Induction of cardioprotection by small netrin-1-derived peptides

We have shown that netrin-1 induces potent cardioprotection via extracellular signal-regulated kinases 1 and 2 (ERK1/2)-dependent endothelial nitric oxide synthase (eNOS)/NO activation. The present study investigated cardioprotective effects of small netrin-1-derived peptides. We synthesized three laminin (Lam) V peptides and found those time dependently increased phosphorylation of ERK1/2 and eNOSs1179 in endothelial cells at the same molar concentration used for netrin-1. Preperfusion with Lam V peptides induced a substantial reduction in infarct size (control 39.3 ± 0.2% vs. 14.6 ± 2.3%, 23.0 ± 2.8%, and 18.8 ± 0.8% for V1, V2, and V3, respectively). Furthermore, reperfusion with all three also induced potent cardioprotection (control 37.6 ± 1.3% vs. 17.6 ± 3.2%, 20.6 ± 1.7%, and 15.8 ± 2.0% for V1, V2, and V3, respectively), implicating that these peptides are consistently beneficial whenever they are delivered to the heart. Based on the sequence alignment, we found a region of high sequence homology and synthesized smaller peptides [V1-9 amino acid (aa), V2-10aa, and V3-11aa]. These smaller peptides markedly reduced infarct size during reperfusion (V1-9aa 16.8 ± 2.2%, V2-10aa 18.6 ± 1.7%, and V3-11aa 16.7 ± 3.0% vs. control 37.6 ± 1.3%). A negative control V3-16aa with no sequence homology failed to protect the heart. Of note, the core area has the characteristic sequence of: Cx(1-2)Cx(3-4)Tx(0-1)G. Furthermore, all three smaller peptides induced NO production in endothelial cells that could in turn diffuse to cardiomyocytes to promote survival. Combined applications of V1-9aa and V2-10aa synergistically induced more NO production. Taken together, these data strongly suggest that small netrin-1-derived peptides are highly effective in protecting the heart against myocardial ischemia-reperfusion injury and has the potential to be developed into peptide drugs directly applicable to the treatment of myocardial infarction.