Abstract 1514: RXP-E: A Connexin43-Binding Peptide that Prevents Action Potential Propagation Block

Abstract

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).

Intracellular Redistribution of Left Ventricular Connexin 43 Contributes to the Remodeling of Electrical Properties of the Heart in Insulin-resistant Elderly Rats

“…63 Overall insight to this subject: innovation is needed for the production of connexin-based therapeutics, although there is little information about the use of connexin-based therapeutics in the clinic for the treatment of either arrhythmia or any other heart disease with an enhancement of gap-junctional communication (i.e., improvement of lowered conduction) via treatment with a connexinbased therapeutic. [65][66][67] Overall, we, here, demonstrated that not only reduced levels of pCx43 and Cx43 but also their apparent internalizations and lateralizations may be associated with abnormal ventricular repolarization, which in turn leads to long-QT in the Aged-group under insulin resistance, similar to the MetS-group. In this regard, although the underlying mechanisms of these two syndromes include alterations in electrophysiological properties of cardiomyocytes besides changes in the heart tissues, further studies are needed to clarify the exact mechanism responsible for the existence of these changes under insulin resistance.…”

“…63 Overall insight to this subject: innovation is needed for the production of connexin-based therapeutics, although there is little information about the use of connexin-based therapeutics in the clinic for the treatment of either arrhythmia or any other heart disease with an enhancement of gap-junctional communication (i.e., improvement of lowered conduction) via treatment with a connexin-based therapeutic. 65–67…”

Cx43 CT domain influences infarct size and susceptibility to ventricular tachyarrhythmias in acute myocardial infarction

“…dual whole-cell voltage clamp analysis in pairs of adult cardiac myocytes internally dialysed with a pipette solution buffered at pH 6.2. 19 Initial whole-cell conductances were not significantly different between cardiomyocyte pairs of either genotype (Cx43/KO: 27.2 + 5.2, NS, n ¼ 5; K258stop/KO: 25.3 + 3.6, NS, n ¼ 6; P . 0.05).…”

“…17 Cells were used for recording within 8 h after isolation. Details on recording of gap junctional currents can be found in Shibayama et al 18 and Lewandowski et al 19 and in Supplementary material online. Estimation of macroscopic junctional conductance (G j ) and series resistance correction followed methods previously described.…”

“…23,37 As such, disruption of these interactions may not have the same impact as the use of a chemical agent that preserves or improves coupling. It should be noted, however, that holding gap junctions open in the setting of intracellular acidification supported slow conduction, 19 which can facilitate re-entrant arrhythmias. 38,39 In summary, we have used a genetically modified murine model to directly characterize, for the first time, the role of the CT domain of Cx43 on cardiac infarct size and arrhythmia susceptibility.…”