Browsing by Author "Smyth, James W."

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    Adenovirus transduction to express human ACE2 causes obesity-specific morbidity in mice, impeding studies on the effect of host nutritional status on SARS-CoV-2 pathogenesis
    Rai, Pallavi; Chuong, Christina; LeRoith, Tanya; Smyth, James W.; Panov, Julia; Levi, Moshe; Kehn-Hall, Kylene; Duggal, Nisha K.; Weger-Lucarelli, James (Elsevier, 2021-11-01)
    The COVID-19 pandemic has paralyzed the global economy and resulted in millions of deaths globally. People with co-morbidities like obesity, diabetes and hypertension are at an increased risk for severe COVID-19 illness. This is of overwhelming concern because 42% of Americans are obese, 30% are pre-diabetic and 9.4% have clinical diabetes. Here, we investigated the effect of obesity on disease severity following SARS-CoV-2 infection using a well-established mouse model of diet-induced obesity. Diet-induced obese and lean control C57BL/6 N mice, transduced for ACE2 expression using replication-defective adenovirus, were infected with SARS-CoV-2, and monitored for lung pathology, viral titers, and cytokine expression. No significant differences in tissue pathology or viral replication was observed between AdV transduced lean and obese groups, infected with SARS-CoV-2, but certain cytokines were expressed more significantly in infected obese mice compared to the lean ones. Notably, significant weight loss was observed in obese mice treated with the adenovirus vector, independent of SARS-CoV-2 infection, suggesting an obesity-dependent morbidity induced by the vector. These data indicate that the adenovirus-transduced mouse model of SARS-CoV-2 infection, as described here and elsewhere, may be inappropriate for nutrition studies.
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    Altered translation initiation of Gja1 limits gap junction formation during epithelial–mesenchymal transition
    James, Carissa C.; Zeitz, Michael J.; Calhoun, Patrick J.; Lamouille, Samy Y.; Smyth, James W. (The American Society for Cell Biology, 2018-02-13)
    Epithelial–mesenchymal transition (EMT) is activated during development, wound healing, and pathologies including fibrosis and cancer metastasis. Hallmarks of EMT are remodeling of intercellular junctions and adhesion proteins, including gap junctions. The GJA1 mRNA transcript encoding the gap junction protein connexin43 (Cx43) has been demonstrated to undergo internal translation initiation, yielding truncated isoforms that modulate gap junctions. The PI3K/Akt/mTOR pathway is central to translation regulation and is activated during EMT, leading us to hypothesize that altered translation initiation would contribute to gap junction loss. Using TGF-β–induced EMT as a model, we find reductions in Cx43 gap junctions despite increased transcription and stabilization of Cx43 protein. Biochemical experiments reveal suppression of the internally translated Cx43 isoform, GJA1-20k in a Smad3 and ERK-dependent manner. Ectopic expression of GJA1-20k does not halt EMT, but is sufficient to rescue gap junction formation. GJA1-20k localizes to the Golgi apparatus, and using superresolution localization microscopy we find retention of GJA1-43k at the Golgi in mesenchymal cells lacking GJA1-20k. NativePAGE demonstrates that levels of GJA1-20k regulate GJA1-43k hexamer oligomerization, a limiting step in Cx43 trafficking. These findings reveal alterations in translation initiation as an unexplored mechanism by which the cell regulates Cx43 gap junction formation during EMT.
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    Alternative mechanisms of translation initiation in modulation of gap junctional coupling
    James, Carissa Chey (Virginia Tech, 2019-04-22)
    Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells.
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    Autoregulation of Connexin43 Gap Junction Formation by Internally Translated Isoforms
    Smyth, James W.; Shaw, Robin M. (Cell, 2013)
    During each heartbeat, intercellular electrical coupling via connexin43 (Cx43) gap junctions enables synchronous cardiac contraction. In failing hearts, impaired Cx43 trafficking reduces gap junction coupling, resulting in arrhythmias. Here we report that internal translation within Cx43 (GJA1) mRNA occurs, resulting in truncated isoforms that autoregulate Cx43 trafficking. We find that at least four truncated Cx43 isoforms occur in the human heart, with a 20 kDa isoform predominating. In-frame AUG codons within GJA1 mRNA are the translation initiation sites and their ablation arrests trafficking of full-length Cx43. The 20 kDa isoform is sufficient to rescue this trafficking defect in trans, suggesting it as a trafficking chaperone for Cx43. Limiting capdependent translation through inhibition of mTOR enhances truncated isoform expression, increasing Cx43 gap junction size. The results suggest that internal translation is a mechanism of membrane protein autoregulation and a potent target for therapies aimed at restoring normal electrical coupling in diseased hearts.
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    Connexin 43 regulates intercellular mitochondrial transfer from human mesenchymal stromal cells to chondrocytes
    Irwin, Rebecca M.; Thomas, Matthew A.; Fahey, Megan J.; Mayán, María D.; Smyth, James W.; Delco, Michelle L. (2024-10-10)
    Background: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Methods: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Results: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Conclusions Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.
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    Determinants of Core Shell Dependent Rotavirus Polymerase Activity
    Steger, Courtney Long (Virginia Tech, 2019-02-22)
    Rotaviruses (RVs) are medically significant gastrointestinal pathogens and are a leading cause of childhood mortality in many countries. The RV RNA-dependent RNA polymerase, VP1, synthesizes RNA during viral replication only in the presence of another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for RV replication, the mechanism by which the core shell regulates polymerase activity remains incompletely understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are required for core shell dependent polymerase activity. First, we used bioinformatics approaches to analyze VP1 and VP2 sequence diversity across many RV strains and identify positional locations of critical amino acid changes within the context of known structural domains and motifs. We next tested how the identified sequence differences influenced VP2-dependent VP1 activity in vitro. These data revealed that VP1 and VP2 protein diversity correlates with functional differences between avian and mammalian RV strains. Then, we used these sequential and functional incompatibilities to map key regions of VP1 important for mediating RNA synthesis. To pinpoint critical interacting regions of VP1 and VP2, we used site directed mutagenesis to engineer several modified VP1 and VP2 proteins. Then, we employed an in vitro RNA synthesis assay to test how the introduced mutations influenced VP2-dependent VP1 activity. Altogether, our results revealed several functionally important VP1 residues critical for in vitro VP2-dependent VP1 activity, either individually or in combination with neighboring residues, including E265/L267, R614, and D971/S978/I980. Structural analyses show VP2 interactions at these surface-exposed VP1 sites, which altogether supports a direct contact model of core shell dependent RV polymerase activity. Moreover, recombinant VP1 proteins containing multiple mutations at buried residues were incapable of facilitating RNA synthesis in vitro under the assay conditions, indicating that an extensive intramolecular signaling network exists to mediate VP1 activity. Taken together, these results suggest that VP2 binding at the VP1 surface may induce intramolecular interactions critical for VP1 activity. Overall, results from these studies provide important insight into VP1-VP2 binding interface(s) that are necessary for RV replication.
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    Dynamic UTR Usage Regulates Alternative Translation to Modulate Gap Junction Formation during Stress and Aging
    Zeitz, Michael J.; Calhoun, Patrick J.; James, Carissa C.; Taetzsch, Thomas; George, Kijana K.; Robel, Stefanie; Valdez, Gregorio; Smyth, James W. (Elsevier, 2019-05-28)
    Connexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-b. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 50 UTR length, of which two are upregulated during TGF-b exposure and hypoxia. Introduction of these transcripts into Gja1/ cells phenocopies the response of Gja1 to TGF-b with reduced internal translation initiation. Inhibiting pathways downstream of TGF-b selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of fulllength Cx43 from shorter Gja1 50 UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication.
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    Effects of constitutive and acute Connexin 36 deficiency on brain-wide susceptibility to PTZ-induced neuronal hyperactivity
    Brunal-Brown, Alyssa Alexandra (Virginia Tech, 2020-10-30)
    Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: a) does Cx36 deficiency affect seizure susceptibility, b) does seizure-like activity affect Cx36 expression patterns, and c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol (PTZ; a GABA(A) receptor antagonist) induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within. 30 minutes and one-hour exposure to 20 mM PTZ significantly reduced the expression of Cx36. This Cx36 reduction persists after one-hour of recovery but recovered after 3-6 hours. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events. 
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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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    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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    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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    Folate regulates RNA m5C modification and translation in neural stem cells
    Xu, Xiguang; Johnson, Zachary; Wang, Amanda; Padget, Rachel L.; Smyth, James W.; Xie, Hehuang (2022-11-23)
    Background Folate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m5C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m5C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m5C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate. Results NSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m5C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m5C methylation and mRNA translation efficiency. Conclusions Altogether, our study reports a transcriptome-wide influence of folate on mRNA m5C methylation and translation in NSCs and reveals a potential link between mRNA m5C methylation and mRNA translation.
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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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    Internally Translated Cx43 Isoform GJA1-20k Affects Epithelial to Mesenchymal Transition and Metastatic Cancer Cell Behavior
    Young, Kenneth Lee, II (Virginia Tech, 2024-08-08)
    Epithelial-mesenchymal transition (EMT) is a trans-differentiation program essential for development and wound healing that is pathologically activated during cancer progression. During this process, cells undergo complex changes at the transcriptional and translational levels leading to dissolution of cell-cell junctions, loss of apical-basal polarity, and cytoskeleton reorganization. Transforming Growth Factor-β (TGF-β) is well-established in driving cancer progression through EMT induction. Remodeling of cellular junctions, including gap junctions, is critical to acquiring migratory and invasive characteristics during EMT. The gene GJA1 encodes for Connexin43 (Cx43), the most ubiquitously expressed gap junction protein where altered regulation of Cx43 is associated with cancer progression. Intriguingly, Cx43 mRNA undergoes alternative ‘internal’ translation initiation, generating N-terminally truncated isoforms, including GJA1-20k, which regulates Cx43 gap junction formation. We have previously demonstrated GJA1-20k expression is inhibited during TGF-β-induced EMT, limiting gap junction formation; however, the relationship between GJA1-20k modulation of gap junction localization and cellular invasion and migration remains unknown. Given the role GJA1-20k has in regulating gap junctions, we hypothesize that suppression of GJA1-20k expression promotes metastatic trait acquisition through limiting gap junction formation. Utilizing lentivirally transduced stable mouse mammary gland epithelial (NMuMG) and triple-negative human breast epithelial (MDA-MB-231) cells expressing GJA1-20k, or Lac Z as control, we tested effects on TGF-β-induced EMT induction and metastatic trait induction. Boyden chambers, would/scratch assays were employed to analyze cell invasion and migration respectively. We found GJA1-20k overexpression during EMT results in decreased cell invasion and migration to LacZ controls. Future directions include evaluation of GJA1-20k restoration in a metastatic breast cancer model in vivo. Investigating the underlying role of GJA1-20k in EMT-induced cell junction remodeling could be promising as a potential pharmacological target process independent of transcriptional or post-translational pathways. Ultimately, by adding novel information in the expanding and compelling field of translational control, this work could aid in developing the future of precision medicine as new therapeutic solutions to treat cancer will require limiting cancer cell’s ability to metastasize.
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    Progression-Mediated Changes in Mitochondrial Morphology Promotes Adaptation to Hypoxic Peritoneal Conditions in Serous Ovarian Cancer
    Grieco, Joseph P.; Allen, Mitchell E.; Perry, Justin B.; Wang, Yao; Song, Yipei; Rohani, Ali; Compton, Stephanie L. E.; Smyth, James W.; Swami, Nathan S.; Brown, David A.; Schmelz, Eva M. (2021-01-13)
    Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.
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    TGF-β-induced activation of mTOR complex 2 drives epithelial–mesenchymal transition and cell invasion
    Lamouille, Samy Y.; Connolly, Erin; Smyth, James W.; Akhurst, Rosemary J.; Derynck, Rik (Company of Biologists, 2012-03-01)
    In cancer progression, carcinoma cells gain invasive behavior through a loss of epithelial characteristics and acquisition of mesenchymal properties, a process that can lead to epithelial–mesenchymal transition (EMT). TGF-b is a potent inducer of EMT, and increased TGF-b signaling in cancer cells is thought to drive cancer-associated EMT. Here, we examine the physiological requirement for mTOR complex 2 (mTORC2) in cells undergoing EMT. TGF-b rapidly induces mTORC2 kinase activity in cells undergoing EMT, and controls epithelial cell progression through EMT. By regulating EMT-associated cytoskeletal changes and gene expression, mTORC2 is required for cell migration and invasion. Furthermore, inactivation of mTORC2 prevents cancer cell dissemination in vivo. Our results suggest that the mTORC2 pathway is an essential downstream branch of TGF-b signaling, and represents a responsive target to inhibit EMT and prevent cancer cell invasion and metastasis.
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    TNF alpha Modulates Cardiac Conduction by Altering Electrical Coupling between Myocytes
    George, Sharon A.; Calhoun, Patrick J.; Gourdie, Robert G.; Smyth, James W.; Poelzing, Steven (Frontiers, 2017-05-23)
    Background: Tumor Necrosis Factor alpha (TNF alpha) upregulation during acute inflammatory response has been associated with numerous cardiac effects including modulating Connexin43 and vascular permeability. This may in turn alter cardiac gap junctional (GJ) coupling and extracellular volume (ephaptic coupling) respectively. We hypothesized that acute exposure to pathophysiological TNF alpha levels can modulate conduction velocity (CV) in the heart by altering electrical coupling: GJ and ephaptic. Methods and Results: Hearts were optically mapped to determine CV from control, TNF alpha and TNF alpha + high calcium(2.5 vs. 1.25 mM) treated guinea pig hearts over 90 mins. Transmission electron microscopy was performed to measure changes in intercellular separation in the gap junction-adjacent extracellular nanodomain-perinexus (W-P). Cx43 expression and phosphorylation were determined by Western blotting and Cx43 distribution by confocal immunofluorescence. At 90 mins, longitudinal and transverse CV (CVL and CVT, respectively) increased with control Tyrode perfusion but TNF alpha slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNF alpha increased W-P relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNF alpha exposure increased CVT within 15 mins. TNF alpha + high calcium also restored CVT at 90 mins and reduced W-P to control values. Interestingly, TNF alpha + high calcium also improved GJ coupling at 90 mins, which along with reduced W-P may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNF alpha exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing.
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    Translating Translation to Mechanisms of Cardiac Hypertrophy
    Zeitz, Michael J.; Smyth, James W. (MDPI, 2020-03-10)
    Cardiac hypertrophy in response to chronic pathological stress is a common feature occurring with many forms of heart disease. This pathological hypertrophic growth increases the risk for arrhythmias and subsequent heart failure. While several factors promoting cardiac hypertrophy are known, the molecular mechanisms governing the progression to heart failure are incompletely understood. Recent studies on altered translational regulation during pathological cardiac hypertrophy are contributing to our understanding of disease progression. In this brief review, we describe how the translational machinery is modulated for enhanced global and transcript selective protein synthesis, and how alternative modes of translation contribute to the disease state. Attempts at controlling translational output through targeting of mTOR and its regulatory components are detailed, as well as recently emerging targets for pre-clinical investigation.
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    Von Hippel-Lindau Syndrome: Characterization of a Potentially Novel VEGF-A Isoform and Elucidation of Molecular and Vascular Mechanisms of Observed Phenotypic Changes
    North, Morgan Hunter (Virginia Tech, 2020-06-17)
    Von Hippel-Lindau (VHL) syndrome is an autosomal dominant predisposition to cancer in neurological tissues, the kidneys, adrenal glands, pancreas, and liver, including neurological hemangioblastoma (HB), pheochromocytoma (PCC), pancreatic neuroendocrine tumors (PNET), pancreatic and renal cysts, and clear cell renal cell carcinoma (ccRCC). The disease process follows Knudson's two-hit model, requiring spontaneous loss or mutation of a normal VHL tumor suppressor allele to induce expression of the disease. VHL syndrome principally involves dysregulation of oxygen sensing pathways including the Hypoxia Inducible Factor (HIF)-Vascular Endothelial Growth Factor-A (VEGF-A) and HIF-Erythropoietin (EPO) pathways. RNA sequencing (RNA-Seq) data from our previously published experiments revealed a potentially novel VEGF-A splice variant with excision of the VEGF Receptor-1 (VEGFR-1)/Flt-1 binding domain, rendering this isoform resistant to native down-regulation. Additionally, phenotypic changes were observed in adult VHL mutant mice, specifically very red appearing extremities with prominently visible vasculature. In order to determine the etiology of this phenotype, we observed red blood cell count, Epo gene expression levels, and arterialization of the blood vessels in these experimental mice as compared to littermate controls. Current research into the VEGF-A isoform is ongoing in the lab, and preliminary evidence for the etiology of the apparent chronic erythema phenotype is inconclusive due to lack of experimental replicates due to COVID-19 quarantine orders.