S-nitrosylation and S-glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca2+ sensitivity in rat fast-twitch muscle fibers.

Nitric oxide is generated in skeletal muscle with activity and decreases Ca2+ sensitivity of the contractile apparatus, putatively by S-nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca2+ sensitivity in mammalian fast-twitch (FT) fibers mediated by S-glutathionylation of Cys134 on fast troponin I (TnIf). Force-[Ca2+] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca2+-buffered solutions. Treatment with S-nitrosylating agents, S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-penicillamine (SNAP), decreased pCa50 ( = -log10 [Ca2+] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca2+ sensitivity decrease was 1) fully reversed with dithiothreitol or reduced glutathione, 2) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and 3) irreversibly blocked by low concentration of the alkylating agent, N-ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused S-nitrosylation of TnIfS-glutathionylation pretreatment blocked the effects of S-nitrosylation on Ca2+ sensitivity, and vice-versa. S-nitrosylation pretreatment prevented NEM from irreversibly blocking S-glutathionylation of TnIf and its effects on Ca2+ sensitivity, and likewise S-glutathionylation pretreatment prevented NEM block of S-nitrosylation. Following substitution of TnIf into rat slow-twitch fibers, S-nitrosylation treatment caused decreased Ca2+ sensitivity. These findings demonstrate that S-nitrosylation and S-glutathionylation exert opposing effects on Ca2+ sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnIf.

Contractile properties and sarcoplasmic reticulum calcium content in type I and type II skeletal muscle fibres in active aged humans.

KEY POINTS: Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres. This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults. The maximum level of force production (per unit cross-sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium. The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast- and slow-twitch muscle fibres in the Old subjects. These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres. ABSTRACT: This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca(2+) content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ∼17% and Ca(2+) sensitivity was also reduced (pCa50 decreased ∼0.05 pCa units) relative to that in Young. S-Glutathionylation of fast troponin I (TnIf ) markedly increased Ca(2+) sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca(2+) content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca(2+) by a combined caffeine and low Mg(2+) stimulus, whereas in fibres of Old the amount of non-releasable Ca(2+) was significantly increased (by > 12% of endogenous Ca(2+) content). Western blotting showed an increased proportion of type I fibres in Old subjects, and increased amounts of calsequestrin-2 and calsequestrin-like protein. The findings suggest that muscle weakness in old age is probably attributable in part to (i) an increased proportion of type I fibres, (ii) a reduction in both maximum specific force and Ca(2+) sensitivity in type II fibres, and also a decreased ability of S-glutathionylation of TnIf to counter the fatiguing effects of metabolites on Ca(2+) sensitivity, and (iii) a reduction in the amount of releasable SR Ca(2+) in both fibre types.

Acute effects of taurine on sarcoplasmic reticulum Ca2+ accumulation and contractility in human type I and type II skeletal muscle fibers.

Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (>10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffected; maximum Ca(2+)-activated force was unchanged in both cases. The effects of taurine on SR Ca2+ accumulation (1) only became apparent after prolonged cytoplasmic exposure, and (2) persisted for some minutes after complete removal of taurine from the cytoplasm, consistent with the hypothesis that the effects were due to an action of taurine from inside the SR. In summary, taurine potentiates the rate of SR Ca2+ uptake in both type I and type II human fibers, possibly via an action from within the SR lumen, with the degree of potentiation being significantly reduced at low physiological taurine levels.

Ca2+-dependent proteolysis of junctophilin-1 and junctophilin-2 in skeletal and cardiac muscle.

Excessive increases in intracellular [Ca(2+)] in skeletal muscle fibres cause failure of excitation-contraction coupling by disrupting communication between the dihydropyridine receptors in the transverse tubular system and the Ca(2+) release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact mechanism is unknown. Previous work suggested a possible role of Ca(2+)-dependent proteolysis in this uncoupling process but found no proteolysis of the dihydropyridine receptors, RyRs or triadin. Junctophilin-1 (JP1; ∼90 kDa) stabilizes close apposition of the transverse tubular system and SR membranes in adult skeletal muscle; its C-terminal end is embedded in the SR and its N-terminal associates with the transverse tubular system membrane. Exposure of skeletal muscle homogenates to precisely set [Ca(2+)] revealed that JP1 undergoes Ca(2+)-dependent proteolysis over the physiological [Ca(2+)] range in tandem with autolytic activation of endogenous µ-calpain. Cleavage of JP1 occurs close to the C-terminal, yielding a ∼75 kDa diffusible fragment and a fixed ∼15 kDa fragment. Depolarization-induced force responses in rat skinned fibres were abolished following 1 min exposure to 40 µm Ca(2+), with accompanying loss of full-length JP1. Supraphysiological stimulation of rat skeletal muscle in vitro by repeated tetanic stimulation in 30 mm caffeine also produced marked proteolysis of JP1 (and not RyR1). In dystrophic mdx mice, JP1 proteolysis is seen in limb muscles at 4 and not at 10 weeks of age. Junctophilin-2 in cardiac and skeletal muscle also undergoes Ca(2+)-dependent proteolysis, and junctophilin-2 levels are reduced following cardiac ischaemia-reperfusion. Junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.

S-glutathionylation of troponin I (fast) increases contractile apparatus Ca2+ sensitivity in fast-twitch muscle fibres of rats and humans.

Oxidation can decrease or increase the Ca2+ sensitivity of the contractile apparatus in rodent fast-twitch (type II) skeletal muscle fibres, but the reactions and molecular targets involved are unknown. This study examined whether increased Ca2+ sensitivity is due to S-glutathionylation of particular cysteine residues. Skinned muscle fibres were directly activated in heavily buffered Ca2+ solutions to assess contractile apparatus Ca2+ sensitivity. Rat type II fibres were subjected to S-glutathionylation by successive treatments with 2,2'-dithiodipyridine (DTDP) and glutathione (GSH), and displayed a maximal increase in pCa50 (−log10 [Ca2+] at half-maximal force) of ∼0.24 pCa units, with little or no effect on maximum force or Hill coefficient. Partial similar effect was produced by exposure to oxidized gluthathione (GSSG, 10 mM) for 10 min at pH 7.1, and near-maximal effect by GSSG treatment at pH 8.5. None of these treatments significantly altered Ca2+ sensitivity in rat type I fibres. Western blotting showed that both the DTDP­GSH and GSSG­pH 8.5 treatments caused marked S-glutathionylation of the fast troponin I isoform (TnI(f)) present in type II fibres, but not of troponin C (TnC) or myosin light chain 2. Both the increased Ca2+ sensitivity and glutathionylation of TnI(f) were blocked by N-ethylmaleimide (NEM). S-nitrosoglutathione (GSNO) also increased Ca2+ sensitivity, but only in conditions where it caused S-glutathionylation of TnI(f). In human type II fibres from vastus lateralis muscle, DTDP­GSH treatment also caused similar increased Ca2+ sensitivity and S-glutathionylation of TnI(f). When the slow isoform of TnI in type I fibres of rat was partially substituted (∼30%) with TnI(f), DTDP­GSH treatment caused a significant increase in Ca2+ sensitivity (∼0.08 pCa units). TnIf in type II fibres from toad and chicken muscle lack Cys133 present in mammalian TnIf, and such fibres showed no change in Ca2+ sensitivity with DTDP­GSH nor any S-glutathionylation of TnI(f) (latter examined only in toad). Following 40 min of cycling exercise in human subjects (at ∼60% peak oxygen consumption), TnI(f) in vastus lateralis muscle displayed a marked increase in S-glutathionylation (∼4-fold). These findings show that S-glutathionylation of TnI(f), most probably at Cys133, increases the Ca2+ sensitivity of the contractile apparatus, and that this occurs in exercising humans, with likely beneficial effects on performance.

Effects of carnosine on contractile apparatus Ca²âº sensitivity and sarcoplasmic reticulum Ca²âº release in human skeletal muscle fibers.

There is considerable interest in potential ergogenic and therapeutic effects of increasing skeletal muscle carnosine content, although its effects on excitation-contraction (EC) coupling in human muscle have not been defined. Consequently, we sought to characterize what effects carnosine, at levels attained by supplementation, has on human muscle fiber function, using a preparation with all key EC coupling proteins in their in situ positions. Fiber segments, obtained from vastus lateralis muscle of human subjects by needle biopsy, were mechanically skinned, and their Ca(2+) release and contractile apparatus properties were characterized. Ca(2+) sensitivity of the contractile apparatus was significantly increased by 8 and 16 mM carnosine (increase in pCa(50) of 0.073 ± 0.007 and 0.116 ± 0.006 pCa units, respectively, in six type I fibers, and 0.063 ± 0.018 and 0.103 ± 0.013 pCa units, respectively, in five type II fibers). Caffeine-induced force responses were potentiated by 8 mM carnosine in both type I and II fibers, with the potentiation in type II fibers being entirely explicable by the increase in Ca(2+) sensitivity of the contractile apparatus caused by carnosine. However, the potentiation of caffeine-induced responses caused by carnosine in type I fibers was beyond that expected from the associated increase in Ca(2+) sensitivity of the contractile apparatus and suggestive of increased Ca(2+)-induced Ca(2+) release. Thus increasing muscle carnosine content likely confers benefits to muscle performance in both fiber types by increasing the Ca(2+) sensitivity of the contractile apparatus and possibly also by aiding Ca(2+) release in type I fibers, helping to lessen or slow the decline in muscle performance during fatiguing stimulation.

Differential effects of peroxynitrite on contractile protein properties in fast- and slow-twitch skeletal muscle fibers of rat.

Oxidative modification of contractile proteins is thought to be a key factor in muscle weakness observed in many pathophysiological conditions. In particular, peroxynitrite (ONOO(-)), a potent short-lived oxidant, is a likely candidate responsible for this contractile dysfunction. In this study ONOO(-) or 3-morpholinosydnonimine (Sin-1, a ONOO(-) donor) was applied to rat skinned muscle fibers to characterize the effects on contractile properties. Both ONOO(-) and Sin-1 exposure markedly reduced maximum force in slow-twitch fibers but had much less effect in fast-twitch fibers. The rate of isometric force development was also reduced without change in the number of active cross bridges. Sin-1 exposure caused a disproportionately large decrease in Ca(2+) sensitivity, evidently due to coproduction of superoxide, as it was prevented by Tempol, a superoxide dismutase mimetic. The decline in maximum force with Sin-1 and ONOO(-) treatments could be partially reversed by DTT, provided it was applied before the fiber was activated. Reversal by DTT indicates that the decrease in maximum force was due at least in part to oxidation of cysteine residues. Ascorbate caused similar reversal, further suggesting that the cysteine residues had undergone S-nitrosylation. The reduction in Ca(2+) sensitivity, however, was not reversed by either DTT or ascorbate. Western blot analysis showed cross-linking of myosin heavy chain (MHC) I, appearing as larger protein complexes after ONOO(-) exposure. The findings suggest that ONOO(-) initially decreases maximum force primarily by oxidation of cysteine residues on the myosin heads, and that the accompanying decrease in Ca(2+) sensitivity is likely due to other, less reversible actions of hydroxyl or related radicals.

Modulation of contractile apparatus Ca2+ sensitivity and disruption of excitation-contraction coupling by S-nitrosoglutathione in rat muscle fibres.

S-Nitrosoglutathione (GSNO) is generated in muscle and may S-glutathionylate and/or S-nitrosylate various proteins involved in excitation­contraction (EC) coupling, such as Na+-K+-ATPases, voltage-sensors (VSs) and Ca2+ release channels (ryanodine receptors,RyRs), possibly changing their properties. Using mechanically skinned fibres from rat extensor digitorum longus muscle, we sought to identify which EC coupling processes are most susceptible to GSNO-modulated changes and whether these changes could be important in muscle function and fatigue. For comparison, we examined the effect of other oxidation, nitrosylation, or glutathionylation treatments (S-nitroso-N-acetyl-penicillamine (SNAP), hydrogen peroxide,2,2-dithiodipyridine and reduced glutathione) on twitch and tetanic force, action potential (AP) repriming, sarcoplasmic reticulum (SR) Ca2+ loading and leakage, and contractile apparatus properties. None of the treatments detectably altered AP repriming, indicating that t-system excitability was relatively insensitive to such oxidative modification. Importantly, the overall effect on twitch and tetanic force of a given treatment was determined primarily by its action on Ca2+ sensitivity of the contractile apparatus. For example, S-nitrosylation with the NO• donor,SNAP, caused matching decreases in the contractile Ca2+ sensitivity and twitch response, and GSNO applied ∼10 min after preparation had very similar effects. The only exception was when GSNO was applied immediately after preparation, which resulted in irreversible decreases in twitch and tetanic responses even though it concomitantly increased Ca2+ sensitivity by∼0.1 pCaunits, the latter evidently due to S-glutathionylation of the contractile apparatus. This decrease in AP-mediated force responses was due to impaired VS­RyR coupling and was accompanied by increased Ca2+ leakage through RyRs. Such oxidation-related impairment of coupling could be responsible for prolonged low frequency fatigue in certain circumstances.

Hydroxyl radical and glutathione interactions alter calcium sensitivity and maximum force of the contractile apparatus in rat skeletal muscle fibres.

Studies on intact muscle fibres indicate that reactive oxygen species (ROS) produced during muscle activity, or applied exogenously, can cause decreased force responses primarily by reducing the Ca(2+) sensitivity of the contractile apparatus. Identification of the molecular basis of this effect is complicated by the fact that studies on skinned muscle fibres in general have not observed reduced contractile Ca(2+) sensitivity when applying ROS, predominantly H(2)O(2). Here, using skinned fibres from rat extensor digitorum longus (EDL) and soleus muscle, it is shown that although H(2)O(2) (> or = 100 microm) has little effect by itself, when added in the presence of myoglobin it causes marked reduction in the Ca(2+) sensitivity of the contractile apparatus, probably due to production of hydroxyl radicals (OH(*)). Maximum force production is also reduced, but only with larger or more prolonged treatments. The effects are not prevented by tempol, a potent superoxide scavenger. Dithiotreitol (DTT) produces little reversal of the sensitivity change if applied afterwards, but it does substantially reverse all the changes if applied before the fibre undergoes an activation sequence. When glutathione (GSH, 5 mM) is present, exposure of EDL fibres to H(2)O(2) and myoglobin causes an increase in Ca(2+) sensitivity, with longer treatments causing a subsequent decrease, whereas in soleus fibres it causes only decreases in sensitivity and maximum force. The increased Ca(2+) sensitivity in EDL fibres is evidently due to the summed actions of (i) a potentiating effect of glutathionylation, which can be reversed by DTT and only occurs in fast-twitch fibres, and (ii) a less reversible reduction in sensitivity. Western blotting showed that reductions in Ca(2+) sensitivity were not due to loss of troponin-C. The present findings help provide a mechanistic basis for diverse findings on the effects of ROS in muscle fibres and implicate OH(*) radicals and glutathione as likely mediators of the effects.

Chloride conductance in the transverse tubular system of rat skeletal muscle fibres: importance in excitation-contraction coupling and fatigue.

Contraction in skeletal muscle fibres is governed by excitation of the transverse-tubular (t-) system, but the properties of the t-system and their importance in normal excitability are not well defined. Here we investigate the properties of the t-system chloride conductance using rat skinned muscle fibres in which the sarcolemma has been mechanically removed but the normal excitation-contraction coupling mechanism kept functional. When the t-system chloride conductance was eliminated, either by removal of all Cl(-) or by block of the chloride channels with 9-anthracene carboxylic acid (9-AC) or by treating muscles with phorbol 12,13-dibutyrate, there was a marked reduction in the threshold electric field intensity required to elicit a t-system action potential (AP) and twitch response. Calculations of the t-system chloride conductance indicated that it constitutes a large proportion of the total chloride conductance observed in intact fibres. Blocking the chloride conductance increased the size of the twitch response and was indicative that Cl(-) normally carries part of the repolarizing current across the t-system membrane on each AP. Block of the t-system chloride conductance also reduced tetanic force responses at higher frequency stimulation (100 Hz) and greatly reduced twitch responses in the period shortly after a brief tetanus, owing to rapid loss of t-system excitability during the AP train. Blocking activity of the Na(+)-K(+) pump in the t-system membrane caused loss of excitability owing to K(+) build-up in the sealed t-system, and this occurred approximately 3-4 times faster when the chloride conductance was blocked. These findings show that the t-system chloride conductance plays a vital role during normal activity by countering the effects of K(+) accumulation in the t-system and maintaining muscle excitability.

Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis.

The Na(+)-K(+) pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K(+) concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na(+)-K(+) pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na(+)-K(+) pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca(2+) release and twitch and tetanic force responses. Conditions were selected such that increased Na(+)-K(+) pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na(+)-K(+) pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level ( approximately 8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na(+)-K(+) pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na(+)-K(+) pump and maintain muscle excitability.

Transverse tubular system depolarization reduces tetanic force in rat skeletal muscle fibers by impairing action potential repriming.

When muscle fibers are repeatedly stimulated, they may become depolarized and force output decline. Excitation of the transverse tubular system (T-system) is critical for activation, but its role in muscle fatigue is poorly understood. Here, mechanically skinned fibers from rat fast-twitch muscle were used, because the sarcolemma is absent but the T-system retains normal excitability and its properties can be studied in isolation. The T-system membrane was fully polarized by bathing the skinned fiber in an internal solution with 126 mM K(+) (control solution) or set at partially depolarized levels (approximately -63 and -58 mV) in solutions with 66 or 55 mM K(+), respectively, and action potentials (APs) were triggered in the sealed T-system by field stimulation. Prolonged depolarization of the T-system reduced tetanic force proportionately more than twitch force, with greater effect at higher stimulation frequency (responses at 20 and 100 Hz reduced to 71 and 62% in 66 mM K(+) and to 54 and 35% in 55 mM K(+), respectively). Double-pulse stimulation showed that depolarization increased the repriming period (estimated minimum time before a second AP can be produced) from approximately 4 ms to approximately 7.5 and 15 ms in the 66 and 55 mM K(+) solutions, respectively. These results demonstrate that T-system depolarization reduces tetanic force by impairing AP repriming, rather than by preventing AP generation per se or by inactivating the T-system voltage sensors. The findings also explain why it is advantageous to reduce the rate of motoneuron stimulation to muscles during repeated or prolonged periods of activity.

Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle.

During vigorous exercise, Pi concentration levels within the cytoplasm of fast-twitch muscle fibers may reach > or =30 mM. Cytoplasmic Pi may enter the sarcoplasmic reticulum (SR) and bind to Ca2+ to form a precipitate (CaPi), thus reducing the amount of releasable Ca2+. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca2+ release mechanism, we investigated the consequences of Pi exposure on normal excitation-contraction coupling. The total amount of Ca2+ released from the SR by a combined caffeine/low-Mg2+ concentration stimulus was reduced by approximately 20%, and the initial rate of force development slowed after 2-min exposure to 30 mM Pi (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by approximately 25% and approximately 45% after 10 and 30 mM Pi exposure, respectively). Tetanic force responses produced after 30 mM Pi exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca2+ by approximately 35%. Ca2+ content assays revealed that the total amount of Ca2+ in the SR was not detectably changed by exposure to 30 mM Pi, indicating that Ca2+ had not leaked from the SR but instead formed a precipitate with the Pi, reducing the amount of available Ca2+ for rapid release. These results suggest that CaPi precipitation that occurs within the SR could contribute to the failure of Ca2+ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca2+ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses.

L(+)-lactate does not affect twitch and tetanic responses in mechanically skinned mammalian muscle fibres.

This study investigated whether a high intracellular concentration of L(+)-lactate (30 mM) affects normal excitation-contraction coupling in skeletal muscle. Electrical stimulation was used to elicit action potentials in the (sealed) transverse-tubular system of mechanically skinned muscle fibres, giving rise to twitch and tetanic force responses. As the sarcolemma was absent, lactate could be applied to the cytoplasmic environment via the bathing solution (at a constant pH of 7.1) and its effect examined independently of other metabolic changes that occur during muscle fatigue. The presence of 30 mM lactate had virtually no effect on direct activation of the contractile apparatus by Ca2+. Lactate also had no significant effect on either the rate of rise or the peak of the twitch response, with the only detectable effect being a slight (13%) slowing in its relaxation rate. As the amplitude of the twitch response (approximately 60% of maximum force) may be regarded as a sensitive indicator of the amount of Ca2+ released by an action potential, there was evidently to change in Ca2+ release in the presence of lactate. Lactate also had no significant effect on the rate of rise and peak force of the tetanic response or on its subsequent relaxation. Additional experiments, in which the sarcoplasmic reticulum was emptied of Ca2+ (in a caffeine solution) and reloaded repeatedly, showed no significant effect of 30 mM lactate on Ca2+ uptake. This study shows that the presence of L(+)-lactate does not inhibit excitation-contraction coupling in mechanically skinned fibres.

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers.

It is unclear whether accumulation of lactate in skeletal muscle fibers during intense activity contributes to muscle fatigue. Using mechanically skinned fibers from rat and toad muscle, we were able to examine the effect of L(+)-lactate on excitation-contraction coupling independently of other metabolic changes. We investigated the effects of lactate on the contractile apparatus, caffeine-induced Ca(2+) release from the sarcoplasmic reticulum, and depolarization-induced Ca(2+) release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable (20-35%) inhibition of caffeine-induced Ca(2+) release, seemingly by a direct effect on the Ca(2+) release channels. However, 15 mM lactate had no detectable effect on Ca(2+) release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect on normal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.