Investigating the role of the extracellular matrix protein agrin in cardioprotection

: production and characterisation of a bioactive recombinant agrin

Abstract

Background: Mature cardiomyocytes (CM) are unable to proliferate, and injury to the myocardium results in permanent damage. The extracellular matrix protein agrin successfully induced myocardial repair in rodent and porcine models of infarct, as published in 2017 and 2019 respectively. Bassat et al. proposed that agrin increases cardiomyocyte proliferation in mice in an age-dependent manner by interacting with the cell surface receptor α-dystroglycan (α-DG). It is unknown whether similar agrin induced, age-related cardiomyocyte proliferation pathways exist in the human heart. We investigated levels of agrin and other proteins involved in the regenerative axis through gene and protein expression analysis in the human heart. To better understand the agrin:α-DG interaction, downstream processes, and to characterise the regenerative capacity of agrin, we produced and purified a recombinant, bioactive form (rec.Agrin) containing only domains interacting with α-DG.
Methods: 1) Biopsies from the right ventricle (RV) were collected from 40 patients who underwent surgery for congenital heart disease. The expression of agrin, dystroglycan, YAP and laminin was investigated. 2) Once produced, rec.Agrin has been characterised using a variety of biochemical and biophysical methods including solid phase binding assays and small angle x ray scattering (SAXS). Based on our molecular model predicting coordination sites for Ca2+ within rec.Agrin, we produced and purified two single mutants within the predicted Ca2+ binding sites.
Results: 1) Agrin transcripts were found in all the biopsy samples analysed; agrin levels were negatively correlated to age (p = 0.026), as were laminin transcripts (p = 0.023). There was no correlation with age for other proteins analysed. Western blotting and immunohistochemistry employed to detect and localise agrin in tissue also indicated its presence in all patient samples. In characterisation of rec.Agrin, SAXS analysis has shown that rec.Agrin is compact, and further compacted in the presence of calcium (Ca2+). Solid phase binding indicated that rec.Agrin binds tightly to α-DG in a Ca2+ dependant manner. Solid phase experiments revealed that α-DG binding is strongly reduced in the mutants.
Conclusions: We have found that agrin is progressively and significantly downregulated with age in human RV tissue, however, not as dramatically as has been shown in mice. We have highlighted similarities and differences to findings in rodents. This has lain groundwork to explore the proposed mechanism and the potential of agrin based cardioprotective therapies. We have developed a bioactive rec.Agrin, with the next step to further investigate its ability to impact cardiomyocyte proliferation through further cellular experiments and a move to in vivo models. By developing rec.Agrin and mutants we have confirmed the involvement of specific residues in coordinating Ca2+, and SAXS experiments further showed that Ca2+ has an effect in compacting the protein.

Date of Award	5 Feb 2024
Original language	English
Awarding Institution	University of Bristol
Sponsors	British Heart Foundation
Supervisor	Giulia Bigotti (Supervisor) & Massimo Caputo (Supervisor)