The Aryl Hydrocarbon Receptor Ligand FICZ Improves Left Ventricular Remodeling and Cardiac Function at the Onset of Pressure Overload-Induced Heart Failure in Mice

Abstract

Adverse ventricular remodeling is the heart's response to damaging stimuli and is linked to heart failure and poor prognosis. Formyl-indolo [3,2-b] carbazole (FICZ) is an endogenous ligand for the aryl hydrocarbon receptor (AhR), through which it exerts pleiotropic effects including protection against inflammation, fibrosis, and oxidative stress. We evaluated the effect of AhR activation by FICZ on the adverse ventricular remodeling that occurs in the early phase of pressure overload in the murine heart induced by transverse aortic constriction (TAC). Cardiac structure and function were evaluated by cardiac magnetic resonance imaging (CMRI) before and 3 days after Sham or TAC surgery in mice treated with FICZ or with vehicle, and cardiac tissue was used for biochemical studies. CMRI analysis revealed that FICZ improved cardiac function and attenuated cardiac hypertrophy. These beneficial effects involved the inhibition of the hypertrophic calcineurin/NFAT pathway, transcriptional reduction in pro-fibrotic genes, and antioxidant effects mediated by the NRF2/NQO1 pathway. Overall, our findings provide new insight into the role of cardiac AhR signaling in the injured heart.

Keywords: aryl hydrocarbon receptor (AhR); cardiac hypertrophy; cardiac remodeling; fibrosis; formyl-indolo [3,2-b] carbazole (FICZ); oxidative stress.

Figure 1

Formyl-indolo [3,2-b] carbazole (FICZ) treatment increases the expression of the aryl hydrocarbon receptor target gene Cyp1a1 in heart tissue, evaluated by real-time PCR analysis. Groups are: Sham (open circles) (N = 6), Sham FICZ (blue circles) (N = 7), transverse aortic constriction (TAC) (black circles) (N = 8) and TAC FICZ (red circles) (N = 5). Data are expressed as mean ± SD. *** p < 0.001.

Figure 2

FICZ administration prevents the increase in left ventricular mass (LVM) and improves cardiac dysfunction in mice subjected to TAC surgery: (A) Representative cardiac magnetic resonance images (CMRI) of mouse hearts before TAC and 3 days after TAC surgery, and treated with vehicle or with FICZ. CMRI analysis of LVM (B), left ventricular end-diastolic volume (LVEDV) (C), left ventricular end-systolic volume (LVESV) (D), and ejection fraction (EF) (E). Groups are: Sham FICZ (blue circles) (N = 8), TAC (black circles) (N = 8) and TAC FICZ (red circles) (N = 8). Data are expressed as mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

FICZ treatment prevents cardiac hypertrophy induced by TAC: (A). Heart weight (HW) (mg). (B). Heart Weight/Tibia Length HW/TL (mg/mm). Groups are: Sham (open circles) (N = 15), Sham FICZ (blue circles) (N = 16), TAC (black circles) (N = 18) and TAC FICZ (red circles) (N = 15). (C). Cardiomyocyte area (µm²). Groups are: Sham (open circles) (n = 106; N = 5), Sham FICZ (blue circles) (n = 200; N = 4), TAC (black circles) (n = 148; N = 5) and TAC FICZ (red circles) (n = 126; N = 4). Data are expressed as mean ± SD. *** p < 0.001. N = number of mice; n = number of cardiomyocytes.

Figure 4

FICZ treatment prevents hypertrophic development by attenuating the calcineurin/RCAN1.4 pathway in mice subjected to TAC surgery. Real-time PCR analysis of mRNA expression of the cardiac hypertrophy biomarker atrial natriuretic peptide (Nppa) (A) and Rcan1.4 (B). Expression was normalized to that of the Rplp0 housekeeping gene. Groups are: Sham (open circles) (N = 5), Sham FICZ (blue circles) (N = 7), TAC (black circles) (N = 8) and TAC FICZ (red circles) (N = 6). Data are expressed as mean ± SD. ** p < 0.01; *** p < 0.001.

Figure 5

FICZ treatment prevents the increased expression of transforming growth factor beta 1 (Tgfb1), collagen type I alpha 1 (Col1a1), and collagen type III alpha 1 (Col3a1) in mice subjected to TAC surgery. Real-time PCR analysis of mRNA expression of Tgfb1 (A), Col1a1 (B), and Col3a1 (C) normalized to that of the Rplp0 housekeeping gene. Groups are: Sham (open circles) (N = 5), Sham FICZ (blue circles) (N = 7), TAC (black circles) (N = 8) and TAC FICZ (red circles) (N = 6). Data are expressed as mean ± SD. *** p < 0.001.

Figure 6

Cardiac staining of 8-hydroxy-2′-deoxyguanosine (8-OHdG) as a biomarker of DNA/RNA damage by oxidative stress: (A). Representative examples of immunofluorescent images obtained in all experimental groups. (B). Levels of 8-OHdG staining in the four groups of mice. Groups are: Sham (open circles) (N = 5), Sham FICZ (blue circles) (N = 8), TAC (black circles) (N = 13) and TAC FICZ (red circles) (N = 6). Data are expressed as mean ± SD. ** p < 0.01.

Figure 7

The antioxidant effect of FICZ on mice subjected to TAC surgery involves activation of the transcription factor nuclear factor erythroid derived 2-like 2 (Nfe2l2) and the antioxidant target gene NADPH/dehydrogenase quinone 1 (Nqo1). Real-time PCR analysis of Nfe2l2 (A) and Nqo1 (B), normalized to the housekeeping gene Rplp0. Groups are: Sham (open circles) (N = 5), Sham FICZ (blue circles) (N = 7), TAC (black circles) (N = 8) and TAC FICZ (red circles) (N = 5). Data are expressed as mean ± SD. *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 8

Scheme of the study design.