The myosuppressin structure-activity relationship for cardiac contractility and its receptor interactions support the presence of a ligand-directed signaling pathway in heart.

The structural conservation and activity of the myosuppressin cardioinhibitory peptide across species suggests it plays an important role in physiology, yet much remains unknown regarding its signaling. We previously reported Drosophila melanogaster myosuppressin (dromyosuppressin, DMS; TDVDHVFLRF-NH) decreases cardiac contractility through a G protein-coupled receptor, DMS-R2. Our study showed the DMS N-terminus amino acids influence its structure-activity relationship (SAR), yet how they act is not established. We predicted myosuppressin N-terminal amino acids played a role in signaling. Here, we tested our hypothesis in the beetle, Zophobas atratus, using a semi-isolated heart bioassay to explore SAR in a different Order and focus on cardiac signaling. We generated a series of myosuppressin truncated analogs by removing the N-terminal residue and measuring the activity of each structure on cardiac contractility. While DVDHVFLRF-NH decreased cardiac contractility, we found VDHVFLRF-NH, DHVFLRF-NH, and HVFLRF-NH increased activity. In contrast, VFLRF- NH decreased activity and FLRF-NH was inactive. Next, we analyzed molecular docking data and found the active truncated analogs interacted with the 3-6 lock in DMS-R2, the myosuppressin cardiac receptor, disrupting the salt bridge between H114 and E369, and K289 and Q372. Further, the docking results showed the inhibitory effect on contractility may be associated with contact to Y78, while the analogs that increased contractility lacked this interaction. The data from our study demonstrated N-terminal amino acids played a role in myosuppressin activity and signaling suggesting the cardiac receptor can be targeted by biased agonists. Our myosuppressin cardiac contractility data and predicted receptor interactions describe the presence of functional selectivity in a ligand-directed signaling pathway in heart.