Thermal instability of rat muscle sarcoplasmic reticulum Ca2+-ATPase function

J. D. Schertzer, H. J. Green, and A. R. Tupling

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

On a given experimental day, aliquots of the muscle homogenates were incubated in vitro for <= 60 min at normal resting body temperature (37°C) or at a temperature indicative of exercise-induced hyperthermia (41°C). At 0, 30, and 60 min of incubation, samples were extracted from each aliquot, and measurements of Ca2+ uptake and Ca2+-ATPase activity were made. To examine the effects of SH oxidation, DTT was added to one-half of the samples, and the samples that did or did not contain DTT were compared at different periods of incubation.

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
Y = Y<SUB>bot</SUB> + (Y<SUB>top</SUB> − Y<SUB>bot</SUB>)/(1 + 10<SUP>(LogCa<SUB>50</SUB>−<IT>X</IT>)</SUP> · n<SUB>H</SUB>)
where Ybot is the value at the bottom of the plateau, Ytop is the value at the top of the plateau, LogCa50 is the logarithm of the concentration that gives a response halfway between Ybot and Ytop (Ca50), and nH is the Hill coefficient.

Oxalate-supported Ca2+ uptake was measured using the Ca2+-fluorescent dye Indo-1 according to the methods of O'Brien and colleagues (25, 27), as modified by Ruell et al. (28) and Tupling et al. (35). Fluorescence measurements were made on a spectrofluorometer (Ratiomaster system, Photon Technology International) equipped with dual-emission monochromators. The measurement of [Ca2+]f with this procedure is based on the difference in maximal emission wavelengths between the Ca2+ bound to Indo-1 and the free Ca2+. The excitation wavelength was 355 nm, and the emission maxima were 485 and 405 nm for free Ca2+ (G) and Ca2+ bound to Indo-1 (F), respectively. The ratio (R) of F to G decreases during SR Ca2+ uptake and is used to calculate [Ca2+]f. Felix software (Photon Technology International) was used to calculate the ionized Ca2+ concentration by the following equation (19)
[Ca<SUP>2+</SUP>]<SUB>f</SUB><IT>=K</IT><SUB>d</SUB> * (G<SUB>max</SUB>/G<SUB>min</SUB>) (R − R<SUB>min</SUB>) / (R<SUB>max</SUB> − R) (1)
where Kd is the equilibrium constant for the interaction between Ca2+ and Indo-1, Rmin is the minimum value of R with the addition of 250 µM EGTA, and Gmax is the maximum value of G with the addition of 1 mM CaCl2. The Kd value for the interaction of Ca2+ and Indo-1 for muscle homogenates is 250 (19).

Simultaneous photon counts per second were recorded for both emission wavelengths. Before the experiment, Ca2+ independent (background) fluorescence was recorded in the reaction medium in the absence of Indo-1. A background fluorescence correction was implemented using the Felix software before each assay was started.

The reaction buffer (pH 7.0) contained (in mM) 200 KCl, 20 mM HEPES, 15 MgCl2, 10 NaN3, 10 PEP, 5 mM oxalate, 5 µM N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Before each assay, 1.5 µM Indo-1 and 18 U/ml each of LDH and PK were added to 2 ml of reaction buffer. Immediately before collection of emission spectra, 55 µl of muscle homogenate were added to the cuvette containing the reaction buffer. After initiation of data collection, 2.5 µl of 10 mM CaCl2 were added to the cuvette, which gave a consistent starting [Ca2+]f of ~3.5 µM. Shortly after the achievement of a constant [Ca2+]f, 5 mM ATP was added to the cuvette to initiate active Ca2+ uptake. All assays were run at 37°C.

The generated curve from Eq. 1, [Ca2+]f vs. time, was smoothed over 21 points by use of the Savitsky-Golay algorithm. The rate of Ca2+ uptake was then analyzed at 1.4 µM [Ca2+]f,. A linear regression was done on a range of values between 1.4 and 1.6 µM [Ca2+]f. Differentiating the linear fit curve allowed determination of Ca2+ uptake rates.

In a subsample (n = 3) from each group, 300 µM ryanodine (RyR) was added before the Ca2+ uptake assay. In this concentration RyR maintains the Ca2+ release channels in the closed state, eliminating bias in the Ca2+ uptake measures.

Protein determination was made by the method of Lowry, as modified by Schacterle and Pollock (29). All samples were analyzed in triplicate.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Rate of Ca2+ uptake in white gastrocnemius (WG) homogenates during in vitro incubations at 37 and 41°C. Values are means ± SE (n = 9). DTT 37, dithiothreitol at 37°C; CON 37, control at 37°C; DTT 41, DTT at 41°C; CON 41, control at 41°C. DTT treatment resulted in generally higher values (P < 0.05, main effect). Inactivation of Ca2+ uptake at both 37 and 41°C (P < 0.05, main effect) was observed. *Significantly lower values at 41°C compared with 37°C.



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Fig. 2.   Rate of Ca2+ uptake in white gastrocnemius (WG) muscle homogenates in the presence of 300 µM ryanodine (RyR) during in vitro incubations at 37°C and 41°C. Values are means ± SE (n = 3). DTT treatment resulted in generally higher values (P < 0.05, main effect). Thermal inactivation of Ca2+ uptake was observed at 41°C (P < 0.05, main effect). *Significantly lower values at 41°C compared with 37°C



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Fig. 3.   Rate of Ca2+ uptake in WG homogenates in the presence and absence of 300 µM RyR during in vitro incubations at 37°C. Values are means ± SE (n >=  3). DTT treatment resulted in generally higher values (P < 0.05, main effect). There was a significant interaction effect for RyR treatment and time of incubation (P < 0.05). *Significantly lower than 0 and 30 min and other conditions at 60 min. #Significantly lower than DTT 37 RyR and 0 min.

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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Fig. 4.   Maximal Ca2+-ATPase activity in WG homogenates during in vitro incubations at 37 and 41°C. Values are means ± SE (n = 9). DTT treatment resulted in generally higher values (P < 0.05, main effect). Greater thermal inactivation of Ca2+-ATPase activity occurred at 41°C compared with 37°C (P < 0.05, main effect). Time-dependent inactivation of Ca2+-ATPase activity (P < 0.05, main effect; 0 > 30 > 60 min)

For WG muscle, Ca2+-ATPase enzyme kinetics following heat stress were assessed (Fig. 5). The Hill slope (nH), a measure of co-operativity, and the pCa50 both decreased at 41°C incubations in CON (Table 1). For both properties, the decrease was maximal at 30 min. At 37°C, no effects were observed in CON for either the nH or pCa50. Importantly, DTT treatment attenuated the alterations in Ca2+-ATPase enzyme kinetics observed at 41°C.


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Fig. 5.   Sample tracings of Ca2+-ATPase enzyme kinetics for WG after 30 min of incubation at 41°C with and without DTT. pCa, the negative logarithm of cytosolic free Ca2+ concentration.


                              
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Table 1.   Ca2+-ATPase enzyme kinetic properties of white gastrocnemius muscle in response to thermal stress

To determine whether SR membrane integrity is altered by heat stress, Ca2+-ATPase activity was assessed during a 30-min incubation at 37 and 41°C with and without the Ca2+ ionophore A-23187. At time 0 and after a 37°C incubation for 30 min, Vmax values were higher with the addition of the Ca2+ ionophore. For both conditions, the ionophore resulted in a 3.6-fold increase in Vmax compared with the "no ionophore" condition (Table 2). A similar effect was observed at 41°C at the start of the incubation. However, after a 30-min incubation at 41°C, the addition of Ca2+ ionophore did not result in a significantly higher Vmax compared with the control or no ionophore condition. As can be seen in Table 2, the 30-min incubation at 41°C, in the absence of Ca2+ ionophore, increased Vmax compared with all other conditions without ionophore. This finding indicates an increased membrane permeability to Ca2+ at the higher incubation temperature.

                              
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Table 2.   Maximal Ca2+-ATPase activity of white gastrocnemius muscle in the presence and absence of A-23187 after thermal stress

Changes in the coupling ratios, defined as the ratio between Ca2+ uptake and Ca2+-ATPase activity, are provided in Fig. 6. As a general effect, coupling ratios were progressively reduced over time. At both 30 and 60 min of incubation, the coupling ratio was lower at 41°C (both CON and DTT) than at 37°C. DTT did not affect the coupling ratios.


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Fig. 6.   Changes in coupling ratios in WG muscle incubated at 37 and 41°C for <= 60 min both with DTT (DTT) and without DTT (CON). A main effect for incubation time (P < 0.05) was found. For time, 0 > 30 > 60 min. An interaction effect for incubation time and temperature (P < 0.05) was found. *Significantly higher for 37°C compared with 41°C. No effect of DTT was observed.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

This study was supported by the Natural Sciences and Engineering Research Council (Canada).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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