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Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle

Published online by Cambridge University Press:  14 January 2020

Heather L. Struckman ,
Stephen Baine Open the ORCID record for Stephen Baine [Opens in a new window] ,
Justin Thomas ,
Louisa Mezache ,
Kirk Mykytyn ,
Sándor Györke ,
Przemysław B. Radwański  and
Rengasayee Veeraraghavan Open the ORCID record for Rengasayee Veeraraghavan [Opens in a new window]
Heather L. Struckman
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA
Stephen Baine
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
Justin Thomas
Affiliation:
Division of Pharmacy Practice and Sciences, College of Pharmacy, The Ohio State University, Columbus, OH, USA
Louisa Mezache
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA
Kirk Mykytyn
Affiliation:
Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, USA
Sándor Györke
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
Przemysław B. Radwański*
Affiliation:
Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA Division of Pharmacy Practice and Sciences, College of Pharmacy, The Ohio State University, Columbus, OH, USA
Rengasayee Veeraraghavan*
Affiliation:
Department of Biomedical Engineering, College of Engineering, The Ohio State University, Columbus, OH, USA Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH, USA Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA
*
*Authors for correspondence: Przemysław B. Radwański, E-mail: [email protected]; Rengasayee Veeraraghavan, E-mail: [email protected]
*Authors for correspondence: Przemysław B. Radwański, E-mail: [email protected]; Rengasayee Veeraraghavan, E-mail: [email protected]
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Abstract

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Keywords

antibodycardiac physiologyexcitation–contraction couplingsodium channelssuper-resolution microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 26 , Issue 1 , February 2020 , pp. 157 - 165
Copyright
Copyright © Microscopy Society of America 2020

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References

Bonilla, IM, Belevych, AE, Baine, S, Stepanov, A, Mezache, L, Bodnar, T, Liu, B, Volpe, P, Priori, S, Weisleder, N, Sakuta, G, Carnes, CA, Radwański, PB, Veeraraghavan, R & Gyorke, S (2019). Enhancement of cardiac store operated calcium entry (SOCE) within novel intercalated disk microdomains in arrhythmic disease. Sci Rep 9(1), 10179.CrossRefGoogle ScholarPubMed
Caldwell, JH, Schaller, KL, Lasher, RS, Peles, E & Levinson, SR (2000). Sodium channel Nav1.6 is localized at nodes of Ranvier, dendrites, and synapses. Proc Natl Acad Sci USA 97(10), 5616–5620.CrossRefGoogle Scholar
Clatot, J, Hoshi, M, Wan, X, Liu, H, Jain, A, Shinlapawittayatorn, K, Marionneau, C, Ficker, E, Ha, T & Deschenes, I (2017). Voltage-gated sodium channels assemble and gate as dimers. Nat Commun 8(1), 2077.CrossRefGoogle ScholarPubMed
Helms, AS, Alvarado, FJ, Yob, J, Tang, VT, Pagani, F, Russell, MW, Valdivia, HH & Day, SM (2016). Genotype-dependent and -independent calcium signaling dysregulation in human hypertrophic cardiomyopathy. Circulation 134(22), 1738–1748.CrossRefGoogle ScholarPubMed
Koleske, M, Bonilla, I, Thomas, J, Zaman, N, Baine, S, Knollmann, BC, Veeraraghavan, R, Gyorke, S & Radwański, PB (2018). Tetrodotoxin-sensitive Navs contribute to early and delayed afterdepolarizations in long QT arrhythmia models. J Gen Physiol 150(7), 991–1002.CrossRefGoogle ScholarPubMed
Maier, LS (2009). A novel mechanism for the treatment of angina, arrhythmias, and diastolic dysfunction: Inhibition of late I(Na) using ranolazine. J Cardiovasc Pharmacol 54(4), 279–286.CrossRefGoogle ScholarPubMed
Maier, SK, Westenbroek, RE, McCormick, KA, Curtis, R, Scheuer, T & Catterall, WA (2004). Distinct subcellular localization of different sodium channel alpha and beta subunits in single ventricular myocytes from mouse heart. Circulation 109(11), 1421–1427.CrossRefGoogle ScholarPubMed
Moreno, JD & Clancy, CE (2012). Pathophysiology of the cardiac late Na current and its potential as a drug target. J Mol Cell Cardiol 52(3), 608–619.CrossRefGoogle ScholarPubMed
Radwański, PB, Brunello, L, Veeraraghavan, R, Ho, HT, Lou, Q, Makara, MA, Belevych, AE, Anghelescu, M, Priori, SG, Volpe, P, Hund, TJ, Janssen, PM, Mohler, PJ, Bridge, JH, Poelzing, S & Gyorke, S (2015). Neuronal Na+ channel blockade suppresses arrhythmogenic diastolic Ca2+ release. Cardiovasc Res 106(1), 143–152.CrossRefGoogle ScholarPubMed
Radwański, PB, Greer-Short, A & Poelzing, S (2013). Inhibition of Na+ channels ameliorates arrhythmias in a drug-induced model of Andersen-Tawil syndrome. Heart Rhythm 10(2), 255–263.CrossRefGoogle Scholar
Radwański, PB, Ho, H-T, Veeraraghavan, R, Brunello, L, Liu, B, Belevych, AE, Unudurthi, SD, Makara, MA, Priori, SG, Volpe, P, Armoundas, AA, Dillmann, WH, Knollmann, BC, Mohler, PJ, Hund, TJ & Györke, S (2016). Neuronal Na+ channels are integral components of pro-arrhythmic Na+/Ca2+ signaling nanodomain that promotes cardiac arrhythmias during β-adrenergic stimulation. JACC 1(4), 251–266.Google ScholarPubMed
Radwański, PB, Johnson, CN, Gyorke, S & Veeraraghavan, R (2018). Cardiac arrhythmias as manifestations of nanopathies: An emerging view. Front Physiol 9, 1228.CrossRefGoogle Scholar
Radwański, PB & Poelzing, S (2011). NCX is an important determinant for premature ventricular activity in a drug-induced model of Andersen-Tawil syndrome. Cardiovasc Res 92(1), 57–66.CrossRefGoogle Scholar
Radwański, PB, Veeraraghavan, R & Poelzing, S (2010). Cytosolic calcium accumulation and delayed repolarization associated with ventricular arrhythmias in a guinea pig model of Andersen-Tawil syndrome. Heart Rhythm 7(10), 1428–1435.CrossRefGoogle Scholar
Sato, D, Clancy, CE & Bers, DM (2017). Dynamics of sodium current mediated early afterdepolarizations. Heliyon 3(9), e00388.CrossRefGoogle ScholarPubMed
van Bemmelen, MX, Rougier, JS, Gavillet, B, Apotheloz, F, Daidie, D, Tateyama, M, Rivolta, I, Thomas, MA, Kass, RS, Staub, O & Abriel, H (2004). Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination. Circ Res 95(3), 284–291.CrossRefGoogle ScholarPubMed
Veeraraghavan, R & Gourdie, R (2016). Stochastic optical reconstruction microscopy-based relative localization analysis (STORM-RLA) for quantitative nanoscale assessment of spatial protein organization. Mol Biol Cell 27(22), 3583–3590.CrossRefGoogle ScholarPubMed
Veeraraghavan, R, Gyorke, S & Radwański, PB (2017). Neuronal sodium channels: Emerging components of the nano-machinery of cardiac calcium cycling. J Physiol.595(12), 3823–3834.CrossRefGoogle ScholarPubMed
Veeraraghavan, R, Hoeker, GS, Alvarez-Laviada, A, Hoagland, D, Wan, X, King, DR, Sanchez-Alonso, J, Chen, C, Jourdan, J, Isom, LL, Deschenes, I, Smyth, J, Gorelik, J, Poelzing, S & Gourdie, RG (2018). The adhesion function of the sodium channel beta subunit (beta1) contributes to cardiac action potential propagation. eLife 7, e37610.CrossRefGoogle ScholarPubMed
Veeraraghavan, R, Lin, J, Hoeker, GS, Keener, JP, Gourdie, RG & Poelzing, S (2015). Sodium channels in the Cx43 gap junction perinexus may constitute a cardiac ephapse: An experimental and modeling study. Pflugers Arch 467(10), 2093–2105.CrossRefGoogle ScholarPubMed
Veeraraghavan, R, Lin, J, Keener, JP, Gourdie, R & Poelzing, S (2016). Potassium channels in the Cx43 gap junction perinexus modulate ephaptic coupling: An experimental and modeling study. Pflugers Arch 468(10), 1651–1661.CrossRefGoogle ScholarPubMed
Veeraraghavan, R & Poelzing, S (2008). Mechanisms underlying increased right ventricular conduction sensitivity to flecainide challenge. Cardiovasc Res 77(4), 749–756.CrossRefGoogle ScholarPubMed
Wang, J, Ou, SW & Wang, YJ (2017). Distribution and function of voltage-gated sodium channels in the nervous system. Channels 11(6), 534–554.CrossRefGoogle Scholar
Weibrecht, I, Leuchowius, KJ, Clausson, CM, Conze, T, Jarvius, M, Howell, WM, Kamali-Moghaddam, M & Soderberg, O (2010). Proximity ligation assays: A recent addition to the proteomics toolbox. Expert Rev Proteomics 7(3), 401–409.CrossRefGoogle ScholarPubMed
Zimmer, T, Haufe, V & Blechschmidt, S (2014). Voltage-gated sodium channels in the mammalian heart. Glob Cardiol Sci Pract 2014(4), 449–463.Google ScholarPubMed
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