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(Circulation Research. 1999;84:1459-1468.)
© 1999 American Heart Association, Inc.

Rapid Communication

Ca²⁺ Waves During Triggered Propagated Contractions in Intact Trabeculae

Determinants of the Velocity of Propagation

Masahito Miura, Penelope A. Boyden, Henk E. D. J. ter Keurs

From the Department of Medicine (M.M., H.E.D.J.K.), University of Calgary, Calgary, Canada, and Department of Pharmacology (P.A.B.), Columbia College of Physicians and Surgeons, New York, NY.

Correspondence and reprint requests to Henk E.D.J. ter Keurs, MD, PhD, Department of Medicine, Health Science Centre, 3330 Hospital Dr NW, Calgary, Alberta, Canada T2N 4N1. E-mail terkeurs{at}ucalgary.ca

	Abstract

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Abstract—During triggered propagated contractions, Ca²⁺ waves travel along cardiac trabeculae with a constant velocity (V_prop) ranging from 0.34 to 5.47 mm/s. To explore the determinants of V_prop, we studied (1) the relationship between [Ca²⁺]_i and V_prop and (2) the effect of low concentrations of caffeine on V_prop. Trabeculae were dissected from the right ventricle of rat hearts. [Ca²⁺]_i was measured using electrophoretically injected fura-2 and an image-intensified CCD camera. Force was measured using a silicon strain gauge, and sarcomere length was measured using laser diffraction techniques. After induction of reproducible Ca²⁺ waves by trains of electrical stimuli (2.5 Hz) at 21.9±0.2°C, the number of stimuli or [Ca²⁺]_o was varied in 9 trabeculae. In 5 trabeculae, the effects of caffeine (0.1 to 1.0 mmol/L) at [Ca²⁺]_o of 2.2±0.3 mmol/L were determined. All images were recorded under stable conditions of wave propagation. The increment in [Ca²⁺]_i during the last electrically stimulated transient (Ca_T) and [Ca²⁺]_i just before onset of the Ca²⁺ waves (Ca_D) were used to estimate the Ca²⁺ loading of the sarcoplasmic reticulum (SR) and the myoplasm, respectively. The ratio (Ca_W/Ca_T) of the [Ca²⁺]_i increment during the waves (Ca_W) to Ca_T was used to estimate the probability of opening of the SR-Ca²⁺ release channel during wave propagation. As a result of an increase of the number of stimuli or [Ca²⁺]_o, V_prop increased in proportion to (1) Ca_T (r=0.82); (2) Ca_D (r=0.88); (3) Ca_W (r=0.85); and (4) Ca_W/Ca_T (r=0.74). The addition of caffeine (0.3 mmol/L) increased V_prop for any Ca_T and any Ca_W, revealing an increased sensitivity of V_prop to Ca_T and Ca_W. In contrast, caffeine had little effect on the relationship between V_prop and Ca_D and no effect on that between V_prop and Ca_W/Ca_T. These results suggest that both the cellular Ca²⁺ loading and open probability of the SR-Ca²⁺ release channels determine the velocity of propagation of Ca²⁺ waves.

Key Words: rat cardiac trabeculae • triggered propagated contraction • Ca²⁺ wave • caffeine

	Introduction

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Local aftercontractions starting from damaged regions of muscle propagate along cardiac trabeculae.^{1 2} The propagated contractions, which can be measured as waves of sarcomere shortening in both rat ventricular^{1 2} and human atrial trabeculae,³ have been denoted as triggered propagated contractions (TPCs). Recently, regional increases in [Ca²⁺]_i have been observed to travel along rat cardiac trabeculae during TPCs and denoted as Ca²⁺ waves.⁴ These Ca²⁺ waves travel along trabeculae at the same velocities as TPCs, ranging from 0.34 to 5.47 mm/s.

Several studies have suggested that the propagation mechanism of TPCs is consistent with a model of Ca²⁺-induced Ca²⁺ release (CICR) from sarcoplasmic reticulum (SR) mediated by Ca²⁺ diffusion to adjacent SR.^{1 4 5 6} The velocity of propagation (V_prop) of TPCs varies depending on the [Ca²⁺]_o, the number and frequency of the electrical stimuli,^{1 2} and the presence or absence of Ca²⁺ channel agonists and antagonists.⁷ These observations are consistent with the assumption that Ca²⁺ loading of the cell (SR Ca²⁺ content and/or cytosolic Ca²⁺) is a main determinant of V_prop. Therefore, we propose that the Ca²⁺ level in the myoplasm and/or SR can determine V_prop via modulation of CICR. Furthermore, computer simulation of CICR and Ca²⁺ diffusion supports the hypothesis that V_prop of Ca²⁺ waves (or TPCs) will be altered depending on the combined effects of an increase in (1) the diastolic [Ca²⁺]_i; (2) the rate of rise of the Ca²⁺ release; and (3) the amount of Ca²⁺ released by the SR.⁸

At a low concentration (0.3 mmol/L), caffeine has been shown to increase V_prop and force.⁷ In addition, 0.5 mmol/L caffeine can increase the amplitude of delayed afterdepolarizations and cause triggered activity.^{9 10 11 12} Recently, caffeine has been shown to enhance the release of Ca²⁺ from the SR by activating a cardiac SR-Ca²⁺ release channel (RyR) incorporated into planar phospholipid bilayers.^{13 14 15 16} This activation of the channel leads to net shift of Ca²⁺ from SR to myoplasm, subsequently modulating Ca²⁺ loading level of the myoplasm and the SR. Thus, we hypothesize that the effect of caffeine on V_prop of TPCs is secondary to drug modulation of CICR due to the changes in Ca²⁺ levels in the myoplasm and SR.

Therefore, in the present study, we investigated (1) the relationship between [Ca²⁺]_i and V_prop of Ca²⁺ waves to evaluate how Ca²⁺ levels in myoplasm and SR can affect V_prop and (2) the effect of caffeine on these relationships to evaluate whether V_prop is sensitive to changes in the probability of opening of the SR-Ca²⁺ release channel (P_o) of RyR.

	Materials and Methods

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Dissection and Mounting of Rat Ventricular Trabeculae
The experiments in the present study were conducted in accordance with the principles outlined in the most recent Guide to the Care and Use of Experimental Animals by the Canadian Council on Animal Care. Lewis Brown Norway rats (Harlan Sprague-Dawley Inc, Indianapolis, Ind; 250 to 300 g) were anesthetized with diethyl ether. Twelve hearts were excised, and the coronary arteries were immediately perfused via the aorta with a Krebs-Henseleit (K-H) solution modified by adding 15 mmol/L KCl. After arrest of the heart, trabeculae (n=12, length 2.07±0.09 mm, width 207±37 µm, thickness 101±4 µm) were dissected from the right ventricle and mounted horizontally between a force transducer and a micromanipulator in a perfusion bath located on the stage of an inverted microscope (Nikon). Trabeculae were stimulated at 0.5 Hz through parallel platinum electrodes in the bath with 5-ms pulses 50% above threshold and superfused with bicarbonate buffered K-H solution containing (in mmol/L) NaCl 120, KCl 5, MgCl₂ 1.2, Na₂SO₄ 1.2, NaH₂PO₄ 2.0, NaHCO₃ 19, glucose 10, and CaCl₂, as specified in the Results. The solutions were in equilibrium with 95% O₂ and 5% CO₂; pH was 7.4.

Fura-2 Loading and Measurement of Fluorescence
[Ca²⁺]_i in the trabeculae was measured as previously described.⁴ Briefly, fura-2 pentapotassium salt was microinjected electrophoretically into one cell and allowed to spread throughout the trabeculae via gap junctions. After the injection, the trabeculae were stimulated at 1 Hz for 30 to 60 minutes until fura-2 had diffused uniformly throughout the preparation. The epifluorescence of fura-2 from the trabeculae at excitation wavelengths of 340 and 380 nm was measured at 510 nm by a photomultiplier tube (PMT) (PMT-R2693 with a C1053-01 socket, Hamamatsu). The signal from the PMT was stored in a personal computer through an analog-digital converter. Alternatively, the fluorescent image of the trabeculae at excitation wavelengths of 360 and 380 nm was recorded by a CCD camera coupled to a 2-stage image intensifier (IIC; model C330, General Scanning Inc) through a 510- to 560-nm bandpass filter. The images were recorded with a videocassette recorder (VCR) for offline analysis. The force of the muscle was measured using a modified silicon semiconductor strain gauge. Sarcomere length (SL) was measured using laser diffraction techniques.¹⁷

Analysis of the Signal From the PMT
[Ca²⁺]_i was determined using the following equation¹⁸ (after subtraction of the autofluorescence of the muscle), as previously described⁴ : [Ca²⁺]_i=K_dxßx(R-R_min)/(R_max-R), where K_d is the effective dissociation constant, R is the ratio of the fluorescence at 340-nm excitation to that at 380-nm excitation (340/380), R_min is R at zero [Ca²⁺], R_max is R at a saturating [Ca²⁺], and ß is the ratio of fluorescence value for Ca²⁺-free dye to fluorescence value for Ca²⁺-bound dye at 380-nm excitation. Because we have previously reported a good correlation between in vitro and in vivo calibrations when free Mg²⁺ was 1 mmol/L in the solutions mimicking the intracellular milieu,¹⁹ values for K_d, R_min, R_max, and ß were determined using in vitro calibrations. R_min and R_max were 0.152 and 4.60, and K_d and ß were 361 nmol/L and 9.18, respectively. This is in agreement with previous data from our laboratory.¹⁹

Analysis of an Image From the IIC
Fura-2 fluorescence images recorded at 30 frames per second on the VCR were analyzed as previously described.⁴ Briefly, fluorescence data of each video frame were digitized with an 8-bit analog-digital converter and stored in a frame buffer memory of 512x480 pixels (Coreco Inc). Therefore, in our optical system, one pixel corresponded to 2.9x2.9 µm in the image plane. For the analysis of the image, a region of interest (ROI) was set horizontally along the long axis of the fluorescence image of trabeculae. The length of the ROI was always 512 pixels (1470 µm) whereas its width was 20 pixels (57.4 µm). To obtain intensity profiles of the fluorescence along the long axis of trabeculae, we calculated an average intensity value from each transverse line of pixels within the ROI. To eliminate high-frequency noise from the intensity profile, we used a low-pass finite impulse response filter (MATLAB) with a cutoff frequency of 5 pixels (14.4 µm). After subtraction of autofluorescence, we calculated the ratio of the fluorescence at 360-nm to that at 380-nm excitation (360/380) at each point on the intensity profiles obtained from the images at 360- and 380-nm excitation light. To correct for the effects of nonuniform illumination of excitation light, we calculated [Ca²⁺]_i at each sampling point after the induction of TPCs using the regression line derived from the relationship between PMT and the IIC ratio determined at the same sampling point. In addition, to avoid noise caused by low-excitation light intensity on the far edges of the profile of fluorescence, we calculated [Ca²⁺]_i only at the regions of the centrally located 250 pixels (719 µm) of the profiles along the trabeculae.

To calculate V_prop, we identified the peak of a Ca²⁺ transient during the Ca²⁺ wave at each pixel along trabeculae and plotted the time of the maximum against the position of the peak. V_prop was calculated from the slope of the fitted line to the plot, when regression analysis showed a linear relationship (r0.9), as described previously.⁴

Experimental Protocol
To induce a Ca²⁺ wave (or TPC), bath temperature was lowered to 20°C to 23°C, and trains of electrical stimuli at 2.5 Hz were applied for 10 seconds at [Ca²⁺]_o of 0.3 mmol/L. SL was set to 2.10 µm for all muscles. [Ca²⁺]_o was then increased in steps of 0.2 mmol/L until a TPC appeared.^{1 2} The measurement of [Ca²⁺]_i started when V_prop of Ca²⁺ waves (or TPCs) triggered by serial trains of stimuli varied by <10%. We regarded such Ca²⁺ waves as reproducible; reproducible conditions lasted at least 30 minutes. In the present study, we induced reproducible Ca²⁺ waves in 12 trabeculae. To change Ca²⁺ loading of the muscle, we varied [Ca²⁺]_o and/or duration of the train of electrical stimuli in 9 trabeculae. The measurement of [Ca²⁺]_i was started again when Ca²⁺ waves (or TPCs) reached a new steady state. Thus, we eventually analyzed 23 reproducible Ca²⁺ waves ([Ca²⁺]_o 2.2±0.3 mmol/L, temperature 21.9±0.2°C).

The effect of caffeine on Ca²⁺ waves was studied using 7 reproducible Ca²⁺ waves elicited in 5 trabeculae (length 2.19±0.19 mm, width 228±57 µm, thickness 101±4 µm, [Ca²⁺]_o 2.4±0.3 mmol/L, temperature 21.6±0.3°C, 10-second electrical stimulation at 2.5 Hz). When the Ca²⁺ waves were reproducible, trabeculae were superfused with K-H solution containing varied concentrations (0.1 to 1.0 mmol/L) of caffeine (Sigma). After the superfusion with caffeine, force development induced by trains of electrical stimuli was monitored and reached a new steady-state level within 5 minutes; measurement of [Ca²⁺]_i was then begun. All measurements made in the presence of caffeine were completed within 30 minutes, and trabeculae were then superfused with caffeine-free K-H solution. After washout, we confirmed that the Ca²⁺ waves were still reproducible.

Data Analysis
To assess Ca²⁺ loading of myoplasm and SR, we calculated the following parameters (see Figure 1). First, we estimated the SR Ca²⁺ loading from the increment in [Ca²⁺]_i during the last stimulated twitch of the trains (Ca_T), ie, the difference between a peak of a Ca²⁺ transient during the twitch and the minimal [Ca²⁺]_i preceding the last twitch. Second, we measured the diastolic [Ca²⁺]_i (Ca_D) just before a Ca²⁺ wave, ie, the minimal [Ca²⁺]_i observed between the last twitch and a subsequent TPC. Third, the amount of Ca²⁺ released during the wave was estimated from the increment in [Ca²⁺]_i during a Ca²⁺ wave (Ca_W), ie, the difference between the peak of a Ca²⁺ wave (Ca_w) and Ca_D. Using these parameters, we calculated Ca_W/Ca_T, the amount of Ca²⁺ released during the wave normalized for the SR Ca²⁺ content. We assumed that this parameter reflects the released fraction of Ca²⁺ inside SR and corresponds to the probability of Ca²⁺ release from SR during the wave (see Discussion). When obtained with use of the PMT (Figure 1), we will refer to these parameters as global changes in [Ca²⁺]_i (gCa_T, gCa_D, and gCa_W). When obtained from images recorded by the IIC, we first calculated these parameters at each pixel position along trabeculae and then averaged the values obtained at each pixel position. In that case, we will refer to the averaged values as regional changes in [Ca²⁺]_i (rCa_T, rCa_D, and rCa_W). Moreover, we measured developed force during the last twitch (F_T) and that during a TPC (F_TPC).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of changes in the duration of electrical stimuli on global changes in [Ca2+]i. Upper panels are changes in [Ca2+]i calculated from the fluorescence signals recorded by the PMT, and the lower panels are 2 force recordings (normalized for cross-sectional area of the muscle) at the 340- and 380-nm excitation wavelength during the last 2 electrically stimulated twitches and a subsequent TPC. The arrows indicate an increase in [Ca2+]i (upper panel) and an aftercontraction (lower panel) during which a TPC was observed. S indicates moment of electrical stimulation (temperature 23.1°C, [Ca2+]o 3.0 mmol/L, experiment 970110). A, Electrical stimulation at 2.5 Hz for 5 seconds. gCaT indicates an increment in [Ca2+]i during the last stimulated twitch; gCaD, a diastolic [Ca2+]i just before a Ca2+ wave; gCaW, an increment in [Ca2+]i during a Ca2+ wave; and gCaW, a peak of [Ca2+]i during a Ca2+ wave. B, Electrical stimulation at 2.5 Hz for 15 seconds. gCaT increased from 508 to 604 nmol/L, gCaD increased from 213 to 256 nmol/L, and gCaW increased from 90 to 204 nmol/L (upper panel). FT increased from 28.6 to 30.2 mN/mm2 and FTPC increased from 10.1 to 13.8 mN/mm2 (lower panel).

Statistics
All averaged values were expressed as mean±SEM. Single-factor ANOVA, unless stated otherwise, was used to detect significant differences (P<0.05).

	Results

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Figures 1 and 2 show clearly that Ca²⁺ waves are accelerated by Ca²⁺ loading.⁴ To change Ca²⁺ loading of the muscle, we increased [Ca²⁺]_o or the duration of the train of electrical stimuli after the induction of reproducible Ca²⁺ waves. Figure 1 shows global [Ca²⁺]_i and force development during the last 2 electrically stimulated twitches of a train at 2.5-Hz stimulation. With a longer period of stimulation, all parameters of global [Ca²⁺]_i (gCa_T, gCa_D, and gCa_W) increased by 19%, 20%, and 127%, respectively, and parameters of force (F_T and F_TPC) increased by 6% and 37%, respectively. In Figure 2, regional changes in [Ca²⁺]_i during comparable TPCs were determined directly after the recordings in Figure 1. With a longer period of stimulation, V_prop of the Ca²⁺ wave increased from 2.92 to 4.40 mm/s. The parameters of regional [Ca²⁺]_i (rCa_T, rCa_D, and rCa_W) also increased in proportion to each other (Figure 3).

View larger version (64K): [in this window] [in a new window]   Figure 2. Effect of changes in the duration of electrical stimuli on regional changes in [Ca2+]i. The regional [Ca2+]i calculated from the images by the IIC during the last electrically stimulated twitch and a TPC a few minutes after the recording of the PMT shown in Figure 1. Abscissa represents time; ordinate, [Ca2+]i; and z-axis, the position along the long axis of the trabecula. s indicates moment of electrical stimulation (experiment 970110). A, Electrical stimulation for 5 seconds. After the end of the clearly uniform stimulated Ca2+ transient, a small Ca2+ transient was observed to move as a wave from position A (*) toward position B at the calculated Vprop of 2.92 mm/s. B, Electrical stimulation for 15 seconds. Vprop increased to 4.40 mm/s. In addition, rCaT increased from 555 to 615 nmol/L, rCaD increased from 165 to 245 nmol/L, and rCaW increased from 157 to 245 nmol/L.

View larger version (22K): [in this window] [in a new window]   Figure 3. Relationships between Vprop and the regional changes in [Ca2+]i under control conditions (n=23). Vprop was tightly and linearly correlated with rCaT (A), rCaD (B), rCaW (C), and rCaW/rCaT (D). Note that all Ca2+ parameters correlate strongly and linearly, indicating that the Ca2+ loading of the muscle caused a proportional increase of rCaD, rCaW, and rCaT. In addition, rCaW/rCaT increased in proportion with the Ca2+ loading of the muscle (for further explanation, see text).

Ca²⁺ loading of the muscle increased the amplitude of TPCs and accelerated their propagation. As a result, TPCs already did occur in between the twitches during the stimulus train (Figure 1), which might cause nonuniformity of the Ca²⁺ release process during the (last) twitch that preceded the TPC, which was analyzed here. The nonuniformity of Ca²⁺ transients after the last stimulus along the analyzed region appeared to be small. The maximal difference in peak amplitude of rCa_T was <40 nmol/L whereas rCa_D differed <20 nmol/L between both ends of the ROI (data not shown). Hence, we believed that activation of the ROI was sufficiently uniform to permit evaluation of the factors that dictate the rate of propagation of the Ca²⁺ waves occurring after the twitch.

Data obtained from 23 reproducible Ca²⁺ waves from 12 trabeculae showed that V_prop correlated strongly with rCa_T (Figure 3A), rCa_D (Figure 3B), and rCa_W (Figure 3C) as well as rCa_W/rCa_T (Figure 3D). In 15 Ca²⁺ waves from 8 trabeculae, V_prop also correlated linearly with F_T (r=0.70) and F_TPC (r=0.67) (data not shown), as we have described previously.⁷ The increase of cytosolic Ca²⁺ correlated with rCa_T (Figure 3) and F_T (not shown). F_T is proportional to the amplitude of rapid cooling contractures (H. Banijamali, H.E.D.J. ter Keurs, unpublished observations, 1994) so that one may conclude that Ca²⁺ loading in our experiments led to proportional increases in cytosolic Ca²⁺ and in the SR Ca²⁺ content (reflected by rCa_T) of the muscle.

It was striking that rCa_W/rCa_T under drug-free conditions increased linearly with both rCa_T and rCa_D (Figure 3). The increase of this ratio suggests that an increase of the Ca²⁺ loading of the muscle increases the probability of opening of the SR-Ca²⁺ channels during the wave. Ca²⁺ wave propagation accelerated with an increase in the latter parameter (rCa_W/rCa_T; Figure 3D). Regional measurements showed that acceleration of the Ca²⁺ wave was not the cause of the larger amplitude of the transient owing to faster wave propagation, as would be observed in the recordings with the PMT, which collected the fluorescence from a larger region in the muscle.⁴

To modify the kinetics of net Ca²⁺ transport from the myoplasm to the SR, 5 trabeculae were superfused with caffeine (0.1 to 1.0 mmol/L) after the induction of a reproducible Ca²⁺ wave. Figure 4 shows an example of the global [Ca²⁺]_i and the force development during the last 2 stimulated twitches in a train and a subsequent TPC in the absence and in the presence of caffeine. The addition of 0.3 mmol/L caffeine decreased the amplitude of gCa_T by 47% while gCa_D and gCa_W increased by 51% and 91%, respectively. F_T also decreased (27%) whereas F_TPC increased (113%). In the presence of 1.0 mmol/L caffeine, we could neither detect an increase in [Ca²⁺]_i nor an aftercontraction following the train of electrical stimuli. gCa_T and F_T decreased by 77% and 55%, respectively. The diastolic diffraction pattern was uniform and stationary whereas twitch force during the train stayed constant. These observations suggested that random spontaneous sarcomere contractions occurred only rarely in the presence of 1 mmol/L caffeine.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effects of caffeine on global changes in [Ca2+]i in a typical trabecula. Upper panels show global changes in [Ca2+]i calculated from the PMT signals, and lower panels show force recordings during the last 2 electrically stimulated twitches in a train and a subsequent TPC in one trabecula using a similar protocol as in Figure 1. S indicates moment of electrical stimulation (temperature 21.9°C, [Ca2+]o 2.0 mmol/L, experiment 970214). A, In the absence of caffeine. The arrows indicate an increase in [Ca2+]i (upper panel) and an aftercontraction (lower panel) during which a TPC was observed. gCaT, gCaD, and gCaW were 697, 163, and 53 nmol/L, respectively (upper panel). FT and FTPC were 28.3 and 1.6 mN/mm2, respectively (lower panel). B, In the presence of 0.3 mmol/L caffeine. The arrows indicate an increase in [Ca2+]i (upper panel) and an aftercontraction (lower panel) during which a TPC was observed. gCaT decreased to 369 nmol/L whereas gCaD and gCaW increased to 246 and 101 nmol/L, respectively (upper panel). FT decreased to 17.3 mN/mm2 whereas FTPC increased to 3.4 mN/mm2 (lower panel). C, In the presence of 1.0 mmol/L caffeine. We could neither detect an increase in [Ca2+]i nor an aftercontraction subsequent to the train of electrical stimuli. gCaT and FT decreased to 158 nmol/L and 11.8 mN/mm2, respectively.

Figure 5 shows the last stimulated Ca²⁺ transient(s) and a cytosolic Ca²⁺ wave during the TPC in the absence (Figure 5A) and in the presence of 0.3 mmol/L caffeine (Figure 5B). The addition of 0.3 mmol/L caffeine increased the calculated V_prop from 1.86 to 6.39 mm/s. rCa_T decreased by 43%, but both rCa_D and rCa_W increased by 40% and 115%, respectively.

View larger version (65K): [in this window] [in a new window]   Figure 5. Effects of caffeine on regional changes in [Ca2+]i in the trabecula of Figure 4. The regional [Ca2+]i calculated from the images by the IIC during the last electrically stimulated twitch and a TPC a few minutes after the recording of the PMT shown in Figure 4. In these 3-dimensional representations, the axes are the same as those in Figure 2. After the end of the clearly uniform stimulated Ca2+ transient, a small Ca2+ transient appeared to move from A (*) toward B (for explanation, see text). s indicates moment of electrical stimulation (experiment 970214). A, Before the addition of caffeine. The calculated Vprop was 1.85 mm/s. B, After the addition of 0.3 mmol/L caffeine. Vprop increased to 6.39 mm/s. rCaD increased from 161 to 226 nmol/L, and rCaW increased from 66 to 142 nmol/L whereas rCaT decreased from 616 to 351 nmol/L.

Figure 6 shows the effects of caffeine on global and regional changes in [Ca²⁺]_i and V_prop. The twitch Ca²⁺ transient declines to 25% of the control value whereas diastolic Ca²⁺ increased monotonically even at 1 mmol/L caffeine. This observation would be expected if caffeine eliminates contribution of the SR to the twitch, leaving only Ca²⁺ transport across the sarcolemma to supply and remove Ca²⁺. Ca²⁺ waves and aftercontractions increased and accelerated with caffeine up to 0.3 mmol/L, but they always disappeared after the addition of 1.0 mmol/L caffeine (n=3). Thus, we measured gCa_T, gCa_D, and gCa_W at caffeine concentrations of 0, 0.1, 0.3, and 0.5 mmol/L (n=7, 3, 7, and 4, respectively) and measured gCa_T at 1.0 mmol/L caffeine (n=3). At these concentrations, gCa_T decreased and gCa_D increased; gCa_W increased at 0.1 mmol/L caffeine but then decreased at 0.5 mmol/L caffeine. Measurement of regional [Ca²⁺]_i changes in 2 of 7 muscles tested at 0.3 mmol/L caffeine and in all 4 muscles tested at 0.5 mmol/L caffeine became inaccurate, because V_prop became too fast to be calculated from video frames obtained at 30 per second. Thus, we calculated rCa_T, rCa_D, rCa_W, and V_prop in control, 0.1 mmol/L, and 0.3 mmol/L caffeine (n=7, 3, and 5, respectively). At these concentrations, rCa_T decreased significantly whereas V_prop increased in the presence of caffeine; rCa_W increased at 0.1 mmol/L caffeine.

View larger version (17K): [in this window] [in a new window]   Figure 6. Effects of caffeine on global () and regional (•) changes in [Ca2+]i and Vprop. gCaD (P<0.05 [B]) and Vprop (P<0.01 [D]) increased significantly in the presence of caffeine whereas gCaT and rCaT decreased (P<0.0001 [A]). gCaW (P<0.005 [C]) and rCaW (P<0.05) increased at 0.1 mmol/L caffeine but then decreased in the presence of 0.5 mmol/L caffeine.

F_T also decreased to 72.8±3.3% (P<0.005) and 62.0±3.5% (P<0.0005) of control F_T in the presence of 0.1 and 0.3 mmol/L caffeine whereas F_TPC increased to 147±11.2% (P<0.05) and 201±21.5% (P<0.01) of control F_TPC, respectively (unpaired t test with unequal variation). The data obtained in each muscle in the presence of the drug were compared, for this analysis, with the data from the same muscle in the drug-free state ([Ca²⁺]_o 2.1±0.2, temperature 21.8±0.2).

The data in Figure 6 suggest that caffeine decreases Ca_T while it increases Ca_W at 0.1 mmol/L. This would be consistent with the effect of caffeine to cause an increase of the probability of opening of the SR-Ca²⁺ channels, increasing Ca²⁺ leak from the SR.^{13 14 15 16 17} The expectation that Ca_W/Ca_T reflects the probability of opening of the SR-Ca²⁺ channels is indeed met by observation of the effect of caffeine. Figure 7A shows that caffeine (0.1 to 0.5 mmol/L) increased both global and regional Ca_W/Ca_T significantly. Changes in force development (F_TPC/F_T) in the same muscles in the presence of caffeine increased significantly with increased caffeine concentration (Figure 7B).

View larger version (11K): [in this window] [in a new window]   Figure 7. Effects of caffeine on global () and regional (•) CaW/CaT (A) and FTPC/FT (B). The global and regional CaW/CaT and FTPC/FT increased significantly (P<0.005) in the presence of caffeine.

Figure 8 shows the effects of caffeine on the relationship between V_prop and regional changes in [Ca²⁺]_i of 8 Ca²⁺ waves from 5 trabeculae. For reference, control data (Figure 3) in the absence of caffeine are reproduced as open circles (). In the presence of caffeine, there remains a reasonable relationship between V_prop and rCa_T (Figure 8A) and rCa_W (Figure 8C). However, if we assume that rCa_T reflects the SR Ca²⁺ content in both drug-free and drug-containing solutions, then we are struck by the observation that the small rCa_T in caffeine are associated with waves with higher V_prop (leftward shift only). This suggests to us that caffeine renders V_prop of Ca²⁺ waves more sensitive to SR Ca²⁺ content. It appears that in the presence of caffeine, Ca²⁺ waves (rCa_W) similar in size to control waves propagate faster (parallel shift upward only), again suggesting that the higher V_prop reflects an increased sensitivity to the SR Ca²⁺ content.

View larger version (24K): [in this window] [in a new window]   Figure 8. Effects of caffeine on the relationships between Vprop and regional changes in [Ca2+]i. In the presence of caffeine (0.1 and 0.3 mmol/L), the relationships between Vprop and rCaT (r=0.71 [A]) and rCaW (r=0.81 [C]) moved leftward and upward whereas the relationships between Vprop and rCaD (r=0.93 [B]) and rCaW/rCaT (r=0.74 [D]) showed little change. indicates in the absence of caffeine; •, in the presence of 0.1 mmol/L caffeine, and , in the presence of 0.3 mmol/L caffeine.

In contrast, caffeine has little effect on the relationships between V_prop and rCa_D (Figure 8B) or rCa_W/rCa_T (Figure 8D) seen in control, although an increase of V_prop correlated with increased rCa_W/rCa_T in the presence of caffeine. The relationship between V_prop and F_TPC/F_T is not altered by caffeine (data not shown). Clearly, Figure 8D shows that the rate of propagation increases in proportion to the increase in fractional Ca²⁺ release during a wave (reflected by rCa_W/rCa_T). Thus, caffeine increases fractional Ca²⁺ release (increased rCa_W/rCa_T) with a commensurate increase of V_prop.

As stated, we assumed that the probability of opening of the SR-Ca²⁺ channels would be reflected by the amount of Ca²⁺ released from the SR relative to the SR Ca²⁺ content (Ca_W/Ca_T). The observation that both the cellular Ca²⁺ loading (as reflected by rCa_T, rCa_D, and rCa_W) and caffeine^{13 14 15 16} increased rCa_W/rCa_T (Figure 9) is consistent with this prediction. In control drug-free conditions, increases in rCa_T, rCa_D, and rCa_W resulted in a concomitant increase in rCa_W/rCa_T. Caffeine increased rCa_W/rCa_T 2-fold (Figure 9B and 9C). Intriguingly, caffeine shifted the relationship between rCa_W/rCa_T and rCa_T more dramatically to the left (Figure 9A), suggesting a stronger dependence of Ca_W/Ca_T on the SR Ca²⁺ content than on Ca_D.

View larger version (19K): [in this window] [in a new window]   Figure 9. Relationships between rCaW/rCaT and regional changes in [Ca2+]i in the absence of caffeine () and after the addition of 0.1 mmol/L (•) and 0.3 mmol/L caffeine (). After the addition of caffeine (0.1 and 0.3 mmol/L), the relationships between Vprop and rCaT (A), rCaD (B), and rCaW (C) moved upward.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
To our knowledge, this is the first study to characterize the determinants of Ca²⁺ wave propagation by direct measurement of the spatial and temporal properties of Ca²⁺ changes in a cardiac multicellular preparation during TPCs. Figure 10 summarizes our observations suggesting that (1) both increased cytosolic Ca²⁺ (Ca_D) and SR Ca²⁺ loading (Ca_T) influence V_prop through the modulation of P_o of RyR (Ca_W/Ca_T) by facilitating CICR, (2) the diastolic [Ca²⁺]_i level (Ca_D) may determine V_prop by facilitating diffusion of Ca²⁺ ions, and (3) at constant cytosolic [Ca²⁺] and SR Ca²⁺ loading, caffeine accelerates V_prop by directly increasing the P_o of RyR (Ca_W/Ca_T).

View larger version (17K): [in this window] [in a new window]   Figure 10. The proposed scheme of events that lead to an acceleration of traveling of Ca2+ waves (and TPCs). Ca2+ loading of the cell increases the SR Ca2+ content and cytosolic Ca2+ level. Both parameters increase the Po of RyR. In addition, the increase of the diastolic [Ca2+]i will increase occupancy of Ca2+ ligands in the cell and increase the effective diffusion coefficient. These effects would decrease the time needed to release Ca2+ from adjacent SR and thus increase the velocity of propagation of the Ca2+ waves. The effect of caffeine can be explained by its direct action on the Po of the RyR (see text for further explanation).

Effect of SR Ca²⁺ Content, Cytosolic Ca²⁺, and Diastolic Ca²⁺ on V_prop
It is obvious that Ca_D only reflects the cytosolic Ca²⁺ in between the contractions and depends on extrusion of Ca²⁺ from the cytosol. Most of the Ca²⁺ (80%) during the twitch in rat cardiac muscle is provided by Ca²⁺ release from SR^{20 21 22 23 24} and leads to the [Ca²⁺]_i rise, Ca_T. Our assumption that Ca_T correlated with the SR Ca²⁺ content was further supported by the observation that the amplitude of the twitch is proportional to that of rapid cooling contractures (data not shown). So, Ca_T eliciting the twitch could be taken to reflect the SR Ca²⁺ content. This interpretation requires caution, because the last twitch of the train nearly always followed a Ca²⁺ wave, which may have caused nonuniformity of Ca²⁺ release during this twitch.²⁵ However, regional measurements of Ca_T showed that this effect was minimal in the range of Ca²⁺ loading, which we have used. We have assumed that Ca²⁺ ions during a Ca²⁺ wave are also released from the SR,^{25 26} since it has been shown that TPCs are abolished by agents that interfere with SR Ca²⁺ loading or release, such as ryanodine and caffeine⁷ (Figure 6). Thus, we assumed that Ca_T reflects the SR Ca²⁺ content available for release, and that Ca_W reflects the amount of Ca²⁺ released from the SR during the Ca²⁺ wave. It follows that the released fraction of the SR Ca²⁺ content during the wave (Ca_W/Ca_T) reflects the probability of opening of SR-Ca²⁺ release channel.

It has been reported that the P_o of cardiac RyR incorporated into planar phospholipid bilayers can be modulated by intraluminal SR Ca^{2+16 27 28 29 30} as well as by cytosolic Ca²⁺.^{31 32} These studies have shown that elevation of [Ca²⁺] on either the luminal or cytosolic side increases P_o of RyR. The relationships between rCa_W/rCa_T and rCa_T on the one hand (Figure 9A) and rCa_D on the other hand (Figure 9B and 9C) suggest that under drug-free conditions, SR Ca²⁺ content and cytosolic Ca²⁺ during a Ca²⁺ wave indeed determine the released fraction of Ca²⁺ from inside the SR, possibly by modulating the probability of opening of the SR-Ca²⁺ channels. If so, our observations are consistent with the features of RyR measured using phospholipid bilayers.^{16 27 28 29 30 31 32}

It follows from the tight correlations between V_prop and rCa_T, rCa_D, rCa_W, and rCa_W/rCa_T described in Figure 3 that V_prop is facilitated under conditions of a higher SR Ca²⁺ content, a higher diastolic [Ca²⁺]_i, and a larger amount of Ca²⁺ released from SR and possibly P_o. These findings were predicted by computer simulation of Ca²⁺ waves using a model of CICR and Ca²⁺ diffusion.⁸ These observations are conceptually summarized in Figure 10, assuming that Ca²⁺ waves (or TPCs) travel along trabeculae, owing to the combination of CICR and Ca²⁺ diffusion.^{1 4 5 6} The simplest explanation of the observations is that SR Ca²⁺ content and cytosolic Ca²⁺ increase P_o of RyR. The latter effect would increase fractional Ca²⁺ release from the SR during a Ca²⁺ wave (rCa_W/rCa_T), thus decreasing the time needed to release Ca²⁺ from adjacent release sites during propagation of the Ca²⁺ wave. We cannot prove from these data that the SR Ca²⁺ content influences P_o, as has been suggested by lipid bilayer studies,^{16 27 28 29 30} but our data are certainly consistent with this hypothesis.

An increase in diastolic Ca²⁺ decreases the buffering capacity for Ca^{2+33 34 35 36} because of the increased Ca²⁺ binding to ligand proteins, as shown in Figure 10. With reduced buffering capacity for Ca²⁺, Ca²⁺ will diffuse faster,³⁷ and Ca²⁺ release in adjacent SR will be "induced" earlier.

Effect of Caffeine
Caffeine has been reported to enhance the sensitivity of the myofilaments to Ca²⁺³⁸ and inhibit net Ca²⁺ uptake by the SR.^{39 40} Recently, it has been concluded from studies using cardiac RyR incorporated into planar lipid bilayers that caffeine can increase the sensitivity of RyR to Ca²⁺¹⁵ and increase P_o of RyR.^{13 14 16 28} The increase in P_o of RyR can enhance the release of Ca²⁺ from SR and result in an increase in [Ca²⁺]_i. On the other hand, the increase in P_o reduces SR Ca²⁺ content and eventually depletes the SR of Ca²⁺, depending on the concentration of caffeine. The observation that caffeine produced an increase in Ca_D and a decrease in Ca_T (or F_T) (Figure 6A and 6B) is consistent with this concept. With caffeine at 1.0 mmol/L, Ca²⁺ waves (and TPCs) disappeared, probably as a result of Ca²⁺ depletion of the SR. We assume that the decrease in Ca_T (and F_T) in the presence of caffeine is due to a net decrease in SR Ca²⁺ content available for release but not due to the preceding spontaneous Ca²⁺ transient (and aftercontraction) for the following reasons. First, gCa_T in the presence of 1.0 mmol/L caffeine was significantly smaller than that of 0.1 to 0.3 mmol/L caffeine (Figure 6A), although spontaneous Ca²⁺ transient (and aftercontractions) had already disappeared at 1.0 mmol/L caffeine. Second, in the presence of 0.1 and 0.3 mmol/L caffeine, the developed force triggered by the last stimulus of a train changed by <10% of that triggered by the first one of the train, which was not preceded by aftercontractions. Therefore, our observations are consistent with the effect of caffeine on RyR observed within lipid bilayers.^{13 14 15 16 29}

In the presence of caffeine, the relationships between V_prop and rCa_D (Figure 8B) and rCa_W/rCa_T (Figure 8D) were almost similar to control. This means that caffeine has little effect on the mechanisms that link rCa_D and rCa_W/rCa_T to V_prop. In contrast, the relationships between V_prop and rCa_T (Figure 8A) and rCa_W (Figure 8C) suggest that caffeine makes V_prop much more sensitive to the SR Ca²⁺ content. This means that caffeine has a substantial effect on the pathway(s) that couple(s) rCa_T or rCa_W to V_prop. As suggested in Figure 10, caffeine increases P_o of RyR similar to SR Ca²⁺ content and cytosolic Ca²⁺. The latter effect of caffeine would increase fractional Ca²⁺ release from the SR during a Ca²⁺ wave (rCa_W/rCa_T), as shown in Figures 7 and 9, and would decrease the time needed to release Ca²⁺ from adjacent release sites during propagation of the Ca²⁺ wave. Hence, for any level of SR Ca²⁺ loading, V_prop would increase, owing to caffeine (Figure 8A). V_prop would also increase, even if the amplitude of the Ca²⁺ propagating transient would be constant, as is shown by Figure 8C.

Limitations of the Study
It is well known that the unitary SR Ca²⁺ release event in both cardiac cells and muscle consists of Ca²⁺ sparks.⁴¹ Ca²⁺ sparks have been described to underlie propagating Ca²⁺ waves^{41 42} at low levels of cellular loading (Ca_o 50% of the EC₅₀). Such Ca²⁺ waves travel a distance of only a few sarcomeres.^{41 42} Although it is tempting to assume that the microscopic waves observed in that study could consist of an avalanche of propagating Ca²⁺ sparks, our methods do not permit statements to this effect for the following reasons. First, we used fura-2, which does not exhibit a high enough photon efficiency to permit visualization of sparks. Second, the use of conventional microscopy precludes evaluation of events, which occur at a submicron scale. Hence, the fundamental question whether Ca²⁺ release by RyR (Ca²⁺ sparks) together with diffusion of Ca²⁺ from terminal cisterna to terminal cisterna (2 µm) causes Ca²⁺ waves as observed in the present study has to await exploration using probes such as confocal microscopy. This approach is important because theoretical modeling of propagation of Ca²⁺ waves has put the challenging constraint on the model that Ca²⁺ release from the SR has to occur in a fraction of a millisecond⁸ to permit V_prop to reach values of several millimeters per second. Even though it has been shown that opening of RyR exhibits rapid kinetics,⁴³ the capacity of the channel to Ca²⁺ release on a submillisecond timescale remains to be proven.

	Acknowledgments

 
This work was supported by grants from the Alberta Heart and Stroke Foundation and a NATO Collaborative Research Grant. Dr ter Keurs is a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). Dr Miura holds a postdoctoral research fellowship from Merck Frosst Canada Inc.

Received March 9, 1999; accepted April 19, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Mulder BJM, de Tombe PP, ter Keurs HEDJ. Spontaneous and propagated contractions in rat cardiac trabeculae. J Gen Physiol. 1989;93:943–961.[Abstract/Free Full Text]

2. Daniels MCG, ter Keurs HEDJ. Spontaneous contractions in rat cardiac trabeculae. Trigger mechanism and propagation velocity. J Gen Physiol. 1990;95:1123–1137.[Abstract/Free Full Text]

3. Daniels MCG, Kieser T, ter Keurs HEDJ. Triggered propagated contractions in human atrial trabeculae. Cardiovasc Res. 1993;27:1831–1835.[Abstract/Free Full Text]

4. Miura M, Boyden PA, ter Keurs HEDJ. Ca²⁺ waves during triggered propagated contractions in intact trabeculae. Am J Physiol. 1998;274:H266–H276.[Abstract/Free Full Text]

5. Daniels MCG, Fedida D, Lamont C, ter Keurs HEDJ. Role of the sarcolemma in triggered propagated contractions in rat cardiac trabeculae. Circ Res. 1991;68:1408–1421.[Abstract/Free Full Text]

6. Zhang YM, Miura M, ter Keurs HEDJ. Triggered propagated contractions in rat cardiac trabeculae. Inhibition by octanol and heptanol. Circ Res. 1996;79:1077–1085.[Abstract/Free Full Text]

7. Daniels MCG, ter Keurs HEDJ. Effects of caffeine, ryanodine, Bay K 8644, and D-600 on propagated contractions in rat cardiac muscle. Biophys J. 1990;57:170a. Abstract.

8. Backx PH, de Tombe PP, van Deen JHK, Mulder BJM, ter Keurs HEDJ. A model of propagating calcium-induced calcium release mediated by calcium diffusion. J Gen Physiol. 1989;93:963–977.[Abstract/Free Full Text]

9. Paspa P, Vassalle M. Mechanism of caffeine-induced arrhythmias in canine cardiac Purkinje fibers. Am J Cardiol. 1984;53:313–319.[Medline] [Order article via Infotrieve]

10. Gennaro MD, Vassalle M. Relationship between caffeine effects and calcium in canine cardiac Purkinje fibers. Am J Physiol. 1985;249:H520–H533.

11. Aronson RS, Cranefield PF, Wit AL. The effects of caffeine and ryanodine on the electrical activity of the canine coronary sinus. J Physiol (Lond). 1985;368:593–610.[Abstract/Free Full Text]

12. Boutjdir M, El-Sherif N, Gough WB. Effects of caffeine and ryanodine on delayed afterdepolarizations and sustained rhythmic activity in 1-day-old myocardial infarction in the dog. Circulation. 1990;81:1393–1400.[Abstract/Free Full Text]

13. Rousseau E, Meissner G. Single cardiac sarcoplasmic reticulum Ca²⁺-release channel: activation by caffeine. Am J Physiol. 1989;256:H328–H333.[Abstract/Free Full Text]

14. Sitsapesan R, Williams AJ. Mechanisms of caffeine activation of single calcium-release channels of sheep cardiac sarcoplasmic reticulum. J Physiol (Lond). 1990;423:425–439.[Abstract/Free Full Text]

15. Meissner G, Rios E, Tripathy A, Pasek DA. Regulation of skeletal muscle Ca²⁺ release channel (ryanodine receptor) by Ca²⁺ and monovalent cations and anions. J Biol Chem. 1997;272:1628–1638.[Abstract/Free Full Text]

16. Xu L, Meissner G. Regulation of cardiac muscle Ca²⁺ release channel by sarcoplasmic reticulum luminal Ca²⁺. Biophys J. 1998;75:2302–2312.[Medline] [Order article via Infotrieve]

17. ter Keurs HEDJ, Rijnsburger WH, van Heuningen R, Nagelsmit MJ. Tension development and sarcomere length in rat cardiac trabeculae. Evidence of length-dependent activation. Circ Res. 1980;46:703–714.[Free Full Text]

18. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450.[Abstract/Free Full Text]

19. Backx PH, ter Keurs HEDJ. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993;264:H1098–H1110.[Abstract/Free Full Text]

20. Schouten VJ, van Deen JK, de Tombe P, Verveen AA. Force–interval relationship in heart muscle of mammals. A calcium compartment model. Biophys J. 1987;51:13–26.[Medline] [Order article via Infotrieve]

21. Wier WG. Cytoplasmic [Ca²⁺] in mammalian ventricle: dynamic control by cellular process. Annu Rev Physiol. 1990;52:467–485.[Medline] [Order article via Infotrieve]

22. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Dordrecht, the Netherlands: Kluwer Academic Publishers; 1991.

23. Han S, Schiefer A, Isenberg G. Ca²⁺ load of guinea-pig ventricular myocytes determines efficacy of brief Ca²⁺ currents as trigger for Ca²⁺ release. J Physiol (Lond). 1994;480:3:411–421.

24. Ginsburg KS, Weber CR, Bers DM. Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes. Effects of thapsigargin and isoproterenol. J Gen Physiol. 1998;111:491–504.[Abstract/Free Full Text]

25. Lakatta EG. Functional implications of spontaneous sarcoplasmic reticulum Ca²⁺ release in the heart. Cardiovasc Res. 1992;26:193–214.[Free Full Text]

26. Miura M, Ishide N, Oda H, Sakurai M, Shinozaki T, Takishima T. Spatial features of calcium transients during early and delayed afterdepolarizations. Am J Physiol. 1993;265:H439–H444.[Abstract/Free Full Text]

27. Sitsapesan R, Williams AJ. Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by luminal Ca²⁺. J Membr Biol. 1994;137:215–226.[Medline] [Order article via Infotrieve]

28. Lukyanenko V, Györke I, Györke S. Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes. Pflügers Arch. 1996;432:1047–1054.

29. Ondrias K, Brillantes A-MB, Scott A, Ehrlich BE, Marks AR. Single channel properties and calcium conductance of the cloned expressed ryanodine receptor/calcium-release channel. Soc Gen Physiol Ser. 1996;51:29–45.[Medline] [Order article via Infotrieve]

30. Chen SRW, Li X, Ebisawa K, Zhang L. Functional characterization of the recombinant type 3 Ca²⁺ release channel (ryanodine receptor) expressed in HEK293 cells. J Biol Chem. 1997;272:24234–24246.[Abstract/Free Full Text]

31. Ashley RH, Williams AJ. Divalent cation activation and inhibition of single calcium release channels from sheep cardiac sarcoplasmic reticulum. J Gen Physiol. 1990;95:981–1005.[Abstract/Free Full Text]

32. Györke S, Vélez P, Suárez-Isla B, Fill M. Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca²⁺. Biophys J. 1994;66:1879–1886.[Medline] [Order article via Infotrieve]

33. Pierce GN, Philipson KD, Langer GA. Passive calcium-buffering capacity of a rabbit ventricular homogenate preparation. Am J Physiol. 1985;249:C248–C255.[Abstract/Free Full Text]

34. Isenberg G, Wendt-Gallitelli M-F. Binding of calcium to myoplasmic buffers contributes to the frequency-dependent inotropy in heart ventricular cells. Basic Res Cardiol. 1992;87:411–417.[Medline] [Order article via Infotrieve]

35. Hove-Madsen L, Bers DM. Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes. Am J Physiol. 1993;264:C677–C686.[Abstract/Free Full Text]

36. Berlin JR, Bassani JWM, Bers DM. Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. Biophys J. 1994;67:1775–1787.[Medline] [Order article via Infotrieve]

37. Phillies GDT. Effects of intermacromolecular interactions on diffusion: I, two component solutions. J Chem Physiol. 1974;60:976–982.

38. Eisner DA, Valdeolmillos M. The mechanism of the increase of tonic tension produced by caffeine in sheep cardiac Purkinje fibres. J Physiol (Lond). 1985;364:313–326.[Abstract/Free Full Text]

39. Niedergerke R, Page S. Analysis of caffeine action in single trabeculae of the frog heart. Proc R Soc Lond B Biol Sci. 1981;213:303–324.[Medline] [Order article via Infotrieve]

40. Hess P, Wier WG. Excitation-contraction coupling in cardiac Purkinje fibers. Effects of caffeine on the intracellular [Ca²⁺] transient, membrane currents, and contraction. J Gen Physiol. 1984;83:417–433.[Abstract/Free Full Text]

41. Wier WG, ter Keurs HEDJ, Marbán E, Gao WD, Balke CW. Ca²⁺ `sparks' and waves in intact ventricular muscle resolved by confocal imaging. Circ Res. 1997;81:462–469.[Abstract/Free Full Text]

42. Cheng H, Lederer MR, Lederer WJ, Cannell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol. 1996;270:C148–C159.[Abstract/Free Full Text]

43. Valdivia HH, Kaplan JH, Ellis-Davies GCR, Lederer WJ. Rapid adaptation of cardiac ryanodine receptors: modulation by Mg²⁺ and phosphorylation. Science. 1995;267:1997–2000.[Abstract/Free Full Text]

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