Low myoplasmic Mg2+ potentiates calcium release during depolarization of frog skeletal muscle fibers.

The role of intracellular free magnesium concentration ([Mg2+]) in modulating calcium release from the sarcoplasmic reticulum (SR) was studied in voltage-clamped frog cut skeletal muscle fibers equilibrated with cut end solutions containing two calcium indicators, fura-2 and antipyrylazo III (AP III), and various concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor calcium transients, whereas fura-2 fluorescence was used to monitor resting calcium. The rate of release (Rrel) of calcium from the SR was calculated from the calcium transient and found to be increased in low internal [Mg2+]. After correcting for effects of calcium depletion from the SR and normalization to SR content, the mean values of the inactivatable and noninactivatable components of Rrel were increased by 163 and 46%, respectively, in low Mg2+. Independent of normalization to SR content, the ratio of inactivatable to noninactivatable components of Rrel was increased in low internal [Mg2+]. Both observations suggest that internal [Mg2+] preferentially modulates the inactivatable component of Rrel, which is thought to be due to calcium-induced calcium release from the SR. This could also explain the observation that, in low internal [Mg2+], the time to the peak of the calcium transient for a 5-ms depolarizing pulse was not very different from the time to the peak of the delta [Ca2+] for a 10-ms pulse of the same amplitude. Finally, in low internal [Mg2+], the calcium transient elicited by a short depolarizing pulse was in some cases clearly followed by a very slow rise of calcium after the end of the pulse. The observed effects of reduced [Mg2+] on calcium release are consistent with a removal of the inhibition that the normal 1 mM myoplasmic [Mg2+] exerts on calcium release in skeletal muscle fibers.     

Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3.

Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36-40). We have injected fluo-3 from a high-purity lot into intact single fibers from frog muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in frog fibers (0.02-0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2-0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.     

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.     

Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibers

Fatigue and recovery from fatigue were related to metabolism in single fibers of the frog semitendinosus muscle. The fibers were held at a sarcomere length of 2.3 microm in oxygenated Ringer solution at 15 degrees C and were stimulated for up to 150 s by a schedule of 10-s, 20-Hz tetanic trains that were interrupted by 1-s rest periods, after which they were rapidly frozen for biochemical analysis. Two kinds of fatigue were produced in relation to stimulus duration. A rapidly reversed fatigue occurred with stimulation for under 40 s and was evidenced by a decline in tetanic tension that could be overcome by 1 s of rest. A prolonged fatigue was caused by stimulation for 100-150 s. It was evidenced during stimulation by a fall in tetanic tension that could not be overcome by 1 s of rest, and after stimulation by a reduction, lasting for up to 82 min, in the peak tension of a 200-ms test tetanus. Fiber phosphocreatine (PCr) fell logarithmically in relation to stimulus duration, from a mean of 121 +/- 8 nmol/mg protein (SEM, n = 12) to 10% of this value after 150 s of stimulation. PCr returned to normal levels after 90-120 min of rest. Stimulation for 150 s did not significantly affect fiber glycogen and reduced fiber ATP by at most 15%. It is suggested that the prolonged fatigue caused by 100-150 s of tetanic stimulation was caused by long-lasting failure of excitation-contraction coupling, as it was not accompanied by depletion of energy stores in the form of ATP. One possibility is that H+ accumulated in fatigued fibers so as to interfere with the action of Ca2+ in the coupling process.     

Role of mitochondrial ATP-sensitive potassium channels on fatigue in mouse muscle fibers

The role of mitochondrial K_ATP (mitoK_ATP) channels on muscle fatigue was assessed in adult mouse skeletal muscle bundles. Muscle fatigue was produced by eliciting short repetitive tetani. Isometric tension and the rate of production of reactive oxygen species (ROS) were measured at room temperature (20-22 °C) using a force transducer and the fluorescent indicator CM-H₂DCFDA. We found that opening mitoK_ATP channels with diazoxide (100 μM) significantly reduced muscle fatigue. Fatigue tension was 34% higher in diazoxide-treated fibers relative to controls. This effect was blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate (5-HD), by the protein kinase C (PKC) inhibitor chelerythrine, and by the nitric oxide (NO) synthase inhibitor N^G-nitro-l-arginine methyl ester hydrochloride (l-NAME) but was not accompanied by a change in the rate of ROS production during fatigue. A physiological role of mitoK_ATP channels on muscle fatigue is proposed.     

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.     

The amplitude and time course of the myoplasmic free [Ca2+] transient in fast-twitch fibers of mouse muscle

Bundles of 10-100 fibers were dissected from the extensor digitorum longus muscle of mouse, mounted in an apparatus for optical recording, and stretched to long sarcomere length (> or = 3.6 microns). One fiber within the bundle was microinjected with furaptra, a fluorescent indicator that responds rapidly to changes in myoplasmic free [Ca2+] (delta [Ca2+]). Twitches and brief tetani were initiated by external stimulation. At myoplasmic furaptra concentrations of approximately 0.1 mM, the indicator's fluorescence signal during fiber activity (delta F/F) was well resolved. delta F/F was converted to delta [Ca2+] under the assumption that furaptra's myoplasmic dissociation constant for Ca2+ is 98 microM at 16 degrees C and 109 microM at 28 degrees C. At 16 degrees C, the peak amplitude of delta [Ca2+] during a twitch was 17.8 +/- 0.4 microM (+/-SEM; n = 8) and the half-width of delta [Ca2+] was 4.6 +/- 0.3 ms. At 28 degrees C, the peak and half-width values were 22.1 +/- 1.8 microM and 2.0 +/- 0.1 ms, respectively (n = 4). During a brief high-frequency tetanus, individual peaks of delta [Ca2+] were also well resolved and reached approximately the same amplitude that resulted from a single shock; the initial decays of delta [Ca2+] from peak slowed substantially during the tetanus. For a single twitch at 16 degrees C, the amplitude of delta [Ca2+] in fast-twitch fibers of mouse is not significantly different from that recently measured in fast- twitch fibers of frog (16.5 +/- 0.9 microM; Zhao, M., S. Hollingworth, and S.M. Baylor. 1996. Biophys. J. 70:896-916); in contrast, the half- width of delta [Ca2+] is surprisingly brief in mouse fibers, only about half that measured in frog (9.6 +/- 0.6 ms). The estimated peak rate at which Ca2+ is released from the sarcoplasmic reticulum in response to an action potential is also similar in mouse and frog, 140-150 microM/ms (16 degrees C).     

Effects of CO2-induced acidification on the fatigue resistance of single mouse muscle fibers at 28C

The role of reduced muscle pH in the development of skeletalmuscle fatigue is unclear. This study investigated the effects oflowering skeletal muscle intracellular pH by exposure to 30% CO₂ on the number of isometrictetani needed to induce significant fatigue. Isolated single mousemuscle fibers were stimulated repetitively at intervals of 4-2.5 sby using 80-Hz, 400-ms tetani at 28°C in Tyrode solution bubbledwith either 5 or 30%CO₂. Stimulation continued until tetanic force had fallen to 40% of the initial value.Exposure to 30% CO₂ caused asignificant fall in intracellular pH of ~0.3 pH unit but did notcause any significant changes in initial peak tetanic force. During thecourse of repetitive stimulation, intracellular pH fell by ~0.3 pHunit in both normal and acidified fibers. The number of tetani neededto reduce force to 40% of the initial value was not significantlydifferent in 5 and 30% CO₂Tyrode. The sole effect of acidosis was to reduce the rate ofrelaxation of force, especially in fatigued fibers. It is concluded that, at 28°C, acidosis per se does not accelerate the development of fatigue during repeated tetanic stimulation of isolated mouse skeletal muscle fibers.

     

Fluctuations in tension during contraction of single muscle fibers.

We have searched for fluctuations in the steady-state tension developed by stimulated single muscle fibers. Such tension "noise" is expected to be present as a result of the statistical fluctuations in the number and/or state of myosin cross-bridges interacting with thin filament sites at any time. A sensitive electro-optical tension transducer capable of resolving the expected fluctuations in magnitude and frequency was constructed to search for the fluctuations. The noise was analyzed by computing the power spectra and amplitude of stochastic fluctuations in the photomultiplier counting rate, which was made proportional to muscle force. The optical system and electronic instrumentation together with the minicomputer software are described. Tensions were measured in single skinned glycerinated rabbit psoas muscle fibers in rigor and during contraction and relaxation. The results indicate the presence of fluctuations in contracting muscles and a complete absence of tension noise in eith rigor or relaxation. Also, a numerical method was developed to simulate the power spectra and amplitude of fluctuations, given the rate constants for association and dissociation of the cross-bridges and actin. The simulated power spectra and the frequency distributions observed experimentally are similar.     

Intersarcomere dynamics of single muscle fibers during fixed-end tetani

The contraction dynamics of end and center regions of single fibers have been measured during fixed-end tetani. Experimental control and data acquisition are provided by a digital system that can acquire diffraction data as fast as every 260 microseconds for 300-700 ms. Tension records are simultaneously displayed on a storage oscilloscope. Resting sarcomere length variation between the end and center regions was analogous to that of Gordon et al. (1966). During the rapid rise in force (less than 45 ms), the end regions contract almost twice as fast as the center regions. During the slow rise in force, the velocity of contraction of the end regions was 3.8 times the velocity of stretch of the center regions. In addition, factors that affected the rate and extent of the slow rise in tension also affected the rate and extent of end shortening. In 58% of the cases studied, the amount of shortening observed in the end region was enough to explain the extent of the slow rise in tension. These data support the explanation of creep first proposed by A. V. Hill (1953) and used by Gordon et al. (1966) to justify their use of the back-extrapolation technique in measuring the isometric force-generating capability of a single fiber. These data also indicate that the laser diffraction technique may provide an effective, noninvasive method for studying sarcomere dynamics during creep and related phenomena.     

Arsenazo III and antipyrylazo III calcium transients in single skeletal muscle fibers

The metallochrome calcium indicators arsenazo III and antipyrylazo III have been introduced individually into cut single frog skeletal muscle fibers from which calcium transients have been elicited either by action potential stimulation or by voltage-clamp pulses of up to 50 ms in duration. Calcium transients recorded with both dyes at selected wavelengths have similar characteristics when elicited by action potentials. Longer voltage-clamp pulse stimulation reveals differences in the late phases of the optical signals obtained with the two dyes. The effects of different tension blocking methods on Ca transients were compared experimentally. Internal application of EGTA at concentrations up to 3 mM was demonstrated to be efficient in blocking movement artifacts without affecting Ca transients. Higher EGTA concentrations affect the Ca signals' characteristics. Differential effects of internally applied EGTA on tension development as opposed to calcium transients suggest that diffusion with binding from Ca++ release sites to filament overlap sites may be significant. The spectral characteristics of the absorbance transients recorded with arsenazo III suggest that in situ recorded signals cannot be easily interpreted in terms of Ca concentration changes. A more exhaustic knowledge of the dye chemistry and/or in situ complications in the use of the dye will be necessary.     

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.     

Recruitment of single muscle fibers during submaximal cycling exercise.

In literature, an inconsistency exists in the submaximal exercise intensity at which type II fibers are activated. In the present study, the recruitment of type I and II fibers was investigated from the very beginning and throughout a 45-min cycle exercise at 75% of the maximal oxygen uptake, which corresponded to 38% of the maximal dynamic muscle force. Biopsies of the vastus lateralis muscle were taken from six subjects at rest and during the exercise, two at each time point. From the first biopsy single fibers were isolated and characterized as type I and II, and phosphocreatine-to-creatine (PCr/Cr) ratios and periodic acid-Schiff (PAS) stain intensities were measured. Cross sections were cut from the second biopsy, individual fibers were characterized as type I and II, and PAS stain intensities were measured. A decline in PCr/Cr ratio and in PAS stain intensity was used as indication of fiber recruitment. Within 1 min of exercise both type I and, although to a lesser extent, type II fibers were recruited. Furthermore, the PCr/Cr ratio revealed that the same proportion of fibers was recruited during the whole 45 min of exercise, indicating a rather constant recruitment. The PAS staining, however, proved inadequate to fully demonstrate fiber recruitment even after 45 min of exercise. We conclude that during cycling exercise a greater proportion of type II fibers is recruited than previously reported for isometric contractions, probably because of the dynamic character of the exercise. Furthermore, the PCr/Cr ratio method is more sensitive in determining fiber activation than the PAS stain intensity method.     

Caffeine and the myoplasmic calcium removal mechanisms in cut frog skeletal muscle fibres

Antipyrylazo III myoplasmic calcium transients were recorded in cut skeletal muscle fibres of the frog (Rana esculenta), using the double vaseline-gap voltage-clamp system. Intracellular calcium removal mechanisms were analysed, using a slightly modified model taken from the literature. Parameter values reported here are generally consistent with those obtained by the original model. Caffeine (0.5 mmol.l-1) moderately enhanced the overall myoplasmic calcium removal. In particular, the rate constant of the non-saturable uptake increased by 51% on the average, but there was a considerable fiber-to-fiber variation. The kinetic features of the binding sites representing the saturable uptake did not change significantly while the concentration of the available sites decreased by 36%. It is concluded that the caffeine-induced changes of the calcium removal components can be explained by supposing an increased resting myoplasmatic Ca2+ concentration in the presence of the drug.     

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.