High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator

Efficiency and reproducibility of gene electrotransfer depend on the electrical specifications provided by the pulse generator, such as pulse duration, pulse number, pulse frequency, pulse combination, and current intensity. Here, we describe the performances of GET42, a pulse generator specifically designed for gene electrotransfer into skeletal muscle. Expression of beta-galactosidase in the Tibialis anterior muscle of Sprague-Dawley male rats was increased 250-fold by GET42 compared to DNA injection alone. Combination of high and low current intensity pulses further increased transfection efficiency (400-fold compared to DNA injection without electrotransfer). Varying degrees of muscle necrosis were observed after gene electrotransfer. Nevertheless, muscle necrosis was dramatically reduced after optimization of cumulated pulse duration without significant reduction in transfection efficiency. Physiological applicability was illustrated by the analysis of cytochrome c promoter transactivation. In conclusion, GET42 has proven to be a reliable and efficient pulse generator for gene electrotransfer experiments, and provides a powerful mean to study in vivo the regulation of gene expression.     

Change of gene expression on protein uptake composition and hindlimb-suspension in rat skeletal muscle

[Purpose]

This study was to investigate changes in BCAT and BCKDH genes by Hindlimb-Suspension (HS) and protein intake composition (casein, Whey protein) in rats.

[Methods]

Following 5-day preliminary feeding, forty-eight male 5 weeks old Sprague Dawley albino rats (110g) divided into 17% protein intake group (24 rats) and 30% protein intake group (24 rats), and each group divided further into Hindlimb-Suspension group (HS; 12 rats) and control group(CON; 12 rats). Eventually, this study was performed with Whey protein intake group (HS; 6 rats, CON; 6 rats) and casein intake group (HS; 6 rats, CON; 6 rats). For analysis purposes, total RNA was extracted from isolated skeletal muscles, and mRNA expression was analyzed using Real Time PCR. Two-way ANOVA was performed to examine the difference in BCATm and BCKDH mRNA expression on protein uptake and myoatrophy. post-hoc test was perform on interaction if any, and significance level was set at p<0.05.

[Results]

In this study, BCATm and BCKDH gene analysis in rat skeletal muscles by hindlimb-suspension and protein intake composition resulted in significant higher BCATm expression in 30% dietary protein group and hindlimb-suspension group that control group. In addition, regarding BCKDH, BCKDH was significantly higher in hindlimb-suspended 30% protein intake group than control group.

[Conclusion]

Overall, protein intake and myoatrophy demonstrated close relationship in skeletal muscles. Therefore, it is likely to affect effectively in prevention or recovery of exercise induced muscle disorder. This effect is considered to be applied to maintain and improve health of not only athletes but also the general public. Additionally it would be applied in convalescent rehabilitation due to skeletal muscle atrophy.     

Protein and collagen content of rat skeletal muscle following space flight

Summary Biochemical determinations of non-collagenous protein and hydroxyproline were made on rat skeletal muscles following 7 days of space flight aboard the NASA space shuttle mission SL-3. Relative to ground-based controls, the wet weight of each experimental muscle was significantly reduced. This was concomitant with a reduction in noncollagenous protein in the muscles. Protein concentration, however, was reduced only in slow-twitch muscles. The effect of space flight on the concentration and hydroxyproline content was different among the muscles. As a result, the loss of muscle mass in some muscles was the consequence of a reduction in both collagenous and non-collagenous proteins, while in others it was primarily the result of a non-collagenous protein loss.     

MicroRNAs in skeletal muscle biology and exercise adaptation

MicroRNAs (miRNAs) have emerged as important players in the regulation of gene expression, being involved in most biological processes examined to date. The proposal that miRNAs are primarily involved in the stress response of the cell makes miRNAs ideally suited to mediate the response of skeletal muscle to changes in contractile activity. Although the field is still in its infancy, the studies presented in this review highlight the promise that miRNAs will have an important role in mediating the response and adaptation of skeletal muscle to various modes of exercise. The roles of miRNAs in satellite cell biology, muscle regeneration, and various myopathies are also discussed.     

Early postnatal growth of skeletal muscle blood vessels of the rat

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Diversification of the myofibrillar M-band in rat skeletal muscle during postnatal development

Summary The fine structure of the M-band in soleus (SOL) and extensor digitorum longus (EDL) muscles in newborn and four-week-old rats was studied using electron-microscopic techniques. In newborn rats, all myotubes and fibres in both muscles had an identical myofibrillar appearance. A five-line M-band pattern was seen in longitudinal sections and distinct M-bridges in cross-sections. The Z-discs were of medium width. On the other hand, in four-week-old rats, different muscle fibre types were observed on the basis of their myofibrillar pattern. In SOL two fibre types were distinguished in longitudinal sections. One had a four-line M-band pattern and very broad Z-discs, whereas the other type had five lines in the M-band and broad Z-discs. In EDL, three different myofibrillar patterns were observed. The M-bands were composed of three, four or five lines. Fibres had either thin, broad or medium Z-disc widths, respectively. In cross-sections of the SOL muscle one group of fibres showed indistinct M-bridges, whereas distinct M-bridges were seen in the other fibres and in all observed EDL muscle fibres. We conclude that initially there seems to be a single intrinsic program for M-band genesis; this program becomes modified upon the induction of functionally differentiated fibres.     

Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues

Fibroblast growth factors are believed to play many distinct roles in vertebrate development, owing to their ability to stimulate cell growth, prevent cell death, determine cell fate, and inhibit terminal differentiation in a variety of in vitro culture systems. We have used in situ hybridization to localize fibroblast growth factor-4 (FGF-4, also termed HST and K-FGF) gene expression in 7.5 to 16.5 day gestation mouse embryos. Seven discrete sites of gene expression were detected: (1) primitive streak (E7.5–8.5); (2) paraxial presomitic mesoderm in the trunk (E7.5–11.5); (3) primitive neuroectoderm (E8.0–8.5); (4) pharyngeal pouch endoderm (E8.5–9.5); (5) branchial arch ectoderm (E8.5–9.5); (6) limb apical ectoderm (E10.5–12.5), and (7) skeletal myoblast groups (E9.5–13.5). FGF-4 gene expression is spatially restricted within many of these sites. The profile of FGF-4 gene expression among skeletal muscle groups is overlapping, but distinct, from that of FGF-5, thereby revealing myoblast heterogeneity at the molecular level and suggesting distinct roles for multiple FGFs in muscle development.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Protocol for rat single muscle fiber isolation and culture

To attain a superior in vitro model of mature muscle fibers, we modified the established protocol for isolating single muscle fibers from rat skeletal muscle. Muscle fiber cultures with high viability were obtained using flexor digitorum brevis muscle and lasted for at least 7 days. We compared the expression levels of adult myosin heavy chain (MyHC) isoforms in these single muscle fibers with myotubes formed from myoblasts; isolated fibers contained markedly more abundant adult MyHC isoforms than myotubes. This muscle fiber model, therefore, will be useful for studying the various functions and cellular processes of mature muscles in vitro.     

Phosphoenolpyruvate-dependent protein kinase activity in rat skeletal muscle

Phosphoenolpyruvate-dependent protein kinase activity has been demonstrated in the soluble fraction of rat skeletal muscle. The reaction was not due to the formation of ATP in the incubation mixture. Cyclic AMP, calcium, ATP and a number of phosphate acceptor proteins did not stimulate the reaction. One 32P-labelled protein (Mr 25000) was observed on SDS gels. The phosphorylated protein contained acid stable phosphoserine as a major phosphorylated amino acid. The phosphorylation reaction in crude extracts was not directly proportional to the amount of protein, but typical of a two-component system; i.e., kinase and substrate. The chromatography of soluble proteins on Ultrogel AcA44 separated the phosphate acceptor protein(s) from the phosphoenolpyruvate-dependent protein kinase activity.     

Resident macrophages (ED2- and ED3-positive) do not phagocytose degenerating rat skeletal muscle fibres

Two soleus muscles with degenerating muscle fibres were serially sectioned and adjacent sections from various levels of the skeletal muscles were stained with antibodies that react with either monocytes and inflammatory macrophages (ED1) or with the major subpopulations of resident macrophages (ED2 and ED3). ED2⁺ and ED3⁺ resident macrophages were abundant throughout the muscles but were not present within the degenerating fibres. ED1⁺ cells, in contrast, were rarely observed within the undamaged regions of the muscles but were abundant within the degenerating fibres and in the perimysium between arterioles and degenerating fibres. It is concluded that the phagocytosis of damaged muscle fibres is not carried out by the major subpopulations of resident macrophages.     

Identification of helical polyribosomes in sections of mature skeletal muscle fibers

Summary The localization and configurations of ribosomes in mature white skeletal muscle fibers of the rat were investigated. Differential visualization of ribosomes and glycogen granules was obtained by fixation with glutaraldehyde only and staining of the sections with uranyl acetate. Ribosomes are then electron dense and glycogen granules electron transparent. Their identity was ascertained by selective extractions of ribonucleic acid and polysaccharide.The vast majority of the ribosomes is not membrane-bound. They are located intermyofibrillarly (predominantly at the level of the I-bands), beneath the sarcolemma, and in the paranuclear cones of sarcoplasm. Occasionally short stretches of granular reticulum occur, often as characteristic double walled vesicles with ribosomes on the inner membrane only.Three main types of polysomal configurations are observed: rosettes of 4 to 6 ribosomes, helical arrays, and whorls of up to about 25 probably membrane-bound ribosomes. The average number of ribosomes in the extended helical configurations is estimated to be about 60. It is argued that these helices represent the polysomes instrumental in the synthesis of the large subunits of myosin. It is emphasized that helical polyribosomes are by no means typical of striated muscle, but probably represent a common configuration of large free polysomes.With the technical assistance of Tineke J. Hoogenboezem.     

盐酸布比卡因、透明质酸酶对大鼠在体肌卫星细胞增殖的影响

目的 :研究盐酸布比卡因和透明质酸酶对成年大鼠肌卫星细胞在体增殖的影响。方法 :免疫组化法 ,H .E染色法 ,光镜和电镜观察。结果 :①正常对照组和生理盐水组肌纤维完整 ,有少量Desmin阳性肌卫星细胞 ,面密度值为 0 .66%± 0 .57%和 2 .48%± 1.13 %。生理盐水组较正常对照组无显著差异 (P >0 .0 5)。②透明质酸酶组肌纤维完整 ,Desmin阳性肌卫星细胞数量增加 ,面密度值为 2 .52 %± 1.41% ,较生理盐水组和正常对照组无显著差异(P >0 .0 5)。③盐酸布比卡因组和盐酸布比卡因 +透明质酸酶混合液组均可见大量坏死和溶解的肌纤维 ,并伴有肌卫星细胞的激活、增殖 ,Desmin阳性肌卫星细胞显著增加 ,并有部分融合形成小肌管。面密度值分别为 19.0 1%± 4.74%和 2 2 .41%± 7.64% ,较生理盐水组显著增加 (P <0 .0 1)。结论 :局麻药盐酸布比卡因能引起在体肌卫星细胞的活化、增殖并形成肌管 ,单独透明质酸酶溶液在本实验条件下对在体肌卫星细胞无明显作用     

Intracellular distribution of fumarase in rat skeletal muscle

The distribution of fumarase activity between the mitochondrial and cytoplasmic compartments of rat skeletal muscle was studied using the method of Fatania and Dalziel (Biochim. Biophys. Acta 631 (1980) 11–19), fractional extraction technique and a method based on the calculation of mitochondrial protein content in the tissue and on the determination of fumarase activity both in the tissue homogenate and in the isolated mitochondria. We found 10%, 5% and 0% of the total fumarase activity in the cytoplasm using these methods, respectively. The results suggest that no more than 10% of the total fumarase activity is present in the cytosolic fraction of rat skeletal muscle. The metabolic consequences of such distribution of fumarase in skeletal muscle are discussed.     

Effect of cold environment on skeletal muscle mitochondria in growing rats

Summary Growing rats (4 weeks old) were kept for 3 weeks at 11° C and 24° C respectively. The cold-adapted animals showed a significantly higher oxygen consumption (64%). Volume density of subsarcolemmal and interfibrillar mitochondria as well as volume density of fat droplets were estimated in M. soleus and the diaphragm of both groups. In cold-adapted animals, the total volume of mitochondria was significantly increased by 24% in diaphragm and 37% in M. soleus. The volume of subsarcolemmal mitochondria was almost doubled in each muscle, but the volume of interfibrillar mitochondria did not change significantly. The surface of the inner mitochondrial membranes per unit volume of mitochondrion in M. soleus was significantly increased both in interfibrillar and subsarcolemmal mitochondria, whereas the surface of the outer mitochondrial membranes per unit volume of mitochondrion was increased only in the subsarcolemmal mitochondria. The volume of fat droplets in the diaphragm and M. soleus of cold adapted animals increased significantly by 62% and 150% respectively.     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。