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First published online 23 November 2004
doi: 10.1242/jcs.01559

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The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction

¹ Laboratory of Molecular Signalling, The Babraham Institute, Babraham Hall, Babraham, Cambridge, CB2 4AT, UK
² Department of Chemistry, School of Chemistry, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK

View larger version (40K): [in a new window]   Fig. 1. Spatially heterogeneous Ca2+ transients during EC-coupling in atrial myocytes. (A) A representative myocyte displaying Ca2+ transients at a series of time points (in mseconds) after depolarisation of the cells. Transients were restricted to the junctional periphery. The solid white line denotes the position of the nucleus (N) and the dotted line indicates the cell periphery. The times noted on top of the images indicate the instances at which the images were taken relative to cell depolarisation. (B) A quantitative analysis of the propagation of electrically evoked Ca2+ signals in an atrial myocyte. The cell is depicted in i and the six regions from which the Ca2+ signal was sampled are indicated running transversely across the cell (denoted `A' to `F'). The average amplitude (ii) and rate of rise of the calcium signal (iii) in each region were calculated from six successive depolarisations, and are shown as mean±s.e.m. *Significantly different (P<0.05) from the response in region A. Please note that before each experimental recording, cells were paced until they had reached a steady-state condition.

View larger version (34K): [in a new window]   Fig. 2. Increased Ca2+ influx enhances the response of the central non-junctional RyRs. The top portion of the figure depicts the experimental protocol. The arrowheads indicate the points at which cells were depolarised. The transformation in the spatial distribution of action potential-evoked Ca2+ signals is shown by the change in the ratio of Ca2+ signal amplitude in the subsarcolemmal space relative to central regions. The filled circles denote the response of the myocyte from which the inset traces were obtained, and the red diamonds show the mean±s.e.m. from six cells (two each from three rats). The inset panels illustrate the time course and amplitude of the Ca2+ signal at subsarcolemmal (black trace) and central (white traces) regions. A pseudo-linescan is shown beneath the traces to illustrate the spatial properties of the recorded Ca2+ signals. The yellow trace under the pseudo-linescan image indicates the extent of cellular contraction and the vertical dashed red line, the onset of depolarisation.

View larger version (27K): [in a new window]   Fig. 3. Sub-millimolar caffeine induces recruitment of central RyRs and enhances contraction during atrial EC-coupling. (A) The experimental protocol used to reversibly sensitise RyRs with 0.1 mM caffeine. (Ba-c) Traces showing the amplitude of electrically evoked Ca2+ responses at a peripheral site (black traces) and a central non-regenerative region (grey traces) before (a), during (b) and after washout (c) of 0.1 mM caffeine. The locations of the subcellular regions that were sampled are shown on the inset cell image. The dashed white line indicates the cell boundary, and the solid white line, the nucleus (N). (Ca-c) Traces depicting the twitch of the cell; a and c were obtained before caffeine application and after washout, respectively and b illustrates the enhancement of contraction observed during caffeine addition. A similar enhancement of contraction was observed in six out of six cells studied.

View larger version (25K): [in a new window]   Fig. 4. Increasing SR Ca2+ load promotes the recruitment of central RyRs during atrial EC-coupling. (A) Cell image showing the locations of the subsarcolemmal site (black circle) and the central non-regenerative region (grey circle) that were analysed. (Ba) The black traces illustrates the response of the subsarcolemmal site before (Bai) and after (Baii) increasing SR Ca2+ load. (Bb) The grey traces in i and ii illustrate the corresponding responses from the central region. The SR Ca2+ load was increased by incubating cells for 1 minute in 10 mM extracellular Ca2+ at the time indicated between the dashed lines. The black arrowheads indicate when the cell was depolarised. Please note, the cell was not paced when the extracellular Ca2+ was increased. Apart from the 1 minute period in 10 mM extracellular Ca2+, the whole experiment was conducted using 1 mM Ca2+. (C) The increase in twitch amplitude during the transients, marked by stars in Bai and Baii. The data shown in this figure are from a single cell representative of seven others (from three rats).

View larger version (38K): [in a new window]   Fig. 5. SERCA pumps retard Ca2+ signal propagation in atrial myocytes. (A) A portion of the atrial myocyte examined. Ca2+ was recorded from a subsarcolemmal site (red circle), and also from a central non-regenerative region (black circle). (B) Summaries of the changes of the contraction amplitude (open black squares) and the subsarcolemmal/central Ca2+ gradient (filled green circles) observed upon acute application of 5 µM CPA (indicated by the black bar). (C) The characteristics of the Ca2+ signals observed in the cell before and during CPA application. The pseudo-linescan images in Caii and Cbii were obtained by sampling Ca2+ across the portion of the cell indicated by the dashed yellow line in A. The contraction of the cell before and during CPA application is shown in Caiii and Cbiii, respectively. Similar results were found in five cells (from three hearts).

View larger version (28K): [in a new window]   Fig. 6. Mitochondria buffer Ca2+ signals in atrial myocytes. (Aa) The experimental protocol. Cells were initially paced under control conditions. The stimulation was then halted and the cells incubated for 3 minutes with 20 µM antimycin + 20 µM oligomycin. After this period, the pacing was continued. The arrowheads indicate the timing of the depolarising pulses. The cell image (Ab) shows the position of the line that was used to generate the pseudo-linescan plots. (B) The response of the cell to electrical pacing under control conditions. The inset plots showing subsarcolemmal and central Ca2+ signals were obtained by sampling the linescan image along the regions shown by the red and black lines. (C) The reaction of the same cell following depolarisation of the mitochondrial membrane potential. The average cellular Ca2+ signal is shown as single black traces in Ba and Ca. The data are representative of 5 cells (from 3 hearts). (D) Ca2+ uptake into mitochondria. An image of a rhod-2-loaded atrial myocyte is depicted in Da. Ca2+ uptake into subsarcolemmal and central mitochondria was assessed by sampling the fluorescence changes within the regions indicated by the filled circles. The changes in mitochondrial Ca2+ are depicted by the correspondingly coloured traces for electrically evoked signals (Db) and a spontaneous Ca2+ wave (Dc), which occurred in the same cell. (Dd) Electrical stimulation only modestly increased rhod-2 fluorescence following application of 20 µM antimycin + 20 µM oligomycin for 3 minutes. The rhod-2 loading depicted in Da is representative of >20 cells from several independent experiments. The responses to electrical stimulation and generation of spontaneous Ca2+ waves are typical of six cells from three independent experiments.

View larger version (19K): [in a new window]   Fig. 7. ß-adrenergic stimulation induces positive inotropy by recruitment of central RyRs. (A) A cell image showing the locations of the subsarcolemmal site (red circle) and the central non-regenerative region (green circle) that were analysed. (B,C) The response of the cell before and following a 3-minute incubation with isoproterenol (0.1 µM). The red and green traces depict the change in Ca2+ concentration at the subsarcolemmal and central region, respectively. The blue traces depict the ratio of the Ca2+ response at the subsarcolemmal site relative to that inside the cell. The upward deflection represents a subsarcolemmal response with a lesser central Ca2+ signal. Note that in the isoproterenol stimulated cell (C), the ratio of subsarcolemmal/central Ca2+ response increases quickly, as in the control situation. However, the ratio more rapidly declines and undershoots, owing to the development of the central Ca2+ response. The contraction of the cell is shown by the black traces. The spatial properties of the Ca2+ signal are illustrated by the pseudo-linescan images in Bb and Cb. The dashed yellow lines and arrowheads indicate the points at which the cell was depolarised. The dashed black line on the cell image in A shows the portion of the cell that was sampled in generating the pseudo-linescan plots. The data shown are from a representative cell typical of eight other cells analysed (from three hearts).

View larger version (41K): [in a new window]   Fig. 8. Endothelin-1 promotes atrial myocyte contraction by increasing the response of non-junctional RyRs. (A) A cell image showing the locations of the subsarcolemmal site (red circle) and the central non-regenerative region (black circle) that were analysed. (B,C) The response of a typical atrial myocyte under control conditions and following a 5-minute incubation with 0.1 µM ET-1. The red and black traces illustrate the Ca2+ response in the subsarcolemmal and central region, respectively. The dashed black line on the cell image shows the portion of the cell that was sampled in generating the pseudo-linescan plots. Similar responses were observed in eight other cells (from three hearts).

View larger version (26K): [in a new window]   Fig. 9. Effect of InsP3 on atrial EC-coupling. (A) The protocol for activating InsP3Rs in atrial myocytes. Cells were initially paced under control conditions to monitor the action potential-evoked Ca2+ response. A membrane permeant form of InsP3 (InsP3BM) was then added to the medium bathing the cells. The arrowheads indicate the points at which cells were depolarised. The filled arrowheads indicate the stimulations that were selected for the analysis shown in Ba-d (i.e. before (Ba) and 2 (Bb), 4 (Bc) or 6 (Bd) minutes after InsP3BM application). The black and red traces in Ba-d show the response of a subsarcolemmal and central site (the location of the sites is marked on the cell image in Bai). The confocal cell images on the right hand side of Ba-d show the spatial pattern of the Ca2+ signals during the experiment. The times at which the images were taken are marked by the correspondingly numbered arrows. The data shown were representative for five cells (from three hearts). (C) Summarises the changes in contraction and in peak Ca2+ amplitudes at subsarcolemmal (black circles) and central (red circles) sites during the InsP3BM application. The data show mean response ± s.e.m. (n=5).

View larger version (81K): [in a new window]   Fig. 10. Atrial myocyte Ca2+ signalling machinery. The cartoon depicts the arrangement of atrial myocyte-specific components of the Ca2+ signalling toolkit that contribute to the generation and modulation of atrial myocyte Ca2+ signals. The green band in the cartoon designates the boundary that the subsarcolemmal Ca2+ signal has to cross in order to recruit central RyRs and trigger contraction.