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.     

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.     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

Influence of inorganic phosphate and pH on ATP utilization in fast and slow skeletal muscle fibers.

The influence of P(i) and pH was studied on myofibrillar ATP turnover and force development during maximally activated isometric contractions, in skinned single fibers from rabbit soleus and psoas muscle. ATP hydrolysis was coupled to the breakdown of NADH, which was monitored photometrically at 340 nm. In psoas the depression by phosphate of force is twice that of ATP turnover, but in soleus force and ATP turnover are depressed equally by P(i). Most, but not all, of the ATPase and force values observed for a combination of high P(i) and low pH could be explained by independent effects of P(i) and pH. The effects of P(i) and pH on ATP turnover can be understood by a three-state cross-bridge scheme. Mass action of phosphate on the reaction from the actomyosin(AM).ADP state to the AM.ADP.P(i) state may largely account for the phosphate dependencies of ATPase activity found. Protons affect cross-bridge detachment from the AM.ADP state and the rate of the AM.ADP.P(i)-to-AM.ADP transition. In this scheme, the effects of P(i) and pH on cross-bridge kinetics appeared to be largely independent.     

Miniature potentials in fast and slow muscle fibers in locusts

Excitatory miniature postsynaptic potentials were studied by an intracellular recording method in fast and slow muscle fibers ofLocusta migratorioides. Statistical analysis showed that liberation of mediator in both types of fibers can be predicted by the formula for a negative binomial distribution with a probability of 85%. This correlation is evidence of some degree of interaction between consecutive liberations of quanta of mediator by nerve endings. It is shown that the fraction of miniature potentials depending on the external calcium concentration is greater in fast muscle fibers. An increase in the magnesium ion concentration from 2 to 40 mM led to a decrease in the frequency of miniature potentials, and this decrease was greater in fast fibers; an increase in the magnesium ion concentration from 1 to 10 mM in calcium-free solutions, on the other hand, led to some increase in frequency, and this also was greater in fast muscle fibers. It is concluded that nerve endings in fast and slow muscle fibers differ in their sensitivity to changes in the ionic composition of the medium.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 13, No. 2, pp. 210–217, March–April, 1981.     

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.     

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.     

Controlled differentiation of myoblast cells into fast and slow muscle fibers

Skeletal muscles are classified into fast and slow muscles, which are characterized by the expression of fast-type myosin heavy chains (fMyHCs) or slow-type myosin heavy chains (sMyHCs), respectively. However, the mechanism of subtype determination during muscle fiber regeneration is unclear. We have analyzed whether the type of muscle is determined in the myoblast cells or is controlled by the environment in which the muscle fibers are formed from myoblast cells. When myoblast cells from 7-day-old chick embryo were cultured and formed into muscle fibers, more than half of the fibers produced only fMyHCs, and the remaining fibers produced both fMyHCs and sMyHCs. However, when myoblast cells were cultured in medium supplemented with a small amount of slow muscle extract, the expression of sMyHCs in muscle fibers increased, whereas the expression of fMyHCs increased in the group supplemented with fast muscle extract compared with the control group. The same results were obtained when cloned mouse myoblast cells (C₂C₁₂ cells) were cultured and formed into muscle fibers. The data presented here thus show that the subtype differentiation of muscle fiber is controlled by the environment in which the muscle fiber forms. This work was funded by the Sasakawa Scientific Research Grant of the Japan Science Society.     

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.     

Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study

Phospholamban, originally described as a cardiac sarcoplasmic reticulum protein, was localized in cryostat sections of three adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with highly specific phospholamban antibodies. Only some myofibers were strongly labeled with phospholamban antibodies. The labeling of myofibers with phospholamban antibodies was compared to the distribution of Type I (slow) and Type II (fast) myofibers as determined by staining adjacent sections cytochemically for the alkali-stable myosin ATPase, a specific marker for Type II myofibers. All the skeletal myofibers labeled for phospholamban above background levels corresponded to Type I (slow) myofibers. The presence of phospholamban in microsomal fractions isolated from canine superficial digitalis flexor (89 +/- 3% Type I) and extensor carpi radialis skeletal muscle (14 +/- 6% Type I) was confirmed by immunoblotting. Antiserum to cardiac phospholamban bound to proteins of apparent Mr values of 25,000 (oligomeric phospholamban) and 5,000-6,000 (monomeric phospholamban) in sarcoplasmic reticulum vesicles from both muscles. Quantification of phospholamban in sarcoplasmic reticulum vesicles from cardic, slow, and fast skeletal muscle tissues following phosphorylation with [gamma-32P] ATP suggested that superficial digitalis flexor and extensor carpi radialis skeletal muscle contained about 16 and 3%, respectively, as much phospholamban as cardiac muscle per unit of sarcoplasmic reticulum. The presence of phospholamban in both Type I (slow) and cardiac muscle fibers supports the possibility that the Ca2+ fluxes across the sarcoplasmic reticulum in both fiber types are similarly regulated, and is consistent with the idea that the relaxant effect of catecholamines on slow skeletal muscle is mediated in part by phosphorylation of phospholamban.     

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.     

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.     

Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

The effect of exercise-training on sarcoplasmic reticulum function in fast and slow skeletal muscle

The effect of endurance exercise on the capacity of crube homogenates (CH) to sequester Ca²⁺ was determined in the slow type I soleus, the fast type IIA deep region of the vastus lateralis (DVL), and the fast type IIB superficial region of the vastus lateralis (SVL). The Ca²⁺ uptake capacity was affected by exercise in a fiber type specific manner. The fast-twitch SVL showed a 35% decrease in the maximal rate of Ca²⁺ uptake (V_max) and a significantly lower K_m while the slow soleus and fast DVL were unaltered. The time course of Ca²⁺ uptake, and the peak amount of Ca²⁺ sequestered was not altered by exercise in any of the muscles studied. The homogenates from the exercise-trained soleus muscles exhibited an increased ability to retain Ca²⁺ and in this capacity became more like fast muscle.     

Distinction of fast fibers and slow fibers in frog abdominal muscle using certain choline esters]

We have registered the contractions of the frog's abdominal muscle in Ringer's solution. The contractions were produced either by acetylcholin or by succinyldicholin. The graphics showed that the succinyldicholin contractions were much slower than the acetylcholin ones. The tracings were not equalized by a preliminary addition of eserin. We conclude that acetylcholine excites the fast fibers and that succinyldicholin excites the slow fibers of the muscle.     

Contractile proteins of fast and slow fibers during differentiation of lobster claw muscle

Contractile protein populations were determined, using gel electrophoresis, during development of the claw closer muscles of the lobster Homarus americanus. In the adult the paired claw closer muscles are asymmetric, consisting of a crusher muscle with all slow fibers and a cutter muscle with a majority of fast and a few slow fibers. The electrophoretic banding pattern of these adult fast and slow fibers shows a similarity in the major proteins including myosin, actin, and tropomyosin which are common to both fiber types. Paramyosin is slightly heavier in fast fibers than in slow. However, fast fibers have three proteins and slow fibers have four proteins which are unique to themselves. Several of these unique proteins belong to the regulatory troponin complexes. In juvenile 4th stage lobster, where the paired closer muscles are undifferentiated, the banding pattern reveals the presence of proteins common to both fiber types including myosin, actin, and tropomysin but the conspicuous absence of all unique fast fiber proteins as well as one unique slow fiber protein. By the juvenile 10th stage most of these unique proteins are present except for one unique slow fiber protein. Thus lobster fast and slow fiber differentiation entails coordinate gene activation to add unique contractile proteins.