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J Appl Physiol 86: 584-591, 1999;
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Vol. 86, Issue 2, 584-591, February 1999

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

Bradley M. Palmer, Joshua M. Lynch, Steven M. Snyder, and Russell L. Moore

Department of Kinesiology and Applied Physiology and The University of Colorado Cardiovascular Institute, University of Colorado at Boulder, Boulder, Colorado 80309


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29°C. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.

sodium/calcium exchange; fura 2; caffeine; sarcolemma; sarcoplasmic reticulum


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CHRONIC RUN TRAINING of the rat has been shown to alter cardiac processes including pressure development, metabolism, and ion handling (3, 5, 8, 30, 31). Alterations in characteristics of pressure development, although possibly due, in part, to changes in mechanical properties (35), have most often been interpreted to reflect changes in the underlying cytosolic Ca2+ concentration ([Ca2+]c) dynamics, which are dictated by the Ca2+ regulatory properties of the sarcolemma (SL), sarcoplasmic reticulum (SR), mitochondria, intracellular buffers, and diffusion (1). Many, but not all, examinations of SL and SR vesicles have demonstrated various training-induced changes in properties of [Ca2+]c regulatory protein composition and/or function (7, 9, 12, 19, 22, 29, 32, 33). In addition, experiments with whole heart preparations, which allow for a more integrated study of the possible training effects on the interdependency of [Ca2+]c regulatory mechanisms, have demonstrated that run training induces an increased sensitivity to changes in external Ca2+ and Na+ concentrations and pacing frequency (3, 8).

The contractile dynamics of left ventricular (LV) myocytes isolated from run-trained rats have also been shown to be more sensitive to external Ca2+ concentration ([Ca2+]o) and pacing frequency than those of sedentary controls (16). At low pacing frequencies (0.067 Hz), the maximal myocyte shortening was greater for the trained rats and increased more as [Ca2+]o increased (0.6-2.0 mM) (16). At higher pacing frequencies (0.5 Hz), the maximal myocyte shortening was greater for the sedentary controls (16, 18). These data represent strong evidence for training-induced modulations of SL Ca2+ influx and efflux mechanisms in such a way that Ca2+ influx is favored (or efflux disfavored) at the low pacing frequency, but efflux is favored (or influx disfavored) at the higher pacing frequency in the myocyte model.

Using fura 2 fluorescence microscopy to detect [Ca2+]c transients in isolated LV myocytes, Laughlin et al. (10) concluded that endurance run training did not affect [Ca2+]c transient characteristics. Moore et al. (16), on the other hand, reported a training-induced decrease in the velocity of [Ca2+]c decline. In a third study, run training was not found to influence temporal characteristics of the fluorescence ratio (R) transient (18). Unfortunately, these three studies do not lead to a clear consensus about the effects of training on [Ca2+]c regulation in intact tissue under laboratory conditions. However, because fura 2 itself may mask differences in [Ca2+]c transient characteristics between populations (18), studies of training-induced changes in [Ca2+]c regulation may have to be more focused on distinct regulatory mechanisms.

In the present report, we describe observations of [Ca2+]c transient characteristics elicited by electrical pacing and by caffeine-induced contractures designed to focus on Na+-dependent Ca2+ efflux mechanisms of the SL. We did not find that run training changed the resting or peak [Ca2+]c of LV myocyte [Ca2+]c transients elicited by electrical pacing, although run training significantly prolonged the temporal characteristics of these [Ca2+]c transients. We also found that run training significantly reduced the rate of Na+-dependent [Ca2+]c clearance as determined by caffeine-induced contractures. These data are the first of their kind to show directly that Na+-dependent Ca2+ efflux, presumably Na+/Ca2+ exchange via the exchanger isoform found in rat cardiac myocytes (NCX1), may be depressed in functional cardiac tissue after chronic run training.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal Model

Female Sprague-Dawley rats were randomly assigned to a sedentary (Sed) group (n = 9) and an exercise trained (Tr) group (n = 9). All animals were housed and cared for as previously described (13). Rats trained for a minimum of 20 wk that included a 12-wk phase during which running intensity and duration were gradual increased. At the end of the first 12 wk, rats ran 5 days/wk for 1 h/day up a 10% grade. The daily training bout consisted of 15 min of running at 20 m/min, 30 min at 28 m/min, and 15 min at 35 m/min. All animals were 9-12 mo of age at time of death. On euthanasia, the animals' adrenal glands and the spleen were dissected and weighed, and plantaris muscles were dissected, homogenized, and assayed for citrate synthase activity as previously described (16, 26).

Animal care and use conformed to the guidelines accepted by the American Physiological Society. This study protocol was reviewed and received prior approval from the Institutional Animal Care and Use Committee at the University of Colorado, Boulder.

Myocyte Isolation

Cardiac myocytes were obtained from the LV free wall and septum from rat hearts by using methods previously described (17). All chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO) except where noted. Animals were heparinized (250 U ip) and then anesthetized with pentobarbital sodium (35 mg/kg body wt ip; Abbott Laboratories, North Chicago, IL). Hearts were rapidly excised and placed in ice-cold saline solution. The aorta was then cannulated, and the heart was retrogradely perfused by using a modified Langendorf perfusion apparatus that could deliver three different solutions maintained at pH 7.4 and 37°C and bubbled with 95% O2-5% CO2 gas. The first solution was a bicarbonate-based modified Krebs-Henseleit buffer, the second solution was a Krebs-Henseleit buffer containing nominal Ca2+, and the third solution contained an additional 375 U/ml collagenase (Worthington, Freehold, NJ) and 420 U/ml hyaluronidase. LV and septal myocardium were minced and placed in a collagenase and hyaluronidase solution. Myocyte isolation continued with mechanical agitation. Isolated LV cardiac myocytes were suspended in bicarbonate-based medium 199, plated onto laminin-coated glass coverslips, and incubated for between 2 and 8 h at 37°C in a humidified 5% CO2-balance room air.

Experimental Protocol

Coverslips were incubated for 5 min in the presence of 0.05% (vol) DMSO + 2 µM fura 2-AM (Molecular Probes, Eugene, OR). Each coverslip was removed from the fura 2-loading medium and used to form the bottom plate of a custom-built flow-through chamber (27). The chamber was placed on the stage of an inverted microscope (Nikon Diaphot) fitted with a ×40 oil-immersion objective. Coverslips were superfused with a normal Tyrode solution (in mM: 140 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 2 pyruvate, and 5 HEPES, pH 7.4) maintained at 29°C. Myocytes were electrically paced via field stimulation by using platinum electrodes with a stimulus duration of 0.5 ms, voltage of 1.5 × stimulation threshold, and at a pacing frequency of 0.5 Hz (Grass Instruments, Boston, MA).

After a myocyte was identified for study, electrical pacing was ceased for 2 min to reduce any possible discrepancies in myocyte contractile states due to differential times to identification. Continuous electrical pacing began again, and fura 2 R and shortening dynamics were recorded at 1, 3, and 5 min. At 7 min, and synchronized to the pacing period, the superfusate was rapidly switched to a Na+-free and Ca2+-free Tyrode solution + 10 mM caffeine (in mM: 140 LiCl, 6 KCl, 1 MgCl2, 10 glucose, 2 pyruvate, 5 HEPES, and 10 caffeine, pH 7.4). After 5 s, during which time [Ca2+]c reached a new steady state, the superfusate was rapidly switched to a Ca2+-free Tyrode solution + 10 mM caffeine (in mM: 140 NaCl, 6 KCl, 1 MgCl2, 10 glucose, 2 pyruvate, 5 HEPES, and 10 caffeine, pH 7.4). During this latter caffeine exposure, Ca2+ was removed from the cytosol via Na+-dependent efflux across the SL. R dynamics were recorded during caffeine exposures and underwent characterization only if the respective myocyte had functionally recovered after the caffeine exposure. The criteria used to indicate functional recovery were the maintenance of resting length and the preservation of monophasic fluorescence and shortening dynamics elicited by electrical stimulation.

Measurements of [Ca2+]c Dynamics

Fura 2 fluorescence was induced with a fluorescence system (IonOptix, Milton, MA) fitted with optical filters of 400 and 360 nm. This choice of filters takes advantage of a linear relationship between [Ca2+]c and R when an excitation wavelength >390 nm is used (28). Fluorescence intensities were recorded as photon-counting rates by using a personal computer. The value for myocyte fluorescence background was determined for each cell by superfusion of Ca2+-free Tyrode + 1 M digitonin for 4 min, which released cytosolic fura 2, and the subsequent measurement of fluorescence with Ca2+-free Tyrode as superfusate. Another measurement of myocyte fluorescence with Ca2+-free Tyrode + 10 mM caffeine as superfusate was also taken and incorporated as fluorescence background for the caffeine exposures.

Custom-made software was used to analyze the R transients recorded during electrical pacing, and the characteristics of resting R (Rrest), peak R (Rpeak), Rpeak minus Rrest (Rdiff), two exponential rate constants, krise and kfall, and time to peak R were determined by nonlinear, least squares fitting of the following double-exponential function to the recorded R transient
R = R<SUB>amp</SUB> (<IT>e</IT><SUP>−<IT>k</IT><SUB>fall</SUB><IT>t</IT></SUP> − <IT>e</IT><SUP><IT>−k</IT><SUB>rise</SUB><IT>t</IT></SUP>) + R<SUB>rest</SUB> (1)
where Ramp is a theoretical amplitude at time 0. Time to peak R was determined from the exponential rate constants as (ln krise - ln kfall)/(krise - kfall), and Rpeak was determined as Eq. 1 evaluated at the time to peak R.

Rdiff during caffeine exposure (Rcaff) was measured as an estimate of SR Ca2+ load. The fraction of SR Ca2+ load released by electrical stimulation was also estimated as the value of Rdiff immediately before caffeine exposure divided by Rcaff. A single-exponential function was fit to the R transients recorded during the last 3.5 s of the first caffeine exposure to determine R at the new [Ca2+]c equilibrium (Requil) and the rate constant to reach that equilibrium (kequil). The first 1.5 s were considered to be strongly influenced by relatively fast Ca2+ regulatory events, i.e., SR uptake, Na+/Ca2+ exchange, and high-affinity Ca2+ buffering. Therefore, analysis of the last 3.5 s was intended to characterize the slower Ca2+-handling mechanisms, i.e., SL Ca2+-ATPase, mitochondrial uptake, and low-affinity Ca2+ buffering. A single-exponential function was also fit to R transients recorded during the subsequent Na+-rich caffeine exposure to determine the rate constant of Na+-dependent [Ca2+]c clearance via Ca2+ efflux across the SL (kefflux). An example of an R transient recorded during electrical stimulation and caffeine exposure, the quality of fits, and the meanings of the R characteristics are illustrated in Fig. 1.


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  		Fig. 1.   Example of fluorescence ratio (R) transient recorded during electrically stimulated and caffeine-induced cytosolic Ca2+ concentration ([Ca2+]c) transients. Bold lines, exponential fits. Caffeine contractures were initiated by rapid application of superfusate containing neither Na+ nor Ca2+ as indicated between 2 and 7 s. A Na+-rich superfusate was then applied, and Na+-dependent Ca2+ efflux ensued. krise and kfall, Exponential rate constants; kefflux, rate constant of Na+-dependent [Ca2+]c clearance via Ca2+ efflux across sarcolemma; kequil, rate constant to reach [Ca2+]c equilibrium; Rrest, resting R; Rcaff, Rpeak minus Rrest during caffeine exposure; Rdiff, Rpeak minus Rrest; Requil, R at [Ca2+]c equilibrium.

Measurement of Myocyte Shortening Dynamics

The positions of myocyte edges were determined by using a video edge-detection device (Crescent Electronics, Sandy, UT) and recorded by using an analog-to-digital converter of the same personal computer that recorded fluorescence. Custom-made software was used to analyze the recorded myocyte shortening transients to determine the following characteristics: peak shortening as a percentage of resting length, maximal shortening velocity as a fraction of peak shortening, maximal relaxation velocity as a fraction of peak shortening, time to peak shortening, and times to 25, 50, and 75% recovery.

Analysis

All analyses were performed by using SPSS version 6.0 (SPSS, Chicago, IL). Contrasts between characteristics of Sed and Tr groups were determined by unpaired, two-tailed t-tests. To test the relative sensitivity of the myocyte groups to "pacing duration," a 2(Sed, Tr) × 3(1, 3, 5 min) repeated- measures ANOVA was performed on all characteristics of the electrically stimulated R and shortening transients. All data are presented as means ± SE. We report statistical significance at the P <=  0.05 and P <=  0.10 levels. Per the principles described by Williams et al. (34), reporting the latter significance level minimizes a propensity to commit a type II (false negative) interpretive error.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animal Model

Training did not significantly affect body weight, adrenal weights, or spleen weight in this study (Table 1). Citrate synthase activity of the plantaris muscle homogenates was significantly increased by training and provides verification that our treadmill training protocol, like those used previously (10, 13, 16, 18), was effective in producing a trained state in the rat.

                              
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  		Table 1.   Animal morphologies and plantaris muscle citrate synthase activity

Myocyte Studies

R dynamics during electrical pacing. Values for Rrest, Rpeak, and Rdiff, were not different between Sed and Tr (Fig. 2). There were, however, significant pacing duration main effects (P < 0.001) for all three values of Rrest, Rpeak, and Rdiff, and a significant "training × pacing duration" interaction (P = 0.013) for Rrest. Collectively, these data suggest that the intracellular Ca2+ load of the myocytes tended to increase with pacing duration under the experimental conditions used in this study. Furthermore, myocytes of the Tr group tended to better defend against this increase in intracellular Ca2+ from influencing diastolic [Ca2+]c.


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  		Fig. 2.   Characteristics of [Ca2+]c dynamics in paced myocytes from sedentary (Sed; open symbols; n = 52) and trained (Tr; closed symbols; n = 48) rats. A: Rrest was not different between groups and increased over time. This increase in diastolic [Ca2+]c was more pronounced in Sed than in Tr rats as indicated by a significant "training × pacing duration" interaction (P = 0.013). B: Rpeak was not different between groups and increased over time. C: Rdiff was also not different between groups and increased over time.

The temporal characteristics of the R transients highlight some important differences in Ca2+ regulation between the Sed and Tr groups (Fig. 3). Although the exponential rate constant krise was not found to be different between the groups, repeated-measures ANOVA revealed a "training" main effect for kfall (P = 0.060). Values for kfall were found to be consistently lower in the Tr group at all time points (Fig. 3B), and time to peak R was longer in the Tr group at 1 min pacing duration (Fig. 3C). There were also pacing duration main effects for krise (P = 0.081) and kfall (P < 0.001) but not for time to peak R. 


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  		Fig. 3.   Temporal characteristics of [Ca2+]c dynamics in paced myocytes from Sed (open symbols; n = 52) and Tr (closed symbols; n = 48) rats. A: krise was not different between groups and tended to decrease over time. B: kfall was significantly lower in Tr group and increased over time. C: time to peak R was greater in Tr group after 1 min of electrical pacing but was not different between groups at later times. dagger  P <=  0.10, * P <=  0.05 for Sed vs. Tr (t-test).

The temporal characteristics of the R transients demonstrate that Ca2+ removal from the cytosol proceeds more slowly in myocytes of the Tr group under the experimental conditions used in this study. In addition, the increase in kfall with pacing duration indicates that the rate of cytosolic Ca2+ removal, which is dominated by SR Ca2+ uptake (1), increased with continuous pacing in both groups. The decrease in krise with pacing frequency is also reflective of an increase in SR Ca2+ load, because krise represents the velocity of Ca2+ influx divided by the total Ca2+ moved.

R dynamics during caffeine exposures. Table 2 presents values for characteristics of the caffeine-induced R transients, which were examined only if the respective myocytes had functionally recovered from caffeine exposure. The subset of myocytes presented in Table 2 represent results from 8 Sed and 7 Tr rats.

                              
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  		Table 2.   Characteristics of caffeine-induced fluorescence ratio transients

Rcaff tended to be higher in the Sed group, therefore suggesting that SR Ca2+ load was greater in myocytes of the Sed group compared with Tr. The value for Rdiff immediately before caffeine exposure was significantly lower in the Tr group (Table 2) and indicates that, of all myocytes that had recovered from caffeine exposure, myocytes of the Tr group had a lower intracellular Ca2+ load compared with Sed. This result is important because it indicates the circumstances under which results from the caffeine exposures can be interpreted. The fraction of SR Ca2+ load released per electrical stimulation was not found to be different between the groups.

The values for kequil and Requil were not different between groups, therefore indicating that the function of Ca2+-handling mechanisms excluding SR uptake, Na+/Ca2+ exchange, and high-affinity Ca2+ buffers were similar and that the [Ca2+]c immediately before Na+-dependent Ca2+ efflux was comparable in both groups. The value of kefflux was significantly lower for myocytes of the Tr group compared with Sed, therefore suggesting that the rate of Na+-dependent [Ca2+]c clearance was lower in the trained state. These results are consistent with the observation that the rate of cytosolic Ca2+ removal during electrical pacing, i.e., kfall, was lower for Tr. The combination of lower kefflux and kfall for the Tr myocytes suggests that the rate of Na+-dependent Ca2+ efflux was suppressed by training and that the rate of SR uptake may have also been suppressed by training under the experimental conditions used in this study.

Shortening dynamics of myocytes. Shortening dynamics were generally different between Sed and Tr groups. Although peak shortening was not significantly different between groups (Fig. 4A), maximum velocities of shortening and relaxation normalized to peak shortening were generally lower in the Tr group (Fig. 4B). Times to peak shortening and to 25, 50, and 75% recovery were also longer in the Tr group (Fig. 4C). These results are consistent with those reported in a study by Palmer et al. (18), where it was demonstrated that myocytes of trained rats displayed a comparatively reduced contractile function and were more sensitive to the contractile-suppressing effects of fura 2 loading.


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  		Fig. 4.   Shortening characteristics of paced myocytes from Sed (open symbols; n = 52) and Tr (closed symbols; n = 48) rats. A: peak shortening was not stastically different between Sed and Tr groups and increased over time. B: maximum rates of shortening and relaxation were generally lower in Tr group compared with Sed. C: times to maximal shortening, 25, 50, and 75% recovery, were generally longer in Tr group and tended to decreases in both groups over time. dagger  P <=  0.10, * P <=  0.05 for Sed vs. Tr (t-test).

The repeated-measures ANOVA revealed significant training main effects for maximal velocity of shortening normalized to peak shortening (P = 0.007), maximal velocity of relaxation normalized to peak shortening (P = 0.091), time to peak shortening (P = 0.002), and time to 25% recovery (P = 0.044) but not for peak shortening. There were also significant pacing duration main effects (P <=  0.05) for all characteristics, including peak shortening, although there were no training × pacing duration interactions. These results demonstrate that contractile function, including relaxation characteristics, of the isolated myocytes generally improved with pacing duration under the experimental conditions used in this study.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Na+-Dependent Ca2+ Efflux Reduced in Run Training

The present study provides direct evidence for a training-induced decrease in the rate of Na+-dependent Ca2+ efflux in functional myocardium. This result in the LV myocyte model, which retains (while possibly altering) the interdependencies of intracellular Ca2+ regulatory mechanisms (14), implies that the intrinsic activity, capacity, or driving force for Na+/Ca2+ exchange is reduced after run training. NCX1 activity in isolated vesicles and density in myocardium have been shown to increase or not change with exercise training (9, 12, 19, 22, 32). Therefore, there is neither a precedent nor evidence to suggest a decrease in intrinsic NCX1 activity or density in the present experimental model.

The driving force for Na+/Ca2+ exchange, which may have been reduced by run training, is theoretically represented as the difference between membrane potential (Vm) and the Nernst equilibrium potential for Na+/Ca2+ exchange (ENa/Ca) defined as ENa/Ca = 3ENa - 2ECa (21, 22, 25). Ca2+ efflux requires that Vm < ENa/Ca during the Na+-rich Ca2+-free caffeine exposure of the present study (21, 22, 25); therefore, a decreased driving force for Na+/Ca2+ exchange due to run training implies one or a combination of the following three conditions: 1) a lower [Ca2+]c, which reduces ENa/Ca by elevating ECa; 2) a higher cytosolic Na+ concentration, which reduces ENa/Ca by reducing ENa; or 3) a higher Vm. Because we found a comparable Requil in both groups, it may be tempting to eliminate the possibility of a lower [Ca2+]c after run training. However, we believe it would be prudent at this time to consider the possibility that Ca2+ concentration in sub-SL space, where NCX1 resides and Na+/Ca2+ exchange evidently takes place (11), may have been lower in the Tr group and yet not discernable through values for R.

One important limitation to the interpretations of the caffeine-induced fluorescence transients is the lower SR Ca2+ load of the myocyte subpopulation that remained functional after caffeine exposure. In both groups the value for Rdiff before Rcaff (Table 2) was substantially lower compared with the Rdiff at 5 min (Fig. 2C). In addition, SR Ca2+ load in this subpopulation may have been lower in the Tr group compared with Sed, as indicated by a trend to a lower Rcaff and a significantly lower Rdiff immediately before caffeine exposure. Therefore, conclusions about training effects on mechanisms investigated with the caffeine-induced fluorescence transients apply only to those myocytes that had relatively low SR Ca2+ release and probably also lower SR Ca2+ load for the Tr group. In addition, the combination of a lower SR Ca2+ load in the Tr group and comparable steady-state Requil between the Sed and Tr groups implies a lower total and bound cytosolic Ca2+ in myocytes of this training model.

Animal Model of Run Training

In this study, training elicited an increase in plantaris muscle citrate synthase activity (~50%), thereby providing a peripheral indicator of the trained state in the rat (13, 16). Myocytes in the Tr group also displayed reduced contractile function compared with Sed, which is in agreement with results reported previously for a similar animal model and experimental conditions (18). The combination of intrinsic decrease in myocyte contractile function in the trained state and increased sensitivity of the Tr group to the depressing effects of fura 2 loading resulted in our observing here a reduction in myocyte contractile function after exercise training (18). The citrate synthase activity and myocyte contractile function data collectively suggest that the treadmill training protocol used in the present study resulted in an animal model of run training that was comparable to that in previous studies (10, 13, 16, 18).

Laboratory treadmill training has been implicated as a stressful training protocol, which could induce catecholamine responses that may subsequently distort training effects (4, 6). Because adrenal and spleen weights have been shown to increase and decrease, respectively, with stress in the rat (24), our finding no change in adrenal or spleen weights due to treadmill training demonstrates that the associated stress is not morphologically significant in our animal model.

Ca2+ Regulation During Electrical Pacing

Measurements of fluorescence R during electrical pacing, i.e., Rrest, Rpeak, and Rdiff, were not significantly different between Sed and Tr groups, therefore providing evidence for no significant changes in the corresponding [Ca2+]c characteristics with training. These results are in agreement with those of previous studies of training effects on myocyte Ca2+ dynamics (10, 16, 18). We additionally found that the values for Rrest, Rpeak, and Rdiff increased with time, suggesting that intracellular Ca2+ load increased with pacing duration.

The accumulation of intracellular Ca2+ load raised diastolic [Ca2+]c less so in the Tr group than in Sed. This result is not visually obvious from Fig. 1 but was found as a significant training × pacing duration interaction. There are at least two possible hypotheses implied by these results: 1) training may alter Ca2+ regulation so as to better protect the intracellular milieu and contractile function from an excess SR Ca2+ load, and 2) training may modify the LV myocyte [Ca2+]c regulation mechanisms so as to reduce the total amount of Ca2+ moved across the SL under the experimental conditions used here. Although L-type Ca2+ current characteristics were found to be similar for Sed and Tr rats (13), the latter hypothesis implies that total Ca2+ influx via SL Ca2+ channels and reverse Na+/Ca2+ exchange (2) may be reduced in the trained state. Considering that Ca2+ efflux would be favored or Ca2+ influx disfavored for the present [Ca2+]o and pacing frequency (16, 18), we speculate that total Ca2+ influx was indeed reduced by run training under the present experimental conditions.

We observed a decreased rate of [Ca2+]c decay during relaxation, as indicated by kfall, and a longer time to peak R for myocytes in the Tr group, therefore suggesting a decrease in one or a combination of the following rates: 1) SR uptake of cytosolic free Ca2+, 2) Na+/Ca2+ exchange, 3) high-affinity Ca2+ buffering, and 4) SR release of Ca2+. This result is in agreement with Moore et al. (16), who reported that the maximum velocity of [Ca2+]c decay was lower in the Tr group myocytes exposed to 1.1 mM [Ca2+]o and paced at 0.067 Hz. However, the decrease in kfall observed here with training is in contrast to reports of Laughlin et al. (10) and Palmer et al. (18), who observed no differences in [Ca2+]c temporal characteristics under conditions similar to those of the present study, i.e., 2 mM [Ca2+]o and 0.2-0.5 Hz. We believe the present study was able to discern the subtle differences in kfall and time to peak R between Sed and Tr for two reasons: 1) the higher number of myocytes used here provided greater statistical power, and 2) the effect of differential pacing durations on kfall and time to peak R, which we found here to be significant, was minimized by ceasing electrical pacing for 2 min before initiating the experimental protocol.

Continuous pacing over 5 min caused significant changes in all temporal characteristics of the [Ca2+]c transients. The rate of SR release, as indicated by krise, decreased with pacing duration. Because krise is defined as the velocity of Ca2+ release divided by the total Ca2+ released, this decrease in krise with pacing duration is likely a reflection of the increase in SR Ca2+ load and release over time. The rate of cytosolic Ca2+ removal, kfall, increased with pacing duration and implies an upregulation of one or more cytosolic Ca2+ removal mechanisms, i.e., SR Ca2+-ATPase, NCX1, or SL Ca2+-ATPase, by a second messenger. Candidates for a second-messenger pathway include, but are not limited to, Ca2+-dependent calmodulin inhibition of phosphodiesterase (23) and stimulation of SR Ca2+ uptake (1, 12), which would also explain the increase in SR Ca2+ load and release with pacing duration. The increase in contractile dynamics with continuous pacing may have also been affected through this same pathway as well as through phosphorylation of myosin light chain via Ca2+-dependent calmodulin activation of myosin light chain kinase (15).

Summary and Conclusions

We have found that run training significantly reduced the rate of Ca2+ removal from the cytosol during electrical pacing as well as the rate of Na+-dependent clearance of [Ca2+]c during caffeine-induced contractures. These data demonstrate that Ca2+ regulatory mechanisms are altered with exercise training and may be specifically reflective of a training-induced decrease in the rate and/or amount of Ca2+ moved across the SL via Na+/Ca2+ exchange. We have also demonstrated here that resting and peak [Ca2+]c of electrically paced LV myocytes did not change with exercise training under our experimental conditions. During 5 min of continuous pacing, resting and peak [Ca2+]c increased significantly in both Sed and Tr groups, therefore indicating an accumulation of intracellular Ca2+ with pacing duration. Diastolic [Ca2+]c in the Tr group was less susceptible to this accumulation of intracellular Ca2+, therefore implying that exercise training better protected the LV myocytes against Ca2+ loading. The rate of Ca2+ removal and contractile function also increased with continuous pacing in both the Sed and Tr groups, suggesting that a second messenger influenced Ca2+ regulatory mechanisms and contraction during these experiments.


    ACKNOWLEDGEMENTS

The authors are grateful for the expert technical assistance of Jinger S. Gottschall, Korinne N. Meyer, Eric A. Mokelke, Sarah J. Nickoloff, and M. Charlotte Olsson.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-40306.

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. §1734 solely to indicate this fact.

Address for reprint requests: B. M. Palmer, Dept. of Kinesiology, Campus Box 354, Univ. of Colorado at Boulder, Boulder, CO 80309 (E-mail: palmerbm{at}spot.colorado.edu).

Received 24 August 1998; accepted in final form 7 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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J APPL PHYSIOL 86(2):584-591
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