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Department of Kinesiology and Applied Physiology University of Colorado at Boulder, Boulder, Colorado 80309
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The effects of run endurance training and fura 2 loading on the contractile function and Ca2+ regulation of rat left ventricular myocytes were examined. In myocytes not loaded with fura 2, the maximal extent of myocyte shortening was reduced with training under our pacing conditions [0.5 Hz at 2.0 and 0.75 mM external Ca2+ concentration ([Ca2+]o)], although training had no effect on the temporal characteristics. The "light" loading of myocytes with fura 2 markedly suppressed (~50%) maximal shortening in the sedentary and trained groups, although the temporal characteristics of myocyte shortening were significantly prolonged in the trained group. No discernible differences in the dynamic characteristics of the intracellular Ca2+ concentration ([Ca2+]) transient were detected at 2.0 mM [Ca2+]o, although peak [Ca2+] and rate of [Ca2+] rise during caffeine contracture were greater in the trained state at 0.75 mM [Ca2+]o. We conclude that training induced a diminished myocyte contractile function under the conditions studied here and a more effective coupling of inward Ca2+ current to sarcoplasmic reticulum Ca2+ release at low [Ca2+]o, and that fura 2 and its loading vehicle DMSO significantly alter the intrinsic characteristics of myocyte contractile function and Ca2+ regulation.
fura 2; dimethyl sulfoxide; caffeine contracture; edge detection
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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THE HYPOTHESIS THAT endurance training elicits adaptations in myocyte Ca2+ regulation has been the focus of a considerable amount of research over the last 25 years (2, 4, 19). Over this time period, the vast majority of the evidence that has been advanced to support or refute this hypothesis is quite indirect (5, 13, 14, 16, 17, 23, 25, 29-34). For example, training-induced alterations in certain characteristics of myocardial contractile function have been interpreted to be suggestive of changes in cytosolic Ca2+ concentration ([Ca2+]c) dynamics (5, 31, 32). Pharmacological and biochemical analyses of [Ca2+]c-regulatory protein composition and/or function in sarcolemmal and sarcoplasmic reticular (SR) vesicles have also been used to address the Ca2+-handling hypothesis (13, 14, 16, 23, 25, 30, 33, 34). Although these types of studies have been very valuable and informative, none has directly examined the issue of whether training alters Ca2+ regulation in the intact cell.
The development and use of intracellular Ca2+ indicators provided investigators with the opportunity to more directly examine the effects of a variety of acute and chronic interventions on Ca2+ regulation in single myocytes in primary culture. In the late 1980s, fluorescent dyes such as fura 2 and indo 1 were successfully employed to reveal a large amount of information about myocardial Ca2+ regulation and its specific role in excitation and contraction (3, 6, 15, 19, 20, 37). The application of this technology to address the issue of whether training elicited adaptations in intrinsic contractile function and intracellular Ca2+ regulation in single ventricular myocytes was first reported in 1992 (15). Laughlin et al. (15) concluded that endurance training affected neither myocyte shortening characteristics nor [Ca2+]c dynamics during electrical stimulation at 0.2 Hz and 2 mM external Ca2+ concentration ([Ca2+]o). In 1993 a similar study from our laboratory concluded that training increased the sensitivity of myocyte shortening characteristics to pacing frequency and [Ca2+]o (19). The basis for the differences in the results of both studies was obscure.
In this study we provide information that may shed light on the reason for the differences between the conclusions drawn in the earlier studies. It appears that the loading of the [Ca2+]c indicator fura 2 may be central in providing these explanations. We demonstrate that fura 2 loading via fura 2-AM dissolved in DMSO significantly affects myocyte contractile function and that training-induced alterations in the extent of myocyte shortening are abolished by the loading of fura 2 into myocytes. Additionally, we provide evidence that the effect of fura 2 loading on the time course of the mechanical response of electrically stimulated myocytes is greatest in left ventricular (LV) myocytes isolated from trained rats. This latter effect occurs in the absence of training-induced differences in the temporal characteristics of the [Ca2+]c transient. We propose that these results may be due to training-induced changes in the viscoelastic properties of single myocytes.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animal Model
Female Sprague-Dawley rats were randomly assigned to a sedentary (Sed, n = 22) and an exercise-trained (Tr, n = 19) group. All animals were housed and cared for as previously described (17). The specific treadmill training protocol has been described in detail (17). Briefly, rats trained for
20 wk, which included a 12-wk phase during which running
intensity and duration were gradually increased. At the end of the
first 12 wk of the protocol, rats ran 5 days/wk for 1 h/day up a 10%
grade. The daily training bout consisted of 45 min of running at 28 m/min with an interspersed 15-min interval of running at 35 m/min. All
animals were 10-14 mo of age at the end of the study. At the time
the animals were killed, plantaris muscles were dissected, homogenized,
and assayed for citrate synthase activity, as previously described (17, 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 at Boulder.
Trabeculae Studies
Trabecular fibers from LV myocardium were prepared from seven Sed and six Tr rat hearts and studied using methods similar to those previously described (9, 11, 12). After trabeculae were isolated and fiber length was optimized, "isometric force-pCa" relationships were determined on permeabilized fibers at 27°C by exposing the fibers to solutions with pCa values of 7.7, 7.0, 6.7, 6.5, 6.3, 6.0, 5.7, and 5.5. Each pCa exposure was interspersed with an exposure to relaxing solution (134 mM potassium gluconate, 20 mM Tris maleate, 2 mM MgCl2, 4 mM EGTA, 2 mM K2ATP, 10 mM creatine phosphate, and 0.5 mg/ml creatine phosphokinase, pH 7.0). The various pCa solutions were prepared by addition of CaCl2 to relaxing solution according to previously described methods by Robertson and Potter (24) and based on concepts developed in part by Fabiato and Fabiato (8). Fibers were permeabilized by 30 min of incubation in a skinning solution, which consisted of relaxing solution containing 300 mg/ml saponin.The position and shape of the normalized isometric force-pCa relationship were characterized for each experiment by a nonlinear least-squares fit to the normalized isometric force, F/Fmax, of a modified Hill equation
![<FR><NU>F</NU><DE>F<SUB>max</SUB></DE></FR> = <FR><NU>[Ca<SUP>2+</SUP>]<SUP><IT>n</IT></SUP><SUB>free</SUB></NU><DE>[Ca<SUP>2+</SUP>]<SUP><IT>n</IT></SUP><SUB>50</SUB> + [Ca<SUP>2+</SUP>]<SUP><IT>n</IT></SUP><SUB>free</SUB></DE></FR>](/content/vol85/issue6/fulltext/2159/img001.gif)
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(1) |
Myocyte Isolation
Cardiac myocytes were obtained from the LV free wall and septum from 15 Sed and 13 Tr rat hearts by use of methods previously described in detail (20). All chemicals and reagents were obtained from Sigma Chemical (St. Louis, MO) except where noted. Briefly, animals were heparinized (250 U ip) and then anesthetized with pentobarbital sodium (35 mg/kg body wt ip; Abbot 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 using a modified Langendorff perfusion apparatus that could deliver three different solutions. All solutions were 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 nominal Ca2+-containing Krebs-Henseleit buffer, 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 2-8 h at 37°C in a humidified atmosphere of 5% CO2 and 20% O2. We have found that myocyte function does not statistically change over the 2- to 8-h incubation time window and that any real effect of incubation time is negligible compared with the differences in the populations being compared.Experimental Protocol
A single coverslip from each day was placed under a microscope, and images of randomly selected myocytes were recorded onto videotape. These video images were used to measure myocyte length and width with use of NIH Image 1.41 video-frame-grabbing software. For studies of [Ca2+]c dynamics, coverslips were incubated for 5 min at 37°C 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 2.0 mM [Ca2+]o Tyrode solution (in mM: 140 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 2 pyruvate, 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 frequency of 0.5 Hz (Grass Instruments, Boston, MA). Fura 2 fluorescence transients were recorded for those myocytes loaded with fura 2, and myocyte shortening transients were recorded for myocytes with and without fura 2 loading under the following five experimental conditions: 1) after2 min of electrical
stimulation in 2.0 mM
[Ca2+]o Tyrode
solution (2CaStim), 2) after an
additional 4 min of electrical stimulation in 0.75 mM
[Ca2+]o
(0.75CaStim), 3) during a 2.5-s
exposure to 10 mM caffeine in 0.75 mM
[Ca2+]o
(0.75CaCaff), 4) after an additional
4 min of electrical stimulation in 2.0 mM
[Ca2+]o
(2CaStimPost), and 5) during a 2.5-s
exposure to 10 mM caffeine in 2.0 mM
[Ca2+]o (2CaCaff).
The initiations of caffeine exposures were synchronized to the pacing period.
Measurements of [Ca2+]c Dynamics
Fura 2 fluorescence was induced with a fluorescence system (IonOptix, Milton, MA) fitted with 400- and 360-nm optical filters. We have found no discernible fluorescence intensity transients during electrical stimulation of fura 2-loaded myocytes excited at 360 nm, justifying the use of 360-nm excitation as the isobestic wavelength in situ. The use of 400-nm excitation wavelength takes advantage of the directly reciprocal relationship between fluorescence intensity and [Ca2+] in vitro for excitation wavelengths >390 nm (28). Despite any subtle differences in the fura 2 fluorescence spectra at 400 nm in situ and in vitro, the resulting fluorescence intensity ratio (I360/I400) was assumed to be linearly related to [Ca2+]c. Fluorescence intensities were recorded as photon counting rates by using a personal computer. The value for myocyte background fluorescence was determined by superfusing myocytes with permeabilizing buffer (Ca2+-free Tyrode solution-4 µM digitonin) for 4 min to eliminate cytosolic fura 2. The subsequent measure of fluorescence in the presence of a Ca2+-free Tyrode superfusate was taken as the true fluorescence background. Quenching of fura 2 fluorescence during the brief 2.5-s exposure to caffeine was not observed, in agreement with O'Neill et al. (21), and was therefore not incorporated into fura 2 calibration. Custom-made software was used to analyze the recorded myocyte R transients to determine the following characteristics: resting R (Rrest), peak R (Rpeak), Rpeak
Rrest
(Rdiff), and two exponential time constants (
rise and
fall) determined by nonlinear
least-squares fitting of the following double-exponential function to
the recorded transient
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(2) |
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Fura 2 fluorescence data are presented as background corrected R values rather than [Ca2+] for several reasons. First, as is the case in virtually every study involving the use of fura 2 in situ, the conversion of R data to [Ca2+]c requires the acceptance of a variety of assumptions, which include (but are not limited to) dissociation constant (Kd) values and fura 2 spectral qualities being identical in all cells across all experimental groups. Our use of R does not formalize these assumptions. As a consequence, speculation about absolute [Ca2+]c based on interpretations of R magnitudes must be made in the context of the limitations inherent in our uncertainty about true in situ Kd values and fura 2 spectra. On the other hand, information about the temporal characteristics of [Ca2+]c dynamics taken from R data is quite reliable and independent of cell type, since our excitation wavelength pairing yields a fluorescence ratio that is linearly related to [Ca2+]c.
Measurement of Myocyte Shortening Dynamics
The positions of myocyte edges were determined using a video edge detection device (Crescent Electronics, Sandy, UT) and recorded 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: resting length, 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. For caffeine-induced contractures, only resting length and peak shortening were recorded.The possible effects of the fura 2-AM vehicle DMSO on shortening dynamics were tested by recording the shortening dynamics of myocytes isolated from one rat after exposure to 1) nothing (control), 2) 5 min of 0.05% (vol) DMSO, or 3) 5 min of 0.05% (vol) DMSO and 2 µM fura 2-AM.
Analysis
All analyses were performed using SPSS (version 6.0). Contrasts between characteristics of Sed and Tr groups were determined by unpaired, two-tailed t-tests. Normalized isometric force-pCa relationships were analyzed by 2 (Sed and Tr) × 3 (pCa 6.7, 6.5, and 6.3) repeated-measures ANOVAs, and between-group contrasts were made at each pCa by using unpaired t-tests. To test the relative sensitivity of the myocyte groups to [Ca2+]o, a 2 (Sed and Tr) × 2 (2CaStim and 0.75CaStim) repeated-measures ANOVA was performed on all characteristics of the electrically stimulated and caffeine-induced R and shortening transients. The effect of a previous caffeine exposure on myocyte shortening characteristics was examined using a 2 (Sed and Tr) × 2 (2CaStim and 2 CaStimPost) repeated-measures ANOVA of the electrically stimulated R and shortening data. The relative sensitivity of the myocyte groups to fura 2 loading was investigated using 2 (Sed and Tr) × 2 (without and with fura 2 loading) ANOVAs performed at each of the 2CaStim and 0.75CaStim conditions. Another 2 (Sed and Tr) × 2 (without and with fura 2 loading) × 2 (2CaStim and 0.75CaStim) repeated-measures ANOVA was also performed to test any change in sensitivity that may have occurred by lowering [Ca2+]o. To test the effects of DMSO on myocyte shortening, a one-way ANOVA and Bonferroni post hoc analyses were applied to all variables of myocyte shortening for the control, DMSO only, and DMSO-fura 2-AM groups.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animal Model
Training did not significantly affect body weight in this study (Table 1). LV weights, recorded from animals used in trabeculae studies, were ~7% greater in the Tr than in the Sed group (Table 1). Training elicited a ~5% increase in myocyte length, whereas myocyte width was not affected (Table 1). Citrate synthase activities of the plantaris muscle homogenates were significantly increased by training (Table 1). Collectively, the central and peripheral markers of training provide unequivocal verification that our treadmill training protocol was effective in producing a trained state.
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Trabeculae Studies
Training did not significantly affect the normalized isometric force-pCa relationship of skinned LV trabeculae (Fig. 2). Hill's coefficients were 3.0 ± 0.4 and 2.8 ± 0.2 for the Sed (n = 11) and Tr (n = 7) groups, respectively (P = 0.730). Values for [Ca2+]50 were 3.8 ± 0.4 × 10
7 and 4.2 ± 0.5 × 107 M, respectively
(P = 0.636). These values were similar
to those found by others investigating skinned cardiac trabeculae under similar conditions (9, 11, 12, 27).
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Myocyte Studies
Cytosolic R dynamics.
Reduction of
[Ca2+]o
from 2.0 to 0.75 mM significantly changed
(P < 0.01) all the
electrically stimulated R characteristics for the Sed and
Tr groups (Fig. 3). Specifically,
Rrest,
Rpeak, and
Rdiff decreased and
rise and
fall increased when
[Ca2+]o
was reduced. Rrest was also higher
in the Tr group under the 2CaStim and 0.75CaStim conditions.
Rpeak and
Rdiff were higher in the Tr group
under the 0.75CaStim condition. When
[Ca2+]o
was increased from 0.75 to 2.0 mM after caffeine exposure, Rrest,
Rpeak,
Rdiff, and
fall increased
(P < 0.01) in Sed and Tr groups
(Fig. 3).
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rise during the
caffeine contracture elicited at 0.75 mM
[Ca2+]o was shorter
in the Tr myocytes. Rrest,
Rpeak, and
Rdiff significantly increased
(P < 0.01) between the 2CaStim and
2CaStimPost conditions in Sed and Tr groups (Fig. 3).
Shortening dynamics of myocytes not loaded with fura 2. All the electrically stimulated shortening characteristics in Sed and Tr groups changed (P < 0.01) with the reduction of [Ca2+]o from 2.0 to 0.75 mM (Figs. 4, A and B, and 5). Specifically, peak shortening and maximal velocities of shortening and relengthening were reduced, and the characteristic times to peak shortening and relengthening were increased when [Ca2+]o was reduced. The most striking difference between the Sed and Tr groups was the lower peak shortening in the Tr group under 2.0CaStim and 0.75CaStim conditions; the temporal characteristics of myocyte contraction were unaffected by training.
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Shortening dynamics of myocytes loaded with fura 2. Peak shortening of fura 2-loaded myocytes in 2.0 mM [Ca2+]o was on the order of 2.5-3%, which is comparable to that reported by Laughlin et al. (15) under similar pacing and superfusate conditions, although without coverslip lamination. The reduction of [Ca2+]o from 2.0 to 0.75 mM significantly changed (P < 0.01) all the electrically paced shortening characteristics for Sed and Tr groups that were loaded with fura 2 (Figs. 4, D and E, and 6). Specifically, maximal velocities were increased, and the characteristic times to peak shortening and recovery were increased with the reduction in [Ca2+]o. The most striking differences between the Sed and Tr groups were the greater times to peak shortening and recovery in the Tr group with 2CaStim and 0.75CaStim; the maximal extent of shortening was not different between groups.
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Effects of fura 2 loading on shortening dynamics. Loading myocytes with fura 2 appreciably diminished the electrically stimulated shortening dynamics (Fig. 4). Loading with fura 2 affected all shortening characteristics in Sed and Tr groups at 2.0 mM [Ca2+]o, including significant decreases (P < 0.01) in peak shortening and maximal velocities and significant increases in times to peak shortening and recovery. "Training × fura" interactions were also found for maximal relaxation velocity and times to 25, 50, and 75% recovery. Fura 2 affected all shortening characteristics for Sed and Tr groups at 0.75 mM [Ca2+]o in a manner similar to that at 2.0 mM [Ca2+]o. Training × fura interactions were also found for time to peak shortening and times to 25 and 50% recovery. These similar results with 2.0 and 0.75 mM [Ca2+]o indicate that the Tr group was more susceptible to the contractile-diminishing effects of fura 2 loading under each of the [Ca2+]o conditions.
The 2 (Sed and Tr) × 2 (without and with fura 2) × 2 (2CaStim and 0.75CaStim) repeated-measures ANOVA revealed a significant (P < 0.05) training × fura 2 × [Ca2+]o interaction for times to 25, 50, and 75% recovery. These results indicate that the sensitivity of the Tr group to the effects of fura 2 loading was more pronounced as [Ca2+]o was reduced.Effects of DMSO exposure on shortening dynamics. Exposing myocytes for 5 min to 0.05% (vol) DMSO significantly changed all characteristics of myocyte shortening dynamics compared with controls (Fig. 7). The loading of myocytes with fura 2, via 5 min of exposure to 0.05% (vol) DMSO-2 µM fura 2-AM, significantly changed all characteristics of myocyte shortening compared with controls and all temporal characteristics, i.e., velocities and times to peak and fractional recovery, compared with myocytes exposed to DMSO only.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Training Model
In this study, training elicited a modest (~7%) LV hypertrophy and increases in LV myocyte length (~5%) and plantaris muscle citrate synthase activity (~50%). These central and peripheral adaptations to endurance training are similar to those reported earlier (17, 19), and they verify the efficacy of the endurance training protocol used in this study.Trabeculae Studies
The trabeculae studies demonstrated no intrinsic differences between Sed and Tr groups in the sensitivity of contractile elements to activation by Ca2+. To our knowledge, this is the first report of its kind using skinned trabeculae. Our findings are consistent with previous work demonstrating that training had no effect on the Ca2+-dependent activation of myofibrillar ATPase activity (32). However, the present results from skinned myocardium are limited in elucidating any possible training-induced modifications in the contractile apparatus Ca2+ sensitivity that may appear in intact myocardium (1).Myocyte Studies
Although the isolated cardiac myocyte is limited as a model of cardiac function because it is examined at nonphysiological pacing frequencies, temperatures, ionic environments, and mechanical loads, examination of the isolated myocyte provides a unique opportunity to differentiate physiological adaptations at the most elemental unit of cardiac function. Before this study the effects of training on myocyte shortening dynamics have been examined in only two reports (15, 19). One study reported that training did not influence intrinsic myocyte shortening characteristics (15), whereas another study, from our laboratory, provided evidence for training-induced changes in intrinsic myocyte shortening dynamics (19). The present study strongly suggests that both fura 2 and DMSO, the vehicle used for loading fura 2, independently and additively affect myocyte shortening dynamics. Therefore, we suggest that fura 2 loading via exposure to fura 2-AM dissolved in DMSO may have been responsible for the differences between the results of the former two studies (15, 19).In the present study, training elicited a reduction in the extent of myocyte shortening without producing significant alterations in the temporal characteristics of myocyte shortening and relengthening in myocytes not loaded with fura 2. In earlier work by Moore et al. (19), training had little effect on the temporal characteristic of myocyte shortening whereas training did have an effect on the extent of myocyte shortening, the magnitude and direction of which varied as a function of myocyte pacing frequency and/or [Ca2+]o. In that study, Tr myocytes shortened more than Sed myocytes when paced at 0.067 Hz and subjected to 2.0 mM [Ca2+]o and less than Sed myocytes when paced at 0.2 Hz and subjected to 0.6 mM [Ca2+]o, whereas between-group differences in myocyte shortening were smaller or absent under conditions that fell between these two extremes (19). In the present study, conducted at 0.5 Hz and 0.75 or 2.0 mM [Ca2+]o, Tr myocytes shortened to a lesser extent that did Sed myocytes. Collectively, these data (19; present study) provide further support for the idea that training alters intrinsic myocyte contractile function and support the speculation that there is an inverse relationship between pacing frequency and the extent of myocyte shortening that is more pronounced after training.
Data from Laughlin et al. (15) and from this study clearly indicate that the maximal extent of myocyte shortening in electrically stimulated, fura 2-loaded myocytes is not significantly affected by training. Fura 2 is a potentially significant intracellular Ca2+ buffer, and our observation that fura 2 loading significantly decreased the extent and prolonged temporal characteristics of myocyte shortening strongly suggests that intracellular [Ca2+] dynamics were altered by loading the fluorescent dye (Fig. 4). Additionally, introduction of the exogenous Ca2+ buffer and exposure to DMSO were responsible for the abolition of the effect of training on the extent of myocyte shortening observed in myocytes containing no fura 2. A wide variety of fura 2-AM-loading conditions appears in the literature, and they involve myocyte incubations in the presence of ~1-2 µM fura 2-AM for ~5-30 min (7, 15, 19, 20, 37). In this context, our loading conditions (5 min in 2 µM fura 2-AM) would be considered to be light to moderate. However, we found that brief exposure to DMSO alone can have significant effects on myocyte contractile function. Our findings clearly underscore the importance of being mindful of the potential effects on myocyte function of intracellular indicators and the vehicles in which they are loaded.
An important finding of this study was that fura 2 loading significantly suppressed the extent of myocyte shortening across both groups of cells. Therefore, fura 2 loading may have masked any discernible differences in the intrinsic intracellular Ca2+ dynamics that were present between the Sed and Tr groups. Consequently, it is possible that the observed Sed vs. Tr differences in myocyte shortening dynamics in cells not loaded with fura 2 may have been due to differences in Ca2+-handling mechanisms that were not reflected in our fura 2 fluorescence measurements. Another important observation was that, whereas there were no significant between-group differences in the temporal characteristics of myocyte shortening in the absence of fura 2, the loading of myocytes with fura 2 elicited a marked prolongation in the temporal characteristics of myocyte contraction that was greatest in the Tr myocytes. The most notable differences in the shortening characteristics between fura 2-loaded myocytes were the increased times to peak shortening and recovery that occur with training. If the [Ca2+]c dynamics at 2.0 mM external Ca2+ are not different between groups loaded with fura 2, as observed here and by Laughlin et al. (15), then the differences in myocyte shortening characteristics in fura 2-loaded cells would implicate training differences in 1) the contractile elements' sensitivity to Ca2+, 2) myocyte stiffness, or 3) myocyte internal dynamic viscosity. Each of these possibilities will be addressed in the context of our results.
Our skinned fiber data and earlier work examining the Ca2+ dependence of myofibrillar ATPase activity (32) provide no evidence that training sensitizes the contractile element to activation by Ca2+. Myocyte stiffness and dynamic viscosity, however, may contribute to training-induced differences in the shortening dynamics. A simple myocyte mechanical model would predict that contraction time characteristics are inversely proportional to myocyte stiffness and directly proportional to dynamic viscosity (22, 35), much the same way that a tighter (i.e., stiffer) string produces a higher pitch (i.e., shorter time period per oscillation). The observed increases in the shortening and relaxation time characteristics, in the absence of differences in the [Ca2+] characteristics and force-pCa relationships, could be indicative of a significant decrease in myocyte stiffness and/or an increase in dynamic viscosity in the Tr group. Such adaptations at the single-cell level would be consistent with findings that training increases myocardial compliance in whole heart preparations (10, 36).
In this study, fura 2 was used to determine whether training elicited
alterations in SR
[Ca2+] dynamics in
myocytes stimulated to contract by electrical pacing and rapid caffeine
application. When taken at face value, our fluorescence ratio data can
be most simply interpreted as follows. First, resting
[Ca2+]c
(Rrest) appeared to be elevated
in Tr myocytes. Second, the [Ca2+]c
elevation (Rdiff) elicited by
electrical stimulation was not different between Sed and Tr groups
during myocyte superfusion with 2.0 mM
[Ca2+]o, whereas the
[Ca2+]c
elevation observed during pacing at 0.75 mM
[Ca2+]o appeared to
be greater in Tr myocytes. Because training does not appear to affect
intrinsic inward Ca2+ current
characteristics in LV myocytes (17), this latter finding may be
indicative of a more effective coupling of inward
Ca2+ current to SR
Ca2+ release or a greater
releasable SR Ca2+ store in Tr
myocytes. Both of these possibilities are consistent with the third
general observation. Specifically, the
[Ca2+]c
elevation (Rdiff) elicited by
rapid caffeine application was faster (i.e.,
rise was lower) in Tr myocytes
with 0.75 mM [Ca2+]o
and tended to be greater in Tr than in Sed myocytes with 0.75 and 2.0 mM [Ca2+]o
superfusion. We readily acknowledge that these quantitative interpretations of our data are based on the assumption that the between-group (Sed vs. Tr) differences in fluorescence ratio
characteristics (Rrest,
Rpeak, and
Rdiff) were accurately
reflective of training-induced alterations in resting and peak
[Ca2+]c
and were not due to training-induced changes in the cytosolic milieu
that differentially influenced the amount and/or fluorescence properties of fura 2 in the isolated myocytes. The latter possibility cannot be ruled out, despite the considerable efforts that have been
made to avoid methodological bias in this and other studies (15,
19).
It is apparent that existing controversies regarding the effect of training on myocyte resting or peak [Ca2+]c during electrical stimulation evade clear resolution. For example, Laughlin et al. (15) found no evidence for an effect of training on resting or peak [Ca2+]c in electrically stimulated myocytes. Moore et al. (19) concluded that resting [Ca2+]c was unaffected, whereas peak [Ca2+]c was attenuated or unaltered, in LV myocytes isolated from trained rats. In this study we found a consistently higher Rrest in the Tr group and higher Rpeak and Rdiff in the Tr group under a number of experimental conditions. The differences in the findings of the three studies (15, 19; present study) are probably due to numerous between-study differences in the methods used to assess [Ca2+] dynamics. For example, when raw fura 2 fluorescence intensities were corrected only for "empty field" (15) and/or "cellular autofluorescence" (18), no detectable effects of training on R transients were found (15, 19). If data from the present study were corrected for cellular autofluorescence only, we would have also observed no training effects (data not shown). An inherent problem with this approach to background fluorescence correction is that it assumes fura 2-AM deesterification and fura 2 loading in nonsarcoplasmic compartments to be identical in all cells. In the study by Moore et al. (19), values for Kd,Ca, Rmax, and Rmin in situ were found for the populations, then applied to the R data on a cell-by-cell basis; however, this technique introduced another assumption of the applicability of the composite values to individual cells. In the present study we used a digitonin-Ca2+-free superfusate treatment (see METHODS) to make fluorescence background determinations on every cell studied. This approach was taken after preliminary studies using our specific fura 2-loading and fluorescence-recording techniques indicated that cytosol Ca2+-insensitive fluorescence on a cell-by-cell basis was variable and significant (~20-40%). The approach presented here, however, also carries with it limitations, including the assumptions that the fura 2 Kd,Ca, fluorescence spectra, and action of digitonin are identical across all cells in Sed and Tr groups.
Summary and Conclusions
The results of our work clearly indicate that training affects the intrinsic contractile function of electrically stimulated cardiac myocytes. Our results corroborate earlier findings that the maximal extent of myocyte shortening is affected by training in ventricular myocytes not loaded with fura 2 (19) and that this specific adaptation to training is absent in myocytes that are loaded with fura 2 (15). The loading of LV myocytes with fura 2 via fura 2-AM dissolved in DMSO markedly reduced the extent of myocyte shortening in Sed and Tr myocytes because of the additive effects of DMSO and fura 2. Additionally, in the absence of Sed vs. Tr differences in the temporal characteristics of the [Ca2+]c transient, fura 2 loading produced a marked slowing of the myocyte mechanical transient that was most pronounced in Tr myocytes. These observations may implicate training-induced changes in the stiffness and/or dynamic viscosity of LV myocytes.This issue of whether training affects the magnitude of the [Ca2+]c transient in paced myocytes continues to evade clear resolution, owing to the complexities and limitations of the fura 2 methodology. Nevertheless, peak [Ca2+]c and the rate of caffeine-induced [Ca2+]c rise were observed to be greater in the trained state at low [Ca2+]o. Collectively, these results imply a more effective coupling of inward Ca2+ current to SR Ca2+ release or a greater releasable SR Ca2+ store in myocytes from trained rats.
The use of fura 2 (and other fluorescent indicators) to study ventricular myocyte [Ca2+] dynamics and associated myocyte contractile function has become increasingly widespread over the last decade. As is clear from this study, the use of fura 2 can significantly affect myocyte contractile function and may also affect the intracellular [Ca2+] transient itself. As a consequence, we conclude that great care should be taken in 1) the choice of questions that are to be answered using the fura 2 methodology and 2) the interpretation of studies where intracellular [Ca2+] and myocyte shortening dynamics are simultaneously examined.
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ACKNOWLEDGEMENTS |
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The authors are grateful for the expert technical assistance of Jinger S. Gottschall, Alexander Hazel, Joshua M. Lynch, Eric A. Mokelke, and M. Charlotte Olsson.
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FOOTNOTES |
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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, University of Colorado at Boulder, Boulder, CO 80309.
Received 23 February 1998; accepted in final form 19 August 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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P < 0.1 vs. Sed for same experimental condition.
