In isolated rat extensor digitorum longus (EDL) muscle mounted for isometric contractions, chronic low-frequency electrical stimulation was found to lead to an increased uptake of45Ca (154% above control after 240 min) and a progressive accumulation of Ca2+ (85% above control after 240 min). In soleus, however, this treatment led to a small, but significant, increase in 45Ca uptake (30% above control after 180 min) but no significant accumulation of Ca2+. In muscles mounted for isotonic contractions without any external load, electrical stimulation gave rise to a larger45Ca uptake and accumulation of Ca2+ in both EDL and soleus. These uptakes of Ca2+ coincided with an accumulation of Na+. During isometric or isotonic contractions, stimulation at 40 Hz increased the initial (60 s) rate of 45Ca uptake in soleus muscle 15- and 30-fold, respectively. The stimulation-induced increase in 45Ca uptake was only reduced by 17% by the Ca2+-channel blockers nifedipine and verapamil but was blocked by tetrodotoxin. The initial rate of stimulation-induced 22Na and45Ca uptake was correlated (r = 0.80;P < 0.003). Stimulation of Na+ channels with veratridine increased 45Ca uptake by 93 and 139% in soleus and EDL, respectively (P < 0.001), effects that were abolished by tetrodotoxin. The results indicate that in skeletal muscle, excitation induces a considerable influx of Ca2+, mediated by Na+ channels.
- extensor digitorum longus
- 45Ca uptake
- electrical stimulation
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
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 10d-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.
45 Ca 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 andresults.
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 (2,800g, 10 min).
Dual label experiments. These were performed as earlier described (10). The muscles were incubated with45Ca (1 μCi/ml) and22Na (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 and22Na 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 of45Ca and22Na activity.
Ca2+contents. Muscles weighing 20–25 mg were homogenized in 3 ml 0.3 M TCA and centrifuged (2,800g, 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 (2,800g, 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-tailedt-test for nonpaired observations. Regression analysis was performed using the method of least squares.
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 of45Ca. 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).
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 in45Ca 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 in45Ca 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.
Because the excitation-induced stimulation of45Ca 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 of45Ca uptake, the experimental error arising from the relatively large extracellular component of45Ca uptake needed to be reduced. To this end, experiments were conducted in which incubation with45Ca 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 of45Ca 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 with45Ca 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 with45Ca 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).
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 of45Ca 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 of45Ca 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 containing45Ca 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 extracellular45Ca.
In the first experiments, soleus muscles were stimulated at a number of different frequencies to determine whether the uptake of45Ca was correlated to the number of depolarizations. The muscles were incubated for 15 min with45Ca 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-induced45Ca 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-induced45Ca uptake was reduced by ∼50% at both 40 and 80 Hz compared with the isotonically contracting muscles.
It should be noted that when expressed per minute, the uptake of45Ca during the 60 s of stimulation at 40 Hz was 15 and 30 times greater than the45Ca uptake at rest, during isometric and isotonic contractions, respectively.
To determine whether the stimulation-induced increase in45Ca 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 in45Ca uptake (10 Hz) increases approximately in proportion to the increase in Ca2+ concentration, with no significant evidence of saturation.
45 Ca-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 in45Ca uptake in the resting muscles. In the stimulated muscles, however, both agents produced a slight but significant reduction of the45Ca 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 in45Ca 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 increase45Ca 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: PCa/PNa= 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 (10−4 M) led to a significant increase in the uptake of45Ca (93 and 139% for soleus and EDL, respectively). Addition of TTX (10−6 M), an Na+-channel blocker, to the incubation medium reduced the veratridine-induced increase in45Ca uptake by 77 and 84% for soleus and EDL, respectively. Treatment with nifedipine (10−5 M), however, could not prevent the stimulating effects of veratridine, again indicating a minor role of the Ca2+ channels in45Ca uptake.
To explore the relations between the influx of Ca2+ and Na+ in more detail, the initial rates of 45Ca and22Na uptake were measured in dual label experiments.
45 Ca and22Na 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 both45Ca and22Na (P < 0.001–0.01). To ascertain that the observed stimulation-induced increase in45Ca 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 in45Ca and22Na uptake.
Next the relation between the uptake of45Ca and22Na was assessed in soleus muscles contracting isometrically. Because this was found to reduce the stimulation-induced increase in45Ca 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 of45Ca (86%;P < 0.01) and22Na (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).
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).
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 of45Ca 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 (10−3 M) and then incubated for 120 min with 45Ca and ouabain (10−3M).
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 in45Ca 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 of45Ca uptake in soleus muscle. Lowering the temperature to 20°C reduced the resting uptake by 60%. The exposure of the muscles to suxamethonium (10−5 M) or carbachol (10−4 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 the45Ca uptake induced by electrical stimulation (40 Hz during the last 60 s).
45 Ca 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 in45Ca was likewise only partially affected by nifedipine (17% decrease) and other Ca2+-channel blockers. Furthermore, dual label experiments showed that the initial rates of45Ca and22Na uptake were closely correlated in both isotonically and isometrically contracting soleus muscles (r = 0.96 and 0.80, respectively). Blocking the excitation-induced22Na+uptake by TTX efficiently suppressed the increase in45Ca 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+(PCa/PNa) increases to 1.25. In our experiments, the ratio between the extracellular concentrations Ca2+and Na+ is 1:112. Assuming a PCa/PNaof 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 and22Na 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 PCa/PNais >1.25 in rat soleus.
Initial rate of45Ca uptake.Another possible mechanism for a combined influx of Ca2+ and Na+ could be damage to the sarcolemma. In soleus muscle, resting45Ca uptake over 15 min (calculated from Table 1) was 3.7–5.6 nmol ⋅ g wet wt−1 ⋅ min−1, which corresponds very well to values previously reported for soleus muscle (5.5 nmol ⋅ g wet wt−1 ⋅ min−1) (19). Assuming a sarcolemma surface area of 1,300 cm2/g wet wt (13), the rate of45Ca uptake corresponds to 0.048–0.072 pmol ⋅ cm−2 ⋅ s−1. These values are more than half that reported for resting frog sartorius muscle at 25°C (0.094 pmol ⋅ cm−2 ⋅ s−1) (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-induced45Ca uptake was 137 nmol ⋅ g wet wt−1 ⋅ min−1or 1.76 pmol ⋅ cm−2 ⋅ s−1, 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 of45Ca 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 in45Ca 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 larger45Ca 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 in45Ca uptake in soleus muscles contracting isotonically was twice that observed in muscles contracting isometrically (Fig. 4).
The cause of this difference in45Ca 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 in45Ca 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 resting45Ca 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 PCa/PNais 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 in45Ca 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 of45Ca, 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+.
We have shown that in skeletal muscle, excitation is accompanied by a substantial increase in Ca2+influx, which can lead to progressive intracellular accumulation of Ca2+. Although SR has a large storage capacity for Ca2+, this may be exceeded during intense or prolonged exercise, resulting in mitochondrial Ca2+ overload and increased cytoplasmic Ca2+. This could impair energy metabolism and induce activation of Ca2+-sensitive enzyme systems involved in muscle autolysis, eventually leading to cellular damage (25).
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.
We thank Ann Charlotte Andersen, Tove Lindahl Andersen, Ebba de Neergaard, and Marianne Stürup-Johansen for skilled technical assistance.
Address reprint requests to H. Gissel.
This study was supported by grants from the Danish Medical Research Council (No. 12–1336) and The Danish Biomembrane Center.
- Copyright © 1999 the American Physiological Society