Mitochondrial and myoplasmic [Ca2+] in single fibres from mouse limb muscles during repeated tetanic contractions

Abstract

Previous studies on single fast-twitch fibres from mouse toe muscles have shown marked fatigue-induced changes in the free myoplasmic [Ca2+] ([Ca2+]i), while mitochondrial [Ca2+] remained unchanged. We have now investigated whether muscle fibres from the legs of mice respond in a similar way. Intact, single fibres were dissected from the soleus and extensor digitorum longus (EDL) muscles of adult mice. To measure [Ca2+]i, indo-1 was injected into the isolated fibres. Mitochondrial [Ca2+] was measured using Rhod-2 and confocal laser microscopy. Fatigue was induced by up to 1000 tetanic contractions (70 Hz) given at 2 s intervals. In soleus fibres, there was no significant decrease in tetanic [Ca2+]i at the end of the fatiguing stimulation, whereas tetanic force was significantly reduced by about 30 %. In 10 out of 14 soleus fibres loaded with Rhod-2 and subjected to fatigue, mitochondrial [Ca2+] increased to a maximum after about 50 tetani; this increase was fully reversed within 20 min after the end of stimulation. The force-frequency curve of the non-responding soleus fibres was shifted to higher frequencies compared to that of the responding fibres. In addition, eight out of nine Rhod-2-loaded EDL fibres showed similar changes in mitochondrial [Ca2+] during and after a period of fatiguing stimulation. The stimulation-induced increase in mitochondrial [Ca2+] was reduced when mitochondria were depolarised by application of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, whereas it was increased by application of an inhibitor of the mitochondrial Na+/Ca2+ exchange (CGP-37157). In conclusion, isolated slow-twitch muscle fibres show only modest changes in tetanic force and [Ca2+]i during repeated contractions. The increase in mitochondrial Ca2+ does not appear to be essential for activation of mitochondrial ATP production, nor does it cause muscle damage.

Figure 1. Typical examples of dual labelling of mitochondria in mouse skeletal muscle fibres (A), the changes in mitochondrial [Ca²⁺] reported by Rhod-2 during a long tetanus (B) and the increase in mitochondrial [Ca²⁺] following localised damage to the sarcolemma (C)

A, R123 (left) and Rhod-2 (right) staining of the subsarcolemmal mitochondria in a soleus fibre before (Con), at the end of 10 tetani (10T) and at 1, 2 and 3 min afterwards (1′, 2′ and 3′, respectively). Mitochondria are observed organised in rows parallel to the long axis of the fibre; the dark oval at the left is a nucleus (length along the major axis 27 mm). B, transverse line scan in a 38 mm diameter soleus fibre before, during and after a 4.4 s, 70 Hz tetanus (indicated by the white bar below the fibre). Time runs from left to right. For ease of identification, the fibre was stimulated with five tetani 30 s before in order to cause a measurable signal in the mitochondria. It can be seen that there was no generalised rise in the myoplasmic fluorescence during the tetanus. C, the same area of a 40 mm soleus fibre loaded with Rhod-2 that did not take up Ca²⁺ in response to tetanic stimulation in images obtained before (Con) and 15 min after (15′ post) local damage was induced. Intense fluorescence can be seen in the damaged area, while less intense fluorescence occurred in the bands of mitochondria that are located more centrally.

Figure 2. Tetanic [Ca²⁺]_i and force changes before, during and after fatigue

Typical [Ca²⁺]_i and force transients recorded from a single soleus fibre injected with indo-1 during stimulation with 1000 tetani at 2 s intervals. The numbers above the traces refer to the number of that tetanus in the series. Note that the [Ca²⁺]_i and force records show little change over the series of tetani.

Figure 3. Plateau [Ca²⁺]_i and peak force during a series of 1000 tetanic contractions

Mean values (± S.E.M.) for tetanic [Ca²⁺]_i (A) and relative force (B) from seven fibres during a series of 1000 tetani. Note the break in the axis between the values for 200 and 500 tetani. *Significantly different from value at time 0, P < 0.05. C, the mean force-[Ca²⁺] curve from six fibres together with the mean data points measured during the first (First) and the last (Last) tetani.

Figure 4. The rates of relaxation of [Ca²⁺]_i and force transients are similar in the first and last tetani

Typical examples of the first (continuous line) and the last (dotted line) tetanic [Ca²⁺]_i (top) and force transients (bottom) in a series of 1000 tetani, superimposed to allow comparison. Note that the decay of [Ca²⁺]_i showed no slowing in the last tetanus. Note also that the last force transient showed a slower rate of force development than the control, while the rate of force relaxation was virtually unchanged in the last compared to the first tetanus.

Figure 5. Typical example of the changes in mitochondrial [Ca²⁺] in a soleus fibre during and after a series of repeated tetani

Image of the mitochondrial Rhod-2 fluorescence before (Con), during a series of 1000 tetani (10T to Last) and for 30 min after (1′ to 30′). The mitochondrial [Ca²⁺] is clearly increased after 10 tetani and reaches its maximum at 50 or 100 tetani. The mitochondrial [Ca²⁺] falls rapidly after the end of stimulation. Fibre diameter is 45 μm. The coloured bar at the bottom right indicates the level of Ca²⁺, with blue representing low [Ca²⁺] and red high [Ca²⁺].

Figure 6. Mean change in mitochondrial [Ca²⁺] before, during and after a series of repeated tetani

A, mean data (± S.E.M.) from 10 soleus fibres (•) and eight EDL fibres (▵) showing the changes in the mitochondrial Rhod-2 F/F₀ during and for 30 min after a series of up to 1000 tetani. The mitochondrial [Ca²⁺] is clearly increased after 10 tetani, reaches its maximum at 50 tetani, and thereafter falls steadily during the remaining period of stimulation. B, after stimulation was stopped, the mitochondrial [Ca²⁺] falls relatively rapidly, with a decline to 50 % of its final value within 3 min.

Figure 7. Force-frequency curves of soleus and EDL fibres

Force-frequency curves for those soleus fibres in which mitochondrial [Ca²⁺] increased as a result of tetanic stimulation (•, n = 10) and those that did not (□, n = 4) are shown together with the force-frequency curve of the EDL fibres in which mitochondria took up Ca²⁺ (▵, n = 8). Values shown are means ± S.E.M.

Figure 8. Typical example of the changes in mitochondrial [Ca²⁺] in an EDL fibre during and after a series of repeated tetani

Image of the mitochondrial Rhod-2 fluorescence before (Con), during a series of 192 tetani (10T to Last) and for 10 min after (1′ to 10′). The mitochondrial [Ca²⁺] is clearly increased after 10 tetani and reaches its maximum after 50 tetani. The mitochondrial [Ca²⁺] falls rapidly after the end of stimulation. Fibre diameter is 39 μm. The coloured bar in the bottom right indicates the level of Ca²⁺, with blue representing low [Ca²⁺] and red high [Ca²⁺].

Figure 9. Elevated mitochondrial [Ca²⁺] was not associated with impaired tetanic force production

Plot of the peak mitochondrial Rhod-2 F/F₀versus the final force recorded in the individual soleus (•) and EDL (▵). Each symbol represents one fibre. The continuous line is a line fitted to the soleus data by least squares regression, whilst the dotted line is a similar line fitted to the EDL data.

Figure 10. Repeated series of 25 tetani in the absence of drugs induce identical changes in mitochondrial [Ca²⁺]

A, changes in the mitochondrial Rhod-2 fluorescence (F/F₀) during a first series of 25 tetani (solid line, filled circles) and 45 min later during a second series of 25 tetani (dotted line, open circles). The two curves are virtually superimposable. Values are mean ± S.E.M. of three fibres. B, changes in mitochondrial Rhod-2 fluorescence during a second series of 25 tetani compared to the first series. Values are mean ± S.E.M. in the absence of any drug (CON, n = 3), in the presence of 0.5 μM FCCP (n = 5), 10 μM CGP (n = 5) or 4 μM CyA (n = 6). *Significantly different from the CON value, p < 0.05, paired t test.