JCS03073 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Digoxin-stimulated positive inotropy in ventricular myocytes. The traces in panels Ai-iv depict Ca²⁺ transients recorded in a ventricular myocyte during continuous superfusion with digoxin (1 μM). The digoxin superfusion was initiated just after the trace in Ai was obtained. The arrows indicate when field stimulation was applied. Panel B shows the normalised response (mean ± s.e.m.; n=8) of ventricular myocytes to electrical pacing over a 25-minute period. *P< 0.05 (indicates that the data are significantly different from control at the same time point); calculated using Student’s t-test). 100% of cells (n=8) responded to 1 μM digoxin. Digoxin caused a progressive increase in the amplitude of field stimulation-evoked Ca²⁺ signals. It also promoted the occurrence of SCTs. As with 100 nM ET-1, 1 μM digoxin increased the amplitude of pacing-evoked Ca²⁺ signals by ∼100% after superfusion for 20 minutes. Although digoxin enhanced the amplitude of field stimulation-evoked Ca²⁺ transients, it did not affect the time for Ca²⁺ increase or recovery.

• Supplemental Figure 2 -

  Fig. S2. Effects of 2 mM and 100 mM 2-APB on field stimulation-evoked Ca²⁺ signals. Panel A depicts the averaged response of electrically-paced cells under control conditions (black line and symbols; n=6), or in the presence of 2 mM 2-APB (red line and red symbols; n=7) or 100 mM 2-APB (green line and green symbols; n=10). *P< 0.05 (indicates that the data are significantly different from control at the same time point); calculated using Student’s t-test). Panel B shows typical responses from cells paced in the presence of 2 mM or 100 mM 2-APB. The arrowheads indicate the times at which the cells were depolarised.

• Supplemental Figure 3 -

  Fig. S3. 2-APB did not alter the ability of isoproterenol or digoxin to trigger SCTs. Panel A depicts the average (mean ± s.e.m.) increase of pacing-induced Ca²⁺ signals in ventricular myocytes following application of isoproterenol (black bars; n=14) or isoproterenol + 2-APB (grey bars; n=12). Panel B shows averaged data for myocytes stimulated with digoxin (black bars; n=5) or digoxin + 2-APB (grey bars; n=5). 2-APB did not have any significant effect on the response of cells to isoproterenol or digoxin when assessed using Student’s t-test.

• Supplemental Figure 4 -

  Fig. S4. Ins(1,4,5)P₃ ester caused positive inotropy in ventricular myocytes, which was antagonised by 2-APB. This figure depicts the average (mean ± s.e.m.) increase of pacing-induced Ca²⁺ signals in ventricular myocytes following application of a membrane-permeant Ins(1,4,5)P₃ ester (black line and symbols; n=14) or Ins(1,4,5)P₃ + 2-APB (red line and symbols; n=15).