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First published online September 20, 2006
doi: 10.1242/10.1242/jcs.03223

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Calcium signalling during excitation-contraction coupling in mammalian atrial myocytes

¹ Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
² Department of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK
³ Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK

View larger version (61K): [in a new window]   Fig. 1. Structure of ventricular, atrial and neonatal rat cardiac myocytes. Panels A and B depict portions of a ventricular and an atrial myocyte, respectively. They illustrate the relative positioning of some of the key elements involved in EC coupling. The network SR elements wrap around both the myofibrils and mitochondria. For additional representations of these cells see Franzini-Armstrong et al. and others (Franzini-Armstrong et al., 2005; Sommer and Jennings, 1992; Yamasaki et al., 1997). Panel C shows the topology of a myofibril relative to the ventricular myocyte section above it in A. The junctional couplings involving the peripheral sarcolemma in the middle of the A-band of ventricular myocytes has clearly been visualised in some studies (Chen-Izu et al., 2006) but are not always evident (Fig. 3Aii). In atrial cells, it appears that junctional coupling exists at the Z-lines and also in the middle of the A-band (Fig. 3Bii).

View larger version (92K): [in a new window]   Fig. 2. Structure of ventricular, atrial and neonatal rat cardiac myocytes. Panels A, B and C depict immunostained adult ventricular, adult atrial and neonatal ventricular myocytes respectively. The left-hand panels (Ai, Bi and Ci) show cells immunostained with a monoclonal antibody raised against -actinin (Sigma). The middle panels (Aii, Bii and Cii) display cells immunostained with a polyclonal antibody raised against type 2 RyRs (a kind gift from Prof. V. Sorrentino, San Raffaele Scientific Institute, Siena, Italy). The right-hand panels (Aiii, Biii and Ciii) depict cells immunostained with a monoclonal antibody raised against the type 1 sodium/calcium exchanger (R3F1; a kind gift from Prof. K. Philipson, UCLA, California). To visualise the NCX staining in the neonatal myocytes, a field of confluent cells is depicted (Ciii) because this provides the best contrast in these flat cells. The neonatal myocyte nuclei were visualised by DAPI staining (pseudocoloured in blue). Specific immunostaining was visualised using secondary antibodies conjugated to Alexa Fluor 405 (blue), 488 (green) or 568 (red). The -actinin staining shows the precise striated structure of the fully differentiated adult ventricular and atrial cells (Ai and Bi). The neonatal cells show -actinin striation (Ci), but they do not have the same degree of organisation as their adult counterparts. RyRs have a regular transverse striated pattern in adult cells (Aii and Bii). Note the two distinct populations of RyRs in adult atrial cells. In addition to the transverse `non-junctional' RyRs, atrial cells express a ring of `junctional' RyRs (Bii). The latter are responsible for the initiation of EC coupling around the circumference of the cell. In the neonatal myocytes (Ci), the degree of RyR expression is lower and these are less organised than in adult cells. In both atrial (Biii) and neonatal cells (Ciii), the NCX staining is prominent only around the circumference of the cells. In ventricular myocytes that have T-tubules, the NCX protein is evident on the sarcolemma and at the Z-lines deep inside the cells (Aiii). Bars, 20 µm.

View larger version (131K): [in a new window]   Fig. 3. Colocalisation of VOCCs and RyRs in ventricular, atrial and neonatal rat cardiac myocytes. Panels A, B and C depict immunostained adult ventricular, adult atrial and neonatal ventricular myocytes, respectively. The left-hand panels (Ai, Bi and Ci) show cells immunostained with a polyclonal antibody raised against the 1c subunit of L-type VOCCs (CNC1; a kind gift from Prof. W. Catterall, Dept. of Pharmacology, University of Washington, Seattle). The middle panels (Aii, Bii and Cii) display cells immunostained with a monoclonal antibody raised against type 2 RyRs (Calbiochem; C3-33). The right-hand panels (Aiii, Biii and Ciii) depict a merge of the VOCC and RyR images. The neonatal myocyte nuclei were visualised using DAPI staining (blue). Specific immunostaining was visualised using secondary antibodies conjugated to Alexa Fluor 488 or 568. Bars, 10 µm.

View larger version (28K): [in a new window]   Fig. 4. Spatially heterogeneous Ca2+ signalling during atrial myocyte EC-coupling. Panel A shows the development of a Ca2+ signal following electrical depolarisation in a single atrial myocyte. The left-hand montage shows pseudocolour-coded images of the entire cell. To indicate the initiation of discrete Ca2+ sparks (designated by the arrowheads) and their lateral spreading, the right-hand column of images depicts part of the cell (the region bounded by the dashed box) at a higher magnification. The time at which the electrical pulse was applied is shown by the open arrow. Three obvious Ca2+ spark sites are marked by black arrowheads. The surface plots in panel B depict the spatial and temporal development of a Ca2+ signal from a different atrial cell. The Ca2+ concentration is indicated by both the colour and the height of the peaks (blue/green indicates low [Ca2+] and yellow/red depicts high [Ca2+]). A clear peripheral ring of Ca2+ is evident with a steep `valley' of low Ca2+ concentration in the centre of the cell. Panel C illustrates the consistency of the spatial gradient of Ca2+ signalling in atrial myocytes during electrical pacing. The traces show the Ca2+ concentration at the two regions depicted in panel A. The peripheral region (red circle and red trace) displays a large Ca2+ rise with each depolarisation (denoted by the black arrowheads) whereas the central region has a lower response (black circle and black trace). This figure was reproduced with permission from Blackwell Publishing and The Physiological Society (Mackenzie et al., 2001).

View larger version (37K): [in a new window]   Fig. 5. Positive inotropic effect of raising extracellular Ca2+. Progressive centripetal propagation of Ca2+ signals promotes contraction of atrial cardiomyocytes. The traces shown were obtained from a single atrial myocyte that was electrically paced and loaded with the Ca2+ indicator fluo3. Ca2+ elevation is denoted by an increase in fluo3 emission. The cell was electrically paced at a constant frequency, just as if it was receiving a regular action potential from the sinoatrial node. To simulate the effect of a positive inotropic agonist, the extracellular Ca2+ concentration was elevated from 1 to 10 mM as indicated. This provides a larger Ca2+ influx signal and ultimately leads to increased SR Ca2+ content, thereby mimicking the effect of ß-adrenergic stimulation (Huser et al., 1996; Mackenzie et al., 2004a), but without changing the phosphorylation status of any of the proteins involved in EC coupling. The inward movement of Ca2+ within the cell was monitored by observing changes in nuclear Ca2+ concentration. Since atrial cell nuclei are centrally positioned, and nuclear Ca2+ derives almost exclusively from the surrounding cytoplasm, they are ideal cellular areas in which the ability of Ca2+ to penetrate into cells can be assessed. The change of extracellular Ca2+ did not affect the amplitude of Ca2+ signals in the sub-sarcolemmal region where EC coupling was initiated, but it caused a progressive increase in Ca2+ signalling in the remainder of the cell, as shown by the increased amplitude of the signals in the nuclear region. Concomitantly with greater inward movement of Ca2+, the cellular contraction was enhanced.