Effects of pH on myofibrillar ATPase activity in fast and slow skeletal muscle fibers of the rabbit.

In permeabilized single fibers of fast (psoas) and slow (soleus) muscle from the rabbit, the effect of pH on isometric myofibrillar ATPase activity and force was studied at 15 degrees C, in the pH range 6.4-7.9. ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH, present in the bathing solution. NADH absorbance at 340 nm was determined inside a measuring chamber. To measure ATP turnover in single soleus fibers accurately, a new measuring chamber (volume 4 microliters) was developed that produced a sensitivity approximately 8 times higher than the system previously used. Under control conditions (pH 7.3), the isometric force was 136 and 115 kN/m2 and the ATP turnover was 0.43 and 0.056 mmol per liter fiber volume per second in psoas and soleus fibers, respectively. Over the pH range studied, isometric force increased monotonically by a factor 1.7 for psoas and 1.2 for soleus fibers. In psoas the isometric ATPase activity remained constant, whereas in soleus it slightly decreased with increasing pH. The pH dependency of relative tension cost (isometric ATPase activity divided by force) was practically identical for psoas and soleus fibers. In both cases it decreased by about a factor 0.57 as pH increased from 6.4 to 7.9. The implications of these findings are discussed in terms of cross-bridge kinetics. For both fiber types, estimates of the reaction rates and the distribution of cross-bridges and of their pH dependencies were obtained. A remarkable similarity was found between fast- and slow-twitch fibers in the effects of pH on the reaction rate constants.     

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.     

Force generation and phosphate release steps in skinned rabbit soleus slow-twitch muscle fibers.

The force-generation and phosphate-release steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibers (STF) were investigated using sinusoidal analysis, and the results were compared with those of rabbit psoas fast-twitch fibers (FTF). Single fiber preparations were activated at pCa 4.40 and ionic strength 180 mM at 20 degrees C. The effects of inorganic phosphate (Pi) concentrations on three exponential processes, B, C, and D, were studied. Results are consistent with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and P is inorganic phosphate. The values determined are k4 = 5.7 +/- 0.5 s-1 (rate constant of isomerization step, N = 9, mean +/- SE), k-4 = 4.5 +/- 0.5 s-1 (rate constant of reverse isomerization), K4 = 1.37 +/- 0.13 (equilibrium constant of the isomerization), and K5 = 0.18 +/- 0.01 mM-1 (Pi association constant). The isomerization step (k4) in soleus STF is 20 times slower, and its reversal (k-4) is 20 times slower than psoas fibers. Consequently, the equilibrium constant of the isomerization step (K4) is the same in these two types of fibers. The Pi association constant (K5) is slightly higher in STF than in FTF, indicating that Pi binds to cross-bridges slightly more tightly in STF than FTF. By correlating the cross-bridge distribution with isometric tension, it was confirmed that force is generated during the isomerization (step 4) of the AMDP state and before Pi release in soleus STF.     

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.     

Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

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.     

Desmin filaments influence myofilament spacing and lateral compliance of slow skeletal muscle fibers

Intermediate filaments composed of desmin interlink Z-disks and sarcolemma in skeletal muscle. Depletion of desmin results in lower active stress of smooth, cardiac, and skeletal muscles. Structural functions of intermediate filaments in fast (psoas) and slow (soleus) skeletal muscle were examined using x-ray diffraction on permeabilized muscle from desmin-deficient mice (Des-/-) and controls (Des+/+). To examine lateral compliance of sarcomeres and cells, filament distances and fiber width were measured during osmotic compression with dextran. Equatorial spacing (x-ray diffraction) of contractile filaments was wider in soleus Des-/- muscle compared to Des+/+, showing that desmin is important for maintaining lattice structure. Osmotic lattice compression was similar in Des-/- and Des+/+. In width measurements of single fibers and bundles, Des-/- soleus were more compressed by dextran compared to Des+/+, showing that intermediate filaments contribute to whole-cell compliance. For psoas fibers, both filament distance and cell compliance were similar in Des-/- and Des+/+. We conclude that desmin is important for stabilizing sarcomeres and maintaining cell compliance in slow skeletal muscle. Wider filament spacing in Des-/- soleus cannot, however, explain the lower active stress, but might influence resistance to stretch, possibly minimizing stretch-induced cell injury.     

Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 ± 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean ± S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 ± 69 vs. 34 ± 21 x 10 ³µm ³) than fast and slow soleus fibers (40 ± 20 vs. 30 ± 14 x 10 ³µm ³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 µm) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 µm) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 ± 51 vs. 55 ± 22 and 44 ± 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

Fiber-type proportions in mammalian soleus muscle during postnatal development

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%–70% being fast and 30%–40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

Fiber-type proportions in mammalian soleus muscle during postnatal development.

We analyzed the fiber-type composition of the soleus muscle in rats and mice to determine whether the adult proportion of fiber types is fixed soon after birth or whether it changes during postnatal maturation. We examined muscles from animals varying in age from 1 week to 1 year using monoclonal antibodies that distinguish between fast and slow isoforms of myosin heavy chains. In cross sections of unfixed muscle containing profiles of all myofibers in the muscle, we counted the fibers that stained with antibodies to fast myosin, and in adjacent sections, those that stained positive with an antibody to slow myosin. We also counted the total number of fibers in each section. Rat soleus contained about 2500 myofibers, and mouse about 1000 at all ages studied, suggesting that myogenesis ceases in soleus by 1 week after birth or sooner. In mouse soleus, the relative proportions of fibers staining positive with fast and slow myosin antibodies were similar at all ages studied, about 60%-70% being fast and 30%-40% slow. In rat soleus, however, the proportions of fast antibody-positive and slow antibody-positive fibers changed dramatically during postnatal maturation. At 1 week after birth, about 50% of rat soleus fibers stained with fast myosin antibodies, whereas between 1 and 2 months this value fell to about 10%. In mouse, about 10% of fibers at 1 week, but none at 1 year, reacted with both fast and slow antibodies, whereas in rat, fewer than 3% bound both antibodies to a significant degree at 1 week. It is puzzling why, in rat soleus, the majority of apparently fast fibers present at 1 week is converted to a slow phenotype, whereas in mouse soleus the predominant change appears to be the suppression of fast myosin expression in a subset of fibers that expresses both myosin types at 1 week. It is possible that this may be related to differences in size and the amount of body growth between these two species.     

大鼠和家兔生后发育各阶段比目鱼肌纤维的比较

为研究大鼠与家兔骨骼肌各类型肌纤维的数量和二维分布以及生后发育对其影响,取生后2d和2、4、6、8、10周龄(体重10g和32、95、190、280、320g)大鼠及生后2d和2、4、8、12、16、20、24周龄(体重100g和220、400、750、1200、1600、2100、2500g)家兔的比目鱼肌做琥珀酸脱氢酶染色。实验结果表明,大鼠和家兔比目鱼肌纤维被分成Ⅰ型(SO),ⅡX型(FO)和ⅡA型(FOG)3型。使用图像分析系统分析每型肌纤维在生后发育各阶段的相关变化,大鼠和家兔比目鱼肌中：Ⅰ型纤维分布于整块肌肉,其数量随着生后发育而增加。幼体ⅡX型纤维分布在整块肌肉中,其数量随生后发育而减少;ⅡA型分布在肌肉中深层,数量几乎无变化;至成体时只有少量的ⅡX和ⅡA分布在肌表层。整个发育期间未见ⅡB型纤维。ⅡA型纤维直径最大,Ⅰ型中等,而ⅡX型最小。家兔3型肌纤维的平均横切面积比大鼠的大。这些结果表明大鼠和家兔后肢肌各种类型肌纤维的数量比例和分布随生长过程发生改变。     

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Abstract. Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the 'slow' area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.     

Isometric training of young rats — Effects upon hind limb muscles

The soleus, rectus femoris, and gastrocnemius muscles of young rats trained isometrically for 4 weeks were studied by light and electron microscopy.--The percentage of fast-twitch oxidative muscle fibers decreased at the cost of the fast-twitch glycolytic fibers in the rectus femoris muscle. The percentages of the slow-twitch oxidative fibers did not change significantly in any of the muscles studied. The changes in the areas of the muscle fibers were specific for the muscle and the fiber type and indicate geometrical rearrangements of the fibers in the trained muscles. The Z and M lines were broader in the soleus (containing about 85% slow-twitch oxidative fibers) than in the rectus femoris muscle (containing about 90% fast-twitch glycolytic fibers), while the sarcomere length and the pseudo-H zone were similar. The length of the myosin filaments appeared to be slightly shorter in the fast rectus femoris than in the slow soleus muscle.--The hypothesis on the temporal progress of muscle adaptation to training (Müller, 1974) was substantiated. Correlations between biochemical (Exner et al., 1973a) and histochemical parameters measuring the oxidative capacity were preserved during adaptation to training. The comparison of the histochemical results with the physiological data on similar animals (Exner et al., 1973a) suggests a complex relationship between the contraction time and the percentage of fast-twitch muscle fibers.     

Myogenic and neurogenic contributions to the development of fast and slow twitch muscles in rat.

The histochemical ATPase activity and the myosin light chains of a rat fast muscle (extensor digitorum longus, EDL) and a rat slow muscle (soleus) during development have been investigated. Both muscles initially synthesize fast myosin light chains and show the intense histochemical ATPase activity characteristic of adult fast muscle fibers. After birth, the soleus begins to accumulate slow fibers with their characteristic low histochemical ATPase activity, and slow myosin light chains begin to appear. Sciatic neurectomy prevents the development of slow fibers and the synthesis of slow myosin light chains in the soleus, while the EDL is unaffected. Similarly, cordotomy of an adult rat results, in the soleus, in the appearance of fibers with more intense staining for ATPase and an increase in fast myosin light chains. The EDL is unchanged by cordotomy. As a result, we suggest that slow muscle development, but not fast muscle development, is dependent upon the functional activity of the nervous system.     

Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle

The myosin isozymes present in the developing rat soleus muscle from 1 week to 6 weeks after birth were investigated using biochemical and immunological methods. Electrophoresis of native myosin reveals that adult slow myosin is present in the soleus as early as 1 week after birth. At this time, embryonic and neonatal myosin can also be demonstrated. Using an immunotransfer technique, the presence of slow myosin heavy chain can be demonstrated at all time points examined whereas neonatal myosin heavy chain diminishes in quantity between 2 and 3 weeks, and is undetectable in the adult soleus. Specific polyclonal antibodies were prepared to embryonic, neonatal, and adult fast and slow myosins. Immunocytochemistry reveals a cellular heterogeneity at all stages examined. Different combinations of myosin isozymes can be found in the soleus fibers depending on the stage of development; these results suggest therefore that myosin isozyme transitions are occurring. Approximately half the fibers contain embryonic and slow myosin at 1 week after birth; these fibers subsequently contain only slow myosin. A second group of fibers contains embryonic and neonatal myosin at 1 week and most of them subsequently accumulate adult fast myosin. A portion of this latter group begins to acquire slow myosin from 4 weeks of age. These data are interpreted to suggest that a preprogrammed sequence of myosin isozymes is embryonic----neonatal----adult fast. At any time during development of an individual fiber, induction of slow myosin accumulation and repression of other types can occur.     

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

We investigated the expression and functional properties of slow skeletal troponin T (sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight (sTnT1, sTnT2) and low-molecular-weight (sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca(2+) activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca(2+) affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.