Browsing by Author "Poelzing, Steven"

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    The adhesion function of the sodium channel beta subunit (beta 1) contributes to cardiac action potential propagation
    Veeraraghavan, Rengasayee; Hoeker, Gregory S.; Alvarez-Laviada, Anita; Hoagland, Daniel T.; Wan, Xiaoping; King, D. Ryan; Sanchez-Alonso, Jose; Chen, Chunling; Jourdan, L. Jane; Isom, Lori L.; Deschenes, Isabelle; Smith, James W.; Gorelik, Julia; Poelzing, Steven; Gourdie, Robert G. (2018-08-14)
    Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that beta 1(SCN1B) - mediated adhesion scaffolds trans-activating Na(V)1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential beta 1 localization at the perinexus, where it co-locates with Na(V)1.5. Smart patch clamp (SPC) indicated greater sodium current density (I-Na) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, beta adp1, potently and selectively inhibited beta 1-mediated adhesion, in electric cell-substrate impedance sensing studies. beta adp1 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, beta adp1 precipitated arrhythmogenic conduction slowing. In summary, beta 1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.
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    Arrhythmogenic mechanisms of acute cardiac infection
    Padget, Rachel Lee (Virginia Tech, 2022-04-06)
    Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection.
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    Building a Better Scar: Re-engineering Extracellular Matrix Structure in Dermal Scars
    Montgomery, Jade (Virginia Tech, 2020-01-27)
    Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes.
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    The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats
    Allen, Mitchell E.; Pennington, Edward Ross; Perry, Justin B.; Dadoo, Sahil; Makrecka-Kuka, Marina; Dambrova, Maija; Moukdar, Fatiha; Patel, Hetal D.; Han, Xianlin; Kidd, Grahame K.; Benson, Emily K.; Raisch, Tristan B.; Poelzing, Steven; Brown, David A.; Shaikh, Saame Raza (2020-07-17)
    Allen and Pennington et al. show that the cardiolipin-binding peptide elamipretide mitigates disease-induced fragmentation of cristae networks following cardiac ischemia reperfusion in rats. This study suggests that elamipretide targets mitochondrial membranes to sustain cristae networks, improving their bioenergetic function. Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.
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    Cellular Size, Gap Junctions, and Sodium Channel Properties Govern Developmental Changes in Cardiac Conduction
    Nowak, Madison B.; Veeraraghavan, Rengasayee; Poelzing, Steven; Weinberg, Seth H. (2021-10-25)
    Electrical conduction in cardiac ventricular tissue is regulated via sodium (Na+) channels and gap junctions (GJs). We and others have recently shown that Na(+)channels preferentially localize at the site of cell-cell junctions, the intercalated disc (ID), in adult cardiac tissue, facilitating coupling via the formation of intercellular Na(+)nanodomains, also termed ephaptic coupling (EpC). Several properties governing EpC vary with age, including Na(+)channel and GJ expression and distribution and cell size. Prior work has shown that neonatal cardiomyocytes have immature IDs with Na(+)channels and GJs diffusively distributed throughout the sarcolemma, while adult cells have mature IDs with preferentially localized Na(+)channels and GJs. In this study, we perform an in silico investigation of key age-dependent properties to determine developmental regulation of cardiac conduction. Simulations predict that conduction velocity (CV) biphasically depends on cell size, depending on the strength of GJ coupling. Total cell Na(+)channel conductance is predictive of CV in cardiac tissue with high GJ coupling, but not correlated with CV for low GJ coupling. We find that ephaptic effects are greatest for larger cells with low GJ coupling typically associated with intermediate developmental stages. Finally, simulations illustrate how variability in cellular properties during different developmental stages can result in a range of possible CV values, with a narrow range for both neonatal and adult myocardium but a much wider range for an intermediate developmental stage. Thus, we find that developmental changes predict associated changes in cardiac conduction.
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    The conduction velocity-potassium relationship in the heart is modulated by sodium and calcium
    King, D. Ryan; Entz, Michael, II; Blair, Grace A.; Crandell, Ian; Hanlon, Alexandra L.; Lin, Joyce; Hoeker, Gregory S.; Poelzing, Steven (2021-03)
    The relationship between cardiac conduction velocity (CV) and extracellular potassium (K+) is biphasic, with modest hyperkalemia increasing CV and severe hyperkalemia slowing CV. Recent studies from our group suggest that elevating extracellular sodium (Na+) and calcium (Ca2+) can enhance CV by an extracellular pathway parallel to gap junctional coupling (GJC) called ephaptic coupling that can occur in the gap junction adjacent perinexus. However, it remains unknown whether these same interventions modulate CV as a function of K+. We hypothesize that Na+, Ca2+, and GJC can attenuate conduction slowing consequent to severe hyperkalemia. Elevating Ca2+ from 1.25 to 2.00 mM significantly narrowed perinexal width measured by transmission electron microscopy. Optically mapped, Langendorff-perfused guinea pig hearts perfused with increasing K+ revealed the expected biphasic CV-K+ relationship during perfusion with different Na+ and Ca2+ concentrations. Neither elevating Na+ nor Ca2+ alone consistently modulated the positive slope of CV-K+ or conduction slowing at 10-mM K+; however, combined Na+ and Ca2+ elevation significantly mitigated conduction slowing at 10-mM K+. Pharmacologic GJC inhibition with 30-mu M carbenoxolone slowed CV without changing the shape of CV-K+ curves. A computational model of CV predicted that elevating Na+ and narrowing clefts between myocytes, as occur with perinexal narrowing, reduces the positive and negative slopes of the CV-K+ relationship but do not support a primary role of GJC or sodium channel conductance. These data demonstrate that combinatorial effects of Na+ and Ca2+ differentially modulate conduction during hyperkalemia, and enhancing determinants of ephaptic coupling may attenuate conduction changes in a variety of physiologic conditions.
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    Detection, Isolation and Quantification of Myocardial Infarct with Four Different Histological Staining Techniques
    Wu, Xiaobo; Meier, Linnea; Liu, Tom X.; Toldo, Stefano; Poelzing, Steven; Gourdie, Robert G. (MDPI, 2024-10-18)
    Background/Objectives: The precise quantification of myocardial infarction is crucial for evaluating 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 of the isolation and quantification of diffuse fibrosis in the heart. Methods: Our method is developed based on the color difference in the infarct and non-infarct regions after histological staining. Mouse cardiac tissues stained with Masson’s trichrome (MTS), hematoxylin and eosin (H&E), 2,3,5-Triphenyltetrazolium chloride and picrosirius red were included to demonstrate the performance of our method. Results: We demonstrate that our algorithm can effectively identify and produce a clear visualization of infarct tissue in the four staining techniques. Notably, the infarct region on an H&E-stained tissue section can be clearly visualized after processing. The MATLAB-based program we developed holds promise for infarct quantification. Additionally, our program can isolate and quantify diffuse fibrotic elements from an MTS-stained cardiac section, which suggests the algorithm’s potential for evaluating pathological cardiac fibrosis in diseased cardiac tissues. Conclusions: 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 diffuse and patchy fibrosis in MTS-stained cardiac tissues.
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    Dissemination of Health Promotion Information in Cooperative Extension:  A multi-study exploration of channels, sources, and characteristics that influence intervention uptake
    Strayer, Thomas Edward III (Virginia Tech, 2019-05-09)
    A translational gap exists between the development of an evidence-based health promotion intervention and its eventual implementation in the intended setting. This lack of translation impacts the uptake of health promotion interventions within delivery systems such as the Cooperative Extension Service (Extension). Within this system, Extension educators serve as the intermediaries addressing needs in the communities in which they are employed with support from Extension health specialists. Previous research has shown that educators utilize other peer educators and specialists to learn and adopt health promotion programming, but these studies are over two decades old (e.g., missing technological advances such as Internet and social media) and often focused on a single state Extension system. The purpose of this research was to understand how evidence-based health promotion intervention information is shared within Cooperative Extension by 1) identifying information sources and channels of Extension specialists and educators while 2) identifying the characteristics of an intervention that aid in the adoption and uptake of these health promotion interventions. The first mixed-methods study aimed to identify information sources and channels used by Extension educators from a national sample and learn their preferences for information delivery. Results of this study (Manuscript 1), identified specialists as the key information source Therefore, the second study (Manuscript 2) focused on Extension health specialists' preferences for information sources and channels while also 1) determine how specialists communicate with educators 2) preliminary thoughts on a dissemination intervention. The final study (Manuscript 3) explored the intervention characteristics that are both educator and specialists deemed most important to their adoption decision-making process. The results of this dissertation inform the development of a dissemination intervention to bridge the translational gap across Extension. The information sources and channels used and trusted by both Extension educators and specialists are highlighted in this work. Additionally, specialists have given insight for consideration for an online repository that can be used on demand to both facilitate the adoption and uptake of health promotion interventions as needed by Extension Educators.
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    Distinguishing between overdrive excited and suppressed ventricular beats in guinea pig ventricular myocardium
    Greer-Short, Amara D.; Poelzing, Steven (Frontiers, 2015-02-18)
    Rapid ventricular pacing rates induces two types of beats following pacing cessation: recovery cycle length (RCL) prolongation (overdrive suppression) and RCL shortening (overdrive excitation). The goals of this study were to compare common experimental protocols for studying triggered activity in whole-heart preparations and differentiate between recovery beats using a new methodology. Post-pacing recovery beat cycle length (RCL) and QRS were normalized to pre-paced R-R and QRS intervals and analyzed using a K-means clustering algorithm. Control hearts only produced suppressed beats: RCL ratio increased with rapid pacing (25 +/- 4.0%, n = 10) without changing QRS duration. Rapid pacing during hypercalcemia + hypothermia (5.5 mM and 34 degrees C) produced significantly earlier excited beats (53 +/- 14%, n = 5) with wider QRS durations (58 +/- 6.3%, n = 5) than suppressed beats. Digoxin + hypothermia (0.75 mu M) produced the most excited beats with significantly earlier RCL (44 +/- 3.2%, n = 6) and wider QRS (60 +/- 3.1%, n = 6) ratios relative to suppressed beats. Increasing pacing further shortened RCL (30 +/- 7.8%, n = 6). In a prospective study, TTX (100 nM) increased RCL ratio (15 +/- 6.0%, n = 10) without changing the QRS duration of excited beats. The algorithm was compared to a cross-correlation analysis with 93% sensitivity and 94% specificity. This ECG based algorithm distinguishes between triggered and automatic activity.
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    Diverse Mechanisms Impair Thalamic Circuit Function in a Dravet Syndrome Mouse Model
    Studtmann, Carleigh (Virginia Tech, 2022-04-06)
    Dravet syndrome (DS) is an infantile epileptic encephalopathy that is caused by loss-of-function mutations in the SCN1A gene, which encodes the voltage-gated sodium channel, NaV1.1. Haploinsufficiency of NaV1.1 in DS patients leads to imbalanced excitability across brain circuits, resulting in a broad phenotypic profile including drug-resistant convulsive and non-convulsive (absence) seizures, cognitive impairment, ataxia, and sleep disruption. Dysfunction in the somatosensory corticothalamic (CT) circuit underlies several DS phenotypes including absence seizures and sleep disturbances. Yet, the precise mechanisms underlying somatosensory CT circuit dysfunction in DS remain unclear. Here, we sought to identify the cellular and synaptic mechanisms underlying somatosensory CT circuit dysfunction in a haploinsufficiency DS mouse model. This work reveals that NaV1.1 haploinsufficiency leads to cell-type-specific changes in the excitability of reticular thalamic (nRT), ventral posterolateral (VPL), and ventral posteromedial (VPM) neurons. Further, we identified alterations in both glutamatergic and GABAergic synaptic connectivity within the somatosensory CT circuit in DS mice. These findings introduce glutamatergic neuron dysfunction and synaptic alterations as novel disease mechanisms underlying thalamic circuit dysfunction in DS, providing new targets for therapeutic intervention. In addition, we reveal that VPL and VPM neurons exhibit distinct firing properties in a healthy CT circuit, suggesting they differentially contribute to circuit-wide function in health and dysfunction in disease.
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    Effects of Perfusate Composition (Na+ and Ca2+) on Cardiac Electrical and Mechanical Function in the Isolated Langendorff-Perfused Heart
    King, David Ryan (Virginia Tech, 2021-02-11)
    Following the landmark studies on scientific reproducibility, or the lack thereof, by Bayer and Amgen in the past decade, there has been a renewed interest in scientific rigor and reproducibility in both the scientific and public media. In several recent reports, the high attrition rate observed in clinical trials has been attributed to irreproducibility at the preclinical level. Cardiology is no exception to this rule. In our systematic review of the ex vivo Langendorff-perfused heart, we found methods reporting to be sparse at best, specifically as it pertains to documenting the ex vivo perfusate compositions employed in the Langendorff heart. Our lab has demonstrated that variation in perfusate compositions can unmask disease states in genetically modified animals. In this dissertation, we exploit this concept with a therapeutic end-point in mind. We show that perfusate variation, specifically sodium and calcium elevations, can attenuate conduction slowing associated with severe hyperkalemia. Likewise, elevating sodium is capable of sustaining intrinsic rhythm where hearts would otherwise go asystolic. In doing so, elevated sodium prevents repolarization prolongation in these hearts. Together, these studies would suggest that elevating extracellular sodium, and calcium, should be considered as therapeutic targets in the context of conduction defects. However, when considering the heart's primary role as a pump, we found that elevating sodium actually depresses cardiac mechanical function. This is both in a pre- and post-ischemic setting. In short, we show that electrolyte variation may influence both cardiac electrophysiology and contraction; however, an improvement in one does not guarantee an improvement in both. Maintaining proper cardiac physiological function is a complex process that is tightly regulated by the ionic makeup of the extracellular environment. To improve insights from preclinical studies at the clinical level it is paramount that researchers properly document methods so that any significant results may be properly interpreted in clinical trial design.
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    Effects of Perfusate Solution Composition on the Relationship between Cardiac Conduction Velocity and Gap Junction Coupling
    Entz, Michael William II (Virginia Tech, 2018-01-16)
    Reproducibility of results in biomedical research is an area of concern that should be paramount for all researchers. Importantly, this issue has been examined for experiments concerning cardiac electrophysiology. Specifically, multiple labs have found differences in results when comparing cardiac conduction velocity (CV) between healthy mice and mice that were heterozygous null for the gap junction (GJ) forming protein, Connexin 43. While the results of the comparison study showed differing extracellular ionic concentrations of the perfusates, specifically sodium, potassium, and calcium ([Na+]o, [K+]o, and [Ca2+]o), there was a lack of understanding why certain combinations of the aforementioned ions led to specific CV changes. However, more research from our lab indicates that these changes can predict modifications to a secondary form of cardiac coupling known as ephaptic coupling (EpC). Therefore the work in this dissertation was twofold, 1) to examine the effects of modulating EpC through perfusate ionic concentrations while also modulating GJC and 2) to investigate the effects of modulating all three of the main ions contributed with cardiac conduction (Na+, K+, Ca2+) and the interplay between them. Firstly I designed and tested changes from the use of 3D printed bath for optical mapping procedures. After verification that the bath did not modify electrophysiological or contrile parameters, I studied the effects of physiologic changes to EpC determinants ([Na+]o and [K+]o) on CV during various states of GJ inhibition using the non-specific GJ uncoupler carbenoxolone (CBX). Multiple pacing rates were used to further modify EpC, as an increased pacing rate leads to a decrease in sodium channel availability through modification of the resting membrane potential. with no to low (0 and 15 µM CBX) GJ inhibition, physiologic changes in [Na+]o and [K+]o did not affect CV, however increasing pacing rate decreased CV as expected. When CBX was increased to 30 µM, a combination of decreasing [Na+]o and increasing [K+]o significantly decreased cardiac CV, specifically when pacing rate was increased. Next, the combinatory effects of cations associated with EpC (Na+, K+, and Ca2+) were tested in to examine how cardiac CV reacts to changes in perfusate solution and how this may explain differences in experimental outcomes between laboratories. Briefly, experiments were run where [K+]o was varied throughout an experiment and the values for [Na+]o and [Ca2+]o were at one of two specific values during an experiment. 30 µM CBX was added to half of the experiments to see the changes in the CV-[K+]o relationship with GJ inhibition. With unaltered GJ coupling, elevated [Na+]o maintains CV during hyperkalemia. Interestingly, both [Na+]o and [Ca2+]o must be increased to maintain normal CV during hyperkalemia with reduced GJ coupling. These data suggest that optimized fluids can sustain normal conduction under pathophysiologic conditions like hyperkalemia and GJ uncoupling. Taken as a whole, this dissertation attempts to shed light on the importance of ionic concentration balance in perfusate solutions on cardiac conduction.
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    Electrophysiologic effects of the I-K1 inhibitor PA-6 are modulated by extracellular potassium in isolated guinea pig hearts
    Hoeker, Gregory S.; Skarsfeldt, Mark A.; Jespersen, Thomas; Poelzing, Steven (The Physiological Society, 2017-01)
    The pentamidine analog PA-6 was developed as a specific inward rectifier potassium current (I-K1) antagonist, because established inhibitors either lack specificity or have side effects that prohibit their use in vivo. We previously demonstrated that BaCl2, an established I-K1 inhibitor, could prolong action potential duration (APD) and increase cardiac conduction velocity (CV). However, few studies have addressed whether targeted I-K1 inhibition similarly affects ventricular electrophysiology. The aim of this study was to determine the effects of PA-6 on cardiac repolarization and conduction in Langendorff-perfused guinea pig hearts. PA-6 (200 nm) or vehicle was perfused into ex-vivo guinea pig hearts for 60 min. Hearts were optically mapped with di-4-ANEPPS to quantify CV and APD at 90% repolarization (APD(90)). Ventricular APD90 was significantly prolonged in hearts treated with PA-6 (115 +/- 2% of baseline; P < 0.05), but not vehicle (105 +/- 2% of baseline). PA-6 slightly, but significantly, increased transverse CV by 7%. PA-6 significantly prolonged APD90 during hypokalemia (2 mmol/L [K+](o)), although to a lesser degree than observed at 4.56 mmol/L [K+](o). In contrast, the effect of PA-6 on CV was more pronounced during hypokalemia, where transverse CV with PA-6 (24 +/- 2 cm/sec) was significantly faster than with vehicle (13 +/- 3 cm/sec, P < 0.05). These results show that under normokalemic conditions, PA-6 significantly prolonged APD90, whereas its effect on CV was modest. During hypokalemia, PA-6 prolonged APD90 to a lesser degree, but profoundly increased CV. Thus, in intact guinea pig hearts, the electrophysiologic effects of the I-K1 inhibitor, PA-6, are [K+](o)-dependent.
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    Elevated perfusate [Na+] increases contractile dysfunction during ischemia and reperfusion
    King, D. Ryan; Padget, Rachel L.; Perry, Justin B.; Hoeker, Gregory S.; Smyth, James W.; Brown, David A.; Poelzing, Steven (2020-10-14)
    Recent studies revealed that relatively small changes in perfusate sodium ([Na+](o)) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+](o) modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+](o) that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia-reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia-reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+](o) decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+](o) at baseline and reduced the time to peak ischemic contracture. Ischemia-reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+](o) further depressed mechanical function. In summary, elevating [Na+](o) by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+](o), with cardiac ischemia may require further investigation.
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    Ephaptic Coupling Is a Mechanism of Conduction Reserve During Reduced Gap Junction Coupling
    Lin, Joyce; Abraham, Anand; George, Sharon A.; Greer-Short, Amara; Blair, Grace A.; Moreno, Angel; Alber, Bridget R.; Kay, Matthew W.; Poelzing, Steven (Frontiers, 2022-05-05)
    Many cardiac pathologies are associated with reduced gap junction (GJ) coupling, an important modulator of cardiac conduction velocity (CV). However, the relationship between phenotype and functional expression of the connexin GJ family of proteins is controversial. For example, a 50% reduction of GJ coupling has been shown to have little impact on myocardial CV due to a concept known as conduction reserve. This can be explained by the ephaptic coupling (EpC) theory whereby conduction is maintained by a combination of low GJ coupling and increased electrical fields generated in the sodium channel rich clefts between neighboring myocytes. At the same time, low GJ coupling may also increase intracellular charge accumulation within myocytes, resulting in a faster transmembrane potential rate of change during depolarization (dV/dt_max) that maintains macroscopic conduction. To provide insight into the prevalence of these two phenomena during pathological conditions, we investigated the relationship between EpC and charge accumulation within the setting of GJ remodeling using multicellular simulations and companion perfused mouse heart experiments. Conduction along a fiber of myocardial cells was simulated for a range of GJ conditions. The model incorporated intercellular variations, including GJ coupling conductance and distribution, cell-to-cell separation in the intercalated disc (perinexal width-W-P), and variations in sodium channel distribution. Perfused heart studies having conditions analogous to those of the simulations were performed using wild type mice and mice heterozygous null for the connexin gene Gja1. With insight from simulations, the relative contributions of EpC and charge accumulation on action potential parameters and conduction velocities were analyzed. Both simulation and experimental results support a common conclusion that low GJ coupling decreases and narrowing W-P increases the rate of the AP upstroke when sodium channels are densely expressed at the ends of myocytes, indicating that conduction reserve is more dependent on EpC than charge accumulation during GJ uncoupling.
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    Extracellular Spaces and Cardiac Conduction
    Raisch, Tristan B. (Virginia Tech, 2019-04-22)
    Despite decades of research and thousands of studies on cardiac electrophysiology, cardiovascular disease remains among the leading causes of death in the United States today. Despite substantially beneficial advances, we have largely shifted cardiovascular disease from an acute to a chronic issue. It is therefore clear that our current understanding of the heart's functions remain inadequate and we must search for untapped therapeutic approaches to eliminate these deadly and costly ailments once and for all. This thesis will focus on the electrophysiology of the heart, specifically the mechanisms of cell-to-cell conduction. Canonically, the understood mechanism of cardiac conduction is through gap junctions (GJ) following a cable-like conduction model. While both experimentally and mathematically, this understanding of conduction has explained cardiac electrical behavior, it is also incomplete, as evidenced by recent conflicting modeling and experimental data. The overall goal of this thesis is to explore a structure modulating an ephaptic, or electric field, cellular coupling mechanism: the GJ-adjacent perinexus, with three specific aims. First, I identified the perinexus – a recently-established structure in rodent myocardium – in human atrial tissue. I also observed a significant tendency for open-heart surgery patients with pre-operative atrial fibrillation to have wider perinexi, indicating a possibly targetable mechanism of atrial fibrillation, one of the costliest, and most poorly-understood cardiac diseases. Next, I developed a high-throughput, high-resolution method for quantifying the perinexus. Finally, I sought to reconcile a major controversy in the field: whether cardiac edema could either be beneficial or harmful to cardiac conduction. Using a Langendorff perfusion model, I added osmotic agents of various sizes to guinea pig hearts and measured electrical and structural parameters. My findings suggest that while cardiac conduction is multifaceted and influenced by several parameters, the strongest correlation is an inverse relationship between conduction velocity and the width of the perinexus. This study is the first to osmotically expand and narrow the perinexus and show an inverse correlation with conduction. Importantly, my conduction data cannot be explained by factors consistent with a cable-like conduction mechanism, indicating once again that the perinexus could be a therapeutic target for a myriad of cardiac conduction diseases.
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    Heart Rate and Extracellular Sodium and Potassium Modulation of Gap Junction Mediated Conduction in Guinea Pigs
    Entz, Michael, II; George, Sharon A.; Zeitz, Michael J.; Raisch, Tristan B.; Smyth, James W.; Poelzing, Steven (Frontiers, 2016-02-02)
    Background: Recent studies suggested that cardiac conduction in murine hearts with narrow perinexi and 50% reduced connexin43 (Cx43) expression is more sensitive to relatively physiological changes of extracellular potassium ([K+](o)) and sodium ([Na+](o)). Purpose: Determine whether similar [K+](o) and [Na+](o) changes alter conduction velocity (CV) sensitivity to pharmacologic gap junction (GJ) uncoupling in guinea pigs. Methods: [K+](o) and [Na+](o) were varied in Langendorff perfused guinea pig ventricles (Solution A: [K+](o) = 4.56 and [N+](o) = 153.3 mM. Solution B: [K+](o) = 6.95 and [Na+](o) = 145.5 mM). Gap junctions were inhibited with carbenoxolone (CBX) (15 and 30 mu M). Epicardial CV was quantified by optical mapping. Perinexal width was measured with transmission electron microscopy. Total and phosphorylated Cx43 were evaluated by western blotting. Results: Solution composition did not alter CV under control conditions or with 15 mu M CBX. Decreasing the basic cycle length (BCL) of pacing from 300 to 160 ms decreased CV uniformly with both solutions. At 30 mu M CBX, a change in solution did not alter CV either longitudinally or transversely at BCL = 300 ms. However, reducing BCL to 160 ms caused CV to decrease more in hearts perfused with Solution B than A. Solution composition did not alter perinexal width, nor did it change total or phosphorylated serine 368 Cx43 expression. These data suggest that the solution dependent CV changes were independent of altered perinexal width or GJ coupling. Action potential duration was always shorter in hearts perfused with Solution B than A. independent of pacing rate and/or CBX concentration. Conclusions: Increased heart rate and GJ uncoupling can unmask small CV differences caused by changing [K+](o) and [Na+](o). These data suggest that modulating extracellular ionic composition may be a novel anti-arrhythmic target in diseases with abnormal GJ coupling, particularly when heart rate cannot be controlled.
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    Interaction of α Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia‐Reperfusion Injury
    Jiang, Jingbo; Hoagland, Daniel T.; Palatinus, Joseph A.; He, Huamei; Iyyathurai, Jegan; Jourdan, L. Jane; Bultynck, Geert; Wang, Zhen; Zhang, Zhiwei; Schey, Kevin; Poelzing, Steven; McGowan, Francis X.; Gourdie, Robert G. (American Heart Association, 2019-08-19)
    Background α Carboxyl terminus 1 (αCT1) is a 25–amino acid therapeutic peptide incorporating the zonula occludens‐1 (ZO‐1)–binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia‐reperfusion injury. Methods and Results To study αCT1‐mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp‐Asp‐Leu‐Glu‐Iso sequence and lysines (Lys345, Lys346) in an α‐helical sequence (helix 2) within the Cx43‐CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO‐1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO‐1–postsynaptic density‐95/disks large/zonula occludens‐1 2 or Cx43‐CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43‐CT, but not ZO‐1–postsynaptic density‐95/disks large/zonula occludens‐1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia‐reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia‐reperfusion. Interestingly, a short 9–amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO‐1, is correlated with cardioprotection. Pharmacophores targeting Cx43‐CT could provide a translational approach to preserving heart function after ischemic injury.
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    Intercalated Disk Extracellular Nanodomain Expansion in Patients With Atrial Fibrillation
    Raisch, Tristan B.; Yanoff, Matthew S.; Larsen, Timothy R.; Farooqui, Mohammed A.; King, D. Ryan; Veeraraghavan, Rengasayee; Gourdie, Robert G.; Baker, Joseph W.; Arnold, William S.; AlMahameed, Soufian T.; Poelzing, Steven (Frontiers, 2018-05-04)
    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 beta 1 subunit, and connexin43 (C x 43) was determined by confocal immunofluorescence. Perinexal width (Wp) from TEM was manually segmented by two blinded observers using ImageJ software. Results: Nav1.5, beta 1, and C x 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.
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    Interplay between Ephaptic and Gap Junctional Coupling in Cardiac Conduction
    George, Sharon Ann (Virginia Tech, 2016-03-24)
    Sudden cardiac death occurs due to aberrations in the multifactorial process that is cardiac conduction. Conduction velocity (CV) and its modulation by several determinants, like cellular excitability, tissue structure and electrical coupling by gap junctions (GJ), have been extensively studied. However, there are several discrepancies in cardiac electrophysiology research that have extended over decades, suggesting elements that are still not completely understood about this complex phenomenon. This dissertation will focus on one such mechanism, ephaptic coupling (EpC). The purpose of this work is twofold, 1) to identify ionic determinants of EpC, and its interactions with gap junctional coupling (GJC) and, 2) to investigate the possible role of serum ion modulation in cardiac arrhythmia therapy. First, the effects of altering extracellular ion concentration – sodium, potassium and calcium at varying GJ protein expression were studied. Briefly, reducing sodium was related to CV slowing under conditions of reduced EpC (wide intercalated disc nanodomains – perinexi) and GJC (reduced GJ protein – Connexin43). On the other hand, increasing potassium slowed CV in hearts with wide perinexi independent of GJC. Elevating calcium, reduced perinexal width and was associated with fast CV during physiologic sodium concentration. However, under conditions associated with disease, like hyponatremia, elevating calcium still reduced perinexal width but slowed CV. These findings are the first to suggest that ionic modulators of EpC could modulate CV during health and disease. Next, the potential of perfusate ion modulation in cardiac arrhythmia therapy was investigated. Briefly, in a model of myocardial inflammation, TNFα, a pro-inflammatory cytokine, slowed CV relative to control conditions and this was associated with widening of the perinexus (reduced EpC). Increasing extracellular calcium restored CV to control values by improving not only EpC but also GJC. Finally, in a model of metabolic ischemia in the heart, CV response due to solutions with varying sodium and calcium concentrations were tested. The solutions that were associated with wider perinexi and elevated sodium performed best during ischemia by attenuating CV slowing, reducing arrhythmias and increasing time to asystole. Taken together, these findings provide evidence for the possibility of ionic determinants of EpC in cardiac arrhythmia therapy.