Epac2-Rap1 Signaling Regulates Reactive Oxygen Species Production and Susceptibility to Cardiac Arrhythmias

Abstract

Aims: In the heart, β₁-adrenergic signaling involves cyclic adenosine monophosphate (cAMP) acting via both protein kinase-A (PKA) and exchange protein directly activated by cAMP (Epac): a guanine nucleotide exchange factor for the small GTPase Rap1. Inhibition of Epac-Rap1 signaling has been proposed as a therapeutic strategy for both cancer and cardiovascular disease. However, previous work suggests that impaired Rap1 signaling may have detrimental effects on cardiac function. The aim of the present study was to investigate the influence of Epac2-Rap1 signaling on the heart using both in vivo and in vitro approaches.

Results: Inhibition of Epac2 signaling induced early afterdepolarization arrhythmias in ventricular myocytes. The underlying mechanism involved an increase in mitochondrial reactive oxygen species (ROS) and activation of the late sodium current (INa_late). Arrhythmias were blocked by inhibition of INa_late or the mitochondria-targeted antioxidant, mitoTEMPO. In vivo, inhibition of Epac2 caused ventricular tachycardia, torsades de pointes, and sudden death. The in vitro and in vivo effects of Epac2 inhibition were mimicked by inhibition of geranylgeranyltransferase-1, which blocks interaction of Rap1 with downstream targets.

Innovation: Our findings show for the first time that Rap1 acts as a negative regulator of mitochondrial ROS production in the heart and that impaired Epac2-Rap1 signaling causes arrhythmias due to ROS-dependent activation of INa_late. This has implications for the use of chemotherapeutics that target Epac2-Rap1 signaling. However, selective inhibition of INa_late provides a promising strategy to prevent arrhythmias caused by impaired Epac2-Rap1 signaling.

Conclusion: Epac2-Rap1 signaling attenuates mitochondrial ROS production and reduces myocardial arrhythmia susceptibility. Antioxid. Redox Signal. 27, 117-132.

Keywords: Ca2+; Epac; ROS; Rap1; arrhythmias; cardiac.

FIG. 1.

Inhibition of Epac2 induces EAD-like [Ca²⁺] oscillations. (A) Typical confocal line-scan recording from a field-stimulated ARVM loaded with fluo-4 (upper), the associated line profile (middle), and selected Ca²⁺ transients on an expended timescale, both individually and normalized/superimposed (lower). Introduction of ESI-05 (25 μM) was followed by prolongation of the descending phase of the Ca²⁺ transient, which subsequently developed a distinct plateau phase with Ca²⁺ oscillations. s.p.: spontaneous SR Ca²⁺ release; black arrow: early prolongation of descending phase; red arrows: plateau/Ca²⁺ oscillations. ‘*’ indicates corresponding Ca²⁺ transient. (B) Cumulative data showing the percentage of cells exhibiting EAD-like Ca²⁺ oscillations following addition of ESI-05 (25 μM), ESI-05 + isoproterenol (ISO, 20 nM), ESI-05 + KN93 (10 μM), and ESI-05 + KN92 (10 μM). No cells exhibited EADs in the absence of ESI-05 (n = 30). (C) Original (left) and cumulative data (right) showing the effects of ESI-05 (25 μM) on the frequency of spontaneous Ca²⁺ sparks in ARVMs. **p < 0.01, n.s., not significant. ARVM, adult rat ventricular myocyte; EAD, early afterdepolarization arrhythmias; ISO, isoproterenol. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Epac2 inhibition decreases basal Rap1GTP and the effects of ESI-05 are mimicked by Rap1GTP inhibition. (A) Original (left) and cumulative data (right) showing that Rap1GTP was detectable under basal conditions (see the Materials and Methods section) in ARVMS and could be reduced by Epac2 inhibition with ESI-05 or increased by the Epac activator 8-CPT (25 μM), ISO (1 μM), or a lower level of ISO (10 nM) in combination with the phosphodiesterase inhibitor, rolipram (20 μM). ISO was added together with the β₂ antagonist CI 118,551 (1 μM). n = 4–12, *p < 0.05, **p < 0.01, ***p < 0.005. (B) Schematic showing that Epac activation leads to increased levels of active Rap1GTP and that geranylgeranylation of Rap1GTP by GGT-1 facilitates membrane association and interactions with effector proteins. Inhibition of GGT-1 with GGTI-298 blocks the downstream effects of Rap1GTP. (C) Typical confocal line-scan recordings from a field-stimulated ARVM loaded with fluo-4 (upper), the associated line profile (middle), and selected Ca²⁺ transients presented on an expended timescale, both individually and normalized/superimposed (lower). Inhibition of GGT-1 with GGTI-298 (0.1 μM) mimicked the effects of ESI-05 on [Ca²⁺]_i transients. s.p.: spontaneous SR Ca²⁺ release; black arrow: early prolongation of descending phase; red arrows: plateau/Ca²⁺ oscillations. ‘*’ indicates corresponding Ca²⁺ transient. Similar effects occurred in 8 of 10 cells from three hearts. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Increased mitochondrial ROS production underlies effects of ESI-05 and GGTI-298. (A) Representative line-scan images (left) and associated line profiles (right) obtained from field-stimulated ARVMs before (upper) and after (lower) introduction of ESI-05 (25 μM) following preincubation with the NOX inhibitor, DPI (20 μM), the reducing agent, dithiothreitol DTT (2 mM), the SOD mimetic, MnTMPyP (100 μM), or the mitochondria targeted antioxidant, mitoTEMPO (30 μM). (B) Cumulative data showing the effects of all inhibitors tested on the percentage of cells exhibiting EAD-like [Ca²⁺] oscillations, additionally including the mitochondrial inhibitor, rotenone (100 nM), and the nitric oxide synthase inhibitor, L-NAME (500 μM). n = 15–30. (C, D) Show the same protocol and presentation of data for ARVMs exposed to GGT-298 (0.1 μM). n = 10–27. (E) Original xy confocal images showing mitoSOX fluorescence recorded at time zero and then after 12 min, under control conditions (n = 15) (upper) or in the presence of ESI-05 (25 μM, middle, n = 17) or GGTI-298 (0.1 μM, lower, n = 13). Yellow broken lines indicate cell outline. (F) Cumulative data showing the time-dependent change in mitoTEMPO fluorescence under control conditions or in the presence of ESI-05 or GGTI-298. *p < 0.05, ** p < 0.01, ***, p < 0.005, n.s., not significant, n = 13–17. DPI, diphenyleneiodonium; DTT, dithiothreitol; NOX, NADPH oxidase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Effects of ESI-05 or GGTI-298 on AP duration. (A) Whole-cell patch-clamp recording of APs obtained from an ARVM stimulated repetitively (arrows) at 1 Hz. During exposure to ESI-05, the descending phase of the AP developed a sustained plateau with oscillations in E_m, characteristic of EADs. Breaks (-//-) indicate 2 and 4 min after initiation of recording. (B) Original (left) and cumulative data (right) showing AP prolongation in the presence of ESI-05 (n = 9). (C) Original (left) and cumulative data (right) showing AP prolongation in the presence of GGTI-298 (n = 13). (D) Original (left) and cumulative data (right) showing that preincubation of cells with either mitoTEMPO (30 μM, n = 6) or ranolazine (20 μM, n = 5) prevented the increase in AP duration with ESI-05. (E) Original (left) and cumulative data (right) showing that preincubation of cells with either mitoTEMPO (30 μM, n = 5) or ranolazine (20 μM, n = 5) prevented the increase in AP duration with GGTI-298. n.s.,no significant difference. ***p < 0.001. AP, action potential. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Effects of ESI-05 or GGTI-298 on INa_late. (A) Representative recordings of INa recorded from ARVMs using whole-cell patch-clamp under control conditions and in the presence of ESI-05 (left and middle) or GGTI-298 (right), showing that both drugs increase INa_late. The effects of ESI-05 or GGTI-298 on INa_late were markedly reduced by preincubation of ARVMs with either the INa_late inhibitor ranolazine (20 μM) or mitoTEMPO (30 μM). (B) Cumulative data showing the effects of ESI-05 on INa_late and the effects of preincubation with ranolazine or mitoTEMPO (n = 5–7). (C) Cumulative data showing the effects of GGTI-298 on INa_late and the effects of preincubation with ranolazine or mitoTEMPO. n = 5–12, * p < 0.05, **p < 0.01, n.s., not significant. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Inhibition of Epac-Rap1 signaling does not affect ICa. (A) L-type Ca²⁺ currents were evoked in isolated rat ventricular myocytes before and during exposure of ESI-05 (upper) or GGTI-298 (lower). Cells were exposed to either drug for 3.3 min. Currents were evoked by step depolarizations from −30 to +10 mV (75 ms, 0.1 Hz). (B) Bar graph showing cumulative data (mean ± SE) with the amplitude expressed as a percentage of the control current amplitude. Neither ESI-05 (p > 0.05, n = 5) nor GGTI-298 (p > 0.05, n = 5) resulted in a significant change in peak ICa. Data were obtained from three hearts.

FIG. 7.

In vivo effects of ESI-05 and GGTI-298. (A) Mean heart rate calculated in vehicle (n = 7), ESI-05 (n = 7), and GGTI-298 groups (n = 5) in baseline conditions, after isoproterenol injection (1 mg/kg ip., in sterile NaCl 0.9% vehicle), and after vehicle (DMSO/NaCl solution ip., 1/1000: v/v), ESI-05 (1 mg/kg ip., in DMSO/NaCl solution, 1/1000: v/v), or GGTI-298 (1 mg/kg ip., in DMSO/NaCl solution, 1/1000: v/v). (B) Ventricular extrasystoles were counted over 180 min from ECGs after vehicle (DMSO/NaCl solution ip., 1/1000: v/v), ESI-05 (1 mg/kg ip., in DMSO/NaCl solution, 1/1000: v/v), or GGTI-298 (1 mg/kg ip., in DMSO/NaCl solution, 1/1000: v/v). (C) Typical example of ventricular extrasystoles recorded after ESI-05 injection. (D) Typical example of torsades de pointes initiated by a ventricular extrasystole and terminated in sudden cardiac death recorded after ESI-05 injection. NS, no significant difference. **p < 0.01 versus vehicle, Student's t-test. n = 5–12, *p < 0.05, **p < 0.01.

FIG. 8.

Diagram summarizing β₁-adrenergic signaling via PKA and Epac-Rap1. The presence of a β₁-ADR agonist increases the production of cAMP via AC. cAMP acts simultaneously via both PKA and Epac pathways to influence the function of intracellular targets that control Ca²⁺ signaling, force production, electrical connectivity, and cell survival. Some effects of Epac are mediated via its role as a guanine nucleotide exchange factor for the small GTPase Rap1, which facilitates the release of guanosine diphosphate, thereby increasing the level of active Rap1 GTP. The GTPase-activating protein, RAP-GAP, opposes this effect by facilitating GTP hydrolysis. Geranylgeranylation of active Rap1 via GGT-1 is required for interaction with downstream membrane targets. Our data suggest that Rap1GTP is a negative regulator of ROS production by the mitochondria. When Epac2 or GGT-1 is inhibited, the increase in ROS is associated with activation of CaMKII and the development of EAD arrhythmias due to increased INa_Late and AP prolongation. Arrhythmias can be prevented by inhibition of CaMKII with KN93, preincubation with the mitochondria-targeted antioxidant mitoTEMPO, or inhibition of INa_Late with ranolazine. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars