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This Article Abstract Full Text (PDF) All Versions of this Article: 90/5/703    most recent expphysiol.2005.030247v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fredsted, A. Articles by Clausen, T. Search for Related Content PubMed PubMed Citation Articles by Fredsted, A. Articles by Clausen, T. Related Collections Muscle

¹ Department of Physiology, University of Aarhus, Århus, Denmark

	Abstract

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Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca²⁺, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O₂, 5% CO₂), anoxia (95% N₂, 5% CO₂) or varying degrees of reduced oxygenation. At varying extracellular Ca²⁺ concentrations ([Ca²⁺]_o), ⁴⁵Ca influx and total cellular Ca²⁺ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca²⁺, 15–75 min of exposure to anoxia increased ⁴⁵Ca influx by 46–129% (P < 0.001) and Ca²⁺ content by 20–50% (P < 0.001). Mg²⁺ (11.2 mM) reduced the anoxia-induced increase in ⁴⁵Ca influx by 43% (P < 0.001). In muscles incubated at 20 and 5% O₂, ⁴⁵Ca influx was also stimulated (P < 0.001). Increasing [Ca²⁺]_o to 5 mM induced a progressive increase in both ⁴⁵Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca²⁺ content and LDH release increased markedly and showed a significant correlation (r² = 0.55, P < 0.001). In conclusion, anoxia or incubation at 20 or 5% O₂ leads to an increased influx of ⁴⁵Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca²⁺. The loss of cell membrane integrity further increases Ca²⁺ influx, which may elicit a self-amplifying process of cell membrane leakage.

(Received 21 February 2005; accepted after revision 13 May 2005; first published online 20 May 2005)
Corresponding author A. Fredsted: Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Århus C, Denmark. Email: af{at}fi.au.dk

	Introduction

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It is well established that prolonged hypoxia or anoxia causes cellular damage in skeletal muscle. This has been documented by measurements of the release of the intracellular enzymes creatine kinase (CK) and lactic acid dehydrogenase (LDH) from isolated muscles in vitro (Jones et al. 1983; McCall & Duncan, 1989) or from muscles to the plasma in vivo in animals and human subjects (Highman & Altland, 1960; McCall & Duncan, 1989; Wilber et al. 2000). Jones et al. (1984) showed that the anoxia-induced loss of LDH from mouse soleus muscle could be reduced by 60–70% by omitting Ca²⁺ from the incubation medium. When exposed to anoxia, skeletal muscles develop contractures (Jones et al. 1983) and in keeping with this, it was shown that anoxia increases the resting cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in single frog muscle fibres (Kohin et al. 2001). Similar observations were made on mouse C2C12 myotubes and porcine primary satellite cells (Lambert et al. 2001; Ørtenblad et al. 2003). It was also shown that the rate of rise in cytoplasmic Ca²⁺ concentration was reduced by lowering the concentration of Ca²⁺ in the incubation medium (Lambert et al. 2001). Taken together these observations indicate that anoxia increases the influx of Ca²⁺ in skeletal muscle and that this triggers cell damage.

We have previously reported that when the uptake of Ca²⁺ in isolated skeletal muscle is increased by electrical stimulation, electroporation or the Ca²⁺ ionophore A23187 [GenBank] , significant cell membrane damage takes place and that this can be correlated with the uptake or content of Ca²⁺ in the muscle (Gissel & Clausen, 2000, 2003).

Prolonged increase in [Ca²⁺]_i may initiate a cascade of degradative mechanisms. Ca²⁺-dependent proteases such as calpains (Duan et al. 1990; Belcastro, 1993; Belcastro et al. 1998) and phospholipases such as phospholipase A₂ (PLA₂) (Duan et al. 1990; Duncan & Jackson, 1987) may be activated leading to damage to the sarcolemma or cytoskeleton. Increased [Ca²⁺]_i may also augment the production of reactive oxygen species (ROS) leading to peroxidation of membrane lipids (Reid & Li, 2001), thus contributing to the muscle damage. Increased plasma concentration of products of lipid peroxidation have been found both in patients with chronic obstructive pulmonary disease (COPD) and patients with chronic heart failure (CHF) (Gosker et al. 2000). This may in part be due to an increased production of ROS, caused by an intracellular Ca²⁺ overload in the muscles of these patients. Elevated production of ROS may in turn lead to increased membrane permeability. Due to these Ca²⁺-activated degradative mechanisms, intracellular enzymes such as LDH or CK may leak out through the damaged sarcolemma.

In an attempt to reduce the cell damage, various agents have been used to inhibit the Ca²⁺ influx. Tetrodotoxin (TTX, a Na⁺ channel inhibitor) has been shown to reduce Ca²⁺ influx in myotubes (Lambert et al. 2001), whereas Mg²⁺ has been shown to inhibit Ca²⁺ channels, therefore reducing the Ca²⁺ influx to cardiomyocytes (Sharikabad et al. 2001a, 2001b).

In the above-mentioned studies on the effects of anoxia on muscle cell damage, the uptake of ⁴⁵Ca or Ca²⁺ content were not measured. Therefore, the present study was performed to test the hypothesis that muscle cell membrane damage induced by anoxia to a large extent is caused by increased influx of Ca²⁺ from the extracellular phase. This was examined in isolated rat extensor digitorum longus (EDL) muscles by comparing ⁴⁵Ca influx and Ca²⁺ content with LDH release during exposure to electrical stimulation or variations in [Ca²⁺]_o. In most experiments, muscles exposed to total anoxia were compared to muscles exposed to the standard gas mixture of 95% O₂ and 5% CO₂. The effects of graded oxygenation (20 or 5% O₂) were tested in a few experiments. Some of the results have previously been presented in a preliminary version (Fredsted et al. 2004).

	Methods

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Animals

All experiments were carried out using 4-week-old female and male Wistar rats (own breed) weighing 60–70 g and had free access to food and water. Animals of this size were chosen to obtain muscles small enough (20–25 mg) to improve diffusion of gas, ions and substrates during incubation. Experiences from this laboratory have shown that relatively large molecules (TTX, tubocurarine and ouabain) exert full effect within 15 min of incubation in this size of muscle. The animals were kept at constant temperature (21°C) and day length (12 h). All handling and use of animals complied with Danish animal welfare regulations.

Muscle preparation and incubation

The animals were killed by cervical dislocation followed by decapitation, and intact EDL muscles were dissected out as previously described (Chinet et al. 1977). The standard incubation medium was a bicarbonate-based Krebs–Ringer (KR) solution (pH 7.3) containing (mM): NaCl 122.1, NaHCO₃ 25.1, KCl 2.8, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 1.3 and D-glucose 5.0. Incubation took place at 30°C in a volume of 5 ml. The buffer was continuously gassed with a mixture of O₂, N₂ and 5% CO₂ depending on the experiment. After preparation, the muscles were mounted at resting length on muscle holders with electrodes allowing electrical stimulation with isometric contractions when indicated. They were equilibrated with a mixture of 95% O₂ and 5% CO₂ in the standard medium (1.3 mM Ca²⁺) for 30 min before further treatment. This procedure has been shown to allow the maintenance of constant K⁺, Na⁺ and Ca²⁺ content for several hours in vitro (Clausen & Flatman, 1977; Everts et al. 1993).

Experimental protocols

This study was performed to investigate ⁴⁵Ca uptake, Ca²⁺ content and muscle cell integrity in muscles exposed to anoxia compared to muscles incubated under standard conditions (95% O₂). The effects of a graded [Ca²⁺]_o as well as a graded reduction in oxygenation from standard conditions were also studied. Three experimental protocols were applied.

(1) Resting muscles (0–20% O₂ versus 95% O₂) exposed to either (a) normal [Ca²⁺]_o (1.3 mM) or (b) high [Ca²⁺]_o (5.0 mM). In these experiments ⁴⁵Ca uptake and efflux, total muscle Ca²⁺ content, and release of LDH were measured. This was done to study the effects of anoxia (and oxygen tensions below our standard conditions) on Ca²⁺ flux and muscle cell integrity.

(2) Rest or 30-min fatiguing stimulation followed by 150 min rest (0 versus 95% O₂ throughout the experiment). Muscles were exposed to either (a) normal or (b) graded [Ca²⁺]_o (0.3–5.0 mM). In these experiments, LDH release and total muscle Ca²⁺ content were measured. This was done to investigate how the muscles tolerate metabolic stress under conditions of an increased oxygen demand as well as an increased Ca²⁺ load due to excitation-induced influx of Ca²⁺.

(3) Rest or 30-min fatiguing stimulation followed by 120 min rest at 0% O₂ followed by 120 min reoxygenation at 95% O₂. This was done at normal [Ca²⁺]_o. LDH release was measured in these experiments to investigate the effects of reoxygenation on muscle cell integrity.

Oxygenation

After equilibration for 30 min the muscles on holders were moved to new tubes containing buffer pre-gassed (30 min) with the indicated mixture of N₂, O₂ and 5% CO₂. The same gas mixture was used during electrical stimulation as well as during the following period of rest in all experiments other than the reoxygenation experiments where gassing was changed to 95% O₂ and 5% CO₂ following 120 min rest. To investigate a possible graded response to a reduction in oxygen tension from standard conditions (95% O₂), 5 and 20% oxygen were chosen.

Electrical stimulation

A previously developed standard fatiguing stimulation protocol was applied in the stimulation-experiments (Mikkelsen et al. 2004). Following equilibration, muscles were stimulated intermittently for 30 min (10 s on, 30 s off) at 40 Hz (1 ms pulses of 10 V). The set-up allowed for simultaneous stimulation of 12 muscles in 5-ml incubation chambers. Stimulation was applied through two platinum electrodes (4 mm apart) placed on the muscle holder on either side of the central part of the muscle.

Resting ⁴⁵Ca uptake

The rate of ⁴⁵Ca uptake was measured over a 15-min period at various time points throughout the experiment. The muscles were incubated in 5 ml buffer containing ⁴⁵Ca (0.5 µCi ml^–1). Immediately after incubation the muscles were washed (4 x 30 min) at 0°C in Ca²⁺-free buffer containing 0.5 mM EGTA in order to remove extracellular ⁴⁵Ca. Finally the tendons were removed, and the muscles were blotted, weighed and soaked overnight in 3 ml 0.3 M trichloroacetic acid (TCA). The next day ⁴⁵Ca activity of the TCA extract was determined by liquid scintillation counting (Packard, Tri-Carb 2100 TR) and ⁴⁵Ca uptake was calculated on the basis of this and the specific activity of ⁴⁵Ca in the incubation medium. The uptake was corrected for loss of intracellular ⁴⁵Ca during the washout by a previously determined correction factor of 1.6 (Gissel & Clausen, 1999). In the experiments where the effect of increasing [Ca²⁺]_o was investigated, muscles were preincubated for 15 min in buffer containing either 1.3 or 5.0 mM Ca²⁺ during oxygenation. In the following anoxic period they were also incubated in buffer with either 1.3 or 5.0 mM Ca²⁺. Further treatment was as described above.

⁴⁵Ca efflux

In order to determine whether the increase in Ca²⁺ uptake during anoxia was due to a decrease in Ca²⁺ efflux, a ⁴⁵Ca efflux experiment was performed using a standard technique described earlier (Clausen et al. 1975). Following equilibration, muscles were loaded with ⁴⁵Ca for 60 min at 30°C in buffer containing ⁴⁵Ca (2 µCi ml^–1). Hereafter the muscles were mounted at resting length on muscle holders and washed for 200 min at 30°C in KR solution. The muscles were exposed to anoxia the last 70 min of the washout period. The muscles on holders were moved to new tubes every 10 or 20 min. At the end of the experiment the tendons were removed, and the muscles were blotted, weighed and soaked overnight in 3 ml 0.3 M TCA. The next day, ⁴⁵Ca activity of the TCA extract of the muscle and samples from every tube of the washout buffer was determined by liquid scintillation counting. From these values the fractional loss of ⁴⁵Ca per minute was calculated.

LDH release

Changes in muscle cell integrity were monitored by measuring the release of LDH into the incubation medium. After the muscles had been mounted on the muscle holders they were prewashed (4 x 30 min) under standard conditions (95% O₂, 5% CO₂ and 1.3 mM Ca²⁺) to wash out any LDH released from cells damaged during excision of the muscles. Buffer samples (500 µl) for determination of the baseline level of LDH activity were taken from the last of the four prewash tubes. Muscles were either stimulated or resting for 30 min, followed by recovery at rest. In experiments with varying [Ca²⁺]_o, the muscles were incubated in buffer containing 0.3, 1.3, 2.5 or 5.0 mM Ca²⁺ only during recovery. The muscles were moved to new tubes every 30 min, and buffer samples for determination of LDH release were taken immediately after removal of the muscles. In brief, the LDH activity in the incubation medium was determined by spectrophotometric measurement of the decrease in the concentration of the substrate NADH by conversion of pyruvate to lactate. Activity was expressed as units per gram muscle wet weight. A buffer sample of 0.25 ml was mixed with 2.65 ml phosphate buffer containing NADH (0.4 mM) and pyruvate (0.4 mM). Absorbance was measured at 340 nm at 30°C (for further details see Gissel & Clausen, 2003). Spontaneous release of LDH was monitored in resting control muscles.

Ca²⁺ content

At the time indicated the muscles were taken off the holders and blotted, and the tendons carefully cut off, weighed, and then soaked overnight in 3 ml 0.3 M TCA to extract Ca²⁺. Previous studies showed that the extraction of Ca²⁺ from the whole muscle was as complete as that achieved by homogenization and subsequent centrifugation of the TCA extract (Clausen et al. 1993; Gissel & Clausen, 2000). Ca²⁺ content was determined by atomic absorption spectrophotometry (Phillips PU 9200, Pye Unicam, Cambridge, UK) using 1.5 ml TCA extract mixed with 150 µl 0.27 M KCl. The muscle extracts were measured against a blank and standards containing 12.5 or 25 µM Ca²⁺ and the same amount of TCA and KCl. The Ca²⁺ contents were corrected for loss of intracellular Ca²⁺ during the ice-cold washout by a previously determined correction factor of 1.6 (Gissel & Clausen, 1999).

Chemicals and isotope

All chemicals were of analytical grade. TTX and nifedipine were purchased from Sigma Chemical Co. (St Louis, MO, USA), and NADH and pyruvate from Boehringer Mannheim (Germany). ⁴⁵Ca (1.31 Ci mmol^–1) was obtained from Amersham International (Aylesbury, UK).

Statistics

Results are given as mean values ± S.E.M. The statistical significance of any difference between groups was ascertained using the two-tailed Student's t test for unpaired observations, while the statistical significance of multiple comparisons was ascertained using a one-way ANOVA followed by Tukey's post hoc test. Significant differences were accepted when *P < 0.05, **P < 0.01 or ***P < 0.001.

	Results

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Ca²⁺ fluxes and cellular Ca²⁺ content

Figure 1 shows the time course of changes in the rate of ⁴⁵Ca uptake (measured in 15-min periods) in resting anoxic or oxygenated muscles. Resting oxygenated muscles showed very little variation in the rate of ⁴⁵Ca uptake during the 150 min of measurements. In contrast, the anoxic muscles showed an increase in ⁴⁵Ca uptake, which was significant after 15 min of anoxia (0.106 ± 0.004 µmol (g wet wt)^–1 (15 min)^–1 compared to 0.072 ± 0.005 µmol (g wet wt)^–1 (15 min)^–1 in the oxygenated controls, P < 0.001). In the following 150 min of anoxia a further progressive increase in ⁴⁵Ca uptake was observed, reaching a rate of 0.191 ± 0.007 µmol (g wet wt)^–1 (15 min)^–1 in the last time interval, 3-fold higher than the uptake in oxygenated muscles (P < 0.001).

View larger version (13K): [in this window] [in a new window]   Figure 1.  Rate of 45Ca uptake in rat EDL muscles exposed to anoxia Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and exposed to oxygenation (95% O2, 5% CO2) or anoxia (95% N2, 5% CO2) for 15–150 min. Muscles were resting throughout treatment and were incubated with 45Ca (0.5 µCi ml–1) for the final 15 min of the incubation period, thereafter they were taken for washout (4 x 30 min) in ice-cold Ca2+-free buffer. The rates of 45Ca uptake are indicated in the middle of the 15-min measurement period. All values are means with error bars indicating 2 x S.E.M., n = 6–12 muscles.

View larger version (14K): [in this window] [in a new window]   Figure 2.  Rate of 45Ca uptake in rat EDL muscles exposed to varying degrees of oxygenation Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and exposed to standard oxygenation (95% O2, 5% CO2), reduced oxygenation (20% O2, 75% N2, 5% CO2) or (5% O2, 90% N2, 5% CO2) or anoxia (95% N2, 5% CO2) for 15 or 90 min. All muscles were resting and incubated with 45Ca (0.5 µCi ml–1) for the final 15 min of the experiment. Finally, the muscles were taken for washout (4 x 30 min) in ice-cold Ca2+-free buffer. Values are means with error bars showing S.E.M., n = 6 muscles. At the top of each bar is% O2. Significant differences from oxygenated controls, **P < 0.01 and ***P < 0.001.

View larger version (13K): [in this window] [in a new window]   Figure 3.  Rate of fractional loss of 45Ca in rat EDL muscles exposed to oxygenation followed by anoxia Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and exposed to standard oxygenation (95% O2, 5% CO2). Muscles were loaded for 60 min at 30°C in buffer containing 45Ca (2 µCi ml–1). Thereafter the muscles were washed for 200 min at 30°C in KR solution, and moved to new tubes every 10 or 20 min. The muscles were exposed to anoxia (95% N2, 5% CO2) in the last 70 min of the washout period. Values are means with error bars indicating 2 x S.E.M., n = 4–12 muscles.

Effects of [Ca²⁺]_o on resting ⁴⁵Ca uptake and LDH release

As anoxia increased the influx of ⁴⁵Ca from the extracellular space, the effects of increasing [Ca²⁺]_o were examined. As shown in Fig. 4, increasing [Ca²⁺]_o from 1.3 to 5.0 mM augmented ⁴⁵Ca uptake by 165% (P < 0.001) after 15 min of anoxia and by 397% (P < 0.001) after 90 min of anoxia. This was associated with significant increases in muscle Ca²⁺ content (data not shown).

View larger version (10K): [in this window] [in a new window]   Figure 4.  Rate of 45Ca uptake in rat EDL muscles exposed to anoxia and different [Ca2+]o Muscles were mounted at resting length in KR solution with either 1.3 or 5.0 mM Ca2+ and exposed to oxygenation (95% O2, 5% CO2) or anoxia (95% N2, 5% CO2) for 15 or 90 min. All muscles were resting and incubated with 45Ca (0.5 µCi ml–1) during the final 15 min of the experiment. Finally, the muscles were taken for washout (4 x 30 min) in ice-cold Ca2+-free buffer. Values are means with error bars showing 2 x S.E.M., n = 3–6 muscles.

Figure 5 shows the effect of varying [Ca²⁺]_o on the LDH release from resting anoxic muscles. Resting anoxic muscles showed no significant increase in LDH release in the first 90 min of incubation, and only in the subsequent 30-min period was LDH release significantly increased (P < 0.05). However if muscles were incubated in the presence of 5.0 mM Ca²⁺, there was a progressive increase in LDH release already after 60 min of anoxia, reaching a plateau of between 4 and 4.5 U (g wet wt)^–1 (30 min)^–1 after 90 min (P < 0.001), corresponding to an approximately 10-fold increase above the pre-anoxic level.

View larger version (12K): [in this window] [in a new window]   Figure 5.  Rate of LDH release in resting rat EDL muscles exposed to anoxia and different [Ca2+]o Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscle. Following prewash, muscles were exposed to anoxia (95% N2, 5% CO2) and incubated in buffer with 1.3 or 5.0 mM Ca2+. Muscles were resting throughout the experiment. The muscles were moved to new tubes every 30 min and after removal of the muscles, buffer samples were taken for determination of LDH activity. The values given in the middle of the 30-min periods are means with error bars indicating 2 x S.E.M., n = 3–24 muscles. Significant differences from the pre-anoxic level, *P < 0.05 and ***P < 0.001.

We have previously shown that in oxygenated muscles electrical stimulation increases Ca²⁺ influx, Ca²⁺ content and LDH release (Gissel, 2000; Gissel & Clausen, 2003). It was of interest therefore to examine whether these effects of electrical stimulation were modified by anoxia.

Ca²⁺ content.  Ca²⁺ content in muscles incubated under standard conditions (95% O₂, 5% CO₂ and 1.3 mM Ca²⁺) and exposed to stimulation for 30 min followed by 15 min of rest, was 1.55 ± 0.08 µmol (g wet wt)^–1 (n = 8). This is a non-significant increase of 0.18 ± 0.10 µmol (g wet wt)^–1 (P = 0.18) compared to resting controls (Table 1). In contrast to this, if the muscles were exposed to anoxia during this treatment, Ca²⁺ content increased to 2.66 ± 0.09 µmol (g wet wt)^–1 (n = 6) which is a significant increase of 0.61 ± 0.17 µmol (g wet wt)^–1 (P < 0.05) compared to resting muscles exposed to 45 min of anoxia (Table 1). Thus the net Ca²⁺ accumulation caused by stimulation and a 15-min recovery period is 3.4-fold higher in the anoxic muscles than in the standard oxygenated muscles (0.61 and 0.18 µmol (g wet wt)^–1, respectively).

LDH release.  Figure 6 shows the LDH release from resting or stimulated anoxic muscles compared with muscles incubated under standard conditions (95% O₂, 5% CO₂ and 1.3 mM Ca²⁺). Resting oxygenated muscles showed no change in LDH release for 180 min. When stimulated for 30 min under oxygenated conditions, LDH release increased from 0.42 ± 0.10 U (g wet wt)^–1 (30 min)^–1 at pre-stimulation level to 2.00 ± 0.15 U (g wet wt)^–1 (30 min)^–1 (P < 0.001) in the first 30 min after cessation of stimulation. No further increase was observed during the following 120 min of recovery. If the muscles were exposed to anoxia and stimulation the LDH release increased from 0.48 ± 0.04 U (g wet wt)^–1 (30 min)^–1 at pre-stimulation level to 1.78 ± 0.22 U (g wet wt)^–1 (30 min)^–1 (P < 0.001) in the first 30 min after cessation of stimulation. This was followed by a further progressive increase, reaching 6.04 ± 0.42 U (g wet wt)^–1 (30 min)^–1 (P < 0.001) after 150 min of rest. In conclusion, the modest increase in LDH release seen in resting anoxic muscles is dramatically augmented (5.7-fold) by electrical stimulation.

View larger version (17K): [in this window] [in a new window]   Figure 6.  Rate of LDH release from rat EDL muscles exposed to anoxia and electrical stimulation Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscles. Following this prewash, muscles were exposed to oxygenation (95% O2, 5% CO2) or anoxia (95% N2, 5% CO2). The first 30 min of this incubation the muscles were either resting or stimulated for 30 min (40 Hz, 10 s on, 30 s off, 10 V, 1 ms pulses) followed by 120 min of rest. The muscles were moved to new tubes every 30 min and after removal of the muscles, buffer samples were taken for determination of LDH activity. The values given in the middle of the 30-min periods are means with error bars showing 2 x S.E.M., n = 3–24 muscles.

View larger version (12K): [in this window] [in a new window]   Figure 7.  Cellular Ca2+ content and LDH release in rat EDL muscles exposed to anoxia and electrical stimulation Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscle. Following this prewash all muscles were exposed to anoxia (95% N2, 5% CO2) and during the first 30-min period they were stimulated (40 Hz, 10 s on, 30 s off, 10 V, 1 ms pulses). The muscles were moved to new tubes every 30 min. After removal of the muscles buffer samples were taken for determination of LDH activity. Muscles were taken at the time points indicated for measurements of total cellular Ca2+ content. Muscles were incubated under anoxic conditions for 120 min (•), 180 min (), 240 min () and 270 min (). A linear regression line is presented, with the corresponding correlation coefficient and the level of significance.

View larger version (15K): [in this window] [in a new window]   Figure 8.  Rate of LDH release in stimulated rat EDL muscles exposed to anoxia and various [Ca2+]o Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscle. Following prewash, muscles were exposed to anoxia (95% N2, 5% CO2) throughout the experiment. The muscles were stimulated for 30 min (40 Hz, 10 s on, 30 s off, 10 V, 1 ms pulses) in buffer containing 1.3 mM Ca2+ and in the following resting period exposed to 0.3, 1.3 or 5.0 mM [Ca2+]o as indicated. The muscles were moved to new tubes every 30 min and after removal of the muscles buffer samples were taken for determination of LDH activity. The values given in the middle of the 30-min periods are means with error bars indicating 2 x S.E.M., n = 3–4 muscles.

View larger version (12K): [in this window] [in a new window]   Figure 9.  Relationship between Ca2+ content and LDH release in rat EDL muscles exposed to anoxia, stimulation and various [Ca2+]o Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscle. Following prewash, muscles were exposed to anoxia (95% N2, 5% CO2) for 180 min. Muscles were initially stimulated for 30 min (40 Hz, 10 s on, 30 s off, 10 V, 1 ms pulses) at 1.3 mM Ca2+ and in the following resting period exposed to 0.3, 1.3, 2.5 or 5.0 mM [Ca2+]o. The muscles were moved to new tubes every 30 min. After removal of the muscles at the end of the experiment, buffer samples were taken for determination of LDH activity. Muscles were analysed for total cellular Ca2+ content. Values are means with error bars indicating 2 x S.E.M., n = 6–7 muscles.

View larger version (16K): [in this window] [in a new window]   Figure 10.  Rate of LDH release in stimulated rat EDL muscles exposed to anoxia and reoxygenation Muscles were mounted at resting length in KR solution (1.3 mM Ca2+) and washed (4 x 30 min) at 30°C in order to remove the LDH released during excision of the muscle. Following prewash, muscles were exposed to anoxia (95% N2, 5% CO2). Muscles were initially stimulated for 30 min (40 Hz, 10 s on, 30 s off, 10 V, 1 ms pulses). Following 150 min of anoxia (30 min stimulation followed by 120 min rest), some muscles were exposed to reoxygenation (95% O2, 5% CO2) for 120 min. The muscles were moved to new tubes every 30 min and after removal of the muscles buffer samples were taken for determination of LDH activity. The values given in the middle of the 30-min periods are means with error bars indicating 2 x S.E.M., n = 4–40 muscles. *Significant difference between anoxic and reoxygenated muscles (P < 0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

Oxygenation

In keeping with many in vitro studies on isolated skeletal muscles in KR solution, we used a mixture of 95% O₂ and 5% CO₂ for gassing. The high-oxygen content was used to allow better oxygenation of the deeper fibres, but the possibility that it may cause the generation of reactive oxygen species (ROS), which in turn could give rise to cellular damage, cannot be excluded. However, as shown in Figs 1 and 6, incubation for 150–180 min showed no increase in ⁴⁵Ca uptake or LDH release, indicating maintenance of cellular integrity. It was also a constant finding that rat EDL muscles could maintain constant tetanic force for many hours in vitro.

The majority of the experiments compared the standard gassing with 95% O₂ and 5% CO₂ with a mixture containing 95% N₂ and 5% CO₂. In a few instances we examined the effects of graded oxygenation, using gas mixtures containing 5 or 20% O₂. It should be noted that with these mixtures, the oxygen tension in the buffer may have been higher than that normally reached in muscle capillaries. In spite of this, there is reason to expect that the supply of oxygen to the deeper fibres would be lower than in the buffer and even lower than in the experiments performed using 95% O₂. Unfortunately, we could not predict the O₂ tension in different layers of the inner muscle fibres, primarily due to the stirring induced by the continuous gassing.

⁴⁵Ca fluxes

Only 15 min exposure of resting muscles to anoxia causes an increase in ⁴⁵Ca uptake. The experiments with varying degrees of oxygenation showed that even when isolated muscles were exposed to buffer gassed with 5 or 20% O₂, ⁴⁵Ca influx increased. As the reduction in oxygenation became more severe, ⁴⁵Ca uptake was augmented, in particular during prolonged exposure. In the anoxic muscles the increase in ⁴⁵Ca uptake was associated with a net increase in total muscle Ca²⁺ content. As the Ca²⁺ content of the muscles depends on two processes, the Ca²⁺ uptake and the Ca²⁺ efflux across the cell membrane, it can be concluded that the influx of Ca²⁺ exceeds the efflux of Ca²⁺ despite an observed increase in both. The mechanism behind the anoxia-induced increase in Ca²⁺ uptake is still unidentified. In a study on myotubes by Lambert et al. (2001) it was found that TTX (which blocks the Na⁺ channels) inhibited the anoxia-induced rise in free cytosolic Ca²⁺ concentration by 93%. However, in our study TTX only reduced the anoxia-induced increase in ⁴⁵Ca uptake by 22–26%. Nifedipine caused no reduction in ⁴⁵Ca uptake, suggesting that the L-type Ca²⁺ channels are not involved. In rat cardiomyocytes, increased extracellular Mg²⁺ concentration (5 mM) has been shown to reduce [Ca²⁺]_i by 40% and LDH release by 90% after 60 min of anoxia followed by 120 min of reoxygenation (Sharikabad et al. 2001a,b). In our experiments, around 40% of the anoxia-induced increase in ⁴⁵Ca uptake was inhibited by 11.2 mM Mg²⁺. However, as there was no additive effect of TTX and Mg²⁺, the mechanism responsible for the remaining 60% of the initial Ca²⁺ influx observed during anoxia still remains to be identified. Recently there has been much focus on the superfamily of transient receptor potential (TRP) channels. Some of these have a high permeability for Ca²⁺, and may be activated by mitochondrial inhibition or under conditions where cellular ATP is decreased (Toescu, 2004). As low ATP levels are expected during anoxia, TRP channels could be involved in the observed influx of Ca²⁺. However, further studies are needed to identify whether TRP channels are actually involved in Ca²⁺ influx.

Membrane integrity

An increased influx of Ca²⁺ may in time lead to muscle Ca²⁺ overload and muscle membrane leakage (Gissel & Clausen, 2000, 2003). As an indicator of this we measured the LDH release from the muscle (Gissel & Clausen, 2000). In resting anoxic muscles, LDH release showed no increase until after 120 min of incubation (Fig. 6), indicating a relatively late onset of membrane leakage. This is important because it shows that the early increase in ⁴⁵Ca uptake induced by anoxia is not due to a general leakage of the cell membrane, but is mediated by a specific transport system. Earlier studies by Jones and colleagues on mouse soleus muscle incubated at 2 mM Ca²⁺ showed that 30 min exposure to anoxia leads to increases in LDH release in resting as well as in stimulated muscles (Jones et al. 1983, 1984). In accordance with our study they found that the elevated LDH release was exacerbated if the muscles were stimulated during exposure to anoxia. In a study by McCall & Duncan (1989), an increase in CK release was observed in resting mouse soleus muscle, incubated at 2.55 mM Ca²⁺, after 90 min of exposure to N₂. Again, stimulation amplified the CK response.

The present study showed that when resting muscles were exposed to 5.0 mM [Ca²⁺]_o, ⁴⁵Ca uptake was further increased. Concomitantly, LDH release and thus membrane leakage occured earlier and was much more pronounced. Jones et al. (1984) showed that the exclusion of calcium from the incubation medium led to a reduced LDH release in stimulated anoxic soleus muscles. In our experiments LDH release from muscles exposed to stimulation was also very dependent on [Ca²⁺]_o during the subsequent recovery. During the stimulation all muscles were incubated at 1.3 mM Ca²⁺ but the higher the [Ca²⁺]_o during recovery, the more LDH was released. Thus only differentiating [Ca²⁺]_o in the recovery period was enough to elicit a large difference in the LDH response. This emphasizes the importance of the resting uptake of Ca²⁺ following stimulation in the development of plasma membrane damage.

It should be mentioned that during the experiments described above, only a modest fraction of the total LDH content was released from the muscles. The cumulated release of LDH from the stimulated muscles incubated at 5.0 mM Ca²⁺ was 45 U (g wet wt)^–1 after 180 min of exposure to anoxia, amounting to only 7.7% of their total LDH content (Gissel & Clausen, 2000). Muscles incubated at 1.3 mM Ca²⁺ released only 4.1% of their total LDH content. However, the values do not permit distinguishing between widespread, but partial cell damage and localized, more complete loss of integrity of individual cells.

Cellular Ca²⁺ content and membrane damage

In the anoxic muscles the total cellular Ca²⁺ content is markedly higher following stimulation and a short recovery period than in the oxygenated muscles. In a study by Tupling et al. (2001) it was found that muscle ATP content was fully depleted in rat skeletal muscle during total ischaemia, and a concomitantly reduced sarcoplasmic reticulum (SR) Ca²⁺ uptake due to impaired Ca²⁺ pump function was observed. In the anoxic muscles, the lack of ATP impaired the clearance of Ca²⁺ from the cytosol by Ca²⁺-ATPases in the SR and the plasma membrane. This would lead to a rise in [Ca²⁺]_i with possible detrimental consequences. If the increase in [Ca²⁺]_i leads to damage to the membrane, further influx of Ca²⁺ will occur and a self-amplifying process may be initiated. The correlation between Ca²⁺ content and LDH release shown in Fig. 7 supports the hypothesis that increased cellular Ca²⁺ concentration, caused by anoxia-induced Ca²⁺ influx from the extracellular space, is the initiator of cell membrane damage indicated by a concomitant increase in LDH release. However, as shown in Fig. 9, it seems that the cells become saturated with Ca²⁺ and LDH release reaches a plateau when [Ca²⁺]_o is raised above 2.5 mM, leading to a cellular Ca²⁺ content above 5 µmol (g wet wt)^–1.

A self-amplifying process?

The data on reoxygenation show that the anoxia-induced membrane damage evaluated by LDH release is not readily reversible. Reoxygenation does not diminish or even stabilize the LDH release following a period of anoxia. The increased permeability of the cell membrane allows further influx of Ca²⁺ resulting in the 2- to 2.5-fold higher Ca²⁺ content observed in these muscles compared to resting oxygenated muscles. Our results seem to indicate that once initiated the damage process continues despite reoxygenation. This is well in agreement with reperfusion injury studies and brings further support to the idea of a self-amplifying process. The increased LDH release during reoxygenation could be due to production of reactive oxygen radicals.

ROS

Several studies have suggested an increased level of ROS during anoxia in skeletal muscle (Mohanraj et al. 1998; Clanton et al. 1999; Tsutsui et al. 2001; Ilavazhagan et al. 2001; Ørtenblad et al. 2003). Loss of Ca²⁺ homeostasis also stimulates the synthesis of superoxide anions from xanthine oxidase (Clanton et al. 1999; Reid, 2001). Superoxide anions undergo electron exchange reactions and the outcome is hydroxyl radicals. Increased levels of ROS can initiate lipid peroxidation and thereby induce changes and disruptions in the cell membrane, which would lead to an increase in the uptake of Ca²⁺. The effect of antioxidant treatment in rats exposed to hypoxia has been investigated in a couple of studies. Ørtenblad et al. (2003) showed that when an antioxidant (BHT) was added to the medium there was an almost total reduction in ROS production in anoxic porcine myotube cultures. Ilavazhagan et al. (2001) have shown a significant reduction in plasma LDH level in rats exposed to simulated hypoxia of 7620 m after vitamin E supplementation. Patients with COPD may also be exposed to increased levels of oxidative stress in the skeletal muscles (Langen et al. 2003; Couillard et al. 2003). ROS may very well be involved in the damage process in our studies. However, we have not tested the effects of antioxidants in our experiments.

Perspectives

General or localized anoxia/hypoxia is a frequently occurring condition during surgery, trauma, transplantations and diabetes. In general these situations can lead to anoxia-induced cell damage in the muscles involved. Patients with circulatory or respiratory insufficiency are also exposed to hypoxia and may thus be at risk of chronic skeletal muscle cell damage (Gosker et al. 2000). Loss of muscle mass and decline in muscle cross-sectional area (sarcopenia) are commonly observed among patients with hypoxia, leading to reduced muscle strength and endurance. Athletes training at high altitudes are also exposed to hypoxia, and it has been shown that this acute hypoxia leads to increased activity (3-fold) of intracellular enzymes in plasma even when training is not intensified (Wilber et al. 2000).

Conclusions

In rat EDL muscle, anoxia induces increased influx and accumulation of Ca²⁺, which is correlated with release of LDH. The degree of muscle cell membrane damage is very dependent on [Ca²⁺]_o. This indicates that influx of external Ca²⁺, possibly combined with a lack of ATP, plays a role in the mechanisms of anoxia-induced loss of cell membrane integrity in skeletal muscle. An increased membrane leakage of Ca²⁺ into the cells may result in a self-amplifying process, continuing long after the cessation of exercise or anoxia.

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	Acknowledgements

 
We thank Vibeke Uhre, Tove Lindahl Andersen, Marianne Stürup-Johansen and Ann-Charlotte Andersen for skilled technical assistance. The study was supported by grants from the Danish Medical Research Council (No. 22-01-0189 and 22-02-0523), the Danish Biomembrane Research Centre and Aarhus Universitets Forskningsfond.

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