Abstract 1514: RXP-E: A Connexin43-Binding Peptide that Prevents Action Potential Propagation Block
Gap junctions (GJ’s) provide a low resistance pathway for cardiac electrical propagation. The role of GJ regulation in arrhythmias is unclear, partly due to the limited availability of pharmacological tools. Recently, we showed that a peptide called “RXP-E” binds to the carboxyl terminal of connexin43 (Cx43) and prevents chemically-induced uncoupling in Cx43-expressing N2a cells. Here, we used pull-down experiments to show that RXP-E binds to adult cardiac Cx43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes (NRVM’s). Separately, RXP-E was concatenated to a cytoplasmic transduction peptide for translocation into the cytoplasm (construct CTP-RXP-E). The effect of RXP-E on action potential (AP) propagation was assessed by high resolution optical mapping in monolayers of NRVMs, containing approximately 20% of randomly distributed myofibroblasts. Conduction velocity (CV) was 164 ± 8mm/sec (avg±SEM; n=12; pacing frequency 2Hz) in untreated cells, and 158±10mm/sec, (n=6) and 180±7mm/sec (n=10) in monolayers treated with CTP-RXPE or a scrambled version of the peptide (CTP-Scr), respectively (pNS). Exposure of either untreated, or CTP-Scr-treated monolayers to heptanol caused propagation block. However, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, AP propagation was maintained, albeit at a slower velocity (87±5mm/sec;n=4; P<0.001). Similarly, intracellular acidification (pHi=6.2) caused a loss of AP propagation in control or CTP-Scr monolayers; however, propagation was maintained in CTP-RXP-E treated cells, though at a slower rate (CV=93 ± 28mm/sec; n=4). Consistent with these results, patch clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium or calcium currents, nor did it alter the voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to:
bind cardiac Cx43;
prevent heptanol and acidification-induced uncoupling of cardiac GJ’s and
preserve AP propagation among cardiac myocytes.
RXP-E can be used to characterize the role of GJ’s in the function of multicellular systems, including the heart.
This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).