Computational modeling indicates that cardiac conduction may involve ephaptic coupling – intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) –mediated adhesion scaffolds trans-activating Na_V1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with Na_V1.5. Smart patch clamp (SPC) indicated greater sodium current density (I_Na) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 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, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-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 β1 subunit, and connexin43 (C×43) was determined by confocal immunofluorescence. Perinexal width (Wp) from TEM was manually segmented by two blinded observers using ImageJ software. Results: Nav1.5, β1, and C×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.

We previously demonstrated that altering extracellular sodium (Na_o) and calcium (Ca_o) can modulate a form of electrical communication between cardiomyocytes termed “ephaptic coupling” (EpC), especially during loss of gap junction coupling. We hypothesized that altering Na_o and Ca_o modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Na_o (147 or 155 mM) and Ca_o (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction-adjacent perinexal width ( W_P), 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 Na_o and 2.0 mM Ca_o; however, theoretically increasing EpC with 155 mM Na_o was arrhythmogenic, and CV could not be measured. Reducing Ca_o to 1.25 mM expanded W_P, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing W_P with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded W_P, 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.

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.

BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43 ^Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43 ^Ser368 are protected against mouse adenovirus type-3–induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3–infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased I _K1 and I _Ks current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43 ^Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.

In recent decades, the scientific community has seen an increased interest in rigor and reproducibility. In 2017, concerns about methodological thoroughness and reporting practices were implicated as significant barriers to reproducibility within the preclinical cardiovascular literature, particularly in studies using animal research. The Langendorff, whole heart technique has proven to be an invaluable research tool, being modified in a myriad of ways to probe questions across the spectrum of physiological and pathophysiological functions of the heart. As a result, significant variability in the application of the Langendorff technique exists. This literature review quantifies the different methods employed in the implementation of the Langendorff technique and provides brief examples of how individual parametric differences can impact the outcomes and interpretation of studies. From 2017 to 2020, significant variability of animal models, anesthesia, cannulation time, perfusate composition, pH, and temperature demonstrate that the technique has diversified to meet new challenges and answer different scientific questions. The review also reveals which individual methods are most frequently reported, even if there is no explicit agreement upon which parameters should be reported. The analysis of methods related to the Langendorff technique suggests a framework for considering methodological approach when interpreting seemingly contradictory results, rather than concluding that results are irreproducible.