Calcium transients in developing mouse skeletal muscle fibres

Abstract

Ca(2)(+) transients elicited by action potentials were measured using MagFluo-4, at 20-22 degrees 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, F(2(10))/F(1), 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 Ca(2)(+) 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 Ca(2)(+)-free solutions with a larger decrease in the transient peak amplitude (25% versus 11% in adult fibres), possibly indicating a contribution of Ca(2)(+) influx to the Ca(2)(+) transient in younger animals. Cyclopiazonic acid (1 mum), an inhibitor of the sarcoplasmic reticulum (SR) Ca(2)(+)-ATPase, abolished the Ca(2)(+) 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 Ca(2)(+) removal model showed that the results are consistent with a larger relative contribution of the SR Ca(2)(+) pump and a lower expression of myoplasmic Ca(2)(+) buffers in fibres of young versus old animals.

Figure 1. Movement artefacts in MagFluo-4 transients

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.

Figure 2. Developmental changes in morphology and kinetics

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.

Figure 3. Change of decay of fluorescence transients with age

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.

Figure 4. Contribution of fast and slow components to Ca²⁺ clearance at different age

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.

Figure 5. Action potentials in 7- and 42-day-old mice

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.

Figure 6. Time course of tetanic fluorescence transients

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.

Figure 7. Ca²⁺ removal fit analysis and calculation of Ca²⁺ release flux

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 k_on,T,Ca,k_off,T,Ca,F and k_uptake 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).

Figure 8. Repriming from inactivation in adult mouse fibres

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.

Figure 9. Variability of the F₂₍₁₀₎/F₁ ratio in fibres from young animals

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.

Figure 10. Time course of repriming from inactivation in low- (▴) and high-inactivation fibres (•) of 7-day-old animals

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).

Figure 11. Effect of external Ca²⁺ removal on fluorescence transients obtained in fibres of 42- and 7-day-old mice

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.

Figure 12. Ca²⁺ inhibition

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.

Figure 13. Modelling of developmental changes and CPA effects on Ca²⁺ transients

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 k_uptake) 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: k_on,T,Ca = 9.15 μm⁻¹ s⁻¹, k_off,T,Ca = 25.4 s⁻¹; P-sites: k_on,P,Ca = 62.6 μm⁻¹ s⁻¹, k_off,P,Ca = 0.75 s⁻¹, k_on,P,Mg = 0.05 μm⁻¹ s⁻¹, k_off,P,Mg = 4.5 s⁻¹, [Mg²⁺]= 1 mm.