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Journal of Physiology (2001), 537.2, pp. 371-378
© Copyright 2001 The Physiological Society

Depolarisation-evoked Ca²⁺ waves in the non-excitable rat megakaryocyte

David Thomas, Michael J. Mason and Martyn P. Mahaut-Smith

	ABSTRACT

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1. A combination of patch clamp, confocal microscopy and immunohistochemistry was used to examine the spatial properties of Ca²⁺ signalling in the rat megakaryocyte, a non-excitable cell type in which membrane potential can markedly modulate agonist-evoked Ca²⁺ release.
2. Intracellular calcium ion concentration ([Ca²⁺]_i) increases, stimulated by both ADP and depolarisation, frequently originated from a peripheral locus and spread as a wave throughout the cell. Spatially restricted [Ca²⁺]_i increases, consistent with elementary Ca²⁺ release events, were occasionally observed prior to ADP-evoked waves.
3. ADP- and depolarisation-evoked Ca²⁺ waves travelled approximately twice as fast around the periphery of the cell compared to across its radius, leading to a curvilinear wavefront. There was no sigificant difference between wave velocities generated by the two stimuli.
4. Immunohistochemical staining of type III IP₃ receptors, the endoplasmic reticulum-specific protein GRP78/BiP and calreticulin indicated a major peripheral location of the cellular Ca²⁺ stores which probably accounts for the accelerated wave velocity at the cell periphery.
5. These data demonstrate that [Ca²⁺]_i increases, stimulated by depolarisation or the agonist ADP, have indistinguishable spatial properties, providing evidence that similar underlying mechanisms are responsible for their generation.

	INTRODUCTION

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Membrane potential plays a fundamental role in regulating release of Ca²⁺ from intracellular stores in a variety of cell types. In skeletal muscle, membrane voltage can directly gate Ca²⁺ release via dihydropyridine receptor:ryanodine receptor (RyR) conformational coupling (Schneider & Chandler, 1973; Rios & Brum, 1987). Alternatively, depolarisation can evoke influx of Ca²⁺ via voltage-gated Ca²⁺ channels leading to RyR-mediated Ca²⁺-induced Ca²⁺ release (CICR), as occurs during excitation- contraction coupling in cardiac muscle (Fabiato, 1983). A similar indirect mechanism for voltage-induced Ca²⁺ release can also result from stimulation of inositol 1,4,5-trisphosphate (IP₃) receptors, which display marked Ca²⁺ sensitivity following an increase in cytosolic IP₃ levels (Bezprozvanny et al. 1991).

Evidence now suggests that IP₃-mediated Ca²⁺ signalling can be modulated by membrane potential via a mechanism independent of Ca²⁺ influx. In both rat megakaryocytes and coronary artery smooth muscle cells, fluctuations in membrane potential during agonist stimulation result in modulation of Ca²⁺ release from intracellular stores (Ganitkevich & Isenberg, 1993; Mahaut-Smith et al. 1999; Mason et al. 2000; Mason & Mahaut-Smith, 2001). This phenomenon is bipolar in nature in that depolarisation produces an increase in Ca²⁺ release whilst hyperpolarisation leads to a decline in [Ca²⁺]_i consistent with re-uptake into the stores. The underlying mechanism is unknown; however, functional IP₃ receptors are required (Ganitkevich & Isenberg, 1993; Mahaut-Smith et al. 1999; Mason & Mahaut-Smith, 2001). A number of reports suggest that a similar voltage-sensitive process exists in skeletal muscle (Jaimovich et al. 2000), gastric pylorus smooth muscle (Van Helden et al. 2000) and the giant alga Chara (Wacke & Thiel, 2001). Voltage control of IP₃ receptor-dependent Ca²⁺ release may therefore be a more ubiquitous process.

The rat megakaryocyte is a non-excitable cell type, lacking voltage-gated Ca²⁺ channels (Somasundaram & Mahaut-Smith, 1994; Mahaut-Smith et al. 1999) and also lacking RyR-dependent Ca²⁺ release (Uneyama et al. 1993; Mason & Mahaut-Smith, 2001). Agonist-evoked [Ca²⁺]_i increases are primarily the result of Ca²⁺ release from an IP₃-sensitive store, sustained by store-dependent Ca²⁺ influx (Uneyama et al. 1993; Somasundaram & Mahaut-Smith, 1994). These properties make the megakaryocyte an excellent cell type in which to further investigate voltage modulation of IP₃-dependent Ca²⁺ stores. The present study has combined confocal laser scanning fluorescence microscopy with whole-cell patch clamp to investigate the spatiotemporal properties of [Ca²⁺]_i signals in the rat megakaryocyte resulting from both agonist application and membrane depolarisation. An immunohistochemical approach was also used to explore the distribution of Ca²⁺ stores within the unique cellular structure of the megakaryocyte in an attempt to explain the spatial properties of the [Ca²⁺]_i signals detected.

	METHODS

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Megakaryocyte isolation

Male Wistar rats were killed by exposure to a rising concentration of CO₂ followed by cervical dislocation. Megakaryocytes were isolated as described previously (Mahaut-Smith et al. 1999) in a saline containing (mM): 145 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 Hepes, 10 D-glucose, pH 7.35 (NaOH) and 0.64 U^.ml^-1 type VII apyrase (Sigma-Aldrich, Poole, UK). Apyrase was present during preparation and storage of cells to degrade spontaneously released adenosine nucleotides and thus limit P2 receptor desensitisation, but was omitted during experiments. Cells were used 3-9 h after isolation with all experiments carried out at room temperature.

Electrophysiology

Conventional whole-cell patch clamp recordings were carried out using an Axopatch 200A amplifier (Axon Instruments, Union City, CA, USA) in voltage clamp mode with 70-75 % series resistance compensation. Membrane potentials have not been corrected for the ~-3 mV offset that results from the liquid : liquid junction potential between pipette and bath saline solutions. The pipette saline contained (mM): 150 KCl, 2 MgCl₂, 0.1 EGTA, 0.05 Na₂GTP, 10 Hepes, 0.05 K₆ Oregon Green 488 BAPTA-1 (Molecular Probes, Leiden, Netherlands) at pH 7.2 (KOH).

[Ca²⁺]_i fluorescence measurements

Confocal fluorescence measurements were made on a Zeiss Axiovert 100M inverted microscope (Carl Zeiss Ltd, Welwyn Garden City, UK) with a Zeiss LSM 510 confocal laser scanning module. Oregon Green 488 BAPTA-1 was excited at 488 nm and emission collected at > 505 nm. Images were typically 128 pixels 30-50 pixels and acquired at 10-15 Hz, with the optical sections set to either 3 or 12.5 µm. ADP was applied by means of a gravity-controlled superfusion system. Fluorescence signals were background-subtracted and expressed as f/f₀ ratios to normalise fluorescence levels (f) against starting fluorescence (f₀). For images and 3-dimensional plots, f/f₀ has been pseudocoloured using a standard rainbow look-up table from blue (low [Ca²⁺]_i) to red (high [Ca²⁺]_i). Ca²⁺ wave velocities are expressed as means ± standard deviation with statistical difference assessed using Student's paired t test.

Immunocytochemistry

Fixation, washing and antibody incubation were carried out in buffer containing (mM): 100 sodium cacodylate, 1 CaCl₂, 1 MgCl₂, 2 % sucrose, pH 7.3 (HCl). All stages were carried out in 0.1 % Triton X-100 at room temperature unless otherwise stated. Washes (15 min) consisted of a 1:15 dilution of the cell suspension prior to centrifugation. Cells were fixed by dilution with an equal volume of fixative (3 % paraformaldehyde and 0.05 % glutaraldehyde, 60 min), washed, and resuspended in 0.5 % NaBH₄ (15 min) to quench reactive aldehyde groups. Cells were washed twice before resuspension in blocking solution (10 µg ml^-1 of unlabelled secondary antibody, 60 min), washed twice more, and then incubated with primary antibody (4 °C, overnight). Following two washes, cells were incubated with fluorescently tagged secondary antibody (10 µg ml^-1, 60 min in darkness), washed twice, and resuspended in Triton X-100-free buffer before confocal fluorescence measurements at 1 µm optical section. Unlabelled primary antibodies were used at the following concentrations: 1 µg ml^-1 anti-GRP78/BiP, 1:100 anti-calreticulin (Cy-3) (both from Affinity BioReagents Inc., Golden, CO, USA), 1 µg ml^-1 anti-IP₃ receptor isoform type III (BD Transduction Laboratories, Lexington, KY, USA). For labelling of -tubulin, 1:100 Cy-3-conjugated anti--tubulin (Sigma-Aldrich, Poole, UK) was used at the primary antibody stage with secondary antibody omitted, otherwise cells were treated identically. Fluorescently tagged secondary antibodies were from the Alexa Fluor series (Molecular Probes) with Alexa Fluor 488 used to detect anti-IP₃ antibodies and Alexa Fluor 546 used to detect anti-GRP78/BiP and anti-calreticulin antibodies. Secondary antibodies were excited at either 488 or 543 nm with emission collected at either > 505 or > 560 nm, respectively. Cy-3 was excited at 543 nm and emission collected at > 560 nm.

	RESULTS

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ADP-evoked [Ca²⁺]_i signals take the form of a curvilinear wave

At a constant holding potential of -75 mV, ADP-stimulated [Ca²⁺]_i increases clearly originated in a peripheral area followed by waves of Ca²⁺ sweeping across the cell volume in 45 of 55 megakaryocytes. These agonist-evoked Ca²⁺ waves traversed the periphery of the cell more rapidly than across its radius leading to the development of a curvilinear wavefront (Fig. 1A). The faster wave progression around the edge of the cell can clearly be observed in plots of average [Ca²⁺]_i for four 16 µm² regions of interest, one at the wave origin (1) and three others (2-4) positioned equidistant from the first. In the remaining 10 cells, where no clear waves were observed, more amorphous [Ca²⁺]_i increases were seen filling in from the periphery of the cell (Fig. 1B). This is consistent with a Ca²⁺ wave that originated outside the optical section and traversed the cell approximately perpendicular to the plane of focus.

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Figure 1. Spatial characteristics of ADP-evoked [Ca2+]i increases Responses from two cells representing the spatial patterns observed during ADP-evoked [Ca2+]i signals. A, the [Ca2+]i increase originates in the cell periphery and spreads as a wave throughout the whole cell. B, a more homogeneous increase in [Ca2+]i is observed filling in from the perimeter of the cell. Plots show f/f0 in A, four equidistant and B, five 16 µm2 regions of interest.

Nuclear polarity and evidence for unitary Ca²⁺ events

In all megakaryocytes, the position of the nucleus could be clearly distinguished from the higher level of raw fluorescence exhibited by intra-nuclear compared with cytoplasmic Oregon Green 488 BAPTA-1 (see f_raw image, Fig. 2A). Such an effect has previously been reported in other cell types and is attributed to an enhancement of indicator fluorescence within the nucleoplasm rather than a higher Ca²⁺ concentration (Perez-Terzic et al. 1997; Thomas et al. 2000). In 38 of 55 cells, the nucleus was positioned within a single hemisphere of the cell and in the majority of these polarised cells (28 of 38) the initial ADP-evoked Ca²⁺ wave originated in the nucleated hemisphere at a locus between the nuclear and plasma membranes. The origin of subsequent Ca²⁺ waves was more variable and often resulted in spatially chaotic Ca²⁺ signals typical of an excitable medium (Lechleiter et al. 1991). The curvilinear spread of the Ca²⁺ waves shown in Fig. 1A was observed regardless of the site of wave origin relative to the nucleus. In many cell types, recruitment of subcellular elementary Ca²⁺ release events precede Ca²⁺ waves (Yao et al. 1995; Bootman et al. 1997). Given the large size of the megakaryocyte, the small optical section (~1 µm thickness) required to clearly image elementary Ca²⁺ signals is equivalent to 5 % of the megakaryocyte volume. Consequently, the probability of capturing elementary Ca²⁺ events in thin sections is very small. However, at the thicker optical sectioning used in this study, localised [Ca²⁺]_i increases were occasionally observed at the periphery of the cell prior to the initial Ca²⁺ wave (5 cells; see for example Fig. 2A). Small, transient [Ca²⁺]_i increases were detected within cytoplasmic regions of interest close to the wave origin (Fig. 2A, black) but not at the opposite side of the cell (Fig. 2A, red). The limited spread of these [Ca²⁺]_i transients is further illustrated by 3-dimensional plots of fluorescence ratio for the entire cell at rest (Fig. 2Bi) and at the peak of three localised increases (Fig. 2Bii-iv) preceding the Ca²⁺ wave. Whilst the number of localised [Ca²⁺]_i increases observed was insufficient for rigorous analysis, these transient increases are consistent with elementary Ca²⁺ puffs and the hierarchical model of [Ca²⁺]_i signalling (Yao et al. 1995; Bootman et al. 1997).

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Figure 2. Localised [Ca2+]i transients precede ADP-evoked Ca2+ waves A, spatially restricted increases in [Ca2+]i (black trace, denoted ii-iv) that fail to propagate to the rest of the cell (red trace) precede the onset of a global Ca2+ wave. The raw fluorescence image (fraw) illustrates the enhanced nuclear fluorescence of the Ca2+ indicator. B, 3-D representations of the whole cell under basal conditions (i) and during the three localised elevations in [Ca2+]i (ii-iv) denoted in A.

Depolarisation-evoked Ca²⁺ waves and comparison with ADP-evoked waves

Depolarisation during ADP stimulation resulted in [Ca²⁺]_i elevations (Fig. 3A) that also took the form of a curvilinear Ca²⁺ wave originating from a peripheral locus (Fig. 3B) in 41 of 55 cells. Furthermore, the curvilinear nature of the Ca²⁺ waves was unaltered when depolarisations were immediately preceded by brief hyperpolarisations (to -100 mV) in order to slightly lower the [Ca²⁺]_i (data not shown). The remaining 14 cells showed more homogeneous increases in [Ca²⁺]_i. In 27 of 38 cells in which the nucleus was located within a single hemisphere, depolarisation-evoked [Ca²⁺]_i increases also originated from that hemisphere. As observed for ADP-evoked waves, the site of origin of individual depolarisation-evoked Ca²⁺ waves was not constant in any one cell and shared the same location as the initial ADP-evoked wave in only 70 of 190 depolarisations. Thus it would appear that ADP and depolarisation-induced Ca²⁺ waves can start from a common site, but are not obliged to do so.

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Figure 3. Comparison of ADP- and depolarisation-evoked Ca2+ waves A, [Ca2+]i oscillations in response to ADP subside to an elevated [Ca2+]i plateau and are enhanced by depolarisation from -75 mV to either 0 or +75 mV. B, representative images of the rising phase of the initial ADP-evoked Ca2+ wave (i) and the final depolarisation-evoked Ca2+ wave (ii) show the similar spatial properties of the curvilinear Ca2+ wave. C, both stumuli induce Ca2+ waves whose velocity around the perimeter of the cell is approximately double that across the cell radius. ADP-evoked velocities: 95 ± 29 µm s-1 peripherally and 41 ± 12 µm s-1 radially. Depolarisation-evoked velocities: 118 ± 39 µm s-1 peripherally and 48 ± 8 µm s-1 radially.

In all cells displaying a clear wave of [Ca²⁺]_i increase, the peripheral wavefront velocity was significantly faster than the radial velocity for both ADP (P < 0.05, n = 11) and depolarisation (P < 0.05, n = 11) (Fig. 3C). There was no significant difference between the ADP- and depolarisation-evoked peripheral (P > 0.05, n = 11) or radial (P > 0.05, n = 11) Ca²⁺ wave velocities.

Ca²⁺ storage and release proteins are peripherally located

In the rat megakaryocyte, the majority of Ca²⁺ mobilised by ADP and depolarisation is derived from IP₃-dependent stores, which are believed to reside in the endoplasmic reticulum (ER). To explore the underlying basis for the spatial properties of [Ca²⁺]_i signals in the megakaryocyte, the localisation of the ER and Ca²⁺ stores was examined by immunohistochemistry. Fixed cells stained with the ER marker GRP78/BiP showed an intense peripheral fluorescence that declined progressively towards the centre of the cell with a number of areas devoid of staining (Fig. 4A). In images of uniform 1 µm thickness, such a pattern suggests that the ER is primarily located around the cell perimeter. Nucleic acid counterstains showed the areas devoid of staining to be sections of the multilobular polyploidic nucleus (data not shown). A higher peripheral density of ER and the intracellular Ca²⁺ store was further supported by staining patterns of antibodies directed against the Ca²⁺ storage protein calreticulin, and the type III isoform of the IP₃ receptor Ca²⁺ release channel (Fig. 4B and C). These staining patterns did not result from poor antibody penetration as fluorescently tagged anti--tubulin antibodies yielded a relatively uniform cytoplasmic stain (Fig. 4D). Hence, the subcellular localisation of the Ca²⁺ signalling machinery correlates well with the preferential path of [Ca²⁺]_i increase during Ca²⁺ waves.

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Figure 4. Immunohistochemical detection of intracellular Ca2+ stores Transmitted and confocal fluorescence images of fixed megakaryocytes show the graded distrubution of the ER marker GRP78/BiP (A), and the peripheral localisation of the Ca2+ storage protein calreticulin (B) and the type III IP3 receptor (C). These peripheral staining patterns were not due to poor antibody penetration as antibodies directed against -tubulin produce a more evenly distributed cytoplasmic fluorescence signal (D).

	DISCUSSION

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In the rat megakaryocyte, membrane potential exerts bipolar control over the IP₃-dependent Ca²⁺ stores by an unknown mechanism but one that is independent of Ca²⁺ influx and Ca²⁺ extrusion via Na⁺-Ca²⁺ or Na⁺-Ca²⁺, K⁺ exchangers (Mahaut-Smith et al. 1999; Mason et al. 2000; Mason & Mahaut-Smith, 2001). This phenomenon is active over the physiological range of potentials and can induce [Ca²⁺]_i oscillations (Mahaut-Smith et al. 1999; Mason et al. 2000). The underlying pathway may be important for control of [Ca²⁺]_i in a variety of cell types. To explore the interaction between membrane potential and Ca²⁺ release in the megakaryocyte further, confocal microscopy was used to compare the spatial properties of ADP- and depolarisation-evoked [Ca²⁺]_i increases and found them to be markedly similar. In the majority of cells (45/55 and 41/55 for ADP and depolarisation, respectively), the [Ca²⁺]_i increase was observed as a wave spreading more rapidly around the periphery compared to radially across the cell, but with no difference in velocities for the two stimuli (Fig. 3C). The Ca²⁺ wave originated at the cell edge in response to either ADP or depolarisation and was frequently located in the nuclear hemisphere for both stimuli when a clear nuclear polarity was visible (74 and 71 % for ADP and depolarisation, respectively). Although the wave origin was indistinguishable between ADP and depolarisation for only 70/190 depolarisations, this apparent difference can be explained by the fact that the site of wave origin was not constant for any one cell. A similar switching of wave origin was also observed in a non-confocal imaging study of ATP-evoked [Ca²⁺]_i signals in the megakaryocyte (Tertyshnikova & Fein, 1997) and suggests that the cytoplasm becomes an excitable medium following an elevation of IP₃ and Ca²⁺ (Lechleiter et al. 1991).

As a consequence of the large cytoplasmic volume of the megakaryocyte, localised Ca²⁺ release events were observed prior to ADP in only a few experiments. However, the restricted spatial spread of these events (Fig. 2) and their detection close to the site of origin of the Ca²⁺ wave are in accordance with a hierarchical model for megakaryocyte agonist-evoked [Ca²⁺]_i signalling as described for other cells (Yao et al. 1995; Bootman et al. 1997). Unfortunately, the rare occurrence of localised Ca²⁺ increases, the lack of voltage-dependent responses in the absence of other stimuli, and the random nature of elementary Ca²⁺ release events complicates their use in exploring further the depolarisation-dependent Ca²⁺ release phenomenon. However, the comparable spatial and temporal properties of the ADP- and depolarisation-evoked [Ca²⁺]_i increases (this study and Mahaut-Smith et al. 1999; Mason et al. 2000) suggests that similar mechanisms are responsible for their generation. In platelets, and by inference megakaryocytes, ADP activates Ca²⁺ mobilisation via P2Y₁ receptors coupled to G_q and phospholipase C (Leon et al. 1999). Therefore, one explanation for the depolarisation-evoked Ca²⁺ release in the megakaryocyte is an increased production of IP₃ (Mahaut-Smith et al. 1999). Voltage-dependent IP₃ generation has been reported in skeletal muscle (Vergara et al. 1985; Jaimovich et al. 2000) and proposed to underlie control of Ca²⁺ release by the membrane potential in several types of smooth muscle cell and the giant alga Chara (Itoh et al. 1992; Ganitkevich & Isenberg, 1993; Van Helden et al. 2000; Wacke & Thiel, 2001). This may result from direct voltage control of the agonist receptor, its G-protein or phospholipase C, or from modulation of the availability of polar substrates and co-factors (Ganitkevich & Isenberg, 1993; Mahaut-Smith et al. 1999). An alternative hypothesis for the effect of membrane voltage on Ca²⁺ release in the megakaryocyte is a form of configurational coupling between plasma membrane and internal membrane proteins, such as that envisaged for excitation- contraction coupling in skeletal muscle (Schneider & Chandler, 1973) or proposed to explain store-mediated Ca²⁺ entry (Irvine, 1992; Kiselyov et al. 1998). The predominantly peripheral location of the Ca²⁺ stores (Fig. 4) gives some credence to this model since it may enhance the ability of plasma membrane proteins to physically interact with the Ca²⁺ stores. However, such a conformational coupling model would have to take into account the delay of 0.6 s observed between depolarisation and initial [Ca²⁺]_i increase in the megakaryocyte (Mahaut-Smith et al. 1999) compared to 10 ms in skeletal muscle (Zhu et al. 1986). Thus, the slow onset of the depolarisation-evoked [Ca²⁺]_i increase and the comparable nature of the ADP and voltage-dependent wave-like characteristics lend support to the hypothesis that production of IP₃ is involved.

Comprehensive experimental evidence relating the density of Ca²⁺ release sites or ER to Ca²⁺ wave velocity or an initiation site have not been reported and the different properties of the three IP₃ receptor subtypes complicate these issues (Ramos-Franco et al. 1998). In model systems, a higher IP₃ receptor density is predicted to increase Ca²⁺ wave velocity (Bugrim et al. 1997), therefore the predominantly peripheral location of the intracellular Ca²⁺ stores in the megakaryocyte (Fig. 4) can account for the more rapid peripheral, compared to radial, Ca²⁺ wave velocity. This store distribution may also explain why waves always started close to the cell perimeter, although a differential distribution of IP₃ receptor subtypes (Ramos-Franco et al. 1998) or cell morphology (Fink et al. 1999) may also account for the frequent juxtanuclear Ca²⁺ wave origin.

Finally, it is worthwhile speculating on the reasons for the robust nature of the depolarisation-evoked Ca²⁺ release response in megakaryocytes considering the prominently peripheral Ca²⁺ store location. Concentration of the Ca²⁺ stores towards the cell edge may allow an innate voltage sensitivity to IP₃ production to be more effectively transduced into Ca²⁺ release. It will therefore be interesting to compare the location of the stores in other cells exhibiting voltage-dependent Ca²⁺ release via IP₃ receptors. The unique membrane cytoarchitecture of the megakaryocyte resulting from the platelet-generating demarcation membrane system (Yamada, 1957) must also be considered in future studies of the voltage-dependent Ca²⁺ release mechanism.

In summary, the indistinguishable spatial characteristics of the [Ca²⁺]_i increases generated in the megakaryocyte in response to ADP and depolarisation support the hypothesis that similar underlying mechanisms are responsible for their generation.

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Acknowledgements

This work was funded by grants from the Medical Research Council (G9900182 and G9901465) and British Heart Foundation (Basic Science Lectureship, BS/10, to M. P. M.-S.). We are also grateful to Jon Holdich for expert technical assistance and Dr Christof Schwiening for image analysis software.

Corresponding author

M. P. Mahaut-Smith: Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

Email: mpm11{at}cam.ac.uk

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