Extracellular Perinexal Separation Is a Principal Determinant of Cardiac Conduction.

BACKGROUND: Cardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in the heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular resistance. By contrast, ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs such as the perinexus. Our objective was to determine the relationship between ventricular CV and structural changes to micro- and nanoscale extracellular spaces. METHODS: Conduction and Cx43 (connexin43) protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with the osmotic agents albumin, mannitol, dextran 70 kDa, or dextran 2 MDa. Peak sodium current was quantified in isolated guinea pig ventricular myocytes. Extracellular resistance was quantified by impedance spectroscopy. Intercellular communication was assessed in a heterologous expression system with fluorescence recovery after photobleaching. Perinexal width was quantified from transmission electron micrographs. RESULTS: CV primarily in the transverse direction of propagation was significantly reduced by mannitol and increased by albumin and both dextrans. The combination of albumin and dextran 70 kDa decreased CV relative to albumin alone. Extracellular resistance was reduced by mannitol, unchanged by albumin, and increased by both dextrans. Cx43 expression and conductance and peak sodium currents were not significantly altered by the osmotic agents. In response to osmotic agents, perinexal width, in order of narrowest to widest, was albumin with dextran 70 kDa; albumin or dextran 2 MDa; dextran 70 kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. CONCLUSIONS: Cardiac conduction does not correlate with extracellular resistance but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

Spatial Pattern Analysis using Closest Events (SPACE)-A Nearest Neighbor Point Pattern Analysis Framework for Assessing Spatial Relationships from Digital Images.

The quantitative description of biological structures is a valuable yet difficult task in the life sciences. This is commonly accomplished by imaging samples using fluorescence microscopy and analyzing resulting images using Pearson's correlation or Manders' co-occurrence intensity-based colocalization paradigms. Though conceptually and computationally simple, these approaches are critically flawed due to their reliance on signal overlap, sensitivity to cursory signal qualities, and inability to differentiate true and incidental colocalization. Point pattern analysis provides a framework for quantitative characterization of spatial relationships between spatial patterns using the distances between observations rather than their overlap, thus overcoming these issues. Here we introduce an image analysis tool called Spatial Pattern Analysis using Closest Events (SPACE) that leverages nearest neighbor-based point pattern analysis to characterize the spatial relationship of fluorescence microscopy signals from image data. The utility of SPACE is demonstrated by assessing the spatial association between mRNA and cell nuclei from confocal images of cardiac myocytes. Additionally, we use synthetic and empirical images to characterize the sensitivity of SPACE to image segmentation parameters and cursory image qualities such as signal abundance and image resolution. Ultimately, SPACE delivers performance superior to traditional colocalization methods and offers a valuable addition to the microscopist's toolbox.

Indirect Correlative Light and Electron Microscopy (iCLEM): A Novel Pipeline for Multiscale Quantification of Structure From Molecules to Organs.

Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform.

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.

Histologic, viral, and molecular correlates of heart disease in fatal COVID-19.

Cardiac manifestations are common in severe COVID-19. This study compared the histologic, viral, and molecular findings in cardiac tissue in fatal COVID-19 (n = 11) and controls (n = 11). In situ hybridization (SARS-CoV2 RNA) and immunohistochemistry for viral proteins and the host response were quantified for the samples and compared with qRTPCR and Western blot data. Control hearts showed a high resident population of macrophages that had variable ACE2 expression. Cardiac ACE2 expression was 10× greater in the heart tissues of cases and controls with obesity or type II diabetes. Multifocal endothelial cell swelling and degeneration, perivascular edema plus microvascular thrombi were unique to the cases. SARS-CoV2 RNA and nucleocapsid protein were rarely detected in situ in any COVID-19 heart. However, in each case abundant SARS-CoV-2 spike protein was evident. Co-expression experiments showed that the spike protein localized mostly to the ACE2+ interstitial macrophages/pericytes that were activated as evidenced by increased IL6 and TNFα expression. Western blots confirmed the presence of the viral spike protein, but not the nucleocapsid protein, in the cardiac homogenates. The intercalated disc proteins connexin 43, the primary cardiac gap junction protein, and NaV1.5, the predominant cardiac sodium channel, each showed marked lateral migration in the myocytes in the cases, which would increase the risk of reentrant arrhythmias. It is concluded that the viral spike protein, endocytosed by macrophages/pericytes, can induce a myocarditis with the possibility of conduction dysfunction due to abnormal localization of key intercalated disc proteins.

Muscarinic-dependent phosphorylation of the cardiac ryanodine receptor by protein kinase G is mediated by PI3K-AKT-nNOS signaling.

Post-translational modifications of proteins involved in calcium handling in myocytes, such as the cardiac ryanodine receptor (RyR2), critically regulate cardiac contractility. Recent studies have suggested that phosphorylation of RyR2 by protein kinase G (PKG) might contribute to the cardioprotective effects of cholinergic stimulation. However, the specific mechanisms underlying these effects remain unclear. Here, using murine ventricular myocytes, immunoblotting, proximity ligation as-says, and nitric oxide imaging, we report that phosphorylation of Ser-2808 in RyR2 induced by the muscarinic receptor agonist carbachol is mediated by a signaling axis comprising phosphoinositide 3-phosphate kinase, Akt Ser/Thr kinase, nitric oxide synthase 1, nitric oxide, soluble guanylate cyclase, cyclic GMP (cGMP), and PKG. We found that this signaling pathway is compartmentalized in myocytes, as it was distinct from atrial natriuretic peptide receptor-cGMP-PKG-RyR2 Ser-2808 signaling and independent of muscarinic-induced phosphorylation of Ser-239 in vasodilator-stimulated phosphoprotein. These results provide detailed insights into muscarinic-induced PKG signaling and the mediators that regulate cardiac RyR2 phosphorylation critical for cardiovascular function.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes.

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease.

Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Neuronal sodium channels: emerging components of the nano-machinery of cardiac calcium cycling.

Excitation-contraction coupling is the bridge between cardiac electrical activation and mechanical contraction. It is driven by the influx of Ca2+ across the sarcolemma triggering Ca2+ release from the sarcoplasmic reticulum (SR) - a process termed Ca2+ -induced Ca2+ release (CICR) - followed by re-sequestration of Ca2+ into the SR. The Na+ /Ca2+ exchanger inextricably couples the cycling of Ca2+ and Na+ in cardiac myocytes. Thus, influx of Na+ via voltage-gated Na+ channels (NaV ) has emerged as an important regulator of CICR both in health and in disease. Recent insights into the subcellular distribution of cardiac and neuronal NaV isoforms and their ultrastructural milieu have important implications for the roles of these channels in mediating Ca2+ -driven arrhythmias. This review will discuss functional insights into the role of neuronal NaV isoforms vis-à-vis cardiac NaV s in triggering such arrhythmias and their potential as therapeutic targets in the context of the aforementioned structural observations.

Potassium channels in the Cx43 gap junction perinexus modulate ephaptic coupling: an experimental and modeling study.

It was recently demonstrated that cardiac sodium channels (Nav1.5) localized at the perinexus, an intercalated disc (ID) nanodomain associated with gap junctions (GJ), may contribute to electrical coupling between cardiac myocytes via an ephaptic mechanism. Impairment of ephaptic coupling by acute interstitial edema (AIE)-induced swelling of the perinexus was associated with arrhythmogenic, anisotropic conduction slowing. Given that Kir2.1 has also recently been reported to localize at intercalated discs, we hypothesized that Kir2.1 channels may reside within the perinexus and that inhibiting them may mitigate arrhythmogenic conduction slowing observed during AIE. Using gated stimulated emission depletion (gSTED) and stochastic optical reconstruction microscopy (STORM) super-resolution microscopy, we indeed find that a significant proportion of Kir2.1 channels resides within the perinexus. Moreover, whereas Nav1.5 inhibition during AIE exacerbated arrhythmogenic conduction slowing, inhibiting Kir2.1 channels during AIE preferentially increased transverse conduction velocity-decreasing anisotropy and ameliorating arrhythmia risk compared to AIE alone. Comparison of our results with a nanodomain computer model identified enrichment of both Nav1.5 and Kir2.1 at intercalated discs as key factors underlying the experimental observations. We demonstrate that Kir2.1 channels are localized within the perinexus alongside Nav1.5 channels. Further, targeting Kir2.1 modulates intercellular coupling between cardiac myocytes, anisotropy of conduction, and arrhythmia propensity in a manner consistent with a role for ephaptic coupling in cardiac conduction. For over half a century, electrical excitation in the heart has been thought to occur exclusively via gap junction-mediated ionic current flow between cells. Further, excitation was thought to depend almost exclusively on sodium channels with potassium channels being involved mainly in returning the cell to rest. Here, we demonstrate that sodium and potassium channels co-reside within nanoscale domains at cell-to-cell contact sites. Experimental and computer modeling results suggest a role for these channels in electrical coupling between cardiac muscle cells via an ephaptic mechanism working in tandem with gap junctions. This new insight into the mechanism of cardiac electrical excitation could pave the way for novel therapies against cardiac rhythm disturbances.

Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: an experimental and modeling study.

It has long been held that electrical excitation spreads from cell-to-cell in the heart via low resistance gap junctions (GJ). However, it has also been proposed that myocytes could interact by non-GJ-mediated "ephaptic" mechanisms, facilitating propagation of action potentials in tandem with direct GJ-mediated coupling. We sought evidence that such mechanisms contribute to cardiac conduction. Using super-resolution microscopy, we demonstrate that Nav1.5 is localized within 200 nm of the GJ plaque (a region termed the perinexus). Electron microscopy revealed close apposition of adjacent cell membranes within perinexi suggesting that perinexal sodium channels could function as an ephapse, enabling ephaptic cell-to-cell transfer of electrical excitation. Acute interstitial edema (AIE) increased intermembrane distance at the perinexus and was associated with preferential transverse conduction slowing and increased spontaneous arrhythmia incidence. Inhibiting sodium channels with 0.5 µM flecainide uniformly slowed conduction, but sodium channel inhibition during AIE slowed conduction anisotropically and increased arrhythmia incidence more than AIE alone. Sodium channel inhibition during GJ uncoupling with 25 µM carbenoxolone slowed conduction anisotropically and was also highly proarrhythmic. A computational model of discretized extracellular microdomains (including ephaptic coupling) revealed that conduction trends associated with altered perinexal width, sodium channel conductance, and GJ coupling can be predicted when sodium channel density in the intercalated disk is relatively high. We provide evidence that cardiac conduction depends on a mathematically predicted ephaptic mode of coupling as well as GJ coupling. These data suggest opportunities for novel anti-arrhythmic therapies targeting noncanonical conduction pathways in the heart.

Mechanisms of cardiac conduction: a history of revisions.

Cardiac conduction is the process by which electrical excitation spreads through the heart, triggering individual myocytes to contract in synchrony. Defects in conduction disrupt synchronous activation and are associated with life-threatening arrhythmias in many pathologies. Therefore, it is scarcely surprising that this phenomenon continues to be the subject of active scientific inquiry. Here we provide a brief review of how the conceptual understanding of conduction has evolved over the last century and highlight recent, potentially paradigm-shifting developments.

Cardiac-Specific Deletion of Scn8a Mitigates Dravet Syndrome-Associated Sudden Death in Adults.

BACKGROUND: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear. OBJECTIVES: In this study, the authors sought to provide evidence implicating remodeling of Na+ - and Ca2+ -handling machinery, including NaV 1.6 and Na+/Ca2+exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias. METHODS: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS. RESULTS: DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death. CONCLUSIONS: These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure.

BACKGROUND: Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. OBJECTIVES: In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. METHODS: The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF. RESULTS: AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with NaV1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1. CONCLUSIONS: These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.