Mn2+ influx in collagenase-isolated fibres: changes from d0 to d5
Individual quenching rates of Fura-PE3 fluorescence in the presence of external Mn2+ ions are shown in Fig. 1. At d0, the influx remained below 5 % min−1 and no significant difference was detected between normal and mdx fibres. At d1, most of the values remained within the 5 % limits, while a small fraction of the fibres (8/27 and 9/25 for normal and mdx respectively) showed larger and widely dispersed values: from 6.1 to 21.5 % min−1 for normal fibres, and from 5.3 to 95 % min−1 for those from mdx muscles (we checked that this was not due to particularly high levels of Fura-PE3 loading). At d2, most values were again concentrated within the 5 % min−1 limit with only 1/7 (normal) and 1/14 (mdx) showing values around 10–13 % min−1. At d3, the mean values were 2.5 and 5 % min−1 for normal and mdx fibres respectively, a significant difference (P < 0.01). These latter rates are very similar to those reported previously for collagenase-isolated fibres after 3 days of culture (Tutdibi et al. 1999); at this age of culture, these authors never observed fluxes over 10 % min−1 in a set of more than 150 individual observations of both normal and mdx fibres (H. Brinkmeier, personal communication). However, when we extended observations to d4 and d5, the difference of Ca2+ influx between normal and mdx fibres was no longer significant.
The correlation between Mn2+ influx and [Ca2+]i
The cytosolic [Ca2+] was measured for each fibre before the Mn2+ quenching rate was determined (see Methods) in order to assess the relationship between Ca2+ influx (estimated from the Mn2+ influx) and the steady-state value of [Ca2+]i. Among the 132 fibres studied (Fig. 1), 130 individual pairs of values were obtained over the 6 days of study (68 mdx, 62 normal). They are displayed in Fig. 2A, without reference to the day of observation. The largest fraction of the results (116/130) is clustered within 0–10 % min−1 quenching rate and 25–85 nm[Ca2+]i, as extreme limits. Results from normal and mdx fibres overlap. This densely populated cluster at the far left of Fig. 2A is presented as an expanded view in Fig. 2B. The whole set of data (Fig. 2A) exhibits an extremely skewed distribution, rendering calculation of arithmetic means inappropriate. Instead, the medians and the 25–75 percentile ranges were determined for both the quenching rates and the cytosolic [Ca2+] as reported in Table 1. Medians were compared by a non-parametric statistical analysis (Mann-Whitney Rank sum test). Although the median of the quenching rate was significantly larger in mdx fibres (P < 0.01), the median of cytosolic [Ca2+] did not differ from normal (P/ 0.2). Outside the main cluster of results, a small fraction of the fibres (12/130) showed both higher [Ca2+]i values in the 100–140 nm range and a wide dispersion of the influx rate from > 5 to 95 % min−1. All but one of the results of this second fraction were obtained at d1. Here, results from normal and mdx no longer overlap, due to the much higher values for Mn2+ influx in four mdx fibres (as already seen in Fig. 1). Notwithstanding the very high influx values in some mdx fibres, [Ca2+]i values in this group were not different from those obtained with the normal fibres of this high Mn2+ influx fraction and remained within the 100–140 nm range, without reaching the contraction threshold, for all fibres were relaxed with visible striations at the time of measurement. We checked that these results did not come from the same animal. Moreover, the four fibres showing high Mn2+ influx rates came from different cultures each of which also contained fibres having quenching rates as low as 2 % min−1. Thus, the dispersion of the results reflected the heterogeneity of the fibre population and not the differences among animals.
The relationship between the Ca2+ influx rate and the cytosolic [Ca2+] was further studied by artificially increasing the Ca2+ influx with low concentrations of the Ca2+ ionophore 4-bromo A23187. We restricted our study to fibres cultured for 2 and 3 days, when those fibres with elevated influx had been naturally eliminated (see below). As seen in Fig. 3, we obtained influx rates ranging from 2 to 44 % min−1 while [Ca2+]i remained within the 30–78 nm limits, with no difference between normal and mdx fibres. The results of Fig. 3 show that, within the duration of the experiment (< 120 min), the ‘robustness’ of the cytosolic Ca2+ homeostasis in response to a large increase of Ca2+ influx is as good in mdx fibres as in normal ones, even when this influx was up to 10-fold above naturally occurring values.
Figure 3. Correlation between Mn2+ quenching rate and cytosolic [Ca2+] in the presence of 4-bromo-A23187
Open symbols, normal fibres; filled symbols, mdx fibres. Key: day of measurement. Fibres were obtained from 11 normal and 3 mdx mice. A23187 concentrations: 25–75 nm.
Download figure to PowerPoint
Fibre survival and Mn2+ influx
The appearance of some fairly high values of Mn2+ influx at d1, indicated that abnormally high Ca2+ influx was occurring for some mdx and normal fibres. This prompted us to examine the survival of the fibres. However, for technical reasons, it was impossible to follow the fate of individual fibres where Mn2+ influx had been measured. We thus had to rely on a study of the survival of a population of fibres. Six cultures were run in parallel and the number of remaining fibres were counted from d0 to d3. Only well elongated fibres (thus maintaining the resting state), showing no structural alterations, were counted. As shown in Fig. 4A, mdx fibres show a 10 % decline in number from d0 to d1, followed by a sharper decline such that survival was reduced to 51 %, at d2. This rate of decline subsequently slowed. In contrast, normal fibres showed a steady decline of 10–12 % per day (Fig. 4A, dotted line) so that survival at d2 was still 78 %. Thus, taking the loss rate of normal fibres as reference, mdx fibres showed an excess loss of 27 % (i.e. the difference between 78 and 51 %) from d0 to d2. For normal fibres, survival declined smoothly in spite of the fact that a fraction of the fibres (8/27) displayed, at d1, values of Ca2+ influx in the > 5 to 21 % min−1 range. In Fig. 4A and B, statistical significance (marked *) concerns the difference between mdx and normal fibres.
To see if the sharp decrease of survival of mdx fibres from d1 to d2 could be attenuated, fibres were isolated and maintained in a medium containing one-tenth of the normal [Ca2+]o of the culture medium, i.e. 0.08 mm, assuming that the reduction of [Ca2+]o would similarly reduce the influx of Ca2+. Under these conditions, survival of mdx fibres decreased regularly from d0 to d3, as seen in Fig. 4B, without any sharp decrease between d1 and d2. The excess of mdx fibre death from d0 to d2 was now reduced from 27 % (at 0.8 mm[Ca2+]o) to 10 %. For normal fibres, the reduction of external [Ca2+] did not affect the survival time course. To facilitate the comparison between the 0.8 and 0.08 mm Ca2+ conditions, data from d1, d2 and d3 of Fig. 4A and B were compiled in Fig. 4C where the statistical analysis now concerns differences of survival in the two [Ca2+]o media.
On return to normal [Ca2+]o, neither type of fibre showed any sudden significant change of survival rate. Both survival profiles from d0 to d4 approximate to a smooth exponential decay, with time constants of 225 for normal and 147 h for mdx fibres. Thus, even at low [Ca2+]o, elimination of mdx fibres proceeded about 1.5 times faster than for normal fibres. When comparing survival at d2 in Fig. 4A and B, it is clear that a fraction of cell death (≈one-third) observed in 0.8 mm[Ca2+]o also occurred at 0.08 mm when the size of the Ca2+ entry was reduced proportionately. From d4 to d5, however, no further significant decrease of survival could be detected for either type of fibre.
Voltage-independent Ca2+ channels: characteristics and progression from d0 to d5
Channel activity was observed on collagenase-isolated FDB fibres, by the patch clamp technique (cell-attached configuration) at holding potential of −60 mV (Fig. 5A). Results displayed in Table 2 give the single-channel conductance and the reversal potential for different solutions within the patch pipette. The I-V relationship is strictly ohmic, showing no sign of voltage activation or inactivation and is identical in mdx and normal fibres (not shown). Cationic selectivity is poor and allows fluxes of Ca2+, Ba2+, Mn2+ and Na+ (and most likely K+ from within the cell). The estimated PCa/PNa ratio (Lee & Tsien, 1984) is around 0.7. Moreover, the open probability is greatly reduced by trivalent cations (e.g. 50 μM La3+). Open times showed a Poisson-like distribution, approximating to an exponential decay, the time constant of which is also reported in Table 2. These characteristics bear strong resemblance to those of the mechanosensitive channels already described in similar preparations (Franco-Obregón & Lansman, 1994). Indeed, when gentle suction was applied to the patches, we observed, in all fibres tested (10 mdx and 10 C57), a significant increase of the mean open probabilities which amounted to 69 ± 8 % and 53 ± 8 % (s.e.m.), for mdx and C57 fibres respectively (with no significant difference between mouse lines, P/ 0.7, t test). Interestingly, the mechanosensitivity of these channels was present in the absence of dystrophin. All these gating properties were identical in normal and mdx fibres. Moreover, we observed that they remained unchanged from d0 to d5.
In contrast with this stability, the occurrence of these channels and the open probabilities are different between normal and mdx fibres and showed typical patterns of change from d0 to d5. Channel occurrence was estimated as the percentage of the fibres that, when sampled by the patch pipette, show channel activity (each fibre was sampled only once). Soon after isolation, channel activity was recorded in 80 % of the fibres, both in normal and mdx fibres. As shown in Fig. 5B, the occurrence steadily decreased from d0 to d5, but the decrease was always less marked for mdx fibres. Eventually, by d5, occurrence had declined to 20 and 32 % for normal and mdx fibres, respectively.
Figure 5C gives the change in the average channel open probabilities from d0 to d5. At d0, the values for normal and mdx fibres are very similar to that reported previously, with a significant difference between the two types of fibres. Surprisingly, for both types of fibres, the average open probability nearly doubled at d1 and recovered the d0 values by d3, remaining stable thereafter. At d5, the average value of mdx fibre was about twice the value for normal fibres (P < 0.05). These transient changes are still better illustrated by the histograms of the open probabilities from d0 to d5 (Fig. 6). At d0, the values did not exceed 0.1, while at d1, values for mdx fibres spread up to 0.27. Gradually both distributions narrowed again and recovered their d0 patterns by d5.
Figure 6. Pattern of change from d0 to d5 of the distribution of the open probabilities of voltage-insensitive Ca2+ channels
Open columns, normal fibres; filled columns, mdx fibres.
Download figure to PowerPoint
By integration of the current records over the 120 s of observation, the quantity of charge (pA s) passing through the membrane patch was calculated, when using a patch pipette filled with 110 mm CaCl2. As seen in Fig. 5D, a large increase at d1 followed by a progressive return towards the d0 values was observed for both types of fibre. For each day, values for mdx fibres were about twice those for normal fibres, except at d1 when the mean quantity of charge was tripled in mdx.
In spite of the large difference in experimental conditions, estimates of Ca2+ influx by either the Mn2+ fluorescence quenching technique (Fig. 1) or by electrical measurements with a Ca2+ filled patch pipette (Fig. 5 and Fig. 6) provide remarkably consistent information on three points: (1) a large increase of Ca2+ influx had developed at d1; (2) this was accompanied by large fibre-to-fibre differences, over the same d1-d2 period; (3) a progressive return towards the low d0 values took place within the next 2 days (d2-d4). However, in contrast with the measurements of charges passing through the patch pipettes which were systematically higher in mdx fibres (Fig. 5D), estimation of Ca2+ influx by the Mn2+ quenching rate detected no difference at d0, d4 and d5 (Fig. 1). We think that the latter measurements better reflect the physiological situation since they were obtained in near-physiological conditions (in the presence of 140 mm Na+), while the patch technique artificially amplified the Ca2+ influx, as pipettes were filled with 110 mm Ca2+.