Cardiac extracellular matrix-derived matrikine for cardiac regeneration following myocardial infarction

Introduction: : Cardiovascular disease (CVD) remains the leading cause of death worldwide, with myocardial infarction (MI) as the primary contributor to CVD-related deaths. Moderate-to-severe MI often progresses to heart failure, for which heart transplantation remains the only definitive treatment. Thus, developing regenerative strategies to repair the injured heart is critically important. A major obstacle to cardiac regeneration is the limited proliferative capacity of postnatal cardiomyocytes (CMs), which transition from hyperplastic to hypertrophic growth shortly after birth. The cardiac extracellular matrix (cECM) has been shown to support CM proliferation and tissue repair; however, the therapeutic potential of cECM-derived matrikines remains largely unexplored. Our group has identified a peptide sequence from fibrillin-1 (F1R1) that promotes CM proliferation in vitro that may act through modulating TGF-beta signaling, which could also alter the remodeling response of cardiac fibroblasts. In this study, we investigate the use of F1R1 as a regenerative therapeutic method to enhance cardiac repair following MI.

Materials and

Methods: : MI was induced in rats by permanent ligation of the left anterior descending (LAD) artery. Animals were randomly assigned to one of three groups: F1R1 peptide, scrambled F1R1 peptide, or non-treatment control. Both F1R1 and scrambled peptides (95% purity, GenScript) were solubilized in phosphate-buffered saline (PBS) and administered via injection immediately following MI. Cardiac function was assessed with echocardiography over a 2-month period. At the study endpoint, left ventricular (LV) pressure-volume (PV) loop measurements were performed to evaluate cardiac functional performance. Following tissue harvest, hearts were processed for histological analysis, bulk RNA sequencing, and ECM structure-function profiling using our novel analytical method.
In addition, in vitro studies were conducted to investigate the action of F1R1. Primary neonatal cardiac fibroblasts (CFs) were isolated from rat pups (P2) and cultured with serum-free media. TGF-β protein (Sino Biological) was added as a simulation of infarction in vitro and mixed with each cultural condition: F1R1 peptide, scrambled F1R1 peptide, and anti-TGF-β neutralizing antibody (eBioscience™). CFs were treated with the corresponding condition for 2 days, fixed and stained with Hoechst (nuclei), ɑ-smooth muscle actin (ɑ-SMA), and phalloidin (F-actin). The expression level of activated fibroblasts (myofibroblasts) was assessed. Further signaling pathways associated with CM proliferation and repair will be performed via western blotting.

Results, Conclusions, and Discussions:: The in vivo study results demonstrated that the F1R1-treated rats showed increased ejection fraction (EF) (%) compared to scrambled F1R1 and non-treatment controls for 2 months (Fig. 1a). Studies have shown that a low EF following MI is associated with an increased risk of heart failure, which supports our hypothesis in F1R1 peptide can promote cardiac repair post-MI. Fast green histology staining showed that scrambled F1R1-treated hearts have a more significant loss of LV wall thickness than the F1R1 group (Fig. 1b), which indicated a more severe damage from the injury.
To investigate the potential mechanism of the action of F1R1 on improving cardiac repair post-injury, the Basic Local Alignment Search Tool (BLAST) sequencing method was utilized to identify the alignment sequence between F1R1 and Latent TGF-β binding protein-2 (LGBT2) (Fig. 2a). The PEP-site Finder was further employed to identify the potential binding site of F1R1 on TGF-β, the finger 2 region (Fig. 2b). These findings indicate that there is a high probability that F1R1 can mitigate fibrosis responses through binding with TGF-β. A series of in vitro experiments will be conducted to support our hypothesis, starting with in vitro stimulation of TGF-β in neonatal rat cardiac fibroblasts and using F1R1 as a treatment, with anti-TGF-β neutralizer antibody as a positive control and non-treatment as a negative control (Fig. 3). Protein-protein interactions will be carried out to identify F1R1 and TGF-β through western blotting. Together, these findings suggest that F1R1 promotes cardiac repair post-injury, potentially by modulating TGF-β signaling and reducing fibrosis.

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