Effect of exercise training on intracellular transients in ventricular myocytes of rats
free Ca2+
M. HAROLD LAUGHLIN, MARY E. SCHAEFER, AND M. STUREK Departments of Veterinary Biomedical Sciences and Physiology and Dalton Research Center, University of Missouri, Columbia, Missouri 65211 LAUGHLIN, M. HAROLD, MARY E. SCHAEFER, AND M. STUREK. Effect of exercise training on, intrczcelLular free Cu2+ transients in ventricular myocytes of rats. J. Appl. Physiol. 73(4): 1441-1448, 1992.-The purpose of this study was to test the hypothesis that exercise training induces enhanced intracellular free Ca2+ (Ca,) availability to the contractile elements of cardiac cells. Cai transients were directly measured in single isolated contracting ventricular myocytes from exercisetrained (EX) and sedentary control (SED) rats. Male SpragueDawley rats underwent 16 wk of progressive treadmill exercise (32 m/min, 8% grade, 1.5 h/day) (EX) or were cage confined (SED). EX rats had lower resting heart rate and elevated skeletal muscle oxidative capacity. Cai was measured with the fluorescent Cai indicator fura-2. Simultaneous video monitoring indicated that myocytes suspended in physiological salt solution were quiescent until stimulated electrically at a frequency of 0.2 Hz (12-36 V, 2-ms duration). Stimulated Ca, transients, measured from changes in fura- fluorescence, were similar in cells from EX and SED groups. Peak shortening, time to peak shortening, velocity of shortening, contraction duration, and time to half-relaxation were also similar in cells from EX and SED rats. Ryanodine (10 PM) was applied to eliminate the contribution of Ca2+ release from sarcoplasmic reticulum to the Ca, transient. Verapamil was applied to eliminate the contribution of voltage-gated Ca2’ channels to Ca, transients. Cai transients were also similar in cells from EX and SED groups after these pharmacological interventions. These results suggest that treadmill training of rats does not alter Ca, availability to the contractile elements in isolated ventricular myocytes.
addition, Tibbits et al. (29) found increased Ca2+ uptake by Na+-Ca2+ exchanger in cardiac sarcolemmal vesicles. Penpargkul et al. (13) reported enhanced binding of Ca2+ by cardiac sarcoplasmic reticulum (SR) of hearts from EX rats. The present study was undertaken to directly examine the Cai dynamics associated with contraction of myocardial cells of EX rats. Therefore a series of experiments was conducted in which systolic (transient) changes in Cai were measured with the fluorescent Cai indicator fura- in isolated ventricular myocytes from endurancetrained and SED control rats. We postulated that exercise training would increase peak amplitude and rate of decay of the Cai transient during activation. The pharmacological tools, ryanodine (226) and verapamil(3), were used to discriminate the contributions of the SR and sarcolemma (SL) to ventricular myocyte Cai transient. METHODS
Animals and training. Male Sprague-Dawley rats of ZOO-250 g body wt were purchased from Charles River. The rats were housed in pairs and kept in a 12:12-h lightdark cycle at a controlled temperature (23 t 2OC). Rat chow and water was provided ad libitum. Rats were exposed to treadmill exercise (lo-20 m/min, 0% grade, 10 min/day) for 1 wk. Rats that were most resistant to treadmill exercise were eliminated from the study. The remaining rats were randomly divided into calcium influx; ryanodine; calcium release channel; verapamil; contractility; voltage-gated calcium channel two groups, SED rats and EX rats. The EX rats began a training program in which running duration and treadmill speed and grade were increased incrementally over the next 5 wk. Training bouts took place 5 days/wk. By ENDURANCE EXERCISE TRAINING produces adaptations in the cardiovascular system as evidenced by an inweek 6, EX rats ran 1.5 h/day at 32 m/min on an 8% creased maximal oxygen transport capacity, more effigrade. Final intensity was maintained for the last 6-10 wk of the training program. Experiments began after EX cient cardiac performance, and enhanced intrinsic functioning of the myocardium (4,18). Although the fact that rats had trained for 12 wk and were conducted over a exercise training results in bradycardia (training brady4-wk period. EX rats continued the training program until they were used in the experiments. The duration of cardia) is well established, the nature of training-induced adaptation of cardiac contraction and/or relaxation is a training ranged from 12 to 16 wk. SED rats remained subject of continuing controversy. Several studies sug- cage-confined during the training period. Two series of experiments were conducted, separated gest that exercise training enhances contractile function of the heart (1, 8, 18). A popular hypothesis is that an by -6 mo. Each series of experiments involved one increased availability of intracellular free Ca2+ (Cai) for group of SED (n = 6) and one group of EX (n = 7) rats as described above. Thus there were a total of 12 SED and excitation-contraction coupling may be an underlying mechanism (1, 8, 11, 13, 14, 27-30). In one study, Ca2+ 14 EX rats used in this study. Training effectiveness was determined by measurebinding sites in papillary muscle preparations from exercise-trained (EX) rats were found to be increased by 63% ments of citrate synthase activity in the long head of the when compared with sedentary (SED) controls (28). In triceps brachii and resting heart rates of SED and EX 0161-7567/92
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CARDIOMYOCYTE
rats. Enzyme activity was determined spectrophotometritally, according to the method of Srere (23). For measurement of resting heart rates, each rat was anesthetized with methoxyflurane (Metofane) and instrumented with subcutaneous stainless steel sutures in the right and left axillary region and left thigh. Electrocardiogram and heart rate were recorded on an oscillographic recorder (model 28009, Gould ECG/Biotach Amplifier, model 13-4615-65) while the rats rested in a black box undisturbed for 65 min. Heart rates declined as a function of time in all rats tested such that a steady state was reached after 30-50 min (resting heart rate). Heart rate measurements were made between 9:00 A.M. and 12:00 P.M. during weeks lo-12 of training. Rats from the EX group were not exercised on the day of testing until after resting heart rate had been measured. Cell isolation procedures. Animals were anesthetized with pentobarbital sodium (0.1 ml/100 g ip) and given sodium heparin (1,000 IU ia) 20 min before excision of the heart. After isolation of hindquarters for a study of vascular transport capacity (14, 26), an incision was made into the thoracic cavity, the heart was rapidly removed and placed in ice-cold buffer containing the following: lo-20 PM ionized Ca2+, minimal essential medium Joklik-modified buffer (GIBCO, Grand Island, NY), 60 mM taurine (Sigma Chemical, St. Louis, MO), 20 mM creatine (Sigma), and 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Fisher Biotech, Fairlawn, NJ). Ca2+-tolerant cardiac myocytes were then prepared by retrograde perfusion through the ascending aorta of the isolated heart using a modification of the methods of Geisbuhler et al. (9). The coronary circulation was perfused at constant flow with perfusate equilibrated with 100% oxygen (pH 7.4) while the hearts were suspended in a water-jacketed chamber maintained at 37.0 z!I 0.5”C. The initial perfusate [buffer supplemented with 0.1% bovine serum albumin, fraction V (BSA, ICN Immunobiologicals, Lisle, IL), and 1 IU heparin/ml] was used to clear the vasculature of blood and was not recirculated. Subsequently, 0.7 mg collagenase/ml (Worthington Biochemical, Freehold, NJ) was added to the perfusate, and this solution was allowed to recirculate for 40-50 min. The ventricles were dissected free, minced with scissors in fresh collagenase-containing perfusate, and agitated in a 37°C water bath under 100% oxygen for 10 min. CaCl, (50 PM) was added, and the suspension was agitated for another 10 min. Tissue was drawn up into a pipette to gently disperse cells. The suspension was filtered through a double layer of gauze and diluted 1:4 with collagenase-free buffer containing 0.1% wt/vol BSA. After the cells settled, the supernatant was removed, and the cells were resuspended in buffer containing 1.5% BSA (15 mg/ml). This was repeated twice, and the cells were resuspended for a final time in buffer containing 1.5% BSA. Ca2+ tolerance was induced slowly over 30 min, during which time the cells were maintained in suspension by gentle agitation and were continually gassed with 100% oxygen. Final Ca2+ concentration was 1 mM. Experiments were completed within 5 h of the final addition of CaCl,. Cells were chosen for inclusion in the study based on
CA,
AND EXERCISE
FIG. 1. A: photomicrograph of 3 cardiac myocytes. Rod-shaped cell represents type of cells used to measure Ca2+ transients and contractile motion in present study. Cell at bottom left is partially contracted and has “frayed” end; cell would not have been included in study. Cell that had undergone irreversible contracture (top left) also would not have been studied. Horizontal calibration bar at lower edge equals 20 Frn. B: fluorescent image of same cell when fura- in cell is excited by 380-nm light.
the following criteria: rod shape; quiescent until stimulated to contract by field stimulation (contraction verified by visual observation via video monitor); when not being stimulated to contract, the ratio of the 340- to the 380-nm fluorescence intensity was required to be cl.0 (which corresponds to Cai of 5100 nM), indicating “normal” resting Cai. Figure 1 is an example of a cardiac myocyte typical of those selected for this study. This micrograph illustrates a typical rod-shaped cell that would be included in our study, a partially contracted cell with “frayed” end (bottom left), and a cell that had undergone an irreversible contracture (top left), both of which would not have been studied. Fura- measurements of Cai. A final fura- concentration of 2.5 PM was obtained by adding 5 ~1 of 1 mM fura-21acetoxymethyl ester (AM) (Molecular Probes, Eugene, OR, lot 9J) stock solution in dimethyl sulfoxide to a 2-ml aliquot of buffer that contained the cells, 1.5% BSA, and 1 mM CaCl,. This was incubated for 15 min at 37°C and then spun at very low speed on a centrifuge (Fisher Centrific). The supernatant was removed, and the cells were resuspended in 2 ml of an Eagle’s minimal essential medium-HEPES buffer solution composed of (in mM) 2.0 CaCl,, 135 NaCl, 1 MgCl,, 5 KCl, 0.44 KH,PO,, 0.34 Na,HPO,, 2.6 NaHCO,, 20 HEPES, and 10 glucose, as well as amino acids, vitamins, and 0.001% phenol red (pH adjusted to 7.4 with NaOH). The cells were then rinsed for 30 min at 37°C to dilute any remaining fura-2. Fura- was selected as the Cai indicator because it has the following properties. I) The shift in the excitation spectrum of fura- caused by Ca2+ binding allows calculation of a ratio of fluorescence intensities. This ratio is related to Ca2+ concentration in a manner relatively independent of dye concentration and optical path length (24, 25, 31) (variables that can change with cell size, width and myocyte contraction). 2) Fura- can be loaded into the cell easily as the cell-permeant ester form, which is deesterified by esterases in the cytoplasm. 3) Furableaching is minimal. In theory, any bleaching that does
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11
1
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1000 [Ca2+],
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2000
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FIG. 2. Ca2+ calibration curves for furameasurements of intracellular free Ca2+. In vitro, measurements in lOO- to 200~PM-diam droplets of fura-2, Ca2+-EGTA standard solutions. Myoplasmic K,, ,, measurements in saponin-treated cell suspensions with furapentapotassium salt added after saponin; means + SE+ = 5. Myoplasmic AM, measurements in saponin-treated cell suspensions pretreated with fura-2/AM; means + SE, n = 5. SE bars are only apparent when Ca2+ = 1,000 nM because they were too small under other conditions. See text for further details.
occur can be compensated for when the ratio of fluorescence intensities at two wavelengths is calculated. A&rofluorometry. Cai transients were measured in a manner similar to that reported previously in more detail (24, 25, 31). Briefly, the cell superfusion chamber consisted of a Plexiglas block machined to accommodate a thin glass cover slip as a bottom. A droplet of cell suspension was placed in the chamber, and a thin sheet of buffer continuously flowed across the cells. The superfusion chamber was mounted on a Nikon Diaphot inverted microscope (Nikon, Garden City, NY), and cells were localized by observation in the microscope or video system. One important feature of the instrumentation is that the rotational period of the interference filter wheel was set at 50 ms so that a ratio was obtained every 50 ms. Two separate signals corresponding to 34O- and 380-nm signals that were fed into separate channels of an analogto-digital converter (Scientific Solutions, Solon, OH) and microcomputer equipped with the pClamp data acquisition system (Axon Instruments, Foster City, CA). After data had been acquired from a given cell, background fluorescence levels were determined by adjusting the microscope stage so that no cells or debris were in the field of view defined by the diaphragm. Fluorescence signals were stored by pClamp and were later transferred into a spreadsheet program (Quattro, Borland), which enabled correction of the signals for background fluorescence and then calculation of the ratio of the 340- to the 380-nm signal. [ Ca2+] calibration of fura- fluorescence ratio. We examined the quantitative relationship between calculated amounts of free calcium in a calibration buffer [containing appropriate Ca2+ -ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) concentrations and (in mM) 126 KCl, 10 NaCl, 20 HEPES, 1 MgCl, 2H,O, pH 7.1 with KOH] and the measured fluorescence ratio. A standard or in vitro curve was generated (Fig. 2) from these data using regression (2nd-order polynomial fit) over a range of free [Ca”‘] from 0 to 1,500 nM. This procedure validated the use of ratio fluorescence (F,,,/F,,) as an estimate of myoplasmic [Ca”‘] as described in detail (24, 25, 31). l
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In vitro calibrations were used to determine whether this ratio method of Ca2+ determination normalized changes in optical path length that may occur during the experimental protocol with contractions of the cardiac myocytes. This was tested in vitro by placing lOO- to 200pm-diam droplets of furaCa2+-EGTA standard solutions on the coverslip. There was a known ratio for these droplets (and associated free Ca2’ concentration). Observation of fluorescence at various positions on the hemispherical droplet varied the path length (mimicking variable thickness of the cell) by lo- to 100-fold. Multiple independent calibrations failed to demonstrate any effect of gross differences in optical path length on fluorescence ratios (data not shown). Calibration curves were also generated using “myoplasmic” furato estimate whether fura-2/AM was properly hydrolyzed to the pentapotassium salt (15, 22, 3 1). Myoplasmic Ca2+ was measured in separate cell suspensions, obtained from the same animal, with either fura-2/AM, the cell permeant ester form of fura-2, or the cell impermeant pentapotassium salt (KJ. Ventricular myocytes were dispersed as outlined above. One-half of the cells were loaded with fura-2/AM by incubating them with 2.5 PM of the permeant ester form, fura-2/AM, for 15 min at 37°C in 2CaNa (see below for composition) saline solution plus 0.2% BSA. The other one-half of the cells was treated similarly, except that no fura-2/AM was added. After incubation, the cells in each of the two suspensions were centrifuged to form pellets, the supernatant removed, and each fraction rinsed at 37°C for 30 min with 2 ml dispersion media containing 2% horse serum with no added fura-2/AM. The two separate suspensions of ventricular myocytes were then each divided further into three fractions. After another centrifugation, the pellets were resuspended in 500 ~1 of final fura[ Ca2+] calibration buffer solution containing either 100, 400, or 1,000 nM free [Ca”‘] and were allowed to rinse for 15 min. The cells were centrifuged, the supernatant was removed, and 25 ~1 of the same calibration buffer solutions containing 100 pg saponin/ml was added to the pellet of cells. Furapentapotassium salt (40 ,uM) was included in the saponin solution added to the pellets that had not been loaded with fura-2/AM. Ratios were then obtained at each [Ca”‘] for cells loaded with the cell-permeant and -impermeant forms of fura-2. Two separate calibration curves were generated and are plotted in Fig. 2. Although the three curves in Fig. 2 are similar, the myoplasmic AM curve tended to show slightly less Ca2+ sensitivity than the pentapotassium salt. The minor difference in Ca2+ sensitivity between the in vitro and the myoplasmic AM curves in Fig. 2 is the result of incomplete cleavage of the ester groups on fura-2/AM by myoplasmic esterases (24, 25, 31). Another aspect of the use of furaas an indicator of intracellular [Ca”‘] is the sequestering of fura-2/AM by mitochondria (22), which results in a lower dynamic range of the furaratio, despite the adequate Ca2’ sensitivity of the myoplasmic fura- we have shown in Fig. 2. These considerations led us to express the fura- data as ratios (15,22,24,25,31). Video monitoring and measurement of cell shortening. Cells were observed during each experiment via a video monitor (Panasonic CT-1301) and recorded with a video
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CARDIOMYOCYTE
cassette recorder (Panasonic AG-1230). Observation of contraction simultaneously with fluorescence recordings was made possible by inserting a binocular head (Science Instrument Shop, University of Missouri) containing a dichroic mirror (Omega Optical) that reflected the furafluorescence to the photomultiplier onto the side port of the Nikon Diaphot. Simultaneously, additional illumination of the cell was provided by placing a 600-nm interference filter in the filter holder of the Diaphot microscope. Only this narrow bandwidth light was then allowed to pass from the halogen lamp of the Diaphot through the dichroic mirror to a the video camera, thus permitting observation of the cell on a video monitor during furamicrofluorometry. A 510-nm interference filter was placed immediately in front of the photomultiplier to further ensure no interference from the 600-nm illumination. Shortening characteristics of the myocytes were measured from video recordings of the cells. A scale with lo-pm divisions was placed on the microscope stage and brought into focus on the monitor. This scale was recorded on a video cassette and was thereafter utilized to quantify the magnitude of contractile movement of each cell. Measurements were restricted to the portion of each cell defined by the diaphragm. Shortening was measured in two ways, distance moved by a landmark on the cell and as the percent of distance traveled by the landmark in reference to the portion of the cell visible on the screen. After a suitable landmark was chosen, the scale was taped to the screen, and the video tape of the cell was advanced frame by frame (33.3 ms/frame). The location of the landmark with respect to its original location was recorded every three frames. Because single cells exhibit beat-to-beat variability in twitch amplitude, average responses from six consecutive contractions, during minutes Z-3 of the experimental protocol (described below) were studied. The average peak distance traveled during the six trials, average time to peak, and average velocity of “shortening,” duration of contraction and time to onehalf relengthening were calculated for each cell. Due to technical constraints, shortening measurements were not made on all cells in which Ca2+ measurements were made. There were two primary reasons for exclusion of shortening measurements for cells in which Ca2+ were included: no video recording was obtained (5 EX cells, 11 SED cells) and lack of clearly definable landmarks and/or loss of focus during contraction (9 EX cells, 12 control cells). Shortening measurements were made in two to three cells per rat. Shortening data were not included in our analysis for a given rat unless acceptable measurements were obtained in two cells from that rat. Solutions and drugs used. Three solutions were used. The first, 2CaNa, contained (in mM) 5 K, 138 Na, 149 Cl, 2 Ca, 1 Mg, 10 HEPES, and 10 glucose. The base, NaOH, was added to adjust pH to 7.4. The remaining two solutions consisted of 2CaNa with the addition of 10 PM ryanodine (CalBiochem, La Jolla, CA), and 2CaNa with 10 PM ryanodine and 10 PM verapamil (CalBiochem). These commonly used supramaximal concentrations of the drugs avoided the possibility of altered drug sensitivity in myocytes of trained animals and ensured maximal
CA1
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1. Resting heart rate and body weights in EX and SED rats after 12 wk of treadmill training TABLE
Resting
Heart beats/min
Body
Rate,
Weight, g
EX SED
301+8 320+6
399t7 448212
P
free Ca2+
M. HAROLD LAUGHLIN, MARY E. SCHAEFER, AND M. STUREK Departments of Veterinary Biomedical Sciences and Physiology and Dalton Research Center, University of Missouri, Columbia, Missouri 65211 LAUGHLIN, M. HAROLD, MARY E. SCHAEFER, AND M. STUREK. Effect of exercise training on, intrczcelLular free Cu2+ transients in ventricular myocytes of rats. J. Appl. Physiol. 73(4): 1441-1448, 1992.-The purpose of this study was to test the hypothesis that exercise training induces enhanced intracellular free Ca2+ (Ca,) availability to the contractile elements of cardiac cells. Cai transients were directly measured in single isolated contracting ventricular myocytes from exercisetrained (EX) and sedentary control (SED) rats. Male SpragueDawley rats underwent 16 wk of progressive treadmill exercise (32 m/min, 8% grade, 1.5 h/day) (EX) or were cage confined (SED). EX rats had lower resting heart rate and elevated skeletal muscle oxidative capacity. Cai was measured with the fluorescent Cai indicator fura-2. Simultaneous video monitoring indicated that myocytes suspended in physiological salt solution were quiescent until stimulated electrically at a frequency of 0.2 Hz (12-36 V, 2-ms duration). Stimulated Ca, transients, measured from changes in fura- fluorescence, were similar in cells from EX and SED groups. Peak shortening, time to peak shortening, velocity of shortening, contraction duration, and time to half-relaxation were also similar in cells from EX and SED rats. Ryanodine (10 PM) was applied to eliminate the contribution of Ca2+ release from sarcoplasmic reticulum to the Ca, transient. Verapamil was applied to eliminate the contribution of voltage-gated Ca2’ channels to Ca, transients. Cai transients were also similar in cells from EX and SED groups after these pharmacological interventions. These results suggest that treadmill training of rats does not alter Ca, availability to the contractile elements in isolated ventricular myocytes.
addition, Tibbits et al. (29) found increased Ca2+ uptake by Na+-Ca2+ exchanger in cardiac sarcolemmal vesicles. Penpargkul et al. (13) reported enhanced binding of Ca2+ by cardiac sarcoplasmic reticulum (SR) of hearts from EX rats. The present study was undertaken to directly examine the Cai dynamics associated with contraction of myocardial cells of EX rats. Therefore a series of experiments was conducted in which systolic (transient) changes in Cai were measured with the fluorescent Cai indicator fura- in isolated ventricular myocytes from endurancetrained and SED control rats. We postulated that exercise training would increase peak amplitude and rate of decay of the Cai transient during activation. The pharmacological tools, ryanodine (226) and verapamil(3), were used to discriminate the contributions of the SR and sarcolemma (SL) to ventricular myocyte Cai transient. METHODS
Animals and training. Male Sprague-Dawley rats of ZOO-250 g body wt were purchased from Charles River. The rats were housed in pairs and kept in a 12:12-h lightdark cycle at a controlled temperature (23 t 2OC). Rat chow and water was provided ad libitum. Rats were exposed to treadmill exercise (lo-20 m/min, 0% grade, 10 min/day) for 1 wk. Rats that were most resistant to treadmill exercise were eliminated from the study. The remaining rats were randomly divided into calcium influx; ryanodine; calcium release channel; verapamil; contractility; voltage-gated calcium channel two groups, SED rats and EX rats. The EX rats began a training program in which running duration and treadmill speed and grade were increased incrementally over the next 5 wk. Training bouts took place 5 days/wk. By ENDURANCE EXERCISE TRAINING produces adaptations in the cardiovascular system as evidenced by an inweek 6, EX rats ran 1.5 h/day at 32 m/min on an 8% creased maximal oxygen transport capacity, more effigrade. Final intensity was maintained for the last 6-10 wk of the training program. Experiments began after EX cient cardiac performance, and enhanced intrinsic functioning of the myocardium (4,18). Although the fact that rats had trained for 12 wk and were conducted over a exercise training results in bradycardia (training brady4-wk period. EX rats continued the training program until they were used in the experiments. The duration of cardia) is well established, the nature of training-induced adaptation of cardiac contraction and/or relaxation is a training ranged from 12 to 16 wk. SED rats remained subject of continuing controversy. Several studies sug- cage-confined during the training period. Two series of experiments were conducted, separated gest that exercise training enhances contractile function of the heart (1, 8, 18). A popular hypothesis is that an by -6 mo. Each series of experiments involved one increased availability of intracellular free Ca2+ (Cai) for group of SED (n = 6) and one group of EX (n = 7) rats as described above. Thus there were a total of 12 SED and excitation-contraction coupling may be an underlying mechanism (1, 8, 11, 13, 14, 27-30). In one study, Ca2+ 14 EX rats used in this study. Training effectiveness was determined by measurebinding sites in papillary muscle preparations from exercise-trained (EX) rats were found to be increased by 63% ments of citrate synthase activity in the long head of the when compared with sedentary (SED) controls (28). In triceps brachii and resting heart rates of SED and EX 0161-7567/92
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Physiological
Society
1441
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CARDIOMYOCYTE
rats. Enzyme activity was determined spectrophotometritally, according to the method of Srere (23). For measurement of resting heart rates, each rat was anesthetized with methoxyflurane (Metofane) and instrumented with subcutaneous stainless steel sutures in the right and left axillary region and left thigh. Electrocardiogram and heart rate were recorded on an oscillographic recorder (model 28009, Gould ECG/Biotach Amplifier, model 13-4615-65) while the rats rested in a black box undisturbed for 65 min. Heart rates declined as a function of time in all rats tested such that a steady state was reached after 30-50 min (resting heart rate). Heart rate measurements were made between 9:00 A.M. and 12:00 P.M. during weeks lo-12 of training. Rats from the EX group were not exercised on the day of testing until after resting heart rate had been measured. Cell isolation procedures. Animals were anesthetized with pentobarbital sodium (0.1 ml/100 g ip) and given sodium heparin (1,000 IU ia) 20 min before excision of the heart. After isolation of hindquarters for a study of vascular transport capacity (14, 26), an incision was made into the thoracic cavity, the heart was rapidly removed and placed in ice-cold buffer containing the following: lo-20 PM ionized Ca2+, minimal essential medium Joklik-modified buffer (GIBCO, Grand Island, NY), 60 mM taurine (Sigma Chemical, St. Louis, MO), 20 mM creatine (Sigma), and 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Fisher Biotech, Fairlawn, NJ). Ca2+-tolerant cardiac myocytes were then prepared by retrograde perfusion through the ascending aorta of the isolated heart using a modification of the methods of Geisbuhler et al. (9). The coronary circulation was perfused at constant flow with perfusate equilibrated with 100% oxygen (pH 7.4) while the hearts were suspended in a water-jacketed chamber maintained at 37.0 z!I 0.5”C. The initial perfusate [buffer supplemented with 0.1% bovine serum albumin, fraction V (BSA, ICN Immunobiologicals, Lisle, IL), and 1 IU heparin/ml] was used to clear the vasculature of blood and was not recirculated. Subsequently, 0.7 mg collagenase/ml (Worthington Biochemical, Freehold, NJ) was added to the perfusate, and this solution was allowed to recirculate for 40-50 min. The ventricles were dissected free, minced with scissors in fresh collagenase-containing perfusate, and agitated in a 37°C water bath under 100% oxygen for 10 min. CaCl, (50 PM) was added, and the suspension was agitated for another 10 min. Tissue was drawn up into a pipette to gently disperse cells. The suspension was filtered through a double layer of gauze and diluted 1:4 with collagenase-free buffer containing 0.1% wt/vol BSA. After the cells settled, the supernatant was removed, and the cells were resuspended in buffer containing 1.5% BSA (15 mg/ml). This was repeated twice, and the cells were resuspended for a final time in buffer containing 1.5% BSA. Ca2+ tolerance was induced slowly over 30 min, during which time the cells were maintained in suspension by gentle agitation and were continually gassed with 100% oxygen. Final Ca2+ concentration was 1 mM. Experiments were completed within 5 h of the final addition of CaCl,. Cells were chosen for inclusion in the study based on
CA,
AND EXERCISE
FIG. 1. A: photomicrograph of 3 cardiac myocytes. Rod-shaped cell represents type of cells used to measure Ca2+ transients and contractile motion in present study. Cell at bottom left is partially contracted and has “frayed” end; cell would not have been included in study. Cell that had undergone irreversible contracture (top left) also would not have been studied. Horizontal calibration bar at lower edge equals 20 Frn. B: fluorescent image of same cell when fura- in cell is excited by 380-nm light.
the following criteria: rod shape; quiescent until stimulated to contract by field stimulation (contraction verified by visual observation via video monitor); when not being stimulated to contract, the ratio of the 340- to the 380-nm fluorescence intensity was required to be cl.0 (which corresponds to Cai of 5100 nM), indicating “normal” resting Cai. Figure 1 is an example of a cardiac myocyte typical of those selected for this study. This micrograph illustrates a typical rod-shaped cell that would be included in our study, a partially contracted cell with “frayed” end (bottom left), and a cell that had undergone an irreversible contracture (top left), both of which would not have been studied. Fura- measurements of Cai. A final fura- concentration of 2.5 PM was obtained by adding 5 ~1 of 1 mM fura-21acetoxymethyl ester (AM) (Molecular Probes, Eugene, OR, lot 9J) stock solution in dimethyl sulfoxide to a 2-ml aliquot of buffer that contained the cells, 1.5% BSA, and 1 mM CaCl,. This was incubated for 15 min at 37°C and then spun at very low speed on a centrifuge (Fisher Centrific). The supernatant was removed, and the cells were resuspended in 2 ml of an Eagle’s minimal essential medium-HEPES buffer solution composed of (in mM) 2.0 CaCl,, 135 NaCl, 1 MgCl,, 5 KCl, 0.44 KH,PO,, 0.34 Na,HPO,, 2.6 NaHCO,, 20 HEPES, and 10 glucose, as well as amino acids, vitamins, and 0.001% phenol red (pH adjusted to 7.4 with NaOH). The cells were then rinsed for 30 min at 37°C to dilute any remaining fura-2. Fura- was selected as the Cai indicator because it has the following properties. I) The shift in the excitation spectrum of fura- caused by Ca2+ binding allows calculation of a ratio of fluorescence intensities. This ratio is related to Ca2+ concentration in a manner relatively independent of dye concentration and optical path length (24, 25, 31) (variables that can change with cell size, width and myocyte contraction). 2) Fura- can be loaded into the cell easily as the cell-permeant ester form, which is deesterified by esterases in the cytoplasm. 3) Furableaching is minimal. In theory, any bleaching that does
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 20, 2019.
CARDIOMYOCYTE
o!
I1
0
r
11
11
500
11
1
r
11
1000 [Ca2+],
“‘1”’
1500
2000
nM
FIG. 2. Ca2+ calibration curves for furameasurements of intracellular free Ca2+. In vitro, measurements in lOO- to 200~PM-diam droplets of fura-2, Ca2+-EGTA standard solutions. Myoplasmic K,, ,, measurements in saponin-treated cell suspensions with furapentapotassium salt added after saponin; means + SE+ = 5. Myoplasmic AM, measurements in saponin-treated cell suspensions pretreated with fura-2/AM; means + SE, n = 5. SE bars are only apparent when Ca2+ = 1,000 nM because they were too small under other conditions. See text for further details.
occur can be compensated for when the ratio of fluorescence intensities at two wavelengths is calculated. A&rofluorometry. Cai transients were measured in a manner similar to that reported previously in more detail (24, 25, 31). Briefly, the cell superfusion chamber consisted of a Plexiglas block machined to accommodate a thin glass cover slip as a bottom. A droplet of cell suspension was placed in the chamber, and a thin sheet of buffer continuously flowed across the cells. The superfusion chamber was mounted on a Nikon Diaphot inverted microscope (Nikon, Garden City, NY), and cells were localized by observation in the microscope or video system. One important feature of the instrumentation is that the rotational period of the interference filter wheel was set at 50 ms so that a ratio was obtained every 50 ms. Two separate signals corresponding to 34O- and 380-nm signals that were fed into separate channels of an analogto-digital converter (Scientific Solutions, Solon, OH) and microcomputer equipped with the pClamp data acquisition system (Axon Instruments, Foster City, CA). After data had been acquired from a given cell, background fluorescence levels were determined by adjusting the microscope stage so that no cells or debris were in the field of view defined by the diaphragm. Fluorescence signals were stored by pClamp and were later transferred into a spreadsheet program (Quattro, Borland), which enabled correction of the signals for background fluorescence and then calculation of the ratio of the 340- to the 380-nm signal. [ Ca2+] calibration of fura- fluorescence ratio. We examined the quantitative relationship between calculated amounts of free calcium in a calibration buffer [containing appropriate Ca2+ -ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) concentrations and (in mM) 126 KCl, 10 NaCl, 20 HEPES, 1 MgCl, 2H,O, pH 7.1 with KOH] and the measured fluorescence ratio. A standard or in vitro curve was generated (Fig. 2) from these data using regression (2nd-order polynomial fit) over a range of free [Ca”‘] from 0 to 1,500 nM. This procedure validated the use of ratio fluorescence (F,,,/F,,) as an estimate of myoplasmic [Ca”‘] as described in detail (24, 25, 31). l
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In vitro calibrations were used to determine whether this ratio method of Ca2+ determination normalized changes in optical path length that may occur during the experimental protocol with contractions of the cardiac myocytes. This was tested in vitro by placing lOO- to 200pm-diam droplets of furaCa2+-EGTA standard solutions on the coverslip. There was a known ratio for these droplets (and associated free Ca2’ concentration). Observation of fluorescence at various positions on the hemispherical droplet varied the path length (mimicking variable thickness of the cell) by lo- to 100-fold. Multiple independent calibrations failed to demonstrate any effect of gross differences in optical path length on fluorescence ratios (data not shown). Calibration curves were also generated using “myoplasmic” furato estimate whether fura-2/AM was properly hydrolyzed to the pentapotassium salt (15, 22, 3 1). Myoplasmic Ca2+ was measured in separate cell suspensions, obtained from the same animal, with either fura-2/AM, the cell permeant ester form of fura-2, or the cell impermeant pentapotassium salt (KJ. Ventricular myocytes were dispersed as outlined above. One-half of the cells were loaded with fura-2/AM by incubating them with 2.5 PM of the permeant ester form, fura-2/AM, for 15 min at 37°C in 2CaNa (see below for composition) saline solution plus 0.2% BSA. The other one-half of the cells was treated similarly, except that no fura-2/AM was added. After incubation, the cells in each of the two suspensions were centrifuged to form pellets, the supernatant removed, and each fraction rinsed at 37°C for 30 min with 2 ml dispersion media containing 2% horse serum with no added fura-2/AM. The two separate suspensions of ventricular myocytes were then each divided further into three fractions. After another centrifugation, the pellets were resuspended in 500 ~1 of final fura[ Ca2+] calibration buffer solution containing either 100, 400, or 1,000 nM free [Ca”‘] and were allowed to rinse for 15 min. The cells were centrifuged, the supernatant was removed, and 25 ~1 of the same calibration buffer solutions containing 100 pg saponin/ml was added to the pellet of cells. Furapentapotassium salt (40 ,uM) was included in the saponin solution added to the pellets that had not been loaded with fura-2/AM. Ratios were then obtained at each [Ca”‘] for cells loaded with the cell-permeant and -impermeant forms of fura-2. Two separate calibration curves were generated and are plotted in Fig. 2. Although the three curves in Fig. 2 are similar, the myoplasmic AM curve tended to show slightly less Ca2+ sensitivity than the pentapotassium salt. The minor difference in Ca2+ sensitivity between the in vitro and the myoplasmic AM curves in Fig. 2 is the result of incomplete cleavage of the ester groups on fura-2/AM by myoplasmic esterases (24, 25, 31). Another aspect of the use of furaas an indicator of intracellular [Ca”‘] is the sequestering of fura-2/AM by mitochondria (22), which results in a lower dynamic range of the furaratio, despite the adequate Ca2’ sensitivity of the myoplasmic fura- we have shown in Fig. 2. These considerations led us to express the fura- data as ratios (15,22,24,25,31). Video monitoring and measurement of cell shortening. Cells were observed during each experiment via a video monitor (Panasonic CT-1301) and recorded with a video
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CARDIOMYOCYTE
cassette recorder (Panasonic AG-1230). Observation of contraction simultaneously with fluorescence recordings was made possible by inserting a binocular head (Science Instrument Shop, University of Missouri) containing a dichroic mirror (Omega Optical) that reflected the furafluorescence to the photomultiplier onto the side port of the Nikon Diaphot. Simultaneously, additional illumination of the cell was provided by placing a 600-nm interference filter in the filter holder of the Diaphot microscope. Only this narrow bandwidth light was then allowed to pass from the halogen lamp of the Diaphot through the dichroic mirror to a the video camera, thus permitting observation of the cell on a video monitor during furamicrofluorometry. A 510-nm interference filter was placed immediately in front of the photomultiplier to further ensure no interference from the 600-nm illumination. Shortening characteristics of the myocytes were measured from video recordings of the cells. A scale with lo-pm divisions was placed on the microscope stage and brought into focus on the monitor. This scale was recorded on a video cassette and was thereafter utilized to quantify the magnitude of contractile movement of each cell. Measurements were restricted to the portion of each cell defined by the diaphragm. Shortening was measured in two ways, distance moved by a landmark on the cell and as the percent of distance traveled by the landmark in reference to the portion of the cell visible on the screen. After a suitable landmark was chosen, the scale was taped to the screen, and the video tape of the cell was advanced frame by frame (33.3 ms/frame). The location of the landmark with respect to its original location was recorded every three frames. Because single cells exhibit beat-to-beat variability in twitch amplitude, average responses from six consecutive contractions, during minutes Z-3 of the experimental protocol (described below) were studied. The average peak distance traveled during the six trials, average time to peak, and average velocity of “shortening,” duration of contraction and time to onehalf relengthening were calculated for each cell. Due to technical constraints, shortening measurements were not made on all cells in which Ca2+ measurements were made. There were two primary reasons for exclusion of shortening measurements for cells in which Ca2+ were included: no video recording was obtained (5 EX cells, 11 SED cells) and lack of clearly definable landmarks and/or loss of focus during contraction (9 EX cells, 12 control cells). Shortening measurements were made in two to three cells per rat. Shortening data were not included in our analysis for a given rat unless acceptable measurements were obtained in two cells from that rat. Solutions and drugs used. Three solutions were used. The first, 2CaNa, contained (in mM) 5 K, 138 Na, 149 Cl, 2 Ca, 1 Mg, 10 HEPES, and 10 glucose. The base, NaOH, was added to adjust pH to 7.4. The remaining two solutions consisted of 2CaNa with the addition of 10 PM ryanodine (CalBiochem, La Jolla, CA), and 2CaNa with 10 PM ryanodine and 10 PM verapamil (CalBiochem). These commonly used supramaximal concentrations of the drugs avoided the possibility of altered drug sensitivity in myocytes of trained animals and ensured maximal
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1. Resting heart rate and body weights in EX and SED rats after 12 wk of treadmill training TABLE
Resting
Heart beats/min
Body
Rate,
Weight, g
EX SED
301+8 320+6
399t7 448212
P
