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Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action, and the potential for these molecules in the treatment of disease.

The relationship between cardiac conduction velocity (CV) and extracellular potassium (K+) is biphasic, with modest hyperkalemia increasing CV and severe hyperkalemia slowing CV. Recent studies from our group suggest that elevating extracellular sodium (Na+) and calcium (Ca2+) can enhance CV by an extracellular pathway parallel to gap junctional coupling (GJC) called ephaptic coupling that can occur in the gap junction adjacent perinexus. However, it remains unknown whether these same interventions modulate CV as a function of K+. We hypothesize that Na+, Ca2+, and GJC can attenuate conduction slowing consequent to severe hyperkalemia. Elevating Ca2+ from 1.25 to 2.00 mM significantly narrowed perinexal width measured by transmission electron microscopy. Optically mapped, Langendorff-perfused guinea pig hearts perfused with increasing K+ revealed the expected biphasic CV-K+ relationship during perfusion with different Na+ and Ca2+ concentrations. Neither elevating Na+ nor Ca2+ alone consistently modulated the positive slope of CV-K+ or conduction slowing at 10-mM K+; however, combined Na+ and Ca2+ elevation significantly mitigated conduction slowing at 10-mM K+. Pharmacologic GJC inhibition with 30-mu M carbenoxolone slowed CV without changing the shape of CV-K+ curves. A computational model of CV predicted that elevating Na+ and narrowing clefts between myocytes, as occur with perinexal narrowing, reduces the positive and negative slopes of the CV-K+ relationship but do not support a primary role of GJC or sodium channel conductance. These data demonstrate that combinatorial effects of Na+ and Ca2+ differentially modulate conduction during hyperkalemia, and enhancing determinants of ephaptic coupling may attenuate conduction changes in a variety of physiologic conditions.

Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action, and the potential for these molecules in the treatment of disease.

Recent studies revealed that relatively small changes in perfusate sodium ([Na+](o)) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+](o) modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+](o) that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia-reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia-reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+](o) decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+](o) at baseline and reduced the time to peak ischemic contracture. Ischemia-reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+](o) further depressed mechanical function. In summary, elevating [Na+](o) by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+](o), with cardiac ischemia may require further investigation.

We previously demonstrated that altering extracellular sodium (Nao) and calcium (Cao) can modulate a form of electrical communication between cardiomyocytes termed "ephaptic coupling" (EpC), especially during loss of gap junction coupling. We hypothesized that altering Nao and Cao modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Nao (147 or 155 mM) and Cao (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( WP), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Nao and 2.0 mM Cao; however, theoretically increasing EpC with 155 mM Nao was arrhythmogenic, and CV could not be measured. Reducing Cao to 1.25 mM expanded WP, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing WP with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded WP, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia.

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that beta 1(SCN1B) - mediated adhesion scaffolds trans-activating Na(V)1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential beta 1 localization at the perinexus, where it co-locates with Na(V)1.5. Smart patch clamp (SPC) indicated greater sodium current density (I-Na) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, beta adp1, potently and selectively inhibited beta 1-mediated adhesion, in electric cell-substrate impedance sensing studies. beta adp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal I-Na, but not whole cell I-Na, in myocyte monolayers. In optical mapping studies, beta adp1 precipitated arrhythmogenic conduction slowing. In summary, beta 1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Aims: Atrial fibrillation (AF) is the most common sustained arrhythmia. Previous evidence in animal models suggests that the gap junction (GJ) adjacent nanodomain - perinexus - is a site capable of independent intercellular communication via ephaptic transmission. Perinexal expansion is associated with slowed conduction and increased ventricular arrhythmias in animal models, but has not been studied in human tissue. The purpose of this study was to characterize the perinexus in humans and determine if perinexal expansion associates with AF. Methods: Atrial appendages from 39 patients (pts) undergoing cardiac surgery were fixed for immunofluorescence and transmission electron microscopy (TEM). Intercalated disk distribution of the cardiac sodium channel Nav1.5, its beta 1 subunit, and connexin43 (C x 43) was determined by confocal immunofluorescence. Perinexal width (Wp) from TEM was manually segmented by two blinded observers using ImageJ software. Results: Nav1.5, beta 1, and C x 43 are co-adjacent within intercalated disks of human atria, consistent with perinexal protein distributions in ventricular tissue of other species. TEM revealed that the GJ adjacent intermembrane separation in an individual perinexus does not change at distances greater than 30 nm from the GJ edge. Importantly, Wp is significantly wider in patients with a history of AF than in patients with no history of AF by approximately 3 nm, and Wp correlates with age (R = 0.7, p < 0.05). Conclusion: Human atrial myocytes have voltage-gated sodium channels in a dynamic intercellular cleft adjacent to GJs that is consistent with previous descriptions of the perinexus. Further, perinexal width is greater in patients with AF undergoing cardiac surgery than in those without.

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that beta 1(SCN1B) - mediated adhesion scaffolds trans-activating Na(V)1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential beta 1 localization at the perinexus, where it co-locates with Na(V)1.5. Smart patch clamp (SPC) indicated greater sodium current density (I-Na) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, beta adp1, potently and selectively inhibited beta 1-mediated adhesion, in electric cell-substrate impedance sensing studies. beta adp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal I-Na, but not whole cell I-Na, in myocyte monolayers. In optical mapping studies, beta adp1 precipitated arrhythmogenic conduction slowing. In summary, beta 1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

Aims: Atrial fibrillation (AF) is the most common sustained arrhythmia. Previous evidence in animal models suggests that the gap junction (GJ) adjacent nanodomain - perinexus - is a site capable of independent intercellular communication via ephaptic transmission. Perinexal expansion is associated with slowed conduction and increased ventricular arrhythmias in animal models, but has not been studied in human tissue. The purpose of this study was to characterize the perinexus in humans and determine if perinexal expansion associates with AF. Methods: Atrial appendages from 39 patients (pts) undergoing cardiac surgery were fixed for immunofluorescence and transmission electron microscopy (TEM). Intercalated disk distribution of the cardiac sodium channel Nav1.5, its beta 1 subunit, and connexin43 (C x 43) was determined by confocal immunofluorescence. Perinexal width (Wp) from TEM was manually segmented by two blinded observers using ImageJ software. Results: Nav1.5, beta 1, and C x 43 are co-adjacent within intercalated disks of human atria, consistent with perinexal protein distributions in ventricular tissue of other species. TEM revealed that the GJ adjacent intermembrane separation in an individual perinexus does not change at distances greater than 30 nm from the GJ edge. Importantly, Wp is significantly wider in patients with a history of AF than in patients with no history of AF by approximately 3 nm, and Wp correlates with age (R = 0.7, p < 0.05). Conclusion: Human atrial myocytes have voltage-gated sodium channels in a dynamic intercellular cleft adjacent to GJs that is consistent with previous descriptions of the perinexus. Further, perinexal width is greater in patients with AF undergoing cardiac surgery than in those without.