Recovery of force during postcontractile depression in single Xenopus muscle fibers

This study examined the relationship between force and cytosolic free calcium concentration ([Ca2+]c) in different fiber types from Xenopus before, during, and after cells underwent postcontractile depression (PCD). During a standardized fatigue run, force in the two fast fatiguing (FF) fiber types (types 1 and 2, n = 10) fell more quickly (5.8 vs. 8.1 min) and to a greater degree [0.36 vs. 0.51 of initial (P(o))] than in the slow fatiguing (SF) fiber type (type 3, n = 11). After the initial fatigue run, both FF and SF experienced a drop in force to <15% P(o) (PCD) at a similar time (20.6 vs. 21.4 min). A second stimulation period, undertaken during PCD, produced significant recovery of force in both groups, but significantly more so in SF than FF (64 +/- 7 vs. 29 +/- 2% P(o)). This force recovery during PCD was accompanied by a significant increase in peak [Ca2+]c, particularly in SF. However, despite the significant recovery of force during stimulation while in PCD, the amount of force produced for a given peak [Ca2+]c was significantly lower in both groups during PCD than at any other point in the experiment. A final stimulation period, initiated when all fibers had recovered from PCD, demonstrated a recovery of both force and peak [Ca2+]c in both groups, but this recovery was significantly greater in SF vs. FF. These data demonstrate that with continuous electrical stimulation, it is possible to produce a significant recovery of force production during the normally quiescent period of PCD, but that it occurs with a decreased muscle force production for a given peak [Ca2+]c. This suggests that factors other than structural alterations of the sarcoplasmic reticulum are likely the cause of PCD in these fibers.     

Phosphorylating pathways and fatigue development in contracting Xenopus single skeletal muscle fibers

To investigate the differential contribution of oxidative and substrate-level phosphorylation to force production during repetitive, maximal tetanic contractions, single skeletal muscle fiber performance was examined under conditions of high-oxygen availability and anoxia. Tetanic force development (P) was measured in isolated, single type-1 muscle fibers (fast twitch; n = 6) dissected from Xenopus lumbrical muscle while being stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz), with each frequency lasting 2 min. Two separate work bouts were conducted, with the perfusate PO(2) being either 0 or 159 mmHg. No significant (P < 0. 05) difference was found in the initial peak tensions (P(0)) between the high (334 +/- 57 kPa) and the low (325 +/- 41 kPa) PO(2) treatment. No significant difference in P was observed between the treatments during the first 50 s. However, a significant difference in force production was observed between the high (P/P(0) = 0.96 +/- 0.02) and the low PO(2) condition (P/P(0) = 0.92 +/- 0.02) by 60 s of work. After 60 s, steady-state force production was maintained during the high compared with the low PO(2) condition until stimulation frequency was increased, at which point developed tension during the high PO(2) condition began to decline. Time to fatigue (P/P(0) = 0.3) was reached significantly sooner during the low (250 +/- 16 s) than the high PO(2) condition (367 +/- 28 s). These results demonstrate that during the first 50 s of 0.25-Hz contractions, substrate-level phosphorylation has the capacity to maintain force and ATP hydrolysis when oxidative phosphorylation is absent. This period was followed by an oxygen-dependent phase in which force generation was maintained during the high PO(2) condition (but not during the low PO(2) condition) until the onset of a final fatiguing phase, at which a calculated maximal rate of oxidative phosphorylation was reached.     

Twitch and tetanic tension during culture of mature Xenopus laevis single muscle fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 microm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days ('stable fibres'), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture ('unstable fibres'). After 7-9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.     

Mechanical properties of muscle spindles in Xenopus laevis

Mechanical properties of isolated living muscle spindles from Xenopus laevis were examined in order to determine their role in sensory transduction. The reticular zone of the intrafusal muscle fibers was identified microscopically by: (1) its position beneath the sensory endings, (2) its length, 50–100 μm, (3) its extension during intrafusal muscle contraction, and (4) its coarse striations with a period of about 1.5 times the normal sarcomere length. The reticular zone in the passive muscle spindle did not extend until the spindle was stretched to about 1.05–1.1 its maximal length in the animal (L _m ). Evidence was obtained that the absence of extension of the reticular zone at normal muscle lengths was due to the presence of the spindle capsule which acted as a stiff element in parallel with the sensory region. At those lengths at which the reticular zone did extend (> L _m ), no rate — sensitive mechanical properties were detected in response to step and ramp extensions. The sensory discharge of the spindle showed no dynamic transient in response to ramp extensions if the reticular zone were not extended. During extension of the reticular zone a dynamic sensory transient appeared. It is concluded that current notions on the mechanical origin of the rate — sensitive properties of the sensory discharge of the muscle spindle do not apply to Xenopus laevis. In addition, it is not likely that the passive spindle in this animal is a sensitive stretch receptor.     

Differential expression of the Ca2+-binding protein parvalbumin during myogenesis in Xenopus laevis

We have used immunocytological techniques to examine the developmental expression of the Ca2+-binding protein parvalbumin in Xenopus laevis embryos. Western blot experiments show that at least three different forms of parvalbumin are expressed during embryogenesis; the tadpole tail expresses one form, adult brain expresses another, mylohyoid muscle expresses both, and gastrocnemius and sartorius muscles express these two plus a third form. Parvalbumin (PV) is first detectable by immunofluorescence at stages 24-25 of development, a time when myotomal muscles are differentiating and contractile activity occurs spontaneously in embryos. At metamorphosis, PV is expressed in developing limb muscles. While the majority of skeletal muscle fibers express high levels of PV in both embryos and adults, a second fiber type has no detectable PV. The arrangement of PV-containing fibers is stereotyped in each muscle group examined. Histochemical staining of tadpole muscles indicate that PV-containing fibers correspond to fast-twitch skeletal muscles, whereas those without PV correspond to slow-twitch muscles. During tail resorption at metamorphosis, PV appears to be extruded from dying tail muscle cells and taken up by phagocytic cells.     

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis.

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin isoenzymes in single muscle fibres of Xenopus laevis: analysis of five different functional types

An analysis has been performed of the native myosin isoenzyme composition of isolated skeletal muscle fibres from Xenopus laevis with well-defined isotonic contraction properties. Fast twitch 'white' (type 1) fibres contained three isomyosins; fast twitch 'red' (type 2) fibres showed two major myosin bands with migration velocities very similar to those of the two slower bands in type 1. Slow twitch (type 3) fibres yielded a single, slowly migrating band as did slow tonic (type 5) fibres, whereas the myosin from type 4 (very slow twitch, 'intermediate') fibres migrated with a somewhat higher mobility. The results suggest that amphibian skeletal muscle may possess the principal fibre types found in mammals and birds.     

Dichloroacetate accelerates the fall in intracellular PO2 at onset of contractions in Xenopus single muscle fibers

mM DCA, whereas the second group [control (Con); n = 10] was incubated for 30 min in Ringer solution only. After incubation, fibers were electrically stimulated to elicit tetanic contractions (0.5 Hz) for 2 min during which PiO2 was monitored. PiO2 before contractions began was 32.0 +/- 1.8 and 29.0 +/- 1.8 Torr for DCA and Con, respectively, and fell to 6.0 +/- 1.3 and 8.8 +/- 2.4 Torr (no significant difference), respectively, after steady state was reached. The kinetics of the fall, determined by both the time delay (from the start of contractions to the initial decrease in PiO2) and the tau (63% of the change to a steady state in PiO2), were calculated. In DCA cells, the tau was significantly (P < 0.05) faster than Con (22.1 +/- 3.6 vs. 39.7 +/- 5.8 s). In contrast, the time delay was not significantly (P > 0.45) different between the two groups (11.4 +/- 1.7 vs. 12.6 +/- 2.3 s, respectively). The amount of fatigue, reflected by a decrease in force production from initial, was not significantly different between groups. These data suggest that by stimulating pyruvate dehydrogenase with DCA in isolated single skeletal muscle cells, the faster fall in PiO2 is indicative of oxidative metabolism being more rapidly activated. This is the first evidence that oxygen uptake at the onset of contractions may be altered by DCA during moderate- to high-intensity contractile activity.     

Characteristics of the sensory discharge of the muscle spindle in Xenopus laevis

Single isolated muscle spindles from the toad Xenopus laevis were studied with regard to their response to different levels of steady stretch and to their response to small precisely controlled length variations. The spectral distribution of the applied variations was designed to be essentially uniform in the region between 0.04 Hz and a number of selectable upper limits none exceeding 20 Hz. The results obtained relate to the statistics of receptor discharge intervals, to receptor transfer functions and to the coding and decoding of sensory information. The conclusion is that spectral analysis techniques can be used to clarify many aspects of muscle spindle behavior.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Identification of myosin heavy chain isoforms in skeletal muscle of four Southern African wild ruminants

The aim was to separate and characterize the myosin heavy chain (MHC) isoforms of four southern African wild ruminants, namely Blesbuck (Damaliscus dorcas phillipsi), Kudu (Tragelaphus strepsiceros), Black Wildebeest (Connochaetes gnou) and Blue Wildebeest (Connochaetes taurinus). Longissimus dorsi muscle samples were subjected to SDS-PAGE and Western blot analyses using antibodies raised against MHC isoforms. The specificity of these antibodies was assessed using immunohistochemistry combined with ATPase histochemistry, Three MHC isoforms were separated and the bands were identified from fastest to slowest migrating as MHC I, MHC IIx and MHC IIa. The mobility of the MHC isoforms was similar for all four species, including that of bovine, but differed from human muscle. Kudu muscle exhibited the lowest proportion of MHC I and the highest proportion of MHC IIx, whereas Blesbuck muscle had the least MHC IIx. The two Wildebeest species were intermediate in isoform content. In conclusion, when new species are studied, existing electrophoretic protocols may need to be modified to achieve quantifiable separation and isoform migration pattern must be verified in order to reach correct interpretations. Furthermore, antibody specificity may differ between techniques as well as species and needs confirmation.     

Intracellular PO2 kinetics at different contraction frequencies in Xenopus single skeletal muscle fibers.

Increasing contraction frequency in single skeletal muscle fibers has been shown to increase the magnitude of the fall in intracellular Po(2) (Pi(O(2))), reflecting a greater metabolic rate. To test whether Pi(O(2)) kinetics are altered by contraction frequency through this increase in metabolic stress, Pi(O(2)) was measured in Xenopus single fibers (n = 11) during and after contraction bouts at three different frequencies. Pi(O(2)) was measured via phosphorescence quenching at 0.16-, 0.25-, and 0.5-Hz tetanic stimulation. The kinetics of the change in Pi(O(2)) from resting baseline to end-contraction values and end contraction to rest were described as a mean response time (MRT) representing the time to 63% of the change in Pi(O(2)). As predicted, the fall in Pi(O(2)) from baseline following contractions was progressively greater at 0.5 and 0.25 Hz than at 0.16 Hz (32.8 +/- 2.1 and 29.3 +/- 2.0 Torr vs. 23.6 +/- 2.2 Torr, respectively) since metabolic demand was greater. The MRT for the decrease in Pi(O(2)) was progressively faster at the higher frequencies (0.5 Hz: 45.3 +/- 4.5 s; 0.25 Hz: 63.3 +/- 4.1 s; 0.16 Hz: 78.0 +/- 4.1 s), suggesting faster accumulation of stimulators of oxidative phosphorylation. The MRT for Pi(O(2)) off-kinetics (0.5 Hz: 84.0 +/- 11.7 s; 0.25 Hz: 79.1 +/- 8.4 s; 0.16 Hz: 81.1 +/- 8.3 s) was not different between trials. These data demonstrate in single fibers that the rate of the fall in Pi(O(2)) is dependent on contraction frequency, whereas the rate of recovery following contractions is independent of either the magnitude of the fall in Pi(O(2)) from baseline or the contraction frequency. This suggests that stimulation frequency plays an integral role in setting the initial metabolic response to work in isolated muscle fibers, possibly due to temporal recovery between contractions, but it does not determine recovery kinetics.     

Identification and quantification of actin isoforms in vertebrate cells and tissues

The cytoskeletal protein actin exists in vertebrates as six different isoforms, which are difficult to identify conclusively because of a high degree (greater than 90%) of overall sequence homology. We have used IEF immunoblotting in combination with a panel of isoform-specific and -selective antibodies to analyze the actin isoform composition of nine tissues from adult rat. In three nonmuscle tissues (lung, spleen, and testis), we detected a previously unreported isoform that we identified as smooth muscle alpha. The IEF immunoblot technique was also used to quantify the proportions of the isoforms expressed in these nine rat tissues.     

Identification and characterisation of Xenopus moesin, a Src substrate in Xenopus laevis oocytes.

The cortical actin cytoskeleton, consisting of actin filaments and actin binding proteins, immediately underlies the inner surface of the plasma membrane and is important both structurally and in relaying signals from the surface to the interior of the cell. Signal transduction processes, initiated in the cortex, modulate numerous cellular changes ranging from modifications of the local cytoskeleton structure, the position in the cell cycle, to cell behaviour. To examine the molecular mechanisms and events associated with cortical changes. We have investigated targets of the protein tyrosine kinase, Src, which is associated with the cortical cytoskeleton, in Xenopus laevis oocytes. When a mRNA encoding an activated form of Src tyrosine kinase (d-Src) is injected into oocytes several changes are observed: proteins are phosphorylated, the rate at which progesterone matures an oocyte to an egg is accelerated, and the cortex at the site of injection appears to contract. Previous studies have implicated actin filaments in the Src-stimulated cortical rearrangements. In this study we identify two actin binding proteins-cortactin and moesin--as Src substrates in Xenopus oocytes that are Src substrates. We cloned and characterised the cDNA encoding one of those, Xenopus moesin, a member of the ezrin/radixin/moesin (ERM) family of actin binding proteins. In addition, we have determined that moesin is recruited to the cortex at the site of Src mRNA injection.