Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
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ABSTRACT |
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To examine the thermal instability and the
role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR)
Ca2+-ATPase function, crude homogenates were prepared from
the white portion of the gastrocnemius (WG) adult rat muscles
(n = 9) and incubated in vitro for 60 min either at a
normal resting body temperature (37°C) or at a temperature indicative
of exercise-induced hyperthermia (41°C) with DTT and without DTT
(CON). In general, treatment with DTT resulted in higher
Ca2+-ATPase and Ca2+ uptake values
(nmol · mg protein
1 · min
1,
P < 0.05), an effect that was not specific to time of
incubation. Incubations at 41°C resulted in lower (P < 0.05) Ca2+ uptake rates (156 ± 18 and 35.9 ± 3.3) compared with 37°C (570 ± 54 and 364 ± 26) at 30 and
60 min, respectively. At 37°C, ryanodine (300 µM), which was used
to block Ca2+ release from the calcium release channel,
prevented the time-dependent decrease in Ca2+ uptake. A
general inactivation (P < 0.05) of maximal
Ca2+-ATPase activity (Vmax) in CON
was observed with incubation time (0 > 30 > 60 min), with
the effect being more pronounced (P < 0.05) at 41°C
compared with 37°C. The Hill slope, a measure of co-operativity, and
the pCa50, the cytosolic Ca2+ concentration
required for half-maximal activation of Ca2+-ATPase
activity, decreased (P < 0.05) at 41°C only.
Treatment with DTT attenuated the alterations in enzyme kinetics. The
increase in Vmax with the Ca2+
ionophore A-23187 was less pronounced at 41°C compared with 37°C. It is concluded that exposure of homogenates to a temperature typically
experienced in exercise results in a reduction in the coupling ratio,
which is mediated primarily by lower Ca2+ uptake and occurs
as a result of increases in membrane permeability to Ca2+.
Moreover, the decreases in Ca2+-ATPase kinetics in WG with
sustained heat stress result from SH oxidation.
muscle; Ca2+ regulation; temperature; Ca2+ uptake; Ca2+-ATPase
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INTRODUCTION |
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SKELETAL MUSCLE CONTRACTION is regulated by the concentration of free cytosolic Ca2+ ([Ca2+]f), which is primarily controlled by the sarcoplasmic reticulum (SR) (23). The SR is an elaborate muscle membrane system residing at the junction between the transverse tubule, the sarcolemmal membrane, and the contractile apparatus (4, 23). Sarcolemmal and T-tubule depolarization induces a rapid release of Ca2+ from the luminal SR store, via the Ca2+ release channel [ryanodine (RyR)], resulting in increased [Ca2+]f and muscle contraction. The SR is also the primary site of Ca2+ resequestration, acting as a sink for the removal of [Ca2+]f after excitation and allowing muscle relaxation to occur. Ca2+ sequestration into the SR is regulated by the Ca2+-ATPase, a 110-kDa transmembrane protein (23, 24).
Repetitive contractile activity ultimately results in an inability to produce a desired force. The fatigue that occurs has been frequently ascribed to failure in excitation-contraction coupling and, in particular, loss of [Ca2+]f regulation by the SR (1, 15, 18). Studies using animal (7, 37, 38) and human models (6, 35) have shown altered SR Ca2+ handling after prolonged exercise to fatigue. Studies published to date indicate that reductions in [Ca2+]f observed at fatigue occur, at least in part, from reductions in Ca2+ release (15). Several studies have also shown that Ca2+ uptake (2, 6-8, 16, 39) and/or Ca2+-ATPase activity (6-8, 16) are depressed. However, many studies, using a number of different animal models including prolonged running (9), prolonged low-frequency stimulation (10), high-frequency stimulation (14), and short-term sprinting (13), have failed to show a difference in SR Ca2+ handling. Clearly, conflicting results have been reported, which may be due to a number of factors, including differences in exercise protocols, environmental conditions, tissue sampling sites, and assay procedures.
One factor that could explain the differences among studies is muscle temperature. Exercise results in a sustained, increased muscle temperature, which can reach 43°C (3, 8). Thermal instability of SR Ca2+-sequestering properties has been demonstrated by incubating SR vesicle preparations at high temperatures (21, 34). In general, these studies demonstrate that the coupling ratio between Ca2+ uptake and Ca2+-ATPase activity is dramatically reduced (12, 17, 34). Although reduction in both Ca2+ uptake (12, 17) and Ca2+-ATPase activity (12, 32) occur with short-term heating, disproportionate reductions in Ca2+ uptake appear to predominate (17, 26). The disproportionate reduction in Ca2+ uptake occurs not as a result of leakage from the Ca2+ release channel but from an apparent aggregation of Ca2+-ATPase monomers in the SR vesicles and an increase in the permeability to Ca2+ (12, 17, 32, 34). As a result, the effects of the Ca2+ ionophore A-23187 are reduced after heat treatment compared with control (17).
Inactivation of muscle Ca2+-ATPase has been shown to occur in native SR vesicles during prolonged incubation to elevated temperatures (12, 32, 36). The increased temperature is believed to result in protein unfolding, which exposes hydrophobic domains and leads to an aggregation of Ca2+-ATPase monomers (12, 32). The exposure of hydrophobic residues has been attributed to oxidation of sulfhydryl (SH) groups by reactive oxygen species (ROS) (12, 32). It has been demonstrated that even a small inactivation of the Ca2+-ATPase pump is sufficient to functionally uncouple the entire vesicle and result in increased vesicle permeability to Ca2+ (12). At least one study, using prolonged exercise, has shown that a similar uncoupling may occur in a selected skeletal muscle of rats (16). Interestingly, ROS and/or nitric oxide (NO) have been credited with inactivating SR Ca2+-ATPase in rat muscle (22).
Dithiothreitol (DTT) is an SH-reducing agent that is frequently added to homogenization buffers to prevent oxidative damage during muscle homogenization. The importance of DTT in attenuating SR impairment has been shown in experiments where SH oxidation was induced by exposure to peroxydisulfate or hydrogen peroxide or by extended storage (30). Accordingly, it might be expected that stability of the SR Ca2+-sequestering function in SR preparations exposed to hyperthermia would be improved with DTT in the incubation medium.
In the present study, we sought to characterize the temperature dependency of the SR muscle Ca2+-sequestering function and to determine the effects of SH oxidation on the changes that occur by use of a protocol that would result in a similar thermal strain experienced by muscle during prolonged exercise. We hypothesized that in vitro incubation of rat skeletal muscle homogenates for up to 60 min at resting body temperature (37°C) compared with a temperature indicative of exercise-induced hyperthermia (41°C) would result in a greater decline in Ca2+ uptake compared with maximal Ca2+-ATPase activity (Vmax). Moreover, because the hyperthermic effects have been previously examined primarily in SR vesicle preparations, we used crude muscle homogenates. Crude muscle homogenates are more physiological than SR vesicle preparations, and it is possible that many compounds may alter the temperature effects on Ca2+-ATPase and Ca2+ uptake behavior.
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METHODS |
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Animal description and care. Adult female Sprague-Dawley rats (237 ± 11 g) were fed laboratory chow and water ad libitum. The animals were housed in an environmentally controlled room (temperature 22-24°C, 40-60% relative humidity) with reversed light-dark cycles. The experimental protocols were initiated at approximately the same time each day to prevent diurnal variations in physiological parameters, including muscle glycogen (11). The Animal Care Committee of the University of Waterloo approved all experimental protocols.
Sample preparation and temperature manipulations.
Sprague-Dawley rats (n = 9) were anesthetized with an
intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt). After anesthetization, the whole gastrocnemius muscle was excised from
each hindlimb and placed in ice-cold buffer and separated into white
(WG) and red portions. The WG tissue from both legs was combined and
diluted 1:11 (wt/vol) in an ice-cold homogenizing buffer (pH 7.5)
containing (in mM) 250 sucrose, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 0.2 phenylmethylsulfonyl fluoride (PMSF), and 0.2% sodium azide (NaN3) (20). The homogenization
buffer was prepared in both the absence (CON) and presence (5 mM) of
the SH-reducing agent DTT. The muscles were mechanically homogenized
with a polytron homogenizer (PT 3100) at 16,500 rpm for 2 × 30-s
bursts separated by a 30-s break. Immediately after homogenization,
aliquots were rapidly frozen in liquid nitrogen and stored at 80°C
until analysis of SR function.
Measurements of SR function.
Ca2+-induced Ca2+-ATPase activity was measured
in whole muscle homogenates by means of the spectrophometric assay
developed by Simonides and van Hardeveld (33), as modified
by our laboratory (35). The reaction buffer contained (in
mM) 200 KCl, 20 HEPES, 15 MgCl2, 10 NaN3, 10 phosphoenolpyruvate (PEP), 5 ATP, and 1 EGTA. The pH of the
reaction buffer was adjusted to 7.0 at 37°C. Immediately before the
reaction was started, 18 U/ml lactate dehydrogenase (LDH), 18 U/ml
pyruvate kinase (PK), 0.3 mM NADH, and 15 µl of WG homogenate were
added to 1 ml of reaction buffer. In addition, assays were run both
with (1 µM) and without the Ca2+ ionophore A-23187
(Sigma, C-7522). Assays were performed at 37°C and 340 nm (Shimadzu
UV 160). After the recording of baseline absorbance and fluorescence of
NADH, the reaction was initiated by adding 3 µl of 100 mM
CaCl2 and monitored for ~2 min. At the end of this
period, additional 0.5-µl additions of 100 mM CaCl2 were
made to assess Ca2+-dependent Ca2+-ATPase
activity and to ensure that the activity was maximal
(Vmax). Basal or Mg2+-ATPase
activity was determined by adding 1 µl of the specific inhibitor of
the Ca2+-ATPase, cyclopiazonic acid. The
[Ca2+]f corresponding to each
CaCl2 addition was assessed separately using dual-emission
spectrofluorometry and the fluorescent Ca2+-binding dye
Indo-1. The [Ca2+]f required for half-maximal
activation of Ca2+-ATPase activity (pCa50) and
the Hill coefficient (nH) were determined by nonlinear
regression after Ca2+-ATPase activity was plotted against
the negative logarithm of [Ca2+]f (pCa). The
Hill coefficient was calculated using values ranging between 20 and
80% of the Vmax according to the following
sigmoidal dose-response equation
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(1) |
Data analysis. To determine the effects of temperature (37°C vs. 41°C), time of incubation (0, 30, 60 min), and treatment (with and without DTT), a repeated-measures three-way analysis of variance (ANOVA) was employed. The same analysis was used to determine the effect of ionophore treatment. Where significant differences were found, Duncan's post hoc tests were used to compare specific means. For all comparisons, the level of significance was set at P < 0.05. All data are presented as means ± SE.
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RESULTS |
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SR Ca2+ uptake.
In vitro incubations of WG homogenates at both 37°C and 41°C
resulted in reductions of SR Ca2+ uptake over time (Fig.
1). The increased heat stress during the 41°C incubations induced a significantly greater depression in Ca2+ uptake compared with 37°C at both 30 and 60 min. The
inclusion of DTT, used to block oxidation, resulted in generally higher Ca2+ uptake rates, an effect that was independent of
incubation time. To examine the effects of release of Ca2+
from the SR Ca2+ release channel on
Ca2+-uptake, RyR was used to block Ca2+
release. The addition of 300 µM RyR, an inhibitor of SR
Ca2+ release, in a subsample did not affect the reduction
in Ca2+ uptake rates during incubations at 41°C (Fig.
2). However, at 37°C, RyR prevented the
reduction in Ca2+ uptake that was observed at 37°C when
incubations were performed without RyR. The result suggests that, at
37°C, release of Ca2+ by the SR Ca2+ release
channel must be considered in the net Ca2+ uptake rates
observed. Interestingly, this effect was observed only at 37°C and
not at 41°C. This influence of RyR at 37°C is illustrated in Fig.
3. In the control condition, RyR
eliminated the time-dependent reduction in Ca2+ uptake.
Such an effect was not observed when the homogenate was treated with
DTT.
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SR Ca2+-ATPase activity.
Maximal Ca2+-ATPase activity (Vmax),
measured in CON, was lower at 41°C than 37°C in WG homogenates, an
effect that was independent of the time of incubation (Fig.
4). In addition, DTT treatment resulted
in generally higher Vmax. This was evident at
the onset of incubation and persisted throughout the incubation period. A main effect of time of incubation was found for
Ca2+-ATPase activity such that Vmax
was progressively reduced (0 > 30 > 60 min). This effect
was not specific to temperature level.
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DISCUSSION |
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The purpose of the study was to assess alterations in SR
Ca2+-sequestering function associated with thermal stress
in rat skeletal muscle. Crude homogenates were prepared from rat WG
muscle and incubated in vitro either at a normal resting body
temperature (37°C) or at a temperature indicative of exercise-induced
hyperthermia (41°C) for 60 min. To examine the mechanisms of
thermal inactivation, homogenates were prepared in the absence (CON) or
presence of the SH-reducing agent DTT.
As hypothesized, we found a more pronounced depression in SR Ca2+ uptake rates after 30 and 60 min of incubation of 41°C compared with 37°C. Interestingly, only modest reductions in Vmax activity were observed during similar incubations. However, as with Ca2+ uptake, the reduction in Vmax was more pronounced at the higher temperature. At 41°C incubation, the reduction in Vmax was also accompanied by reductions in nH and pCa50. No changes in these properties were found at 37°C, regardless of the period of incubation. As a result of the greater depression in Ca2+ uptake compared with Ca2+ transport, the coupling ratio was significantly reduced across conditions. At 41°C, the coupling ratios were lower during 30 and 60 min of incubation compared with 37°C.
Our general findings are consistent with previous studies investigating thermal stress in SR vesicle preparations (12, 17). As with our study, these investigations have reported either no (17) or modest reductions (12) in Vmax behavior of the Ca2+-ATPase, depending on the protocol employed. However, regardless of the protocol, thermal stress results in pronounced reductions in net Ca2+ uptake. Our results also illustrate the potent effect of increased temperature on Ca2+ sequestration. At 41°C, a temperature stress not uncommon in exercising muscle, the reduction in net Ca2+ uptake was fully manifested by 30 min. The primary mechanism underlying the reduction in Ca2+ uptake has been commonly attributed to Ca2+ leakage from the SR lumen to the cytosol through the Ca2+ pumps (12, 17, 32). According to this reasoning, Ca2+-ATPase pumping ability remains relatively intact, the inefficiency resulting from the increase in membrane permeability to Ca2+. Under such circumstances, increased vesicle permeability is indicated by a decrease in passive loading (no requirement for ATP) of Ca2+ into the SR in the absence of oxalate (12). Collectively, our results suggest that Ca2+ uptake may not be compromised; rather, the reduced net Ca2+ uptake results from the efflux of Ca2+ from the lumen of the SR. Under such conditions, the coupling ratio per se is not the primary problem. However, as a result of the leaky SR, more energy is needed to achieve a given net Ca2+ uptake.
The increase in vesicle permeability to Ca2+ appears to be secondary to the thermal effects on the Ca2+ pump. Increased temperature has been proposed to induce unfolding and aggregation of the Ca2+-ATPase, which can form channels for Ca2+ leakage (12). As has been shown previously (17), we have found that, after heat treatment, the effects of the Ca2+ ionophore A-23187 are considerably reduced. At 37°C, the Ca2+ ionophore resulted in a 3.6-fold increase in Vmax of the Ca2+-ATPase compared with no ionophore both at 0 min and after 30 min of incubation. However, at 41°C, the effects of the ionophore were reduced to 1.3-fold at 30 min. This change provides clear evidence of the effects of thermal stress on SR membrane permeability. We have also used RyR in a subsample at a concentration known to inhibit opening of the Ca2+ release channel to determine whether release of Ca2+ from the Ca2+ release could bias our findings. Our results indicate that, at 41°C, Ca2+ release did not alter the thermal effects on Ca2+ uptake. At 37°C, however, the inclusion of RyR abolished the reductions in Ca2+ uptake observed with time of incubation. These findings would indicate that, at the lower temperature, release of Ca2+ from the Ca2+ release channel is contributing to the low Ca2+ uptake rates observed.
Senisterra et al. (32) demonstrated that thermal denaturation of the Ca2+-ATPase enzyme resulted in unfolding of the protein, exposure of hydrophobic residues of the enzyme, and oligomerization. Oligomerization was defined as aggregation from more active, low-molecular-weight aggregates to less active, high-molecular-weight aggregates. Interestingly, at temperatures below 43°C, the unfolding of the protein was slower than at higher temperatures and did not approach completion after 50 min of exposure, whereas protein unfolding was not observed at temperatures below 30°C. These effects were attributed to SH oxidation, since they were attenuated by DTT treatment (32). We also investigated the affects of SH oxidation during our heat stress protocols.
A decrease in the co-operative behavior of the Ca2+-ATPase enzyme (nH) and an increase in the pCa50 were shown after 30- and 60-min incubations at 41°C. Treatment of the muscle homogenates with an SH-reducing agent (DTT) attenuated these changes, suggesting that SH oxidation, which leads to enzyme oligomerization, is the mechanism responsible for alterations in Ca2+-ATPase enzyme kinetics induced by heat stress. Our results also indicate that SH oxidation during sustained heat stress significantly decreases Vmax. Although we found higher Vmax activities with DTT compared with CON, these effects cannot be explained by prolonged heat stress, because they occurred during homogenate preparation. This suggests that other mechanisms in addition to SH oxidation are involved in mediating the decreases in Ca2+-ATPase activity observed as a general effect of time regardless of condition. At present, it is not clear what mechanisms promote the instability.
Treatment with DTT also resulted in generally higher values for Ca2+ uptake, an effect that could also be explained by SH oxidation during the homogenization procedure. This finding is consistent with previous work from our laboratory using skeletal muscle (31) and from other groups using cardiac muscle (5). Interestingly, at 37°C, DTT reduced the decline on Ca2+ uptake observed at 60 min. This suggests that SH oxidation can progress during incubation at this temperature.
In summary, we found that in vitro incubation of white gastrocnemius rat skeletal muscle homogenates at a temperature indicative of exercise-induced hyperthermia (41°C) for up to 60 min results in large reductions in SR Ca2+ uptake and only modest reductions in maximal Ca2+-ATPase activity. The thermal inactivation of Ca2+ uptake at 41°C was not due to SH oxidation of the Ca2+-ATPase enzyme or alterations in SR Ca2+-release channel activity. The increased heat stress employed in this study caused significant reductions membrane integrity and permeability to Ca2+, resulting in a leaky SR. In addition, alterations in Ca2+-ATPase enzyme kinetics are evident following increased heat stress, an effect that is due to SH oxidation.
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ACKNOWLEDGEMENTS |
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This study was supported by the Natural Sciences and Engineering Research Council (Canada).
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON Canada N2L 3G1 (E-mail: green{at}healthy.uwaterloo.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00204.2002
Received 9 May 2002; accepted in final form 23 June 2002.
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1999[ISI][Medline].