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First published online 18 December 2007
doi: 10.1242/jcs.021386

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Endothelin-1 enhances nuclear Ca²⁺ transients in atrial myocytes through Ins(1,4,5)P₃-dependent Ca²⁺ release from perinuclear Ca²⁺ stores

¹ Department of Cardiology and Pneumology, University Medicine Göttingen, Germany
² Department of Cardiology, Medical University of Graz, Auenbruggerplatz 15, A-8036 Graz, Austria

View larger version (57K): [in this window] [in a new window]   Fig. 1. Simultaneous confocal imaging of the cytoplasmic and nuclear [Ca2+] transient (CaT) in an electrically stimulated rabbit atrial myocyte. (A) 2D confocal images of an atrial myocyte. Numbers indicate milliseconds after electrical stimulation. (B, left) CaTs of distinct subcellular regions, as indicated in the schematic representation of the cell (same cell as in A): black trace: whole cytoplasm (wc); green traces: subsarcolemmal regions (ss); red trace: nucleus (nuc); blue traces: central regions adjacent to the nucleus (ct). The vertical dashed line marks the peak of the CaTs of the two central regions. (B, right) Average values of peak systolic [Ca2+], time-to-peak and the time constant for decay from 13 atrial myocytes. Colours refer to the subcellular regions as shown in the cell scheme (bar, 5 µm). *, P<0.05 versus subsarcolemmal regions; #, P<0.05 versus central regions. (C) A pair of atrial myocytes (a,b). Left: Fluo-4 fluorescence (Ca2+) during the decaying phase of an electrically stimulated CaT. Right: same cells after cessation of stimulation and loading with Syto-16 (DNA). Because of the large increase in fluorescence caused by Syto-16 loading, laser energy was attenuated to 10% of the initial value. Bars, 10 µm. The results in C are representative for a total of seven cells studied.

View larger version (23K): [in this window] [in a new window]   Fig. 2. The nuclear CaT amplitude varies between atrial myocytes. (A) Pairs of cytoplasmic (black) and nuclear (red) CaTs from three different atrial myocytes. For better comparison, CaTs were normalised to the cytoplasmic CaT. (B) Histogram showing the distribution of the ratio of nuclear to cytoplasmic systolic [Ca2+] obtained under control conditions from a total of 95 atrial myocytes studied.

View larger version (63K): [in this window] [in a new window]   Fig. 3. ET enhances nuclear CaTs. (A) 2D Fluo-4 fluorescence images of an atrial myocyte during an electrically stimulated CaT before (Control) and during exposure to 10 nM ET. Bar, 20 µm. (B) Cytoplasmic (black traces) and nuclear (red traces) CaTs recorded before (left) and during (right) ET treatment. (C) The means ± s.e.m. of ET-induced changes in systolic [Ca2+]. Nuc/Cyto, ratio of nuclear to cytoplasmic [Ca2+]. (D) ET-induced changes in CaT kinetics. (Left) Normalised original CaTs before and during (blue) ET exposure. (Right) The means ± s.e.m. of time-to-peak and the time constant for CaT decay. The data were obtained from 24 atrial myocytes.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Line-scan imaging of the ET-induced enhancement of nuclear CaTs. (A) Original line-scan images and normalised fluorescence traces of the cytoplasmic (black) and nuclear (red) CaTs in an atrial myocyte before (Control) and following a 10 minute exposure to 10 nM ET. The cell scheme illustrates the position of the scan line. Bars (at right), 5 µm. (B) Average values of the effects of 10 nM ET on peak systolic [Ca2+] (normalised to the pre-ET control), time to peak and the time constant for decay in the cytoplasm (black) and the nucleus (red). Means ± s.e.m. of five atrial myocytes.

View larger version (16K): [in this window] [in a new window]   Fig. 5. The ET-induced enhancement of nuclear CaTs depends neither on the basal systolic [Ca2+] in the nucleus nor on the ratio of nuclear to cytoplasmic peak [Ca2+]. The ET-induced increase in peak [Ca2+] in the nucleus as a function of basal systolic [Ca2+] in the nucleus (A) or the ratio of nuclear to cytoplasmic systolic [Ca2+] (B) before ET exposure. The data are from 24 atrial myocytes challenged with ET.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Elevation of cytoplasmic CaTs is not sufficient to enhance nuclear CaTs. (A) Original recordings of cytoplasmic (black) and nuclear CaTs (red) of two atrial myocytes before and during exposure to 10 nM ET (left) or 10 nM ISO (right). (B) Average values of peak systolic [Ca2+] in the cytoplasm (black) and in the nucleus (red) following application of ET (n=24), ISO (n=10) or 4 mM extracellular Ca2+ (`4Ca'; n=6). (C) Normalised ratio of nuclear to cytoplasmic [Ca2+] following application of ET (n=24), ISO (n=10) or 4 mM extracellular Ca2+ (n=6). In B and C, values were normalised to the initial control (=100%). **, P<0.01 versus initial control.

View larger version (35K): [in this window] [in a new window]   Fig. 7. Low concentrations of ET augment nuclear CaTs in the absence of increases in cytoplasmic CaTs. (A) Original recording of cytoplasmic (black) and nuclear (red) CaTs of an atrial myocyte before and during exposure to 0.1 nM ET. (B) Changes of peak systolic [Ca2+] in the cytoplasm (left) and nucleus (right) induced by 0.1 nM ET. (C) Normalised ratio of nuclear to cytoplasmic [Ca2+] following application of 0.1 nM ET. In B and C, values are normalised to the initial control (=100%). Means ± s.e.m. of six atrial myocytes (N.S.=not significant).

View larger version (31K): [in this window] [in a new window]   Fig. 8. Signalling pathway underlying the ET-induced enhancement of nuclear CaTs. (A) Original recordings of cytoplasmic (black) and nuclear CaTs (red) before and after application of 0.25 µM BQ-123 (left) or 3 µM 2-APB (right) and following additional application of 10 nM ET. (B) Means ± s.e.m. of peak cytoplasmic and nuclear [Ca2+] following exposure to ET for 10 minutes in the absence and presence of BQ-123 (n=10), U-73122 (n=7), 2-APB (n=12) and xestospongin C (n=7).

View larger version (29K): [in this window] [in a new window]   Fig. 9. ET increases fractional Ca2+ release from SR and perinuclear stores. (A) Original recording of cytoplasmic (black) and nuclear (red) CaTs before (left) and after (right) ET application. Each time, two electrically stimulated CaTs and a caffeine-induced CaT are shown. The ET effect on electrically stimulated CaTs shown here was the largest effect recorded in this series of experiments. Still there was no increase in Ca2+ load of either SR or perinuclear (PN) stores, indicating that ET augmented the CaT by increased fractional release rather than elevation of Ca2+ store content. (B) Average values of Ca2+ store content (left, normalised to control) and fractional Ca2+ release (FCR) from SR and PN stores (middle) before and following application of ET (n=7) or ISO (n=7). Right, ET- and ISO-induced increases in fractional release from SR and PN stores (FCR). Means ± s.e.m. of seven atrial myocytes for each case.