The use of skeletal muscle to express genes for the treatment of cancer.
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Adenoviruses, Human↗
Biomedical subjects
G Goldspink
Publications and source records attributed to G Goldspink.
Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish.
To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.
Adenosine Triphosphatases↗
Postprandial resynthesis of myofibrillar proteins is translationally rather than transcriptionally regulated in human skeletal muscle.
Feeding stimulates protein synthesis in skeletal muscles, although the regulatory mechanisms are incompletely understood. The aim of this study was to determine whether this could be detected at the gene transcription level for postprandial stimulation of the synthesis of muscle proteins. Healthy male volunteers were investigated after an overnight fast. Open muscle biopsies were performed in the starved state and 3 h after meal intake, consisting of 0.15 gN/kg, 12 kcal/kg. Blood samples were drawn every 15 to 30 min for 5 h. Myosin mRNA and insulin growth factor-I (IGF-I) mRNA were measured by solution hybridization assay in homogenized muscle specimens. After food intake, plasma glucose concentrations increased from 5.0 +/- 0.1 to 7.3 +/- 0.3 (P < or = 0.001), and insulin concentration rose from 3.8 +/- 0.5 mU/L before to 75.3 +/- 11.4 15 min after the meal (P < or = 0.001). Plasma concentration of free fatty acids declined after food intake (P < or = 0.001). Plasma concentrations of amino acids increased from basal values (2864 +/- 128 microM) to 4419 +/- 262 microM (P < or = 0.05) 90 min after meal ingestion. Myosin mRNA concentration in the biopsied muscle tissue was higher during starvation and was reduced by 20% after food intake: 10.8 +/- 1.3 amol mRNA/microgram DNA in the starved state and 8.5 +/- 1.3 amol mRNA/microgram DNA after food intake (P < or = 0.05). Feeding did not alter IGF-I mRNA concentrations in muscle: 0.51 +/- 0.05 and 0.55 +/- 0.06 amol/microgram DNA in the starved and fed state, respectively (P < or = 0.48). Improved protein balance by stimulation of protein synthesis has been related to increased plasma amino acids. Interestingly, in the short term, this was not related to increases in gene transcription of either myofibrillar proteins (myosin) or muscle IGF-I. Thus, postprandial stimulation of protein synthesis appears not to be regulated by increased gene transcription but by increased translation using the increased concentrations of amino acids. In contrast, as far as the 2X myosin mRNA level is concerned, this is enhanced during starvation, which facilitates rapid recovery once the availability of substrate is resumed.
Adult↗
Protein and mRNA levels of the myosin heavy chain isoforms Ibeta, IIa, IIx and IIb in type I and type II fibre-predominant rat skeletal muscles in response to chronic alcohol feeding.
Alcoholic myopathy occurs in between one and two-thirds of all alcohol misusers and is thus one of the most prevalent muscle disorders (2000 cases per 100,000 population). It is characterised by myalgia, muscle weakness and loss of lean tissue mass. Histological features include a reduction in the diameter of Type II muscle fibres, particularly the IIb fibre subset. In contrast, Type I fibres are relatively protected. It is possible that the myopathy is due to perturbations in myosin protein and mRNA expression. To test this hypothesis, we fed rats a liquid diet containing 35% of calories as ethanol. Control rats were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At the end of 6 weeks, total myofibrillary proteins and myosin heavy chain (MyoHC) Ibeta, IIa, IIx and IIb protein and mRNA were analysed in the plantaris (Type II fibre-predominant) and soleus (Type I fibre-predominant) muscles. The data showed that there were significant reductions in the total myofibrillary protein content in the plantaris of ethanol fed rats compared to pair-fed controls (P < 0.05). These changes in the plantaris were accompanied by reductions in total myosin (P < 0.025), as a consequence of specific reductions in the Ibeta, (P < 0.01), IIx (P < 0.05) and IIb (P < 0.05) protein isoforms. The mRNA levels of Ibeta were significantly reduced in the plantaris (P < 0.05). However, mRNA levels of IIa, IIx and IIb in the plantaris were not significantly affected by alcohol feeding. Other changes in the plantaris included significant reductions in desmin (P < 0.01), actin (P < 0.025), and troponin-I (P < 0.05) compared to pair-fed controls. In the soleus, the only significant changes related to a fall in Ibeta mRNA levels and a decline in troponin-C content. We conclude that in the rat, alcoholic myopathy is a feature of Type II fibre rich muscles and is accompanied by multiple protein changes. The decline in specific myosin protein levels, such as IIx and IIb in the absence of corresponding reductions in their mRNAs, is probably due to altered proteolysis or more likely reductions in translational efficiencies, rather than changes in transcription.
Alcohol Drinking↗
Semi-starvation alters myofibrillar mRNA concentrations to expedite rapid recovery of muscle protein stores following feeding.
BACKGROUND: Protein synthesis in skeletal muscle is reduced following starvation and restored by feeding. The mediators and mechanisms are incompletely understood. The aim of this study was to evaluate whether prolongation of undernutrition induced changes in muscle gene expression at the level of mRNA and protein. MATERIALS AND METHODS: The changes in myosin heavy-chain 2X mRNA in adult partially starved (50% of ad libitum standard rodent chow intake for 4 or 7 days) C57BL mice or subsequently refed mice were studied. Ad libitum-fed mice were used as controls. Protein synthesis, total RNA and myosin heavy-chain 2X mRNA concentrations were determined. Plasma concentrations of amino acids were measured by high-performance liquid chromatography. RESULTS: Partial starvation of 4 and 7 days reduced bodyweight by 15.6 +/- 1% and 17.1 +/- 2.1% (P < or = 0.05) vs. ad libitum fed controls. Protein synthesis was reduced by 32 +/- 9% and protein content by 20 +/- 4% (P < or = 0.05) following 7 days of partial starvation. Plasma amino acid concentrations were increased (6297 +/- 853) in refed animals vs. ad libitum-fed controls (3057 +/- 141, P < or = 0.05). Total RNA concentration (micrograms RNA micrograms(-1) DNA) in skeletal muscle was unchanged. Myosin heavy-chain 2X mRNA concentration did not change following 4 days of partial starvation but increased by 24 +/- 5% (P < or = 0.05) following 7 days of partial starvation, hence suggesting that expression of myosin mRNA was nutritionally altered. CONCLUSION: Postprandial stimulation of protein synthesis following starvation may thus be a combination of increased mRNA availability and increased translation. This effect may be activated by peak concentrations of amino acids in plasma following feeding.
Amino Acids↗
Cloning of local growth factors involved in the determination of muscle mass.
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Cloning, Molecular↗
Expression of myosin heavy-chain isoforms in the respiratory muscles following inspiratory resistive breathing.
We investigated the effect of inspiratory resistive breathing (IRB) on the expression of the genes encoding fast and slow isoforms of myosin heavy chain (MyHC) in respiratory muscles. Eleven mongrel dogs were studied for baseline MyHC messenger RNA (mRNA) expression, seven of which were also used to study the effects of IRB. For this latter objective, awake and spontaneously breathing animals were subjected to 2 h of IRB (80 cm H(2)O/L/s) per day for four consecutive days. mRNA expression was assessed in the diaphragm, external intercostal muscle, and a limb muscle, using both slot- blot and in situ hybridizations with isoform-specific probes. A current semiquantitative scoring method (from 0 to 4) was used to quantify the in situ mRNA expression levels, and slot-blot data were analyzed with densitometry. Prior to IRB, slow- and fast-MyHC mRNA expression was moderate, similar, and homogeneous throughout the different regions of the diaphragm, with scores of 1.50 +/- 0.54 (mean +/- SD) for slow and 2.13 +/- 0.35 for fast mRNAs in the costal region of the diaphragm, and of 1.81 +/- 0.37 for slow and 2. 13 +/- 0.64 for fast mRNAs in the crural region of the diaphragm. Although expression of fast-MyHC mRNA remained unchanged after IRB, the relative expression of the mRNA for the slow isoform increased in costal (+30%), crural (+12%), and external intercostal (+27%) muscles. MyHC mRNA expression did not change in limb muscles. We conclude that breathing with a moderate inspiratory resistance for a short period induces the expression of slow MyHC in respiratory muscles.
Animals↗
Effects of environmental temperature on the development of the myotomal white muscle in larval carp (Cyprinus carpio L.).
A study was conducted on common carp (Cyprinus carpio L.) to determine the effects of environmental temperature experienced by embryos and larvae on the development of myotomal white muscle. Eggs from one female were divided into two groups following fertilisation and incubated at constant pre-hatch temperatures of 18 or 28 degrees C. At hatching, larvae from the 18 degrees C-incubated eggs were divided into two groups and either reared at the same temperature of 18 degrees C ('cold' group) or transferred over a period of 5 days (at 2 degrees C per day) to 28 degrees C ('transferred' group). Larvae hatched from eggs incubated at 28 degrees C were reared at the same temperature of 28 degrees C ('warm' group). Larvae were sampled at two developmental stages (stage 1, inflation of the back chamber of the swimbladder; stage 2, inflation of the front chamber of the swimbladder) and at 26 days post-hatching. The maturation of myotome shape during larval life was studied in parallel with the changes occurring in the organisation of white fibres. At stage 1, the epaxial part of the myotomes surrounding the vent had the shape of lamellae inclined backwards, and only one central layer of white fibres was present. At stage 2, the epaxial part of the myotomes began to acquire a V-shape, which was well developed at 26 days post-hatch. At stage 2 and at 26 days post-hatch, two layers of white fibres were identified: the initial central layer and a second apical layer. These differ in their orientation, the initial central layer being orientated backwards and the apical layer forwards, and in the mean fibre diameter, which is greater in the initial central layer. Studies on the effects of temperature (constant 18 degrees C, constant 28 degrees C, transfer from 18 to 28 degrees C at hatching) were carried out according to both the developmental stage and the length of the larvae. At stage 1, no significant differences were found between the three groups for larval standard length and muscle variables. The number of fibres in one quadrant of epaxial white muscle sectioned at the level of the vent was 100-111. At stage 2, there were significant differences between groups. Larval standard length and mass were higher in the cold group than in the warm group. The transferred larvae were of intermediate standard length but had a significantly higher cross-sectional area of white muscle than either of the other two groups. This increase in surface area was related to a 50 % greater fibre number (233) in the transferred larvae compared with the cold (165) or the warm (152) larvae. The increase in fibre number was more marked for large-diameter ( >20 microm) white fibres located in the initial central fibre layer (+58-72 % in transferred larvae) than in small-diameter ((less than equal to) 10 microm) white fibres mainly located in the apical layer (+18-35 %). In 26 days post-hatch samples, transferred larvae still showed a higher total number of white fibres than warm larvae, but the difference was no longer significant when the total number of white fibres was regressed against larval standard length, suggesting that this stimulation may be temporary.
Animals↗
Optimisation of growth hormone production by muscle cells using plasmid DNA.
The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm. The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0.82 O.D. units+/-0.06 vs 0.57+/-0.05 respectively, P<0.001). This was consistent with reduced CMV promoter activity in myotubes. Myoblasts transfected with pcDNA3-GH secreted more bioactive GH than 10-day transfected myotubes (1.1+/-0. 1 vs 0.77+/-0.07 respectively). However, the responses were indistinguishable (both 1.0+/-0.09) if both the myotubes and myoblasts had been transfected with pcDNA3E-GH. Substitution of the vector pMHLC-GH, which used a muscle-specific truncated rabbit myosin heavy chain promoter, and the myosin enhancer resulted in a marked decrease in the responses to the conditioned medium from fused myotubes compared with the vectors pcDNA3-GH and pcDNA3E-GH (0. 24+/-0.02 vs 0.57+/-0.05 vs 0.82+/-0.06 respectively). We concluded that the combination of CMV promoter and myosin light chain enhancer in pcDNA3E-GH had the greatest expression efficiency of the several plasmid vectors which we investigated.
Animals↗
Effect of rate of distraction on loss of range of joint movement, muscle stiffness, and intramuscular connective tissue content during surgical limb-lengthening: a study in the rabbit.
Surgical lengthening of limbs often results in loss of range of joint movement and this has been shown to be associated with an increase in passive tension and an increase in collagen content of the muscles. In this study, we have investigated the length/tension properties and the connective tissue component of muscle distracted at three different rates in order to determine whether low rates of distraction would enable the connective tissue component, as well as the contractile component (number of serial sarcomeres), to adapt more completely to the increased functional length of the muscle and thus lead to improved range of joint movement. It was found that loss of range of movement varied with rate of distraction. At the low rate, there was no change in the passive tension or collagen content compared to muscles from sham-operated animals, and range of movement was significantly greater than at the other rates. At the medium rate, although the muscles showed good adaptation in terms of serial sarcomere number, passive tension and collagen content was increased and range of movement reduced, indicating that changes in the connective tissue component are important factors in loss of joint movement. In the case of muscle distracted at a high rate, failure of the muscle fibres to add on sufficient sarcomeres, combined with changes in the connective tissue, resulted in almost total loss of joint movement.
Animals↗
Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation.
1. Skeletal muscle is a major source of circulating insulin growth factor-1 (IGF-1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF-1 isoforms (muscle L.IGF-1) is similar to the main liver IGF-1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3' exon sequence and is apparently designed for an autocrine/paracrine action (mechano-growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF-1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. 2. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF-1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF-1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham-operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF-1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. 3. The expression of the beta actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF-1 may initially be a response to local damage. 4. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF-1 and beta actin, the decreased expression of the 2X myosin mRNA was less marked in the 'stretch only' muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF-1.
Actins↗
Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload.
The study of the underlying mechanisms by which cells respond to mechanical stimuli, i.e. the link between the mechanical stimulus and gene expression, represents a new and important area in the morphological sciences. Several cell types ('mechanocytes'), e.g. osteoblasts and fibroblasts as well as smooth, cardiac and skeletal muscle cells are activated by mechanical strain and there is now mounting evidence that this involves the cytoskeleton. Muscle offers one of the best opportunities for studying this type of mechanotransduction as the mechanical activity generated by and imposed upon muscle tissue can be accurately controlled and measured in both in vitro and in vivo systems. Muscle is highly responsive to changes in functional demands. Overload leads to hypertrophy, whilst decreased load force generation and immobilisation with the muscle in the shortened position leads to atrophy. For instance it has been shown that stretch is an important mechanical signal for the production of more actin and myosin filaments and the addition of new sarcomeres in series and in parallel. This is preceded by upregulation of transcription of the appropriate genes some of which such as the myosin isoforms markedly change the muscle phenotype. Indeed, the switch in the expression induced by mechanical activity of myosin heavy chain genes which encode different molecular motors is a means via which the tissue adapts to a given type of physical activity. As far as increase in mass is concerned, our group have cloned the cDNA of a splice variant of IGF-1 that is produced by active muscle that appears to be the factor that controls local tissue repair, maintenance and remodelling. From its sequence it can be seen that it is derived from the IGF-1 gene by alternative splicing but it has different exons to the liver isoforms. It has a 52 base insert in the E domain which alters the reading frame of the 3' end. Therefore, this splice variant of IGF-1 is likely to bind to a different binding protein which exists in the interstitial tissue spaces of muscle, neuronal tissue and bone. This would be expected to localise its action as it would be unstable in the unbound form which is important as its production would not disturb the glucose homeostasis unduly. This new growth factor has been called mechano growth factor (MGF) to distinguish it from the liver IGFs which have a systemic mode of action. Although the liver is usually thought of as the source of circulating IGF-1, it has recently been shown that during exercise skeletal muscle not only produces much of the circulating IGF-1 but active musculature also utilises most of the IGF-I produced. We have cloned both an autocrine and endocrine IGF-1, both of which are upregulated in cardiac as well as skeletal muscle when subjected to overload. It has been shown that, in contrast to normal muscle, MGF is not detectable in dystrophic mdx muscles even when subjected to stretch and stretch combined with electrical stimulation. This is true for muscular dystrophies that are due to the lack of dystrophin (X-linked) and due to a laminin deficiency (autosomal), thus indicating that the dystrophin cytoskeletal complex may be involved in the mechanotransduction mechanism. When this complex is defective the necessary systemic as well as autocrine IGF-1 growth factors required for local repair are not produced and the ensuing cell death results in progressive loss of muscle mass. The discovery of the locally produced IGF-1 appears to provide the link between the mechanical stimulus and the activation of gene expression.
Adaptation, Physiological↗
Identification and expression analysis of two developmentally regulated myosin heavy chain gene transcripts in carp (Cyprinus carpio).
Whilst developmentally regulated genes for the myosin heavy chain (MyoHC) have been characterised in mammalian, avian and amphibian species, no developmental MyoHC gene has previously been characterised in a species of fish. In this study, we identify two developmentally regulated MyoHC gene transcripts (named Eggs22 and Eggs24) in carp (Cyprinus carpio) and characterise their expression patterns during embryonic and larval development. The transcripts showed an identical temporal pattern of expression commencing 22 h post-fertilisation (18 degrees C incubation temperature), coincident with the switch from exclusive expression of genes for beta-actin to expression of genes for both beta- and alpha-actin, and continuing for 2 weeks post-hatching. No expression of these myosin transcripts was detected in juvenile or adult carp. Wholemount in situ hybridisation showed that both transcripts are expressed initially in the rostral region of the developing trunk and progress caudally. Both are expressed in the developing pectoral fin and protractor hyoideus muscles. However, the muscles of the lower jaw express only the Eggs22 transcript. No expression of either transcript was detected in cardiac or smooth muscle. A distinct chevron pattern of expression was observed in the myotomal muscle. This was shown to be caused by localisation of the mRNAs to the myoseptal regions of the fibres, the sites of new sarcomere addition during muscle growth, suggesting transport of MyoHC mRNA transcripts. The 3' untranslated region of the Eggs24 transcript contains a 10 base pair motif (AAAATGTGAA) which is shown to be also present in the 3' untranslated regions of MyoHC genes from a wide range of species. Possible reasons for the need for developmental isoforms of myosin heavy chain isoforms are discussed.
Amino Acid Sequence↗
Using skeletal muscle as an artificial endocrine tissue.
Gene transfer into muscle tissue is currently being developed as a method for the production, secretion and delivery of therapeutic proteins. This methodology has been used to produce a variety of physiologically active proteins and may ultimately be applied to the treatment of several diseases. In this review, we consider several applications of this methodology and discuss approaches for modulating therapeutic protein production and secretion from muscle, using growth hormone as an example. In addition, factors limiting the effectiveness of muscle gene transfer are also discussed, as these shall determine the efficacy of muscle gene transfer when applied to humans.
Gene Expression Regulation↗
Partial cardiomyectomy--an animal model of gastro-oesophageal reflux.
Various procedures have been adopted to provide experimental models of gastro-oesophageal reflux. Although some procedures have produced better results than others, previous literature does not assert that experimental gastro-oesophageal reflux can be produced reliably in all animals. This study attempts to compare two procedures to produce a working model of gastro-oesophageal reflux in the rabbit: (i) cardiomyotomy and (ii) cardiomyectomy. The animals underwent intraoesophageal pH monitoring one week before and one week following surgery. The mean changes in % reflux time from preoperative to one week postoperative were compared. The cardiomyotomy group did not demonstrate significant gastro-oesophageal reflux (p > 0.2, p < 0.5), the mean change in % reflux time was 10.5 +/- 14.11%, whereas the cardiomyectomy group did demonstrate significant reflux (p < 0.001) with a mean change in % reflux time of 22.35 +/- 3.05%. A further study investigated the cardiomyectomy procedure in ten rabbits. Animals underwent preoperative, one-week and four-week postoperative intraoesophageal manometry and pH studies. The mean change in values from preoperative to four weeks postoperative were compared. There was a significant increase in mean % reflux time from a preoperative value of 3.05 +/- 2.29% to 42.08 +/- 12.05% at four weeks after surgery (p < 0.001). There was also a significant decrease in mean basal LOS pressure from a preoperative value of 15.00 +/- 5.49 mmHg to 4.45 +/- 1.90 mmHg four weeks following surgery (p < 0.001). This study showed that cardiomyectomy alone produces effective gastro-oesophageal reflux in the rabbit, and thus provides an experimental model to test the effectiveness of antireflux procedures.
Animals↗
Differential expression of myosin heavy chain mRNA and protein isoforms in four functionally diverse rabbit skeletal muscles during pre- and postnatal development.
Myosin heavy chains (hcs) are the major determinant in the speed of contraction of skeletal muscle, and various isoforms are differentially expressed depending on the functional activity of the muscle. Using the rapid amplification of cDNA ends (3' RACE) method, we have characterised the 3' end of the embryonic, perinatal, type 1, 2a, 2x, and 2b myosin hc genes in rabbit skeletal muscle and used them as probes in RNase protection assays to quantitatively monitor their expression in different type of skeletal muscles just before and after birth. SDS PAGE was used to study the changes in the expression level of their respective protein and to determine the relative abundance of each myosin hc isoform in the muscles studied. The results show that for each anatomical muscle, the developmental changes in myosin hc gene expression at the mRNA level correlate strongly to those observed at the protein level. By studying their developmental expression in four functionally diverse skeletal muscles (semimembranosus proprius, diaphragm, tibialis anterior, and semimembranosus accessorius), it was shown that all muscles express the embryonic, perinatal, and type 1 isoform during prenatal development up to the E27 stage. In the diaphragm, low levels of the type 2a and 2x transcripts, which are adult fast isoforms, were also detected at the E27 stage. During the first week of postnatal growth the myosin hc transition leading to the expression of the adult isoforms is complex, and as many as five different myosin heavy chains are concurrently expressed in some muscles at around birth. As the animal matures, individual muscles become adapted to perform highly specialised functions, and this is reflected in the myosin hc composition within these muscles. Accordingly, the expression of the type 1 isoform, and the sequence of appearance and the expression levels of the type 2 isoforms, were exclusively dependent on the muscle type and largely reflect the functional activity of each muscle during the postnatal growth period.
Animals↗
Selective gene expression during adaptation of muscle in response to different physiological demands.
Muscle is a very adaptable tissue in which gene expression is to a large extent influenced by physical signals. Adaptation to a different work regime is brought about by changes in fibre type and fibre cross-sectional area. We have shown both mass and phenotype are markedly altered by stretch and force production within a period as short as 4 days. This is associated with quantitative as well as qualitative changes in gene expression. The latter involves the expression of myosin heavy chain isogenes which encode different types of molecular motors. Some species of fish have exploited this and they are able to rebuild their myofibrillar systems for warm and cold temperature swimming by selective myosin gene expression. To understand how the different myosin isoform confer different contractile properties methods have been developed for cloning, sequencing and visualizing the structure of the ATPase site to explain how the molecular motors are designed. With regard to the chemical link between the physical signal and the upregulation of certain muscle genes we have cloned a new growth factor that is only expressed in muscles subjected to stretch and/or exercise and which is designed for autocrine/paracrine action. Experiments indicate that the expression of a local growth factor which induces repair, remodelling and hypertrophy is one of the ways cells respond to mechanical strain.
Adaptation, Biological↗
Myosin regulatory elements as vectors for gene transfer by intramuscular injection.
Intramuscular injection of plasmid constructs promises to be an effective way of carrying out gene therapy for muscle disorders as well as using muscle as an in vivo expression system for disorders that involve the gene product being secreted into the bloodstream. The effectiveness of this method depends on the design of the cassette used for the expression of the cDNA of the introduced gene. We tested the levels of expression achieved by a number of muscle-specific promoters and a myosin light chain enhancer when spliced to the reporter gene chloramphenicol acetyltransferase (CAT), in vitro and in vivo by injection into fast and slow muscles of the mouse. The results show that the highest levels of expression are achieved by a combination of a truncated myosin heavy chain promoter and the enhancer, and that a whole range of expression levels is obtained with the other combinations tested. The data show that a cassette based on these elements should provide efficient vectors for the introduction and expression of genes following intramuscular injection of naked DNA.
Animals↗