title: Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.
abstract: BACKGROUND
  Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.
  
  
  RESULTS
  Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Jiajie
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Justin K
authors.initials: JK
authors.lastname: Thomson
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Pollard
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Xun
authors.initials: X
authors.lastname: Ai
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging; D000818:Animals; D015820:Cadherins / Q000032:analysis; D003094:Collagen / Q000032:analysis*; D018031:Connexin 43 / Q000032:analysis; D045604:Extracellular Fluid / Q000737:chemistry; D005260:Female; D017629:Gap Junctions / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure*; D006325:Heart Atria / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D008875:Middle Aged; D011817:Rabbits; D055815:Young Adult
chemical_list: D015820:Cadherins; D018031:Connexin 43; D003094:Collagen
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