Electrical properties of frog skeletal muscle fibers interpreted with a mesh model of the tubular system

This paper presents the construction, derivation, and test of a mesh model for the electrical properties of the transverse tubular system (T-system) in skeletal muscle. We model the irregular system of tubules as a random network of miniature transmission lines, using differential equations to describe the potential between the nodes and difference equations to describe the potential at the nodes. The solution to the equations can be accurately represented in several approximate forms with simple physical and graphical interpretations. All the parameters of the solution are specified by impedance and morphometric measurements. The effect of wide circumferential spacing between T-system openings is analyzed and the resulting restricted mesh model is shown to be approximated by a mesh with an access resistance. The continuous limit of the mesh model is shown to have the same form as the disk model of the T-system, but with a different expression for the tortuosity factor. The physical meaning of the tortuosity factor is examined, and a short derivation of the disk model is presented that gives results identical to the continuous limit of the mesh model. Both the mesh and restricted mesh models are compared with experimental data on the impedance of muscle fibers of the frog sartorius. The derived value for the resistivity of the lumen of the tubules is not too different from that of the bathing solution, the difference probably arising from the sensitivity of this value to errors in the morphometric measurements.     

Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers

In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.     

Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers

Values of skeletal muscle intracellular PO2 during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular PO2 in isolated single skeletal muscle fibers by using O2-dependent quenching of a phosphorescent-O2 probe is described. Intact single skeletal muscle fibers from Xenopus laevis were dissected from the lumbrical muscle and mounted in a glass chamber containing Ringer solution at 20 degreesC. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium-meso-tetra (4-carboxyphenyl) porphine bound to bovine serum albumin was injected into single fibers by micropipette pressure injection. Phosphorescence-decay curves (average of 10 rapid flashes) were recorded every 7 s from single cells (n = 24) in which respiration had been eliminated with NaCN, while the PO2 of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculated at the varied extracellular PO2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 microseconds at an extracellular PO2 of 159 Torr to 650-700 microseconds at 0 Torr. The phosphorescent lifetimes for the varied PO2 were used to calculate, by using the Stern-Volmer relationship, the phosphorescence-quenching constant (100 Torr-1. s-1), and the phosphorescence lifetime in a zero-O2 environment (690 microseconds) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular PO2 in isolated single skeletal muscle fibers.     

Alterations in contractility and intracellular Ca2+ transients in isolated bundles of skeletal muscle fibers from rats with chronic heart failure

To determine if chronic heart failure (CHF) leads to functional or structural alterations of skeletal muscle, we compared intracellular Ca2+ signaling, contractility, and the rate of fatigue development, together with electron microscopy (EM), in skeletal muscle preparations from rats with myocardial infarction-induced CHF versus sham-operated control rats. Bundles of 100 to 200 cells were dissected from the extensor digitorum longus (EDL) muscle of control (n = 13) and CHF (n = 19) rats and were either loaded with aequorin or fixed for EM. Muscles from CHF rats exhibited depressed tension development compared with control muscles during twitches (1.4 +/- 0.2 versus 2.8 +/- 0.7 g/mm2, P < .05) and maximal tetani (5.3 +/- 1.4 versus 10.7 +/- 2.4 g/mm2, P < .05). Depressed tension in CHF was accompanied by reduced quantitative [Ca2+]i release during twitches (0.7 +/- 0.1 versus 0.4 +/- 0.1 microM, P < .05) and during maximal tetani (1.8 +/- 0.3 versus 0.9 +/- 0.2 microM, P < .05). Skeletal muscle from CHF rats also demonstrated prolonged intracellular Ca2+ transients during twitches and tetani and accelerated fatigue development. EM revealed a lack of cellular atrophy in the CHF rats. In conclusion, EDL skeletal muscle from rats with CHF had intrinsic abnormalities in excitation-contraction coupling unrelated to cellular atrophy. These findings indicate that CHF is a condition accompanied by EDL skeletal muscle dysfunction.     

Optical rotation signals recorded from a single skeletal muscle fibre of a frog

The decreased availability of economic resources requires the use of methods to assess hospital efficiency. The aim of our study was to retrospectively evaluate the applicability to the Italian reality of a catalogue of medical acts (CdAM-champs beta) set up for the French Health System. We wanted to evaluate the amount of resource utilization of a Cardiothoracic Operating Room of a IRCCS regional general hospital. The study included 83 admissions, occurring in October 1996, to the cardiac surgery department. Medical acts were recorded for all admissions taking into account both the number of acts and the weight of acts expressed as standard cost index or ICR. This takes into account the use of human (medical and nursing staff) and technical resources. Calculation of ICR beta scores was achieved by means of two different expressions. We observed a lack of correlation between the two values of each ICR. The previous formula (1991) gives more emphasis on the different conditions in which the operation was carried out (American Society of Anesthesiology score), duration of anesthesia and patients disease with a statistical significant difference. The last formula (1995) only evaluate patients' disease. None of the two formula of ICR beta turns out to be completely appropriate index of resource utilization during anesthesia; therefore this analysis still remains a difficult problem. Yet it must be recognized that this system deserves the merit of having evaluated the anesthesiological duties, by separating them from the surgical ones.     

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.     

Steady state relation between cytoplasmic free Ca2+ concentration and force in intact frog skeletal muscle fibers

The steady state relation between cytoplasmic Ca2+ concentration ([Ca2+]i) and force was studied in intact skeletal muscle fibers of frogs. Intact twitch fibers were injected with the dextran-conjugated Ca2+ indicator, fura dextran, and the fluorescence signals of fura dextran were converted to [Ca2+]i using calibration parameters previously estimated in permeabilized muscle fibers (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150). In the first series of experiments, [Ca2+]i and isometric force were simultaneously measured during high K+ depolarization. Slow changes in [Ca2+]i and force induced by 15-30 mM K+ appeared to be in equilibrium, as instantaneous [Ca2+]i versus force plot tracked the common path in the rising and relaxation phases of K+ contractures. In the second series of experiments, 2,5-di-tert-butylhydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, was used to decrease the rate of decline of [Ca2+]i after tetanic stimulation. The decay time courses of both [Ca2+]i and force were dose-dependently slowed by TBQ up to 5 micro M; the instantaneous [Ca2+]i- force relations were nearly identical at >/=1 micro M TBQ, suggesting that the change in [Ca2+]i was slow enough to reach equilibrium with force. The [Ca2+]i-force data obtained from the two types of experiments were consistent with the Hill curve using a Hill coefficient of 3.2-3.9 and [Ca2+]i for half activation (Ca50) of 1.5-1.7 micro M. However, if fura dextran reacts with Ca2+ with a 2.5-fold greater Kd as previously estimated from the kinetic fitting (Konishi and Watanabe. 1995. J. Gen. Physiol. 106:1123-1150), Ca50 would be 3.7-4.2 micro M. We also studied the [Ca2+]-force relation in skinned fibers under similar experimental conditions. The average Hill coefficient and Ca50 were estimated to be 3.3 and 1.8 microM, respectively. Although uncertainties remain about the precise levels of [Ca2+]i, we conclude that the steady state force is a 3rd to 4th power function of [Ca2+]i, and Ca50 is in the low micromolar range in intact frog muscle fibers, which is in reasonable agreement with results obtained from skinned fibers.     

Some properties of late after-potential in frog skeletal muscle fiber

Some properties of late after-potentials which appear following a train of impulses were examined in frog skeletal muscle fiber. The decay of the late after-potential followed a simple exponential time course. The time constant of the decay was larger in a viscous solution than in normal Ringer solution. It was proved by physical experiments that the diffusion of K ions was delayed in the viscous medium at the same rate as the decay. The effect of temperature on the decay was low and the Q10 for the time constant was 1.2. When the late after-potentials were recorded at membrane potentials variedly controlled by the polarizing current, the reversal potential shifted in the positive direction with the increase of impulses. These results suggest that the late after-potential may be dependent on K ions accumulated in the T system. During the initial 300 msec period immediately after the onset of the decay, the amplitude was smaller than expected by a simple exponential time course. This effect was especially apparent in the sucrose hypertonic Ringer solution in which the decay was extremely extended. The cause of this non-exponential component was discussed with respect to the K accumulation hypothesis.     

Caveolin-3 is associated with the T-tubules of mature skeletal muscle fibers

Caveolae are abundant in skeletal muscle and their coat contains a specific isoform of caveolin, caveolin-3. It has been suggested that during muscle development, caveolin-3 is associated with the T-tubules, but that in adult muscle it is found on the plasma membrane only. We have studied the distribution of caveolin-3 in single skeletal muscle fibers from adult rat soleus by confocal immunofluorescence and by immunogold electron microscopy. We found that caveolin-3 occurs at the highest density on the plasma membrane but is also present in the core of the fibers, at the I-band/A-band interface where it is associated with the T-tubules. In neither domain of the muscle surface does caveolin-3 colocalize with the glucose transporter GLUT4 and there is no evidence for internalization of the caveolae in muscle.     

The temperature dependence of intracellular pH in isolated frog skeletal muscle: lessons concerning the Na(+)-H+ exchanger

The behavior of cultured rat bone cells growing on modified polyethylene terephthalate (mPET), glass, and machinable ceramic substrates containing enstatite (MgO, SiO2) and glass (CaO-P2O5-Al2O3) was studied. Cell attachment was measured directly on the substrates using an image analysis system. Electron microscopy observations and the MTT test revealed that cells are able to spread and proliferate on the material surface, keeping a healthy ultrastructure on all materials tested in the present study. After having colonized the surface of the materials, as shown by immunocytochemistry, the cells synthesize an osteoid-like matrix composed of osteocalcin, type I collagen, and fibronectin fibrils. The titration of alkaline phosphatase activity showed that the cells grown on the ceramic exhibit a greater osteogenic activity than those grown on controls (glass and mPET). This osteogenic activity results in a mineralization of the extracellular matrix in cultures on ceramic or plastic whereas only few calcium phosphate crystallite traces were revealed by Von Kossa staining on glass. Enstatite constitutes, therefore, an environment compatible with in vitro bone cell life.     

Transient outward K+ channels in vesicles derived from frog skeletal muscle plasma membranes

Whole-cell voltage-clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes. Capacitance measurements showed that these vesicles lack invaginations. In solutions containing K+, transient outward currents with reversal potentials close to EK were recorded with a maximum potassium conductance of 0.3 mS/cm2. These currents inactivated in a voltage-dependent manner with a time constant of decay that reached a limiting value of 26 ms at large depolarizations. The steady-state inactivation reached half-maximum values at -66 mV. Transient currents were completely blocked with 5 mM 4-aminopyridine. Single-channel recordings made in inside-out excised patches from the vesicles had ensemble averages with characteristics similar to those of the macroscopic currents, although with significantly faster inactivation time constants. The single-channel chord conductance was 21 pS when the pipette and bath solutions contained 2.5 mM and 120 mM KCl, respectively. It is concluded that these vesicles contain potassium channels that are very similar to A channels found in neurons and other cells.     

Effect of caffeine on K+ efflux in frog skeletal muscle

The exposure of frog skeletal muscle to caffeine (3-4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (kK,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 microM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 microM tetracaine significantly reduced the increase in kK,o (DeltakK,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced DeltakK,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect DeltakK,o. Fifth, tolbutamide (800 microM), an inhibitor of KATP channels, reduced DeltakK,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced DeltakK,o. Seventh, omitting Na+ from the external medium reduced DeltakK,o by about 40%. Eight, amiloride (5 mM) decreased DeltakK,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.     

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.     

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.     

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.     

Tau protein immunoreactivity in muscle fibers with rimmed vacuoles differs from that in regenerating muscle fibers

To determine whether tau protein found in muscle fibers with rimmed vacuoles and in regenerating fibers was phosphorylated, we examined eight muscle biopsy samples containing rimmed vacuoles (from five patients with distal myopathy with rimmed vacuole formation and three patients with inclusion body myositis) and three muscle biopsy samples from patients with Duchenne muscular dystrophy containing numerous regenerating fibers. Although both rimmed vacuolated and regenerating fibers had increased immunoreactivity against tubulin and tau protein, tau protein in the former was more highly phosphorylated than that in the latter. While very few microtubules in muscle fibers with rimmed vacuoles were recognizable by electron microscopy, regenerating fibers, especially immature ones, contained numerous microtubules. Since tau protein found in vacuolated fibers is hyperphosphorylated, it can be considered to have reduced ability to bind tubulin molecules. Thus, the tau protein cannot stabilize microtubules, resulting in their depolymerization even in the presence of tubulin molecules.     

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.     

Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana

In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgalpha1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgalpha1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5-10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgalpha1S was difficult, the chimera consisting of Fgalpha1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the alpha1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997) J. Gen. Physiol. 110, 379-389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression.     

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.