Lysosomal proteinase-sensitive regions in fast and slow skeletal muscle myosins

We investigated the limited proteolysis of fast and slow myosins purified from rabbit psoas major and semimembranosus proprius muscles, respectively, by the main lysosomal proteinases: cathepsins B, H, L, and D. In EDTA containing buffer, cathepsin D cleaved both myosins only at the rod-S1 junction leading to the formation of two S1 fragments of slightly higher Mr than the three forms obtained with chymotrypsin. On addition of MgCl2 instead of EDTA, myosin hydrolysis was markedly reduced. In contrast, irrespective of the presence of either MgCl2 or EDTA, cathepsin B hydrolysed both myosins into HMM and LMM. Cathepsin L digested myosins more extensively than cathepsins B and D and the main fragments generated were, in decreasing order of importance, rod, S1, S2, HMM, and LMM. In the incubation conditions tested, cathepsin H displayed nondetectable action on myosins. As fast and slow myosin digest patterns were compared, the main differences observed concerned the size of the proteolytic products and the rate of hydrolysis, which was about 4-fold higher for the fast than for the slow isoform. This appeared consistent whatever enzyme was considered.     

Calcium-related abnormalities in fast and slow denervated skeletal muscle in rats

Muscle weights, Ca-ATPase activity and calcium-binding proteins were studied after denervation in rat extensor digitorum longus (EDL) and soleus (Sol) muscles. Muscle weights decreased progressively as a function of denervation time: after 28 days EDL weight diminished by 70% and Sol weight by 47%. Ca-ATPase activity and calsequestrin were quite reduced in control Sol as compared to the control EDL. Denervation caused a considerable reduction in Ca-ATPase and calsequestrin in EDL, making it resemble the control Sol.     

Glucose uptake after immobilization in fast and slow skeletal muscle in rats

1. 3-O-methylglucose uptake was studied after immobilization in rat extensor digitorum longus (EDL) and soleus (Sol) muscles. 2. The immobilization of the ankle was done in one of extreme positions by plaster casts. 3. In both positions, 3-O-methylglucose uptake in EDL increased and that in Sol decreased after immobilization. 4. When immobilization was released uptake returned to control level. 5. The change in uptake after immobilization and after release of immobilization was earlier in Sol.     

Seasonal changes in the response of fast and slow mammalian skeletal muscle fibers to zero potassium.

While investigating the decline in resting membrane potential (RMP) of rat skeletal muscle fibers in zero potassium solution, we discovered that there is seasonal variation in the response of the extensor digitorum longus muscle (EDL). In January, most EDL fibers hyperpolarize in zero K+; in September, most depolarize; the distribution of RMPs recorded in May is bimodal, with some fibers hyperpolarizing and some depolarizing. Fibers from the soleus muscle depolarize in zero K+ irrespective of the season. The ability of EDL fibers to hyperpolarize appears during the 7th and 8th weeks postpartum, and is dependent upon the presence of a functional nerve, since denervation abolished the response. As possible explanations for these findings, inactivation of K(+)-channels and inhibition of the Na-K pump by zero K+ are discussed.     

Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.

We have investigated (a) effects of varying proton concentration on force and shortening velocity of glycerinated muscle fibers, (b) differences between these effects on fibers from psoas (fast) and soleus (slow) muscles, possibly due to differences in the actomyosin ATPase kinetic cycles, and (c) whether changes in intracellular pH explain altered contractility typically associated with prolonged excitation of fast, glycolytic muscle. The pH range was chosen to cover the physiological pH range (6.0-7.5) as well as pH 8.0, which has often been used for in vitro measurements of myosin ATPase activity. Steady-state isometric force increased monotonically (by about threefold) as pH was increased from pH 6.0; force in soleus (slow) fibers was less affected by pH than in psoas (fast) fibers. For both fiber types, the velocity of unloaded shortening was maximum near resting intracellular pH in vivo and was decreased at acid pH (by about one-half). At pH 6.0, force increased when the pH buffer concentration was decreased from 100 mM, as predicted by inadequate pH buffering and pH heterogeneity in the fiber. This heterogeneity was modeled by net proton consumption within the fiber, due to production by the actomyosin ATPase coupled to consumption by the creatine kinase reaction, with replenishment by diffusion of protons in equilibrium with a mobile buffer. Lactate anion had little mechanical effect. Inorganic phosphate (15 mM total) had an additive effect of depressing force that was similar at pH 7.1 and 6.0. By directly affecting the actomyosin interaction, decreased pH is at least partly responsible for the observed decreases in force and velocity in stimulated muscle with sufficient glycolytic capacity to decrease pH.     

Responses of muscle spindles of fast and slow muscles to mechanical stimulation during hypokinesia

Activity of muscle spindles of fast (extensor digitorum longus) and slow (soleus) muscles was studied in cats during hypokinesia of the limb immobilized in a plaster cast. Spontaneous activity of muscle spindles of the fast and slow muscles was unchanged during hypokinesia. Spontaneous activity of primary and secondary endings evoked by passive stretching of the muscle exceeded normal. During stretching of the muscles at different speeds and of different amplitudes, the discharge frequency of the primary and secondary endings was much greater than normally during both the dynamic and the static phase of stretching. These changes were more marked in the slow muscles.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 186–192, March–April, 1978.     

Isolation and characterization of the surface membranes of fast and slow mammalian skeletal muscle.

Fast (extensor digitorum longus) and slow (soleus) rat skeletal muscles served as the source for isolation and biochemical comparison of two distinct surface membrane fractions with properties of the sarcolemma and transverse tubular system. Enriched sarcolemmal membrane from soleus demonstrated a lighter density after sucrose density centrifugation. Sialic acid content was 1.5-fold higher in soleus (62 nmol/mg) than extensor (40 nmol/mg). The specific activity of (Na+ + K+ + Mg2+)-ATPase was similar (1.40 and 1.65 micronmol Pi/mg per 5 min) with the soleus enzyme displaying a (1) greater resistance to inhibition by ouabain, and (2) broader ionic ratio (Na+/K+) requirement than extensor enzyme. The polypeptide and phospholipid composition showed no major differences between the two muscle types. The second surface membrane fraction, tentatively identified as transverse tubule, differed in membrane composition. The major polypeptide of extensor was of 95 000 molecular weight whereas for soleus a Mr=28 000 species was dominant. Total phospholipid content of soleus was 1.5-fold greater than extensor due mostly to increased levels of phosphatidylcholine and phosphatidylethanolamine. Endogenous membrane protein kinase for the 28 000 molecular weight polypeptide was found exclusively in this membrane. The reaction conditions were identical for extensor and soleus since both required divalent cations (Ca2+ and Mg2+) and neither was affected by cyclic AMP. Soleus showed a 2-fold higher capacity for phosphate incorporation than extensor. These studies show that surface membrane fractions derived from fast and slow muscles differ in terms of functional and compositional properties. These differences are specific not only for the surface membrane but for the muscle type and may relate to the known physiological differences observed between fast and slow mammalian muscle.     

Development of ionic currents of zebrafish slow and fast skeletal muscle fibers

Voltage-gated Na+ and K+ channels play key roles in the excitability of skeletal muscle fibers. In this study we investigated the steady-state and kinetic properties of voltage-gated Na+ and K+ currents of slow and fast skeletal muscle fibers in zebrafish ranging in age from 1 day postfertilization (dpf) to 4-6 dpf. The inner white (fast) fibers possess an A-type inactivating K+ current that increases in peak current density and accelerates its rise and decay times during development. As the muscle matured, the V50s of activation and inactivation of the A-type current became more depolarized, and then hyperpolarized again in older animals. The activation kinetics of the delayed outward K+ current in red (slow) fibers accelerated within the first week of development. The tail currents of the outward K+ currents were too small to allow an accurate determination of the V50s of activation. Red fibers did not show any evidence of inward Na+ currents; however, white fibers expressed Na+ currents that increased their peak current density, accelerated their inactivation kinetics, and hyperpolarized their V50 of inactivation during development. The action potentials of white fibers exhibited significant changes in the threshold voltage and the half width. These findings indicate that there are significant differences in the ionic current profiles between the red and white fibers and that a number of changes occur in the steady-state and kinetic properties of Na+ and K+ currents of developing zebrafish skeletal muscle fibers, with the most dramatic changes occurring around the end of the first day following egg fertilization.     

Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle

The appearance of collagen around individual fast twitch (FT) and slow twitch (ST) muscle fibres was investigated in skeletal muscles with different contractile properties using endurance trained and untrained rats as experimental animals. The collagenous connective tissue was analyzed by measuring hydroxyproline biochemically and by staining collagenous material histochemically in M. soleus (MS), M. rectus femoris (MRF), and M. gastrocnemius (MG). The concentration of hydroxyproline in the ST fibres dissected from MS (2.72 +/- 0.35 micrograms X mg-1 d.w.) was significantly higher than that of the FT fibres dissected from MRF (1.52 +/- 0.33 micrograms X mg-1 d.w.). Similarly, the concentration of hydroxyproline was higher in ST (2.54 +/- 0.51 micrograms X mg-1 d.w.) than in FT fibres (1.60 +/- 0.43 micrograms X mg-1 d.w.), when the fibres were dissected from the same muscle, MG. Histochemical staining of collagenous material agreed with the biochemical evidence that MS and the slow twitch area of MG are more collagenous than MRF and the fast twitch area of MG both at the level of perimysium and endomysium. The variables were not affected by endurance training. When discussing the role of collagen in the function of skeletal muscle it is suggested that the different functional demands of different skeletal muscles are also reflected in the structure of intramuscular connective tissue, even at the level of endomysial collagen. It is supposed that the known differences in the elastic properties of fast tetanic muscle compared to slow tonic muscle as, e.g., the higher compliance of fast muscle could at least partly be explained in terms of the amount, type, and structure of intramuscular collagen.     

Subcellular responses of p53 and Id2 in fast and slow skeletal muscle in response to stretch-induced overload.

Tumor suppressor p53 and inhibitor of DNA-binding/differentiation Id2 were examined after 7 or 21 days of wing weighting in fast patagialis (PAT) and slow anterior latissimus dorsi (ALD) wing muscles of young adult and old Japanese quails. The contralateral wing served as the intra-animal control. Seven days of loading increased PAT and ALD muscle weight by 28 and 96%, respectively, in young birds. PAT and ALD muscle weight was 49 and 179% greater, respectively, than control muscles after 21 days of loading in young birds. In aged birds, no PAT or ALD hypertrophy was found after 7 days of loading; however, PAT and ALD muscle weight increased by 29 and 102%, respectively, after 21 days of loading. Id2 protein in the nuclear muscle fraction increased in both PAT and ALD muscles from young adult and old birds that were loaded for 7 days and in ALD muscles after 21 days of loading relative to contralateral control muscles. Nuclear p53 protein was greater in 7- or 21-day loaded PAT and ALD muscles relative to control muscles in both age groups. Cytosolic Id2 and p53 protein contents were not changed in loaded PAT or ALD muscles relative to control muscles at any time point. These data suggest that nuclear, but not cytosolic, Id2 and p53 are responsive to stretch-induced muscle overload. Moreover, the attenuated ability of the aged skeletal muscle to achieve hypertrophy does not appear to be explained by the subcellular changes in Id2 and p53 content with overload.     

Substrate and enzyme profile of fast and slow skeletal muscle fibers in rhesus monkeys

Results from the Russian Cosmos program suggest that the rhesusmonkey is an excellent model for studying weightlessness-induced changes in muscle function. Consequently, the purpose of this investigation was to establish the resting levels of selected substrateand enzymes in individual slow- and fast-twitch muscle fibers of therhesus monkey. A second objective was to determine the effect of an18-day sit in the Spacelab experiment-support primate facility[Experimental System for the Orbiting Primate (ESOP)].Muscle biopsies of the soleus and medial gastrocnemius muscles wereobtained 1 mo before and immediately after an 18-day ESOP sit. Thebiopsies were freeze-dried, and individual fibers were isolated andassayed for the substrates glycogen and lactate and for the high-energyphosphates ATP and phosphocreatine. Fiber enzyme activity was alsodetermined for the glycolytic enzymes phosphofructokinase and lactatedehydrogenase (LDH) and for the oxidative markers 3-hydroxyacyl-CoAdehydrogenase (-OAC) and citrate synthase. Consistent with otherspecies, the fast type II fibers contained higher glycogen content thandid the slow type I fibers. The ESOP sit had no significant effects onthe metabolic profile of the slow fibers of either muscle or the fast fibers of the soleus. However, the fast gastrocnemius fibers showed asignificant decline in phosphocreatine and an increase in lactate. Also, similar to other species, the fast fibers contained significantly higher LDH activities and lower 3-hydroxyacyl-CoA dehydrogenase activities. For the muscle enzymes, the quantitatively most important effect of the ESOP sit occurred with LDH where activities increased inall fiber types postsit except the slow type I fiber of the medial gastrocnemius.

     

Differential response of fast and slow myosin ATPase from skeletal muscle to F-actin and to phalloidin F-actin

Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89±0.34 μM with F-actin and 0.90±0.71 μM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95±1.27 μM with F-actin and 0.27±0.16 μM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.     

Divalent cation binding properties of slow skeletal muscle troponin in comparison with those of cardiac and fast skeletal muscle troponins

1. New methods of preparing troponins from slow skeletal and cardiac muscle of the chicken have been developed. The electrophoretic mobilities of slow skeletal muscle troponin subunits were different from those of the corresponding fast skeletal muscle subunits. 2. A new method for determining the amount of divalent cations bound to troponin was developed. The principle of the method is to immobilize troponin by conjugating it with Sepharose 4B resin, thus making it readily sedimentable. 3. The numbers of Sr and Ca ions bound to slow muscle troponin at concentrations sufficient to produce maximum contraction were 1.73 and 1.36 mol per mol, respectively, being nearly equal to those of cardiac troponin but half of those of fast muscle troponin. 4. The concentrations of Sr and Ca ions giving half-maximal ion binding to slow muscle troponin (K50%) were 5.5 X 10(-6) M and 4.6 X 10(-7) M, respectively. 5. K50% for Sr of cardiac troponin was significantly higher than that of slow muscle troponin. Although K50% for Sr of cardiac troponin was the same as that of fast muscle troponin, cardiac troponin bound more Sr ions than fast muscle troponin at lower Sr ion concentrations. The mechanism underlying the high sensitivity of cardiac muscle contraction to Sr ions is discussed in comparison with that of slow muscle.