Satellite cells in the regenerated and regrafted skeletal muscles of rats

Soleus (SOL) muscles were grafted into extensor digitorum longus (EDL) muscle beds (EDL-first-graft). Sixty days later, some mature EDL-first-grafts were regrafted into their own beds (EDL-second-grafts). Fully regenerated muscle fibers and satellite cells were observed in both types of mature grafts. The ratios of satellite cell nuclei per total nuclei (myonuclei and satellite cell nuclei) were 4.81 +/- 0.47% for EDL-2nd graft, 4.26 +/- 0.51% for EDL-1st-graft, 4.30 +/- 0.33% for control SOL, and 3.30 +/- 0.18% for control EDL. It is thought that satellite cells are required for the repeated activity of muscle fiber regeneration. The persistance of satellite cells in EDL-second-grafts suggests that satellite cells are not depleted during the first grafting, making second-grafts possible.     

Oxidative modifications of low-density lipoproteins (LDL) by the human endothelial cell line EA.hy 926

Modifications of LDL by the EA.hy 926 cell line were compared to those generated by human umbilical vein endothelial cells (HUVEC). Thiobarbituric acid reactive substances (TBARS) index values (TBARS sample/TBARS cell-free control ratio) were 2.64±0.18 (m±SE, n=11) and 3.12±0.24 (n=11), for HUVEC and EA.hy 926, respectively. The percentage of the most electronegative modified LDL fraction (fraction C), assessed by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC), represented 14±3% (n=34) and 22±13% (n=10) of total modified LDL in HUVEC and EA.hy 926, respectively. LDL modified by both cell lines showed increased agarose electrophoretic mobility and apo B100 fragmentation on SDS-PAGE. None of the results were significantly different between the two cell lines. Superoxide anion production was 0.12±0.04 (n=11) and 0.07±0.01 nmol/min/mg cell protein (n=11) in HUVEC and EA.hy 926, respectively. Cell-specific effects on LDL were abrogated in cysteine-free medium. Moreover, cell-modified LDL were similarly degraded by J774 macrophage-like cells. We conclude that EA.hy 926 cells are a good model for investigating endothelial cell-induced modifications of LDL. Advantages include ready availability and less individual variability than with HUVEC.     

Effects of the spinal cord section and of subsequent denervation on the mechanical properties of fast and slow muscles

Summary The Soleus muscle of the rat, 3–6 months old, becomes significantly faster than in the controls, if the spinal cord is cut at birth. Mechanical properties of Extensor Digitorum Longus (EDL) muscle are not altered by spinal cord section. In cordotomized animals Soleus muscle always remains slower than EDL muscle. Denervation, performed 3–6 months after birth, has the same slowing effects in the Soleus and EDL muscles, both in cordotomized and in the control animals.     

Culture in low levels of oxygen enhances in vitro proliferation potential of satellite cells from old skeletal muscles

The proliferation ability of satellite cells (considered the 'stem cells' of mature myofibers) declines with increasing age when cultured under standard cell culture conditions of 21% oxygen. However, actual oxygen levels in the intact myofiber in vivo are an order of magnitude lower. No studies to date have addressed the issue of whether culturing satellite cells from old muscles under more 'physiologic' conditions would enhance their proliferation and/or differentiation ability. Therefore, we analyzed satellite cells derived from 31-month-old rats in standard cultures with 21% O₂ and in lowered (∼3%) O₂. Under the lowered O₂ conditions, we noted a remarkable increase in the percentage of large-sized colonies, activation of cell cycle progression factors, phosphorylation of Akt, and downregulation of the cell cycle inhibitor p27^Kip1. These data suggest that lower O₂ levels provide a milieu that stimulates proliferation by allowing continued cell cycle progression, to result ultimately in the enhanced in vitro replicative life span of the old satellite cells. Such a method therefore provides an improved means for the ex vivo generation of progenitor satellite cell populations for potential therapeutic stem cell transplantation. Received 20 April 2001; received after revision 28 May 2001; accepted 31 May 2001     

Pleiotropic effects of sonic hedgehog on muscle satellite cells

Muscle satellite cells are believed to form a stable, self-renewing pool of stem cells in adult muscle where they function in tissue growth and repair. A regulatory disruption of growth and differentiation of these cells is assumed to result in tumor formation. Here we provide for the first time evidence that sonic hedgehog (Shh) regulates the cell fate of adult muscle satellite cells in mammals. Shh promotes cell division of satellite cells (and of the related model C2C12 cells) and prevents their differentiation into multinucleated myotubes. In addition, Shh inhibits caspase-3 activation and apoptosis induced by serum deprivation. These effects of Shh are reversed by simultaneous administration of cyclopamine, a specific inhibitor of the Shh pathway. Taken together, Shh acts as a proliferation and survival factor of satellite cells in the adult muscle. Our results support the hypothesis of the rhabdomyosarcoma origin from satellite cells and suggest a role for Shh in this process.Received 23 February 2005; received after revision 2 May 2005; accepted 9 June 2005     

The growth of Atlantic salmon (Salmo salar L.) myosatellite cells in culture at two different temperatures

Temperature is known to affect fish growth, and in Atlantic salmon there is an influence on muscle cellularity. Primary muscle cell culture makes it possible to investigate direct effects of temperature on myogenic cells. Salmon myosatellite cells were cultured for the first time in this study. The cells were cultured at either 5°C or 11°C. Increased temperature led to an increase in differentiation rate and especially hypertrophic growth (Q₁₀=4.0). No nuclear proliferation was evident in the satellite cell population isolated at either temperature. This may be due to the presence of different subpopulations of myogenic cells at different developmental ages or the presence of indirect factors in vivo.     

Structural characteristics and distribution of satellite cells along crayfish muscle fibers.

The distribution of satellite cells (sc) in long-sarcomere muscle fibers from the carpopod extensor muscle of the crayfish (Astacus fluviatilis) has been studied electron-microscopically. The sc are spindle-shaped and are oriented parallel to the long axis of a fiber. The mean lengths of sc nuclei (17.00 microns) and that of myonuclei (18.35 microns) differ non-significantly. In older animals, the mean ratio of the number of sc nuclei to the total number of nuclei (sc nuclei + myonuclei) is 0.0716, 0.0848, and 0.034 for the tendon, central and shell segments, respectively. The corresponding values for younger animals are 0.158, 0.166, and 0.081. The mean numbers of sc nuclei per mm of a fiber are 94, 117, and 47 (older animals), and 164, 117, and 94 (younger animals) for the tendon, central and shell segments, respectively. The high incidence of sc per unit fiber length in crayfish may be related to the fact that crayfish muscle fibers have a much larger diameter than vertebrate muscle cells.     

The effect of shock on blood oxidation-reduction potential

Oxidation-reduction (redox) potential measurements were made in the blood of rabbits subjected to hemorrhagic shock followed by treatment with a mild oxidizing agent (albumin). Control redox potential reading corrected for pH was –8.8±1.3 millivolts (mV) in arterial blood (A) and –18.0±2.0 mV in venous blood (V). This A-V difference indicated that hydrogen equivalents coming from muscle and other tissues were partially consumed in the lungs. A 20-mV drop on the V and a 13 mV on the A side was seen after shock. This did not fully return to control 2 h after return of the shed blood. Infusion of 2 g of albumin/kg/h raised the V redox potential to control, but it returned to untreated levels when the albumin was discontinued. The reductive load imposed on the animal by shock appeared to be large and not readily reversed by reperfusion or by the quantity of albumin given. Thus, it may be concluded that cellular respiration had not been adequately restored. This reductive load may impede recovery by suppression of cellular respiration and other cell and organ functions.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

A tonic component in the motility of the upper urinary tract (renal pelvis-ureter)

Summary In isolated muscle strips of porcine renal pelvis and ureter, the calcium antagonist nifedipine (3·10^–7 moles/l) completely suppressed spontaneous phasic mechanical activity and the phasic components of an adrenaline-induced activation (P-component). In the presence of nifedipine, adrenaline induced in pelvis preparations (but not in the ureter) a tonic contraction (T-component) which was on average 61% of the control reaction (SD±26%; n=35).The experiments were supported by a grant from the Deutsche Forschungsgemeinschaft (Go 130/20).     

Structural characteristics and distribution of satellite cells along crayfish muscle fibers

The distribution of satellite cells (sc) in long-sarcomere muscle fibers from the carpopod extensor muscle of the crayfish (Astacus fluviatilis) has been studied electron-microscopically. The sc are spindle-shaped and are oriented parallel to the long axis of a fiber. The mean lengths of sc nuclei (17.00 m) and that of myonuclei (18.35 m) differ non-significantly. In older animals, the mean ratio of the number of sc nuclei to the total number of nuclei (sc nuclei + myonrclei) is 0.0716, 0.0848, and 0.034 for the tendon, central and shell segments, respectively. The corresponding values for younger animals are 0.158, 0.166, and 0.081. The mean numbers of sc nuclei per mm of a fiber are 94, 117, and 47 (older animals), and 164, 117, and 94 (younger animals) for the tendon, central and shell segments, respectively. The high incidence of sc per unit fiber length in crayfish may be related to the fact that crayfish muscle fibers have a much larger diameter than vertebrate muscle cells.     

Effects of the spinal cord section and of subsequent denervation on the mechanical properties of fast and slow muscles.

The Soleus muscle of the rat, 3--6 months old, becomes significantly faster than in the controls, if the spinal cord is cut at birth. Mechanical properties of Extensor Digitorum Longus (EDL) muscle are not altered by spinal cord section. In cordotomized animals Soleus muscle always remains slower than EDL muscle. Denervation, performed 3--6 months after birth, has the same slowing effects in the Soleus and EDL muscles, both in cordotomized and in the control animals.     

Muscle lipofuscin content and satellite cell volume is increased after high altitude exposure in humans

Summary Muscle ultrastructural changes during a typical expedition to the Himalayas were analyzed by taking muscle biopsies from seven climbers before and after their sojourn at high altitude (over 5000 m for 8 weeks). M. vastus lateralis samples were analyzed morphometrically from electron micrographs. A quantitative evaluation was made of lipofuscin, satellite cells and myonuclei. Significant increases of the volume densities of lipofuscin (+235%) and satellite cells (+215%) were observed.     

Muscle lipofuscin content and satellite cell volume is increased after high altitude exposure in humans

Muscle ultrastructural changes during a typical expedition to the Himalayas were analyzed by taking muscle biopsies from seven climbers before and after their sojourn at high altitude (over 5000 m for 8 weeks). M. vastus lateralis samples were analyzed morphometrically from electron micrographs. A quantitative evaluation was made of lipofuscin, satellite cells and myonuclei. Significant increases of the volume densities of lipofuscin (+ 235%) and satellite cells (+ 215%) were observed.     

Evaluation of the ability of intact platelets to accumulate acridine organge

Summary A new approach to the evaluation of the uptake of fluorescent, probes by intact, cells is described. Acridine organe (AO) was used because it can be selectively accumulated by serotonin-containing granules of platelets. Analysis of the fluorescence signal allows the estimation of the relative volume of the granules and the equilibrium coefficients for AO transport across the cytoplasm and granule membranes. The following results were obtained for human and rabbit platelets: the relative volumes of the granules were 14±1% and 29±2%, the ratios of intragranular-extracellular probe concentration were 2260±382 and 30,000±5500, and the cytoplasm-extracellular medium concentration ratios were 375±60 and 225±60, respectively.     

Insulin action in cultured human skeletal muscle cells during differentiation: assessment of cell surface GLUT4 and GLUT1 content

In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, cultured myotubes are a useful tool to facilitate biological and molecular validation of novel pharmacological agents aimed to improve glucose metabolism in skeletal muscle.     

Satellite cells in denervated muscles.

It is known that, in a denervated striated muscle, the satellite cells multiply by mitotic division. A liaison between these satellite cells and the Schwann cell in front of the post-synaptic membrane in denervated frog muscle has been observed. It is probable that such cell connections help in the subsistence of the Schwann cell in a denervated muscle.     

Heat evolution of cultured human keratinocytes

Summary The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm ^tm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83±12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134±35) pW/cell was obtained when the transformed kerationocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Satellite cells in denervated muscles

Summary It is known that, in a denervated striated muscle, the satellite cells multiply by mitotic division. A liaison between these satellite cells and the Schwann cell in front of the post-synaptic membrane in denervated frog muscle has been observed. It is probable that such cell connections help in the subsistence of the Schwann cell in a denervated muscle.     

Stimulatory effect of methyl jasmonate on the ethylene production in tomato fruits

Summary Methyl jasmonate at a concentration of 0.5% in lanolin paste was applied to detached mature green and red ripe tomatoes cv. Tempo. One to 10 days after treatment, slices were cut at a depth of about 2 mm for ethylene determination. It was found that methyl jasmonate strongly stimulated ethylene production both in green and red fruits, production was about 1.6 to 7.9 times higher than in control tissue.Acknowledgments. We wish to thank Dr E. Demole, Firmenich SA, Geneva, Switzerland, for the gift of authentic (±)-methyl jasmonate and Mrs H. Sas for her excellent technical assistance.