Plasticity in mammalian skeletal muscle.

While it has been recognized for many years that different limb muscles belonging to the same mammal may have markedly differing contractile characteristics, it is only comparatively recently that it has been demonstrated that these differences depend upon the motor innervation. By appropriately changing the peripheral nerve innervating a mammalian skeletal muscle, it is possible to change dramatically the contractile behaviour of the reinnervated muscle. The manner by which the motor innervation determines the nature of a muscle fibre's contractile machinery is not completely understood, but it appears that the number and pattern of motor nerve impulses reaching the muscle play an important role. The biochemical changes occurring within muscle fibres whose contractile properties have been modified by altered motor innervation include the synthesis of different contractile proteins.     

Carbonic anhydrase activity in mammalian skeletal and cardiac muscle.

1. The presence of extravascular carbonic anhydrase activity in skeletal muscle, and its absence from cardiac muscle, were demonstrated in the rat. 2. The activity in skeletal muscle is approximately correlated with the proportion of dark fibres present in the middle fibre bundles.     

Glycerol treatment in mammalian skeletal muscle

Summary Potassium (K-) contractures were recorded from slow-twitch (mouse soleus) and fast-twitch (mouse extensor digitorum longus (EDL) and rat sternomastoid) muscles. The mouse limb muscles responded to a maintained increase in external potassium concentration with a rapid increase in tension (fast contracture) which inactivated and was followed by a slow contracture. Rat sternomatoid muscles responded with fast contractures only. The threshold potassium concentration for contraction was higher in fast-twitch muscles than in soleus muscles, at 22 and at 37°C. After corrections had been made for the more rapid depolarization of soleus fibers, the threshold potential for soleus fiber contraction was 15 mV closer to the resting membrane potential than the threshold for fast-twitch fiber contraction. The K-contracture results were confirmed by two microelectrode voltage-clamp experiments. Activation of fast twitch fibers required depolarizing pulses that were 15 to 20 mV greater than the pulses required to activate soleus fibers. When the time courses of K-contractures were compared it was evident that inactivation with prolonged depolarization was much faster in the fast-twitch muscles than in the soleus muscles. The results suggest that the voltage dependence and kinetics of the process coupling T-tubule depolarization with calcium release from the sarcoplasmic reticulum may depend on fiber type in mammalian skeletal muscle.     

On the relationship of ultrastructural and cytochemical features to color in mammalian skeletal muscle

Summary The semitendinosus muscle of the albino rat is divided grossly into two clearly distinguishable parallel longitudinal bands, one red (anterior) and the other white (posterior). By using mitochondrial content as a criterion for distinguishing fiber types, it is demonstrated that the red portion of the muscle is composed predominantly of red (52%) and intermediate (40%) fibers, while the white portion consists primarily of white fibers (82%). Red fibers have the smallest and white fibers have the largest average diameter. Ultrastructural characteristics of the three fiber types resemble closely those previously described for the rat diaphragm. Red fibers are rich in large mitochondria with abundant cristae, and possess the widest Z lines. In red fibers, the H-band region of the sarcoplasmic reticulum consists of an elaborate network of narrow tubules. In white fibers, mitochondria are smaller, less numerous, and have fewer cristae; Z lines are about half as wide as in red fibers. In the H-band region of the sarcoplasmic reticulum there is a more compact arrangement of broad more or less parallel tubules. Intermediate fibers are similar to red fibers except that their diameters are larger; mitochondria are somewhat smaller and cristae are less abundant; the width of the Z lines is close to that of white fibers. The consistent difference in Z line width establishes this dimension as an important criterion for distinguishing fiber types and facilitates ultrastructural identification, especially of the intermediate fiber.The clear relationship between color of the semitendinosus and cytological features of its component fibers supports the use of the terms red, white, and intermediate as simple and valid designations for fiber types in mammalian skeletal muscle. Measurement of the cross-sectional area contributed by each fiber type to the total area indicates that both red and intermediate fibers may contribute to redness in mammalian skeletal muscle.An early portion of this work was carried out with MissSharon Whelan (Mrs.Bernard Weiss). The author acknowledges the important contribution of Mr.Richard Stearns through his skillful work on the photographic illustrations and the technical assistance of MissAnn Campbell and Mrs.Joan Normington. — This study was supported by Grant No. HD 01026-04 from the United States Public Health Service.     

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Caffeine and length dependence of staircase potentiation in skeletal muscle

Skeletal muscle sensitivity to Ca2+ is greater at long lengths, and this results in an optimal length for twitch contractions that is longer than optimal length for tetanic contractions. Caffeine abolishes this length dependence of Ca2+ sensitivity. Muscle length (ML) also affects the degree of staircase potentiation. Since staircase potentiation is apparently caused by an increased Ca2+ sensitivity of the myofilaments, we tested the hypothesis that caffeine depresses the length dependence of staircase potentiation. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 10 s of 10-Hz stimulation were analyzed at seven different lengths to evaluate the length dependence of staircase potentiation. In the absence of caffeine, length dependence of Ca2+ sensitivity was observed, and the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased length (DT = 1.47 - 0.05 ML, r2 = 0.95, where DT is developed tension). Length dependence of Ca2+ sensitivity was decreased by caffeine when caffeine was administered in amounts estimated to result in 0.5 and 0.75 mM concentrations. Furthermore, the negative slope of the relationship between staircase potentiation and muscle length was diminished at the lower caffeine dose, and the slope was not different from zero after the higher dose (DT = 1.53 - 0.009 ML, r2 = 0.43). Our study shows that length dependence of Ca2+ sensitivity in intact skeletal muscle is diminished by caffeine. Caffeine also suppressed the length dependence of staircase potentiation, suggesting that the mechanism of this length dependence may be closely related to the mechanism for length dependence of Ca2+ sensitivity.     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of pH on the length-dependent twitch potentiation in skeletal muscle.

When muscle is elongated, there is a length dependence of twitch potentiation and an increased Ca(2+) sensitivity of the myofilaments. Changes in the charge potential of myofilaments, induced by a decrease in pH, are known to abolish the length dependence of Ca(2+) sensitivity. This study was aimed at testing the hypothesis that a decrease in pH, and the concomitant loss of length dependence of Ca(2+) sensitivity, depresses the length dependence of staircase potentiation. In vitro, isometric twitch contractions of fiber bundles dissected from the mouse extensor digitorum longus, performed before and after 10 s of 10-Hz stimulation (i.e., the staircase potentiation protocol) were analyzed at five different lengths, ranging from optimal length for maximal force production (L(o); = 12 +/- 0.7 mm) to L(o) + 1.2 mm (L(o) + 10%). These measurements were made at an extracellular pH of 6.6, 7.4, and 7.8 (pH changes induced by altering the CO(2) concentration of the bath solution). At pH 7.4 and 7.8, the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased fiber bundle length (r(2) = 0.95 and r(2) = 0.99, respectively). At pH 6.6, the length dependence of potentiation was abolished, and the slope of the length-potentiation relationship was not different from zero (r(2) = 0.05). The results of this study indicate that length dependence of potentiation in intact skeletal muscle is abolished by lowering the pH. Because decreasing the pH decreases Ca(2+) sensitivity and changes the charge potential of the filaments, the mechanism of length-dependent potentiation may be closely related to the length dependence of Ca(2+) sensitivity, and changes in the charge potential of the myofilaments may be important in regulating this relationship.     

Purification of desmin from adult mammalian skeletal muscle.

A method has been developed for preparation of purified desmin from mature mammalian (porcine) skeletal muscle. A crude desmin-containing fraction was prepared by modification of procedures used for isolation of smooth-muscle intermediate-filament protein [Small & Sobieszek (1977) J. Cell Sci. 23, 243-268]. The desmin was extracted in 1 M-acetic acid/20 mM-NaCl at 4 degrees C for 15h from the residue remaining after actomyosin extraction from washed myofibrils. Successive chromatography on hydroxyapatite and DEAE-Sepharose CL-6B in 6M-urea yielded desmin that was routinely more than 97% 55 000-dalton protein and that had no detectable actin contamination. Removal of urea by dialysis against 10mM-Tris/acetate (pH 8.5)/1 mM dithioerythritol and subsequent clarification at 134 000 g (rav. 5.9 cm) for 1 h results in a clear desmin solution. Dialysis of purified desmin against 100 mM-NaCl/1 mM-MgCl2/10 mM-imidazole/HCl, pH 7.0, at 2 degrees C resulted in the formation of synthetic desmin filaments have an average diameter of 9-11.5 nm. The present studies demonstrate that the relatively small amount of desmin in mature skeletal muscle can be isolated in sufficient quantity and purity to permit detailed studies of its properties and function. Although 10nm filaments have not been unequivocally demonstrated in mature muscle in vivo, that the purified skeletal-muscle desmin will form 10 nm filaments in vitro lends support to their possible existence and cytoskeletal function in mature skeletal-muscle cells.     

Indomethacin and meclofenamate potentiation of skeletal muscle active hyperemia

The effects of indomethacin and meclofenamate on active hyperemia following sustained, maximal isometric contractions were studied in free-flowing dog gracilis muscles. Muscles were stimulated to contract in situ for 1, 4, 7, and 10 s durations in the absence and presence of indomethacin (62.5 micrograms/ml blood), meclofenamate (50 micrograms/ml blood), or appropriate vehicles. Drugs were administered by continuous intra-arterial infusion into the muscle. Cyclo-oxygenase inhibition was verified by intra-arterial injection of arachidonic acid. Resting vascular conductance decreased by 28% with meclofenamate but not with indomethacin. Meclofenamate and indomethacin increased active hyperemia excess flows by 49% and 101%, respectively, following 10 s of contraction. These results differ markedly from previous studies. We suggest that non-specific actions of both drugs, unrelated to their effect on prostaglandin synthesis, result in potentiation of normal vasodilator responses to muscle contraction.