β-Arrestin-biased AT1R stimulation promotes cell survival during acute cardiac injury

Abstract

Pharmacological blockade of the ANG II type 1 receptor (AT1R) is a common therapy for treatment of congestive heart failure and hypertension. Increasing evidence suggests that selective engagement of β-arrestin-mediated AT1R signaling, referred to as biased signaling, promotes cardioprotective signaling. Here, we tested the hypothesis that a β-arrestin-biased AT1R ligand TRV120023 would confer cardioprotection in response to acute cardiac injury compared with the traditional AT1R blocker (ARB), losartan. TRV120023 promotes cardiac contractility, assessed by pressure-volume loop analyses, while blocking the effects of endogenous ANG II. Compared with losartan, TRV120023 significantly activates MAPK and Akt signaling pathways. These hemodynamic and biochemical effects were lost in β-arrestin-2 knockout (KO) mice. In response to cardiac injury induced by ischemia reperfusion injury or mechanical stretch, pretreatment with TRV120023 significantly diminishes cell death compared with losartan, which did not appear to be cardioprotective. This cytoprotective effect was lost in β-arrestin-2 KO mice. The β-arrestin-biased AT1R ligand, TRV120023, has cardioprotective and functional properties in vivo, which are distinct from losartan. Our data suggest that this novel class of drugs may provide an advantage over conventional ARBs by supporting cardiac function and reducing cellular injury during acute cardiac injury.

Fig. 1.

TRV120023 (TRV and TRV023) decreased mean arterial pressure (MAP) in a dose-dependent manner. A steady hypertensive state was established by continuous intravenous infusion of ANG II (0.1 µg·kg⁻¹·min⁻¹). After establishing a hypertensive state, a continuous intravenous infusion of TRV120023 decreased mean arterial pressure in 1 mg·kg⁻¹·min⁻¹ and 100 μg·kg⁻¹·min⁻¹ infusion groups (†P < 0.05; *P < 0.01; n = 3).

Fig. 2.

Representative pressure-volume loops and changes in end-systolic elastance (Ees) and maximal elastance (Emax) in wild-type (WT) and β-arrestin-2 knockout (KO) mice. WT mice before and after infusion with: A: saline, B: TRV120023 (10 μg·kg⁻¹·min⁻¹), C: TRV120023 (100 μg·kg⁻¹·min⁻¹), and D: losartan (5 mg·kg⁻¹·min⁻¹). β-Arrestin-2 KO mice before and after infusion with: E: TRV120023 (10 μg·kg⁻¹·min⁻¹) and F: TRV120023 (100 μg·kg⁻¹·min⁻¹). LV, left ventricle.

Fig. 3.

Enhanced contractility and cardiac function by TRV120023 is β-arrestin-2 (βarr2) dependent. A and B: myocardial contractility measures (Ees and Emax) were increased significantly in WT treated with TRV120023 [10 μg·kg⁻¹·min⁻¹ (TRV10) and 100 μg·kg⁻¹·min⁻¹ (TRV100)]. In β-arrestin-2 KO mice, Ees and Emax were decreased in a dose-dependent manner (*P < 0.001 for the saline-infused group vs. TRV120023-infused groups of the corresponding genotype; WT, n = 6; β-arrestin-2 KO, n = 5). C: LV systolic pressure was decreased significantly in the TRV120023-infusion group (*P < 0.001 for the saline-infused group vs. TRV120023-infused groups of the corresponding genotype and for saline-infused WT vs. β-arrestin-2 KO; WT, n = 6; β-arrestin-2 KO, n = 5). D: ejection fraction was not changed in TRV10- and TRV100-infusion groups in WT but was decreased in β-arrestin-2 KO mice (†P < 0.01 for β-arrestin-2 KO infused with TRV100 vs. saline-treated β-arrestin-2 KO and TRV100-treated WT; WT, n = 6; β-arrestin-2 KO, n = 5).

Fig. 4.

Recruitment of β-arrestin-2 with TRV120023 with no activation of G-protein-mediated signaling. A: dose response of TRV120023 in human embryonic kidney (HEK)293 cells overexpressing ANG II type 1 receptor (AT1R) for recruitment of β-arrestin and activation of G-protein [inositol monophosphate 1 (IP-One)] reveals selective recruitment of β-arrestin. Data are mean ± SE for 15–20 independent experiments. B: dose response of losartan (Los) in HEK293 cells overexpressing AT1R reveals an absence of β-arrestin recruitment and G-protein activation. C and D: administration of TRV120023 to HEK293 cells expressing high (600 fM/mg) and very high (2 pM/mg) levels of AT1R did not result in accumulation of diacylglycerol (DAG) as revealed by the fluorescence resonance energy transfer-based DAG reporter (DAGR) (31).

Fig. 5.

TRV120023 enhances ERK1/2 and Akt signaling after ischemia reperfusion (IR). A and C: ERK1/2 and Akt phosphorylation were increased significantly in TRV120023-treated WT mice undergoing IR (**P < 0.05 for saline IR vs. saline Sham and losartan IR vs. losartan Sham; †P < 0.01 for saline IR vs. TRV IR; *P < 0.001 for TRV IR vs. saline and losartan IR; n = 5–6 in each group). B and D: Akt and ERK1/2 phosphorylation were not changed significantly in β-arrestin-2 KO mice after IR (n = 4 in each group). TRV120023 = 100 μg·kg⁻¹·min⁻¹ infusion; losartan = 5 mg·kg⁻¹·min⁻¹ infusion. p-ERK and p-AKT, phosphorylated ERK and Akt, respectively; t-ERK and t-AKT, total ERK and Akt, respectively.

Fig. 6.

IR-induced cardiomyocyte apoptosis was attenuated by TRV120023 pretreatment. A: representative images of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) staining. White arrowheads indicate TUNEL-positive nuclei (green). B: the number of TUNEL-positive nuclei was increased significantly in IR groups compared with Sham groups. TUNEL-positive nuclei were significantly attenuated in TRV120023-treated groups (*P < 0.001 for all IR vs. Sham conditions and for TRV IR vs. losartan and saline IR; n = 5–6/group). C: the antiapoptotic effect of TRV120023 was lost in β-arrestin-2 KO animals undergoing IR (**P < 0.05 for TRV Sham vs. IR condition; n = 4–6/group). TRV = 100 μg·kg⁻¹·min⁻¹ infusion; losartan = 5 mg·kg⁻¹·min⁻¹ infusion.

Fig. 7.

TRV120023 promotes cytoprotective signaling after mechanical stretch injury. A: ERK1/2 phosphorylation was increased significantly in TRV120023-treated WT mice undergoing ex vivo balloon stretch (Str) injury compared with losartan-treated mice [†P < 0.01 for untreated perfusion control (PC) vs. ANG; **P < 0.05 for untreated PC vs. untreated Str and TRV PC; *P < 0.001 for TRV Str vs. untreated PC and losartan Str; n = 5/group]. B: representative images of TUNEL-stained histologic sections revealing diminished apoptosis in TRV120023 animals undergoing balloon stretch. White arrowheads indicate TUNEL-positive nuclei (green). C: the percent of TUNEL-positive nuclei was increased significantly in balloon-stretched “Untreated” and losartan-treated hearts compared with PC hearts (†P < 0.01 for untreated Str vs. untreated PC; *P < 0.001 for losartan Str vs. losartan PC; n = 4/group). TRV120023-treated hearts had significantly less TUNEL-positive nuclei compared with losartan-treated hearts (*P < 0.001 for TRV Str vs. losartan Str; n = 4/group).