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      Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc

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          Abstract

          Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and ‘angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC Na V1.5. We show that the N-cadherin-Na V1.5 association is not random, that Na V1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of Na V1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.

          Abstract

          In myelinated fibres conduction and adhesion proteins aggregate at discrete foci, but it is unclear if this organization is present in other excitable cells. Using nanoscale visualization and in silico techniques, the authors show that adhesion/excitability nodes exist at the intercalated discs of adult cardiac muscle.

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          Most cited references51

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          NIH Image to ImageJ: 25 years of image analysis.

          For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.
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            Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy.

            Recent advances in far-field fluorescence microscopy have led to substantial improvements in image resolution, achieving a near-molecular resolution of 20 to 30 nanometers in the two lateral dimensions. Three-dimensional (3D) nanoscale-resolution imaging, however, remains a challenge. We demonstrated 3D stochastic optical reconstruction microscopy (STORM) by using optical astigmatism to determine both axial and lateral positions of individual fluorophores with nanometer accuracy. Iterative, stochastic activation of photoswitchable probes enables high-precision 3D localization of each probe, and thus the construction of a 3D image, without scanning the sample. Using this approach, we achieved an image resolution of 20 to 30 nanometers in the lateral dimensions and 50 to 60 nanometers in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures.
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              HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.

              We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                20 January 2016
                2016
                : 7
                : 10342
                Affiliations
                [1 ]The Leon H Charney Division of Cardiology, New York University School of Medicine (NYU-SoM) , 522 First Avenue, Smilow 805, New York, New York 10016, USA
                [2 ]Imperial College, National Heart and Lung Institute, Department of Cardiac Medicine, Imperial Center for Translational and Experimental Medicine, Hammersmith Campus , Du Cane Road, London W12 0NN, UK
                [3 ]Center for Health Informatics and Bioinformatics, NYU-SoM , Translational Research Building, 227 East 30th Street, New York, New York 10016, USA
                [4 ]Microscopy Core, NYU-SoM , 522 First Avenue, Skirball Institute, 2nd Floor, New York, New York 10016, USA
                [5 ]Division of Medicine, Imperial College, Hammersmith Campus , Du Cane Road, London, London W12 0NN, UK
                [6 ]Department of Biochemistry and Molecular Pharmacology, NYU-SoM , 522 First Avenue, MSB 3rd Floor, New York, New York 10016, USA
                Author notes
                [*]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0002-7579-9764
                Article
                ncomms10342
                10.1038/ncomms10342
                4735805
                26787348
                05220722-7948-4385-8256-b1c767bbf003
                Copyright © 2016, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 07 May 2015
                : 01 December 2015
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