S5 - Targeted Agrin Delivery for Cardiomyocyte Regeneration: The Role of ECM-Binding Peptides

Introduction: : The pathological accumulation of extracellular matrix (ECM) in cardiac fibrosis leads to impaired myocardial function and contractility. Although cardiomyocyte (CM) proliferation is essential for cardiac repair, it is markedly reduced in fibrotic hearts, highlighting the need for effective therapeutic strategies. Localized delivery of the C-terminal fragment of agrin (C-term agrin) has shown promise in stimulating CM proliferation (Bassat et al., Nature 2017). To enhance this effect by specifically targeting fibrotic tissue, we explore the potential of a polypeptide sequence from placental growth factor-2 (PlGF2(123-144)). Its demonstrated high affinity for ECM proteins (Martino et al., Science 2014), which are prevalent in fibrotic hearts, suggests its utility as a delivery vehicle for C-term agrin. We aim to maximize C-term agrin's ability to promote CM proliferation by exploiting this targeting capability for localized delivery and sustained signaling specifically within fibrotic regions. Thus, we hypothesize that delivering C-term agrin conjugated with the PlGF2(123-144) peptide can enhance CM proliferation within the microenvironment of cardiac fibrosis.

Materials and

Methods: : Predicting LG (laminin globular) domain-matriglycan binding: Glycowork (Thomès et al., Glycobiology 2021) was employed for the computational prediction of LG domain binding to matriglycan.

PlGF2(123-144)-agrin conjugation: PIGF2(123-144) (Ac-RRPKGRGKRRREKQRPTDCHL-CONH2, 2672 Da) was synthesized in the LSU AgCenter Biotechnology Laboratory. Rat agrin (R&D Systems, cat# 550-AG/CF) was conjugated to PIGF2(123-144) using Sulfo-SMCC [sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate] crosslinker.

SDS-PAGE: Protein electrophoresis was performed using NuPAGETM MOPS SDS running buffer and run for 1 h at 150 V. Protein gels were imaged using IBright CL1500 Imaging System (Invitrogen).

ELISA: The binding of PlGF2(123-144)-agrin conjugates to coated ECM proteins (collagen I, III, fibronectin) and ECM mixtures (normal and fibrotic ratios) was quantified using an ELISA. Following blocking, the conjugate was incubated with the coated wells, and bound agrin was detected with an HRP-conjugated anti-agrin antibody. Absorbance was measured at 450 nm after TMB substrate addition.

Injection to the mouse heart: To confirm the delivery of PlGF2(123-144)-agrin, we cryosectioned samples and assessed the localization of PlGF2(123-144)-agrin by immunohistochemistry (IHC) and fluorescence microscopy. CM proliferation in vivo was examined in wild-type (WT) mice after myocardial infarction (MI). Mice were injected with PlGF2(123-144)-agrin, free agrin, or PBS, and proliferating CMs (EdU+/cTnT+ or MF20+) in the infarct region were quantified by IHC and fluorescence microscopy.

Results, Conclusions, and Discussions:: We used Glycowork to predict lectin-glycan interactions, specifically investigating how the length of the matriglycan repeating unit (GlcA(b1-3)Xyl(a1-3))n GlcA(b1-4)Xyl affects these interactions. LG3 negatively impacted binding, whereas LG1 and LG2 facilitated high-affinity interactions (Figure 1B). SDS-PAGE analysis (Figure 2A) confirmed successful production of PlGF2(123-144)-agrin conjugates, with a major band around 120-130 kDa. As shown in Figure 2B, the conjugate significantly enhanced binding to fibronectin (FN) and collagen I (Col I) compared to baseline, with the strongest affinity for Col I. Binding to collagen III (Col III) was lower. Under various ECM conditions (Figure 2C), PlGF2(123-144)-agrin binding to normal ECM (10 µg coating) was similar to fibrotic ECM at the same concentration. However, increasing fibrotic ECM coating to 20 µg reduced conjugate binding. To confirm in vivo delivery, immunofluorescence imaging of mouse heart tissue was performed (Figure 3A). A distinct signal confirmed PlGF2(123-144)-agrin’s presence and distribution within heart tissue, primarily in the interstitial space. Finally, we evaluated the conjugate's impact on CM proliferation in vivo. Representative images (Figure 3B) revealed significantly more EdU-positive (proliferating) CMs in the PlGF2(123-144)-agrin-treated group than in agrin-alone and PBS control groups. Quantitative analysis (Figure 3C) showed a statistically significant increase in proliferating CMs in the PlGF2(123-144)-agrin group, while controls showed minimal proliferation.

These preliminary findings indicate that the PlGF2(123-144)-agrin conjugate was successfully produced and delivered to heart tissue. Its enhanced binding to fibronectin and especially collagen I, key components of the fibrotic cardiac microenvironment, suggests potential for targeted delivery and action in fibrotic regions. The observed increase in CM proliferation highlights the conjugate's potential to stimulate cardiac regeneration in the context of fibrosis.

Acknowledgements and/or References (Optional):: This research was funded in part by the NSF CAREER award (JPJ).