James W. Smyth

Heart Rate and Extracellular Sodium and Potassium Modulation of Gap Junction Mediated Conduction in Guinea Pigs

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 [Na+]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 μM). 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 15μM CBX. Decreasing the basic cycle length (BCL) of pacing from 300 to 160 ms decreased CV uniformly with both solutions. At 30 μM 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.

Extracellular sodium dependence of the conduction velocity-calcium relationship: evidence of ephaptic self-attenuation.

Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca(2+)]o). Here, we test the hypothesis that increasing [Ca(2+)]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na(+)]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca(2+)]o (1 to 3.4 mM) significantly decreased WP Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca(2+)]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (∼50% Cx43) hearts was more sensitive to changes in [Ca(2+)]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca(2+)]o nor [Na(+)]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca(2+)]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression.

TNFα Modulates Cardiac Conduction by Altering Electrical Coupling between Myocytes

Background: Tumor Necrosis Factor α (TNFα) 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 TNFα 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, TNFα and TNFα + 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 (WP). 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 TNFα slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNFα increased WP relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNFα exposure increased CVT within 15 mins. TNFα + high calcium also restored CVT at 90 mins and reduced WP to control values. Interestingly, TNFα + high calcium also improved GJ coupling at 90 mins, which along with reduced WP may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNFα exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing.

The adhesion function of the sodium channel beta subunit (β1) contributes to cardiac action potential propagation

Computational modeling indicates that cardiac conduction may involve ephaptic coupling – intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that β1(SCN1B) –mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential β1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, βadp1, potently and selectively inhibited β1-mediated adhesion, in electric cell-substrate impedance sensing studies. βadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, βadp1 precipitated arrhythmogenic conduction slowing. In summary, β1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.

STORM Localizations - Part 1/3

Zip file containing single molecule localization data from STORM. The zip file is divided into 3 volumes. Volume 1/3

Alternative mechanisms of translation initiation: An emerging dynamic regulator of the proteome in health and disease

ABSTRACT Eukaryotic mRNAs were historically thought to rely exclusively on recognition and binding of their 5′ cap by initiation factors to effect protein translation. While internal ribosome entry sites (IRESs) are well accepted as necessary for the cap‐independent translation of many viral genomes, there is now recognition that eukaryotic mRNAs also undergo non‐canonical modes of translation initiation. Recently, high‐throughput assays have identified thousands of mammalian transcripts with translation initiation occurring at non‐canonical start codons, upstream of and within protein coding regions. In addition to IRES‐mediated events, regulatory mechanisms of translation initiation have been described involving alternate 5′ cap recognition, mRNA sequence elements, and ribosome selection. These mechanisms ensure translation of specific mRNAs under conditions where cap‐dependent translation is shut down and contribute to pathological states including cardiac hypertrophy and cancer. Such global and gene‐specific dynamic regulation of translation presents us with an increasing number of novel therapeutic targets. While these newly discovered modes of translation initiation have been largely studied in isolation, it is likely that several act on the same mRNA and exquisite coordination is necessary to maintain ‘normal’ translation. In this short review, we summarize the current state of knowledge of these alternative mechanisms of eukaryotic protein translation, their contribution to normal and pathological cell biology, and the potential of targeting translation initiation therapeutically in human disease.

STORM and TEM Identify the Cardiac Ephapse: An Intercalated Disk Nanodomain with Previously Unanticipated Functions in Cardiac Conduction

1. Virginia Tech Carilion Research Institute, and Center for Heart and Regenerative Medicine, Virginia Polytechnic University, Roanoke, VA. 2. School of Biomedical Engineering and Sciences, Virginia Polytechnic University, Blacksburg, VA. 3. Dept. of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH. 4. Heart and Vascular Research Center, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH. 5. Dept. of Medicine, National Heart & Lung Institute, Imperial College London, London, UK.

Abstract 4765: Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide

Glioblastoma (GBM) is a highly malignant and lethal cancer of the central nervous system. Despite intensive research efforts, there has been limited progress in improving patient outcome. The current therapy for GBM patients includes surgical resection, radiotherapy and cytotoxic chemotherapy with temozolomide (TMZ), conferring a median survival time of only 14.6 months. Failure to generate more effective treatment strategies is due in part to the cellular heterogeneity within GBM tumors, which comprise a sub-population of GBM cancer stem cells (GSCs) characterized by self-renewal characteristics and resistance to TMZ. Recent work, including our own, has demonstrated that levels of the gap junction protein Connexin43 (Cx43) correlate with GBM TMZ resistance. Increased Cx43 levels occur in GSCs compared to parental GBM cells, and patients with high levels of Cx43 mRNA and low levels of MGMT, an enzyme that repairs TMZ-induced DNA lesions, have a significantly shorter life span. In contrast, Cx43 has also been associated with anti-proliferative effects in glioma and reduced levels of Cx43 protein are reported in high-grade gliomas. In addition to forming gap junctions, Cx43 can regulate cell proliferation, migration, and apoptosis, through distinct channel-independent mechanisms. Therefore, altering the localization and/or activity of Cx43 rather than Cx43 expression alone may represent a more targeted strategy in GBM treatment. Using super-resolution microscopy, we find intracellular Cx43 decorating microtubules in GSCs demonstrating for the first time such clustering in situ. Regulation of Cx43 function is primarily associated with multiple sites for post-translational modification and protein-protein interaction within the Cx43 carboxy-terminus (CT) which includes a tubulin binding domain. We have developed a peptide named JM2 (juxtamembrane 2) composed of the Cx43 CT amino acids encompassing the microtubule-binding sequence fused to an antennapedia cell penetration domain for cellular uptake. Super-resolution microscopy and biochemical analysis confirm JM2 specific interaction with microtubules concomitantly with a loss of Cx43 interaction with microtubules in GSCs derived from patient tumors. In addition, we observe that JM2 decreases Cx43 gap junction plaque formation and cell-cell communication in GSCs and limits microtubule dynamics. Importantly, JM2 decreases cell survival in TMZ-resistant GSCs and GSC neurosphere formation in vitro, and GSC-derived tumor growth in vivo. Our current research includes the development of JM2-loaded biodegradable nanoparticles for sustained JM2 delivery in preparation for future clinical trials. In conclusion, we have developed a Cx43 mimetic peptide that significantly decreases the tumorigenic potential of GSCs and represents a novel and potent therapeutic opportunity to alter Cx43 activity in targeting chemoresistant GSCs for GBM treatment. Citation Format: Samy Lamouille, James W. Smyth, Laurie O9Rourke, Pratik Kanabur, Sujuan Guo, Jane Jourdan, Zhi Sheng, Robert G. Gourdie. Targeting glioblastoma cancer stem cells with a novel Connexin43 mimetic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2017-4765