Biology subjects

Gourdie, R. G.

Publications and source records attributed to Gourdie, R. G..

12 recordsLinked to original sources

bioRxiv2026

Concealed Conduction Vulnerability in Scn1b Haploinsufficiency Emerges with Osmotic Stress

RationaleSCN1B encodes the {beta}-subunits of the main cardiac voltage-gated sodium channel, NaV1.5. Variants are linked to cardiac conduction disease, often with concealed phenotypes. Whether {beta}1-subunits regulate conduction through nanoscale intercalated disc (ID) structures, e.g. perinexi, and ephaptic coupling remains unresolved. ObjectiveTest whether Scn1b haploinsufficiency induces latent conduction abnormalities that are unmasked by perturbations in extracellular nanodomains. Methods and ResultsAdult Scn1b+/- mice and wild-type (WT) littermates underwent multiscale phenotyping (qRT-PCR, Western blot, patch clamp, transmission electron microscopy (TEM), ex vivo optical mapping, in vivo ECG). Scn1b+/- hearts showed [~]50% reductions in Scn1b mRNA and {beta}1 protein without changes in canonical conduction proteins. Peak sodium current, baseline conduction velocity ex vivo, and baseline QRS duration in vivo were unchanged. However, TEM revealed increased baseline perinexal width in Scn1b+/- hearts. Osmotic expansion of the perinexus with mannitol slowed conduction to a greater extent in Scn1b+/- hearts and prolonged QRS duration in vivo. In contrast, perinexal narrowing with dextran 2MDa selectively increased conduction velocity in Scn1b+/- hearts. ConclusionsScn1b haploinsufficiency preserves baseline excitability and conduction but structurally remodels the ID at the nanoscale, increasing sensitivity to extracellular nanodomain perturbations. These data support a structural role for {beta}1-subunits in ephaptic coupling, and that conduction is maintained over a range of perinexal widths with pathological conduction slowing occurring beyond a critical width. Importantly, osmotic stress unmasks a concealed conduction phenotype, identifying extracellular nanodomain stability as a potential therapeutic target to mitigate arrhythmia risk in SCN1B-associated disease.

biophysics↗

bioRxiv2026

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.

biophysics↗

bioRxiv2025

Hypernatremia Enhances Transient Outward Potassium and Late Sodium Currents in a Mouse Model of Long QT Syndrome Type 3

Cardiac voltage-gated sodium channel gain-of-function (NavGOF) is characterized by action potential duration (APD) prolongation. Hypernatremia and perinexal widening synergistically prolong cardiac APD in guinea pig. However, guinea pig lack the transient outward potassium current (Ito), which could be increased by hypernatremia and thereby shorten APD. ObjectiveDetermine whether hypernatremia and perinexal expansion synergistically prolong APD in an animal model functionally expressing Ito. MethodsWhole-cell Ito was measured in isolated genetically-modified NavGOF ({Delta}KPQ) mouse ventricular myocytes. Ventricular APD at 30 (APD30) and 90 (APD90) percent repolarization were measured from optically mapped, Langendorff-perfused wild-type (WT) and {Delta}KPQ mouse hearts at different perfusate sodium concentrations (145 or 160mM), without and with the perinexal adhesion antagonist {beta}adp1. ResultsIn isolated myocytes, hypernatremia (160mM sodium) increased Ito. In whole-heart, hypernatremia significantly decreased both APD30 and APD90 in WT but only APD30 in {Delta}KPQ preparations. Perinexal disruption with {beta}adp1 did not change APD30 or APD90 in WT hearts, however it decreased APD30 and increased APD90 in {Delta}KPQ hearts. Combination of hypernatremia and {beta}adp1 did not synergistically change APD in {Delta}KPQ hearts. Computational models predict that Ito activation can prevent synergistic APD prolongation in mouse during hypernatremia and perinexal expansion that was observed previously in a guinea pig NavGOF model lacking Ito. ConclusionsHypernatremia during NavGOF prevents early ventricular repolarization due to Ito activation (mouse) and prolongs repolarization in the absence of Ito (guinea pig). Future studies in animal models electrophysiologically similar to humans are needed to determine if hypernatremia and perinexal expansion are proarrhythmic during NavGOF.

physiology↗

bioRxiv2025

Orally Delivered Milk-Derived Nanovesicles Loaded with Connexin 43 Peptides forTargeted Cardiac Ischemia-Reperfusion Therapy

Extracellular vesicles have emerged as promising nanocarriers for targeted drug delivery, but their therapeutic potential is limited by challenges related to administration route, loading, targeted delivery and production at scale. Here, we report an innovative approach for targeted delivery of therapeutic peptides to injured tissues using milk-derived small extracellular vesicles (mEVs) as an abundant, safe, orally administrable nanoplatform. We demonstrate that a sub-population of mEVs naturally contain Connexin 43 (Cx43) and its Carboxyl-Terminal (CT) polypeptides, which have been shown to play crucial roles in wound healing and tissue repair. Leveraging this intrinsic property, we developed an esterification method to efficiently and uniformly load mEVs with enhanced levels of an exogenous Cx43 CT peptide (CT11 - RPRPDDLEI), as assessed by flow cytometry-based vesicle quantification and mass spectrometry. These engineered mEVs exhibited remarkable injury targeting capabilities, with > 30-fold increases in uptake by injured cells compared to non-wounded cells in vitro and preferential accumulation in wounded tissues in vivo. Notably, CT11-loaded mEVs orally administered after myocardial infarction reduced infarct size by >60% and preserved heart function in a mouse model of ischemia-reperfusion injury. This study represents a significant advance in nanomedicine, demonstrating the utilization of naturally occurring milk-derived extracellular vesicles as an oral delivery system for therapeutic peptides, achieving unprecedented targeting efficiency and efficacy in the treatment of myocardial ischemia-reperfusion injury.

bioengineering↗

bioRxiv2024

Detection, Isolation and Quantification of Myocardial Infarct with Four Different Histological Staining Techniques

Precise quantification of myocardial infarction is crucial for evaluating the therapeutic strategies. We developed a robust, color-based semi-automatic algorithm capable of infarct region detection, isolation and quantification with four different histological staining techniques, and the isolation and quantification of diffuse fibrosis in the heart. Our method is developed based on the color difference in the infarct and non-infarct regions after histological staining. Mouse cardiac tissues stained with Massons trichrome (MTS), hematoxylin and eosin (H&E), 2,3,5-Triphenyltetrazolium chloride and picrosirius red were included to demonstrate the performance of our method. We demonstrate that our algorithm can effectively identify and produce a clear visualization of infarct tissue for the four staining techniques. Notably, the infarct region on a H&E-stained tissue section can be clearly visualized after processing. The MATLAB-based program we developed holds promise in the infarct quantification. Additionally, our program can isolate and quantify the diffuse fibrotic elements from an MTS-stained cardiac section, which suggested the algorithms potential for evaluating pathological cardiac fibrosis in diseased cardiac tissues. In conclusion, we demonstrate that this color-based algorithm is capable of accurately identifying, isolating and quantifying cardiac infarct regions with different staining techniques, as well as the diffuse and patchy fibrosis in MTS-stained cardiac tissues.

pathology↗

bioRxiv2024

Increased interaction between connexin43 and microtubules is critical for glioblastoma stem-like cell maintenance and tumorigenicity.

Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells hold the ability to self-renew or differentiate following treatment, participating in tumor recurrence. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 with microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully ablated Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro, and limited GSC-derived tumor growth in vivo. Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.

cancer biology↗

bioRxiv2023

Development and Characterization of the Mode-of-Action of Inhibitory and Agonist Peptides Targeting the Voltage-Gated Sodium Channel SCN1B/β1 Subunit

Treatment of cardiac arrhythmias by targeting ion channels is challenging, with safe and effective therapies remaining an unmet clinical need. Modeling and experimental studies have shown that a voltage-gated sodium channel (VGSC)-rich nanodomain at edge of the gap junction (GJ) called the perinexus could provide new mechanistic insights into normal and abnormal conduction of action potentials in the heart. We have reported that a 19 amino acid SCN1B ({beta}1/{beta}1B) mimetic peptide derived from the immunoglobulin domain of the VGSC subunit called {beta}adp1 acutely disrupts {beta}1-mediated adhesive interactions at cardiac perinexii, prompting arrhythmogenic changes during time courses of up to an hour. In the present study, we sought to gain further insight on {beta}adp1 mode-of-action, as well as identifying new SCN1B ({beta}1/{beta}1B) mimetic peptides, with potential for inhibiting and/or promoting {beta}1-mediated adhesion. This included studies of the effect of {beta}adp1 and related peptides on SCN1B ({beta}1/{beta}1B) Regulated Intramembrane Proteolysis (RIP) - a signaling pathway that has been shown to effect gene transcription, including that of VGSC subunits. Using patch clamp to assay cell-cell contact-associated VGSC activity in cardiomyocytes, and electric cell substrate impedance sensing (ECIS) to assess intercellular adhesion in cells heterologously expressing {beta}1, we find that inhibitory effects of {beta}adp1 can persist for up to 5 hours. However, this acute inhibition is not sustained, with {beta}adp1 effects on {beta}1-mediated adhesion lost after 24 hours. We also determined that a short peptide (LQLEED) near the carboxyl-terminal portion of {beta}adp1 inhibited adhesion in {beta}1-expressing cells in a manner similar to {beta}adp1. Paradoxically, dimeric peptides incorporating a repeat of the LQLEED sequence promoted intercellular adhesion at all time points studied over a 2-day time course. Inhibitory and agonistic peptides were found to effect {beta}1 RIP, with {beta}adp1increasing RIP continuously over 48 hours, whilst dimeric agonists acutely increased RIP at 6 hours post-treatment, but not thereafter. In the presence of DAPT, an inhibitor of RIP, the effects of {beta}adp1 on ECIS-measured intercellular adhesion were lost, suggesting a relationship between RIP and inhibitory effects of the peptide. In sum, we identify novel SCN1B ({beta}1/{beta}1B) mimetic peptides with potential to inhibit and promote intercellular {beta}1-mediated adhesion, possibly including by effects on {beta}1 RIP, suggesting paths to development of anti-arrhythmic drugs targeting the perinexus.

cell biology↗

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↗

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↗

bioRxiv2020

Connexin 43 confers chemoresistance through activating PI3K

Circumventing chemoresistance is crucial for effectively treating glioblastoma due to limited therapeutic options. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to the frontline chemotherapy temozolomide; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we show that Cx43, but not other connexins, is highly expressed in glioblastoma and strongly correlates with poor patient prognosis and chemoresistance, making Cx43 the prime therapeutic target among all connexins. The intracellular carboxyl terminus of Cx43 binds to phosphatidylinositol 3-kinase (PI3K) catalytic subunit {beta} (PIK3CB, also called PI3K{beta} or p110{beta}), thereby activating PI3K signaling independent of Cx43-channels and subsequently inducing temozolomide resistance. A combination of CT1, a Cx43-targeting peptide inhibitor, and PIK3CB-selective inhibitors restores temozolomide sensitivity in vitro and in vivo. This study not only reveals novel mechanistic insights into chemoresistance in glioblastoma, but also demonstrates that targeting Cx43 and PIK3CB/p110{beta} is an effective approach for overcoming chemoresistance.

cancer biology↗

bioRxiv2020

The Connexin 43 Carboxyl Terminal Mimetic Peptide alphaCT1 Prompts Differentiation of a Collagen Scar Matrix Resembling Unwounded Skin

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide αCT1 improves cutaneous scar appearance by 47% 9-months post-surgery – though mode-of-action remains unknown. Scar matrix structure in biopsies 2 to 6 weeks post-wounding treated topically with αCT1 or control treatments from human subjects, Sprague-Dawley rats, and IAF hairless guinea pigs were compared. The sole effect on scar structure in humans was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds, a state that resembles unwounded skin. This more random alignment was recapitulated in both animal models, together with transient increases in collagen density, although the guinea pig was found to more closely replicate the pattern of response to αCT1 in human scars, compared to rat. Fibroblasts treated with αCT1 in vitro showed decreased directionality and an agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix post-wounding that is similar to unwounded skin – changes that correlate with long-term improvement in scar appearance.Competing Interest StatementGSG is CEO and President of FirstString Research Inc. CLG is Senior Director of Research and Development at FirstString Research Inc. RGG is a non-remunerated member of the Scientific Advisory Board of FirstString Research, which licensed α carboxyl terminus 1 peptide. GSG, RGG, LJJ, and CLG have ownership interests in FirstString Research Inc. The remaining authors have no disclosures to report.View Full Text

cell biology↗

bioRxiv2019

Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

BackgroundCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that CT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKC{varepsilon} phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of CT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function.\n\nMethods and ResultsTo determine the basis of CT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKC{varepsilon} phosphorylation, identifying an interaction between negatively charged amino acids in the CT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short -helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that CT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of CT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid CT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury.\n\nConclusionsInteraction of CT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.

cell biology↗