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First published online 1 August 2006
doi: 10.1242/jcs.03073

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Inositol 1,4,5-trisphosphate supports the arrhythmogenic action of endothelin-1 on ventricular cardiac myocytes

¹ Calcium Group, Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
² Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK
³ School of Chemistry, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK
⁴ GE Healthcare, Forest Farm, Whitchurch, Cardiff, CF14 7YT, UK

View larger version (22K): [in a new window]   Fig. 1. Ca2+ transients in electrically-paced ventricular myocytes. (A) Recordings depict the typical response of a single ventricular myocyte immediately after isolation, loaded with indo-1 to repetitive field stimulation. Arrows indicate field stimulation. (B) Normalised response (mean ± s.e.m.; n=28 cells) of ventricular myocytes to electrical pacing over a 25-minute period. (C) Traces show typical Ca2+ responses from a single cell at the start of an experiment (black trace) and after 25 minutes of stimulation (grey trace). There were no significant changes in the time of increase, peak amplitude or recovery of the Ca2+ transient.

View larger version (49K): [in a new window]   Fig. 2. ET-1 stimulates positive inotropy and SCTs in ventricular myocytes. (Ai-iv) Traces depict the effect of continuous superfusion of ventricular myocytes with ET-1 (100 nM). The ET-1 superfusion was initiated just after the trace in i was recorded. Arrows indicate field stimulation. (B) Normalised response (mean ± s.e.m.; n=14 cells) of ventricular myocytes to electrical pacing over a 25-minute period. *P<0.05; calculated using Student's t-test). (C) Montages of confocal images collected at 210-millisecond intervals obtained from a single ventricular myocyte loaded with Fluo-4 either (i) before, (ii) 5 minutes after or 12.5 minutes (iii) after the start of superfusion with ET-1. The cell was field-stimulated at the times indicated. Bar, spatial scale of 15 µm.

View larger version (43K): [in a new window]   Fig. 3. Isoproterenol stimulates positive inotropy and SCTs in ventricular myocytes. (Ai-iv) Traces depict the effect of continuous superfusion of ventricular myocytes with isoproterenol (100 nM). The isoproterenol superfusion was initiated just after the trace in i was recorded. Arrows indicate field stimulation. (B) Normalised response (mean ± s.e.m.; n=12 cells) of ventricular myocytes to electrical pacing over a 25-minute period. *P<0.05; calculated using Student's t-test). (C) Montages of confocal images obtained from a single ventricular myocyte loaded with Fluo-4 either (i) before, (ii) 5 minutes after or 12.5 minutes (iii) after the start of superfusion with isoproterenol. The cell was field stimulated at the times indicated by the white arrows. N.B. In panel Cii, the initial image of the cell (i.e. at 0 milliseconds) shows elevated Ca2+ at the extreme ends of the cell. This reflects the final stage of a Ca2+ wave that had propagated horizontally throughout the myocyte. Bar, spatial scale of 15 µm.

View larger version (20K): [in a new window]   Fig. 4. Characteristics of SCTs observed during control pacing. (A) Average frequency (mean ± s.e.m.; n=28 cells) of SCTs during control pacing of isolated ventricular myocytes. To calculate the SCT frequency, the number of events occurring within defined stimulation periods of 30-seconds was counted. (B) Time of occurrence of the SCTs relative to the previous field-stimulation-evoked Ca2+ signal. Since the cells were electrically paced at 0.33 Hz, the maximal time of occurrence was 3 seconds. (C,D) Number of spontaneous Ca2+ sparks recorded using confocal imaging of electrically (C) paced or (D) unpaced cells.

View larger version (24K): [in a new window]   Fig. 5. Characteristics of SCTs observed during ET-1 superfusion. (A) Average frequency (mean ± s.e.m.; n=14 cells) of SCTs during continuous superfusion of isolated ventricular myocytes with ET-1 (100 nM). (B) Time of occurrence of the SCTs relative to the previous field-stimulation-evoked Ca2+ signal. (C,D) Number of spontaneous Ca2+ sparks recorded using confocal imaging of electrically (C) paced or (D) unpaced cells.

View larger version (25K): [in a new window]   Fig. 6. Characteristics of SCTs observed during superfusion with digoxin. (A,B) Average frequency of SCTs (mean ± s.e.m.; n=8 cells) during continuous superfusion of isolated ventricular myocytes with either (A) 1 µM or (B) 10 µM digoxin. (C) Proportion of cells that displayed SCTs in response to 1 or 10 µM digoxin. (D) Time of occurrence of the SCTs relative to the previous field-stimulation-evoked Ca2+ signal.

View larger version (29K): [in a new window]   Fig. 7. Characteristics of SCTs observed during superfusion with isoproterenol. (A) Average frequency of SCTs (mean ± s.e.m.; n=12 cells) of SCTs during continuous superfusion of isolated ventricular myocytes with isoproterenol (100 nM). (B) Time of occurrence of the SCTs relative to the previous field-stimulation-evoked Ca2+ signal. (C,D) Number of spontaneous Ca2+ sparks recorded using confocal imaging of electrically (C) paced or (D) unpaced cells.

View larger version (14K): [in a new window]   Fig. 8. Bar graph, showing the average latency for the initiation of SCTs during superfusion with control medium or the agonists shown. * indicates data significantly different from control (determined using Kruskal-Wallis one-way ANOVA with Bonferroni correction). The data represent mean ± s.e.m. (n=60 SCTs for each condition).

View larger version (30K): [in a new window]   Fig. 9. Differences in the characteristics of SCTs stimulated by ET-1, digoxin and isoproterenol. (A,B) Parts of photometry recordings from cells stimulated with either (Ai) 100 nM ET-1 or (Bi) 100 nM isoproterenol. (Aii, Bii) Traces on an expanded time scale. Arrows indicate instances when the cells were field-stimulated. The dashed grey lines illustrate the rate of increase of the field-stimulation-evoked Ca2+ signals and the subsequent SCTs. It is evident that for the ET-1-stimulated cell (Aii) the rate of increase of the SCT was comparable to the field-stimulation-evoked event (i.e. it was due to an action potential). By contrast, in the isoproterenol-stimulated cell, the SCT developed at a much slower rate (i.e. it was due to a Ca2+ wave). (C) Peak rate of Ca2+ signal increase during SCTs recorded from six cells incubated with ET-1, and six cells treated with isoproterenol. indicates the typical rate of increase of field-stimulation-evoked Ca2+ transients (Em). , SCTs that arose with a similar rate to those triggered by field stimulation. , SCTs that were consistent with the development of Ca2+ waves. Italicised numbers indicate the number of SCTs measured for each data point, they also illustrate the relative proportions of events observed in each of the individual cells. (D) Proportion of SCTs that occurred as an action potential (i.e. that had a rate of increase >2000 nM s-1) for ET-1, digoxin or isoproterenol.

View larger version (29K): [in a new window]   Fig. 10. The phospholipase C antagonist U73122 inhibits ET-1-evoked positive inotropy and SCTs. (A-C) (i) normalised amplitude of field-stimulation-evoked Ca2+ transients during superfusion with the reagents shown on the upper bars. (ii) Corresponding SCT frequencies.

View larger version (33K): [in a new window]   Fig. 11. ET-1 and Ins(1,4,5)P3 ester both stimulate SCTs that are inhibited by 2-APB. (A) Response of a single ventricular myocyte to continuous superfusion with ET-1 (100 nM). The cell was stimulated with ET-1 following the acquisition of the top (0 seconds) trace. Positive inotropy and SCT frequency developed progressively during the application of ET-1. (B) Similar experiment to that illustrated in A, except that the cell was superfused simultaneously with ET-1 (100 nM) and 2-APB (2 µM). (C) Average SCT frequencies for cells superfused with either ET-1 alone (black bars) or ET-1 + 2-APB (grey bars). (D,E) Similar experiments to those illustrated in A and B, except that the cells were stimulated with a membrane-permeant Ins(1,4,5)P3 ester (2 µM) instead of ET-1. (F) Average SCT frequencies for cells superfused with either Ins(1,4,5)P3 ester alone (black bars) or Ins(1,4,5)P3 ester + 2-APB (grey bars). *P<0.05 (significantly different from control); calculated using Student's t-test).

View larger version (14K): [in a new window]   Fig. 12. Inhibition of Ins(1,4,5)P3-evoked SCTs by 2-APB. (A) Proportion of cells that displayed one or more SCTs during a 25-minute photometry experiment while being superfused with the reagents shown. (B) Average latency until the first SCT for cells stimulated with the reagents shown. *P<0.05 (significantly different from control); calculated using Student's t-test).

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