Calcium transients in developing mouse skeletal muscle fibres
1 Centro de Biofisica y Bioquímica, Instituto Venezolano de Investigaciones Cientificas IVIC, Apartado 21827, Caracas 1020A, Venezuela
2 Department of Applied Physiology, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
Abstract
Ca2+ transients elicited by action potentials were measured using MagFluo-4, at 20–22°C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days). The rise time of the transient (time from 10 to 90% of the peak) was 2.4 and 1.1 ms in fibres of 7- and 42-day-old mice, respectively. The decay of the transient was described by a double exponential function, with time constants of 1.8 and 16.4 ms in adult, and of 4.6 and 105 ms in 7-day-old animals. The fractional recovery of the transient peak amplitude after 10 ms, F2(10)/F1, determined using twin pulses, was 0.53 for adult fibres and ranged between 0.03 and 0.60 in fibres of 7-day-old animals This large variance may indicate differences in the extent of inactivation of Ca2+ release, possibly related to the difference in ryanodine receptor composition between young and old fibres. At the 7 and 10 day stages, fibres responded to Ca2+-free solutions with a larger decrease in the transient peak amplitude (25% versus 11% in adult fibres), possibly indicating a contribution of Ca2+ influx to the Ca2+ transient in younger animals. Cyclopiazonic acid (1 μM), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase, abolished the Ca2+ transient decay in fibres of 7- and 10-day-old animals and significantly reduced its rate in older animals. Analysis of the transients with a Ca2+ removal model showed that the results are consistent with a larger relative contribution of the SR Ca2+ pump and a lower expression of myoplasmic Ca2+ buffers in fibres of young versus old animals.
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Introduction
The development and structural organization of the molecular elements that participate in excitation–contraction coupling (ECC) of skeletal muscle constitute complex processes that in mammals may continue for several weeks after birth. Changes in the orientation of the intracellular membrane systems are followed by the formation of the characteristic structures in the junction between the membranes of the transverse tubules and the sarcoplasmic reticulum (SR), finally leading to the assembly of functional Ca2+ release units (Veratti, 1961; Walker & Schrodt, 1968; Franzini-Armstrong, 1991; Flucher et al. 1993; Flucher & Franzini-Armstrong, 1996).
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These morphological changes are accompanied by biochemical and physiological changes occurring at various levels. For instance, the resting membrane potential increases from about –42 mV at day 4 to –76 mV at day 27 after birth, accompanied by a reduction of the intracellular sodium and an increase in the potassium activity (Ward & Wareham, 1985), probably related to the progressive expression of the Na+–K+ pump in the sarcolemma. The formation of ryanodine receptor (RyR) and dihydropyridine (DHP) receptor clusters is accompanied by the expression of Ca2+-ATPase in the SR membrane (Holland & Perry, 1969; Boland et al. 1974; Gauthier & Hobbs, 1986). At birth, all muscle fibres are of a slowly contracting type, but soon after birth a process starts that replaces neonatal myosin isoforms by isoforms found in adult slow and fast twitch fibres, leading to changes in the contractile properties (Close, 1964; Whalen et al. 1981). In embryonic and neonatal muscle fibres, two types of Ca2+ currents (ICa) are present: one fast and DHP insensitive, the other slow and DHP sensitive. The fast ICa disappears during the first 3 weeks of postnatal development (Beam & Knudson, 1988).
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Recently it has been shown that a stretch–release cycle causes a marked, transient increase in [Ca2+]i in intact neonatal rat muscle fibres, but not in adult fibres, indicating changes in the Ca2+ handling mechanisms (Mutungi et al. 2003). However, a detailed physiological analysis of the changes in Ca2+ release and Ca2+ clearance properties during development is lacking.
In this investigation we used enzymatically dissociated mouse muscle fibres to study the properties of generation and termination of calcium transients associated with normal contractile activity during postnatal development.
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Methods
Fibre preparation
The enzymatic dissociation method was similar to the one previously published by Carroll et al. (1995). Briefly, flexor digitorum brevis (FDB) muscles were dissected from mice (NMR IVIC) of 7–42 days of age. The mice were killed by rapid cervical dislocation. Procedures were approved by the local animal care committee. Muscles were incubated in a modified mammalian Ringer solution (see below) containing 1 mM Ca2+ and 4 mg ml–1 collagenase (Worthington CLS2) for 1 h at 36°C. After incubation with collagenase, the muscles were washed three times with the 1 mM Ca2+ Ringer solution and gently separated from tendons and remaining tissue with a fire-polished Pasteur pipette. We did not use animals younger than 7 days due to difficulties in obtaining viable fibres with this isolation procedure.
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Fluorescence recording
The composition of the basic experimental solution (mammalian Ringer solution) was as follows (mM): 145 NaCl, 2.5 KCl, 1.0 MgSO4, 2.5 CaCl2, 10 glucose, 10 Hepes, pH 7.4. For the 0 Ca2+ experiments the solution was prepared without CaCl2 and with 0.5 mM EGTA. MagFluo-4 AM (the acetoxymethyl ester form of MagFluo-4; Molecular Probes) was loaded into fibres by immersing them for 30–40 min at room temperature (21–23°C) in a mammalian Ringer solution containing 10 μM of the dye. Fibres adhering spontaneously to the glass bottom of the experimental chamber were selected for recording fluorescence transients. The experimental chamber was mounted on the stage of an inverted Nikon Diaphot TMD microscope equipped for epifluorescence. The fibres were illuminated with a xenon lamp (100 W) only during recording of the light signals, to avoid photo-bleaching of the dye. The characteristic wavelengths (in nm) of the filter combination (excitation/dichroic/barrier) were 450–490/510/520. The light signals were collected from a spot of approximately 12 μm diameter, with a photomultiplier connected to a Nikon P1 amplifier. This procedure allowed recording from numerous fibres within the microscope field.
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Action potentials leading to intracellular Ca2+ transients were elicited by applying supra-threshold rectangular current pulses (0.2–0.4 ms duration) through two platinum plate electrodes placed on either side along the experimental chamber. The amplifier output was fed into an Axon Instruments TL1 DMA interface. The data were acquired and analysed using the Axon Instruments pCLAMP 6 program.
In aqueous solutions the reported dissociation constant of MagFluo-4 AM for calcium is 22 μM and that for magnesium is 4.7 mM (Molecular Probes: Handbook of Fluorescent Probes and Research Products. Ninth Edition). Although the dye concentration in the myoplasm was not measured, it is likely to be considerably higher than the extracellular loading concentration of 10 μM, since the AM ester is hydrolysed intracellularly, allowing build-up of the free dye in the myoplasm. On the other hand, we have shown that in toad muscle fibres, twitch tension is not diminished during or after the dye loading procedure, indicating that the dye in the myoplasm did not interfere with the contractile proteins or with the Ca2+ release mechanism (Caputo & Bolaos, 2001; Caputo et al. 2004). Due to uncertainties regarding the concentration and the dissociation constant of the dye in the fibres, we present the Ca2+ transients as F/F ((Fmax – Frest)/Frest) values.
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Influence of contraction on fluorescence recordings
Control experiments were carried out to assess the extent by which movement artefacts could affect the Ca2+ transient waveform. In these experiments butanedionemonoxime (BDM; 2 mM) was used to abolish contractions. Figure 1 shows two examples of such recordings. The records in the upper panel of Fig. 1A were obtained before (thin trace) and after (thick trace) addition of BDM for a fibre that showed a moderate movement artefact, causing an undershoot in the fluorescence transient. Normally, records of this type received no further consideration. It can be seen that in the presence of 2 mM BDM the transient amplitude is reduced and the undershoot disappears, without changes in the transient time course, as shown in the normalized traces in the lower panel. The records in Fig. 1B were obtained from a fibre that did not show a movement artefact. The recordings in Fig. 1B are representative of the transients that were routinely studied in this investigation. The figure demonstrates that BDM, apart from reducing the Ca2+ transient amplitude, caused no further changes in the transient characteristics. Although it is not possible to totally exclude the presence of movement artefacts they were small and affected our fluorescence traces only marginally.
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The distortion of Ca2+ transients due to the presence of movement artefacts could be suppressed in the presence of 2 mM butanedionemonoxime (BDM, A). BDM reduced the amplitude of the transients without affecting their time course as shown in the lower, normalized records.
Repetitive stimulation experiments
Two stimulation procedures were used. Fibres were either repetitively stimulated at different frequencies to determine the time course of decay of Ca2+ transients, or stimulated with a double stimulus protocol (Simon & Schneider, 1988; Simon et al. 1991; Jong et al. 1995) to investigate the time course of repriming after rapid inactivation. In the repriming experiments the second fluorescence transient rose from the falling phase of the first transient for pulse intervals up to 190 ms. The rapid change of fluorescence on pulse application was defined as the amplitude of the second Ca2+ transient in these cases.
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Optical recording of action potentials
Some experiments were carried out to measure action potentials in fibres of 7- and 42-day-old animals, using the potentiometric fluorescent dye Di-8-ANEPPS. This dye reports rapid membrane potential changes. For these experiments the fibres were exposed to 20 μM Di-8-ANEPPS AM (Molecular Probes) for about 45 min. Then the dye was removed from the extracellular solution and the fibres were mounted in the experimental chamber to record fluorescence signals. The filter combination (characteristic wavelengths, in nm) used for this dye was 450–490/580/590 (excitation/dichroic/barrier). Due to the small amplitude five consecutive signals were averaged.
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Removal model analysis and Ca2+ release calculation
In part of the analysis we used a model-based approach to characterize Ca2+ removal properties and to estimate SR Ca2+ release flux (Melzer et al. 1986, 1987) that utilizes the recovery kinetics of the Ca2+ transient following action potential-triggered Ca2+ release. Here, measured fluorescence changes were assumed to be proportional to changes in free Ca2+ concentration and release was assumed to have ceased 1 ms after the peak of the transient. For the amplitude of the free Ca2+ transient resulting from a single action potential, we used the mean value of 18.5 μM reported by Baylor & Hollingworth (2003). Concentrations [Ca2+]0, [T]tot and [P]tot for resting free Ca2+, T-sites (Ca2+-specific sites of troponin C) and P-sites (parvalbumin-like Ca2+–Mg2+ sites), respectively, in adult fibres were adapted from that study. The values were 100 nM, 0.240 mM and 1.5 mM, respectively. For model differential equations to describe the kinetics of Ca2+ binding to T- and P-sites see Baylor et al. (1983). Baylor & Hollingworth (2003) present and justify rate constant values at 16°C and assume values at 28°C used in their calculations to be twice as high. For consistency with this publication, we made the same assumption and calculated the values for our mean experimental temperature (22°C) by linear interpolation between the values at 16 and 28°C. The resulting values are as follows. T-sites: kon,T,Ca = 133 μM–1 s–1, koff,T,Ca = 173 s–1; P-sites: kon,P,Ca = 62.6 μM–1 s–1, koff,P,Ca = 0.75 s–1, kon,P,Mg = 0.05 μM–1 s–1, koff,P,Mg = 4.5 s–1. Fast binding other than to troponin C (indicator dye, ATP, etc.) was described by a component proportional to free Ca2+ with proportionality constant F. In addition, the transport rate of the SR Ca2+-ATPase was modelled for simplicity to be proportional to the free Ca2+ concentration and described by a single rate constant kuptake. kon,T,Ca, koff,T,Ca, F and kuptake were adjusted by least squares fitting of model-calculated free Ca2+ decays to measured decays (for an explanation of the general procedure see also Timmer et al. 1998). After the best fit was obtained, the Ca2+ occupancies of all model compartments (T-sites, P-sites, F-sites (fast binding sites) and uptake) were summed and the release flux calculated as the time derivative of the sum. Model calculations were performed using Euler's method with software written in Delphi (see Schuhmeier & Melzer, 2004, for more details).
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Statistics
Statistical analysis of differences in mean values was performed using Student's t test for two independent populations. For the calculations, the program Origin 7 (Microcal Software Inc., Northampton, MA, USA) was used. Differences were considered statistically significant at P < 0.05.
Results
Responses to single stimuli
Micrographs documenting the morphology of enzymatically dissociated FDB muscle fibres, obtained from animals ranging in age from 7 to 42 days after birth, are shown in Fig. 2A. Figure 2B depicts the corresponding Ca2+ transients recorded at different ages. The signal amplitudes are normalized to highlight the profound changes in the decay time course. The inset of Fig. 2B shows that the time course of the rising phase of the transient also became faster with age.
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A, morphological changes occurring during postnatal development of enzymatically dissociated fibres from mouse flexor digitorum brevis (FDB) muscles. In younger animals, fibres have a smaller diameter and appear to be wavy. Calibration bar: 20 μm. B, MagFluo-4 fluorescence transients measured in fibres of different ages. The amplitudes of the records have been normalized to give a better demonstration of the changes in the decay phase of the transient. Further information is given in Table 1. The inset depicts the same records at a higher time resolution to show the changes in the rising phase of the transients.
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In agreement with previous work (Caputo & Bolaos, 2001; Caputo et al. 2004), in adult fibres ( 42 days), the time course of decay could be described by a double exponential function of the type:
Here, A and B are the amplitudes of the two components that decay with different time constants 1 and 2, respectively.
For adult fibres the values obtained for 1 and 2 were 1.8 and 16.4 ms, while for the 7-day-old animals they were 4.6 and 105 ms, respectively. The table also shows that the amplitude of the transients, expressed in terms of F/F, increased with age. It is noteworthy that the most profound changes occurred later than 10 days after birth. From day 7 to day 10 none of the parameters, with the exception of the slow decay time constant, showed a statistically significant difference.
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Figure 3 shows that the distribution of the values of the time constants 1 and 2 changed with the age of the animal. In the younger animals the values of the fast and slow time constants showed a wide dispersion that diminished with age. The biphasic decay of the Ca2+ transients suggests that two different components contribute to lowering free [Ca2+]i during a twitch. The graph of Fig. 4 shows the relative magnitude of these two components plotted against age of the animal. It can be seen that the fast component () increases with age while the slow component () decreases. Thus, while in 7-day-old animals the relative contributions of the fast and slow components to the decay phase were 22 and 78%, respectively, in adult animals they were 55 and 45%, respectively.
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Histograms showing the distribution of the values of fast and slow time constants of decay of fluorescence transients. In younger animals the values are more widely distributed.
Change with age of the relative contribution of the fast and slow decay components to the falling phase of fluorescence transients. The magnitudes of the two components A and B in eqn (1) were obtained by fitting this function to the transient decay phase.
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Action potentials in developing fibres
In view of the marked difference in the resting membrane potential between young and adult mouse fibres (Ward & Wareham, 1985) it also seemed important to perform measurements of the action potentials to test whether differences in excitation between fibres of young and adult animals might be responsible for differences in the time course of the fluorescence transients. Thus, several experiments were carried out using the potentiometric dye Di-8-ANEPPS to record fluorescence transients associated with membrane action potentials. Figure 5 shows examples of optically recorded action potentials in fibres from 7- (upper row) and 42-day-old animals (lower row). Table 2 summarizes the spike parameters obtained with several fibres of each type. With the exception of the transient half-width, significant differences were found between the parameters of young and adult fibres. In addition younger fibres showed a clear after-hyperpolarization (undershoot) not observed in adult fibres. Adult fibres often showed clear signs of movement artefacts (the two records on the left of Fig. 5). Such artefacts were more conspicuous than in the case of MagFluo-4 transients due to the higher amplification and the averaging procedure used in this case. The movement artefacts could be abolished by blocking fibre contraction with BDM, as shown in the two records on the right. It can also be noticed that the movement artefacts started when the membrane potential had practically regained its resting value and thus did not affect the estimation of action potential parameters. The results indicate that the prolonged Ca2+ transient relaxation in fibres of young animals cannot be explained in terms of a prolongation of the action potential.
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Di-8-ANEPPS fluorescence transients reporting membrane potential changes associated with action potentials in 7- (upper records) and 42-day-old (lower records) mouse fibres. Further details are given in the text and in Table 2.
Responses to tetanic stimulation
The upper records in Fig. 6 show MagFluo-4 responses to 100 Hz stimulation in three fibres from mice of different ages. In younger animals (7 and 10 days), there was a pronounced summation of the individual transients due to their slower decay phases, as shown in Fig. 2. In the fibre from the adult animal there was considerably less summation of the individual transients. More interestingly, the young fibres showed a single exponential decay after the last stimulus, whereas in adult fibres this decay appeared to follow a bi-exponential function, described by eqn (1). For 12 adult fibres the values of the two time constants were estimated to be 25 and 330 ms, respectively. The relative contributions of the fast (A) and slow (B) components to the decay phase of tetani were 64 and 36%, respectively. The graph in Fig. 6 shows that the single time constant of younger animals () changes with age from a value of about 334 ms for the 7-day-old animals to about 70 ms for the 15-day-old animals. Thus, this value tends to approach the value of the fast time constant of the adult animals, 23 ms, represented by the filled circle and marked 1 in the graph. As mentioned above, in fibres from adult animals the decay of the fluorescence transient after tetanic stimulation showed a second and slower decay phase with a time constant of about 300 ms (, labelled 2).
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Upper records in the figure show normalized fluorescence transients in response to a 200 ms, 100 Hz tetanic stimulation of fibres from 7-, 10- and 42-day-old animals. In younger animals the decay phase of the tetanus can be described by a single exponential, while in the adult animals it is better described by two exponentials. The insets next to the tetanic responses show the three first individual transients at a higher time resolution. The graph shows how the time constants of decay of the tetani change with the age of the animals.
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In adult animals, the value of the ratio between the amplitudes of the second and first transients in the tetanus was larger than in younger animals, as it is shown in the insets next to each transient. However, in younger mice we found a large variability between fibres. Whereas in adult animals the ratio varied between 0.51 and 0.67, in the younger animals it varied between 0.23 and 0.63.
Removal model analysis and Ca2+ release calculation
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The kinetics of the fluorescence transients reported by the indicator dye are the result of Ca2+ release, Ca2+ binding to intrinsic sites and transport by the SR Ca2+-ATPase. We performed a calculation of the Ca2+ release flux underlying the Ca2+ transients by using a modified version of the ‘removal model fit’ approach originally developed for voltage-clamped frog fibres (Melzer et al. 1986, 1987). This procedure uses model-calculated Ca2+ transients and least squares optimization of some of the model parameters to make calculated Ca2+ transients consistent with the measured data with regard to the relaxation time interval after the end of release. We used kinetic parameters of troponin C and parvalbumin and a model structure similar to the one applied in a recent comprehensive analysis of action potential-triggered Ca2+ transients in mouse EDL fibres (Baylor & Hollingworth, 2003). We added only one further component to model Ca2+ reuptake to the SR (rate constant kuptake, see Methods). Figure 7A shows measurements of a single Ca2+ transient and a tetanic response obtained in the same fibre. The thick lines show the best simultaneous fit of the kinetic removal model to the single transient and to nine decays within the tetanic response. The fit describes well both the individual time courses and the slowing of the kinetics of decay from the single response to the end of the tetanic response. For four experiments, the mean values (± S.E.M.) of the free fit parameters kon,T,Ca, koff,T,Ca, F and kuptake (see Methods) were 30.4 ± 19.1 μM–1 s–1, 44.2 ± 14.9 s–1, 7.92 ± 2.86 and 295 ± 177 s–1, respectively.
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A, free Ca2+ transients derived from fluorescence recordings using assumptions described in Methods. The recording on the left was induced by a single action potential, the one on the right by a series of spikes at 100 Hz stimulation frequency. Superimposed thick lines show the results of a simultaneous ‘removal model fit’ describing well the observed changes in relaxation kinetics. The fit used kon,T,Ca,koff,T,Ca,F and kuptake as free parameters. Best parameter values obtained after convergence were 3.95 μM–1 s–1, 25.4 s–1, 8.2 and 205 s–1, respectively. B, Ca2+ release flux derived from the removal analysis in A. C, release flux time course in the intervals indicated by horizontal lines at a higher time resolution. Data were smoothed using a local adaptive filter (Schuhmeier et al. 2003).
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Figure 7B shows the calculated Ca2+ release rates derived from the removal analysis in Fig. 7A. During the sequence of 21 individual transients of the tetanic response, peak release flux decreased to about 25% of the initial value of 225 μM ms–1. This fractional change in the Ca2+ release flux amplitude corresponds well to the alterations in the observed amplitudes of the rapid changes in the fluorescence transient, confirming that the decrease in the Ca2+ transient amplitudes results largely from a decrease in the release rate. Figure 7C depicts sections of the release rate traces at an expanded time scale for a better display of the release flux time course. The duration at half-maximum flux was about 1.0 ms at the first pulse and did not change appreciably during the course of the pulse series.
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Recovery from inactivation
The progressive decrease in the amplitude of the individual Ca2+ transients during a 100 Hz tetanus probably results from a fast inactivation of the Ca2+ release mechanism (Schneider & Simon, 1988). In a further series of experiments, we explored recovery from inactivation using a double stimulation procedure as used previously by different authors (Simon & Schneider, 1988; Simon et al. 1991; Jong et al. 1995). Figure 8A demonstrates the double pulse procedure for an adult mouse muscle fibre. Figure 8B shows the superimposed records of a complete run. The fibre was allowed to rest for 20 s between recordings. Finally, the graph in Fig. 8C shows the time course of recovery obtained from several fibres. Each point represents the mean ± S.E.M. of seven experiments. The graph shows the fractional recovery of the fluorescence peak amplitude (F2/F1) plotted against the interval between pulses. The data could be well fitted by a single exponential function with a time constant of 33 ms. In adult fibres, the value of F2(10)/F1, i.e. the fractional amplitude of the second transient (at a 10 ms interval) with respect to the first one, was 0.53. The extrapolated value of the ratio F2/F1 at time zero (F2(0)/F1) was 0.36. When the same type of experiment was performed on fibres from younger animals, a large variability was observed regarding the extent of inactivation caused by the first response. The records in Fig. 9A show examples of this behaviour when fibres from animals of 7, 10, 15 and 42 days, were stimulated with two pulses separated by a 10 ms interval. The records on the left are examples of fibres with a higher degree of inactivation, while those on the right are examples of fibres with lower inactivation. The numbers next to the records indicate the fraction of fibres (percentage) of each type. For the 7-day-old animals 50% of the fibres belonged to each group. With increasing age, however, the percentage value of the first group diminished whereas that of the second group increased, reaching 100% in adult fibres. The histograms in Fig. 9B show the corresponding distribution of the F2(10)/F1 values obtained at each age for several fibres, suggesting the presence of two distinct fibre groups in younger animals. The segregation criterion for the groups was based on the mean F2(10)/F1 value of 0.576 ± 0.099 (mean ± S.D., n = 87) obtained in our laboratory with adult fibres, which were considered to be of the low inactivation type. We considered fibres from young animals exhibiting F2(10)/F1 values in an interval of two standard deviations from this mean value (i.e. between 0.38 and 0.76) to be of the low inactivation type, while fibres showing F2(10)/F1 values lower than 0.38 were considered to belong to the high inactivation type.
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The records in A show fluorescence transients elicited by two action potentials separated by varying stimulus intervals. B shows the superimposed traces of 10 pairs of fluorescence signals. The stimulus intervals varied between 10 and 190 ms. The graph in C summarizes the results obtained with several fibres of adult mice. The graph shows the mean values (± S.E.M.; n = 7) of F2/F1 plotted as a function of the interval between the two stimuli. The mean value of F2/F1 obtained at 10 ms (F2(10)/F1) is 0.53. The value of the intersect at 0 time (F2(0)/F1) is 0.36. The continuous line shows a single exponential function fitted to the experimental points. The curve represents the time course of recovery (repriming) from inactivation of the fluorescence transient associated with the first response. The time constant of repriming was 32.6 ms.
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The records in A were obtained in double-stimulus experiments with a stimulus interval of 10 ms in fibres from 7-, 10-, 15- and 42-day-old animals. The two columns of records show examples of variability in the F2(10)/F1 ratio obtained in fibres of the same age. The histograms in B show the distribution of the values of F2(10)/F1, suggesting the presence of two groups of fibres, one showing characteristics similar to adult fibres.
Finally, Fig. 10 compares the time course of recovery from inactivation in these two types of fibres (filled circles and triangles, respectively) in 7-day-old mice, with that of adult fibres (dashed line). In addition to the difference in the initial fractional inactivation, the time course of recovery is different in the two fibre types, with time constant values of 42.5 ms for the low inactivation type and 28.9 ms for the high inactivation type.
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The symbols represent the mean values (± S.E.M.) obtained in several experiments. The lines show single exponential functions fitted to the experimental data points. Data are shown in comparison with results obtained in adult fibres (dashed line taken from the graph of Fig. 8).
The effect of external Ca2+
The upper records of Fig. 11 show the effect on the fluorescence transients when the fibres were exposed to a medium prepared without Ca2+. In adult fibres the transient amplitude decreased by 12 ± 4% (n = 4) (Fig. 11Aa). Fibres of younger animals showed a larger decrease of 25 ± 7% (n = 7) (Fig. 11Ba). In both cases, no substantial changes in the response time course could be seen, as shown by superimposition of normalized records before and after Ca2+ removal (Fig. 11Ab and Bb). The graph in Fig. 11C shows the effect of 0 Ca2+ on the recovery from inactivation in adult fibres. In this case, there is little or no difference caused by the absence of external Ca2+. Notice that in both cases the F2(10)/F1 value is higher than 0.50. The graph in Fig. 11D shows the results obtained with 7-day-old fibres, belonging to the ‘high inactivation’ type. In this case, fibres in the presence of normal Ca2+ showed a higher degree of inactivation (F2(10)/F1 = 0.30), which considerably diminished in the absence of external Ca2+ (F2(10)/F1 = 0.57).
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The records in Aa and Ba show the effect of 0 Ca2+ in the external medium on the transient amplitude. The same records, normalized to the peak, are shown in Ab and Bb, respectively, to demonstrate that the time course of the transient is not affected by external Ca2+ deprivation. The graphs C and D show the effect of 0 Ca2+ on repriming from inactivation. The graph in C shows the repriming from inactivation in fibres from adult animals obtained in the presence () and absence () of external Ca2+. The graph in D shows repriming curves obtained under the same conditions in fibres of the high-inactivation type of 7-day-old fibres.
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Role of SR Ca2+-ATPase
Figure 12 shows the effect of cyclopiazonic acid (CPA) on the time course of decay of Ca2+ transients at different ages. As shown above for control conditions, the fluorescence transients in all fibres exhibited a fast decay component followed by a slower phase. After poisoning the SR Ca2+-ATPase with CPA, the fluorescence transient of the 7 day fibre depicted in this figure showed a considerably reduced fast decay component, followed by a sustained or plateau phase with a slight secondary rise and no sign of decay for almost 300 ms. The 10 day fibre shows a similar response, but a slow decay starts after the secondary rise. In the 15- and 45-day-old animals the fast component of decay is followed by a slower phase showing that the effect of CPA, although noticeable, diminishes with the age of the animal. For the 15-day-old animals the time constants 1 and 2 showed values of 3.6 ± 0.7 and 41.4 ± 15 in the absence of CPA and values of 11.5 ± 1.3 and 141.8 ± 20 (n = 5) in its presence. In 42-day-old animals the 1 and 2 values were, respectively, 1.5 ± 0.1 and 18.7 ± 2.1 before, and 2.1 ± 0.2 and 78.8 ± 10.5 (n = 8) after, application of CPA.
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Effect of cyclopiazonic acid (CPA) on the time course of fluorescence transients obtained in fibres of 7-, 10-, 15- and 42- (adult) day-old animals.
Discussion
In adult mouse fibres, Ca2+ transients elicited by action potentials and measured with a low affinity Ca2+ indicator dye show a rapid rising phase that leads to a peak in less than 1.5 ms, followed by a decaying phase that can be described by a two-exponential process with time constants of 1.8 and 16 ms (Caputo et al. 2004). Similar properties have been reported by other authors (Hollingworth et al. 1996; Carroll et al. 1997; Baylor & Hollingworth, 2003). In our experiments, the activation of Ca2+ release in adult muscle fibres was probably synchronized all along the fibre length due to the field stimulation and transversally due to the regenerative inward spread of the sodium action potential along the transverse tubules. Due to the rapid repolarization of the action potential, the release mechanism is rapidly deactivated; inactivation may also have contributed to the termination of Ca2+ release and to the early onset of the decay phase (Baylor et al. 1983; Schneider & Simon, 1988; Simon et al. 1991; Jong et al. 1993, 1995; Hollingworth et al. 1996; Caputo & Bolaos, 2002; Baylor & Hollingworth, 2003). Ca2+ binding to intracellular Ca2+ buffers and the SR Ca2+-ATPase contribute in variable amounts to the decay of the transients.
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The results presented here demonstrate that, compared with adult fibres, young mouse fibres show a slower rising phase of the Ca2+ transients and a much slower time course of decay. The experiments carried out with the potentiometric dye Di-8-ANEPPS indicate some differences between action potentials of young (7 days) and adult animals. The action potential amplitudes in adult fibres appear to be about 30% larger than those in younger fibres, which were characterized by a sizeable after-hyperpolarization. The amplitude difference is probably due to a lower value of the resting membrane potential in young fibres expected from the lower K+ and higher Na+ activities in the intracellular space reported by Ward & Wareham (1985). This could cause inactivation of the Na+ channels, and therefore a reduction in the action potential amplitude and a prolongation of its time course, which was also observed as shown in Table 2. The after-hyperpolarization could be due to differences in the expression level of the channels, a larger deviation of the resting potential from the K+ equilibrium potential, or a less-developed transverse tubular system. The amplitude of action potentials in rat myotubes in culture has been reported to be rather small, about 47 mV on day 6 and to increase to 120 mV by day 14 (Bakker et al. 1996). Interestingly most of these myotubes also exhibited pronounced after-hyperpolarizations. The consequences of partial inactivation of the Na+ conductance could be a delayed inward spread of the action potential, which might explain the slower rising phase of Ca2+ transients. Poor development of Ca2+-releasing units in the young animals, due to incomplete structural arrangement of internal membrane systems (Veratti, 1961; Walker & Schrodt, 1968; Franzini-Armstrong, 1991; Flucher et al. 1993; Flucher & Franzini-Armstrong, 1996) might also contribute to this effect. However, it seems clear from our results that the differences observed in the action potentials between younger and older animals is not sufficient to explain the prolonged time course of decay of the Ca2+ transients. This latter effect rather points to a reduced Ca2+ clearance capacity of young fibres. This is supported by the results obtained with the removal analysis carried out for some of the fibres (see below).
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It is known that different elements involved in ECC become functional at different times during development (Franzini-Armstrong, 1991; Flucher et al. 1993). This agrees with our finding that kinetic characteristics develop with different timing in different fibres, even from the same animal. In fact, fibres from younger animals do not behave as a homogeneous population with regard to certain parameters of the fluorescent transients. In particular, the values of the fast and slow time constants of decay showed a broad dispersion that disappeared with age. Also, the double pulse experiments indicated that the extent of inactivation caused by a single transient may vary widely between fibres of young animals. In about half of the 7-day-old fibres the extent of inactivation was similar to that observed in adult fibres, showing a fractional inactivation value of 0.36 estimated for time zero (F2(0)/F1), and of 0.50 when measured after 10 ms (F2(10)/F1). The other half of the fibres showed a higher degree of inactivation, with a F2(0)/F1 value of 0.03, and a F2(10)/F1 value of 0.27.
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One might argue that the stronger inactivation observed in a group of fibres from young animals could be the consequence of fibre damage. However, as shown in Fig. 10 the difference in the extent of inactivation became evident only for the shortest stimulus interval used (i.e. 10 ms) and disappeared at longer recovery periods. Both groups of fibres recovered to the same extent indicating that the high-inactivation fibres exhibit a faster initial recovery. This would not be expected from unhealthy fibres, which in our experience typically show a rapid run-down and finally complete loss of responsiveness to stimuli.
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Our conclusion is that different fibres, even from the same animal, may reach adult characteristics with a different time course. This is compatible with the observation that in rat myotubes in culture a large variation of Ca2+ transients, ranging from small and slow to large and rapid, could be found during the early days of development (Bakker et al. 1996).
It is worthwhile to note that mouse fibres of the high-inactivation type behaved similarly to amphibian muscle fibres in which the corresponding values of F2(0)/F1 and F2(10)/F1 were 0.06 and 0.27, respectively (Caputo et al. 2004). The greater extent of inactivation observed in some of the younger fibres might be linked to a different composition of ryanodine receptor isoforms. Similar to adult amphibian muscle (Murayama & Ogawa, 1992; Franzini-Armstrong & Protasi, 1997; Sorrentino & Reggiani, 1999), isoform RyR3, located in extra-junctional regions of the SR, is expressed in addition to RyR1 in young but not in adult mammalian muscle fibres (Felder & Franzini-Armstrong, 2002). Comparison of mammalian and amphibian muscle fibres suggested the presence of different release mechanisms (Rios & Pizarro, 1991; Shirokova et al. 1996; Ogawa et al. 2000; Murayama & Ogawa, 2002).
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When external Ca2+ was omitted, the response peak amplitude decreased to a greater extent in fibres from young than from adult animals (25% versus 11%). The decrease in 0 Ca2+ is compatible with the demonstration of robust fast Ca2+ currents in younger animals (Beam & Knudson, 1988). When Ca2+ release units have not yet fully developed, Ca2+ influx through sarcolemma Ca2+ channels might contribute to Ca2+ signalling. It has been shown that during in vitro skeletal myogenesis the fraction of contractile activity that depends on the presence of calcium currents diminishes progressively with age while a Ca2+ current-insensitive component becomes predominant. In adult fibres the extent of inactivation of Ca2+ release was not affected by removal of external Ca2+, while in young fibres, of the high-inactivation type, inactivation was reduced to the level of adult fibres. These results might indicate that entry of Ca2+ contributes to the fast inactivation phenomenon.
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Removal analysis and release calculation
As we showed in Fig. 7, the decline in Ca2+ transient amplitude during a 100 Hz tetanus was caused by a corresponding decline in the Ca2+ release flux amplitude. Our approach to calculating release differed from previous detailed analyses in adult mouse muscle fibres (Hollingworth et al. 1996; Baylor & Hollingworth, 2003) by also taking into account the decay of free Ca2+ after turn-off of release. We found that model parameters of Baylor & Hollingworth (2003) had to be altered somewhat to account for the measured kinetics of decay. The main changes concerned fast myoplasmic buffering. Fast buffering was described in the present model (as it was in the model by Baylor & Hollingworth) by a single non-instantaneous and non-linear binding component (T-sites, see Methods and Results) in combination with an instantaneous and linear binding component (F-sites). The rate constants of the T-sites had to be lowered for the fit to describe the data. However, to draw conclusions regarding the kinetics of troponin C in muscle from these results would be premature because different fast sites (troponin, ATP, pump sites, etc.) were lumped into just two simple model components.
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The decay of a single Ca2+ transient could be described by a double exponential process, with a fast initial phase, followed by a slower component. The relative contribution of the two components changed with age in a reciprocal way: although the slow component accounted for most of the decay phase (80%) in fibres of 7-day-old animals, it made up only 45% in adult fibres. Considering single transients of adult animals, the fast component may be caused by Ca2+ binding to intracellular buffers like parvalbumin, as suggested by the increase in its relative contribution with age and in agreement with previous work (Carroll et al. 1997). The slow component may be associated with the operation of the SR Ca2+ pump, as suggested by the experiments in which CPA was used to block the pump.
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Despite the simplicity of the model, the removal analysis shown in Fig. 7 provided an almost perfect fit to the measured Ca2+ transients and described well the kinetic changes in the relaxation time course occurring during repetitive stimulation. In the model, the slowing of relaxation results from both the T- and P-sites, which substantially contribute to the more rapid relaxation in the single Ca2+ transient but become progressively saturated during the course of the tetanic stimulation.
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Kinetic differences of Ca2+ transients in neonatal and adult animals
The differences among Ca2+ transients in specimens of different ages revealed a larger relative contribution of the SR Ca2+ pump to the relaxation kinetics in young fibres. A possible explanation for the different shapes of the Ca2+ transients is therefore the lower expression of myoplasmic Ca2+ buffers in the young fibres. The records of Fig. 13 show the results of a removal model analysis in another set of experiments which support this view.
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Top row: action potential-activated Ca2+ transients recorded from an adult muscle fibre before (A) and after (B) blocking the SR pump with CPA. Superimposed thick lines are the results of a removal model calculation (see Results for explanation). Bottom row: action potential-activated Ca2+ transients recorded from a muscle fibre of a 7-day-old mouse before (C) and after (D) pump block by CPA with corresponding removal model description. Values of the model parameters ([T]tot, [P]tot and kuptake) that were changed to simulate the kinetic differences were as follows. A: 0.240 mM, 1.5 mM, 1188 s–1; B: 0.240 mM, 1.5 mM, 0 s–1; C: 0.120 mM, 0 mM, 364 s–1; D: 0.120 mM, 0 mM, 0 s–1. Fixed model parameters were as follows. F-sites: F = 28.9; T-sites: kon,T,Ca = 9.15 μM–1 s–1, koff,T,Ca = 25.4 s–1; P-sites: kon,P,Ca = 62.6 μM–1 s–1, koff,P,Ca = 0.75 s–1, kon,P,Mg = 0.05 μM–1 s–1, koff,P,Mg = 4.5 s–1, [Mg2+] = 1 mM.
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The traces on the left (Fig. 13A and C) present Ca2+ transients obtained with single action potentials under normal conditions in an adult fibre and in a fibre of a 7-day-old mouse, respectively. The traces on the right (Fig. 13B and D) show the altered transients of the same fibres after blocking the SR Ca2+ pump with CPA. The superimposed thick lines show Ca2+ transient decays calculated with the removal model (see Methods and Fig. 13 legend). The differences in the calculated traces in the four panels result only from changes in the concentration of T- and P-sites and the uptake rate constant (kuptake). The arrows indicate the sequence of the analysis. Starting with the adult fibre and the pump blocked (Fig. 13B), we used the best-fit parameters of the analysis in Fig. 13 as initial values and adjusted kon,T,Ca and F (thus accounting for fibre variability), with kuptake set to zero (assuming complete block), to fit the decay. The resulting set of best-fit parameters was then kept constant and only kuptake adjusted to fit the trace on the left (Fig. 13A). Proceeding from this result, we tried to fit the trace obtained from the young fibre (Fig. 13C) by altering the P-site concentration. However, even complete elimination of the P-sites was not sufficient to model the slower kinetics. A reduction of T-site concentration to 50%, and of kuptake to 31%, led to the fitted trace in (Fig. 13C). The kinetic change from panel C to panel D could then simply be simulated by setting kuptake to zero. The result is compatible with lack of expression of slow parvalbumin-like sites (P-sites) and reduced expression of fast binding sites and pump activity in young fibres compared to adult fibres. Since kuptake varied substantially even among normal adult fibres, the difference in P-site concentration most likely constitutes the decisive factor. Even though this theoretical analysis is based on a simplified model for myoplasmic Ca2+ turnover, it provides a quite plausible explanation for the substantial changes in kinetics that were observed in these experiments.
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In summary, the present study describes Ca2+ transient kinetics in skeletal muscle fibres at different stages of postnatal development. It revealed characteristic changes that are probably linked both to the maturation and reorganization of the Ca2+ release machinery and to the changing expression levels of Ca2+ buffering and transport sites.
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2 Department of Applied Physiology, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
Abstract
Ca2+ transients elicited by action potentials were measured using MagFluo-4, at 20–22°C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days). The rise time of the transient (time from 10 to 90% of the peak) was 2.4 and 1.1 ms in fibres of 7- and 42-day-old mice, respectively. The decay of the transient was described by a double exponential function, with time constants of 1.8 and 16.4 ms in adult, and of 4.6 and 105 ms in 7-day-old animals. The fractional recovery of the transient peak amplitude after 10 ms, F2(10)/F1, determined using twin pulses, was 0.53 for adult fibres and ranged between 0.03 and 0.60 in fibres of 7-day-old animals This large variance may indicate differences in the extent of inactivation of Ca2+ release, possibly related to the difference in ryanodine receptor composition between young and old fibres. At the 7 and 10 day stages, fibres responded to Ca2+-free solutions with a larger decrease in the transient peak amplitude (25% versus 11% in adult fibres), possibly indicating a contribution of Ca2+ influx to the Ca2+ transient in younger animals. Cyclopiazonic acid (1 μM), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase, abolished the Ca2+ transient decay in fibres of 7- and 10-day-old animals and significantly reduced its rate in older animals. Analysis of the transients with a Ca2+ removal model showed that the results are consistent with a larger relative contribution of the SR Ca2+ pump and a lower expression of myoplasmic Ca2+ buffers in fibres of young versus old animals.
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Introduction
The development and structural organization of the molecular elements that participate in excitation–contraction coupling (ECC) of skeletal muscle constitute complex processes that in mammals may continue for several weeks after birth. Changes in the orientation of the intracellular membrane systems are followed by the formation of the characteristic structures in the junction between the membranes of the transverse tubules and the sarcoplasmic reticulum (SR), finally leading to the assembly of functional Ca2+ release units (Veratti, 1961; Walker & Schrodt, 1968; Franzini-Armstrong, 1991; Flucher et al. 1993; Flucher & Franzini-Armstrong, 1996).
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These morphological changes are accompanied by biochemical and physiological changes occurring at various levels. For instance, the resting membrane potential increases from about –42 mV at day 4 to –76 mV at day 27 after birth, accompanied by a reduction of the intracellular sodium and an increase in the potassium activity (Ward & Wareham, 1985), probably related to the progressive expression of the Na+–K+ pump in the sarcolemma. The formation of ryanodine receptor (RyR) and dihydropyridine (DHP) receptor clusters is accompanied by the expression of Ca2+-ATPase in the SR membrane (Holland & Perry, 1969; Boland et al. 1974; Gauthier & Hobbs, 1986). At birth, all muscle fibres are of a slowly contracting type, but soon after birth a process starts that replaces neonatal myosin isoforms by isoforms found in adult slow and fast twitch fibres, leading to changes in the contractile properties (Close, 1964; Whalen et al. 1981). In embryonic and neonatal muscle fibres, two types of Ca2+ currents (ICa) are present: one fast and DHP insensitive, the other slow and DHP sensitive. The fast ICa disappears during the first 3 weeks of postnatal development (Beam & Knudson, 1988).
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Recently it has been shown that a stretch–release cycle causes a marked, transient increase in [Ca2+]i in intact neonatal rat muscle fibres, but not in adult fibres, indicating changes in the Ca2+ handling mechanisms (Mutungi et al. 2003). However, a detailed physiological analysis of the changes in Ca2+ release and Ca2+ clearance properties during development is lacking.
In this investigation we used enzymatically dissociated mouse muscle fibres to study the properties of generation and termination of calcium transients associated with normal contractile activity during postnatal development.
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Methods
Fibre preparation
The enzymatic dissociation method was similar to the one previously published by Carroll et al. (1995). Briefly, flexor digitorum brevis (FDB) muscles were dissected from mice (NMR IVIC) of 7–42 days of age. The mice were killed by rapid cervical dislocation. Procedures were approved by the local animal care committee. Muscles were incubated in a modified mammalian Ringer solution (see below) containing 1 mM Ca2+ and 4 mg ml–1 collagenase (Worthington CLS2) for 1 h at 36°C. After incubation with collagenase, the muscles were washed three times with the 1 mM Ca2+ Ringer solution and gently separated from tendons and remaining tissue with a fire-polished Pasteur pipette. We did not use animals younger than 7 days due to difficulties in obtaining viable fibres with this isolation procedure.
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Fluorescence recording
The composition of the basic experimental solution (mammalian Ringer solution) was as follows (mM): 145 NaCl, 2.5 KCl, 1.0 MgSO4, 2.5 CaCl2, 10 glucose, 10 Hepes, pH 7.4. For the 0 Ca2+ experiments the solution was prepared without CaCl2 and with 0.5 mM EGTA. MagFluo-4 AM (the acetoxymethyl ester form of MagFluo-4; Molecular Probes) was loaded into fibres by immersing them for 30–40 min at room temperature (21–23°C) in a mammalian Ringer solution containing 10 μM of the dye. Fibres adhering spontaneously to the glass bottom of the experimental chamber were selected for recording fluorescence transients. The experimental chamber was mounted on the stage of an inverted Nikon Diaphot TMD microscope equipped for epifluorescence. The fibres were illuminated with a xenon lamp (100 W) only during recording of the light signals, to avoid photo-bleaching of the dye. The characteristic wavelengths (in nm) of the filter combination (excitation/dichroic/barrier) were 450–490/510/520. The light signals were collected from a spot of approximately 12 μm diameter, with a photomultiplier connected to a Nikon P1 amplifier. This procedure allowed recording from numerous fibres within the microscope field.
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Action potentials leading to intracellular Ca2+ transients were elicited by applying supra-threshold rectangular current pulses (0.2–0.4 ms duration) through two platinum plate electrodes placed on either side along the experimental chamber. The amplifier output was fed into an Axon Instruments TL1 DMA interface. The data were acquired and analysed using the Axon Instruments pCLAMP 6 program.
In aqueous solutions the reported dissociation constant of MagFluo-4 AM for calcium is 22 μM and that for magnesium is 4.7 mM (Molecular Probes: Handbook of Fluorescent Probes and Research Products. Ninth Edition). Although the dye concentration in the myoplasm was not measured, it is likely to be considerably higher than the extracellular loading concentration of 10 μM, since the AM ester is hydrolysed intracellularly, allowing build-up of the free dye in the myoplasm. On the other hand, we have shown that in toad muscle fibres, twitch tension is not diminished during or after the dye loading procedure, indicating that the dye in the myoplasm did not interfere with the contractile proteins or with the Ca2+ release mechanism (Caputo & Bolaos, 2001; Caputo et al. 2004). Due to uncertainties regarding the concentration and the dissociation constant of the dye in the fibres, we present the Ca2+ transients as F/F ((Fmax – Frest)/Frest) values.
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Influence of contraction on fluorescence recordings
Control experiments were carried out to assess the extent by which movement artefacts could affect the Ca2+ transient waveform. In these experiments butanedionemonoxime (BDM; 2 mM) was used to abolish contractions. Figure 1 shows two examples of such recordings. The records in the upper panel of Fig. 1A were obtained before (thin trace) and after (thick trace) addition of BDM for a fibre that showed a moderate movement artefact, causing an undershoot in the fluorescence transient. Normally, records of this type received no further consideration. It can be seen that in the presence of 2 mM BDM the transient amplitude is reduced and the undershoot disappears, without changes in the transient time course, as shown in the normalized traces in the lower panel. The records in Fig. 1B were obtained from a fibre that did not show a movement artefact. The recordings in Fig. 1B are representative of the transients that were routinely studied in this investigation. The figure demonstrates that BDM, apart from reducing the Ca2+ transient amplitude, caused no further changes in the transient characteristics. Although it is not possible to totally exclude the presence of movement artefacts they were small and affected our fluorescence traces only marginally.
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The distortion of Ca2+ transients due to the presence of movement artefacts could be suppressed in the presence of 2 mM butanedionemonoxime (BDM, A). BDM reduced the amplitude of the transients without affecting their time course as shown in the lower, normalized records.
Repetitive stimulation experiments
Two stimulation procedures were used. Fibres were either repetitively stimulated at different frequencies to determine the time course of decay of Ca2+ transients, or stimulated with a double stimulus protocol (Simon & Schneider, 1988; Simon et al. 1991; Jong et al. 1995) to investigate the time course of repriming after rapid inactivation. In the repriming experiments the second fluorescence transient rose from the falling phase of the first transient for pulse intervals up to 190 ms. The rapid change of fluorescence on pulse application was defined as the amplitude of the second Ca2+ transient in these cases.
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Optical recording of action potentials
Some experiments were carried out to measure action potentials in fibres of 7- and 42-day-old animals, using the potentiometric fluorescent dye Di-8-ANEPPS. This dye reports rapid membrane potential changes. For these experiments the fibres were exposed to 20 μM Di-8-ANEPPS AM (Molecular Probes) for about 45 min. Then the dye was removed from the extracellular solution and the fibres were mounted in the experimental chamber to record fluorescence signals. The filter combination (characteristic wavelengths, in nm) used for this dye was 450–490/580/590 (excitation/dichroic/barrier). Due to the small amplitude five consecutive signals were averaged.
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Removal model analysis and Ca2+ release calculation
In part of the analysis we used a model-based approach to characterize Ca2+ removal properties and to estimate SR Ca2+ release flux (Melzer et al. 1986, 1987) that utilizes the recovery kinetics of the Ca2+ transient following action potential-triggered Ca2+ release. Here, measured fluorescence changes were assumed to be proportional to changes in free Ca2+ concentration and release was assumed to have ceased 1 ms after the peak of the transient. For the amplitude of the free Ca2+ transient resulting from a single action potential, we used the mean value of 18.5 μM reported by Baylor & Hollingworth (2003). Concentrations [Ca2+]0, [T]tot and [P]tot for resting free Ca2+, T-sites (Ca2+-specific sites of troponin C) and P-sites (parvalbumin-like Ca2+–Mg2+ sites), respectively, in adult fibres were adapted from that study. The values were 100 nM, 0.240 mM and 1.5 mM, respectively. For model differential equations to describe the kinetics of Ca2+ binding to T- and P-sites see Baylor et al. (1983). Baylor & Hollingworth (2003) present and justify rate constant values at 16°C and assume values at 28°C used in their calculations to be twice as high. For consistency with this publication, we made the same assumption and calculated the values for our mean experimental temperature (22°C) by linear interpolation between the values at 16 and 28°C. The resulting values are as follows. T-sites: kon,T,Ca = 133 μM–1 s–1, koff,T,Ca = 173 s–1; P-sites: kon,P,Ca = 62.6 μM–1 s–1, koff,P,Ca = 0.75 s–1, kon,P,Mg = 0.05 μM–1 s–1, koff,P,Mg = 4.5 s–1. Fast binding other than to troponin C (indicator dye, ATP, etc.) was described by a component proportional to free Ca2+ with proportionality constant F. In addition, the transport rate of the SR Ca2+-ATPase was modelled for simplicity to be proportional to the free Ca2+ concentration and described by a single rate constant kuptake. kon,T,Ca, koff,T,Ca, F and kuptake were adjusted by least squares fitting of model-calculated free Ca2+ decays to measured decays (for an explanation of the general procedure see also Timmer et al. 1998). After the best fit was obtained, the Ca2+ occupancies of all model compartments (T-sites, P-sites, F-sites (fast binding sites) and uptake) were summed and the release flux calculated as the time derivative of the sum. Model calculations were performed using Euler's method with software written in Delphi (see Schuhmeier & Melzer, 2004, for more details).
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Statistics
Statistical analysis of differences in mean values was performed using Student's t test for two independent populations. For the calculations, the program Origin 7 (Microcal Software Inc., Northampton, MA, USA) was used. Differences were considered statistically significant at P < 0.05.
Results
Responses to single stimuli
Micrographs documenting the morphology of enzymatically dissociated FDB muscle fibres, obtained from animals ranging in age from 7 to 42 days after birth, are shown in Fig. 2A. Figure 2B depicts the corresponding Ca2+ transients recorded at different ages. The signal amplitudes are normalized to highlight the profound changes in the decay time course. The inset of Fig. 2B shows that the time course of the rising phase of the transient also became faster with age.
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A, morphological changes occurring during postnatal development of enzymatically dissociated fibres from mouse flexor digitorum brevis (FDB) muscles. In younger animals, fibres have a smaller diameter and appear to be wavy. Calibration bar: 20 μm. B, MagFluo-4 fluorescence transients measured in fibres of different ages. The amplitudes of the records have been normalized to give a better demonstration of the changes in the decay phase of the transient. Further information is given in Table 1. The inset depicts the same records at a higher time resolution to show the changes in the rising phase of the transients.
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In agreement with previous work (Caputo & Bolaos, 2001; Caputo et al. 2004), in adult fibres ( 42 days), the time course of decay could be described by a double exponential function of the type:
Here, A and B are the amplitudes of the two components that decay with different time constants 1 and 2, respectively.
For adult fibres the values obtained for 1 and 2 were 1.8 and 16.4 ms, while for the 7-day-old animals they were 4.6 and 105 ms, respectively. The table also shows that the amplitude of the transients, expressed in terms of F/F, increased with age. It is noteworthy that the most profound changes occurred later than 10 days after birth. From day 7 to day 10 none of the parameters, with the exception of the slow decay time constant, showed a statistically significant difference.
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Figure 3 shows that the distribution of the values of the time constants 1 and 2 changed with the age of the animal. In the younger animals the values of the fast and slow time constants showed a wide dispersion that diminished with age. The biphasic decay of the Ca2+ transients suggests that two different components contribute to lowering free [Ca2+]i during a twitch. The graph of Fig. 4 shows the relative magnitude of these two components plotted against age of the animal. It can be seen that the fast component () increases with age while the slow component () decreases. Thus, while in 7-day-old animals the relative contributions of the fast and slow components to the decay phase were 22 and 78%, respectively, in adult animals they were 55 and 45%, respectively.
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Histograms showing the distribution of the values of fast and slow time constants of decay of fluorescence transients. In younger animals the values are more widely distributed.
Change with age of the relative contribution of the fast and slow decay components to the falling phase of fluorescence transients. The magnitudes of the two components A and B in eqn (1) were obtained by fitting this function to the transient decay phase.
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Action potentials in developing fibres
In view of the marked difference in the resting membrane potential between young and adult mouse fibres (Ward & Wareham, 1985) it also seemed important to perform measurements of the action potentials to test whether differences in excitation between fibres of young and adult animals might be responsible for differences in the time course of the fluorescence transients. Thus, several experiments were carried out using the potentiometric dye Di-8-ANEPPS to record fluorescence transients associated with membrane action potentials. Figure 5 shows examples of optically recorded action potentials in fibres from 7- (upper row) and 42-day-old animals (lower row). Table 2 summarizes the spike parameters obtained with several fibres of each type. With the exception of the transient half-width, significant differences were found between the parameters of young and adult fibres. In addition younger fibres showed a clear after-hyperpolarization (undershoot) not observed in adult fibres. Adult fibres often showed clear signs of movement artefacts (the two records on the left of Fig. 5). Such artefacts were more conspicuous than in the case of MagFluo-4 transients due to the higher amplification and the averaging procedure used in this case. The movement artefacts could be abolished by blocking fibre contraction with BDM, as shown in the two records on the right. It can also be noticed that the movement artefacts started when the membrane potential had practically regained its resting value and thus did not affect the estimation of action potential parameters. The results indicate that the prolonged Ca2+ transient relaxation in fibres of young animals cannot be explained in terms of a prolongation of the action potential.
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Di-8-ANEPPS fluorescence transients reporting membrane potential changes associated with action potentials in 7- (upper records) and 42-day-old (lower records) mouse fibres. Further details are given in the text and in Table 2.
Responses to tetanic stimulation
The upper records in Fig. 6 show MagFluo-4 responses to 100 Hz stimulation in three fibres from mice of different ages. In younger animals (7 and 10 days), there was a pronounced summation of the individual transients due to their slower decay phases, as shown in Fig. 2. In the fibre from the adult animal there was considerably less summation of the individual transients. More interestingly, the young fibres showed a single exponential decay after the last stimulus, whereas in adult fibres this decay appeared to follow a bi-exponential function, described by eqn (1). For 12 adult fibres the values of the two time constants were estimated to be 25 and 330 ms, respectively. The relative contributions of the fast (A) and slow (B) components to the decay phase of tetani were 64 and 36%, respectively. The graph in Fig. 6 shows that the single time constant of younger animals () changes with age from a value of about 334 ms for the 7-day-old animals to about 70 ms for the 15-day-old animals. Thus, this value tends to approach the value of the fast time constant of the adult animals, 23 ms, represented by the filled circle and marked 1 in the graph. As mentioned above, in fibres from adult animals the decay of the fluorescence transient after tetanic stimulation showed a second and slower decay phase with a time constant of about 300 ms (, labelled 2).
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Upper records in the figure show normalized fluorescence transients in response to a 200 ms, 100 Hz tetanic stimulation of fibres from 7-, 10- and 42-day-old animals. In younger animals the decay phase of the tetanus can be described by a single exponential, while in the adult animals it is better described by two exponentials. The insets next to the tetanic responses show the three first individual transients at a higher time resolution. The graph shows how the time constants of decay of the tetani change with the age of the animals.
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In adult animals, the value of the ratio between the amplitudes of the second and first transients in the tetanus was larger than in younger animals, as it is shown in the insets next to each transient. However, in younger mice we found a large variability between fibres. Whereas in adult animals the ratio varied between 0.51 and 0.67, in the younger animals it varied between 0.23 and 0.63.
Removal model analysis and Ca2+ release calculation
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The kinetics of the fluorescence transients reported by the indicator dye are the result of Ca2+ release, Ca2+ binding to intrinsic sites and transport by the SR Ca2+-ATPase. We performed a calculation of the Ca2+ release flux underlying the Ca2+ transients by using a modified version of the ‘removal model fit’ approach originally developed for voltage-clamped frog fibres (Melzer et al. 1986, 1987). This procedure uses model-calculated Ca2+ transients and least squares optimization of some of the model parameters to make calculated Ca2+ transients consistent with the measured data with regard to the relaxation time interval after the end of release. We used kinetic parameters of troponin C and parvalbumin and a model structure similar to the one applied in a recent comprehensive analysis of action potential-triggered Ca2+ transients in mouse EDL fibres (Baylor & Hollingworth, 2003). We added only one further component to model Ca2+ reuptake to the SR (rate constant kuptake, see Methods). Figure 7A shows measurements of a single Ca2+ transient and a tetanic response obtained in the same fibre. The thick lines show the best simultaneous fit of the kinetic removal model to the single transient and to nine decays within the tetanic response. The fit describes well both the individual time courses and the slowing of the kinetics of decay from the single response to the end of the tetanic response. For four experiments, the mean values (± S.E.M.) of the free fit parameters kon,T,Ca, koff,T,Ca, F and kuptake (see Methods) were 30.4 ± 19.1 μM–1 s–1, 44.2 ± 14.9 s–1, 7.92 ± 2.86 and 295 ± 177 s–1, respectively.
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A, free Ca2+ transients derived from fluorescence recordings using assumptions described in Methods. The recording on the left was induced by a single action potential, the one on the right by a series of spikes at 100 Hz stimulation frequency. Superimposed thick lines show the results of a simultaneous ‘removal model fit’ describing well the observed changes in relaxation kinetics. The fit used kon,T,Ca,koff,T,Ca,F and kuptake as free parameters. Best parameter values obtained after convergence were 3.95 μM–1 s–1, 25.4 s–1, 8.2 and 205 s–1, respectively. B, Ca2+ release flux derived from the removal analysis in A. C, release flux time course in the intervals indicated by horizontal lines at a higher time resolution. Data were smoothed using a local adaptive filter (Schuhmeier et al. 2003).
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Figure 7B shows the calculated Ca2+ release rates derived from the removal analysis in Fig. 7A. During the sequence of 21 individual transients of the tetanic response, peak release flux decreased to about 25% of the initial value of 225 μM ms–1. This fractional change in the Ca2+ release flux amplitude corresponds well to the alterations in the observed amplitudes of the rapid changes in the fluorescence transient, confirming that the decrease in the Ca2+ transient amplitudes results largely from a decrease in the release rate. Figure 7C depicts sections of the release rate traces at an expanded time scale for a better display of the release flux time course. The duration at half-maximum flux was about 1.0 ms at the first pulse and did not change appreciably during the course of the pulse series.
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Recovery from inactivation
The progressive decrease in the amplitude of the individual Ca2+ transients during a 100 Hz tetanus probably results from a fast inactivation of the Ca2+ release mechanism (Schneider & Simon, 1988). In a further series of experiments, we explored recovery from inactivation using a double stimulation procedure as used previously by different authors (Simon & Schneider, 1988; Simon et al. 1991; Jong et al. 1995). Figure 8A demonstrates the double pulse procedure for an adult mouse muscle fibre. Figure 8B shows the superimposed records of a complete run. The fibre was allowed to rest for 20 s between recordings. Finally, the graph in Fig. 8C shows the time course of recovery obtained from several fibres. Each point represents the mean ± S.E.M. of seven experiments. The graph shows the fractional recovery of the fluorescence peak amplitude (F2/F1) plotted against the interval between pulses. The data could be well fitted by a single exponential function with a time constant of 33 ms. In adult fibres, the value of F2(10)/F1, i.e. the fractional amplitude of the second transient (at a 10 ms interval) with respect to the first one, was 0.53. The extrapolated value of the ratio F2/F1 at time zero (F2(0)/F1) was 0.36. When the same type of experiment was performed on fibres from younger animals, a large variability was observed regarding the extent of inactivation caused by the first response. The records in Fig. 9A show examples of this behaviour when fibres from animals of 7, 10, 15 and 42 days, were stimulated with two pulses separated by a 10 ms interval. The records on the left are examples of fibres with a higher degree of inactivation, while those on the right are examples of fibres with lower inactivation. The numbers next to the records indicate the fraction of fibres (percentage) of each type. For the 7-day-old animals 50% of the fibres belonged to each group. With increasing age, however, the percentage value of the first group diminished whereas that of the second group increased, reaching 100% in adult fibres. The histograms in Fig. 9B show the corresponding distribution of the F2(10)/F1 values obtained at each age for several fibres, suggesting the presence of two distinct fibre groups in younger animals. The segregation criterion for the groups was based on the mean F2(10)/F1 value of 0.576 ± 0.099 (mean ± S.D., n = 87) obtained in our laboratory with adult fibres, which were considered to be of the low inactivation type. We considered fibres from young animals exhibiting F2(10)/F1 values in an interval of two standard deviations from this mean value (i.e. between 0.38 and 0.76) to be of the low inactivation type, while fibres showing F2(10)/F1 values lower than 0.38 were considered to belong to the high inactivation type.
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The records in A show fluorescence transients elicited by two action potentials separated by varying stimulus intervals. B shows the superimposed traces of 10 pairs of fluorescence signals. The stimulus intervals varied between 10 and 190 ms. The graph in C summarizes the results obtained with several fibres of adult mice. The graph shows the mean values (± S.E.M.; n = 7) of F2/F1 plotted as a function of the interval between the two stimuli. The mean value of F2/F1 obtained at 10 ms (F2(10)/F1) is 0.53. The value of the intersect at 0 time (F2(0)/F1) is 0.36. The continuous line shows a single exponential function fitted to the experimental points. The curve represents the time course of recovery (repriming) from inactivation of the fluorescence transient associated with the first response. The time constant of repriming was 32.6 ms.
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The records in A were obtained in double-stimulus experiments with a stimulus interval of 10 ms in fibres from 7-, 10-, 15- and 42-day-old animals. The two columns of records show examples of variability in the F2(10)/F1 ratio obtained in fibres of the same age. The histograms in B show the distribution of the values of F2(10)/F1, suggesting the presence of two groups of fibres, one showing characteristics similar to adult fibres.
Finally, Fig. 10 compares the time course of recovery from inactivation in these two types of fibres (filled circles and triangles, respectively) in 7-day-old mice, with that of adult fibres (dashed line). In addition to the difference in the initial fractional inactivation, the time course of recovery is different in the two fibre types, with time constant values of 42.5 ms for the low inactivation type and 28.9 ms for the high inactivation type.
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The symbols represent the mean values (± S.E.M.) obtained in several experiments. The lines show single exponential functions fitted to the experimental data points. Data are shown in comparison with results obtained in adult fibres (dashed line taken from the graph of Fig. 8).
The effect of external Ca2+
The upper records of Fig. 11 show the effect on the fluorescence transients when the fibres were exposed to a medium prepared without Ca2+. In adult fibres the transient amplitude decreased by 12 ± 4% (n = 4) (Fig. 11Aa). Fibres of younger animals showed a larger decrease of 25 ± 7% (n = 7) (Fig. 11Ba). In both cases, no substantial changes in the response time course could be seen, as shown by superimposition of normalized records before and after Ca2+ removal (Fig. 11Ab and Bb). The graph in Fig. 11C shows the effect of 0 Ca2+ on the recovery from inactivation in adult fibres. In this case, there is little or no difference caused by the absence of external Ca2+. Notice that in both cases the F2(10)/F1 value is higher than 0.50. The graph in Fig. 11D shows the results obtained with 7-day-old fibres, belonging to the ‘high inactivation’ type. In this case, fibres in the presence of normal Ca2+ showed a higher degree of inactivation (F2(10)/F1 = 0.30), which considerably diminished in the absence of external Ca2+ (F2(10)/F1 = 0.57).
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The records in Aa and Ba show the effect of 0 Ca2+ in the external medium on the transient amplitude. The same records, normalized to the peak, are shown in Ab and Bb, respectively, to demonstrate that the time course of the transient is not affected by external Ca2+ deprivation. The graphs C and D show the effect of 0 Ca2+ on repriming from inactivation. The graph in C shows the repriming from inactivation in fibres from adult animals obtained in the presence () and absence () of external Ca2+. The graph in D shows repriming curves obtained under the same conditions in fibres of the high-inactivation type of 7-day-old fibres.
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Role of SR Ca2+-ATPase
Figure 12 shows the effect of cyclopiazonic acid (CPA) on the time course of decay of Ca2+ transients at different ages. As shown above for control conditions, the fluorescence transients in all fibres exhibited a fast decay component followed by a slower phase. After poisoning the SR Ca2+-ATPase with CPA, the fluorescence transient of the 7 day fibre depicted in this figure showed a considerably reduced fast decay component, followed by a sustained or plateau phase with a slight secondary rise and no sign of decay for almost 300 ms. The 10 day fibre shows a similar response, but a slow decay starts after the secondary rise. In the 15- and 45-day-old animals the fast component of decay is followed by a slower phase showing that the effect of CPA, although noticeable, diminishes with the age of the animal. For the 15-day-old animals the time constants 1 and 2 showed values of 3.6 ± 0.7 and 41.4 ± 15 in the absence of CPA and values of 11.5 ± 1.3 and 141.8 ± 20 (n = 5) in its presence. In 42-day-old animals the 1 and 2 values were, respectively, 1.5 ± 0.1 and 18.7 ± 2.1 before, and 2.1 ± 0.2 and 78.8 ± 10.5 (n = 8) after, application of CPA.
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Effect of cyclopiazonic acid (CPA) on the time course of fluorescence transients obtained in fibres of 7-, 10-, 15- and 42- (adult) day-old animals.
Discussion
In adult mouse fibres, Ca2+ transients elicited by action potentials and measured with a low affinity Ca2+ indicator dye show a rapid rising phase that leads to a peak in less than 1.5 ms, followed by a decaying phase that can be described by a two-exponential process with time constants of 1.8 and 16 ms (Caputo et al. 2004). Similar properties have been reported by other authors (Hollingworth et al. 1996; Carroll et al. 1997; Baylor & Hollingworth, 2003). In our experiments, the activation of Ca2+ release in adult muscle fibres was probably synchronized all along the fibre length due to the field stimulation and transversally due to the regenerative inward spread of the sodium action potential along the transverse tubules. Due to the rapid repolarization of the action potential, the release mechanism is rapidly deactivated; inactivation may also have contributed to the termination of Ca2+ release and to the early onset of the decay phase (Baylor et al. 1983; Schneider & Simon, 1988; Simon et al. 1991; Jong et al. 1993, 1995; Hollingworth et al. 1996; Caputo & Bolaos, 2002; Baylor & Hollingworth, 2003). Ca2+ binding to intracellular Ca2+ buffers and the SR Ca2+-ATPase contribute in variable amounts to the decay of the transients.
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The results presented here demonstrate that, compared with adult fibres, young mouse fibres show a slower rising phase of the Ca2+ transients and a much slower time course of decay. The experiments carried out with the potentiometric dye Di-8-ANEPPS indicate some differences between action potentials of young (7 days) and adult animals. The action potential amplitudes in adult fibres appear to be about 30% larger than those in younger fibres, which were characterized by a sizeable after-hyperpolarization. The amplitude difference is probably due to a lower value of the resting membrane potential in young fibres expected from the lower K+ and higher Na+ activities in the intracellular space reported by Ward & Wareham (1985). This could cause inactivation of the Na+ channels, and therefore a reduction in the action potential amplitude and a prolongation of its time course, which was also observed as shown in Table 2. The after-hyperpolarization could be due to differences in the expression level of the channels, a larger deviation of the resting potential from the K+ equilibrium potential, or a less-developed transverse tubular system. The amplitude of action potentials in rat myotubes in culture has been reported to be rather small, about 47 mV on day 6 and to increase to 120 mV by day 14 (Bakker et al. 1996). Interestingly most of these myotubes also exhibited pronounced after-hyperpolarizations. The consequences of partial inactivation of the Na+ conductance could be a delayed inward spread of the action potential, which might explain the slower rising phase of Ca2+ transients. Poor development of Ca2+-releasing units in the young animals, due to incomplete structural arrangement of internal membrane systems (Veratti, 1961; Walker & Schrodt, 1968; Franzini-Armstrong, 1991; Flucher et al. 1993; Flucher & Franzini-Armstrong, 1996) might also contribute to this effect. However, it seems clear from our results that the differences observed in the action potentials between younger and older animals is not sufficient to explain the prolonged time course of decay of the Ca2+ transients. This latter effect rather points to a reduced Ca2+ clearance capacity of young fibres. This is supported by the results obtained with the removal analysis carried out for some of the fibres (see below).
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It is known that different elements involved in ECC become functional at different times during development (Franzini-Armstrong, 1991; Flucher et al. 1993). This agrees with our finding that kinetic characteristics develop with different timing in different fibres, even from the same animal. In fact, fibres from younger animals do not behave as a homogeneous population with regard to certain parameters of the fluorescent transients. In particular, the values of the fast and slow time constants of decay showed a broad dispersion that disappeared with age. Also, the double pulse experiments indicated that the extent of inactivation caused by a single transient may vary widely between fibres of young animals. In about half of the 7-day-old fibres the extent of inactivation was similar to that observed in adult fibres, showing a fractional inactivation value of 0.36 estimated for time zero (F2(0)/F1), and of 0.50 when measured after 10 ms (F2(10)/F1). The other half of the fibres showed a higher degree of inactivation, with a F2(0)/F1 value of 0.03, and a F2(10)/F1 value of 0.27.
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One might argue that the stronger inactivation observed in a group of fibres from young animals could be the consequence of fibre damage. However, as shown in Fig. 10 the difference in the extent of inactivation became evident only for the shortest stimulus interval used (i.e. 10 ms) and disappeared at longer recovery periods. Both groups of fibres recovered to the same extent indicating that the high-inactivation fibres exhibit a faster initial recovery. This would not be expected from unhealthy fibres, which in our experience typically show a rapid run-down and finally complete loss of responsiveness to stimuli.
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Our conclusion is that different fibres, even from the same animal, may reach adult characteristics with a different time course. This is compatible with the observation that in rat myotubes in culture a large variation of Ca2+ transients, ranging from small and slow to large and rapid, could be found during the early days of development (Bakker et al. 1996).
It is worthwhile to note that mouse fibres of the high-inactivation type behaved similarly to amphibian muscle fibres in which the corresponding values of F2(0)/F1 and F2(10)/F1 were 0.06 and 0.27, respectively (Caputo et al. 2004). The greater extent of inactivation observed in some of the younger fibres might be linked to a different composition of ryanodine receptor isoforms. Similar to adult amphibian muscle (Murayama & Ogawa, 1992; Franzini-Armstrong & Protasi, 1997; Sorrentino & Reggiani, 1999), isoform RyR3, located in extra-junctional regions of the SR, is expressed in addition to RyR1 in young but not in adult mammalian muscle fibres (Felder & Franzini-Armstrong, 2002). Comparison of mammalian and amphibian muscle fibres suggested the presence of different release mechanisms (Rios & Pizarro, 1991; Shirokova et al. 1996; Ogawa et al. 2000; Murayama & Ogawa, 2002).
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When external Ca2+ was omitted, the response peak amplitude decreased to a greater extent in fibres from young than from adult animals (25% versus 11%). The decrease in 0 Ca2+ is compatible with the demonstration of robust fast Ca2+ currents in younger animals (Beam & Knudson, 1988). When Ca2+ release units have not yet fully developed, Ca2+ influx through sarcolemma Ca2+ channels might contribute to Ca2+ signalling. It has been shown that during in vitro skeletal myogenesis the fraction of contractile activity that depends on the presence of calcium currents diminishes progressively with age while a Ca2+ current-insensitive component becomes predominant. In adult fibres the extent of inactivation of Ca2+ release was not affected by removal of external Ca2+, while in young fibres, of the high-inactivation type, inactivation was reduced to the level of adult fibres. These results might indicate that entry of Ca2+ contributes to the fast inactivation phenomenon.
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Removal analysis and release calculation
As we showed in Fig. 7, the decline in Ca2+ transient amplitude during a 100 Hz tetanus was caused by a corresponding decline in the Ca2+ release flux amplitude. Our approach to calculating release differed from previous detailed analyses in adult mouse muscle fibres (Hollingworth et al. 1996; Baylor & Hollingworth, 2003) by also taking into account the decay of free Ca2+ after turn-off of release. We found that model parameters of Baylor & Hollingworth (2003) had to be altered somewhat to account for the measured kinetics of decay. The main changes concerned fast myoplasmic buffering. Fast buffering was described in the present model (as it was in the model by Baylor & Hollingworth) by a single non-instantaneous and non-linear binding component (T-sites, see Methods and Results) in combination with an instantaneous and linear binding component (F-sites). The rate constants of the T-sites had to be lowered for the fit to describe the data. However, to draw conclusions regarding the kinetics of troponin C in muscle from these results would be premature because different fast sites (troponin, ATP, pump sites, etc.) were lumped into just two simple model components.
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The decay of a single Ca2+ transient could be described by a double exponential process, with a fast initial phase, followed by a slower component. The relative contribution of the two components changed with age in a reciprocal way: although the slow component accounted for most of the decay phase (80%) in fibres of 7-day-old animals, it made up only 45% in adult fibres. Considering single transients of adult animals, the fast component may be caused by Ca2+ binding to intracellular buffers like parvalbumin, as suggested by the increase in its relative contribution with age and in agreement with previous work (Carroll et al. 1997). The slow component may be associated with the operation of the SR Ca2+ pump, as suggested by the experiments in which CPA was used to block the pump.
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Despite the simplicity of the model, the removal analysis shown in Fig. 7 provided an almost perfect fit to the measured Ca2+ transients and described well the kinetic changes in the relaxation time course occurring during repetitive stimulation. In the model, the slowing of relaxation results from both the T- and P-sites, which substantially contribute to the more rapid relaxation in the single Ca2+ transient but become progressively saturated during the course of the tetanic stimulation.
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Kinetic differences of Ca2+ transients in neonatal and adult animals
The differences among Ca2+ transients in specimens of different ages revealed a larger relative contribution of the SR Ca2+ pump to the relaxation kinetics in young fibres. A possible explanation for the different shapes of the Ca2+ transients is therefore the lower expression of myoplasmic Ca2+ buffers in the young fibres. The records of Fig. 13 show the results of a removal model analysis in another set of experiments which support this view.
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Top row: action potential-activated Ca2+ transients recorded from an adult muscle fibre before (A) and after (B) blocking the SR pump with CPA. Superimposed thick lines are the results of a removal model calculation (see Results for explanation). Bottom row: action potential-activated Ca2+ transients recorded from a muscle fibre of a 7-day-old mouse before (C) and after (D) pump block by CPA with corresponding removal model description. Values of the model parameters ([T]tot, [P]tot and kuptake) that were changed to simulate the kinetic differences were as follows. A: 0.240 mM, 1.5 mM, 1188 s–1; B: 0.240 mM, 1.5 mM, 0 s–1; C: 0.120 mM, 0 mM, 364 s–1; D: 0.120 mM, 0 mM, 0 s–1. Fixed model parameters were as follows. F-sites: F = 28.9; T-sites: kon,T,Ca = 9.15 μM–1 s–1, koff,T,Ca = 25.4 s–1; P-sites: kon,P,Ca = 62.6 μM–1 s–1, koff,P,Ca = 0.75 s–1, kon,P,Mg = 0.05 μM–1 s–1, koff,P,Mg = 4.5 s–1, [Mg2+] = 1 mM.
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The traces on the left (Fig. 13A and C) present Ca2+ transients obtained with single action potentials under normal conditions in an adult fibre and in a fibre of a 7-day-old mouse, respectively. The traces on the right (Fig. 13B and D) show the altered transients of the same fibres after blocking the SR Ca2+ pump with CPA. The superimposed thick lines show Ca2+ transient decays calculated with the removal model (see Methods and Fig. 13 legend). The differences in the calculated traces in the four panels result only from changes in the concentration of T- and P-sites and the uptake rate constant (kuptake). The arrows indicate the sequence of the analysis. Starting with the adult fibre and the pump blocked (Fig. 13B), we used the best-fit parameters of the analysis in Fig. 13 as initial values and adjusted kon,T,Ca and F (thus accounting for fibre variability), with kuptake set to zero (assuming complete block), to fit the decay. The resulting set of best-fit parameters was then kept constant and only kuptake adjusted to fit the trace on the left (Fig. 13A). Proceeding from this result, we tried to fit the trace obtained from the young fibre (Fig. 13C) by altering the P-site concentration. However, even complete elimination of the P-sites was not sufficient to model the slower kinetics. A reduction of T-site concentration to 50%, and of kuptake to 31%, led to the fitted trace in (Fig. 13C). The kinetic change from panel C to panel D could then simply be simulated by setting kuptake to zero. The result is compatible with lack of expression of slow parvalbumin-like sites (P-sites) and reduced expression of fast binding sites and pump activity in young fibres compared to adult fibres. Since kuptake varied substantially even among normal adult fibres, the difference in P-site concentration most likely constitutes the decisive factor. Even though this theoretical analysis is based on a simplified model for myoplasmic Ca2+ turnover, it provides a quite plausible explanation for the substantial changes in kinetics that were observed in these experiments.
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In summary, the present study describes Ca2+ transient kinetics in skeletal muscle fibres at different stages of postnatal development. It revealed characteristic changes that are probably linked both to the maturation and reorganization of the Ca2+ release machinery and to the changing expression levels of Ca2+ buffering and transport sites.
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