come from increases in intramuscular enzymes and myo- globin in the blood (2, ... ulate production of free radicals (3), which cause perox- idation of membrane ...
Rat skeletal muscle mitochondrial and injury from downhill walking
[ Ca2+]
C. DUAN, M. D. DELP, D. A. HAYES, P. D. DELP, AND R. B. ARMSTRONG Exercise Biochemistry Laboratory, Department of Physical Education, University of Georgia, Athens, Georgia 30602
DUAN, C., M. D. DELP, D. A. HAYES, P. D. DELP, AND R. B. ARMSTRONG. Rat skeletal muscle mitochondrial [Ca*+] and injury from downhill walking. J. Appl. Physiol. 68(3): 12411251, 1990.-The purpose of this study was to evaluate the relationship between mitochondrial Ca*+ concentration (MCC) and the extent of muscle injury in rats that have performed prolonged downhill walking (eccentric exercise). MCC was used as an indicator of elevated [Ca*+] in the muscles, and injury was estimated from histochemical analysis of muscle cross sections by determining the numbers of intact fibers per unit area in the muscles. Elevations in MCC in the soleus and vastus intermedius muscles over time postexercise were inversely related (P < 0.05) to the number of intact fibers per square millimeter in the respective muscles after downhill walking. Verapamil administration attenuated the elevation in MCC and injury in histochemical sections resulting from the downhill walking in soleus muscle, but intraperitoneal injection of the chelators EDTA or ethylene glycol-bis(P-aminoethylether)-N, N,N’,N’-tetraacetic acid significantly attenuated the increases in MCC and injury to both the vastus intermedius and soleus muscles in the downhill walkers. The chelators appear to exert their “protective” effects within the specific muscles that show the injury and do not significantly affect serum [Ca’+]. It is concluded that increases in MCC occur during exercise-induced fiber injury and that elevations in cellular Ca*’ may have a role in the etiology of the injury process. mitochondria; damage
plasma
enzymes;
verapamil;
EDTA;
EGTA;
muscle damage has been documented in humans (9, 21) and animals (2, 16). Unaccustomed exercise, particularly that involving eccentric contractions (2, 25), is accompanied by injury to fibers in the active skeletal muscles. Evidence for the injury has come from increases in intramuscular enzymes and myoglobin in the blood (2, 9, 28), direct histological (2, 16, 24) or electron-microscopic (2, 10, 21) examination, biochemical analysis (2, 9), and urinary excretion of muscular proteins (9). The mechanisms underlying exercise-induced muscle injury are not known. However, a loss of Ca2+ homeostasis in the muscle cells may be implicated in the etiology (1, 7, 13), as it is in a variety of other muscle injury (7, 13) and disease (36) models. In exercise-induced injury, elevated intracellular Ca2’ theoretically could result from a variety of alterations in cell function or morphology caused by the exercise. Any structural disruption in the cell membrane or sarcoplasmic reticulum or alteration in EXERCISE-INDUCED
0161-7567/90
$1.50 Copyright
Ca2+ influx through channels in sarcolemma could allow the ion to move into the cytosol down its concentration gradient. Alternatively, lowered ATP levels could initiate a cycle involving attenuated ability to extrude Ca2+from the sarcoplasm via ATP-dependent pumps, followed by further impedance of respiration as the result of Ca2+ accumulation in the mitochondria (6, 36). In diseased muscle, up to 50 times the normal concentration of Ca2+ may be found in the mitochondria (33); these large quantities of the ion primarily come from the extracellular space, where Ca2+ concentration is several orders of magnitude higher than in the cytosol. Regardless of the mechanism of entry, high Ca2’ concentrations in the cell can have a number of detrimental effects. The mitochondria take up large amounts of Ca2+, which impairs respiration and ATP production (6, 36). High intracellular Ca2+ concentration increases the activation of phospholipase A2 (7, 13), which results in production of lysophospholipids, leukotrienes, and prostaglandins that may promote degradation of cellular structures. High Ca2’ concentration increases the activation of Ca2+-activated proteases (19) and might stimulate production of free radicals (3), which cause peroxidation of membrane lipids. Also, elevated Ca2+ may produce muscle contracture (24). Thus loss of Ca2’ homeostasis in the muscle cell can potentially initiate a number of destructive processes and serve as a central mechanism in exercise-induced muscle fiber injury. Mitochondrial Ca2’ concentration (MCC) may be used as an indicator of elevated Ca2+ in muscle fibers (32). Sembrowich and co-workers (30) reported that the Ca2+ uptake kinetics of mitochondria in slow-twitch muscles are adequate for the mitochondria to have an important role in regulation of Ca2+ concentration in the fibers. In normal muscle cells, the MCC is relatively low compared with that in the sarcoplasmic reticulum, and even after sustained increases of cytosolic Ca2’ within the physiological range in maximally contracting muscle, MCC is not elevated (32). However, abnormally high cystolic Ca2+ levels are buffered by the mitochondria. For example, Tate et al. (34) reported that the MCC of muscles in trained rats exercised to exhaustion increased 132% above that in sedentary rested rats (the possibility of associated muscle damage was not investigated in that study). The purpose of the present study was to determine the relationship between MCC and muscle injury in rats that have performed downhill walking (2). Injury was evalu-
0 1990 the American
Physiological
Society
1241
1242
CA*+
AND
MUSCLE
ated in sections of the muscles assayed with histochemistry. The relationship between injury and MCC was studied in normal animals and in rats in which Ca2’ accumulation in the muscle fibers was antagonized by verapamil, EDTA, or ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetate (EGTA). Verapamil blocks slow Ca2+ channels (11), whereas EDTA and EGTA chelate Ca2+ and are excreted, thus lowering body Ca2+ (17,33). The results provide evidence for a relationship between elevated MCC and muscle injury after exercise. The observations that the Ca2’ antagonists attenuate the incidence of damage suggest that Ca2’ may have a causal role in the injury process.
INJURY
3) 2 days postexercise (48 h). The muscles studied were the soleus and vastus intermedius because these were previously shown to sustain the greatest injury during walking down an incline (2). Histochemical analysis of the muscles was used to visualize injury for comparison with the measurements of MCC. Protocol II. The results of protocol I demonstrated a relationship between MCC and muscle injury in the exercised rats. Protocol II was designed to pharmacologically attenuate the accumulation of Ca2’ in the muscles resulting from the exercise by blocking slow channels with verapamil HCl; it was hypothesized this would be associated with a reduction in the incidence of injury in the muscles. Twelve male rats with an average body weight of 590 MATERIALS AND METHODS t 27 g were randomly divided into two groups of six: 1) downhill walkers with saline and 2) downhill walkers Animals and Animal Care with verapamil. Rats in the drug group had 750 mg/l Male and female Sprague-Dawley rats were used in verapamil HCl in their drinking water (27) from 12 h the experiments (Charles River Laboratories). They were before exercise to the time they were killed, and they housed four per cage in a room maintained at 23 t 2°C were also injected intraperitoneally with verapamil (0.75 and provided with food (commercial rat chow) and water mg/kg) in saline (1.5 ml/kg) (31) 20 min before exercise ad libitum. At the time of the experiments the females and once every 8 h after walking (i.e., 6 times) until they weighed 279-374 g and the males 410-622 g. None of the were killed. Because a dose-response relationship was rats had been on a treadmill before the acute experiment. not established, we used the largest doses encountered in the literature for both oral and injection routes in an Exercise Protocol effort to maximize the verapamil effect (e.g., the injection alone represents -10 times the normal intravenous huIn all experiments two to four rats were exercised man dose). In preliminary experiments it was observed simultaneously on a motor-driven treadmill (Stanhope that this intraperitoneal dose of verapamil resulted in a Scientific) at 15 m/min down a 17” incline for 130 min 13% reduction in heart rate within 6 min in anesthetized (26 bouts of 5 min each of walking separated by 2-min rats. The rats in the saline group drank normal tap water rests). Brushing the tail was used to encourage the aniand were injected with saline (1.5 ml/kg) at the same mals to exercise; no electrical stimulation was employed. time points. Two days after exercise, the soleus and A similar exercise protocol was previously shown to vastus intermedius muscles of both legs were used for result in marked injury to fibers in the deep slow extensor histochemistry and measurement of MCC. (soleus and vastus intermedius) muscles of rats (2, 24, Protocol III. Protocol III was also designed to antago28) in the muscles of rats that perAfter exercise, the rats were placed back in their cages nize Ca2+ accumulation formed the downhill walking exercise through chelation with free access to food and water until they were killed of the ion with EGTA, a specific chelator of Ca2+. The for tissue sampling. hypothesis tested was that EGTA would prevent the increase in MCC in the muscles and lower the extent of Experimental Design muscle injury. Thirty-two male rats with an average weight of 547 t A total of six separate protocols were performed in this 12 g were randomly divided into four groups: 1) nonexstudy. Protocol I. This set of experiments was designed to ercise saline controls, 2) nonexercise with EGTA, 3) downhill walkers with saline, and 4) downhill walkers investigate the relationship between MCC and muscle with EGTA. Rats in the EGTA groups were injected injury immediately and 2 days after the downhill walk. intraperitoneally with EGTA (150 mg/kg) in saline (1.5 Previous research (2) showed that muscle injury induced ml/kg) at 24 h before, 20 min before, and 24 h after by this exercise is more apparent 2 days after than exercise. Animals in the saline groups were injected with immediately after the exercise. Whereas local disruptions an equal volume of saline at the same times. Two days of the banding pattern in some fibers can be observed after downhill walking, soleus and vastus intermedius immediately after exercise, by 2 days postexercise there muscles from the nonexercise and exercise groups were are obvious regions of degeneration marked by accumuremoved for measurement of MCC and determination of lations of macrophages (2, 24). This protocol allowed muscle injury from histochemistry. Also, a blood sample testing of the hypothesis that time-dependent changes was obtained from each rat at the time the animals were in MCC after exercise would be related to the incidence killed for measurement of creatine kinase (CK) activity of muscle damage. Nine female rats with an average weight of 322 t 34 g in the plasma. were randomly assigned to three groups of three each: 1) Protocol IV. The purpose of protocol IV was similar to sedentary control, 2) immediate postexercise (0 h), and that of protocol III as described above. However, in
cA2+
AND
MUSCLE
protocol IV Ca2+ accumulation in the muscles was antagonized with the chelator EDTA to elucidate the association between MCC and muscle injury. EDTA has been used therapeutically to chelate Ca2+ and other heavy metals (33); it was of interest to compare the effects of this chelator with those of EGTA. Twenty-four male rats with a mean body weight of 465 t 31 g were randomly divided into three groups of eight: 1) nonexercise controls with saline, 2) downhill walkers with saline, and 3) downhill walkers with EDTA. Animals in the EDTA group were given intraperitoneal injections of the chelator (150 mg/kg) in saline (1.5 ml/kg) 20 min before and 24 h after exercise. Rats in the saline groups were given injections of saline (1.5 ml/kg) at the same times. Two days after exercise the soleus and vastus intermedius muscles of one leg were used for histochemistry; the same muscles of the other leg were used for measurement of MCC. Protocol V. In the previous protocols it was demonstrated that the Ca2+ chelators EGTA and EDTA attenuated the incidence of muscle injury 2 days after downhill walking. Because the muscles had only been examined at the one time period [the time period showing the peak response in normal rats (2)], the possibility existed that the chelation was simply delaying the injury response. The purpose of this protocol was to follow the injury in the muscles out to 6 days after downhill walking in rats treated with EDTA to test this possibility. Eighteen male rats (454 t 24 g) were randomly divided into three groups of six rats each: 1) 2 days postexercise, 2) 4 days postexercise, and 3) 6 days postexercise. At these times after downhill walking the soleus and vastus intermedius muscles were removed for histochemistry, and a blood sample was taken for plasma CK determination. Protocol VI. EGTA and EDTA treatments were effective in lowering MCC and the incidence of muscle injury. The purpose of protocol VI was to estimate where the chelators might exert their effect in attenuating the injury to the muscles. Two possibilities were that they lower extracellular [Ca”‘], thus diminishing the concentration gradient from extra- to intracellular compartments, or that they serve to buffer the Ca2+ levels within the affected muscles. Protocol VI was designed to provide information about these two possibilities. Twenty male rats with a mean body weight of 536 t 31 g were randomly assigned to four groups of five each: 1) nonexercise with saline, 2) nonexercise with EDTA, 3) downhill walkers with saline, and 4) downhill walkers with EDTA. Two days after exercise a blood sample was removed for analysis of serum-free [Ca”‘], and soleus, vastus intermedius, and tibialis anterior muscles were removed for measurement of [EDTA] and for histochemistry. The tibialis anterior muscle was also studied as a further control, because it has previously been observed that this flexor muscle sustains negligible injury during the downhill walking regimen (24). Liver, plasma, and urine samples were also taken to measure [EDTA]. Analytic
Procedures
Muscle IMCC. Mitochondria
by Ernster
and Nordenbrand
were isolated as described (8). The muscle sample
INJURY
1243
was freed from fat and connective tissue, weighed, and immersed in cold 0.15 M KCl. The tissue was minced and rinsed with the KC1 solution. The minced tissue was then rinsed with Chappell-Perry medium and suspended in -1 vol of the same medium. Homogenization was carried out with a Potter-Elvehjem homogenizer for l-2 min. The homogenization and all following operations were performed at 0-4°C. The homogenate was diluted with Chappell-Perry medium to a volume of -10 times the initial weight of the muscle and centrifuged twice each at 650 and 14,000 g for 10 min. The mitochondrial pellet was then suspended in 0.15 KC1 (6-10 mg mitochondrial protein/ml). Of the mitochondrial suspension, 1 ml was used to analyze the total [Ca”‘] in an atomic absorption spectrophotometer (Mark II Janell-Ash 965). Mitodhondrial protein was measured with the Lowry method (18), and MCC was expressed as milligrams Ca2+/g mitochondrial protein. Muscle histochemistry. Muscle samples were mounted on a specimen holder in OCT Compound (Miles Laboratories) and frozen in 2methylbutane cooled in liquid N2. Cross sections (10 pm) were cut in a microtome cryostat at -20°C and stained for NADH-tetrazolium reductase (NADH-TR) activity (23). Muscle injury was quantified by determining the number of intact normalappearing fibers per mm2 of tissue in each muscle cross section by means of a microscope viewing screen with a calibrated square grid. The evaluation was done in singleblind fashion. In each muscle section, five I-mm2 areas were analyzed, and the average number of intact fibers/ mm2 was computed for each muscle. The number of intact fibers per unit area is inversely related to the amount of injury in the tissue. Injury-related decreases in intact fibers/mm2 theoretically could result from three pathological processes: 1) degeneration of injured fibers; 2) tissue edema, which spreads fascicles and fibers within fascicles; and 3) swelling of fibers. The first two in particular were apparent in the injured muscles (see Fig. 2). This method of analysis was adopted because it was not possible to quantify the numbers of injured fibers in the damaged muscles. In areas of heavy damage, which were filled with invading cells and debris, it was not possible to determine the number of muscle fibers that originally occupied the sites (see Fig. 2). Another complication was the dissolution of the normal fascicular and fiber arrangements in the injured muscles, presumably as the result of edema in the tissue (Fig. 2). Plasma CK actiuity. After methoxyflurane anesthesia, the abdominal cavity of the rat was opened and a 5- to lo-ml blood sample was obtained from the bifurcation of the abdominal aorta for CK determination. The blood sample was immediately placed in a tube with EDTA, mixed, and centrifuged at room temperature to obtain plasma, which was refrigerated until the time of assay. CK activity in the plasma was spectrophotometrically determined with a commercial kit (Sigma 45-5). CK analyses were completed within 4 h after blood withdrawal. Data are presented in units per liter. Serum [Ca2+/. Rats were anesthetized with methoxyflurane, and blood (l-2 ml) was obtained by cardiac puncture. Serum was extracted from the blood samples
1244
CA’+
AND
MUSCLE
and fluorometrically analyzed for free [ Ca2+] (Technicon Autoanalyzer) with the methods of Wallach et al. (35) and Kepner and Hercules (15). Serum free [Ca”‘] data are expressed in mM. Muscle /EDTAl. After removal of the blood sample for serum- [Ca”+j determination as described above; a tourniquet was placed around the hip of one hindlimb to occlude blood flow. Muscles from this limb were subsequently sampled for histochemistry. One milliliter of heparinized (200 U/ml) 0.1% procaine hydrochloride in saline was gravitationally perfused via an 18-gauge Luer stub adapter inserted through the apex of the heart into the left ventricle. A nick was made in the inferior vena cava at the level of the diaphragm for perfusate exit. The purpose of this perfusion was to remove blood from the muscles of the limb without the tourniquet so that EDTA in the blood would not contaminate the tissue samples in the subsequent determinations of muscle [EDTA]. Complete removal of blood was not verified independently, but the perfusion resulted in marked blanching of the muscles that were studied. Muscle samples (soleus, vastus intermedius, and tibialis anterior) were removed from the perfused leg, weighed, minced in cold 0.9% saline, and homogenized in 1 vol of saline. The homogenate was diluted with saline to a volume of 10 times the initial weight of the tissue. Then 1 ml of this suspension was used to analyze [EDTA] by the method of Paris et al. (26). This spectrophotometric procedure is based on measurement of the decrease in absorbance (at 551 nm) of copper l-(2-pyridylazo)-2-naphthol (PAN) as copper is dissociated from the PAN and chelated by EDTA; the decrease in absorbance is proportional to the concentration of EDTA, because copper-EDTA has a higher stability constant than copper-PAN. [EDTA] values are presented in milligrams per gram muscle wet weight.
INJURY
MITOCHONDRIAL [Co ‘+I (mg/g protein)
I
b
18
C
P&T-EXERCISE (W
FIG. 1. Mitochondrial Ca2+ concentrations (means t SE) in muscles of control rats that did not exercise (C) and in muscles of rats that walked downhill for 3 h at 0 and 48 h postexercise. S, soleus; VI, vastus intermedius. a Postexercise value different from respective control value (P < 0.05). b 48-h postexercise value different from both respective control and O-h values (P < 0.05).
1. Numbers of intact fibers of vastus lateralis muscle cross sections assayed for NADH tetrazolium reductase TABLE
Protocol
Treatment
I
II, III,
V
RESULTS
Protocol I. The MCC in soleus muscles of the animals taken immediately after exercise (0 h rats) was higher than that of nonexercised rats, and in both soleus and vastus intermedius muscles the MCC of the rats taken 48 h after exercise was higher than that of the controls (Fig. 1). Histochemical analysis of muscle cross sections indicated that the number of intact fibers/mm2 in the two muscles of the three groups was inversely related to the MCC of the muscles (r = 0.94, y = 2.31~ + 31.60, P < 0.05, n = 6); i.e., the higher the MCC, the greater the incidence of injury in the muscles. Immediately after exercise (0 h) there was evidence of muscle injury in both muscles, and by 2 days after exercise, a larger incidence of muscle injury (Tables 1 and 2). Protocols II, III, and IV. Average MCC values for muscles from sedentary control saline-injected rats and
s Muscle
lssa VI Muscle
Statistical Analyses
Data were analyzed using one-way analysis of variance with nonrepeating measures and Duncan’s multiple range test. Differences were considered significant at the 0.05 level.
a
10
VI
n
Control Postexercise Oh 48 h IV
Control Saline EGTA Postexercise Saline Verapamil EGTA EDTA Postexercise, 2 days 4 days 6 days Control Saline EDTA Postexercise Saline EDTA
Intact
Fibers/mm*
3
289t14
3 3
187t08 139+14
16 8
228klO 220t08
22 6 8 8
144tO8 134t13 185tlO 182203
6 6 6
180t07 216kO4 223205
5 5
263k12 216tlO
5 5
146k12 180t08
EDTA
Values are means * SE. Within each protocol, to the same vertical lines are not different from Refer to text for description of treatments.
mean values adjacent each other (P > 0.05).
the Z-day postexercise saline-injected rats, respectively, were the same (P > 0.05) from protocols II, III, and IV, so the data were combined for comparison with the data from the verapamil-, EGTA-, and EDTA-injected groups (Fig. 3). As observed in protocol I, MCC in both soleus (+139%) and vastus intermedius (+207%) muscles increased in the saline-injected rats 2 days after downhill
cA2+
AND
MUSCLE
2. Numbers of intact fibers of soleus muscle cross sections assayedfor NADH-TR
TABLE
Protocol
I
Treatment
II,
VI
3 3 3
311t16 193t20 167tlO
16 8
253t06 251t07
22 6 8 8
170tlO 184tll 214t12 206r413
III, IV Control Saline EGTA Postexercise Saline Verapamil EGTA EDTA
V
Intact Fibers/mm2
n
Control 0 h postexercise 48 h postexercise
Postexercise, 2 days 4 days 6 days
EDTA Samples
Control Saline EDTA Postexercise Saline EDTA
Values are means k SE. Within each protocol, to the same vertical lines are not different from Refer to text for description of treatments.
lost
269t15 233tlO 147tll 202t07 mean values adjacent each other (P > 0.05).
walking. Histochemical examination of the muscles confirmed that there was a relatively small number of intact fibers/mm2 in these animals (Tables 1 and 2; Fig. 2). In vastus intermedius muscle, verapamil treatment did not attenuate the increase in MCC in the exercised rats (Fig. 3). Also, there was a relatively high incidence of injury in the vastus intermedius muscles of verapamiltreated rats that exercised as indicated by histochemistry (Table 1, Fig. 2). On the other hand, verapamil treatment did decrease the elevation in MCC in the soleus muscle, insofar as the mean value was not statistically different from that in the nonexercised saline control rats. Although the number of intact fibers/mm2 in the soleus muscle of the verapamil-treated rats was not different from that in the saline-injected exercised rats, it also was not different from the means for the EGTA- and EDTAinjected animals that exercised (Table 2). EGTA treatment significantly reduced the MCC of both soleus (-41%) and vastus intermedius (-44%) muscles in the downhill walkers compared with that in the saline-injected walkers (Fig. 3). EGTA treatment also altered the injury process as evidenced in histochemical cross sections (Tables 1 and 2). In both muscles, the number of intact fibers/mm2 was higher than in the saline-injected walkers, although the values for both muscles were lower than in nonexercised control rats. EGTA treatment in nonexercised control rats had no influence on either MCC or histochemistry. MCC values in nonexercised rats given EGTA were 2.77 t 0.60 and 1.77 t 0.45 mg/g protein, respectively, in soleus and vastus intermedius muscles, compared with 2.29 t 0.40 and 2.17 t 0.33 mg/g in the same muscles in salineinjected control animals. Both saline- and EGTA-treated nonexercised animals had relatively high numbers of
INJURY
1245
intact fibers/mm2 in histochemical sections in both muscles (Tables 1 and 2, Fig. 2). Plasma CK activity was elevated 2 days after downhill walking in both the saline(+105%) and the EGTA- (+213%) injected animals, so EGTA did not provide “protection” from injury from the downhill exercise for this variable (Fig. 4). The magnitude of the mean CK activity for the EGTA exercised group is the result in large part of the high value (270 U/ 1) measured in one rat in that group. The effects of EDTA treatment in reducing MCC and the incidence of muscle injury as indicated by histochemistry were similar to those of EGTA (Tables 1 and 2, Fig. 2). After downhill walking, MCC in rats injected with EDTA was 32% lower than in animals injected with saline in the soleus muscle and 43% lower in the vastus intermedius muscle (Fig. 3); in both muscles MCC in the EDTA-injected rats was not different from that in the nonexercised saline-treated animals (P > 0.05). The main goal of this study was to determine the relationship between MCC and muscle injury. When the numbers of intact fibers/mm2 are regressed on the MCCs of the vastus intermedius and soleus muscles from the six groups of animals included in protocols 11, III, and IV, a significant inverse relationship is observed (Fig. 5). When the relationship is determined for the two muscles separately, the regression coefficients are even higher: vastus intermedius, r = -0.98, y = -1.85~ + 25.75, P < 0.05, n = 6; soleus, r = -0.93, y = -2.85x + 31.50, P c 0.05, n = 6. These findings, with those in protocol I, indicate a close relationship between MCC in the muscles and the amount of injury. Protocol V. Evaluation of histochemical sections of vastus intermedius muscles showed the lowest number of intact fibers/mm2 in downhill walkers treated with EDTA 2 days after exercise (Table 1). Four days after walking, there was an increase in the number of intact fibers/mm2 in the muscles over that in the 2day group. Six days after exercise, the amount of injury evident was similar to that at 4 days postexercise, although in absolute terms, the mean number of intact fibers/mm2 was higher at 6 days postexercise than at 4 days. These observations indicated that EDTA attenuated the magnitude of muscle injury and did not simply delay the process. Also there was no delay in an elevated plasma CK activity in the downhill walkers treated with EDTA (Fig. 4). There were no differences in plasma CK activity at 2, 4, and 6 days after exercise in EDTA-treated rats; the absolute mean values decreased over this time period (Fig. 5). There was a significant elevation in plasma CK in the EGTA- and EDTA-treated rats at 2 days after exercise. Protocol VI. EDTA treatment did not affect the serumfree [Ca”‘] measured on the 2nd day after downhill walking (Table 3). However, there was a large increase in [EDTA] in the soleus and vastus intermedius muscles of downhill walkers treated with the chelator over the levels observed in nonexercised rats injected with EDTA or exercise and nonexercised rats injected with saline (Fig. 6). [EDTA] in the tibialis anterior muscle, which does not perform eccentric contractions or sustain injury
1246
CA*+
AND
MUSCLE
INJURY
CA*+
AND
MUSCLE
INJURY
1247
1248
cA2+
MITOCHONDRIAL (mg/g
AND
MUSCLE
INTACT
[Ca2+]
protein)
a
FIBERS/MM
*
MUSCLE
300
a
8
INJURY
I
s Muscle
m
VI Muscle
I 200
b b T
4 -
100
Y = -21.9x -0.9 L 7 0.05
+ 281 .I 1
0
0 0
2
4
6
8
l
C SAL
‘-E SAL
P-E VERA
P-E EGTA
MITOCHONDRIAL [Ca*T
P-t EDTA
3. Mitochondrial Ca2’ concentrations (means k SE) in muscles of control nonexercised (C) rats given saline (SAL) and rats at 2 days postexercise (P-E) given saline, verapamil (VERA), EDTA, or EGTA. S, soleus; VI, vastus intermedius. a Postexercise value different from respective nonexercise control value (P < 0.05). b Postexercise value different from respective postexercise saline value (P < 0.05). FIG.
PLASMA CK ACTIVITY (u/I) 150 r 125
Mean numbers of intact fibers/mm2 for vastus intermedius muscles from 6 groups of rats included in protocols II-IV 2 for list of groups) regressed on mean MCC for the same each group.
TABLE 3. Serum-free [ca2+] in control rats given saline and in downhill walkers 2 days postexercise given saline or EDTA
a [Ca”+],
a Control Saline Postexercise Saline EDTA
i
F
100 F L
mM
2.44 t 0.08 2.54 k 0.04 2.43 zk 0.05
Values are means t SE; n = 4/group. among means (P > 0.05).
T
75 -
There
were
no differences
MUSCLE [EDTA]
50 T,
(mg/g
-1
protein
x
10) a
18
25 O*
FIG. 5. and soleus (see Table muscles in
SA
EG
SA 2
CONTROL
ED # ED 2
DAYS POST-EXERC
I lxsl
-
4
6
SE
FIG. 4. Plasma CK activities (means t SE) in nonexercised control rats given saline (SA) or EGTA (EG) and in rats postexercise given saline, EGTA, or EDTA (ED). a Postexercise value different from saline and EGTA control values (P < 0.05).
during downhill walking (23), was not different in the EDTA-treated rats that exercised from that in the salineinjected nonexercised control animals (data not shown). As described above in previous protocols, histochemical examination of cross sections of soleus and vastus intermedius muscles verified that EDTA treatment markedly reduced the amount of fiber damage (Tables 1 and 2). These results indicate that the chelation treatment does not reduce the extracellular pool of Ca2+ (as indicated by serum [Ca2+] measurements), but that specific muscles that perform eccentric contractions during the downhill walking, i.e., soleus and vastus intermedius, accumulate EDTA, which may serve to buffer Ca2+ in the injured fibers and attenuate the degenerative processes. There
s Muscle VI Muscle
12
6
T
L
T
0+ cs
CE
P-ES
P-EE
FIG. 6. Muscle EDTA concentrations (means t SE) in control nonexercised rats (C) given saline (S) or EDTA (E) and in rats 2 days postexercise (P-E) given saline or EDTA. a Different from other 3 values for respective muscle (P < 0.05).
was no difference in liver, plasma, or urine [EDTA] among the groups of rats (control saline, saline exercised, EDTA control, or EDTA exercised) (P > 0.05; data not shown).
cA2+
AND
MUSCLE
DISCUSSION
The main purpose of this series of experiments was to study the relationship between MCC and the injury in muscles of rats resulting from downhill walking. We hypothesized that injury in the muscles in the postexercise period would be associated with elevated MCC. This hypothesis was based on several previous observations: 1) downhill walking in untrained rats results in a predictable muscle injury in the deep slow extensor muscles with the peak response occurring 2 days after the exercise (2), 2) muscle damage and degeneration in several injury models are associated with elevations in Ca2’ concentration in the injured muscle fibers (7, l3), and 3) measurement of mitochondrial Ca2+ concentration provides an indicator of increased Ca2+ in the muscle cell (33). The results support the hypothesis, because the magnitude of Ca2+ in the muscle mitochondria under various conditions was related fairly closely to the degree of muscle fiber injury as indicated by histochemistry. These observations raise the question of whether the elevated Ca2’ in the muscle fibers is simply one manifestation of the injury process or whether Ca2+ is mechanistically involved in the etiology of the injury. The preponderance of evidence in the literature would indicate the latter. As indicated above, elevated muscle cell Ca2+ is evident in fibers damaged by various diseases (32, 36), chemicals (7, l3), or mechanical influences (unpublished observations). Furthermore, the injury process in several of these models can be inhibited by preventing the elevation in Ca2+ from occurring (7,13). These observations suggest, but do not prove, that Ca2+ elevations per se cause loss of viability in the injured muscle fibers. The mechanism(s) by which Ca2+ is elevated in the muscle cells after the downhill walking exercise is not known. Several possibilities can be suggested. First, the source of the Ca2’ could either be intracellular or extracellular. Under various conditions reuptake of Ca2’ by sarcoplasmic reticulum is compromised (5); cystolic accumulation of Ca2’ from sarcoplasmic reticulum in the downhill walkers could have resulted in the elevated MCC in the present study. If the source of the Ca2+ was extracellular, two mechanisms of entry can be proposed. First, the Ca2’ could have entered the cells through Ca2+ channels in the sarcolemma. Increasing tension in the sarcolemma during eccentric contractions conceivably could allow ions to move through the membrane down their concentration gradient; e.g., stretch-activated channels for several other ions have been described for skeletal muscle (12). We have unpublished data showing that static stretch beyond optimal length at physiological lengths (within anatomic limits) causes Ca2+ influx through verapamil-sensitive channels in isolated muscles and that eccentric contractions involve stretching muscles over their physiological length range while they are active. In the present study, verapamil attenuated the elevation in MCC in soleus muscle (but not vastus intermedius muscle) in the downhill walkers but did not completely block the increase in [Ca”‘] in the mitochondria. As described before, these data must be interpreted with care, because dose-response relationships have not been worked out on this model. However, our observa-
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tions suggest that in the downhill walkers some of the Ca2+ may have entered the cells in soleus muscle through verapamil-sensitive channels. Another complication in interpreting the verapamil data is that relatively high concentrations of verapamil can have deleterious effects on muscle fibers. In both cardiac (4) and skeletal (unpublished observations) muscles, verapamil induces a dose-dependent release of intramuscular enzymes during in vitro incubation; during 2-h incubation in 0.75 mM verapamil, both fast and slow rat muscles may lose up to 50% of their CK activity to the incubation medium (unpublished observations). These apparent deleterious effects of verapamil may partially explain the fact that verapamil attenuated the elevation in MCC in the downhill walkers but did not lower the incidence of histological injury. The second mechanism by which extracellular Ca2+ could gain entry to the cytosol is through ruptures in the cell membrane. Any disruption to the structure of the sarcolemma resulting from the exercise presumably would cause elevated MCC, because the concentration gradient from interstitium to cytosol is quite steep (extracellular fluids contain three to four orders of magnitude higher [ Ca2+] than cytoplasm). The histological appearance of the injured fibers immediately after downhill walking suggests that there may be disruption of the sarcolemma (2, 24), which is also supported by the presence of intracellular enzymes (e.g., CK and lactate dehydrogenase) in the blood (2, 28). Structural damage to muscle cell membranes may result from high specific tensions developed in the active fibers during the eccentric contractions (1). McCully and Faulkner (19) reported that lengthening contractions of mouse muscle produce higher forces than isometric or shortening contractions and result in significantly more injury to the fibers after the contractions. Disruption of the sarcolemma could result from 1) mechanical tension, 2) activation of phospholipase A2 (25), 3) lipid peroxidation from free oxygen radicals (l4), or 4) high local temperatures produced during the exercise (21). One technical concern in this study is that, in isolating the mitochondria from the control and treatment groups, altered stability of the organelles resulting from the treatment may have resulted in preferential isolation of different components, which in turn could influence the Ca2+ measurements. There is ultimately no way to estimate that possibility from the data obtained, but the similarity of the mitochondrial protein fractions isolated from the muscles of the different groups argues against this being a major problem. For example, the percent of the total muscle wet weight composed of mitochondrial protein in the vastus intermedius muscle of the control sedentary group in protocol I was 2.8 t 0.2% compared with 2.3 t 0.1% for the &day postexercise group. It is improbable that the fivefold difference in mitochondrial Ca2’ between the two groups can be explained by altered mitochondrial separations. Also the values for MCC obtained for the rat muscles in this study were similar to those reported by Tate et al. (34), who used a different mitochondrial isolation procedure. For example, MCC in control vastus intermedius muscle in protocol I (Fig. 1)
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CA*+
AND
MUSCLE
was 1.5 mg/g mitochondrial protein (or 0.04 pmol/mg protein), which compares with their value of 0.05 pmol/ mg protein (34). They (34) reported that MCC in muscles from trained rats run to exhaustion was 0.12 pmol/mg protein, which was similar to MCC in vastus intermedius muscle immediately after exercise (Fig. 1, 5.1 mg/g or 0.13 pmol/mg protein). The possibility that Ca2+ may be a causative factor in the muscle injury is supported by the drug treatments employed in this study. The Ca2+ chelators EDTA and EGTA had a marked effect in preventing the accumulation of Ca2+ in the mitochondria and the injury evident in the sections assayed with histochemistry. The mechanism by which the chelators lowered mitochondrial Ca2+ accumulation (and muscle fiber injury) is of interest. Presumably, the EDTA and EGTA effectively bound Ca2+ in some compartment(s) so that the ion was prevented from entering the mitochondria in the affected muscle cells. It is not clear where this chelation occurred, but the data suggest that it took place within the muscle fibers per se. First, injection of EDTA did not appear to affect the long-term concentration of free Ca2+ in the plasma (serum [Ca”‘] was only measured 2 days postexercise). Therefore, reduction of the extra- to intracellular concentration gradient presumably was not the explanation, if it is assumed that the free Ca2+ concentration in the serum is representative of the interstitial levels. Spencer et al. (33) reported that slow intravenous infusion of EDTA in human subjects resulted in marked calcinuria without altering serum Ca2’ levels. We could detect no EDTA in samples of plasma or urine from either control or exercised rats. On the other hand, EDTA concentrations were markedly elevated specifically in the eccentrically contracting muscles of the animals that received injections of EDTA and exercised downhill. These muscles were perfused with saline before removal for homogenization, so the EDTA presumably was not located in blood trapped in the muscles. It is possible that the EDTA was localized in the interstitial fluids, as opposed to within the fibers per se. Technically, the method (26) used to measure EDTA was nonspecific; it is based on measurement of decreases in absorbance of copper-PAN as copper is chelated by EDTA, because copper-EDTA has a higher stability constant than copper-PAN. Presumably other chelating factors in the muscle homogenates could also compete for the copper, so the measurement should more appropriately be considered as an indicator of “chelating power.” Nonetheless, the results indicate that the EDTA accumulated within the affected muscles (either intracellularly or within the intersitium) and exerted a protective influence locally at the sites of potential injury. Clinical application of EDTA as a Ca2+ chelator in treatment of degenerative vascular disease has been advocated for a number of years but has not been accepted by the medical community (29). It does not appear that a mechanistic rationale for this clinical treatment has been elucidated. Alternative explanations for the effect of the chelators are possible. For example, chelation conceivably could influence recruitment of motor units in the muscles, because transmitter release from cu-motoneurons is de-
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pendent on Ca2+ influx from the interstitium. Diminished acetylcholine release from a given population of neuron terminals would necessitate recruitment of additional motor units to produce the required force to maintain locomotory speed, which would effectively lower the specific tension generated within any given motor unit and decrease the potential for mechanical damage to the constituent fibers. In conclusion, the present study indicates that injury to skeletal muscles resulting from downhill walking in rats is associated with elevations in Ca2+ in the mitochondria of the injured muscles. Furthermore, the data suggest that elevated Ca2+ in the muscle fibers may have a central role in the injury process, because treatment with chelators (EDTA and EGTA) reduced the accumulation of Ca2’ in the mitochondria and attenuated the muscle fiber injury as evidenced by histochemistry. The authors thank Brenda Arnold for help in preparing the manuscript. This research was supported by National Institutes of Health Grant AM-37098 and Biomedical Research Support Grant S07RR-07025-21. Address reprint requests to R. B. Armstrong. Received
14 October
1988; accepted
in final
form
8 November
1989.
REFERENCES 1. ARMSTRONG, R. B. Mechanisms of exercise-induced delayed onset muscular soreness: a brief review. Med. Sci. Sports Exercise 16: 529-538,1984. R. B., R. W. OGILVIE, AND J. A. SCHWANE. Eccentric 2. ARMSTRONG, exercise-induced injury to rat skeletal muscle. J. Appl. Physiol. 54: 80-93,1983. 3. BRAUGHLER, J. M. Calcium and lipid peroxidation. In: Oxygen Radicals and Tissue Injury, edited by B. Halliwell. Bethesda, MD: Fed. Am. Sot. Exp. Biol., 1988, p. 99-104. 4. COHEN, L., Z. GILULA, P. MEIER, B. LAZARON, AND D. HERBSTMAN. Competitive effects of verapamil and calcium ion as regulators of myocardial enzyme leakage. J. Cardiouasc. Pharmacol. 3: 581-597,198l. 5. DHALLA, N. S., P. K. DAS, AND G. P. SHARMA. Subcellular basis of cardiac contractile failure. J. Mol. Cell. Cardiol. 10: 363-385, 1978. 6. DRAHOTA, Z., E. CARAFOLI, C. S. ROSSI, R. L. GAMBLE, AND A. L. LEHNINGER. The steady state maintenance of accumulated Ca*+ in rat liver mitochondria. J. Biol. Chem. 240: 2712-2720, 1965. 7. DUNCAN, C. J., AND M. J. JACKSON. Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle. J. Cell Sci. 87: 183-188, 1987. 8. ERNSTER, L., AND K. NORDENBRAND. Skeletal muscle mitochondria. Methods Enzymol. 10: 86-94, 1967. 9. EVANS, W. J., C. N. MEREDITH, J. G. CANNON, C. A. DINARELLO, W. R. FRONTERA, V. A. HUGHES, B. H. JONES, AND H. G. KNUTTGEN. Metabolic changes following eccentric exercise in trained and untrained men. J. Appl. Physiol. 61: 1864-1868, 1986. 10. FRIDI~N, J., M. SJ~STR~M, AND B. EKBLOM. A morphological study of delayed muscle soreness. Experientia Base1 37: 506-507, 1981. 11. GONZALEZ-SERRATOS, J., R. VALLE-AGUILERA, D. LATHROP, AND M. CEL CARMEN GARCIA. Slow inward calcium currents have no obvious role in muscle excitation-contraction coupling. Nature Lond. 298: 292-294,1982. 12. GUHARAY, F., AND F. SACHS. Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J. Physiol. Lond. 352: 685-701, 1984. 13. JACKSON, M. J., D. A. JONES, AND R. H. T. EDWARDS. Experimental skeletal muscle damage: the nature of the calcium-activated degenerative processes. Eur. J. Clin Inuest. 14: 369-374, 1984. 14. JENKINS, R. R. Free radical chemistry. Sports Med. 5: 156-170, 1988. 15. KEPNER, B. L., AND D. M. HERCULES. Fluorometric determination
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MUSCLE
of calcium in blood serum. Anal. Chem. 35: 1238-1240, 1963. 16. KUIPERS, H., 3. DRUKKER, P. M. FREDERICK, P. GEURTEN, AND G. KRANENBURG. Muscle degeneration after exercise in rats. Int. J. Sports Med. 4: 45-51, 1983. 17. LAMAR, C. P. Chelation therapy of occlusive arteriosclerosis in diabetic patients. Angiology 15: 379-395, 1964. A. L. FARR, AND R. J. RAN18. LOWRY, 0. H., N. J. ROSEBROUGH, DALL. Protein measurement with the Folin phenol reagent. J. BioZ. Chem. 193: 267-275,195l. 19. MCCULLY, K. K., AND J. A. FAULKNER. Injury to skeletal muscle fibers of mice following lengthening contractions. J. Appl. Physiol. 59: 119-126, 1985. 20. MELLGREN, R. L. Calcium-dependent proteases: an enzyme system active at cellular membranes? FASEB J. 1: 110-115, 1987. 21. NADEL, E. R., U. BERGH, AND B. SALTIN. Body temperatures during negative work exercise. J. Appl. Physiol. 33: 553-558, 1972. D. J., G. MCPHAIL, K. R. MILLS, AND R. H. T. ED22. NEWHAM, WARDS. Ultrastructural changes after concentric and eccentric contractions of human muscle. J. Neural. Sci. 61: 109-122, 1983. A. B., W. SHIN, AND J. DRUCKER. Mitochondrial 23 NOVIKOFF, localization of oxidative enzymes. Staining results with two tetrazolium enzymes. Staining results with two tetrazolium salts. J. Biophys. Biochem. CytoZ. 9: 47-61, 1961. R. W., R. B. ARMSTRONG, K. E. BAIRD, AND C. L. 24. OGILVIE, BOTTOMS. Lesions in the rat soleus muscle following eccentrically biased exercise. Am. J. Anat. 182: 335-346, 1988. R. M., P. J. REEDS, T. ATKINSON, AND R. H. SMITH. 25. PALMER, The influence of changes in tension on protein synthesis and prostaglandin release in isolated rabbit muscles. Biochem. J. 214: 1011-1014, 1963. Microdosage spectropho26. PARIS, F., D. PRADEAU, AND M. HAMON.
27.
28.
29. 30.
31.
32. 33.
34.
35.
36.
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tometrique de l’acide ethylene-diaminotetracetique dans les preparations pharmaceutiques. Ann. Pharm. Fr. 43: 133-138, 1985. RUSKOAHO, H. J., AND E. SAVOLAINEN. Effects of long-term verapamil treatment on blood pressure, cardiac hypertrophy and collagen metabolism in spontaneously hypertensive rats. Cardiowsc. Res. 19: 335-362, 1985. SCHWANE, J. A., AND R. B. ARMSTRONG. Effect of training on skeletal muscle injury from downhill running in rats. J. Appl. Physiol. 55: 969-975, 1983. SCOTT, P. J. Chelation therapy for degenerative vascular disease. N. 2. Med. J. 95: 583-589,1982. SEMBROWICH, W. L., J. J. QUINTINSKIE, AND G. LI. Calcium uptake in mitochondria from different skeletal muscle types. J. Appl. Physiol. 59: 137-141, 1985. SKIRBOLL, L. R., R. A. HOWARD, AND K. L. DRETCHEN. The effect of verapamil on the gastrocnemius and soleus muscles of the cat in vivo. Eur. J. Pharmacol. 60: 15-21, 1979. SOMLYO, A. P. Cellular site of calcium regulation. Nature Lond. 309: 516-517,1984. SPENCER, H., V. VANKINSCOTT, I. LEWIN, AND D. LASZLO. Removal of calcium in man by ethylenediamide tetra-acetic acid: metabolic study. J. Clin. Inuest. 31: 1023-1027, 1952. TATE, C. A., H. W. BONNER, AND S. W. LESLIE. Calcium uptake in skeletal muscle mitochondria. Eur. J. AppZ. Physiol. Occup. Physiol. 39: 111-116, 1978. WALLACH, D. F. H., D. M. SURGENOR, J. SODERBERG, AND E. DELANO. Preparation and properties of 3,6-dihydroxy-2,4-bis-[N, N’-di(carboxymethyl)aminomethyl] fluoran. Anal. Chem. 31: 456460,1959. WROGEMANN, K., AND S. D. J. PENA. Mitochondrial calcium overload: a general mechanism for cell-necrosis in muscle diseases. Lancet 2: 672-673,1976.
