Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice

Abstract

1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.

Figure 1. Changes in membrane potential recorded in response to voltage-clamp depolarizations

The noisy traces correspond to the changes in membrane potential, recorded with a single microelectrode, in response to 20 ms voltage-clamp command steps (superimposed continuous lines) of 20, -20 and -40 mV applied to the skeletal muscle fibre with the patch-clamp amplifier. The inset shows the same records after scaling by the corresponding change in command voltage.

Figure 2. Initial resting [Ca²⁺]_i in control and mdx muscle fibres

The top panels show the individual values of initial resting [Ca²⁺]_i measured in 72 control (left) and 57 mdx fibres (right). [Ca²⁺]_i values are plotted versus the age of the mice from which muscle fibres were isolated. The bottom panels show the corresponding mean and s.e.m. values of resting [Ca²⁺]_i from mice of similar ages; these values were obtained from averaging data points over a period arbitrarily set to every consecutive 10 weeks. The number of fibres is indicated above each corresponding bar.

Figure 3. Indo-1 calcium transients elicited by short depolarizing pulses in a control and in an mdx fibre exhibiting a similar resting [Ca²⁺]_i

A, indo-1 percentage saturation traces from a control (left) and an mdx fibre (right) in response to 5, 15 and 25 ms duration command pulses to 0 mV (V_c, bottom traces). The control and the mdx fibre were from a 13- and a 7-week-old mouse, respectively. B, [Ca²⁺]_i transients calculated from the above corresponding indo-1 percentage saturation traces. For clarity, [Ca²⁺]_i traces elicited by pulses of increasing duration are shifted in the x and y direction. The smooth curves superimposed on the decay of the [Ca²⁺]_i traces correspond to the results from fitting a single exponential plus constant function. The value of the corresponding time constant is indicated next to each trace.

Figure 4. Average properties of indo-1 calcium transients elicited by short depolarizing pulses in a batch of control and mdx muscle fibres exhibiting similar resting [Ca²⁺]_i levels

A, mean (continuous trace) ±s.e.m. (vertical lines) indo-1 percentage saturation traces elicited by 5 and 15 ms depolarizing pulses at 0 mV in 10 control (left) and 7 mdx fibres (right). B, from top to bottom, mean ±s.e.m. values of peak [Ca²⁺]_i, time constant of [Ca²⁺]_i decay and final [Ca²⁺]_i level measured from the individual control and mdx[Ca²⁺]_i traces on which exponential fits were performed. The number of fibres used for each pulse duration is indicated at the top of B.

Figure 5. Indo-1 calcium transients elicited by 50 ms depolarizing pulses of increasing amplitude in a control and in an mdx fibre exhibiting a similar resting [Ca²⁺]_i

A, indo-1 percentage saturation traces from a control (left) and an mdx fibre (right) in response to depolarizing command steps at -40, -30, -20, -10, 0 and +10 mV (V_c, bottom traces). The control and the mdx fibre were from a 13- and an 11-week-old mouse, respectively. B, [Ca²⁺]_i transients calculated from the above corresponding indo-1 percentage saturation traces; traces are displayed for pulses of increasing amplitude from left to right. C, mean ±s.e.m. indo-1 peak percentage saturation measured in response to 50 ms depolarizations at various command potentials. •, control fibres (n= 14); ^, mdx fibres (n= 6).

Figure 6. Indo-1 calcium transients elicited by depolarizing pulses at 0 mV of increasing duration in a control and in an mdx fibre from a batch of mice older than ≈35 weeks

The mdx fibres from this batch displayed a significantly lower mean resting [Ca²⁺]_i than control ones. A, a series of indo-1 percentage saturation traces from a control (left) and an mdx fibre (right) in response to 5, 15 and 45 ms duration command pulses at 0 mV. The control and mdx fibre were from a 50- and a 41-week-old mouse, respectively. B, [Ca²⁺]_i transients calculated from the above corresponding indo-1 percentage saturation traces. The smooth curves superimposed on the decay of the [Ca²⁺]_i traces correspond to the results from fitting a single exponential plus constant function. The value of the corresponding time constant is indicated next to each trace.

Figure 7. Average properties of indo-1 calcium transients elicited by depolarizing pulses of increasing duration in fibres from a batch of control and mdx mice older than ≈35 weeks

A, mean (continuous trace) ±s.e.m. (vertical lines) indo-1 percentage saturation traces elicited by 5, 15, 25, 35 and 45 ms depolarizing pulses at 0 mV in 13 control (left) and 11 mdx fibres (right). B, from top to bottom, mean ±s.e.m. values of peak [Ca²⁺]_i, time constant of [Ca²⁺]_i decay and final [Ca²⁺]_i level, measured from the individual control and mdx[Ca²⁺]_i traces on which exponential fits were performed. The number of fibres used for each pulse duration is indicated next to the peak [Ca²⁺]_i data points in B.

Figure 8. Indo-1 calcium transients elicited by 50 ms depolarizing pulses of increasing amplitude in control and in mdx fibres from a batch of mice older than ≈35 weeks

A, indo-1 percentage saturation traces from a control (left) and an mdx fibre (right) in response to depolarizing command steps at -40, -30, -20, -10, 0 and +10 mV. The control and mdx fibre were from a 50- and a 38-week-old mouse, respectively. B, mean ±s.e.m. indo-1 peak percentage saturation measured in response to 50 ms depolarizations at various command potentials (V_c). •, control fibres (n= 11); ^, mdx fibres (n= 11).