Old Dominion University ODU Digital Commons Biological Sciences Theses & Dissertations Biological Sciences Summer 2017 The Chondrocyte Channelome: A Novel Ion Channel Candidate in the Pathogenesis of Pectus Deformities Anthony J. Asmar Old Dominion University, [email protected] Follow this and additional works at: https://digitalcommons.odu.edu/biology_etds Part of the Biology Commons, Molecular Biology Commons, and the Physiology Commons Recommended Citation Asmar, Anthony J.. "The Chondrocyte Channelome: A Novel Ion Channel Candidate in the Pathogenesis of Pectus Deformities" (2017). Doctor of Philosophy (PhD), Dissertation, Biological Sciences, Old Dominion University, DOI: 10.25777/pyha-7838 https://digitalcommons.odu.edu/biology_etds/19 This Dissertation is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion in Biological Sciences Theses & Dissertations by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. THE CHONDROCYTE CHANNELOME: A NOVEL ION CHANNEL CANDIDATE IN THE PATHOGENESIS OF PECTUS DEFORMITIES by Anthony J. Asmar B.S. Biology May 2010, Virginia Polytechnic Institute M.S. Biology May 2013, Old Dominion University A Dissertation Submitted to the Faculty of Old Dominion University in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY BIOMEDICAL SCIENCES OLD DOMINION UNIVERSITY August 2017 Approved by: Christopher Osgood (Co-Director) Michael Stacey (Co-Director) Lesley Greene (Member) Andrei Pakhomov (Member) Jing He (Member) ABSTRACT THE CHONDROCYTE CHANNELOME: A NOVEL ION CHANNEL CANDIDATE IN THE PATHOGENESIS OF PECTUS DEFORMITIES Anthony J. Asmar Old Dominion University, 2017 Co-Directors: Dr. Christopher Osgood Dr. Michael Stacey Costal cartilage is a type of rod-like hyaline cartilage connecting the ribs to the sternum. The chest wall deformities pectus excavatum (PE) and pectus carinatum (PC) involve displacement of the sternum causing a depression or protrusion of the chest. There is little knowledge about costal cartilage and pectus deformities with much of its understanding based on assumptions from articular cartilage. Chondrocytes are subjected to a constantly changing environment with fluctuations in pH and osmolarity. Ion channels detect these changes and in turn regulate proliferation, differentiation, and extracellular matrix production. Using ion channel qPCR arrays, we produced expression profiles for normal, fetal, PE-affected, and PC-affected costal chondrocytes as well as articular chondrocytes. Costal and articular chondrocytes had many commonly expressed ion channels with certain channels specific to each cartilage type. The discrepancy in ion channel expression is likely to be a reflection of the functional differences between the two cartilage types. Additionally, fetal costal chondrocytes had several other distinct ion channels possibly due to the differentiation status of the cells. In PC and PE chondrocytes, ACCN1 (ASIC2) and KCNN2 (SK2) were consistently down-regulated compared to normal costal chondrocytes. However, Western blot analysis found deceases only in ASIC2 protein levels. ASIC2 is a proton-gated ion channel involved in cell response to extracellular pH changes. Calcium monitoring revealed a delay in the formation calcium transients in PC cells when challenged with low pH which may be caused by aberrant signaling from ASIC channels. Immunofluorescent analysis of connexins found that Cx43 was present in chondrocytes with phosphorylated Cx43 localizing in and around the nucleus. Analysis of ATP release found that release is likely a connexin-mediated process, though external acidosis did not induce ATP release. Analysis of microRNAs found upregulation and down-regulation of several microRNAs in PC versus control cells, though further studies are needed to identify a possible microRNA signature for pectus deformities. Overall, we have generated a comprehensive ion channel profile for the costal chondrocytes, as well as identified a possible contributing factor for pectus deformities. iv Copyright, 2017, by Anthony J. Asmar, All Rights Reserved. v This thesis is dedicated to my parents, Leila and Tony, and sister, Rita, for all their encouragement, support, and patience enabling me to make it to this point. vi ACKNOWLEDGMENTS I would especially like to acknowledge Dr. Michael Stacey, for all his assistance and the opportunity to perform this research under his guidance. I would also like to acknowledge Dr. Christopher Osgood for his advice and support throughout my graduate experience. For their input and help in developing my dissertation project, I would like to acknowledge my committee members, Drs. Leslie Greene, Andrei Pakhomov, and Jing He, as well as Drs. Patrick Sachs, Loree Heller, and Robert Bruno. For their continued friendship and support, I would like to thank Drs. Robert Milletich, Peter Mollica, Wassim Obeid, Hany Salaheldeen, Charalampos Brimpas, and John Reid. I would also like to thank everyone at the Frank Reidy Research Center for Bioelectrics. vii TABLE OF CONTENTS Page LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES .........................................................................................................................x Chapter 1. INTRODUCTION .......................................................................................................................1 1.1 CARTILAGE .........................................................................................................................1 1.2 HYALINE CARTILAGE ......................................................................................................1 1.3 EXTRACELLULAR MATRIX .............................................................................................2 1.4 ECM MAINTENANCE .........................................................................................................6 1.5 COSTAL CARTILAGE .........................................................................................................9 1.6 PECTUS DEFORMITIES ....................................................................................................10 1.7 ION CHANNELS ................................................................................................................12 1.8 CONNEXINS .......................................................................................................................31 1.9 MICRORNAS ......................................................................................................................36 2. METHODS ................................................................................................................................41 2.1 SAMPLES ............................................................................................................................41 2.2 RNA EXTRACTION AND PCR ANALYSIS ....................................................................42 2.3 WESTERN BLOTTING ......................................................................................................43 2.4 CALCIUM IMAGING .........................................................................................................43 2.5 METABOLIC ASSAY ........................................................................................................44 2.6 MICRORNA MICROARRAY ............................................................................................46 2.7 MICROARRAY ANALYSIS ..............................................................................................45 2.8 IMMUNOCYTOCHEMISTRY ...........................................................................................46 2.9 ATP MEASUREMENTS .....................................................................................................47 2.10 STATISTICAL ANALYSIS ..............................................................................................48 3. RESULTS AND DISCUSSION ................................................................................................49 3.1 ION CHANNELS IN NORMAL CARTILAGE .................................................................49 viii Page 3.2 ION CHANNELS IN PECTUS DEFORMITIES ................................................................63 3.3 ACID SENSITIVITY IN COSTAL CHONDROCYTES ...................................................74 3.4 OSMOTIC SENSITIVITY IN COSTAL CHONDROCYTES ...........................................81 3.5 CONNEXINS .......................................................................................................................87 3.6 MICRORNAS ......................................................................................................................97 4. CONCLUSIONS......................................................................................................................108 REFERENCES ............................................................................................................................110 APPENDICES .............................................................................................................................138 VITA ........................................................................................................................................... 163 ix LIST OF TABLES Table Page 1. Summary of transient curve properties