Hoagland, D. | Search bioRxiv

bioRxiv2023

Development and Characterization of the Mode-of-Action of Inhibitory and Agonist Peptides Targeting the Voltage-Gated Sodium Channel SCN1B/β1 Subunit

Treatment of cardiac arrhythmias by targeting ion channels is challenging, with safe and effective therapies remaining an unmet clinical need. Modeling and experimental studies have shown that a voltage-gated sodium channel (VGSC)-rich nanodomain at edge of the gap junction (GJ) called the perinexus could provide new mechanistic insights into normal and abnormal conduction of action potentials in the heart. We have reported that a 19 amino acid SCN1B ({beta}1/{beta}1B) mimetic peptide derived from the immunoglobulin domain of the VGSC subunit called {beta}adp1 acutely disrupts {beta}1-mediated adhesive interactions at cardiac perinexii, prompting arrhythmogenic changes during time courses of up to an hour. In the present study, we sought to gain further insight on {beta}adp1 mode-of-action, as well as identifying new SCN1B ({beta}1/{beta}1B) mimetic peptides, with potential for inhibiting and/or promoting {beta}1-mediated adhesion. This included studies of the effect of {beta}adp1 and related peptides on SCN1B ({beta}1/{beta}1B) Regulated Intramembrane Proteolysis (RIP) - a signaling pathway that has been shown to effect gene transcription, including that of VGSC subunits. Using patch clamp to assay cell-cell contact-associated VGSC activity in cardiomyocytes, and electric cell substrate impedance sensing (ECIS) to assess intercellular adhesion in cells heterologously expressing {beta}1, we find that inhibitory effects of {beta}adp1 can persist for up to 5 hours. However, this acute inhibition is not sustained, with {beta}adp1 effects on {beta}1-mediated adhesion lost after 24 hours. We also determined that a short peptide (LQLEED) near the carboxyl-terminal portion of {beta}adp1 inhibited adhesion in {beta}1-expressing cells in a manner similar to {beta}adp1. Paradoxically, dimeric peptides incorporating a repeat of the LQLEED sequence promoted intercellular adhesion at all time points studied over a 2-day time course. Inhibitory and agonistic peptides were found to effect {beta}1 RIP, with {beta}adp1increasing RIP continuously over 48 hours, whilst dimeric agonists acutely increased RIP at 6 hours post-treatment, but not thereafter. In the presence of DAPT, an inhibitor of RIP, the effects of {beta}adp1 on ECIS-measured intercellular adhesion were lost, suggesting a relationship between RIP and inhibitory effects of the peptide. In sum, we identify novel SCN1B ({beta}1/{beta}1B) mimetic peptides with potential to inhibit and promote intercellular {beta}1-mediated adhesion, possibly including by effects on {beta}1 RIP, suggesting paths to development of anti-arrhythmic drugs targeting the perinexus.

cell biology↗

bioRxiv2019

Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

BackgroundCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that CT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKC{varepsilon} phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of CT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function.\n\nMethods and ResultsTo determine the basis of CT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKC{varepsilon} phosphorylation, identifying an interaction between negatively charged amino acids in the CT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short -helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that CT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of CT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid CT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury.\n\nConclusionsInteraction of CT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.

cell biology↗