Dopamine D1/D5 Receptor Signaling Is Involved in Arrhythmogenesis in the Setting of Takotsubo Cardiomyopathy

Figure 1. The dopamine D1/D5 receptor activation contributes to occurrence of arrhythmic events. hiPSC-CMs were treated by vehicle (Control) or 500 μM Epi or Epi plus 10 μM SCH 23390 or 50 μM (±)-SKF 38393 or 5 μM fenoldopam for 1 h. Spontaneous APs of spontaneously beating hiPSC-CMs were measured in the first 100 s after the whole cell configuration was obtained. The arrhythmic events including EAD-like (black arrows) or DAD-like and failed AP (red arrows) events or triggered activity (green arrows) in each cell were counted and divided by total events to obtain the percentage of arrhythmic events. Data were summarized in Table 1. (A) Representative traces of spontaneous APs recorded in a cell of control group. (B) Representative AP traces in a cell of Epi-group. (C) Representative AP traces in a cell of Epi+SCH 23390 group. (D) Representative AP traces in a cell of (±)-SKF 38393 group. (E) Representative AP traces in a cell of fenoldopam group.

Figure 2. Dopamine D1/D5 receptor signaling contributed to effect of epinephrine on action potential. hiPSC-CMs were challenged by either vehicle (Control) or 500 μM epinephrine (Epi) or epinephrine plus 10 μM SCH 23390 or SCH 23390 alone for 1 h. Action potentials (APs) were recorded at 1 Hz. (A) Examples of APs in a cell of each group. (B) Mean values of APD10 in each group cells. (C) Mean values of APD50 in each group cells. (D) Mean values of APD90 in each group cells. (E) Mean values of Vmax of APs in each group cells. (F) Mean values of AP amplitude (APA) in each group cells. (G) Mean values of resting potential (RP) in each group cells. “n” numbers in (B) are for (B–G). The p-values vs. Control were decided by the analysis of one-way ANOVA with Holm-Sidak post-test.

Figure 3. Dopamine D1/D5 receptor agonists mimicked epinephrine effects on the action potential (AP). hiPSC-CMs were challenged by either vehicle (Control) or 50 μM (±)-SKF 38393 or 5 μM fenoldopam for 1 h and action potentials (APs) were recorded. (A) Examples of AP traces in each group cells. (B) Mean values of APD10 in each group cells. (C) Mean values of APD50 in each group cells. (D) Mean values of APD90 in each group cells. (E) Mean values of AP Vmax in each group cells. (F) Mean values of AP amplitude (APA) in each group cells. (G) Mean values of resting potential (RP) in each group cells. “n” numbers in (B) are for (B–G). The p-values vs. Control were determined by the analysis of one-way ANOVA with Holm-Sidak post-test.

Figure 4. ROS mediated the effects of (±)-SKF 38393 or fenoldopam on action potentials. hiPSC-CMs were challenged by vehicle (Control) or 50 μM (±)-SKF 38393 and 5 μM fenoldopam or 100 μM H2O2 (H2O2) in the presence or absence of a ROS-blocker (1 mM NAC), or NAC alone for 1 h. APs were recorded at 1 Hz. Shown are examples of APs in each group cells. Statistical data were summarized in Table 2.

Figure 5. Dopamine D1/D5 receptor activation increased ROS-production. hiPSC-CMs were treated with or without antibody against troponin T (cTnT) and ROS fluorescence dye was applied to measure ROS production. Vehicle (Control) (C) or 50 μM (±)-SKF 38393 or 5 μM fenoldopam or (±)-SKF 38393 plus 10 μM SCH 23390 or fenoldopam plus 10 μM SCH 23390 (D–G) was applied to cells for 1 h before measurements. ROS-generation was analyzed by FACS. (A,B) A representative recoding indicating cardiac troponin T (cTnT) in hiPSC-CMs. (D,E) A representative recoding indicating enhancement of ROS generation by (±)-SKF 38393 or fenoldopam. (F,G) A representative recoding indicating that SCH 23390 prevented the effect of (±)-SKF 38393 or fenoldopam on ROS generation. (H) Averaged values of fluorescence intensity showing the ROS-levels in cells. “blank” means control measurements in cells that were not treated by the ROS fluorescence dye. “n” numbers in (H) are numbers of different independent experiments using cells from different differentiations. The p-values vs. Control were determined by the analysis of one-way ANOVA with Holm-Sidak post-test.

Figure 6. Protein kinases and NADPH oxidases participating in dopamine D1/D5 receptor and ROS signaling. hiPSC-CMs were challenged for 1 h by vehicle [Control, (A)] or 50 μM (±)-SKF 38393 (B) or 5 μM fenoldopam (C) or 100 μM H2O2 [H2O2, (D)] or 10 μM PKC activator PMA [PMA, (E)] or 10 μM Chele (F) or (±)-SKF 38393 plus 10 μM chelerythrine (Chele), an inhibitor of protein kinase C (G), or fenoldopam plus Chele (H) or H2O2 plus Chele (I) or (±)-SKF38393 plus 10 μM DPI (J) or fenoldopam plus DPI (K) or (±)-SKF 38393 plus 1 μM H89, an inhibitor of protein kinase A (L). Action potentials paced at 1 Hz were recorded. Statistical data are summarized in Table 3. (A–L) Examples of AP-traces in each group cells.

Figure 7. Effects of (±)-SKF 38393 or fenoldopam on ion channel currents. hiPSC-CMs were challenged for 1 h by vehicle (Control) or 50 μM (±)-SKF 38393 and 5 μM fenoldopam. Sodium channel current (INa) was recorded with the patch clamp (voltage-clamp) protocol indicated in (A) (inset). The L-type calcium channel current (ICa−L) was recorded with the voltage-clamp protocol indicated in (F) (inset). (A,B) Representative traces of INa in absence (Control) and presence of (±)-SKF38393. (C) I–V curves of peak INa. (D) Mean values of peak INa at −40 mV. (E,F) Examples of traces of ICa−L in absence (Control) and presence of (±)-SKF38393. (G) I–V curves of ICa−L. (H) Mean values of ICa−L at 10 mV. “n” numbers represent the number of analyzed cells. The p-values vs. Control were determined by t-test.