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

Abstract

Background: Previous studies suggested involvement of non-ß-adrenoceptors in the pathogenesis of Takotsubo cardiomyopathy (TTC). This study was designed to explore possible roles and underlying mechanisms of dopamine D1/D5 receptor coupled signaling in arrhythmogenesis of TTC.

Methods: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were challenged by toxic concentration of epinephrine (Epi, 0.5 mM for 1 h) for mimicking the catecholamine excess in setting of TTC. Specific receptor blockers and activators were used to unveil roles of D1/D5 receptors. Patch clamp, qPCR, and FACS analyses were performed in the study.

Results: High concentration Epi and two dopamine D1/D5 receptor agonists [(±)-SKF 38393 and fenoldopam] reduced the depolarization velocity and prolonged the duration of action potentials (APs) and caused arrhythmic events in iPSC-CMs, suggesting involvement of dopamine D1/D5 receptor signaling in arrhythmogenesis associated with QT interval prolongation in the setting of TTC. (±)-SKF 38393 and fenoldopam enhanced the reactive oxygen species (ROS)-production. H₂O₂ (100 μM) recapitulated the effects of (±)-SKF 38393 and fenoldopam on APs and a ROS-blocker N-acetylcysteine (NAC, 1 mM) abolished the effects, suggesting that the ROS-signaling is involved in the dopamine D1/D5 receptor actions. A NADPH oxidases blocker and a PKA- or PKC-blocker suppressed the effects of the dopamine receptor agonist, implying that PKA, NADPH oxidases and PKC participated in dopamine D1/D5 receptor signaling. The abnormal APs resulted from dopamine D1/D5 receptor activation-induced dysfunctions of ion channels including the Na⁺ and L-type Ca²⁺ and I_Kr channels.

Conclusions: Dopamine D1/D5 receptor signaling plays important roles for arrhythmogenesis of TTC. Dopamine D1/D5 receptor signaling in cardiomyocytes might be a potential target for treating arrhythmias in patients with TTC.

Keywords: D1/D5 dopamine receptor; Takotsubo cardiomyopathy; arrhythmia; catecholamine excess; human-induced pluripotent stem cell-derived cardiomyocytes.

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 (I_Na) was recorded with the patch clamp (voltage-clamp) protocol indicated in (A) (inset). The L-type calcium channel current (I_Ca−L) was recorded with the voltage-clamp protocol indicated in (F) (inset). (A,B) Representative traces of I_Na in absence (Control) and presence of (±)-SKF38393. (C) I–V curves of peak I_Na. (D) Mean values of peak I_Na at −40 mV. (E,F) Examples of traces of I_Ca−L in absence (Control) and presence of (±)-SKF38393. (G) I–V curves of I_Ca−L. (H) Mean values of I_Ca−L at 10 mV. “n” numbers represent the number of analyzed cells. The p-values vs. Control were determined by t-test.