Voltage-dependent calcium release in human malignant hyperthermia muscle fibers

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics

The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.     

Bed rest increases the amount of mismatched fibers in human skeletal muscle

The effects of a 37-day period of bed rest on myosin heavy chain (MHC) expression on both mRNA and protein level in human skeletal muscle fibers were studied. Muscle biopsies from vastus lateralis muscle were obtained from seven healthy young male subjects before and after the bed-rest period. Combined in situ hybridization, immunocytochemistry, and ATPase histochemistry analysis of serial sections of the muscle biopsies demonstrated that fibers showing a mismatch between MHC isoforms at the mRNA and protein level increased significantly after the bed-rest period, suggesting an increase in the amount of muscle fibers in a transitional state. Accordingly, fibers showing a match in expression of MHC-1 and of MHC-2A at the mRNA and protein level decreased, whereas fibers showing a match between MHC-2X mRNA and protein increased after bed rest. Overall, there was an increase in fibers in a transitional state from phenotypic type 1 --> 2A and 2A --> 2X. Furthermore, a number of fibers with unusual MHC mRNA and isoprotein combinations were observed after bed rest (e.g., type 1 fibers with only mRNA for 2X and type 1 fibers negative for mRNA for MHC-beta/slow, 2A, and 2X). In contrast, no changes were revealed after an examination at the protein level alone. These data suggest that the reduced load-bearing activity imposed on the skeletal muscles through bed rest will alter MHC gene expression, resulting in combinations of mRNA and MHC isoforms normally not (or only rarely) observed in muscles subjected to load-bearing activity. On the other hand, the present data also show that 37 days of bed rest are not a sufficient stimulus to induce a similar change at the protein level, as was observed at the gene level.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Chronic administration of taurine to aged rats improves the electrical and contractile properties of skeletal muscle fibers

Fimbriae or pili are essential adherence factors usually found in pathogenic bacteria to aid colonization of host cells. Three major structural pilin genes, fimA, sfaA, and papA, from Escherichia coli natural isolates were examined and nucleotide sequence data revealed elevated levels of both synonymous and nonsynonymous site variation at these loci. Examination of synonymous site variation shows a fivefold increase in fimA sites, relative to the housekeeping gene mdh; and similarly the sfaA and papA genes have increased synonymous sites variation relative to fimA. Nonsynonymous site variation is also elevated at all three loci but, in particular, at the papA locus (kN = 0.44). The kN/kS ratio for the three genes are among the highest yet reported for E. coli genes. Regional variation in nucleotide polymorphism within each of the genes reveal hypervariable segments where nonsynonymous substitutions exceed synonymous substitutions. We propose that at the fimA, papA, and sfaA genes, diversifying selection has brought about the increase levels of polymorphism.     

Contractile studies of single human skeletal muscle fibers: a comparison of different muscles, permeabilization procedures, and storage techniques

The study of single muscle fibers has improved our understanding of muscle physiology and pathology. To compare three techniques for fiber preparation and storage, biopsies were obtained from the tibialis anterior and vastus lateralis muscles of a hemiparetic patient and a control subject. Single fibers were prepared with: (1) chemical skinning (CS) and storage at -20 degrees C; (2) chemical skinning followed by sucrose (SU) incubation and storage at -80 degrees C; or (3) freeze-drying (FD) and -80 degrees C storage. Cross-sectional area (CSA), resting, maximal (P0), and specific tension (P0/CSA), and maximum shortening velocity (V0) were determined in 189 cells. CSA was similar in all groups. Resting tension was higher and P0 and P0/CSA lower after FD. In general, V0 was the same in all groups. Our data suggest that CS and SU preserve the properties of single muscle fibers better than FD. SU may allow longer storage of fibers.     

Functional morphology of serially linked skeletal muscle fibers

In the skeletal muscle fiber organization of many vertebrate muscles, serial arrangements or linkages of muscle fibers along the muscle or fascicle are commonly found. These serially linked muscle fibers employ distinct junctional morphologies from muscle to muscle. Notable are the end-to-end linkages of muscle fibers through tendinous intersections (TIs), where many fibers end onto a continuous connective tissue plate with folded terminations similar to myotendinous junctions. Besides this end-to-end linkage, overlapping linkages or arrangements occur among nonspanning fibers terminating intrafascicularly. These nonspanning fibers bear tapering terminations with direct cell-cell (myomuscular) junctions or without any specialized junctions. Despite their overlapping linkages or tapering profiles, nonspanning fibers maintain a uniform sarcomere length along the linked fibers, suggesting that the overlapping-linked nonspanning fibers are equivalent to the end-to-end linked fibers in their mechanical capacity. However, the junctional compliance could differ in their extracellular elastic components and their organization at junctional sites, e.g., direct mechanical (myomuscular) junctions vs. indirect linkages through connective tissue. Increasing evidence suggests that the elastic components, including muscle fibers as well as connective tissues, are more critical than previously thought for the mode and/or the efficiency of tension transmission among serially arranged fibers and thus for the mechanical properties of the muscle.     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Two mechanisms of quantized calcium release in skeletal muscle

Skeletal muscle uses voltage sensors in the transverse tubular membrane that are linked by protein-protein interactions to intracellular ryanodine receptors, which gate the release of calcium from the sarcoplasmic reticulum. Here we show, by using voltage-clamped single fibres and confocal imaging, that stochastic calcium-release events, visualized as Ca2+ sparks, occur in skeletal muscle and originate at the triad. Unitary triadic Ca(2+)-release events are initiated by the voltage sensor in a steeply voltage-dependent manner, or occur spontaneously by a mechanism independent of the voltage sensor. Large-amplitude events also occur during depolarization and consist of two or more unitary events. We propose a 'dual-control' model for discrete Ca2+ release events from the sacroplasmic reticulum that unifies diverse observations about Ca(2+)-signalling in frog skeletal muscle, and that may be applicable to other excitable cells.     

Capillary supply of skeletal muscle fibers in untrained and endurance-trained men

Muscle fiber diameters and numbers of capillaries per fiber, per square millimeter, and around each fiber were determined in needle biopsies from the lateral part of the quadriceps muscle of 23 young men. Twelve subjects were untrained (UT) and eleven were endurance-trained (ET) athletes. Average values for maximal oxygen uptake were 51.3 (UT) and 72.0 ml/kg-min (ET). Mean fiber diameters were not significantly different in the two groups (48.8 and 49.1 micron). The capillaries per fiber ratios were 1.77+/-0.10 and 2.49+/-0.08 (mean+/-SE) in the UT and ET groups, respectively. The numbers of capillaries around each fiber were 4.43+/-0.19 (UT) and 5.87+/-0.18 (ET). The numbers of capillaries per mm2 were 585+/-40 (UT) and 821+/-28 (ET). Fiber diameters were 28% smaller in ultrathin than in fresh-frozen sections from the same biopsies. After correction for this difference, the numbers of capillaries per mm2 were 305 and 425 in the UT and ET, respectively. The capillaries per fiber ratio increased with increasing fiber diameter, but not sufficiently to maintain the number of capillaries per mm2. Fibers containing many mitochondria are surrounded by more capillaries than fibers with few mitochondria.     

Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle

OBJECT: Ependymomas in children continue to generate controversy regarding their histological diagnosis and grading. optimal management, and possible prognostic factors. To increase our knowledge of these tumors the authors addressed these issues in a cohort of children with prospectively staged ependymomas treated with radiotherapy and chemotherapy. METHODS: Children between the ages of 2 and 17.3 years harboring an intracranial ependymoma confirmed by a central review of the tumor's pathological characteristics were treated according to Children's Cancer Group Protocol 921 from 1986 to 1992. Treatment following surgery and postoperative tumor staging (including brain computerized tomography or magnetic resonance [MR] imaging, spinal MR imaging or myelography, and cerebrospinal fluid cytological investigation) included craniospinal irradiation with a local boost to the primary tumor and patient randomization to receive adjuvant chemotherapy with either 1) CCNU, vincristine, and prednisone, or 2) the eight-drugs-in-1-day regimen. Centralized review of the tumor pathological characteristics revealed 20 ependymomas and 12 anaplastic ependymomas in the 32 children included in the study. Diagnoses made at the individual institutions included anaplastic (malignant) ependymoma (15 patients), ependymoma (four patients), ependymoblastoma (nine patients), ependymoastrocytoma (one patient), and primitive neuroectodermal tumor (three patients), which were discordant with the centralized review diagnosis in 22 of 32 cases. Only three of the 32 patients had metastatic disease (two with M and one with M3 stages). At surgery, 47% of tumors were estimated to be totally resected. Among the 14 of 17 patients who suffered a relapse and were evaluated for site of relapse, 10 (71%) had an isolated local relapse, three (21%) had concurrent local and metastatic relapse, and only one (7%) had an isolated metastatic relapse. Kaplan-Meier estimates of 5-year progression-free survival (PFS) and overall survival rates were 50 +/- 10% and 64 +/- 9%, respectively. CONCLUSIONS: Predictors of PFS duration included an estimate of the extent of resection made at surgery (total compared with less than total, p = 0.0001) and the amount of residual tumor on postoperative imaging as verified by centralized radiological review (< or = 1.5 cm2 compared with > 1.5 cm2, p < 0.0001). No other factors, including centrally reviewed tumor histopathological type, location, metastasis and tumor (M and T) stages, patient age, race, gender, or chemotherapy treatment regimen significantly correlated with PFS duration. The pattern of predominantly local relapse and the important influence of residual tumor or the extent of resection on PFS duration confirms a prevailing impression that local disease control is the major factor in the prediction of outcome of ependymoma. Survival rates were comparable with those reported by other investigators who have treated patients with similar doses of radiation and no chemotherapy.     

Imaging elementary events of calcium release in skeletal muscle cells

In skeletal muscle cells, calcium release to trigger contraction occurs at triads, specialized junctions where sarcoplasmic reticulum channels are opened by voltage sensors in the transverse tubule. Scanning confocal microscopy was used in cells under voltage clamp to measure the concentration of intracellular calcium, [Ca2+]i, at individual triads and [Ca2+]i gradients that were proportional to calcium release. In cells stimulated with small depolarizations, the [Ca2+]i gradients broke down into elementary events, corresponding to single-channel currents of about 0.1 picoampere. Because these events were one-tenth to one-fifth the size of calcium sparks (elementary release events of cardiac muscle), skeletal muscle control mechanisms appear to be fundamentally different.     

The effect of autografting on the myosin composition in skeletal muscle fibers

The purpose of the study was to investigate the changes in myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following autotransplantation of extensor digitorum longus muscles. Muscles were grafted in "standard" and "nerve-intact" conditions. MHC and MLC isoforms were analyzed by sodium dodecyl sulphate gel electrophoresis. Changes in MHC isoforms 10, 30, and 60 days after grafting were similar in the "standard" and the "nerve-intact" grafts. In contrast to MHC, changes in MLC were different in the 10th day groups, but the same in the 30th day groups. Sixty days after grafting the content of MLC isoforms was the same as the control muscles. These data indicate that transient loss of functional innervation, even for a short time, has permanent effect on the composition of MHC but not MLC isoforms in regenerating skeletal muscle fibers.     

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.     

Degeneration and regeneration of striated skeletal muscle fibers in Duchenne muscular dystrophy

Like all other muscular dystrophies, Duchenne muscular dystrophy is characterized by the coexistence of degenerative lesions of the muscle fibers and of regenerative changes. The present study has been carried out in order to precise the degree of regeneration at different stages of the disease, by analyzing the expression of several markers of cell proliferation and of muscular differentiation. In the two affected foetuses of our series, the m. quadriceps is histologically normal, except for the absent expression of immunoreactive dystrophin. The quadriceps from the eight children of our series (20 months-16 years) all present clear dystrophic changes. Muscle regeneration is characterized by activation of the satellite cells, by their multiplication followed by their fusion giving birth to regenerative fibers. By studying the expression of muscular markers (vimentin, desmin, isoforms of the myosin heavy chains), it has been possible to define more precisely the degree of maturation and of differentiation of these regenerative fibers. Our results suggest that an abortive regeneration of the muscle fibers in Duchenne muscular dystrophy can explain, at least partly, the progressive evolution of this disease.     

Differential titin isoform expression in human skeletal muscle

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW approximately 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (mean +/- SE; age = 25.4 +/- 2.9 years, height = 177.7 +/- 1.8 cm, weight = 76.5 +/- 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n = 10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n = 5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P > 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.     

Functional role of vacuolization of the T-system of skeletal muscle fibers

T-tubules of skeletal muscle fibres easily transform into large vacuoles under the influence of various factors. These include osmotic shock produced by the efflux of small molecular weight molecules (e.g. glycerol), hypertonic shock, muscle fatigue and muscle damage. In most cases, vacuolation is reversible but the molecular mechanisms involved are not clear. Also, the functional role of reversible vacuolation has not been established. However, three possibilities may be considered. (1) Redistribution of ions and water between the cytoplasm and the extracellular space comprised by the T-system. Thus, the formation of large vacuoles may be a mechanisms for rapid osmoregulation that corresponds to regulated volume decrease in other types of cell. However, in our hands, inhibitors of various pathways that participate in volume regulation had no effect on reversible vacuolation. (2) Resealing of mechanical damage of the plasma membrane. This is usually accompanied by the development near the damaged membrane of numerous vacuoles which we have observed by confocal microscopy and use of a hydrophobic dye (RH414), to arise in part from T-tubules. (3) By confocal microscopy, it has also been shown that extracellular fluorescein dextran (Mr = 10,000), and both plasmid DNA (pUC18) and sonicated high molecular weight DNA stained with YOYO, enter vacuoles derived from T-tubules. This finding may indicate that reversible vacuolation, in the absence of membrane damage, could provide a pathway from the extracellular environment to the cytoplasm that is additional or complimentary to endocytosis; it may also be particularly relevant to the ability of muscle to be transfected by the direct injection of DNA. These several observations strongly indicate that the function of the T-system in skeletal muscle fibers is not restricted to excitation-contraction coupling.     

Sonoelastic determination of human skeletal muscle elasticity

It is not currently practical to directly measure viscoelastic parameters in human muscles in situ. Methods used in vitro cannot readily be applied, and motion analysis provides only a gross estimate. We report on the application of a hybrid approach, sonoelastography, which uses ultrasound to measure the propagation of shear waves induced by externally applied vibrations. Because shear waves predominate in incompressible viscoelastic media at low frequencies, sonoelastic data should be comparable to those obtained using conventional means. We recorded vibration propagation speeds as a function of applied load in the quadriceps muscles of ten volunteers as they underwent a series of static contractions. Data collection during dynamic contractions, not possible with the current equipment, will be the subject of future experimentation. Although statistically significant correlations were not uniformly obtained above 60 Hz nor for propagation perpendicular to the muscle fibers, this is felt to have resulted from deviations from the applied plane wave model. Calculated values of Young's modulus for 30 Hz propagation parallel to the muscle fibers were 7 +/- 3, 29 +/- 12 and 57 +/- 37 x 10(3) Nm-2 for applied loads of 0, 7.5 and 15 kg, respectively. The corresponding values at 60 Hz were 25 +/- 6, 75 +/- 61 and 127 +/- 65. These values were statistically significant and linearly correlated with the applied load, as expected. Our data represent the first in situ human measurements of their kind. It is anticipated that sonoelastography will provide a useful adjunct to the study of human biomechanics.     

Quantitative analysis for histochemical grouping of muscle fibers after skeletal muscle transplantation by microneurovascular anastomoses

A poly-histochemical quantitative assay was carried out on fibers of grafted rectus femoris muscles of rabbit which had undergone simulated free muscle transplantation by microneurovascular anastomoses in speciments at 3, 6, 12 months postoperatively. At the same time, the same assay was done for two experimental control groups: either the motor nerve or the patella tendon was simply severed and immediately sutured. It was found that the contractile characteristics of whole muscle were depended on relative number of different muscle fibers. That meant it was depended on the relative number of different motor fibers which had run through the anastomosed site. The caliber change among different types of muscle fibers mainly represented there was cross reneurotization during nerve regeneration.