Excitation-induced Ca2+uptake in rat skeletal muscle

Excitation-induced Ca21 uptake in rat skeletal muscle H. GISSEL AND T. CLAUSEN ˚ rhus C, Denmark Department of Physiology, University of Aarhus, DK-8000 A Gissel, H., and T. Clausen. Excitation-induced Ca21 uptake in rat skeletal muscle. Am. J. Physiol. 276 (Regulatory Integrative Comp. Physiol. 45): R331–R339, 1999.—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 of 45Ca (154% above control after 240 min) and a progressive accumulation of Ca21 (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 Ca21. In muscles mounted for isotonic contractions without any external load, electrical stimulation gave rise to a larger 45Ca uptake and accumulation of Ca21 in both EDL and soleus. These uptakes of Ca21 coincided with an accumulation of Na1. 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 Ca21-channel blockers nifedipine and verapamil but was blocked by tetrodotoxin. The initial rate of stimulation-induced 22Na and 45Ca uptake was correlated (r 5 0.80; P , 0.003). Stimulation of Na1 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 Ca21, mediated by Na1 channels. soleus; extensor digitorum longus; stimulation; Na1 channels

45Ca

uptake; electrical

of skeletal muscles in vivo has been shown to lead to a progressive net uptake of Ca21 in rabbits (36) and in rats (20). A recent study showed that also in vitro, tetanic contractions were associated with an increase in Ca21 content of hamster diaphragm (23). This excitation-induced net accumulation of Ca21 is considerable and is only reversed slowly (20). Early studies on frog sartorius muscle demonstrated that excitation is associated with an influx of Ca21 (5), but little is known about the mechanisms involved and the long-term effects of this Ca21 uptake. Because the transport capacity of the Ca21-ATPase of the sarcoplasmic reticulum (SR) exceeds that of the Ca21-ATPase in the sarcolemma by far (7), Ca21 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 Na1/Ca21 exchange seems to be of minor importance for the net extrusion of Ca21 at low cytoplasmic Ca21 (16, 24). Cellular damage arising from various types of contractile activity has often been attributed to increased cytoplasmic Ca21 (17, 25). We therefore found it important to explore possible mechanisms for the excitationinduced accumulation of Ca21 in muscle. We have characterized the effects of electrical stimulation on the ELECTRICAL STIMULATION

uptake of 45Ca and the net accumulation of Ca21 in isolated rat soleus and extensor digitorum longus (EDL) muscles, typical examples of predominantly slowtwitch 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 Na1 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 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, K1, Na1, and Ca21 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 Ca21-free Krebs-Ringer bicarbonate buffer containing 0.5 mM EGTA to remove extracellular Ca21. Control experiments were conducted to estimate the amount of Ca21 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).

0363-6119/99 $5.00 Copyright r 1999 the American Physiological Society

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EXCITATION-INDUCED CA21 ACCUMULATION

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 3 15 min in 2 ml Na1-Ca21-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. Ca21 contents. Muscles weighing 20–25 mg were homogenized in 3 ml 0.3 M TCA and centrifuged (2,800 g, 10 min). Ca21 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. Na1 contents. Muscles weighing 20–25 mg were homogenized in 3 ml 0.3 M TCA and centrifuged (2,800 g, 10 min). The Na1 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 Ni21, Gd31, 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 6 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

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). In the same experiment, the increase in total Ca21 content was measured. In soleus, stimulation resulted in a slight but nonsignificant increase in the Ca21

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. l, EDL stimulated; n, EDL resting; r, soleus stimulated; s, soleus resting. All the stimulated groups differ significantly from resting controls (P , 0.01). Mean values 6 SE are shown (n 5 6 muscles).

content from 1.16 6 0.08 (resting controls) to 1.30 6 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 Ca21 content from 1.41 6 0.04 to 2.61 6 0.19 µmol/g wet wt (P , 0.001). The net gain in total Ca21 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 Ca21 content. This was evident after 120 min (33 and 38% for soleus and EDL, respectively; P , 0.01), and after 180 min the Ca21 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 of 45Ca uptake was more pronounced and therefore easier to


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). l, EDL stimulated; n, EDL resting; r, soleus stimulated, s, soleus resting. Mean values 6 SE are shown (n 5 6–17 muscles).

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 Ca21 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 3 30 min in Ca21-free buffer containing 0.5 mM EGTA. This served to remove extracellular Ca21 and reduce the amount of 45Ca bound to the muscle cell surface (10). To determine the loss of intracellular Ca21 during this 4 3 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 Ca21-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 Ca21, 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

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represent the washout of intracellular Ca21. 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 Ca21 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). 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 Ca21 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 3 30 min in Ca21-free buffer containing 0.5 mM EGTA to remove extracellular 45Ca.

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 3 30 min 1 12 3 15 min at 0°C in Ca21-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). r, Soleus stimulated; s, soleus resting.

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Fig. 4. Effect of increasing frequency on the initial stimulationinduced 45Ca uptake. Soleus muscles were mounted for isotonic (r) or isometric (j) 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 3 30 min in Ca21-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 6 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 6 SE are shown (n 5 3–25 muscles).

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 3 30 min in Ca21-free buffer containing 0.5 mM EGTA. Figure 4 shows the initial stimulation-induced 45Ca uptake (uptake in stimulated muscles 2 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. 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 Ca21 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 Ca21 concentration, with no significant evidence of saturation. 45Ca-uptake mechanisms. The lack of saturation with increasing extracellular Ca21 suggested that a channel could be mediating the uptake. To identify this channel we analyzed the effects of four agents known to block Ca21 channels: nifedipine and verapamil, both known specific blockers of the L-type Ca21 channels; Ni21, a well known blocker of Ca21 channels in skeletal muscle (2); and Gd31, a blocker of stretch-activated Ca21 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 (217%; P , 0.05). Despite this reduction, the 45Ca uptake in the stimulated muscles was still twice that observed in resting muscles. Ni21 or Gd31 caused no reduction in 45Ca uptake, either in the muscles at rest or in stimulated muscles. It should be noted that neither Ni21 nor Gd31 produced any change in muscle contractions induced by direct stimulation. Taken together, these experiments indicate that Ca21-channels only play a minor role in mediating the stimulationinduced increase 45Ca uptake. The results are shown in Table 1. Already in 1976, it was observed that in frog muscle the permeability of the Na1 channel to Ca21 was quite high (permeability ratio: PCa/PNa 5 0.1) (6). We there-

Fig. 5. Stimulation-induced 45Ca uptake at different concentrations of extracellular Ca21. Soleus muscles were mounted for isotonic contractions and incubated in buffer of varying Ca21 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 3 30 min Ca21-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 6 SE are shown (n 5 5–22 muscles).

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Table 1. Effects of Ca21-channel blocking agents on 45Ca uptake in resting and stimulated soleus muscles 45Ca

uptake, µmol · g wet wt21 · 15 min21

Resting

Control 0.058 6 0.007 (6) Nifedipine (1025 M) 0.083 6 0.011 (6) Control 0.084 6 0.014 (6) Verapamil (1025 M) 0.076 6 0.012 (6)

Stimulated

0.205 6 0.008 (6) NS

0.170 6 0.010 (6) P , 0.05 0.196 6 0.007 (6)

NS

0.163 6 0.009 (6) P , 0.05

Control 0.056 6 0.003 (3) 0.220 6 0.026 (6) Ni21 (0.2 mM) 0.061 6 0.013 (3) NS 0.224 6 0.008 (3) Gd31 (0.1 mM) 0.094 6 0.007 (3) P , 0.05 0.261 6 0.015 (3)

NS NS

Values are given as means 6 SE with no. of observations in parentheses. After equilibration for 60 min and preincubation for 15 min with agents indicated, muscles were incubated for 15 min in buffer containing 45Ca (0.5 µCi/ml) and the agents indicated followed by washout at 0°C for 4 3 30 min in Ca21-free buffer containing 0.5 mM EGTA. Muscles were mounted so as to allow isotonic contraction and were either at rest throughout the entire incubation period or stimulated directly the last 60 s at 40 Hz (10 V, 1.0 ms). NS, not significant.

fore explored the possibility that the excitation-induced 45Ca uptake was mediated by Na1 channels. For this purpose we stimulated the uptake of Na1 in the muscles by adding veratridine, an Na1-channel agonist known to reduce the normal fast inactivation of the Na1 channel. As can be seen from Table 2 incubation of resting soleus and EDL muscles for 15 min with veratridine (1024 M) led to a significant increase in the uptake of 45Ca (93 and 139% for soleus and EDL, respectively). Addition of TTX (1026 M), an Na1-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 (1025 M), however, could not prevent the stimulating effects of veratridine, again indicating a minor role of the Ca21 channels in 45Ca uptake. To explore the relations between the influx of Ca21 and Na1 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 stimulationinduced increase in 45Ca uptake was not an effect of the stimulation pulse affecting voltage-sensitive Ca21 channels in the triad junctions or the sarcolemma, muscles were also stimulated in the presence of TTX. As expected, this entirely suppressed the stimulationinduced 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 stimulationinduced uptake (uptake in stimulated muscles 2 uptake in resting muscles) of the two isotopes (r 5 0.80, P , 0.003). In soleus muscles stimulated to contract isotonically for 60–180 min at 0.5 Hz, measurements of the Na1 content showed a close and highly significant correlation between the Ca21 and the Na1 contents (r 5 0.80, P , 0.001) (Fig. 7). For EDL, a similar correlation was obtained (r 5 0.69, P , 0.001). It should be noted that in the resting muscles, no significant correlation between Ca21 and Na1 content could be detected (P . 0.3) (data not shown). The increase in the Ca21 content found during electrical stimulation in muscles stimulated for 60–180 min might be secondary to increased Na1 content. To examine whether a raised Na1 content alone could cause an increased net uptake of 45Ca via the Na1/Ca21- exchanger, ouabain was used to induce a rise in intracellular Na1 content. Soleus muscles were pretreated for 15 min with ouabain (1023 M) and then incubated for 120 min with 45Ca and ouabain (1023 M). As can be seen from Table 4, treatment with ouabain produced a much larger increase in the intracellular Na1 content (194%) than 120 min of electrical stimula-

Table 2. Effects of veratridine, TTX, and nifedipine on 45Ca uptake in soleus and EDL muscle 45Ca

uptake, µmol · g wet wt21 · 15 min21

Soleus

EDL

Control Veratridine

0.056 6 0.004 (8) 0.108 6 0.005 (8)

P , 0.001

0.062 6 0.005 (8) 0.148 6 0.013 (8)

P , 0.001

TTX TTX 1 veratridine

0.048 6 0.006 (7) 0.060 6 0.008 (8)

NS

0.048 6 0.002 (8) 0.062 6 0.002 (8)

NS

Nifedipine Nifedipine 1 veratridine

0.070 6 0.006 (8) 0.106 6 0.005 (8)

P , 0.001

0.059 6 0.002 (4) 0.102 6 0.004 (3)

P ,0.001

Values are given as means 6 SE with no. of observations in parentheses. After equilibration for 30 min and preincubation for 15 min in normal buffer without or with TTX (1026 M) or nifedipine (1025 M), the muscles were incubated for 15 min in buffer containing 45Ca (0.5 µCi/ml) and the agents indicated followed by washout at 0°C for 4 3 30 min in Ca21-free buffer containing 0.5 mM EGTA. Veratridine was used at the concentration 1024 M. EDL, extensor digitorum longus.

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Table 3. Effects of TTX on 45Ca and 22Na uptake in resting and stimulated soleus muscles Uptake, µmol · g wet wt21 · 5 min21 Resting 45Ca

Control TTX 22Na

Control TTX

Stimulated

0.042 6 0.002 (6) 0.052 6 0.007 (6)

NS

0.078 6 0.005 (6) 0.058 6 0.003 (6)

P , 0.01

3.0 6 0.3 (6) 2.7 6 0.3 (6)

NS

5.2 6 0.2 (6) 3.0 6 0.1 (6)

P , 0.001

Values are given as means 6 SE with no. of observations in parentheses. After equilibration for 30 min and preincubation for 15 min with or without TTX (1027 M), the muscles were incubated in buffer containing 45Ca (1 µCi/ml) and 22Na (1 µCi/ml) for 5 min followed by washout at 0°C for 4 3 15 min in Na1-Ca21 free buffer containing 0.5 mM EGTA. The muscles were mounted so as to allow isotonic contraction and were either at rest throughout the entire incubation period or stimulated at 60 Hz 5 s every min for 5 min (10 V, 0.1 ms).

tion at 0.5 Hz. Despite this there was no significant change in Ca21 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 Ca21 content observed during electrical stimulation. Finally the role of temperature and endplate ion channels was explored.

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 3 15 min in Na1-Ca21-free buffer. The resting 45Ca and 22Na uptake during the incubation period (15 min) averaged 0.069 6 0.003 and 4.0 6 0.2 µmol/g wet wt, respectively (n 5 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 5 0.80; P , 0.003; n 5 11).

Fig. 7. Relation between total Na1 and Ca21 content in soleus muscles stimulated at 0.5 Hz for 60–180 min. Each point represents corresponding values for Na1 and Ca21 measured in the same muscle. Same experiment as described in Fig. 2. Regression analysis was performed using the method of least squares (n 5 25).

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 (1025 M) or carbachol (1024 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 6 0.007 µmol/g wet wt (n 5 7 segments) compared with 0.079 6 0.006 µmol/g wet wt (n 5 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). DISCUSSION

uptake via Na1 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 Ca21 in both muscles. This coincides with an increased uptake and net accu45Ca

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Table 4. Effects of ouabain and electrical stimulation on the uptake of 45Ca, Ca21, and Na1 contents in soleus muscle 45Ca

Control Ouabain (1023 M) Stimulation (0.5 Hz)

Total Ca21 Content

Uptake

0.77 6 0.02 (10) 0.87 6 0.03 (10) 1.45 6 0.14 (8)

P , 0.05 P , 0.001

1.21 6 0.04 (10) 1.24 6 0.05 (10) 1.74 6 0.04 (8)

Intracellular Na1 Content

NS P , 0.001

17.0 6 1.5 (10) 50.0 6 1.5 (10) 19.6 6 2.6 (8)

P , 0.001 NS

Values are given as means 6 SE (in µmol/g wet wt) with no. of observations in parentheses. After equilibration for 30 min and preincubation for 15 min with or without ouabain (1023 M), the muscles were incubated with or without ouabain (1023 M) for 120 min in buffer containing 45Ca (0.1 µCi/ml). One group was mounted to contract isotonically and stimulated at 0.5 Hz for 120 min. Intracellular Na1 contents were calculated by deducting the Na1 residing in the extracellular space, measured using [14C]sucrose, from the total Na1 content. Treated groups are compared with resting controls.

mulation of Na1. Addition of veratridine, an Na1channel agonist, increased resting uptake of 45Ca. This effect was almost completely blocked by TTX, but could not be blocked by nifedipine, a Ca21-channel blocker. The stimulation-induced increase in 45Ca was likewise only partially affected by nifedipine (17% decrease) and other Ca21-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 5 0.96 and 0.80, respectively). Blocking the excitation-induced 22Na1 uptake by TTX efficiently suppressed the increase in 45Ca uptake. Ca21 flux via Na1 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 Ni21, but it is completely blocked by TTX. During ‘‘slip mode’’ the permeability of

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 (1025 M); carbachol (1024 M); tubocurarine (1025 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 3 30 min in Ca21-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 6 SE are shown (n 5 3–20).

the Na1 channel to Ca21 (PCa/PNa ) increases to 1.25. In our experiments, the ratio between the extracellular concentrations Ca21 and Na1 is 1:112. Assuming a PCa/PNa of 1.25, the ratio between the influx of Ca21 and Na1 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 PCa/PNa is .1.25 in rat soleus. Initial rate of 45Ca uptake. Another possible mechanism for a combined influx of Ca21 and Na1 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 wt21 · min21, which corresponds very well to values previously reported for soleus muscle (5.5 nmol · g wet wt21 · min21 ) (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 · cm22 · s21. These values are more than half that reported for resting frog sartorius muscle at 25°C (0.094 pmol · cm22 · s21 ) (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 wt21 · min21 or 1.76 pmol · cm22 · s21, 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 Ca21 uptake. Role of Ca21 channels. That Ca21 channels do not represent a major route for Ca21 uptake is surprising. Earlier studies on skeletal muscle using the voltage clamp technique have demonstrated Ca21 currents and Ca21 action potentials that were all blocked by nifedipine or Ni21 (2, 9, 15, 29). In one study, however, electrical stimulation was found to produce an increase in cellular Ca21 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 Ca21 contents in soleus but led to a progressive and pro-

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nounced (up to 4-fold) accumulation of Ca21 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 Ca21 content in EDL increased almost twofold, whereas no net increase in total Ca21 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 Ca21 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 Na1 than isometric contractions (34). The similarity between the excitation-induced increases in Ca21 and Na1 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 Ca21. 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 Ca21 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 Ca21 into the SR by the SR Ca21-ATPase has a high temperature coefficient, this suggests that uptake and accumulation of Ca21 in the muscle are dependent on uptake and storage in the SR. The capacity for accumulation of Ca21 is larger in EDL than in soleus. First, EDL contains more SR (31) and around six times as much Ca21-ATPase as soleus (18). Second, it has been shown that at resting myoplasmic Ca21, the SR of slow-twitch fibers is saturated with Ca21, whereas the SR of fast-twitch fibers is only one-third saturated (22). In EDL, therefore, the excitation-induced increase in Ca21 influx may readily become accumulated in the SR, whereas in soleus, SR cannot contribute much to the clearance of the Ca21 entering the cytoplasm. During isometric contractions, we find, in keeping with previous observations in vivo (20), that EDL accumulates much more Ca21 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% fasttwitch 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 stimu-

lation-induced accumulation of Ca21 in soleus could be confined to the fast-twitch fiber component of the muscle. In soleus muscles contracting isometrically, most of the Ca21 taken up during excitation seems to be reextruded. During isotonic contractions, however, the Ca21 influx per twitch is considerably larger, possibly resulting in a Ca21 load exceeding the capacity for reextrusion from the cells. This gives rise to the observed accumulation of Ca21, 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 Ca21-ATPase pump rate and thereby the uptake of Ca21. In contrast to cardiac muscle, however, phosphorylation of phospholamban in SR vesicles from slow-twitch muscle produces only a minor stimulation of Ca21 uptake (26). Role of endplate channels. The nAchr channel of the endplate excludes anions but is otherwise unselective. The PCa/PNa is near 0.3 (1), which leaves a good possibility of a Ca21 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 Ca21 during electrical stimulation. Muscle damage. It has been proposed that cellular damage arising from contractile activity could be attributed to increased cytoplasmic Ca21 (3, 17) and that after intense exercise, major alterations in the structure and function of the SR occur, possibly as a result of increased Ca21 content (32). Increases in the cytoplasmic free Ca21 concentration have been reported as a result of fatiguing stimulation (30). On the other hand, it has been proposed that localized increase in cytoplasmic Ca21 may stop further release of Ca21 from SR, causing excitation-contraction uncoupling (28). This Ca21-dependent uncoupling might protect the muscle against further deleterious increases in cytoplasmic Ca21. Perspectives We have shown that in skeletal muscle, excitation is accompanied by a substantial increase in Ca21 influx, which can lead to progressive intracellular accumulation of Ca21. Although SR has a large storage capacity for Ca21, this may be exceeded during intense or prolonged exercise, resulting in mitochondrial Ca21 overload and increased cytoplasmic Ca21. This could impair energy metabolism and induce activation of Ca21-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 Ca21 is more pronounced in EDL (predominantly


fast-twitch fibers) than in soleus (predominantly slowtwitch fibers), also in vivo (20). This adds further support to the idea that exercise-induced cellular damage is the result of excitation-induced Ca21-influx. We thank Ann Charlotte Andersen, Tove Lindahl Andersen, Ebba de Neergaard, and Marianne Stu¨rup-Johansen for skilled technical assistance. This study was supported by grants from the Danish Medical Research Council (No. 12–1336) and The Danish Biomembrane Center. Address reprint requests to H. Gissel.

18.

19. 20. 21.

Received 29 December 1997; accepted in final form 7 October 1998. REFERENCES 1. Adams, D. J., T. M. Dwyer, and B. Hille. The permeability of endplate channels to monovalent and divalent metal cations. J. Gen. Physiol. 75: 493–510, 1980. 2. Almers, W., and P. T. Palade. Slow calcium and potassium currents across frog muscle membrane: measurements with a vaseline-gap technique. J. Physiol. (Lond.) 312: 159–176, 1981. 3. Armstrong, R. B. Initial events in exercise-induced muscular injury. Med. Sci. Sports Exerc. 22: 429–435, 1990. 4. Ben Bachrir-Lamrini, L., B. Sempore, M. Mayet, and R. J. Favier. Evidence of a slow-to-fast fiber type transition in skeletal muscle from spontaneously hypertensive rats. Am. J. Physiol. 258 (Regulatory Integrative Comp. Physiol. 27): R352–R357, 1990. 5. Bianchi, C. P., and A. M. Shanes. Calcium influx in skeletal muscle at rest, during activity, and during potassium contracture. J. Gen. Physiol. 42: 803–815, 1959. 6. Campbell, D. T. Ionic selectivity of the sodium channel of frog skeletal muscle. J. Gen. Physiol. 67: 295–307, 1976. 7. Carafoli, E. Intracellular calcium homeostasis. Annu. Rev. Biochem. 56: 395–433, 1987. 8. Chinet, A., T. Clausen, and L. Girardier. Microcalorimetric determinations of energy expenditure due to active sodiumpotassium transport in the soleus muscle and brown adipose tissue of the rat. J. Physiol. (Lond.) 265: 43–61, 1977. 9. Chirandini, D. J., and E. Stefani. Calcium action potentials in rat fast-twitch and slow-twitch muscle fibers. J. Physiol. (Lond.) 335: 29–40, 1983. 10. Clausen, T., A. B. Dahl-Hansen, and J. Elbrink. The effect of hyperosmolarity and insulin on resting tension and calcium fluxes in rat soleus muscle. J. Physiol. (Lond.) 292: 505–526, 1979. 11. Clausen, T., and M. E. Everts. K1-induced inhibition of contractile force in rat skeletal muscle: role of active Na1-K1 transport. Am. J. Physiol. 261 (Cell Physiol. 30): C799–C807, 1991. 12. Clausen, T., and J. A. Flatman. The effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle. J. Physiol. (Lond.) 270: 383–414, 1977. 13. Clausen, T., and O. Hansen. Ouabain binding and Na1-K1 transport in rat muscle cells and adipocytes. Biochim. Biophys. Acta 345: 387–404, 1974. 14. Danieli-Betto, D., R. Betto, A. Megighian, M. Midro, G. Salviati, and L. Larsson. Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles. Acta Physiol. Scand. 154: 59–64, 1995. 15. Delbono, O., and B. A. Kotsias. Calcium action potentials in innervated and denervated rat muscle fibers. Pflu¨gers Arch. 418: 284–291, 1991. 16. Donoso, P., and C. Hidalgo. Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle. Biochim. Biophys. Acta 978: 8–16, 1989. 17. Duan, C., M. D. Delp, D. A. Hayes, P. D. Delp, and R. B. Armstrong. Rat skeletal muscle mitochondrial [Ca21] and in-

22. 23. 24. 25. 26.

27.

28.

29. 30.

31.

32. 33. 34. 35. 36.

R339

jury from downhill walking. J. Appl. Physiol. 68: 1241–1251, 1990. Everts, M. E., J. P. Andersen, T. Clausen, and O. Hansen. Quantitative determination of Ca21- dependent Mg21-ATPase from sarcoplasmic reticulum in muscle biopsies. Biochem. J. 260: 443–448, 1989. Everts, M. E., and T. Clausen. Effects of thyroid hormones on calcium contents and 45Ca exchange in rat skeletal muscle. Am. J. Physiol. 251 (Endocrinol. Metab. 14): E258–E265, 1986. Everts, M. E., T. Lomø, and T. Clausen. Changes in K1, Na1 and calcium contents during in vivo stimulation of rat skeletal muscle. Acta Physiol. Scand. 147: 357–368, 1993. Fride´n, J., M. Sjo¨stro¨m, and B. Ekblom. Myofibrillar damage following intense eccentric exercise in man. Int. J. Sports Med. 4: 170–176, 1983. Fryer, W. M., and D. G. Stephenson. Total and sarcoplasmic reticulum calcium contents of skinned fibers from rat skeletal muscle. J. Physiol. (Lond.) 493: 357–370, 1996. Howell, S. Compartmental analysis of Ca21 kinetics in longlasting diaphragm fatigue: loss of t-tubular membrane Ca21. J. Appl. Physiol. 80: 2009–2018, 1996. Hoya, A., and R. A. Venosa. Characteristics of the Na1-Ca21 exchange in frog skeletal muscle. J. Physiol. (Lond.) 486: 615– 627, 1995. Jackson, M. J., D. A. Jones, and R. H. T. Edwards. Experimental muscle damage: the nature of the calcium activated degenerative processes. Eur. J. Clin. Invest. 14: 369–374, 1984. Kirchberger, M. A., and M. Tada. Effects of adenosine 38:58monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles. J. Biol. Chem. 251: 725–729, 1976. Kohn, P. G., and T. Clausen. The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VI. The effect of insulin, ouabain, and metabolic inhibitors on the transport of 3-O-methylglucose and glucose in rat soleus muscles. Biochim. Biophys. Acta 225: 277–290, 1971. Lamb, G. D., P. R. Junakar, and D. G. Stephenson. Raised intracellular [Ca21] abolishes excitation-contraction coupling in skeletal muscle fibers of rat and toad. J. Physiol. (Lond.) 489: 349–362, 1995. Lamb, G. D., and T. Walsh. Calcium currents, charge movement and dihydropyridine binding in fast- and slow-twitch muscles of rat and rabbit. J. Physiol. (Lond.) 393: 595–617, 1987. Lee, J. D., H. Westerblad, and D. G. Allen. Changes in tetanic and resting [Ca21]i during fatigue and recovery of single muscle fibres from Xenopus laevis. J. Physiol. (Lond.) 433: 307–326, 1991. Luff, A. R., and H. L. Atwood. Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal muscles of the mouse during postnatal development. J. Cell Biol. 51: 369–383, 1971. McCutcheon, L. J., S. K. Byrd, and D. R. Hodgson. Ultrastructural changes in skeletal muscle after fatiguing exercise. J. Appl. Physiol. 72: 1111–1117, 1992. Nielsen, H. G., and T. Clausen. Excitation induced accumulation in rat skeletal muscle. Does the muscle function as a ‘‘Ca21 trap’’? (Abstract). Acta Physiol. Scand. 157: 30A, 1996. Nielsen, O. B., and T. Clausen. Regulation of Na1-K1 pump activity in contracting rat muscle. J. Physiol. (Lond.) 503: 571–581, 1997. Santana, L. F., A. M. Go´mez, and W. J. Lederer. Ca21 flux through promiscuous cardiac Na1 channels: slip-mode conductance. Science 279: 1027–1033, 1998. Sreter, F. A., K. Mabuchi, A. Ko¨ver, I. Gesztelyi, Z. Nagy, and I. Furka. Effect of chronic stimulation on cation distribution and membrane potential in fast-twitch muscles of rabbit. In: Plasticity of Muscle, edited by D. Pette. New York: de Gruyter, 1980, p. 441–451.