development of chicken skeletal muscle using a group of monoclonal antibodies raised and characterised against purified chicken brain Thy-1. The immunoreactivity attributable to bona fide Thy-1 in muscle was present in nerves, connective tissue associated with the intrafusal capsule and blood vessels, and the extracellular matrix of the muscle fibres. During development there was no change in the staining of nerves, blood vessels or intrafusal capsules. However, the extracellular staining of muscle first appeared around hatching and gradually increased in intensity reaching maximal levels in the adult. The intensity of staining varied within and between the muscles examined. One of the antibodies (SB1 20.11) recognised an additional molecule that is not Thy-1 and that was localised in the cytoplasm of slow muscle fibres. This immunoreactivity was first detectable at E10 in all myotubes that contained both alkali and acid stable myosin ATPase activity (presumptive slow), but not in those myotubes with only alkali-stable myosin ATPase activity (presumptive fast). Thereafter, the staining increased to a maximum in the newly hatched animal and then decreased until reactivity was undetectable in the adult (greater than 25 weeks). All positive fibres initially stained with a uniform intensity but the time of commencement and the rate of loss of staining was variable. Those fibres that contained both acid stable and acid labile myosin ATPase activity lost the antigen much faster than the fibres containing only acid-stable myosin ATPase activity, which also tended to increase in intensity for a longer period. These may represent, respectively, the slow tonic type III fibres and the slow twitch type I fibres classified by Barnard et al. (1982).

Animals↗

In vitro developmental expression of dystroglycan and laminin-alpha2 in human skeletal muscle.

The alpha-subunit of dystroglycan, a member of the dystrophin associated protein complex, binds to extracellular laminin-alpha2, while its beta-subunit interacts with cytoskeletal dystrophin. The exact biological role of dystroglycan, especially during human skeletal muscle development, has not been fully explored. Here, we analysed the distribution and expression characteristics of both dystroglycan subunits and laminin-alpha2 in primary human skeletal muscle cells. During development, expression levels of all three proteins increased with differentiation. The proteins were relocated from the sarcoplasm to the sarcolemma. The size of alpha-dystroglycan decreased from 150-220 kDa at the proliferation stage to 100-120 kDa at the late developmental stage. Both alpha- and beta-dystroglycan were involved in forming a complex with their respective partners laminin-alpha2 and dystrophin/utrophin. Our data show that, during development, cells may employ tightly regulated post-translational species-specific modification to produce different isoforms of alpha-dystroglycan to participate in appropriate functions.

Cell Culture Techniques↗

Inhibition of the cardiac alpha-actin gene in embryonic cardiac myocytes by dominant-negative serum response factor.

Serum response factor (SRF), a transcription factor ubiquitously involved in the processes of cellular proliferation and differentiation, has been implicated in cardiac and skeletal muscle development because of its strong expression in embryonic muscle lineages, and its necessity for the transcription of transiently transfected muscle genes that contain SRF binding sites. This study was designed to ascertain whether SRF is required for the expression of an endogenous SRF-dependent gene during differentiation of early embryonic cardiac myocytes by introducing a dominant-negative SRF construct via retroviral delivery. Although no effect on overt cellular differentiation was detected, semi-quantitative RT-PCR revealed that expression of the SRF-dependent gene cardiac alpha-actin was inhibited, whereas expression of the non-SRF-dependent genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cardiac troponin-C was unaffected. No effect on myocyte proliferation was detected. Curiously, immunohistochemical localization of SRF protein suggested that whereas endogenous SRF was homogeneously dispersed throughout the cytoplasm and nucleus, the dominant-negative SRF protein was concentrated in the nucleus. These results extend previous findings using transiently transfected genes to the endogenous level, indicating that SRF is required for the full expression of muscle genes that contain SRF binding sites during cardiac myocyte differentiation.

Actins↗

Muscle trophic factor: II. Ontogenic development of activity of a muscle trophic factor in chicken serum.

The activity of chicken skeletal muscle cell trophic factor was greater in chicken serum than in other tissues examined. The variation of this activity in chicken serum was studied pre- and post hatching. The activity of the factor in the embryo was high from day 11 to day 13 of incubation and then decreased, reaching a minimum on day 16. It subsequently increased again, reaching a peak around the day of hatching. After hatching, the activity in serum began to decrease on day 4, reaching a minimum at day 18. In the hen, activity did not very significantly beyond day 18. In the rooster, however, activity increased gradually from about day 50, reached a new maximum by day 110, and was maintained at this level thereafter. The ratio of the average activity of serum from roosters older than 110 days to that of hens of the same age was about 4:3. The time course of activity of the trophic factor closely parallels that of the growth and differentiation of the muscle in vivo.

Animals↗

Morphogenesis of the human gluteus maximus muscle arising from two muscle primordia.

In human embryos and fetuses a supernumerary muscle was found situated on the distal margin of the gluteus maximus muscle and supplied by the most distal main branch of the inferior gluteus nerve. According to its origin and insertion it is being named the coccygeofemoralis muscle. In embryos and fetuses of up to 40 mm in CR length the coccygeofemoralis muscle is separated by loose connective tissue from neighbouring fetal muscles. Later on, close contact between the coccygeofemoralis and the distal margin of the fetal gluteus maximus muscle develops, and during the prenatal period both fetal muscles gradually fuse. Postnatally, the coccygeofemoralis muscle is incorporated into the gluteus maximus muscle of which the pars sacroiliaca corresponds to the fetal gluteus maximus itself and the pars coccygea represents the fetal coccygeofemoralis muscle. With respect to the general process of muscle morphogenesis, the developmental pattern described for the gluteus maximus muscle demonstrates that adult muscles may be formed by a fusion of several fetal muscles.

Buttocks↗

Identification of regulatory modules mediating specific expression of the roughest gene in Drosophila melanogaster.

Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily with pleiotropic functions during the development of Drosophila melanogaster. It has been shown to be involved in cell sorting before apoptosis in the developing compound eye, in fusion processes of embryonic muscle development and in axonal pathfinding. In accordance with its multiple functions, the rst gene shows a dynamic expression pattern throughout the development of Drosophila. In order to understand the transcriptional regulation of rst expression we have identified rst cis regulatory sequences in an enhancer detection screen. By dissection of the identified rst cis regulatory sequences we identified several distinct rst regulatory modules. Among others these include elements for expression in interommatidial cells of the pupal eye disc at a time when apoptotic decisions are made in these cells and elements for expression in the embryonic mesoderm. The expression of rst in the embryonic mesoderm is regulated by at least two separate modules.

Animals↗