EDL Fibers Research Articles

Impact of lengthening velocity on the generation of eccentric force by slow-twitch muscle fibers in long stretches

After an initial increase, isovelocity elongation of a muscle fiber can lead to diminishing (referred to as Give in the literature) and subsequently increasing force. How the stretch velocity affects this behavior in slow-twitch fibers remains largely unexplored. Here, we stretched fully activated individual rat soleus muscle fibers from 0.85 to 1.3 optimal fiber length at stretch velocities of 0.01, 0.1, and 1 maximum shortening velocity, vmax, and compared the results with those of rat EDL fast-twitch fibers obtained in similar experimental conditions. In soleus muscle fibers, Give was 7%, 18%, and 44% of maximum isometric force for 0.01, 0.1, and 1 vmax, respectively. As in EDL fibers, the force increased nearly linearly in the second half of the stretch, although the number of crossbridges decreased, and its slope increased with stretch velocity. Our findings are consistent with the concept of a forceful detachment and subsequent crossbridge reattachment in the stretch’s first phase and a strong viscoelastic titin contribution to fiber force in the second phase of the stretch. Interestingly, we found interaction effects of stretch velocity and fiber type on force parameters in both stretch phases, hinting at fiber type-specific differences in crossbridge and titin contributions to eccentric force. Whether fiber type-specific combined XB and non-XB models can explain these effects or if they hint at some not fully understood properties of muscle contraction remains to be shown. These results may stimulate new optimization perspectives in sports training and provide a better understanding of structure–function relations of muscle proteins.

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching.

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin-deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this, we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model, which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse, we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%), and this correlates with a graded force loss over three eccentric contractions for dKO muscles (~36% after first contraction, ~66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (~75% after first contraction, ~89% after three contractions). In dKO, protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

Krüppel-like factor 10 regulates the contractile properties of skeletal muscle fibers in mice.

Klf10 is a member of the Krüppel-like family of transcription factors, which is implicated in mediating muscle structure (fiber size, organization of the sarcomere), muscle metabolic activity (respiratory chain), and passive force. The aim of this study was to further characterize the roles of Klf10 in the contractile properties of skeletal muscle fibers. Fifty-two single fibers were extracted from female wild-type (WT) and Klf10 knockout (KO) oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skinned muscles. Each fiber was immersed successively in relaxing (R), washing (W), and activating (A) solutions. Calcium was included in the activating solution to induce a maximum contraction of the fiber. The maximum force (Fmax ) was measured and normalized to the cross-sectional area to obtain the maximum stress (Stressmax ). After a steady state in contraction was reached, a quick stretch-release was performed; the force at the maximum stretch (Fstretch ) was measured and the stiffness was assessed. Deletion of the Klf10 gene induced changes in the contractile parameters (Fmax , Stressmax , Stiffness), which were lower and higher for soleus and EDL fibers compared with littermates, respectively. These measurements also revealed changes in the proportion and resistance of attached cross-bridges. Klf10 plays a major role in the homeostasis of the contractile behavior of skeletal muscle fibers in a muscle fiber type-specific manner. These findings further implicate important roles for Klf10 in skeletal muscle function and shed new light on understanding the molecular processes regulating the contractility of skeletal muscle fibers.

Contractions dynamic of “fast” and “slow” rat muscle under spinal shock and modulators of contraction

Aim. To study the dynamics of neuromotor regulation of the contractile function of fast and slow muscles in rodents during spinal shock by spinal cord transection at the level Тh11Тh12.
 Methods. The experiments were carried out on laboratory rats weighing 140180 g. The animals were divided into two groups: Control (8 rats) and Spinal shock (6 rats). The lower leg muscles, m. soleus and m. extensor digitorum longus (m. EDL), were dissected by partially isolating without disrupting the connection with the body's circulatory system. The sciatic nerve was stimulated with single electrical impulses (10 V, 0.5 ms). Contractions of both muscles caused by electrical stimulation of the sciatic nerve before and after the injection of the substances into the femoral artery tubocurarine (1 mM) or norepinephrine (10 mM) were recorded in animals of both groups. After spinalization, muscle contractions were re-recorded during electrical stimulation of the sciatic nerve before and 10 minutes after the injection of tubocurarine or noradrenaline into the femoral artery in the same concentrations.
 Results. After spinalization of the animal, the contraction force of the muscle m. EDL fibers increased to 0.430.03 g (p=0.040), but the temporal parameters remained unchanged. M. soleus, on the contrary, showed a decrease in the contraction time to 0.0530.005 s (p=0.045), and no change in the contraction force was observed under these conditions. Intra-arterial administration of norepinephrine in the control group resulted in an increase of m. soleus contractions up to 1.210.17 g (p=0.048), and m. EDL up to 0.570.07 g (p=0.043). The administration of norepinephrine in spinalized animals caused an increase in the contraction of m. soleus up to 1.210.09 g (p=0.047), and m. EDL up to 0.660.05 g (p=0.043). The blocker of postsynaptic cholinergic receptors tubocurarine administration reduced the force of contraction of both muscle types in both control [m. soleus up to 0.390.03 g (p=0.039), m. EDL up to 0.110.02 g (p=0.042)] and spinalized [m. soleus up to 0.340.05 g (p=0.039), m. EDL up to 0.150.04 g (p=0.040)] animals.
 Conclusion. The data obtained demonstrate the presence of significant differences in the mechanisms of control of contractile activity in the fast and slow skeletal muscles of warm-blooded animals; the persistence of the similar effect of the basic modulators on the contraction of both muscles with such a striking reaction to spinalization highlights the contribution of neurotrophic control to the functioning of fast and slow motor units.

Exercise Prevents Formation of Tubular Aggregates in Ageing Skeletal Muscle Fibers of Wild-Type Mice

Tubular aggregates (TAs) are ordered arrays of sarcoplasmic reticulum (SR) tubes found in muscle biopsies from patients affected by TA myopathy (TAM). TAM is a disorder linked to mutations in STIM1 and Orai1, the two main players in store-operated Ca2+ entry (SOCE), a mechanism that allows recovery of extracellular Ca2+ during repetitive muscle activity. TAs are also found in ageing 24 month old C57Bl6 mice in about 50% of fast-twitch EDL fibers (while they are not present in adult 4 month old mice) and, using immunofluorescence, we verified that such TAs are strongly positive for both STIM1 and Orai1. Measurements of contractile force during repetitive stimulation (30 consecutive tetanic pulses of 1 sec at 60 Hz applied every 5 seconds) revealed that 24 month old EDL muscles exhibit a faster decay of contractile force than adult (relative force at the 15th tetanus: 40.7±2.1% vs. 48.3.6±2.8% respectively). In addition, experiments performed in presence or absence of 2.5 mM extracellular Ca2+ suggest that the faster force decay of ageing EDLs is likely caused by their inability to recruit extracellular Ca2+. We finally analyzed EDL muscles from 24 month old mice that exercised voluntarily in wheel cages for 15 months (average of 396±29 km; n=3): formation of TAs was greatly reduced in trained vs. untrained age-matched mice (found in only 7% of fibers), while the capability of EDL muscles to maintain contractile force during repetitive stimulation was significantly rescued (relative force after 15th tetanus: 52.4±4.7%). Our findings suggest that: a) TAs accumulate dysfunctional STIM1 and Orai1; b) exercise limits formation of TAs and improves muscle function during repetitive stimulation, possibly improving the capability of aged muscle to use external Ca2+ via SOCE.

The DHPR Calcium Current in Mammalian Skeletal Muscle: Physiological Necessity or Tolerated Evolutionary Remnant?

In contrast to cardiac excitation-contraction coupling (ECC), skeletal-muscle ECC is based on Ca2+-influx-independent inter-channel communications between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). The role of the small Ca2+ influx through the DHPR in mammalian skeletal muscle, which is not (immediately) required for ECC, is still enigmatic. Previously, we discovered that zebrafish, as well as all higher teleost fish, lack DHPR Ca2+ conductivity in skeletal muscle (Schredelseker et al., PNAS, 2010). Point mutation N617D in pore loop II of zebrafish DHPRα1S-b explained non-conductivity in fast muscle. To investigate the fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon we generated a non-conducting-DHPR knock-in mouse (n.c.DHPR) by introducing the N→D mutation into gene CACNA1S. Interestingly, homozygous n.c.DHPR mice are viable, fertile, visually indistinguishable, and identical in body-weight development to wild-type (WT) siblings. Myotubes isolated from newborn n.c.DHPR mice display complete lack of DHPR Ca2+ influx without altered ECC. No difference in locomotor activity (home cage activity), motor coordination (rotarod, beam walking), and muscle strength (endurance test, wire hang test) is observed in 3-7 months-old homozygous n.c.DHPR mice compared to WT. Identical results were obtained from forced frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle fibers. As soon feasible, tests will be repeated on aged (18 months-old) mice to test for putative age-related accumulative effects on muscle performance, in order to understand if DHPR Ca2+ influx in mammalian skeletal muscle is a physiological necessity or just a tolerated evolutionary remnant of the ancestral pure Ca2+-influx dependent ECC of early chordates and phylogenetic branches below.Supported: FWF P-23229-B09; DK-W1101-B12.

Disease-Related Modification of Chloride Conductance in Skeletal Muscle of Dystrophic Mice: Expression of CLC-1 Channel and Role of Inflammation and Oxidative Stress-Related Signaling

A decrease in resting chloride channel conductance (gCl) occurs in dystrophin-deficient myofibers of mdx mouse as a consequence of both spontaneous degeneration, as in diaphragm (DIA), or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). Alterations in gCl, in parallel with markers of inflammation, can be contrasted by small restoration in dystrophin expression (De Luca et al., Neurobiol Dis, 2008). We focused on the molecular mechanisms underlying gCl impairment and its relation to primary defect. Preliminary qRT-PCR experiments showed a 30-35% reduction of CLC-1 mRNA in both DIA and EDL muscles of mdx mice, irrespective to exercise regimen. The reduction was consistent with the impairment of gCl detected in concomitant electrophysiological experiments in DIA and EDL myofibers; however the selective alteration of gCl in exercised mdx EDL muscle remained unexplained. Further experiments are ongoing to evaluate the possible outcome of pathology and mechanical stress on CLC-1 channel protein level and on the expression of other chloride channel types. Recent results showed that in vivo inhibition of angiotensin (Ang)-II contrasts the decrease in gCl in mdx EDL muscle, disclosing a possible role of this pro-inflammatory and pro-oxidative mediator in chloride channel function (Cozzoli et al., Pharmacol Res 2011). The application of Ang-II to wt EDL fibers reduced gCl in a concentration-dependent manner, with a half-maximal concentration of 67nM. The effect was inhibited by the AT1-receptor antagonist losartan, as well as by the PKC-inhibitor chelerythrine, the antioxidant N-acetyl-cysteine and the inhibitor of NADPH-oxidase apocynin. The results demonstrate that CLC-1 channel expression is affected in dystrophinopathies and that further modulation of gCl is related to inflammation and oxidative stress in skeletal muscle.

Non-crossbridge calcium-dependent stiffness in slow and fast skeletal fibres from mouse muscle

We showed previously that force development in frog and FDB mouse skeletal muscle fibres is preceded by an increase of fibre stiffness occurring well before crossbridge attachment and force generation. This stiffness increase, referred to as static stiffness, is due to a Ca(2+)-dependent stiffening of a non-crossbridge sarcomere structure which we suggested could be attributed to the titin filaments. To investigate further the role of titin in static stiffness, we measured static stiffness properties at 24 and 35°C in soleus and EDL mouse muscle fibres which are known to express different titin isoforms. We found that static stiffness was present in both soleus and EDL fibres, however, its value was about five times greater in EDL than in soleus fibres. The rate of development of static stiffness on stimulation increased with temperature and was slightly faster in EDL than in soleus in agreement with previously published data on the time course of the intracellular Ca(2+) transients in these muscles. The present results show that the presence of a non-crossbridge Ca(2+)-dependent stiffening of the muscle fibre is a physiological general characteristic of skeletal muscle. Static stiffness depends on fibre type, being greater and developing faster in fast than in slow fibres. Our observations are consistent with the idea that titin stiffening on contraction improves the sarcomere structure stability. Such an action in fact seems to be more important in EDL fast fibre than in soleus slow fibres.

Altered fast- and slow-twitch muscle fibre characteristics in female mice with a (S248F) knock-in mutation of the brain neuronal nicotinic acetylcholine receptor

We generated a mouse line with a missense mutation (S248F) in the gene (CHRNA4) encoding the alpha4 subunit of neuronal nicotinic acetylcholine receptor (nAChR). Mutant mice demonstrate brief nicotine induced dystonia that resembles the clinical events seen in patients with the same mutation. Drug-induced dystonia is more pronounced in female mice, thus our aim was to determine if the S248F mutation changed the properties of fast- and slow-twitch muscle fibres from female mutant mice. Reverse transcriptase-PCR confirmed CHRNA4 gene expression in the brain but not skeletal muscles in normal and mutant mice. Ca(2+) and Sr(2+) force activation curves were obtained using skinned muscle fibres prepared from slow-twitch (soleus) and fast-twitch (EDL) muscles. Two significant results were found: (1) the (pCa(50) - pSr(50)) value from EDL fibres was smaller in mutant mice than in wild type (1.01 vs. 1.30), (2) the percentage force produced at pSr 5.5 was larger in mutants than in wild type (5.76 vs. 0.24%). Both results indicate a shift to slow-twitch characteristics in the mutant. This conclusion is supported by the identification of the myosin heavy chain (MHC) isoforms. Mutant EDL fibres expressed MHC I (usually only found in slow-twitch fibres) as well as MHC IIa. Despite the lack of spontaneous dystonic events, our findings suggest that mutant mice may be having subclinical events or the mutation results in a chronic alteration to muscle neural input.

Skeletal Muscle Chloride Channel, a Biophysical Sensor of Dystrophic Progression in Mdx Mouse, is a Potential Target of Pro-inflammatory Mediators

A decrease in resting chloride channel conductance (gCl) characterizes myofibers of dystrophic mdx mouse in relation to both spontaneous degeneration, as in diaphragm, or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al., J. Pharmacol. Exp. Ther. 2003). The molecular mechanism underlying gCl impairment might involve change in ClC-1 channel expression/turnover and/or function. Considering the role of inflammation in dystrophic damage, we tested if pro-inflammatory mediators may have ClC-1 channel as a target, through phosphorylating/dephosphorylating pathways. Two microelectrodes current clamp recordings were used to measure resting gCl in EDL and diaphragm muscle fibers from adult wild-type (wt) and mdx mice. In line with previous evidences, the in vitro application of phorbol dibutyrate (50 microM) reduced gCl of wt EDL myofibers from 2610±240 microS/cm2 (n=30) to 1265±180 microS/cm2 (n=15). The application of TNF-alpha (1-30 ng/ml), a cytokine highly expressed in dystrophic muscle, to wt EDL fibers reduced gCl in a concentration-dependent manner with a maximal significant 20% decrease at 10-30 ng/ml. Angiotensin-II (10-100nM), possibly involved in muscle degeneration and oxidative stress, produced a concentration-dependent decrease of gCl in wt EDL myofibers, with a 40 % decrease at 100 nM. The PKC-inhibitor chelerythrine (1 microM) contrasted the effect of either phorbol ester, TNF-alpha or angiotensin-II. The application of 3.3 nM IGF-1 to diaphragm and EDL muscle fibers from exercised mdx mice, significantly counteracted the 40% impairment of gCl. Okadaic acid (0.25 microM) fully prevented IGF-1 effect, supporting the involvement of a serine-threonine phosphatase in IGF-1 activity (De Luca et al., Br. J. Pharmacol. 1998). Angiotensin receptor antagonists and other tools are in use to gain further insight in the mechanisms involved in inflammation-sensitive ClC-1 impairment in muscular dystrophy (Telethon-Italy GGP05130).

Prediction of Twitch and High Frequency Local Calcium Dynamics in Mouse EDL Fibers at 15-35°C

Sites of calcium release, uptake and action are highly organized and densely packed in skeletal muscle cells. This organization suggests an important role for the spatial distribution of organelles and calcium sensitive proteins in muscle function. The current measurement techniques are not able to measure cytosolic [Ca2+] with both high temporal and high spatial resolution. In addition, it is known that all processes are highly temperature dependent. Therefore, the model of Groenendaal et al. [1], that describes local calcium dynamics at physiological frequencies at 35°C, was extended with Q10 correction factors and calcium-fluorescent dye binding kinetics, to predict local calcium dynamics at 15-35°C.For model validation, simultaneously calcium fluorescent dye and force kinetic measurements were performed. Hereto, murine EDL muscles were isolated and mounted in the set up. Muscle length was adjusted to optimal resting length and stimulation voltage was adjusted to generate maximal force. Rhod-2 AM was loaded in whole muscle at 37°C for 30 minutes or until force was decreased with >25%. Muscles were stimulated at 15-35°C at frequencies between twitch and tetanus.A significant relation was found between the temperature and the fluorescence characteristics, e.g. decrease in decay time with increasing temperature (one-way ANOVA, p-value<0.05). Model simulations predicted a comparable range. In addition, simulations showed an approximately five-fold difference in calcium gradient throughout the sarcomere between 15 and 35°C.The validated model is now able 1. to explain calcium fluorescent dye measurements, 2. to translate data at unphysiological to physiological temperature and 3. to predict local calcium dynamics at low and high frequency stimulations at a wide temperature range.[1] Groenendaal et al. IET Systems Biology, in press.

The expression of NFATc1 in adult rat skeletal muscle fibres

Although numerous studies have recently implicated the calcineurin-nuclear factor of activated T-cells (Cn-NFAT) signalling pathway in the regulation of activity-dependent fibre type switching in adult mammalian skeletal muscles, little is known about the endogenous expression of NFAT proteins in the various fibre types present in these muscles. In this study, the immunolocalization of NFATc1 (also known as NFATc or NFAT2) in the extensor digitorum longus (EDL; a mainly fast-twitch muscle) and the soleus (a predominantly slow-twitch muscle) muscles of adult ( approximately 90-day-old) Wistar rats was investigated. The results show that NFATc1 is expressed only in oxidative fibres (i.e. type I and type IIA fibres) that stain intensely for succinate dehydrogenase activity irrespective of whether they are from the fast- or slow-twitch muscle. Thus, 99 +/- 4% (n = 7 rats) of the muscle fibres in the soleus and 42 +/- 2% (n = 7 rats) of those in the EDL expressed NFATc1. In the soleus muscle fibres, NFATc1 was localized mainly in the fibre nuclei, whereas in the EDL fibres it was localized in both the cytoplasm and the nuclei. However, no difference in its localization was observed between type I and type IIA fibres in both muscles. Western blot experiments showed that the soleus expressed more NFATc1 proteins than the EDL. From these results, we suggest that NFATc1 controls the number and distribution of both type I and type IIA fibres, as well as the oxidative capacity of adult mammalian skeletal muscles.

Evaluation of a Hill based muscle model for the energy cost and efficiency of muscular contraction

The purpose of this study was to evaluate a Hill-based mathematical model of muscle energetics and to disclose inconsistencies in existing experimental data. For this purpose, we simulated iso-velocity contractions of mouse fast twitch EDL and slow twitch SOL fibers, and we compared the outcome to experimental results. The experimental results were extracted from two studies published in the literature, which were based on the same methodology but yielded different outcomes (B96 and B93). In the model, energy cost was modeled as the sum of heat and work. Parameters used to model heat rate were entirely independent of the experimental data-sets. Parameters describing the mechanical behavior were derived from both experimental studies. The model was found to accurately predict the muscle energetics and mechanical efficiency of data-set B96. The model could not, however, replicate the energetics and efficiency of SOL and EDL that were found in data-set B93. The model overestimated the shortening heat rate of EDL but, surprisingly, also the mechanical work rate for both muscles. This was surprising since mechanical characteristics of the model were derived directly from the experimental data. It was demonstrated that the inconsistencies in data-set B93 must have been due to some unexplained confounding artifact. It was concluded that the presented model of muscle energetics is valid for iso-velocity contractions of mammalian muscle since it accurately predicts experimental results of an independent data-set (B96). In addition, the model appeared to be helpful in revealing inconsistencies in a second data-set (B93).

Rigor force responses of permeabilized fibres from fast and slow skeletal muscles of aged rats.

1. Ageing is generally associated with a decline in skeletal muscle mass and strength and a slowing of muscle contraction, factors that impact upon the quality of life for the elderly. The mechanisms underlying this age-related muscle weakness have not been fully resolved. The purpose of the present study was to determine whether the decrease in muscle force as a consequence of age could be attributed partly to a decrease in the number of cross-bridges participating during contraction. 2. Given that the rigor force is proportional to the approximate total number of interacting sites between the actin and myosin filaments, we tested the null hypothesis that the rigor force of permeabilized muscle fibres from young and old rats would not be different. 3. Permeabilized fibres from the extensor digitorum longus (fast-twitch; EDL) and soleus (predominantly slow-twitch) muscles of young (6 months of age) and old (27 months of age) male F344 rats were activated in Ca2+-buffered solutions to determine force-pCa characteristics (where pCa = -log(10)[Ca2+]) and then in solutions lacking ATP and Ca2+ to determine rigor force levels. 4. The rigor forces for EDL and soleus muscle fibres were not different between young and old rats, indicating that the approximate total number of cross-bridges that can be formed between filaments did not decline with age. We conclude that the age-related decrease in force output is more likely attributed to a decrease in the force per cross-bridge and/or decreases in the efficiency of excitation-contraction coupling.

Age-dependent fatigue in single intact fast- and slow fibers from mouse EDL and soleus skeletal muscles

In the present work, we investigate age-dependent changes in isometric endurance in response to repetitive stimulation in single intact fast- and slow-twitch muscle fibers from young and old mice. To examine this issue we performed in vitro experiments in manually dissected EDL and soleus muscle fibers. We examined the force generation capacity of fibers in response to two stimulation protocols characterized by different inter-tetanic intervals, named short (1-s) and long interval (3.65-s). Fatigability was measured according to the fatigue index (FI, ratio between the maximum tension recorded in the last over the first tetanus in a train of pulses), the time course of the FI and sag (gradual decrease in force during a partially fused tetanic contraction). Fibers were classified according to the FI using two different criteria previously used in the literature (first criterion: FI≥1, 075–099, 0.5–074 and <0.5; second criterion: FI≥1, 0.75–0.99, 0.25–0.74 and <0.25). The fatigue index distribution recorded in the population of fibers corresponding to EDL and soleus muscles from young and old mice studied with the short and long interval protocols was not statistically different. In summary, these results support the concept that the decline in mechanical performance with aging is not related with changes in fatigability of individual fast- or slow twitch muscles fibers.

Effects of the PKA inhibitor H-89 on excitation-contraction coupling in skinned and intact skeletal muscle fibres.

This study investigated the effects of the protein kinase A (PKA) inhibitor, H-89, in mechanically-skinned muscle fibres and intact muscle fibres, in order to determine whether PKA phosphorylation is essential for normal excitation-contraction (E-C) coupling. In skinned EDL fibres of the rat, force responses to depolarization (by ion substitution) were inhibited only slightly by 10 microM H-89, a concentration more than sufficient to fully inhibit PKA. Staurosporine (1 microM), a potent non-specific kinase inhibitor, also had little if any effect on depolarization-induced responses. At 1-2 microM, H-89 significantly slowed the repriming rate in rat skinned fibres, most likely due to it deleteriously affecting the T-system potential. With 100 microM H-89, the force response to depolarization by ion substitution was completely abolished. This inhibitory effect was reversed by washout of H-89 and was not due to block of the Ca2+ release channel in the sarcoplasmic reticulum (SR). In intact single fibres of the flexor digitorum longus (FDB) muscle of the mouse, 1-3 microM H-89 had no noticeable effect on action-potential-mediated Ca2+ transients. Higher concentrations (4-10 microM) caused Ca2+ transient failure in fibres stimulated at 20 Hz in a manner indicative of action-potential failure. At 10-100 microM, H-89 also inhibited net Ca2+ uptake by the SR and affected the Ca2+-sensitivity of the contractile apparatus in rat skinned fibres. All such effects were proportionately greater in toad muscle fibres. These results do not support the hypothesis that phosphorylation is essential for the Ca2+ release channel to open in response to voltage-sensor activation in skeletal muscle fibres.

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers.

Fast-twitch and slow-twitch rat skeletal muscles produce dissimilar contractures with caffeine. We used digital imaging microscopy to monitor Ca2+ (with fluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes in Ca2+ in individual fibers were markedly different from average responses of a population. All fibers showed discrete, nonpropagated, local Ca2+ transients occurring randomly in spots about one sarcomere apart. Caffeine increased local Ca2+ transients and sarcomere motion initially at 4 mM in soleus and 8 mM in extensor digitorum longus (EDL; approximately 23 degrees C). Ca2+ release subsequently adapted or inactivated; this was surmounted by higher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction in both types of fiber. In EDL fibers, 16 mM caffeine moderately increased local Ca2+ transients. In soleus fibers, 16 mM caffeine greatly increased Ca2+ release and produced propagated waves of Ca2+ (approximately 1.5-2.5 microns/s). Ca2+ waves in slow-twitch fibers reflect the caffeine-sensitive mechanism of Ca2(+)-induced Ca2+ release. Fast-twitch fibers possibly lack this mechanism, which could account for their lower sensitivity to caffeine.

Regulation of myosin heavy chain expression in adult rat hindlimb muscles during short-term paralysis: comparison of denervation and tetrodotoxin-induced neural inactivation

The extent to which myosin profiles within adult fast and slow muscles are altered by short-term paralysis remains equivocal. We used an array of specific antibodies to identify adult and developmental MHC isoforms within EDL and soleus muscle fibers, and show a marked multiple expression of MHCs with a general shift towards slower and more energy efficient MHC profiles after 2 weeks of denervation or TTX nerve conduction block. Paralysis also induced marked expression of an embryonic MHC within most EDL cell types, and a subtle, paralysis-sensitive, expression of α-cardiac MHC within specific EDL and soleus extrafusal fibers. Comparison of treatment groups also permitted assessment of the relative influence of neural activity versus trophic factors on these isoforms, and confirmed activity as a major, but not sole, regulator of MHC expression.

Changes of macromolecular organizations in nonjunctional sarcolemmas after cross-innervation— a study of fast-and slow-twitch muscle fibres in rats

The purpose of the present study was to analyse the changes of macromolecular organizations in nonjunctional sarcolemmas of different types of skeletal muscle fibres after cross-innervation. In normal rats the mean density of square arrays (6 nm particles organized in orthogonal arrays) was 9.02 / μm2 for the nonjunctional sarcolemmas of fast-twitch extensor digitorum longus (control EDL, CE) muscle fibres and 0.34 / μm2 for the nonjunctional sarcolemmas of slow-twitch soleus (control SOL, CS) muscle fibres. After cross-innervation between the fast-twitch EDL and slow-twitch SOL muscle fibres by slow and fast muscle nerves respectively for three months, the mean density was 0.45/ μm2 for the nonjunctional sarcolemmas of the operated EDL (OE) and 8.3/ μm2 for the nonjunctional sarcolemmas of the operated SOL (OS), This indicates that the cross-innervation causes a reciprocal transformation of the number and distribution of such macromolecular organizations in the electrically excitable nonjunctional sarcolemmas.