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Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

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.

Methods and 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.

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Schematic diagram of the blur algorithm with a dilated N-Cad unit (DNCU) radius of 1 inter-pixel distance (IPD) and 2 IPDs.A&a. An N-Cad pixel at the center of a DNCU (C&c). Dilation radius equals to 1 IPD and 2 IPD (B&b). D&d. 3 overlapping adjacent DCNUs (R = 1 IPD & 2 IPDs, respectively) containing 3 adjacent N-Cad pixels. E&e. Three Cx43 pixels that are pixel-by-pixel matched with three N-Cad pixels, respectively. F-G & f-g. Two Cx43 pixels that do not co-localize with any N-Cad pixel-by-pixel but are included within the area of one of DCNU. All the DNCU covered Cx43 pixels (E-G & e-g) are defined as Cx43E-E. H&h. Two Cx43 pixels that are located outside of all DCNU covered areas and are therefore categorized as Cx43S-S.
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pone-0104357-g003: Schematic diagram of the blur algorithm with a dilated N-Cad unit (DNCU) radius of 1 inter-pixel distance (IPD) and 2 IPDs.A&a. An N-Cad pixel at the center of a DNCU (C&c). Dilation radius equals to 1 IPD and 2 IPD (B&b). D&d. 3 overlapping adjacent DCNUs (R = 1 IPD & 2 IPDs, respectively) containing 3 adjacent N-Cad pixels. E&e. Three Cx43 pixels that are pixel-by-pixel matched with three N-Cad pixels, respectively. F-G & f-g. Two Cx43 pixels that do not co-localize with any N-Cad pixel-by-pixel but are included within the area of one of DCNU. All the DNCU covered Cx43 pixels (E-G & e-g) are defined as Cx43E-E. H&h. Two Cx43 pixels that are located outside of all DCNU covered areas and are therefore categorized as Cx43S-S.

Mentions: To account for the possible lack of one-to-one matched abundance, we developed a blur algorithm to identify Cx43E-E signals including both N-Cad associated stellate Cx43 signals and N-Cad pixel-by-pixel matched Cx43 signals. This was achieved by using an N-Cad base area that covered the stellate Cx43 pixels. To form the base area each positive-stained N-Cad pixel (Figs. 3A, 3a) was computationally dilated at a certain radius (number of inter-pixel distance (IPD); Figs. 3B, 3b) to create a Dilated N-Cadherin Unit (DNCU; Figs. 3C, 3c) using a disk-shaped structuring element pre-coded in Matlab [32], [33]. An ID area between two adjacent myocytes could be composed of a group of adjacent DNCUs. Figs. 3A-3H are diagrams that schematically define a single DNCU and multiple DNCUs with a radius of 1 IPD, and Figs 3a-3h show diagrams of DNCUs with a radius of 2 IPDs. Cx43 pixels (green dots; Figs. 3E-3G and 3e-3g) that were covered by either a single DNCU or a group of adjacent DNCUs were considered as N-Cad co-localized Cx43E-E. These DNCU covered Cx43 pixels included both N-Cad pixel-by-pixel matched Cx43 (Figs. 3E,3e) and stellate Cx43 pixels (Figs. 3F-3G, 3f-3g). Cx43 pixels (Figs. 3H, 3h) located outside of the DNCUs were defined as Cx43S-S.


Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Schematic diagram of the blur algorithm with a dilated N-Cad unit (DNCU) radius of 1 inter-pixel distance (IPD) and 2 IPDs.A&a. An N-Cad pixel at the center of a DNCU (C&c). Dilation radius equals to 1 IPD and 2 IPD (B&b). D&d. 3 overlapping adjacent DCNUs (R = 1 IPD & 2 IPDs, respectively) containing 3 adjacent N-Cad pixels. E&e. Three Cx43 pixels that are pixel-by-pixel matched with three N-Cad pixels, respectively. F-G & f-g. Two Cx43 pixels that do not co-localize with any N-Cad pixel-by-pixel but are included within the area of one of DCNU. All the DNCU covered Cx43 pixels (E-G & e-g) are defined as Cx43E-E. H&h. Two Cx43 pixels that are located outside of all DCNU covered areas and are therefore categorized as Cx43S-S.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126721&req=5

pone-0104357-g003: Schematic diagram of the blur algorithm with a dilated N-Cad unit (DNCU) radius of 1 inter-pixel distance (IPD) and 2 IPDs.A&a. An N-Cad pixel at the center of a DNCU (C&c). Dilation radius equals to 1 IPD and 2 IPD (B&b). D&d. 3 overlapping adjacent DCNUs (R = 1 IPD & 2 IPDs, respectively) containing 3 adjacent N-Cad pixels. E&e. Three Cx43 pixels that are pixel-by-pixel matched with three N-Cad pixels, respectively. F-G & f-g. Two Cx43 pixels that do not co-localize with any N-Cad pixel-by-pixel but are included within the area of one of DCNU. All the DNCU covered Cx43 pixels (E-G & e-g) are defined as Cx43E-E. H&h. Two Cx43 pixels that are located outside of all DCNU covered areas and are therefore categorized as Cx43S-S.
Mentions: To account for the possible lack of one-to-one matched abundance, we developed a blur algorithm to identify Cx43E-E signals including both N-Cad associated stellate Cx43 signals and N-Cad pixel-by-pixel matched Cx43 signals. This was achieved by using an N-Cad base area that covered the stellate Cx43 pixels. To form the base area each positive-stained N-Cad pixel (Figs. 3A, 3a) was computationally dilated at a certain radius (number of inter-pixel distance (IPD); Figs. 3B, 3b) to create a Dilated N-Cadherin Unit (DNCU; Figs. 3C, 3c) using a disk-shaped structuring element pre-coded in Matlab [32], [33]. An ID area between two adjacent myocytes could be composed of a group of adjacent DNCUs. Figs. 3A-3H are diagrams that schematically define a single DNCU and multiple DNCUs with a radius of 1 IPD, and Figs 3a-3h show diagrams of DNCUs with a radius of 2 IPDs. Cx43 pixels (green dots; Figs. 3E-3G and 3e-3g) that were covered by either a single DNCU or a group of adjacent DNCUs were considered as N-Cad co-localized Cx43E-E. These DNCU covered Cx43 pixels included both N-Cad pixel-by-pixel matched Cx43 (Figs. 3E,3e) and stellate Cx43 pixels (Figs. 3F-3G, 3f-3g). Cx43 pixels (Figs. 3H, 3h) located outside of the DNCUs were defined as Cx43S-S.

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

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.

Methods and 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.

Show MeSH
Related in: MedlinePlus