RXP-E: a connexin43-binding peptide that prevents action potential propagation block.
Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.
Lewandowski
Rebecca
R
Departments of Pharmacology, State University of New York Upstate Medical University, Syracuse, USA.
Procida
Kristina
K
Vaidyanathan
Ravi
R
Coombs
Wanda
W
Jalife
José
J
Nielsen
Morten S
MS
Taffet
Steven M
SM
Delmar
Mario
M
D000143:Acids / Q000494:pharmacology; D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D015195:Drug Design; D017629:Gap Junctions / Q000502:physiology*; D019850:Heptanol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology; D011485:Protein Binding; D051381:Rats; D015222:Sodium Channels / Q000502:physiology
D000143:Acids; D002352:Carrier Proteins; D018031:Connexin 43; C490968:Kcnj4 protein, rat; C489461:Kir2.1 channel; D062554:NAV1.5 Voltage-Gated Sodium Channel; D024661:Potassium Channels, Inwardly Rectifying; C532149:RXP-E peptide; C568322:Scn5a protein, rat; D015222:Sodium Channels; D019850:Heptanol