HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 543/3/859    most recent 2002.021519v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loughrey, C. M. Articles by Smith, G. L. Search for Related Content PubMed PubMed Citation Articles by Loughrey, C. M. Articles by Smith, G. L.

Journal of Physiology (2002), 543.3, pp. 859-870
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.021519

The relationship between intracellular [Ca²⁺] and Ca²⁺ wave characteristics in permeabilised cardiomyocytes from the rabbit

C. M. Loughrey *, K. E. MacEachern *, P. Neary † and G. L. Smith ‡

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

Spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release and propagated intracellular Ca²⁺ waves are a consequence of cellular Ca²⁺ overload in cardiomyocytes. We examined the relationship between average intracellular [Ca²⁺] and Ca²⁺ wave characteristics. The amplitude, time course and propagation velocity of Ca²⁺ waves were measured using line-scan confocal imaging of -escin-permeabilised cardiomyocytes perfused with 10 µM Fluo-3 or Fluo-5F. Spontaneous Ca²⁺ waves were evident at cellular [Ca²⁺] > 200 nM. Peak [Ca²⁺] during a wave was 2.0-2.2 µM; the minimum [Ca²⁺] between waves was 120-160 nM; wave frequency was ~0.1 Hz. Raising mean cellular [Ca²⁺] caused increases in all three parameters, particularly Ca²⁺ wave frequency. Increases in the rate of SR Ca²⁺ release and Ca²⁺ uptake were observed at higher cellular [Ca²⁺], indicating calcium-sensitive regulation of these processes. At extracellular [Ca²⁺] > 2 µM, the mean [Ca²⁺] inside the permeabilised cell did not increase above 2 µM. This extracellular-intracellular Ca²⁺ gradient could be maintained for periods of up to 5 min before the cardiomyocyte developed a sustained and irreversible hypercontraction. Inclusion of mitochondrial inhibitors (2 µM carbonyl cyanide m-chlorophenylhydrazone and 2 µM oligomycin) while perfusing with > 2 µM Ca²⁺ abolished the extracellular-intracellular Ca²⁺ gradient through the generation of Ca²⁺ waves with a higher peak [Ca²⁺] compared to control conditions. Under these conditions, cardiomyocytes rapidly (< 2 min) developed a sustained and irreversible contraction. These results suggest that mitochondrial Ca²⁺ uptake acts to delay an increase in [Ca²⁺] by blunting the peak of the Ca²⁺ wave.

(Received 28 March 2002; accepted after revision 11 June 2002; first published online 12 July 2002)
Corresponding author G. L. Smith: West Medical Building, University of Glasgow, UK. Email: g.smith{at}bio.gla.ac.uk

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Intracellular Ca²⁺ waves arising from Ca²⁺ release from internal stores have been observed in a range of mammalian cell types. In many cases the Ca²⁺ waves constitute a physiological signal that triggers downstream cellular processes (Marchant & Parker, 2000). In cardiomyocytes, Ca²⁺ waves arise from a local spontaneous release of Ca²⁺ from the sarcoplasmic reticulum (SR) that under certain circumstances propagate throughout the cell (Ishide et al. 1989; Takamatsu & Wier, 1990; Williams et al. 1990; Trafford et al. 1995a; Cheng et al. 1996; Lukyanenko et al. 1998). Spontaneous Ca²⁺ waves occur during the diastolic period and have been linked to the generation of arrhythmic electrical activity (Miura et al. 1993). Previous studies have suggested that spontaneous Ca²⁺waves are beneficial in: (1) minimising diastolic tone (Stern et al. 1988) and (2) stimulating Ca²⁺ extrusion from the cell (Diaz et al. 1997a). These studies have suggested that increasing cellular [Ca²⁺] leads to Ca²⁺ waves of uniform amplitude but increasing frequency. In contrast, a more recent study monitoring Ca²⁺ waves in intact rat myocardium using rapid confocal imaging identified three distinct types of Ca²⁺ waves differing in frequency, time course and amplitude depending on the cellular Ca²⁺ load (Kaneko et al. 2000). Apart from these two studies (Diaz et al. 1997a; Kaneko et al. 2000), no other has addressed the specific issue of Ca²⁺ wave characteristics at varying cellular Ca²⁺ load. Furthermore, these previous studies arrive at quite different conclusions. The present study was designed to address this dichotomy by examining the characteristics of spontaneous Ca²⁺ waves under standardised intracellular conditions using permeabilised cardiomyocytes.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Cell isolation and permeabilisation

New Zealand White rabbits (2-2.5 kg) were given an intravenous injection of 500 U heparin together with an overdose of sodium pentobarbitone (100 mg kg^-1). The hearts were rapidly excised, weighed and cannulated onto a Langendorff perfusion column via the aorta. The hearts were perfused retrogradely at a perfusion rate of 25 ml min^-1 (37°C), initially with Krebs-Henseliet solution containing 0.75 mM CaCl₂ for 3 min, and then with a nominally calcium-free Krebs-Henseliet solution containing 0.1 mM EGTA for a further 4 min. Thereafter, the hearts were perfused with recirculated Krebs-Henseliet solution supplemented with 1.4 mg ml^-1 collagenase (type 1, Worthington Chemical), 0.1 mg ml^-1 protease (type XIV, Sigma Chemical) and 80 µM CaCl₂ for 10-17 min. The atria and right ventricle were dissected free and discarded. The left ventricular free wall was removed from the column, cut into chunks and incubated (37 °C) sequentially for 5 min in 3 ml recirculated enzyme solution plus 1 ml of Krebs-Henseliet solution containing 80 µM CaCl₂ and 4 % bovine serum albumin (BSA, fraction V, Sigma). The cell suspensions obtained at the end of each incubation period were filtered (250 µm mesh) onto Krebs-Henseliet solution containing 0.1 mM CaCl₂ and 1.5 % BSA, and centrifuged at 5 g for 1 min. The pellet of cells was resuspended in modified Krebs-Henseliet solution buffered with 1 mM EGTA at a concentration of approximately 10⁴ cells ml^-1 until use. The cells were allowed to settle onto the coverslip at the base of a small bath. -Escin (Sigma) was added from a freshly prepared stock solution to the cell suspension to give a final concentration of 0.1 mg ml^-1 for 1-2 min and the -escin was subsequently removed by perfusion with a mock intracellular solution (see below).

Solutions

Permeabilised cells were perfused with a mock intracellular solution with the following composition (mM): 100 KCl, 5 Na₂ATP, 10 disodium creatine phosphate, 5.5 MgCl₂, 25 Hepes, 0.05 K₂EGTA, pH 7.0 (20-21 °C). The [Ca²⁺] in the perfusing solution was varied by the addition of known amounts of 1 M CaCl₂ stock solution (BDH). The fluorescent Ca²⁺ indicators Fluo-3 or Fluo-5F (Molecular Probes) were added to the solution to give a nominal final concentration of 10 µM. All other chemicals were supplied by Sigma (UK).

Data recording and analysis

Confocal line-scan images were recorded using a BioRad Radiance 2000 confocal system. Fluo-3 (or Fluo-5F) in the perfusing solution was excited at 488 nm and measured above 515 nm using the epifluorescence optics of a Nikon Eclipse inverted microscope with a Fluor 60 water objective lens (NA 1.2). The iris diameter was set at 1.9, providing an axial (z) resolution of about 0.9 µm and x-y resolution of about 0.5 µm based on full-width, half-maximal amplitude measurements of images of 0.1 µm fluorescent beads (Molecular Probes). Data was acquired in line-scan mode at 2 ms line^-1; the pixel dimension was 0.3 µm (512 pixels scan^-1; zoom = 1.4). The scanning laser line was oriented parallel with the long axis of the cell and placed approximately equidistant between the outer edge of the cell and the nucleus/nuclei, to ensure the nuclear area was not included in the scan line. As illustrated in Fig. 2A, the LaserScan (BioRad) software saved the data as a series of image files, each containing 30 000 line scans (i.e. 1 min of continuous recording). An experimental record typically comprised four to five line-scan image files; these were reviewed off-line and a single intracellular region (20 pixels wide) was selected on the basis of the earliest events in the majority of Ca²⁺ waves (as indicated in Fig. 2B). This ensured that any movement artefact following the increase in [Ca²⁺] did not affect the estimation of peak [Ca²⁺]. The wave front was taken to be the mid-point of the upstroke. Using a 50 pixel region flanking the 20 pixel region, the gradient was used to calculate wave velocity. An average velocity (5-10 waves) was calculated for each cell.

Calibration of fluorescence indicators

The Ca²⁺ sensitivity of the fluorescent dyes was measured using a series of calcium-buffered solutions based on a mock intracellular solution containing 10 mM EGTA. The equilibrium concentrations of metal ions in the calibration solutions were calculated using a computer program with known affinity constants for H⁺, Ca²⁺ and Mg²⁺ for EGTA (Smith & Miller, 1985) and for ATP and creatine phosphate (Fabiato & Fabiato, 1979). Corrections for ionic strength, details of pH measurement, allowance for EGTA purity and the principles of the calculations are detailed elsewhere (Miller & Smith, 1984). Under these conditions the apparent affinity constant of Fluo-3 for Ca²⁺ was 558 ± 14 nM (n = 6); that of Fluo-5F was 1035 ± 16 nM (n = 4). To establish the behaviour of the dyes in the permeabilised myocytes, simultaneous fluorescence measurements were made from a 6 µm (x) 0.5 µm (y) 0.9 µm (z) volume (20 pixels) within a myocyte and in the solution adjacent to the myocyte. In these calibration experiments, SR Ca²⁺ uptake was inhibited by prior treatment with thapsigargin (10 µM, 20 min). Figure 1A (i) and (ii) shows that a higher fluorescence was recorded from within the cell compared to an equivalent region in free solution, suggesting either: (1) a higher Fluo-3 concentration within the cell or (2) differing Ca²⁺ affinity of the dye within the cytosolic space. Rapidly increasing the [Ca²⁺] perfusing the cell from 150 nM (50 µM EGTA, 10 µM Fluo-3) to 1.12 µM (10 mM EGTA, 10 µM Fluo-3) caused proportionate changes in fluorescence in both regions, as implied by the almost constant ratio of intracellular/extracellular fluorescence (F_IC/F_EC; Fig. 1A (ii)). Similarly, reducing [Ca²⁺] in the perfusing solution to < 1 nM (10 mM EGTA) caused a proportionate decrease in fluorescence but no change in F_IC/F_EC. Using the fluorescence at 1.12 µM and < 1 nM as calibration values, the [Ca²⁺] inside the cell and in the extracellular solution can be calculated by assuming the Fluo-3 Ca²⁺ affinity is the same in both compartments. This yields a value that indicates an equivalent [Ca²⁺] (~150 nM) in both compartments prior to the solution change (Fig. 1A (iii)). In a number of calibration experiments F_IC/F_EC was measured in solutions containing 150 nM Ca²⁺ and on rapid application of solutions containing 375 nM or 1.12 µM Ca²⁺. The F_IC/F_EC value in 375 nM Ca²⁺ was 0.99 ± 0.4 (n = 8) of that in 150 nM Ca²⁺, while the F_IC/F_EC value in 1.12 µM Ca²⁺ was 1.01 ± 0.06 (n = 4) of that in 150 nM Ca²⁺. This constancy of F_IC/F_EC values over a wide range of [Ca²⁺] indicates that Fluo-3 inside the cardiomyocyte has a Ca²⁺ affinity indistinguishable from that of free solution. While the F_IC/F_EC value was constant over a range of [Ca²⁺] in any one preparation, the absolute value varied between cells; on average, the fluorescence within a cardiac myocyte was 140 ± 9 % (n = 14) of that in free solution. Similar behaviour was observed for the lower affinity dye Fluo-5F. Figure 1B (i) shows the fluorescence signals from a cardiomyocyte (in the absence of thapsigargin) perfused with a [Ca²⁺] that was subthreshold for Ca²⁺ waves. Calibration of both intracellular and extracellular signals was performed by perfusing with 375 nM Ca²⁺ in the presence of 10 mM EGTA to clamp the [Ca²⁺] in both compartments. Minimum fluorescence values were obtained by perfusion with a [Ca²⁺] of < 1 nM (10 mM total EGTA). Thus the [Ca²⁺] could be calculated assuming an identical affinity of Ca²⁺ for the dye in both intracellular and extracellular compartments (Fig. 1B (ii)). With 50 µM EGTA present, the [Ca²⁺] was identical in both regions, additional noise in the intracellular signal being attributable to local release of Ca²⁺ from the SR (Ca²⁺ sparks).

		View larger version [in this window] [in a new window]
Figure 1. Line-scan epifluorescence imaging of cardiomyocytes A (i), line-scan epifluorescence image of a single permeabilised cardiomyocyte after incubation with thapsigargin (10 µM for 10 min). The brighter central region is due to the presence of Fluo-3 in the cell; dimmer flanking signals are from the Fluo-3 in the perfusing solutions. The calculated [Ca2+] in the perfusion solution is shown above the trace; control solutions contained a total concentration of EGTA ([EGTA]) and Fluo-3 of 50 µM and 10 µM, respectively. [Ca2+] marked by * indicates solutions containing a total [EGTA] of 10 mM. A (ii), mean fluorescence signal of a 20 pixel region from the intracellular (IC) and extracellular compartments (EC); these regions are indicated by dashed lines in A (i). The ratio of IC fluorescence/EC fluorescence (FIC/FEC) is plotted above (see right-hand axis). A (iii), IC and EC signals converted to [Ca2+] on the basis of the signals in 1.12 µM and <1 nM Ca2+. B (i), mean fluorescence signals from within a cardiomyocyte (IC, grey trace) and a similar region outside the cell (EC, black trace) on raising the external [Ca2+] from 40 nM to 170 nM (in 50 µM EGTA, 10 µM Fluo-3). At the points indicated above the trace, the solution was switched to one containing 375 nM Ca2+, then < 1 nM Ca2+ (10 mM EGTA). B (ii), IC and EC signals converted to [Ca2+] on the basis of the signals in 375 nM and < 1 nM Ca2+.

Statistics

The relationship between extracellular [Ca²⁺] and Ca²⁺ wave parameters was investigated using linear regression. The best-fit gradients are expressed with the asymptotic standard error (a measure of the uncertainty of parameter estimates). Where appropriate, curve fits were compared using an F-test, based on the sum-of-squares difference between the fitted curve and data values. Student's t test or ANOVA, and Tukey-Kramer Multiple comparisons test were used where appropriate.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Measurement of Ca²⁺ waves in permeabilised single myocytes

Figure 2A shows a section of a line-scan image recorded from a single permeabilised cardiac myocyte exposed to a solution containing 260 nM Ca²⁺. Due to the presence of Fluo-3 (10 µM) in the perfusing solution, the fluorescence signal recorded from the complete line scan contains signals from both intracellular and extracellular compartments. Intracellular and extracellular areas were distinguished by the higher fluorescence from cardiomyocyte under quiescent conditions (see Fig. 1) and the presence of Ca²⁺ waves. As described above, the complete series of line scans recorded in the protocol (Fig. 2B) was analysed by selecting a 20 pixel region from a site inside a permeabilised cardiomyocyte and an extracellular site ~6 µm away from the cardiomyocyte (the fluorescence signals are shown in Fig. 2C (i)). Increasing [Ca²⁺] in the perfusing solution from 40 to 260 nM caused an increase in Fluo-3 fluorescence at both sites. The raised intracellular [Ca²⁺] initiated a series of Ca²⁺ waves recorded as transient increases fluorescence at regular intervals at the intracellular site. The delay between the increased cytosolic [Ca²⁺] and the first Ca²⁺ wave reflects a period of net SR Ca²⁺ uptake. The frequency of Ca²⁺ waves quickly (within 1-2 cycles) reached a steady state, suggesting a period of 10-15 s before a new steady SR Ca²⁺ content is achieved. The average of the last five Ca²⁺ waves is shown in the adjacent panel (Fig. 2C (ii)). At the point indicated, the perfusing solution was changed to one containing 375 nM free [Ca²⁺] (buffered by 10 mM EGTA). The high concentration of EGTA effectively clamped the [Ca²⁺], buffering Ca²⁺ release and uptake by the SR. The [Ca²⁺] was subsequently reduced to < 1 nM by perfusion with a solution containing 10 mM EGTA (no added Ca²⁺). Figure 2D shows the trace converted to [Ca²⁺]. There appears to be a large variation in the peak [Ca²⁺] of the Ca²⁺ wave. Some of this variation could be due to the noise associated with the photomultiplier signal combined with the low Ca²⁺ sensitivity of the Fluo-3 fluorescence signal at high [Ca²⁺]. A more reliable estimate of peak [Ca²⁺] can be obtained by converting the averaged fluorescence signal shown in Fig. 2C (ii) to [Ca²⁺] (Fig. 2D (ii)).

		View larger version [in this window] [in a new window]
Figure 2. Analysis of confocal line-scan images of Ca2+ waves A, line-scan image of Ca2+ waves measured on perfusion of a permeabilised cardiomyocyte with an extracellular [Ca2+] of ~260 nM Ca2+. The sections marked with the dashed lines are 20 pixel wide regions within the cell (IC) and in the extracellular solution (EC). B, a longer record of line-scan images from the permeabilised cardiomyocyte while raising the [Ca2+] in the perfusate from 40 nM to 260 nM (50 µM EGTA, 10 µM Fluo-3). The section marked by an asterisk is the region corresponding to the expanded trace in A. Subsequently, the cardiomyocyte was perfused with 375 nM and < 1 nM Ca2+ (* indicates total [EGTA] = 10 mM, 10 µM Fluo-3), as indicated above the trace. C (i), superimposed IC (grey trace) and EC fluorescence signals (black trace) from the line-scan shown in B. C (ii), average fluorescence signal of the last five Ca2+ waves (IC, grey trace) and the corresponding EC signal (black trace); the period averaged is indicated by the open box. D (i), IC and EC [Ca2+] signals calculated from the fluorescence signals obtained in 375 nM and < 1 nM Ca2+. D (ii), the averaged IC (grey) and EC (black) [Ca2+] calculated from the averaged fluorescence signals during the period indicated by the open box. E (i), higher-resolution IC and EC [Ca2+] signals. E (ii), averaged IC (grey) and EC (black) [Ca2+] signals displayed at a higher resolution during the period indicated by the open box. F (i), averaged Ca2+ signals over 10 s periods from IC (grey) and EC (black) signals. F (ii) mean IC and EC [Ca2+] signals during the period indicated by the open box.

In Fig. 2E the minimum intracellular [Ca²⁺] signal between Ca²⁺ waves and the extracellular signal have been plotted on a different scale to show the relationship more clearly. When [Ca²⁺] in the perfusing solution was increased the [Ca²⁺] inside the cell was consistently lower than outside, suggesting the occurrence of active Ca²⁺ uptake by the cell. Ca²⁺ waves began approximately 15 s after an increase in extracellular [Ca²⁺] with a frequency about 0.2 Hz. The minimum inter-wave [Ca²⁺] was maintained at approximately 140 nM. To assess the mean [Ca²⁺] within the cell, average [Ca²⁺] over a series of 10 s periods was calculated for both intracellular and extracellular signals (Fig. 2F (i)). Despite the presence of large-amplitude waves, mean intracellular [Ca²⁺] over a 10 s period was comparable to that in the extracellular space (Fig. 2F (ii)). In several experiments, thapsigargin (10 µM) was applied after a period of spontaneous Ca²⁺ waves. The resulting rapid inhibition of the SR Ca²⁺ pump abolished Ca²⁺ waves and caused both intracellular and extracellular signals to become superimposed (results not shown). Thus, SR Ca²⁺ uptake and release results in a minimum interwave [Ca²⁺] that is considerably lower than the average value. This maintained concentration gradient was balanced by intermittent Ca²⁺ waves that reversed the concentration gradient and generated a net Ca²⁺ efflux from the cell.

Ca²⁺ wave characteristics measured at high cellular [Ca²⁺]

Elevating [Ca²⁺] in the extracellular solution led to Ca²⁺ waves arising more frequently (Fig. 3A, B and C). These signals were recorded using Fluo-5F to ensure adequate sensitivity at higher [Ca²⁺]. Each panel shows sections of records of early and late periods (separated by ~60 s) of a continuous 90 s exposure to high [Ca²⁺]. In Fig. 3A, stable Ca²⁺ wave activity was maintained with a mean extracellular and intracellular [Ca²⁺] of 1.3 µM. In Fig. 3B, in a separate cell, when the extracellular [Ca²⁺] was increased to > 2 µM, larger amplitude and more frequent Ca²⁺ waves occurred. We consistently observed that the mean intracellular and extracellular [Ca²⁺] did not equalize over the 90 s recording period. In the presence of an extracellular [Ca²⁺] of 2.12 ± 0.08 µM, mean intracellular [Ca²⁺] was 1.46 ± 0.06 µM (n = 4, P < 0.01). In a separate set of measurements, cardiomyocytes were exposed to an extracellular [Ca²⁺] > 3 µM. As shown in Fig. 3C, mean intracellular [Ca²⁺] remained at ~2 µM over the 60-90 s period of the recording. Furthermore, the characteristics of Ca²⁺ waves were not constant. Over the period of exposure (90 s) the time course of the Ca²⁺ waves slowed, peak [Ca²⁺] decreased and the waves were of irregular amplitude (see Fig. 3C). During short periods (90-120 s) at these high cellular [Ca²⁺], the myocyte retained a normal relaxed cell length. However, if the period was prolonged (> 2 min) cardiomyocytes developed an irreversible hypercontracture (i.e. did not relax when exposed to < 1 nM [Ca²⁺]). This phenomenon is illustrated in Fig. 4A. Marked cell shortening prevented the use of the calibration signals at 375 nM and < 1 nM to calculate the intracellular [Ca²⁺] inside the previously relaxed cell. However, the calibration protocol could be applied to regions of the shortened cell. As shown in Fig. 4B, intracellular [Ca²⁺] in a cardiomyocyte after a sustained hypercontracture indicated that the SR within the cell was capable of generating low-amplitude Ca²⁺ waves. Mean [Ca²⁺] measurements indicated equilibration between intracellular and extracellular [Ca²⁺] at these high cellular [Ca²⁺].

		View larger version [in this window] [in a new window]
Figure 3. Records of Ca2+ waves at different values of extracellular [Ca2+] Records of EC (black trace) and IC [Ca2+] (grey trace) derived from 20 pixel regions of line-scan images of cardiomyocytes perfused with solutions containing 50 µM EGTA, 10 µM Fluo-5F and the following [Ca2+]: A, 1.3 µM; B, 2.26 µM; C, 3.4 µM. The grey dashed line indicates the calculated mean intracellular [Ca2+].

		View larger version [in this window] [in a new window]
Figure 4. Effect of prolonged exposure to high extracellular [Ca2+] on Ca2+ waves in cardiomyocytes A, series of eight sequential line-scan images recorded from a single cardiomyocyte perfused with 2.6 µM Ca2+ (50 µM EGTA and 10 µM Fluo-5F). The gaps between images represent ~5 s breaks between acquisition periods. Images during perfusion with 375 nM Ca2+ (10 mM EGTA*) and 1 nM Ca2+ (10 mM EGTA*) are indicated above the images. B, EC (black) and IC (grey) [Ca2+] signals derived from line-scan records of a hypercontracted cardiomyocyte during exposure to 2.6 µM Ca2+ (50 µM EGTA, 10 µM Fluo-5F). The grey dashed line indicates the calculated mean intracellular [Ca2+].

Mitochondrial Ca²⁺ uptake affects Ca²⁺ wave characteristics

Agents that dissipate the mitochondrial membrane potential such as carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 µM), inhibit Ca²⁺ uptake by mitochondria. Under these conditions, the mitochondrial F1,F0 ATP synthase can work in reverse and break down ATP. This ATPase is inhibited by oligomycin (2 µM). Figure 5A shows line-scan images recorded from two cardiomyocytes exposed to a short (90 s) period of high (2.3 µM) Ca²⁺. As Figs 3B, 4A and 5A show, this [Ca²⁺] normally produced high-frequency waves that were stable over short periods. However, in the presence of mitochondrial inhibitors (CCCP and oligomycin), the cardiomyocyte initially produced high-frequency waves, but very quickly developed an irreversible hypercontracture. Signals recorded from cardiomyocytes exposed briefly (50-60 s) to ~2.3 µM Ca²⁺ and subsequent calibration solutions without developing a sustained hypercontracture are shown in Fig. 5B. Ca²⁺ waves observed during mitochondrial inhibition reached higher peak values than that seen under control conditions (compare Fig. 5B (i) and (ii)). Furthermore, the significant extracellular- intracellular [Ca²⁺] gradient observed under control conditions was absent after mitochondrial inhibition.

		View larger version [in this window] [in a new window]
Figure 5. Effect of mitochondrial inhibitors on Ca2+ waves at high extracellular [Ca2+] A (i) and (ii), sequential line-scan images recorded from permeabilised cardiomyocytes perfused with 2.3 µM Ca2+ (50 µM EGTA, 10 µM Fluo-5F) followed by perfusion with 375 nM* Ca2+ and 1 nM* Ca2+ (*10 mM [EGTA], 10 µM Fluo-5F). The gaps between images represent ~5 s breaks between acquisition periods. B, records of EC (black trace) and IC (grey trace) [Ca2+] signals derived from 20 pixel regions of line-scan images of a cardiomyocyte perfused with 2.3 µM Ca2+ (50 µM EGTA, 10 µM Fluo-5F) under control conditions (i) and including mitochondrial inhibitors (+ MI) carbonyl cyanide m-chlorophenylhydrazone (CCCP, 2 µM) and oligomycin (2 µM) (ii). The grey dashed lines represent the mean intracellular [Ca2+] over the period shown.

The effects of mitochondrial inhibitors on Ca²⁺ waves were studied in detail at two levels of cellular [Ca²⁺]: at ~400 nM, a level known to allow equilibration of [Ca²⁺] between the intracellular and extracellular compartments, and ~2.0-2.2 µM, which does not normally result in equilibration unless mitochondrial inhibitors are used (as shown in Fig. 5B). The mean values (± S.E.M.) of maximum [Ca²⁺], minimum [Ca²⁺] during a Ca²⁺ wave and wave frequency along with mean extracellular and intracellular values are given in Fig. 6A and B. At ~400 nM, mitochondrial inhibition had no effect on any of the Ca²⁺ wave characteristics studied (Fig. 6A). However, the mean data shown in Fig. 6B confirms that mitochondrial inhibition permits equilibration of Ca²⁺ between extracellular and intracellular compartments by increasing the maximum [Ca²⁺] reached during a Ca²⁺ wave. This suggests that mitochondrial Ca²⁺ uptake normally buffers the peak of the Ca²⁺ wave.

		View larger version [in this window] [in a new window]
Figure 6. Effect of mitochondrial inhibitors on [Ca2+] and Ca2+ wave parameters Averages (± S.E.M.) of Ca2+ signals recorded from line-scan images of cardiomyocytes after perfusion with ~0.4 µM (A) and ~2.1 µM Ca2+ (B) in the absence (Control) and presence (+MI) of 2 µM CCCP, 2 µM oligomycin (n = 4 in each group). For each parameter (i.e. mean extracellular [Ca2+] (here denoted [Ca2+]ec), mean intracellular [Ca2+] (here denoted [Ca2+]ic), minimum [Ca2+], maximum [Ca2+] and wave frequency) significant differences between control and the + MI conditions are indicated (B). Significant differences existed between all of the individual Ca2+ wave characteristics measured at ~0.4 µM and ~2.3 µM [Ca2+](P < 0.001).

Relationship between extracellular and intracellular [Ca²⁺]

Figure 7 plots results from 15 cells using a range of extracellular [Ca²⁺]. No Ca²⁺ waves were recorded in cardiomyocytes exposed to < 200 nM over a 90 s period, but raising mean [Ca²⁺] to approximately 240 nM caused low-frequency Ca²⁺ waves with the minimum [Ca²⁺] between the waves reaching ~140 nM. Using extracellular [Ca²⁺] > 200 nM and < 2 µM, Ca²⁺ waves with nearly uniform amplitude and frequency occurred. At extracellular [Ca²⁺] > 3 µM, Ca²⁺ wave characteristics were not stable. A common feature in experiments using high (> 2 µM) Ca²⁺ was the maintained [Ca²⁺] difference between intracellular and extracellular compartments over the 60-90 s of recording. To examine the relationship between peak [Ca²⁺] or minimum [Ca²⁺] and the extracellular [Ca²⁺], the values obtained at between 200 and 1000 nM Ca²⁺ are plotted on a linear scale in panels (ii) and (iii). The straight lines represent the best-fit linear correlation. A similar format is used in Fig. 7B to indicate the relationship between Ca²⁺ wave frequency and velocity at a range of extracellular [Ca²⁺]. Frequency was highly dependent on intracellular [Ca²⁺], but no clear relationship existed for wave velocity (Fig. 7B (ii) and (iii)).

		View larger version [in this window] [in a new window]
Figure 7. Relationship between various mean intracellular [Ca2+] and Ca2+ wave parameters, and mean extracellular [Ca2+] A (i), a log-log plot of extracellular [Ca2+] vs mean intracellular [Ca2+] from 15 cardiomyocytes (Fluo-3, , Fluo-5F, ), maximum [Ca2+] (wave peak), (Fluo-3, ; Fluo-5F, ), minimum [Ca2+] (Fluo-3, , Fluo-5F, ). The line indicates the unity relationship. A (ii), linear plot of extracellular [Ca2+] (300-900 nM range) vs maximum [Ca2+]. The continuous line is the best-fit linear correlation with gradient 0.77 ± 0.18 (P < 0.01). A (iii), linear plot of extracellular [Ca2+] (300-900 nM range) vs minimum [Ca2+] during a Ca2+ wave. The continuous line is the best-fit linear correlation, and has a gradient of 0.35 ± 0.03 (P < 0.001). B (i), a log-log plot of extracellular [Ca2+] vs. frequency of Ca2+ waves () and wave velocity (). B (ii), linear plot of extracellular [Ca2+] (300-900 nM range) vs Ca2+ wave frequency. The continuous line is the best-fit linear correlation, with gradient 5.5 105 ± 1 105 (waves s-1 M-1; P < 0.01). B (iii), linear plot of extracellular [Ca2+] (300-900 nM range) vs wave velocity (no correlation). C (i), semilog plot of extracellular [Ca2+] vs rate of rise of Ca2+ wave measured at 1 µM Ca2+. The line shows the best-fit linear correlation. C (ii), semilog plot of the rate of decline of the Ca2+ wave measured at various values of extracellular [Ca2+]. The line shows the best-fit linear correlation.

Time course of the Ca²⁺ wave is influenced by the mean cellular [Ca²⁺]

Figure 7C (i) and (ii) indicates the relationship between the rate of rise and rate of decline of the averaged Ca²⁺ wave measured (rates measured at 1 µM intracellular [Ca²⁺]). Both measurements show a weak but significant positive correlation, indicating that the rate of release of Ca²⁺ and rate of uptake are increased at higher extracellular [Ca²⁺]. Similar results were observed at 0.5 and 1.5 µM intracellular [Ca²⁺]. This indicates that the time course of Ca²⁺ waves is sensitive to the mean cellular [Ca²⁺] and, as a consequence, the duration of the Ca²⁺ wave decreases as mean cellular [Ca²⁺] is increased.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

In this study, the permeabilised cardiomyocyte preparation is used as a simplified system with which to study the characteristics of propagated Ca²⁺ waves. A simple set of measurements revealed that the fluorescence from Ca²⁺ indicators Fluo-3 and Fluo-5F was higher within permeabilised cardiac myocytes than in an equivalent extracellular volume. This may be explained by either: (1) indicator binding to structures within the permeabilised cell or (2) an interaction between the cytosol and indicator resulting in increased fluorescence. Regardless of the cause, measurements in this study indicated that the affinity of Ca²⁺ for the dye in the cytosol is not significantly different to that measured in free solution.

Changes in Ca²⁺ wave characteristics at a mean cellular [Ca²⁺] < 1.2 µM

This is the first study to report the relationship between Ca²⁺ wave characteristics and mean cellular [Ca²⁺] in single cardiac muscle cells. Spontaneous Ca²⁺ waves were recorded at a mean intracellular [Ca²⁺] of > 200 nM. Below this value no spontaneous release was evident, although individual Ca²⁺ sparks could be detected. Since the test period was only 90-120 s, it is conceivable that at 150-180 nM, propagated Ca²⁺ waves occurred at frequencies less that 1-2 min^-1 (~0.01 s^-1). However, this frequency is much lower than the value of 0.1 s^-1 expected at 150 nM Ca²⁺, based on a linear extrapolation of the relationship shown in Fig. 7B (ii). In a recent study of intact rat myocardium at room temperature (Kaneko et al. 2000), 'sporadic' Ca²⁺ waves occurred at very low frequencies (0.02-0.2 Hz) with a distinctly lower velocity and amplitude from those observed at higher cellular [Ca²⁺]. Waves with these characteristics were not observed in the present study. At moderate intracellular [Ca²⁺] (250-300 nM), the peak [Ca²⁺] of waves observed in this study was 2.1-2.2 µM. These values are higher than those reported by some other studies (Williams et al. 1990; Smith & O'Neill, 2001), but are similar to others (e.g. Takamatsu & Wier, 1990). The peak of the Ca²⁺ waves increased by approximately 0.4 µM (i.e. from ~2.1 to ~2.5 µM (120 % increase) when cellular [Ca²⁺] was raised from 0.3 µM to 0.9 µM (300 % increase). Minimum [Ca²⁺] increased by 200 % over the same range of cellular [Ca²⁺] (from ~150 nM to ~300 nM), while Ca²⁺ wave frequency increased from ~0.15 to ~0.45 Hz (300 %). Interestingly, Ca²⁺ wave velocity was constant over the entire range of cellular [Ca²⁺] despite marked increases in the peak [Ca²⁺] during a wave. The mean value of 122 ± 6 µm s^-1 (n = 8) is similar the values reported in other studies (Kort et al. 1985; Takamatsu & Wier, 1990; Williams et al. 1990; Trafford et al. 1995b; Kaneko et al. 2000) and faster than others (Cheng et al. 1996; Lukyanenko & Györke, 1999; Smith & O'Neill, 2001). A positive correlation between peak [Ca²⁺] during a wave and velocity has been noted previously (Trafford et al. 1995a; Smith & O'Neill, 2001), but these measurements were made at a constant cellular [Ca²⁺] (and therefore constant minimum [Ca²⁺]). As shown in Fig. 8, a positive correlation between velocity and Ca²⁺ wave amplitude (peak - minimum) was observed at each cellular [Ca²⁺], (mean gradient of 33.4 ± 0.4 µm s^-1µM^-1, n = 8). Approximately parallel increases in minimum and peak [Ca²⁺] occur as mean intracellular [Ca²⁺] is increased. This results in a relatively constant wave amplitude (2.1 ± 0.04 µM, n = 8) over the range of cellular [Ca²⁺] 0.2-1.2 µM. This constant amplitude may explain the lack of marked changes in wave velocity despite increases in the peak [Ca²⁺] during a Ca²⁺ wave. The significant correlation between maximum [Ca²⁺], minimum Ca²⁺ and Ca²⁺ wave frequency and mean cellular [Ca²⁺] was confirmed when the characteristics of the Ca²⁺ waves were studied in a number of cardiomyocytes at two specific values of extracellular [Ca²⁺] (~400 nM and ~2.1 µM, Fig. 6).

		View larger version [in this window] [in a new window]
Figure 8. Relationship between wave velocity and wave amplitude at different values of intracellular [Ca2+] Plot of Ca2+ wave amplitude (i.e. peak [Ca2+] - minimum [Ca2+]) vs. wave velocity for a range of mean intracellular [Ca2+] values (here denoted [Ca2+]ic). Amplitude measurements were made from four to six Ca2+ waves from five cardiomyocytes. Each myocyte was exposed to an extracellular [Ca2+] ranging from 240 nM to 796 nM. The average (± S.E.M., n = 8) Ca2+ wave amplitude and velocity for cardiomyocytes exposed to < 1 µM extracellular [Ca2+] is plotted on the same graph (). The line through the data is the best-fit linear correlation to the data, with a gradient of 33 ± 5 µm s-1 µM-1 (P < 0.03).

Ca²⁺ wave frequency is the parameter most sensitive to mean [Ca²⁺]; an increase in the frequency and, to a lesser extent, the minima and maxima of Ca²⁺ waves accounts for the equilibration of intracellular and extracellular compartments up to a mean cellular [Ca²⁺] of 1.2 µM. In this study, the highly permeable surface membrane allowed equilibration of intracellular and extracellular [Ca²⁺]. In myocytes with intact membranes, a similar mean cellular [Ca²⁺] is a consequence of influx and efflux of Ca²⁺ by transmembrane proteins. In both situations, Ca²⁺ waves serve to increase [Ca²⁺] transiently and stimulate net extrusion from the cell. The resultant 'buffering' of the minimum [Ca²⁺] will limit the development of resting contractile tone in the calcium-overloaded myocyte (Stern et al. 1988; Diaz et al. 1997a). SR Ca²⁺ load was not assessed in this study, therefore it is unclear whether the changes in Ca²⁺ wave frequency were accompanied by increased intra-SR Ca²⁺. Diaz et al. (1997a) noted a higher frequency of spontaneous Ca²⁺ release in rat cardiac myocytes at higher extracellular [Ca²⁺], but no change in either spontaneous release amplitude or response to caffeine (a measure of SR Ca²⁺ content). This led the authors to conclude that spontaneous release occurred when intra-SR Ca²⁺ reached a set point; increasing cytosolic Ca²⁺ increased the rate of uptake by the SR, therefore shortening the time to reach the set point.

As indicated in Fig. 7C, the rate of rise and rate of decay of the Ca²⁺ wave increased as the mean cellular [Ca²⁺] increased. The cause of these changes in time course are unknown; an increased rate of rise of [Ca²⁺] could arise from a higher intra-SR [Ca²⁺] at higher cellular [Ca²⁺]. But an increased rate of Ca²⁺ uptake is more difficult to explain, simply because a higher intra-SR [Ca²⁺] would be expected to reduced SR calcium-pump activity (Mermier & Hasselbach, 1975). This would suggest that the mechanism underlying the higher Ca²⁺ uptake rates is based in the cytoplasm, for example via activation of a calcium-calmodulin kinase (Jackson & Colyer, 1996).

Ca²⁺ wave characteristics at high mean cellular [Ca²⁺] levels (> 2 µM)

When permeabilised cells were exposed to an extracellular [Ca²⁺] close to the maximum [Ca²⁺] during a Ca²⁺ wave (> 2 µM), they produced high-frequency waves of larger amplitude, but the mean intracellular [Ca²⁺] within the cells failed to equilibrate with the extracellular space. All cardiomyocytes studied were able to maintain stable Ca²⁺ wave characteristics over a 2-3 min period when perfused with 2-2.5 µM Ca²⁺. When 3.2-3.4 µM extracellular [Ca²⁺] was used, uniform Ca²⁺ waves were not maintained over the 2 min period, due to slowing in the time course, a fall in maximum and a rise in minimum [Ca²⁺]. Myocytes that developed an irreversible hypercontracture after prolonged periods of high [Ca²⁺] exhibited low-amplitude oscillations of intracellular [Ca²⁺] and equilibration of extracellular and intracellular [Ca²⁺] (Fig. 4B). The frequency of Ca²⁺ waves under these circumstances was variable, but not significantly different from that observed in myocytes prior to a sustained hypercontracture. For this reason, these low-amplitude Ca²⁺ waves are not consistent with the 'agonal waves' reported recently in intact rat myocardium at very high cellular Ca²⁺ loads, which achieved frequencies of > 2 s^-1 (Kaneko et al. 2000). Possible reasons for this disparity include: (1) the high cellular Ca²⁺ loads in intact cells would almost certainly be accompanied by changes in other intracellular factors (e.g. ATP and pH), which may affect Ca²⁺ wave characteristics and (2) individual cardiomyocytes within the syncytium would not develop the same degree of hypercontracture as an isolated cell.

Mitochondrial inhibitors modify Ca²⁺ wave characteristics in high [Ca²⁺]

The absence of any significant effect of mitochondrial inhibitors on the characteristics of Ca²⁺ waves at low (~0.4 µM) extracellular [Ca²⁺] suggests that mitochondrial Ca²⁺ uptake/release does not contribute to the characteristics of the Ca²⁺ wave at this level of Ca²⁺ overload. However, in the continued presence of mitochondrial inhibitors, perfusion with a higher extracellular [Ca²⁺] (~2 µM) resulted in Ca²⁺ waves that reached significantly higher peak values (5.1 ± 0.2 µM, n = 4) to those observed without inhibitors (3.6 ± 0.08 µM, n = 4). Calculation of mean intracellular [Ca²⁺] in the presence of mitochondrial inhibitors generated a value close to the extracellular [Ca²⁺], which indicates there was no sustained gradient of [Ca²⁺]. These results suggest strongly that mitochondrial Ca²⁺ uptake attenuates the peak of a Ca²⁺ wave generated at an intracellular [Ca²⁺] > 2 µM. It is unlikely that this effect is due to a direct effect of CCCP and oligomycin on the SR, since perfusion with these mitchondrial inhibitors had no effect on Ca²⁺ wave characteristics at lower cellular [Ca²⁺]. Although this is the first report of an effect in cardiac muscle, previous studies of Ca²⁺ waves in acinar cells (Straub et al. 2000) and pituitary cells (Kaftan et al. 2000) have shown increases in the amplitude of Ca²⁺ waves after disruption of the mitochondrial membrane potential. Isolated rat heart mitochondria injected into oocytes were able to blunt and slow the intracellular Ca²⁺ transient evoked in response to thapsigargin application (Sheu & Sharma, 1999). In the present study, it is unlikely that these effects resulted from altered intracellular metabolite levels or pH as a result of mitochondrial inhibition, since the permeabilised cardiomyocytes were continuously perfused with a buffered mock intracellular solution. Previous studies have suggested that mitochondrial Ca²⁺ uptake and release occurs during Ca²⁺ sparks and waves/transients in cardiac muscle via the calcium uniporter (Duchen et al. 1998; Sharma et al. 2000). These measurements were made at low cellular [Ca²⁺] and the kinetics of mitochondrial Ca²⁺ efflux were rapid and unlikely to generate the net Ca²⁺ uptake. The apparent threshold for mitochondrial Ca²⁺ uptake observed in this study (extracellular [Ca²⁺] = 1.5-2 µM) is in agreement with previous studies of the passive buffering by mitochondria in permeabilised cardiomyocytes (Fry et al. 1984; Hove Madsen & Bers, 1993). An estimate of total Ca²⁺ released into the cytosol during a Ca²⁺ wave can be made using previously published values for the passive Ca²⁺ buffering capacity of digiton-treated rabbit cardiomyocytes (Hove Madsen & Bers, 1993). These calculations would suggest that at an extracellular [Ca²⁺] of ~0.4 µM, the increase of [Ca²⁺] during a wave (see Fig. 6) would result from the release of 178 µmol Ca²⁺ per litre of non-mitochondrial cell volume. At the higher cellular [Ca²⁺] of ~2 µM, the Ca²⁺ wave characteristics described in Fig. 6 would predict a release of only 165 µM l^-1 Ca²⁺ from the SR. However, the larger Ca²⁺ waves measured in the presence of mitochondrial inhibitors suggest that the amount of Ca²⁺ released by the SR is actually 176 µM l^-1 (similar to that at 0.4 µM), of which ~10 µM l^-1 is taken up by the mitochondria at every Ca²⁺ wave.

Net mitochondrial Ca²⁺ accumulation via the Ca²⁺ uniporter has been reported in stimulated (intact) cardiomyocytes (Di Lisa et al. 1993; Zhou et al. 1998). In the latter study, net mitochondrial Ca²⁺ uptake was observed during depolarisation only when diastolic cytoplasmic [Ca²⁺] exceeded 300-500 nM. This value agrees well with the value of minimum intracellular [Ca²⁺] measured in this study at which mitochondrial Ca²⁺ uptake was first evident (mean extracellular [Ca²⁺] > 2 µM). The results of this study suggest that mitochondrial Ca²⁺ uptake is not maintained, since cardiomyocytes that develop a hypercontracture showed no extracellular-intracellular [Ca²⁺] gradient (at > 2 µM). The use of the single-wavelength dyes (Fluo-3/Fluo-5F) allowed accurate measurement of the intracellular [Ca²⁺] signal before and after, but not during hypercontracture. On this basis, it would appear that an event associated with the exhaustion of a mitochondrial Ca²⁺ uptake mechanism caused a rise in mean intracellular [Ca²⁺] and irreversible cardiomyocyte shortening. In a series of cell types, cell death has been linked to the collapse of the mitochondrial membrane potential and the inhibition of mitochondrial Ca²⁺ uptake (Duchen, 2000). Two possible causes of this event are (1) the opening of the mitochondrial permeability transition pore (Cromton, 2000) or (2) damage to the respiratory chain (Duchen, 2000).

In summary, this study suggests that at cellular [Ca²⁺] between 0.2 and 1.2 µM, spontaneous Ca²⁺ release and the generation of spontaneous Ca²⁺ waves can effectively buffer the minimum [Ca²⁺] between Ca²⁺ waves in cardiac cells. Higher cellular Ca²⁺ levels result in more frequent Ca²⁺ waves with only minor changes in their time course. Attempts to increase intracellular [Ca²⁺] > 2 µM is resisted by the mitochondrial Ca²⁺ accumulation occurring predominately at the peak of the Ca²⁺ wave. Working in this way, mitochondrial Ca²⁺ uptake will extend the period of time that cardiomyocytes can maintain a low diastolic [Ca²⁺] and resist the development of a sustained and irreversible hypercontracture when exposed to high intracellular [Ca²⁺] loads.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

CHENG, H., LEDERER, M. R., LEDERER, W. J. & CANNELL, M. B. (1996). Calcium sparks and [Ca2+]i waves in cardiac myocytes. American Journal of Physiology 270, C148-159	[Medline]
CROMTON, M. (2000). Mitochondrial intermembrane junctional complexes and their role in cell death. Journal of Physiology 529, 11-21	[Abstract/Full Text]
DI LISA, F., GAMBASSI, G., SPURGEON, H. & HANSFORD, R. G. (1993). Intramitochondrial free calcium in cardiac myocytes in relation to dehyrogenase activation. Cardiovascular Research 27, 1840-1844	[Medline]
DIAZ, M. E., TRAFFORD, A. W., O'NEILL, S. C. & EISNER, D. A. (1997a). Measurement of sarcoplasmic reticulum Ca2+ content and sarcolemmal Ca2+ fluxes in isolated rat ventricular myocytes during spontaneous Ca2+ release. Journal of Physiology 501, 3-16	[Abstract]
DIAZ, M. E., TRAFFORD, A. W., O'NEILL, S. C. & EISNER, D. A. (1997b). A measurable reduction of s. r. Ca content follows spontaneous Ca release in rat ventricular myocytes. Pflügers Archiv 434, 852-854	[Medline]
DUCHEN, M. R. (2000). Mitochondria and calcium: from cell signalling to cell death. Journal of Physiology 529, 57-68	[Abstract/Full Text]
DUCHEN, M. R., LEYSSENS, A. & CROMTON, M. (1998). Transient mitochondrial depolarisations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes. Journal of Cell Biology 142, 975-988	[Abstract/Full Text]
FABIATO, A. & FABIATO, F. (1979). Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. Journal of Physiology 75, 463-505
FRY, C. H., POWELL, T., TWIST, V. W. & WARD, J. P. T. (1984). Net calcium exchange in adult rat ventricular myocytes: an assessment of mitochondrial calcium accumulating capacity. Proceedings of the Royal Society 223, 223-238
HOVE MADSEN, L. & BERS, D. M. (1993). Passive Ca buffering and SR Ca uptake in permeabilized rabbit ventricular myocytes. American Journal of Physiology 264, C677-686	[Medline]
ISHIDE, N., URAYAMA, T., INOUE, K., KOMARU, T. & TAKISHIMA, T. (1989). Propagation and collision characteristics of calcium waves in rat myocytes. American Journal of Physiology 259, H940-950
JACKSON, W. A. & COLYER, J. (1996). Translation of Ser16 and Thr17 phospholamban into Ca2+-pump stimulation. Biochemical Journal 316, 201-207	[Medline]
KAFTAN, E. J., XU, T., ABERCROMBIE, R. F. & HILLE, B. (2000). Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis. Journal of Biological Chemistry 275, 25465-25470	[Abstract/Full Text]
KANEKO, T., TANAKA, H., OYAMADA, M., KAWATA, S. & TAKAMATSU, T. (2000). Three distinct types of ca2+ waves in Langendorff-perfused rat heart revealed by real-time confocal microscopy. Circulation Research 86, 1093-1099	[Abstract/Full Text]
KORT, A. A., CAPOGROSSI, M. C. & LAKATTA, E. G. (1985). Frequency, amplitude, and propagation velocity of spontaneous Ca++-dependent contractile waves in intact adult rat cardiac muscle and isolated myocytes. Circulation Research 57, 844-855	[Abstract]
LUKYANENKO, V. & GYÖRKE, I. (1999). Ca2+ sparks and Ca2+waves in saponin-permeabilized rat ventricular myocytes. Journal of Physiology 521, 575-585	[Abstract/Full Text]
LUKYANENKO, V., SUBRAMANIAN, S., GYÖRKE, I. & WIESNER, T. F. (1998). The role of luminal Ca2+ in the generation of Ca2+ waves in rat ventricular myocytes. Journal of Physiology 518, 173-186	[Abstract/Full Text]
MARCHANT, J. S. & PARKER, I. (2000). Functional interactions in Ca2+ signaling over different time and distance scales. Journal of General Physiology 116, 691-695	[Full Text]
MERMIER, P. & HASSELBACH, W. (1975). The effect of calcium and phosphate on the biphasic calcium uptake by the sarcoplasmic reticulum. Zeitschrift fur Naturforschung C 30, 777-780
MILLER, D. J. & SMITH, G. L. (1984). EGTA purity and the buffering of calcium ions in physiological solutions. American Journal of Physiology 246, C160-166	[Medline]
MIURA, M., ISHIDE, N., ODA, H., SAKURAI, M., SHINOZAKI, T. & TAKISHIMA, T. (1993). Spatial features of calcium transients during early and delayed afterdepolarizations. American Journal of Physiology 265, H439-444	[Medline]
SHARMA, V. K., RAMESH, V., FRANZINI-ARMSTRONG, C. & SHEU, S. S. (2000). Transport of Ca from sarcoplasmic reticulum to mitochondria in rat ventricular myocytes. Journal of Bioenergetics and Biomembranes 31, 97-104
SHEU, S. S. & SHARMA, V. K. (1999). A novel technique for quantitative measurement of free Ca2+ concentration in rat mitochondria. Journal of Physiology 518, 577-584	[Abstract/Full Text]
SMITH, G. L. & MILLER, D. J. (1985). Potentiometric measurements of stoichiometric and apparent affinity constants of EGTA for protons and divalent ions including calcium. Biochimica et Biophysica Acta 839, 287-299	[Medline]
SMITH, G. L. & O'NEILL, S. C. (2001). A comparison of ATP and tetracaine on spontaneous Ca2+ release from permeabilised rat cardiac myocytes. Journal of Physiology 534, 37-47	[Abstract/Full Text]
STERN, M. D., CAPOGROSSI, M. C. & LAKATTA, E. G. (1988). Spontaneous calcium release from the sarcoplasmic reticulum in myocardial cells: mechanisms and consequences. Cell Calcium 9, 247-256	[Medline]
STRAUB, S. V., GIOVANNIUCCI, D. R. & YULE, D. I. (2000). Calcium wave propagation in pancreatic acinar cells. Journal of General Physiology 116, 547-559	[Abstract/Full Text]
TAKAMATSU, T. & WIER, W. G. (1990). Calcium waves in mammalian heart: quantification of origin, magnitude, waveform, and velocity. FASEB Journal 4, 1519-1525	[Abstract]
TRAFFORD, A. W., DIAZ, M. E., O'NEILL, S. C. & EISNER, D. A. (1995b). Comparison of subsarcolemmal and bulk calcium concentration during spontaneous calcium release in rat ventricular myocytes. Journal of Physiology 488, 577-586	[Abstract]
TRAFFORD, A. W., LIPP, P., O'NEILL, S. C., NIGGLI, E. & EISNER, D. A. (1995a). Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes. Journal of Physiology 489, 319-326	[Abstract]
WILLIAMS, D. A., DELBRIDGE, L. M. D., CODY, S. H., HARRIS, P. & MORGAN, T. O. (1992). Spontaneous and Propagated Calcium Release in Isolated Cardiac Myocytes Viewed by confocal Microscopy. American Journal of Physiology 262, C731-742	[Medline]
ZHOU, Z., MATLIB, M. A. & BERS, D. M. (1998). Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes. Journal of Physiology 507, 379-403	[Abstract/Full Text]

Acknowledgements

The British Heart Foundation and Scottish Hospitals Endowment supported this research financially. An EPSRC studentship and the School of Veterinary Medicine, Glasgow University, support C.L.; P.N. is a British Heart Foundation lecturer. The authors wish to thank Dr F. L. Burton for reading an earlier version of this manuscript.

This article has been cited by other articles:

				C.M. Loughrey, N. Otani, T. Seidler, M.A. Craig, R. Matsuda, N. Kaneko, and G.L. Smith K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes Cardiovasc Res, November 1, 2007; 76(2): 236 - 246. [Abstract] [Full Text] [PDF]

				N. MacQuaide, J. Dempster, and G. L. Smith Measurement and Modeling of Ca2+ Waves in Isolated Rabbit Ventricular Cardiomyocytes Biophys. J., October 1, 2007; 93(7): 2581 - 2595. [Abstract] [Full Text] [PDF]

				Y. Chen-Izu, S. L. McCulle, C. W. Ward, C. Soeller, B. M. Allen, C. Rabang, M. B. Cannell, C. W. Balke, and L. T. Izu Three-Dimensional Distribution of Ryanodine Receptor Clusters in Cardiac Myocytes Biophys. J., July 1, 2006; 91(1): 1 - 13. [Abstract] [Full Text] [PDF]

				D. Wernicke, C. Thiel, C. M. Duja-Isac, K. V. Essin, M. Spindler, D. J. R. Nunez, R. Plehm, N. Wessel, A. Hammes, R.-J. Edwards, et al. {alpha}-Tropomyosin mutations Asp175Asn and Glu180Gly affect cardiac function in transgenic rats in different ways Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2004; 287(3): R685 - R695. [Abstract] [Full Text] [PDF]

				C. M. Loughrey, G. L. Smith, and K. E. MacEachern Comparison of Ca2+ release and uptake characteristics of the sarcoplasmic reticulum in isolated horse and rabbit cardiomyocytes Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1149 - H1159. [Abstract] [Full Text] [PDF]

				C. M. Loughrey, T. Seidler, S. L. W. Miller, J. Prestle, K. E. MacEachern, D. F. Reynolds, G. Hasenfuss, and G. L. Smith Over-expression of FK506-binding protein FKBP12.6 alters excitation-contraction coupling in adult rabbit cardiomyocytes J. Physiol., May 1, 2004; 556(3): 919 - 934. [Abstract] [Full Text] [PDF]

				D. L. Wokosin, C. M. Loughrey, and G. L. Smith Characterization of a Range of Fura Dyes with Two-Photon Excitation Biophys. J., March 1, 2004; 86(3): 1726 - 1738. [Abstract] [Full Text] [PDF]

				T. Seidler, S. L.W. Miller, C. M. Loughrey, A. Kania, A. Burow, S. Kettlewell, N. Teucher, S. Wagner, H. Kogler, M. B. Meyers, et al. Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes Circ. Res., July 25, 2003; 93(2): 132 - 139. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 543/3/859    most recent 2002.021519v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loughrey, C. M. Articles by Smith, G. L. Search for Related Content