Dissecting Gap Junctional and Ephaptic Contributions to Electrical Conduction in a Novel Cardiomyocyte Pair Model.

Electrical communication between excitable cells depends on both direct gap junction (GJ) currents and field-mediated ephaptic interactions, but their relative contributions have remained difficult to quantify experimentally, limiting mechanistic insight into arrhythmia and other disorders of bioelectric signaling in excitable tissues. Building on the concept of a nanoscale, sodium channel-rich perinexus at the cardiac intercalated disc, we developed a 'Single-on-Paired' (SoP) preparation in which whole-cell sodium current is recorded from one adult ventricular myocyte that remains end-to-end coupled to an intact partner. This configuration revealed a composite two-cell sodium current characterized by a unique pre-peak waveform which exhibits two slopes in the rising phase, and a pronounced activation jump in sodium current amplitude between closely spaced voltage steps. This feature was absent in isolated single myocytes and interpretable as an 'Intercalated Disc Signature' of intercellular activation. By combining graded GJ inhibition, perinexal widening via a Scn1b-derived competitive adhesion peptide, and controlled modulation of extracellular sodium, we show that low sodium conditions favor GJ-dominated activation, whereas at higher, more physiological sodium levels, perinexus-centered ephaptic mechanisms provide substantial support for intercellular activation. A complementary two-cell computational model, simulating our SoP model and incorporating lateral and junctional sodium channels, reproduces the two-cell 'Intercalated Disc Signature' and predicts a shift from GJ-dominated activation at low sodium to ephaptic-dominated support at higher, more physiological sodium concentrations when GJ conductance is reduced. Together, these results provide direct experimental evidence for a specific structural and molecular substrate of ephaptic coupling in the heart and establish a framework for dissecting how nanoscale extracellular cleft geometry, channel organization, sodium level, and GJ conductance jointly tune electric-field-based mechanisms of activation in excitable tissues, with implications for paradoxical clinical responses to anti-arrhythmic interventions.