Cardiovascular and brain effects of liraglutide in transthyretin amyloidosis (ATTR) mice models

Abstract

Aim: The effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs) in hereditary transthyretin amyloidosis (ATTRv) remain uncertain. This study aims to investigate whether liraglutide interacts with transthyretin protein (TTR) and thereby exerts therapeutic effects for ATTRv. Methods: High throughput screening was conducted to characterize the drug targets of liraglutide, and microscale thermophoresis was used to observe direct binding of liraglutide to TTR. Humanized RBP4/TTR (normal)and RBP4/TTR^Val50Met (ATTRv) mice were constructed, and treated with liraglutide (0.3mg/kg/d) or placebo for 28 days. Fasting plasma glucose, intraperitoneal glucose tolerance test (IPGTT), and plasma brain natriuretic peptide (BNP) were measured. Brain and cardiac tissues were processed with western blot, enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (PCR), and pathological staining to evaluate the lesion status in corresponding organs. Results: Liraglutide exhibited high affinity and direct combination ability to TTR. In ATTRv mice, liraglutide significantly decreased the contents of TTR protein in brain compared with placebo. However, the cardiovascular prognosis measurements including heart failure (plasma BNP concentrations), cardiac fibrosis (the relative expression levels of Cola1 and TGFβ1 in cardiac tissues), and pathological changes (right ventricular collagen percentage, ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter) were statistically comparable between mice receiving liraglutide and placebo treatment. Conclusion: Liraglutide could decrease the deposition of TTR in brain tissues, while it did not improve cardiovascular outcomes in ATTRv mice compared to placebo. More researches regarding the mechanisms and therapeutic effects of GLP-1RAs to ATTRv are still required.

Keywords: ATTRv; brain; heart failure; liraglutide; transthyretin amyloidosis polyneuropathy; transthyretin cardiac amyloidosis.

Figure 1

(A) The model diagram of liraglutide and TTR, made by Pymol. Red indicates TTR protein, blue indicates liraglutide, and yellow indicates mutual binding. This model aids in understanding how liraglutide interacts with TTR, offering insights into its potential therapeutic effects at the molecular level. (B) Liraglutide and TTR microscale thermography experiment. Kd(M): 1.569E-07 ± 6.37E-08; Signal to Noise: 10.7. The determined equilibrium dissociation constant (Kd) is 1.569E-07 ± 6.37E-08 M, indicating a strong binding affinity between liraglutide and TTR. The signal-to-noise ratio of 10.7 suggests high reliability of the experimental results.

Figure 2

(A) The intraperitoneal glucose tolerance test (IPGTT) curve of mice on Day29 0-120min (n=6). The curve shows blood glucose levels at various time points, assessing glucose tolerance in mice. (B) Histogram of Day29 IPGTT area under the curve (AUC) data of mice (*: p<0.05; n=6). Statistically significant differences are marked with asterisks (: p<0.0.5), indicating significant variations in glucose tolerance among different treatment groups.

Figure 3

(A) Non-reducing SDS-PAGE of TTR monomer in mice brain (β-actin was boiled and used with SDS-PAGE). β-actin, boiled as an internal control, was used with SDS-PAGE to ensure consistent protein denaturation and quantification. (B) Results of TTR monomer protein expression in mice brain (*: p<0.05; **: p<0.01; n=6). Statistically significant differences are marked with asterisks (: p<0.05; **: p<0.01), indicating significant variations in TTR monomer expression among different treatment groups. (C) Congo Red positive area percentage of mouse brain tissue. Each experiment was repeated three times (n=6). Congo Red staining detects amyloid protein deposits, and the percentage of positive area reflects the severity of amyloidosis.

Figure 4

(A) Results of BNP protein expression in mice heart (n=6). (B) Results of TGF-β protein expression in mice heart (n=6). (C) Results of COL1A protein expression in mice heart (n=6). (D) Results of Day29 BNP concentration in mice serum (n=6). (E) Results of collagen fiber deposition area in the right ventricle of mice (n=6). (F) Results of ventricular septum thickness (n=6). (G) Results of left ventricular wall thickness (n=6). (H) Results of left ventricular internal diameter (n=6). These results assess the effects of liraglutide on cardiac function and structure.

Figure 5

(A) Results of TTR mRNA expression in mice liver (n=6). (B) Results of RBP4 mRNA expression in mice liver (n=6). (C) Results of TTR monomer protein expression in mice liver (n=6). These results evaluate the effects of liraglutide on liver TTR and RBP4 synthesis. These results evaluate the effects of liraglutide on liver TTR and RBP4 synthesis.

Figure 6

Results of TTR stability experiment. The experiment measures changes in optical density of TTR tetramers in the presence of various concentrations of liraglutide, assessing the impact of liraglutide on TTR stability.