INTRODUCTION

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ELECTRICAL STIMULATION of skeletal muscles in vivo has been shown to lead to a progressive net uptake of Ca²⁺ in rabbits (36) and in rats (20). A recent study showed that also in vitro, tetanic contractions were associated with an increase in Ca²⁺ content of hamster diaphragm (23).

This excitation-induced net accumulation of Ca²⁺ is considerable and is only reversed slowly (20). Early studies on frog sartorius muscle demonstrated that excitation is associated with an influx of Ca²⁺ (5), but little is known about the mechanisms involved and the long-term effects of this Ca²⁺ uptake. Because the transport capacity of the Ca²⁺-ATPase of the sarcoplasmic reticulum (SR) exceeds that of the Ca²⁺-ATPase in the sarcolemma by far (7), Ca²⁺ ions entering the cytoplasm are likely to be trapped in the SR rather than to be reextruded from the cell across the sarcolemma. In addition, in skeletal muscle, sarcolemmal Na⁺/Ca²⁺ exchange seems to be of minor importance for the net extrusion of Ca²⁺ at low cytoplasmic Ca²⁺ (16, 24).

Cellular damage arising from various types of contractile activity has often been attributed to increased cytoplasmic Ca²⁺ (17, 25). We therefore found it important to explore possible mechanisms for the excitation-induced accumulation of Ca²⁺ in muscle. We have characterized the effects of electrical stimulation on the uptake of ⁴⁵Ca and the net accumulation of Ca²⁺ in isolated rat soleus and extensor digitorum longus (EDL) muscles, typical examples of predominantly slow-twitch and fast-twitch muscles, respectively. We find that electrical stimulation induces a marked increase in the initial rate of ⁴⁵Ca uptake. A major fraction of this increase seems to be mediated by Na⁺ channels. Part of these results have been presented in a preliminary form (33).

	MATERIALS AND METHODS

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Animals. All experiments were carried out using fed female and male Wistar rats weighing 60-70 g (4 wk old). The animals had free access to food and water and were kept in a thermostated environment (21°C) with constant day length (12 h). Animals of this size were chosen to obtain muscles of sufficiently small size (20-25 mg) to ensure adequate oxygenation and diffusion of substrate during incubation.

Muscle preparation and incubation. The animals were killed by decapitation, and intact soleus or EDL muscles were dissected out as previously described (8, 27). The standard incubation medium was a Krebs-Ringer bicarbonate buffer (pH 7.4) containing (in mM) 120.2 NaCl, 25.1 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂, and 5 or 10 D-glucose. Incubations took place at 30°C under continuous bubbling with a mixture of 95% O₂ and 5% CO₂ in a volume of 2-4 ml. After preparation, the muscles were equilibrated in the standard medium for 30-60 min before further incubation. This procedure has been shown to allow the maintenance of constant membrane potential, K⁺, Na⁺, and Ca²⁺ contents for several hours in vitro (12, 19).

Electrical stimulation. An experimental setup allowing for the simultaneous direct stimulation of 12 muscles was used. Each muscle was placed between two platinum electrodes surrounding the central part of the muscle. The muscles were either mounted at resting length so as to allow isometric contractions or allowed to shorten in an isotonic contraction without any load. Single pulses with an amplitude of 10 V and a duration of 0.1 or 1.0 ms were used. Frequency, amplitude, and pulse duration were checked with a HAMEG HM 207 oscilloscope.

⁴⁵Ca uptake. This was measured essentially as earlier described (10, 19). After equilibration in unlabeled buffer the muscles were incubated in buffer containing ⁴⁵Ca (0.1-6.0 µCi/ml) for 5-240 min, depending on the experiment. Stimulation either occurred throughout the entire incubation period or during the last 1 min. In some experiments this was followed by four consecutive washes (each lasting 30 min) at 0°C in 2-3 ml Ca²⁺-free Krebs-Ringer bicarbonate buffer containing 0.5 mM EGTA to remove extracellular Ca²⁺. Control experiments were conducted to estimate the amount of Ca²⁺ lost from the intracellular compartment during washout. In these experiments the washout time was 240 min. For more details, see legend to Fig. 3 and RESULTS.

Finally, the muscles were blotted, weighed, and homogenized in 3 ml 0.3 M TCA using an ULTRA-TURRAX (T25) tissue homogenizer. ⁴⁵Ca activity was measured by liquid scintillation counting (Packard, Tri-Carb 2100 TR) of the clear supernatant obtained after centrifugation (2,800 g, 10 min).

Dual label experiments. These were performed as earlier described (10). The muscles were incubated with ⁴⁵Ca (1 µCi/ml) and ²²Na (1 µCi/ml) for 5 min at rest or stimulated at 60 Hz (5-20 s each min). This was followed by washout at 0°C for 4 × 15 min in 2 ml Na⁺-Ca²⁺-free Tris sucrose buffer containing 0.5 mM EGTA. After incubation, the muscles were blotted, weighed, and homogenized in 3 ml 0.3 M TCA. ⁴⁵Ca and ²²Na activity was measured by liquid scintillation counting of the clear supernatant obtained after centrifugation (2,800 g, 10 min), with channels set for the separation of ⁴⁵Ca and ²²Na activity.

Ca²⁺ contents. Muscles weighing 20-25 mg were homogenized in 3 ml 0.3 M TCA and centrifuged (2,800 g, 10 min). Ca²⁺ was determined by atomic absorption spectrophotometry (Philips PU 9200, Pye Unicam, Cambridge, UK) using 1.5 ml of the clear supernatant after addition of KCl to a final concentration of 2.4 mM. The muscle extracts were measured against a blank and standards (12.5, 25, and 50 µM CaCl₂) containing the same amount of TCA and KCl.

Na⁺ contents. Muscles weighing 20-25 mg were homogenized in 3 ml 0.3 M TCA and centrifuged (2,800 g, 10 min). The Na⁺ content of the clear supernatant was determined using a Radiometer FLM3 flame photometer (Copenhagen, Denmark) with lithium as internal standard.

Force development. In control experiments the effects on contractility of Ni²⁺, Gd³⁺, and long-term low-frequency stimulation were tested using force transducers as previously described in detail (11).

Chemicals and isotopes. All chemicals used were of analytic grade. Carbachol, gadolinium chloride (GdCl₃), TTX, tubocurarine, ouabain, nifedipine, verapamil, and veratridine were purchased from Sigma Chemical (St. Louis, MO). Nickel chloride (NiCl₂) was from Merck (Whitehouse Station, NJ). ⁴⁵Ca (0.59 Ci/mmol Ca) was from the Danish Atomic Energy Commission (Risø, Denmark), and ²²Na (4.1 Ci/mmol Ca) was from Amersham International (Aylesbury, Bucks, UK).

Statistics. Results are given as mean values ± SE. The statistical significance of any difference between two groups was ascertained using the two-tailed t-test for nonpaired observations. Regression analysis was performed using the method of least squares.

	RESULTS

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Long-term stimulation. Isolated soleus and EDL muscles were mounted for isometric contractions and stimulated directly at 1.0 Hz (1.0-ms pulses) for 120 or 240 min. The maintenance of excitability was examined in long-term stimulation experiments (1 Hz for 240 min) using a force-displacement transducer. Force development declined during this long period, but even during the last 60 min of stimulation, soleus and EDL muscles maintained 72 and 36% of the initial force, respectively, indicating that at least corresponding fractions of the muscle fiber population were excitable. As shown in Fig. 1 electrical stimulation induced a significant increase in the uptake of ⁴⁵Ca. In soleus, the stimulation induced an increase of 23 (P < 0.01) and 30% (P < 0.001) after 120 and 240 min, respectively. In EDL, however, the stimulation induced an increase in ⁴⁵Ca uptake that was around fivefold larger than that of soleus (111 and 154% after 120 and 240 min, respectively; P < 0.001).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of electrical stimulation and isometric mounting on the uptake of 45Ca in soleus and extensor digitorum longus (EDL) muscles. Muscles were mounted on electrodes at resting length, so as to allow isometric contractions, and incubated for 120 or 240 min in buffer containing 45Ca (0.1 µCi/ml). Muscles were either resting or stimulated at 1 Hz (10 V, 1.0 ms) during the entire incubation period. After incubation the muscles were blotted, weighed, and homogenized in 3 ml 0.3 M TCA. After centrifugation, the 45Ca activity of the clear supernatant was determined and, on the basis of the specific activity of the medium, the uptake was expressed as µmol/g wet wt. , EDL stimulated; , EDL resting; , soleus stimulated; , soleus resting. All the stimulated groups differ significantly from resting controls (P < 0.01). Mean values ± SE are shown (n = 6 muscles).

In the same experiment, the increase in total Ca²⁺ content was measured. In soleus, stimulation resulted in a slight but nonsignificant increase in the Ca²⁺ content from 1.16 ± 0.08 (resting controls) to 1.30 ± 0.04 µmol/g wet wt after 240 min of stimulation. In EDL, on the other hand, 240 min of stimulation resulted in a large and highly significant increase in total Ca²⁺ content from 1.41 ± 0.04 to 2.61 ± 0.19 µmol/g wet wt (P < 0.001). The net gain in total Ca²⁺ content of EDL muscles (1.20 µmol/g wet wt) was not significantly different from the increase in ⁴⁵Ca uptake (1.26 µmol/g wet wt).

For comparison, similar experiments were performed using soleus and EDL muscles mounted without any attachment so as to allow isotonic contraction without any load. Under these conditions electrical stimulation produced a more pronounced increase in ⁴⁵Ca uptake even though the frequency was lower (0.5 Hz).

As shown in Fig. 2, a significant increase in ⁴⁵Ca uptake was evident within the first 60 min (P < 0.001), and after 180 min of stimulation ⁴⁵Ca uptake had increased by 156 and 151% for soleus and EDL, respectively (P < 0.001). This stimulation also resulted in an increase in total Ca²⁺ content. This was evident after 120 min (33 and 38% for soleus and EDL, respectively; P < 0.01), and after 180 min the Ca²⁺ content had increased by 68 and 59% for soleus and EDL, respectively (P < 0.001), compared with values for controls at rest.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effect of stimulation and isotonic mounting on the uptake of 45Ca in soleus and EDL muscles. Muscles were mounted on electrodes without any load and incubated for 60-180 min in buffer containing 45Ca (0.1 µCi/ml). Muscles were either resting or stimulated at 0.5 Hz (10 V, 1.0 ms) during the entire incubation period. 45Ca activity was determined as described in Fig. 1. All the stimulated groups differ significantly from resting controls (P < 0.01). , EDL stimulated; , EDL resting; , soleus stimulated, , soleus resting. Mean values ± SE are shown (n = 6-17 muscles).

Because the excitation-induced stimulation of ⁴⁵Ca uptake was more pronounced and therefore easier to detect in muscles undergoing isotonic contractions without any load we found this setup useful for measuring the initial rate of ⁴⁵Ca uptake. Also because soleus tolerates stimulation better than EDL, most of the following experiments were performed with soleus muscle performing isotonic contractions.

Washout experiments. In the experiments described above, ⁴⁵Ca uptake and Ca²⁺ content were measured directly after incubation. To study the initial phase of ⁴⁵Ca uptake, the experimental error arising from the relatively large extracellular component of ⁴⁵Ca uptake needed to be reduced. To this end, experiments were conducted in which incubation with ⁴⁵Ca was followed by washout at 0°C for 4 × 30 min in Ca²⁺-free buffer containing 0.5 mM EGTA. This served to remove extracellular Ca²⁺ and reduce the amount of ⁴⁵Ca bound to the muscle cell surface (10). To determine the loss of intracellular Ca²⁺ during this 4 × 30-min standard washout and to see whether there was any change in the loss as a result of electrical stimulation, control experiments were conducted. After incubation for 15 min with ⁴⁵Ca with or without stimulation at 10 Hz during the final 60 s (isotonic contractions), muscles were washed for 240 min at 0°C in Ca²⁺-free buffer containing 0.5 mM EGTA.

As shown in Fig. 3 it appears that after an initial rapid washout, which probably represents the extracellular fraction of Ca²⁺, the washout curves showed a steady exponential decline. This is reflected in the tight fit of the points to the linear regression lines in the semilogarithmic plot. The linear phase is thought to represent the washout of intracellular Ca²⁺. The regression lines were based on the measurements taken over the last 100 min of washout. From these lines correction factors for the loss of intracellular Ca²⁺ during the 120-min standard washout (see below) were calculated by dividing the regression line intercept (in nmol/g wet wt) by the value at 120 min (in nmol/g wet wt). For soleus muscles incubated with ⁴⁵Ca for 15 min, the correction factor was 1.3 for both muscles at rest and after stimulation. Similar experiments were conducted with EDL muscles, giving a correction factor of 1.6 for both muscles at rest and after stimulation (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effect of stimulation on the washout of 45Ca at 0°C. Soleus muscles were mounted on electrodes without any load and incubated in buffer containing 45Ca (6 µCi/ml) for 15 min, at rest or stimulated at 10 Hz (10 V, 1.0 ms) the last 60 s. This was followed by washout for 2 × 30 min + 12 × 15 min at 0°C in Ca2+-free buffer containing 0.5 mM EGTA. Results were calculated as nmol 45Ca/g muscle wet wt retained in the muscle. Each point represents the mean of 4 observations. Regression lines were constructed by linear regression analysis using values obtained during the late slow phase of washout (140-240 min). , Soleus stimulated; , soleus resting.

Initial ⁴⁵Ca uptake. It was shown that long-term electrical stimulation of isotonically mounted soleus and EDL muscles leads to an increased uptake of ⁴⁵Ca and an increase in total Ca²⁺ content. This net accumulation might reflect stimulation of ⁴⁵Ca influx or, conversely, inhibition of efflux. Alternatively, it might be caused by progressive damage to the muscle. It was of interest, therefore, to conduct a series of experiments of shorter duration where the early phase of ⁴⁵Ca uptake could be quantified. To be able to study the ⁴⁵Ca uptake during the first 60 s of stimulation it was necessary to incubate the muscles in buffer containing ⁴⁵Ca for 15 min with the stimulation taking place during the last 60 s. This treatment allowed diffusion of tracer into the core of the muscle before stimulation. The incubation was followed by a standard washout at 0°C for 4 × 30 min in Ca²⁺-free buffer containing 0.5 mM EGTA to remove extracellular ⁴⁵Ca.

In the first experiments, soleus muscles were stimulated at a number of different frequencies to determine whether the uptake of ⁴⁵Ca was correlated to the number of depolarizations. The muscles were incubated for 15 min with ⁴⁵Ca and stimulated during the last 60 s at 5-80 Hz (10 V, 1.0 ms, isotonic and isometric contractions), followed by washout at 0°C for 4 × 30 min in Ca²⁺-free buffer containing 0.5 mM EGTA.

Figure 4 shows the initial stimulation-induced ⁴⁵Ca uptake (uptake in stimulated muscles  uptake at rest) as a function of frequency. The resting uptake over 15 min averaged 0.07 µmol/g wet wt in all groups. The initial stimulation-induced ⁴⁵Ca uptake increased almost linearly with frequency up to 40 Hz, and therefore, in this physiological range, it appears to be proportional with the number of depolarizations. At 80 Hz, however, ⁴⁵Ca uptake only showed a modest further increase. For comparison the same experiment was performed on muscles mounted for isometric contractions and stimulated at 40 and 80 Hz. Under such conditions stimulation-induced ⁴⁵Ca uptake was reduced by ~50% at both 40 and 80 Hz compared with the isotonically contracting muscles.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of increasing frequency on the initial stimulation-induced 45Ca uptake. Soleus muscles were mounted for isotonic () or isometric () contractions and incubated in buffer containing 45Ca (0.5 µCi/ml) for 15 min. Muscles were either resting or stimulated at the given frequency during the last 60 s, followed by washout at 0°C for 4 × 30 min in Ca2+-free buffer containing 0.5 mM EGTA. This procedure allowed diffusion of tracer into the core of the muscle before stimulation. Resting 45Ca uptake during the incubation period (15 min) was 0.067 ± 0.003 µmol/g wet wt. This value was subtracted from the total 45Ca uptake measured at each frequency to obtain the stimulation-induced 45Ca uptake. All the stimulated groups differ significantly from the resting controls (P < 0.001). Mean values ± SE are shown (n = 3-25 muscles).

It should be noted that when expressed per minute, the uptake of ⁴⁵Ca during the 60 s of stimulation at 40 Hz was 15 and 30 times greater than the ⁴⁵Ca uptake at rest, during isometric and isotonic contractions, respectively.

To determine whether the stimulation-induced increase in ⁴⁵Ca uptake was mediated by a saturable transporter, the concentration of Ca²⁺ in the buffer was varied from 0.6 to 5.0 mM. As shown in Fig. 5 the stimulation-induced increase in ⁴⁵Ca uptake (10 Hz) increases approximately in proportion to the increase in Ca²⁺ concentration, with no significant evidence of saturation.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Stimulation-induced 45Ca uptake at different concentrations of extracellular Ca2+. Soleus muscles were mounted for isotonic contractions and incubated in buffer of varying Ca2+ concentration containing 45Ca (0.5 µCi/ml) for 15 min. Muscles were either resting or stimulated at 10 Hz (10 V, 1.0 ms) during the last 60 s, followed by washout at 0°C for 4 × 30 min Ca2+-free buffer containing 0.5 mM EGTA. Resting 45Ca uptake at each concentration was subtracted from the total 45Ca uptake of the stimulated muscles to obtain the stimulation-induced 45Ca uptake. Mean values ± SE are shown (n = 5-22 muscles).

⁴⁵Ca-uptake mechanisms. The lack of saturation with increasing extracellular Ca²⁺ suggested that a channel could be mediating the uptake. To identify this channel we analyzed the effects of four agents known to block Ca²⁺ channels: nifedipine and verapamil, both known specific blockers of the L-type Ca²⁺ channels; Ni²⁺, a well known blocker of Ca²⁺ channels in skeletal muscle (2); and Gd³⁺, a blocker of stretch-activated Ca²⁺ channels. Nifedipine and verapamil caused no significant reduction in ⁴⁵Ca uptake in the resting muscles. In the stimulated muscles, however, both agents produced a slight but significant reduction of the ⁴⁵Ca uptake (17%; P < 0.05). Despite this reduction, the ⁴⁵Ca uptake in the stimulated muscles was still twice that observed in resting muscles. Ni²⁺ or Gd³⁺ caused no reduction in ⁴⁵Ca uptake, either in the muscles at rest or in stimulated muscles. It should be noted that neither Ni²⁺ nor Gd³⁺ produced any change in muscle contractions induced by direct stimulation. Taken together, these experiments indicate that Ca²⁺-channels only play a minor role in mediating the stimulation-induced increase ⁴⁵Ca uptake. The results are shown in Table 1.

Already in 1976, it was observed that in frog muscle the permeability of the Na⁺ channel to Ca²⁺ was quite high (permeability ratio: P_Ca/P_Na = 0.1) (6). We therefore explored the possibility that the excitation-induced ⁴⁵Ca uptake was mediated by Na⁺ channels. For this purpose we stimulated the uptake of Na⁺ in the muscles by adding veratridine, an Na⁺-channel agonist known to reduce the normal fast inactivation of the Na⁺ channel.

As can be seen from Table 2 incubation of resting soleus and EDL muscles for 15 min with veratridine (10⁴ M) led to a significant increase in the uptake of ⁴⁵Ca (93 and 139% for soleus and EDL, respectively). Addition of TTX (10⁶ M), an Na⁺-channel blocker, to the incubation medium reduced the veratridine-induced increase in ⁴⁵Ca uptake by 77 and 84% for soleus and EDL, respectively. Treatment with nifedipine (10⁵ M), however, could not prevent the stimulating effects of veratridine, again indicating a minor role of the Ca²⁺ channels in ⁴⁵Ca uptake.

To explore the relations between the influx of Ca²⁺ and Na⁺ in more detail, the initial rates of ⁴⁵Ca and ²²Na uptake were measured in dual label experiments.

⁴⁵Ca and ²²Na uptake. The duration of the incubation was reduced to 5 min to avoid major reextrusion of the ²²Na that had been taken up by the muscles (10). In the first experiment, soleus muscles were stimulated to contract isotonically at 60 Hz for 5 s every 1 min. As shown in Table 3, this produced a highly significant increase in the uptake of both ⁴⁵Ca and ²²Na (P < 0.001-0.01). To ascertain that the observed stimulation-induced increase in ⁴⁵Ca uptake was not an effect of the stimulation pulse affecting voltage-sensitive Ca²⁺ channels in the triad junctions or the sarcolemma, muscles were also stimulated in the presence of TTX. As expected, this entirely suppressed the stimulation-induced increase in ⁴⁵Ca and ²²Na uptake.

Next the relation between the uptake of ⁴⁵Ca and ²²Na was assessed in soleus muscles contracting isometrically. Because this was found to reduce the stimulation-induced increase in ⁴⁵Ca uptake, the stimulation time was increased from 5 to 20 s. Mounting the muscles for isometric contraction and stimulating them at 60 Hz for 20 s every min for 5 min resulted in a significant increase in the mean uptake of ⁴⁵Ca (86%; P < 0.01) and ²²Na (73%; P < 0.05). As shown in Fig. 6, there was a close correlation between the stimulation-induced uptake (uptake in stimulated muscles  uptake in resting muscles) of the two isotopes (r = 0.80, P < 0.003).

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Stimulation-induced 45Ca and 22Na uptake in isometrically mounted soleus muscles. Muscles were incubated with 45Ca (1 µCi/ml) and 22Na (1 µCi/ml) for 5 min either resting or stimulated at 60 Hz (10 V, 0.1 ms) for 20 s every 1 min for 5 min. This was followed by washout at 0°C for 4 × 15 min in Na+-Ca2+-free buffer. The resting 45Ca and 22Na uptake during the incubation period (15 min) averaged 0.069 ± 0.003 and 4.0 ± 0.2 µmol/g wet wt, respectively (n = 15 muscles). These values were subtracted from the total 45Ca and 22Na uptake measured to show the stimulation-induced uptake. Each point represents the corresponding 45Ca and 22Na values measured in the same muscle. Regression analysis was performed using the method of least squares (r = 0.80; P < 0.003; n = 11).

In soleus muscles stimulated to contract isotonically for 60-180 min at 0.5 Hz, measurements of the Na⁺ content showed a close and highly significant correlation between the Ca²⁺ and the Na⁺ contents (r = 0.80, P < 0.001) (Fig. 7). For EDL, a similar correlation was obtained (r = 0.69, P < 0.001). It should be noted that in the resting muscles, no significant correlation between Ca²⁺ and Na⁺ content could be detected (P > 0.3) (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Relation between total Na+ and Ca2+ content in soleus muscles stimulated at 0.5 Hz for 60-180 min. Each point represents corresponding values for Na+ and Ca2+ measured in the same muscle. Same experiment as described in Fig. 2. Regression analysis was performed using the method of least squares (n = 25).

The increase in the Ca²⁺ content found during electrical stimulation in muscles stimulated for 60-180 min might be secondary to increased Na⁺ content. To examine whether a raised Na⁺ content alone could cause an increased net uptake of ⁴⁵Ca via the Na⁺/Ca²⁺- exchanger, ouabain was used to induce a rise in intracellular Na⁺ content. Soleus muscles were pretreated for 15 min with ouabain (10³ M) and then incubated for 120 min with ⁴⁵Ca and ouabain (10³ M).

As can be seen from Table 4, treatment with ouabain produced a much larger increase in the intracellular Na⁺ content (194%) than 120 min of electrical stimulation at 0.5 Hz. Despite this there was no significant change in Ca²⁺ content. There was a slight, but significant increase (13%) in the uptake of ⁴⁵Ca. This, however, is not enough to explain the large increases in ⁴⁵Ca uptake and Ca²⁺ content observed during electrical stimulation.

Finally the role of temperature and endplate ion channels was explored.

Figure 8 shows the effects of lowered temperature, suxamethonium, carbachol, tubocurarine, and electrical stimulation on the initial rate of ⁴⁵Ca uptake in soleus muscle. Lowering the temperature to 20°C reduced the resting uptake by 60%. The exposure of the muscles to suxamethonium (10⁵ M) or carbachol (10⁴ M), both agents known to activate the nicotinic acetylcholine receptor (nAchr) cation channels, increased resting uptake by 112 and 107%, respectively. This increase could not be blocked by TTX and was of the same magnitude as that elicited by electrical stimulation at 10 Hz during the last 60 s of the incubation period. Addition of tubocurarine, a known blocker of the nAchr cation channel, efficiently suppressed the effect of carbachol. Cutting the carbachol-treated muscles into three segments so that the middle segment contained the endplate region, showed that the uptake in the middle segment was more than twice that of the other two segments 0.215 ± 0.007 µmol/g wet wt (n = 7 segments) compared with 0.079 ± 0.006 µmol/g wet wt (n = 14 segments). In the muscles treated with both carbachol and tubocurarine, as well as in the control muscles, there was no significant difference between the uptake in the three segments. The same was true for electrically stimulated muscles (40 Hz the last 60 s). However, tubocurarine produced no significant decrease in the ⁴⁵Ca uptake induced by electrical stimulation (40 Hz during the last 60 s).

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Effects of lowered temperature, suxamethonium, carbachol (Car), tubocurarine (Tubo), and electrical stimulation on 45Ca uptake in soleus muscle. All muscles were mounted for isotonic contractions and incubated in buffer containing 45Ca (0.5 µCi/ml) for 15 min. Resting muscles were incubated in normal buffer or with the additions indicated [suxamethonium (105 M); carbachol (104 M); tubocurarine (105 M)] during the entire incubation period. Stimulated muscles were incubated for 15 min and stimulated at the given frequency during the last 60 s. All treatments were followed by washout at 0°C for 4 × 30 min in Ca2+-free buffer containing 0.5 mM EGTA. Incubation temperature was 30°C for all groups, except 1 group incubated at 20°C. * Values significantly different (P < 0.001) from controls. Mean values ± SE are shown (n = 3-20).

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

⁴⁵Ca uptake via Na⁺ channels. The central observations are that electrical stimulation in vitro produces a marked increase in the initial rate of ⁴⁵Ca uptake and a progressive net accumulation of Ca²⁺ in both muscles. This coincides with an increased uptake and net accumulation of Na⁺. Addition of veratridine, an Na⁺-channel agonist, increased resting uptake of ⁴⁵Ca. This effect was almost completely blocked by TTX, but could not be blocked by nifedipine, a Ca²⁺-channel blocker. The stimulation-induced increase in ⁴⁵Ca was likewise only partially affected by nifedipine (17% decrease) and other Ca²⁺-channel blockers. Furthermore, dual label experiments showed that the initial rates of ⁴⁵Ca and ²²Na uptake were closely correlated in both isotonically and isometrically contracting soleus muscles (r = 0.96 and 0.80, respectively). Blocking the excitation-induced ²²Na⁺ uptake by TTX efficiently suppressed the increase in ⁴⁵Ca uptake.

Ca²⁺ flux via Na⁺ channels has recently been reported in cardiac muscle (35). This so-called slip-mode conductance is activated by protein kinase A and is not affected by nifedipine or Ni²⁺, but it is completely blocked by TTX. During "slip mode" the permeability of the Na⁺ channel to Ca²⁺ (P_Ca/P_Na) increases to 1.25. In our experiments, the ratio between the extracellular concentrations Ca²⁺ and Na⁺ is 1:112. Assuming a P_Ca/P_Na of 1.25, the ratio between the influx of Ca²⁺ and Na⁺ should be 1:90 in the absence of a membrane potential. During excitation the ratio between the initial rates of ⁴⁵Ca and ²²Na uptake was 1:40 (Fig. 6). This may be simply explained if the mean membrane potential is slightly more negative during the slip mode or if the ratio P_Ca/P_Na is >1.25 in rat soleus.

Initial rate of ⁴⁵Ca uptake. Another possible mechanism for a combined influx of Ca²⁺ and Na⁺ could be damage to the sarcolemma. In soleus muscle, resting ⁴⁵Ca uptake over 15 min (calculated from Table 1) was 3.7-5.6 nmol · g wet wt¹ · min¹, which corresponds very well to values previously reported for soleus muscle (5.5 nmol · g wet wt¹ · min¹) (19). Assuming a sarcolemma surface area of 1,300 cm²/g wet wt (13), the rate of ⁴⁵Ca uptake corresponds to 0.048-0.072 pmol · cm² · s¹. These values are more than half that reported for resting frog sartorius muscle at 25°C (0.094 pmol · cm² · s¹) (5). The difference may be species related. Also, the sartorius muscle experiments were performed using a shorter washout period without EGTA in the washout medium.

In soleus muscles stimulated at 40 Hz for 60 s and undergoing isotonic contractions, the initial rate of stimulation-induced ⁴⁵Ca uptake was 137 nmol · g wet wt¹ · min¹ or 1.76 pmol · cm² · s¹, which is ~30-fold higher than that measured in resting muscle. In soleus muscles undergoing isometric contraction, 40-Hz stimulation increased the initial rate of ⁴⁵Ca uptake 15-fold. Because such a large increase in the uptake of ⁴⁵Ca occurs even during a very short period of stimulation, damage to the sarcolemma does not seem a likely explanation for the observed rise in Ca²⁺ uptake.

Role of Ca²⁺ channels. That Ca²⁺ channels do not represent a major route for Ca²⁺ uptake is surprising. Earlier studies on skeletal muscle using the voltage clamp technique have demonstrated Ca²⁺ currents and Ca²⁺ action potentials that were all blocked by nifedipine or Ni²⁺ (2, 9, 15, 29). In one study, however, electrical stimulation was found to produce an increase in cellular Ca²⁺ content that was not blocked by verapamil or nifedipine (25).

Long-term stimulation. Earlier experiments showed that long-term in vivo stimulation had no effect on Ca²⁺ contents in soleus but led to a progressive and pronounced (up to 4-fold) accumulation of Ca²⁺ in EDL that could be detected after 2 h (20). In keeping with this, we here find that in isolated muscles contracting isometrically, long-term electrical stimulation produces a much larger increase in ⁴⁵Ca uptake in EDL (1.425 µmol/g wet wt) than in soleus (0.250 µmol/g wet wt). Total Ca²⁺ content in EDL increased almost twofold, whereas no net increase in total Ca²⁺ content was observed in soleus.

Isotonic versus isometric mounting. Isotonic contractions at zero load compared with isometric contractions, gave rise to a larger ⁴⁵Ca uptake and accumulation of Ca²⁺ in soleus as well as in EDL. When measured over 120 min and expressed per contraction, the increase in ⁴⁵Ca uptake during isotonic contractions was 2.6 times larger in EDL and 6.8 times larger in soleus (compared with the increase during isometric contractions). The initial stimulation-induced increase in ⁴⁵Ca uptake in soleus muscles contracting isotonically was twice that observed in muscles contracting isometrically (Fig. 4).

The cause of this difference in ⁴⁵Ca uptake between the two modes of contraction is at present unknown. It is in keeping, however, with the recent observation that in rat soleus muscle, isotonic contractions produce a fivefold larger increase in intracellular Na⁺ than isometric contractions (34). The similarity between the excitation-induced increases in Ca²⁺ and Na⁺ uptake during the two modes of contraction further supports the idea of a common pathway for the influx of the two ions.

Role of SR. SR plays an important role in the accumulation of Ca²⁺. Treatment with thyroid hormones causes a proliferation of the SR and this was found to lead to a considerable increase in ⁴⁵Ca uptake and net Ca²⁺ accumulation in rat soleus muscles (19). In the present study we have shown that lowering the temperature to 20°C reduces resting ⁴⁵Ca uptake by 60%. Because transport of Ca²⁺ into the SR by the SR Ca²⁺-ATPase has a high temperature coefficient, this suggests that uptake and accumulation of Ca²⁺ in the muscle are dependent on uptake and storage in the SR.

The capacity for accumulation of Ca²⁺ is larger in EDL than in soleus. First, EDL contains more SR (31) and around six times as much Ca²⁺-ATPase as soleus (18). Second, it has been shown that at resting myoplasmic Ca²⁺, the SR of slow-twitch fibers is saturated with Ca²⁺, whereas the SR of fast-twitch fibers is only one-third saturated (22). In EDL, therefore, the excitation-induced increase in Ca²⁺ influx may readily become accumulated in the SR, whereas in soleus, SR cannot contribute much to the clearance of the Ca²⁺ entering the cytoplasm. During isometric contractions, we find, in keeping with previous observations in vivo (20), that EDL accumulates much more Ca²⁺ than soleus (Fig. 1). This is likely to reflect the difference between the capacity and saturability of the SR in the two muscles (22). It should be noted that although soleus muscles from mature rats contain <10% fast-twitch fibers (14), soleus from 4-wk-old rats contain a higher percentage of fast-twitch fibers (4). It is possible, therefore, that during isometric contractions, the stimulation-induced accumulation of Ca²⁺ in soleus could be confined to the fast-twitch fiber component of the muscle.

In soleus muscles contracting isometrically, most of the Ca²⁺ taken up during excitation seems to be reextruded. During isotonic contractions, however, the Ca²⁺ influx per twitch is considerably larger, possibly resulting in a Ca²⁺ load exceeding the capacity for reextrusion from the cells. This gives rise to the observed accumulation of Ca²⁺, which could be envisaged to become sequestered in the mitochondria.

Repetitive activation of the muscle can cause phosphorylation of phospholamban in slow-twitch fibers, which in turn could increase the SR Ca²⁺-ATPase pump rate and thereby the uptake of Ca²⁺. In contrast to cardiac muscle, however, phosphorylation of phospholamban in SR vesicles from slow-twitch muscle produces only a minor stimulation of Ca²⁺ uptake (26).

Role of endplate channels. The nAchr channel of the endplate excludes anions but is otherwise unselective. The P_Ca/P_Na is near 0.3 (1), which leaves a good possibility of a Ca²⁺ influx via this channel as well. Activating the endplate channels with suxamethonium or carbachol led to a significant increase in ⁴⁵Ca uptake. This increase was mainly located to the endplate region, was not affected by TTX, but was efficiently suppressed by tubocurarine. Tubocurarine, however, did not reduce the stimulation-induced uptake of ⁴⁵Ca, eliminating the endplate region as the major entry route for Ca²⁺ during electrical stimulation.

Muscle damage. It has been proposed that cellular damage arising from contractile activity could be attributed to increased cytoplasmic Ca²⁺ (3, 17) and that after intense exercise, major alterations in the structure and function of the SR occur, possibly as a result of increased Ca²⁺ content (32). Increases in the cytoplasmic free Ca²⁺ concentration have been reported as a result of fatiguing stimulation (30). On the other hand, it has been proposed that localized increase in cytoplasmic Ca²⁺ may stop further release of Ca²⁺ from SR, causing excitation-contraction uncoupling (28). This Ca²⁺-dependent uncoupling might protect the muscle against further deleterious increases in cytoplasmic Ca²⁺.

Perspectives

It has been reported that the muscle cell damage seen after intense or extensive exercise primarily affects fast-twitch fibers (21). We have found that during isometric contractions, the excitation-induced uptake of Ca²⁺ is more pronounced in EDL (predominantly fast-twitch fibers) than in soleus (predominantly slow-twitch fibers), also in vivo (20). This adds further support to the idea that exercise-induced cellular damage is the result of excitation-induced Ca²⁺-influx.