Induction of cardiac fibrosis by β-blocker in G protein-independent and G protein-coupled receptor kinase 5/β-arrestin2-dependent Signaling pathways

Abstract

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β(1)-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β(1)-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β(1)-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers.

FIGURE 1.

Metoprolol-induced cardiac fibrosis. A, picrosirius red staining of perivascular region of left ventricles of ddY mice after treatment of saline (Control (Con)) or β-blockers (Metoprolol (Met), Propranolol (Pro), Carvedilol (Car)). Paraffin sections of hearts (3 μm thickness) were prepared from mice, to which each β-blocker was administered. The sections were stained with picrosirius red and observed under microscopy. Representative images are shown. Scale bar, 15 μm. B, quantification of collagen volume fraction from A (n = 5). **, p < 0.01. C, up-regulation of mRNA expressions of angiotensin converting enzyme (ACE), Periostin, Collagen 1a (Col1a), α-skeletal actin, and Ctgf (CTGF) in mouse hearts by β-blockers. The expression levels of the fibrotic genes were evaluated by real-time RT-PCR. The fold-increases were calculated by the values of saline-treated mice set as 1 (n = 3–5). **, p < 0.01. D, Masson's trichrome staining of perivascular and interstitial regions of left ventricles from saline- or metoprolol-treated C57BL/6J mice (n = 5–6). Typical images are shown. Arrows indicate the fibrotic region. Scale bar: 15 μm on perivascular images, 30 μm on interstitial images. Collagen volume fraction on each image was quantified and the data were shown below the images. *, p < 0.05. E, hemodynamic parameters (dP/dt_max or dP/dt_min) of the saline- or metoprolol-treated C57BL/6J mice. Saline or metoprolol was administered to mice for 3 months. After the administration, they were subjected to hemodynamic measurements. The parameters dP/dt_max or dP/dt_min represent the maximal rate of pressure development, maximal rate of decay of pressure, respectively. Error bars show S.E. (n = 15). *, p < 0.05; **, p < 0.01.

FIGURE 2.

Metoprolol-induced fibrotic responses did not depend on G protein signaling. A, cAMP accumulation in HEK293 cells expressing β₁-adrenergic receptors by β-blockers (10 μm) in the presence of ICI118551 (50 μm). HEK293 cells stably expressing β₁-adrenergic receptor were transiently transfected with a FRET-based cAMP biosensor. After 15 min of pre-treatment with ICI118551, β-blocker stimulation was performed on the cells and the changes of the fluorescence were followed under microscopy. non, nonstimulated cell; Iso, isopreterenol; Met, metoprolol; CGP, CGP20712A; Bis, bisoprolol; ICI, ICI18,551; Pro, propranolol; Car, carvedilol; Lab, labetalol; Pin, pindolol. ΔFRET ratios were determined at 5 min after the stimulation. The experiments were repeated three times and each result was obtained from 13 to 20 cells. **, p < 0.01; ***, p < 0.001. B, pertussis toxin (PTX) treatment did not influence cAMP accumulation by metoprolol. PTX treatment (100 ng/ml) was performed 16 h before the co-addition of 3-isobutyl-1-methyxanthine (IBMX) (1 mm) and β-blockers (Metoprolol, CGP-20712A) (10 μm) on HEK293 cells stably expressing β₁-adrenergic receptor, and cAMP accumulation was determined by FRET using microscopy. The experiments were repeated three times and each result was obtained from 6 cells.

FIGURE 3.

Metoprolol up-regulated expressions of fibrotic genes in rat neonatal cardiomyocytes but not in cardiac fibroblasts. Metoprolol-induced induction of fibrotic genes in rat neonatal cardiomyocytes (A) or cardiac fibroblasts (B) were determined (n = 3 for cardiomyocytes, and n = 8 for cardiac fibroblast). Rat neonatal cardiomyocytes or cardiac fibroblasts were stimulated with 10 μm metoprolol. The expression levels of fibrotic genes (TGF-β, Ctgf (CTGF), and Col1a (Col1a)) after the stimulation were determined by real time RT-PCR. *, p < 0.05.

FIGURE 4.

Requirement of β-arrestin2 for metoprolol-induced responses. A–C, the changes in BRET ratios by metoprolol or isoproterenol stimulation for 5 min in HEK293 cells (A), H9c2 cells (B), or cardiomyocytes (C). A, HEK293 cells were transfected with β₁-adrenergic receptor-Rluc and GFP²-β-arrestin1 (left panel, βArr1-β₁AR) or β₁-adrenergic receptor-Rluc and β-arrestin2-GFP² (right panel, βArr2-β₁AR) (n = 4). B and C, H9c2 cells (B) or rat neonatal cardiomyocytes (C) were transfected with β₁-adrenergic receptor-Rluc and β-arrestin2-GFP² (n = 3). They were harvested 48 h after the transfection, washed with PBS, and split into 96-well plates. Then, they were incubated with isoproterenol or metoprolol at the indicated concentrations for 5 min. After the incubation, Rluc substrate, DeepBlueC^TM was added to a final concentration of 5 μm. BRET signal was determined by calculating the ratio of the light emitted by the GFP² and the light emitted by RLuc. D and E, the cells were transfected with Rluc-β-arrestin2-GFP² (n = 4 for HEK293 cells and n = 3 for H9c2 cells). Then, the changes in BRET ratios of Rluc-β-arrestin2-GFP² were determined after metoprolol (10 μm) or isoproterenol stimulation (10 μm) for 5 min in HEK293 cells expressing β₁-adrenergic receptor (D) or H9c2 cells expressing β₁-adrenergic receptor (E). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Requirement of β-arrestin2 for metoprolol-induced cardiac fibrosis. A and B, cardiac fibrosis in perivascular (A) or interstitial (B) regions of left ventricles of wild type or β-arrestin2-KO mice by metoprolol. Metoprolol was orally administered to wild type or β-arrestin2-KO mice for 3 months. The degrees of their cardiac fibrosis were evaluated by picrosirius staining using paraffin sections of their hearts. *, p < 0.05; ***, p < 0.001. C, CTGF mRNA expression by metoprolol in wild type or β-arrestin2-KO mice. *, p < 0.05. Mouse heart total RNA was prepared from wild-type or β-arrestin2 knock-out mice (n = 3), which underwent metoprolol administration for 3 months. CTGF expression levels were evaluated by real time RT-PCR. D, determination of dP/dt_min of wild type or β-arrestin2-KO mice. The metoprolol-administered wild type or β-arrestin2-KO mice were subjected to hemodynamic measurements (n = 7–12). ***, p < 0.001.

FIGURE 6.

Requirement of GRK5 for metoprolol-induced cardiac fibrosis. A, interaction between β₁-adrenergic receptor and β-arrestin2 by metoprolol (10 μm) or isoproterenol (1 nm, 100 nm) stimulation in GRK5- or GRK6-knockdown cells (n = 4). HEK293 cells were transiently co-transfected with siRNA (control siRNA, GRK5 siRNA, or GRK6 siRNA), β₁-adrenergic receptor-Rluc, and β-arrestin2-GFP². Two days after transfection, BRET measurement was performed. *, p < 0.05. B, the changes of dP/dt_min in GRK5- or GRK6-KO mice (n = 7–14). Metoprolol was administered to wild type, GRK5-KO, or GRK6-KO mice for 3 months. After the administration, they were subjected to hemodynamic measurements. *, p < 0.05; **, p < 0.01. C and D, cardiac fibrosis in perivascular (C) or interstitial (D) regions of left ventricles of metoprolol-treated wild type or GRK5-KO mice (n = 5). The degrees of cardiac fibrosis in metoprolol-administered wild type or GRK5-KO mice were evaluated by picrosirius staining using paraffin sections of their hearts. *, p < 0.05; ***, p < 0.001.

FIGURE 7.

Schematic diagram depicting the model of metoprolol-mediated cardiac fibrosis. Metoprolol induces cardiac fibrosis through β₁-adrenergic receptor depending on GRK5 and β-arrestin2.