Biology subjects

Veeraraghavan, R.

Publications and source records attributed to Veeraraghavan, R..

10 recordsLinked to original sources

bioRxiv2026

Cardiac unloading via a transvalvular pump prevents arrhythmias following myocardial ischemia / reperfusion by preserving coronary microcirculation and cardiomyocyte nanostructure

Cardiac damage in the aftermath of myocardial ischemia / reperfusion (MI/R) compromises both electrical and mechanical function, predisposing patients to arrhythmia and heart failure. Microvascular obstruction (MVO) is increasingly recognized as a sequela of MI/R and a contributor to proarrhythmic cardiac remodeling. While transvalvular unloading (TVU) using a percutaneously delivered pump is garnering appreciation as a means of limiting infarct size and preserving mechanical function following percutaneous coronary intervention, its benefits for arrhythmia prevention remain unclear. We used a porcine model to test the hypothesis that cardiac unloading prior to and through reperfusion mitigates MVO, preserves cardiomyocyte nanostructure, and alleviates arrhythmias following MI/R. Anesthetized domestic swine undergoing acute ischemia (LAD occlusion, 90 mins) and reperfusion (120 mins) were randomized to TVU (n=5) and no treatment (control; n=6) treatments. Thioflavin-S was infused into the mid-LAD to assess coronary microcirculation and quantify MVO. Arrhythmia burden was assessed by in vivo electrocardiography using 3 lead EKG. Cardiac tissues were preserved for histology and optical microscopy studies. Compared to no treatment controls, TVU hearts evidenced significantly reduced infarct area (48 +/- 5 % vs. 59 +/- 11 % p = 0.03) and MVO (19 +/- 4 % vs. 27 +/- 5% , p = 0.03) normalized to area at risk and a higher myocardial salvage index (52 +/- 5 % vs. 41 +/- 11% p = 0.03). Extensive perivascular edema, and neutrophil infiltration observed in controls was markedly reduced in TVU hearts. Confocal microscopy and spatial pattern analysis revealed significantly mitigated disruption of gap junctions and NaV1.5 dissociation from N-cadherin in TVU hearts compared to controls. Importantly, severity-adjusted arrhythmia burden during reperfusion was significantly lower in TV-P hearts (8+/-8 vs. 12+/-5 episodes lasting greater than 5seconds in controls). Our results demonstrate that TVU effectively prevents arrhythmias during reperfusion by preserving coronary microvasculature and cardiomyocyte nanostructure.

physiology↗

bioRxiv2025

CLUSTERING DYNAMICALLY MODULATE THE BIOPHYSICS OF VOLTAGE-GATED SODIUM CHANNELS: HOW NANOSCALE PHENOMENA DETERMINE HEALTH AND DISEASE

Precise regulation of ion channel biophysics is an essential life process that governs electrical signaling in excitable tissues. Many ion channels including voltage-gated Na+ channels (NaVs) exist in the membrane as clusters, which show distinct biophysical behavior not predicted by single-channel measurements. In both heterologous and native systems, we report that single-channel-based predictions significantly overestimated Na+ current (INa) amplitudes from multi-channel clusters. Computational modeling suggested that these observations could reflect interactions between adjacent channels, such as recently reported between NaVs, and identified specific biophysical consequences thereof. This updated model not only accurately predicted behaviors observed from NaV clusters and consequent cellular physiology, but also suggested the possibility that clustered NaVs may respond differently to use-dependent pharmacological agents. Experiments validated the latter prediction and further identified modulation of clustering as a novel approach to correcting macroscopic electrophysiological dysfunction resulting from NaV defects linked to life-threatening arrhythmias and seizures. Thus, our study not only motivates a fundamental revision of how ion channels behave when clustered but also highlights resulting biophysical effects as important considerations for pharmacology and a potential therapeutic target to address human disease.

physiology↗

bioRxiv2025

Selectivity filter mutation in NaV1.5 promotes ventricular tachycardia

Loss-of-Function (LoF) mutations in the SCN5A gene, which encodes for the predominant cardiac NaV isoform, NaV1.5 result in either deficiency in the channel expression or function. Impaired NaV1.5 expression and function underlie reduced peak Na+ current (INa) and result in ventricular conduction velocity slowing, predisposing the heart to conduction block and ventricular arrhythmias clinically associated with Brugada syndrome (BrS). Recently, a missense mutation in NaV1.5 selectivity filter (DEKA motif), K1419E (DEEA) has been identified in patients with BrS. Despite early characterization of mutations in selectivity filter of other NaV isoforms, little is known about the impact of DEEA on NaV1.5 function as well as on cardiac electrophysiology. Therefore, we generated a mouse heterozygous for NaV1.5 DEEA to characterize the mutation and investigate the outcome of this functionally deficient NaV1.5 variant on cardiac electrophysiology and arrhythmias. Heterologous expression system and isolated cardiomyocytes revealed lower current density and unchanged NaV1.5 expression in DEEA vs. wild type (DEKA). On the organ level, optical mapping revealed conduction velocity slowing in DEEA hearts, which was accentuated by flecainide resulting in vivo ventricular arrhythmias. Overall, to our knowledge, we provide the first mechanistic insight into the proarrhythmic consequences of a functionally deficient BrS mutation in NaV1.5. Condensed abstractNaV1.5 mutations have been associated with life-threatening arrhythmias. Recently, a selectivity filter mutation (K1419E-NaV1.5, DEKA[->]DEEA), has been linked to Brugada Syndrome (BrS). While DEKA mutations in other NaV isoforms affected channel conductance, the impact of DEEA on NaV1.5 and arrhythmogenesis is unknown. Therefore, we generated mice heterozygous for NaV1.5-DEEA. Cardiomyocytes isolated from DEEA hearts exhibited substantial reduction in sodium current, ventricular conduction slowing and susceptibility to ventricular arrhythmias in vivo that were unmasked by flecainide. Together, DEEA murine model is the first to recapitulate a functional deficiency in NaV1.5, and thus offers insight into the proarrhythmic mechanism of BrS.

physiology↗

bioRxiv2023

Vascular Endothelial Barrier Protection Prevents Atrial Fibrillation by Preserving Cardiac Nanostructure

Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting [~]3% of the US population. It is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). The mechanism underlying AF is not well understood and current treatments are limited to managing this progressive disease, rather than arresting the underlying pathology. We previously identified edema-induced disruption of sodium channel (NaV1.5) -rich intercalated disk (ID) 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. We identified two molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak. AF incidence was increased in untreated mice exposed to VEGF relative to vehicle controls. VEGF also increased the average number of AF episodes. VEGF shifted NaV1.5 signal to longer distances from Cx43 gap junctions (GJs), measured by a distance transformation-based spatial analysis of 3D confocal images of IDs. Similar effects were observed with NaV1.5 localized near mechanical junctions (MJs) composed of N-cad. Blocking connexin43 hemichannels (CT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared to VEGF alone with no treatment. Concurrently, both peptide therapies preserved NaV1.5 distance from GJs to control levels and reduced MJ-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small molecule inhibitors of Cx43 and Panx1.

physiology↗

bioRxiv2023

EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

RationaleCardiac conduction is understood to occur through gap junctions. Recent evidence supports ephaptic coupling as another mechanism of electrical communication in heart. Conduction via gap junctions predicts a direct relationship between conduction velocity (CV) and bulk extracellular volume. Ephaptic theory is premised on the existence of a biphasic relationship between CV and the volume of specialized extracellular clefts within intercalated discs. ObjectiveDetermine the relationship between ventricular CV and structural changes to micro and nano-scale extracellular spaces. MethodsConduction and connexin43 (Cx43) gap junction protein expression were quantified from optically mapped guinea pig whole-heart preparations perfused with albumin, mannitol, dextran 70kDa, or dextran 2MDa. Peak sodium current was quantified from isolated guinea pig ventricular myocytes. Extracellular resistance (Re) 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. ResultsCV was significantly reduced by mannitol, and increased by albumin, dextran 70kDa and 2MDa. The combination of albumin and dextran 70kDa decreased CV relative to albumin alone. Re was reduced by mannitol, not significantly changed by albumin, and increased by both dextran 70kDa and dextran 2MDa. Cx43 gap junction expression and conductance, and peak sodium current were not significantly altered by the osmotic agents. The perinexal width in response to osmotic agents, in order of narrowest to widest, was: albumin with dextran 70kDa, albumin or dextran 2MDa alone, dextran 70kDa or no osmotic agent, and mannitol. When compared in the same order, CV was biphasically related to perinexal width. ConclusionsCardiac conduction does not correlate with bulk tissue impedance, but is biphasically related to perinexal separation, providing evidence that the relationship between CV and extracellular volume in ventricular myocardium is determined by ephaptic mechanisms under conditions of normal gap junctional coupling.

biophysics↗

bioRxiv2023

Spatial Pattern Analysis using Closest Events (SPACE): A Nearest Neighbor Point Pattern Analysis Framework for Assessing Spatial Relationships from Image Data

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 Pearsons 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 microscopists toolbox.

bioengineering↗

bioRxiv2023

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 which 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.

systems biology↗

bioRxiv2023

Unraveling Chamber-specific Differences in Intercalated Disc Ultrastructure and Molecular Organization and Their Impact on Cardiac Conduction

During each heartbeat, the propagation of action potentials through the heart coordinates the contraction of billions of individual cardiomyocytes and is thus, a critical life process. Unsurprisingly, intercalated discs, which are cell-cell contact sites specialized to provide electrical and mechanical coupling between adjacent cardiomyocytes, have been the focus of much investigation. Slowed or disrupted propagation leads to potentially life-threatening arrhythmias in a wide range of pathologies, where intercalated disc remodeling is a common finding. Hence, the importance and urgency of understanding intercalated disc structure and its influence on action potential propagation. Surprisingly, however, conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, owing to lack of quantitative structural data at subcellular through nano scales. In order to address this critical gap in knowledge, we sought to quantify intercalated disc structure at these finer spatial scales in the healthy adult mouse heart and relate them to function in a chamber-specific manner as a precursor to understanding the impacts of pathological intercalated disc remodeling. Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by inter-chamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. These data provide the first stepping stone to elucidating chamber-specific impacts of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.

physiology↗

bioRxiv2021

The Cx43 Carboxyl-Terminal Mimetic Peptide alphaCT1 Protects Endothelial Barrier Function in a ZO1 Binding-Competent Manner

The Cx43 CT mimetic peptide, CT1, originally designed to bind to ZO1 and thereby inhibit Cx43/ZO1 interaction, was used as a tool to probe the role of Cx43/ZO1 association in regulation of epithelial/endothelial barrier function. Using both in vitro and ex vivo methods of barrier function measurement, including Electric Cell-Substrate Impedance Sensing(ECIS), a FITC-dextran transwell permeability assay, and a FITC-dextran cardiovascular leakage protocol involving Langendorff-perfused mouse hearts, CT1 was found to protect the endothelium from thrombin-induced breakdown in cell-cell contacts. Barrier protection was accompanied by significant remodeling of the F-actin cytoskeleton, characterized by a redistribution of F-actin away from the cytoplasmic and nuclear regions of the cell, towards the endothelial cell periphery, in association with alterations in cellular orientation distribution. In line with observations of increased cortical F-actin, CT1 upregulated cell-cell border localization of endothelial VE-cadherin, the Tight Junction protein Zonula Occludens 1 (ZO1), and the Gap Junction Protein (GJ) Connexin43 (Cx43). A ZO1-binding-incompetent variant of CT1, CT1-I, indicated that these effects on barrier function and barrier-associated proteins, were likely associated with Cx43 CT sequences retaining ability to interact with ZO1. These results implicate the Cx43 CT and its interaction with ZO1, in the regulation of endothelial barrier function, while revealing the therapeutic potential of CT1 in the treatment of vascular edema.

cell biology↗

bioRxiv2021

Intercalated Disk Nanoscale Structure Regulates Cardiac Conduction

The intercalated disk (ID) is a specialized subcellular region that provides electrical and mechanical connections between myocytes in the heart. The ID has a clearly defined passive role in cardiac tissue, transmitting mechanical forces and electrical currents between cells. Recent studies have shown that Na+ channels, the primary current responsible for cardiac excitation, are preferentially localized at the ID, particularly within nanodomains around mechanical and gap junctions, and that perturbations of ID structure alter cardiac conduction. This suggests that the ID may play an important, active role in regulating conduction. However, the structure of the ID and intercellular cleft are not well characterized, and to date, no models have incorporated the influence of ID structure on conduction in cardiac tissue. In this study, we developed an approach to generate realistic finite element model (FEM) meshes replicating ID nanoscale structure, based on experimental measurements from transmission electron microscopy (TEM) images. We then integrated measurements of the intercellular cleft electrical conductivity, derived from the FEM meshes, into a novel cardiac tissue model formulation. FEM-based calculations predict that the distribution of cleft conductances are sensitive to regional changes in ID structure, specifically the intermembrane separation and gap junction distribution. Tissue-scale simulations demonstrated that ID structural heterogeneity leads to significant spatial variation in electrical polarization within the intercellular cleft. Importantly, we find that this heterogeneous cleft polarization regulates conduction by desynchronizing the activation of post-junctional Na+ currents. Additionally, these heterogeneities lead to a weaker dependence of conduction velocity on gap junctional coupling, compared with prior modeling formulations that neglect or simplify ID structure. Further, we find that disruption of local ID nanodomains can lead to either conduction slowing or enhancing, depending on gap junctional coupling strength. Overall, our study demonstrates that ID nanoscale structure can play a significant role in regulating cardiac conduction.

bioengineering↗