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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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Determining an optimal radius of DNCU for Cx43E-E and Cx43S-S quantification. A-B & L-M.Representative Cx43 (green) and N-Cad (red) double immunostaining confocal images (A, L) and grayscale images (B, M) of enlarged single myocytes containing Cx43E-E only (A) or both Cx43E-E and Cx43S-S (L). C-J & N-U. Grayscale images of the enlarged single myocytes with an incrementally increased DNCU radius (1, 5, 7, 30 IPD, respectively) for both N-Cad (left column) and Cx43 (right column). Arrows (U) indicate the inclusion of lateralized Cx43 by an extensively enlarged radius of the DNCU. K & V. Summarized data of quantitative Cx43 values corresponding to a continuously increased radii of DNCU when myocytes present Cx43E-E only (K) or present both Cx43E-E and Cx43S-S (V). The Y-axis value of the first plateau reflects quantitative amount of ID located Cx43E-E (red label) and the difference of the Y-axis value between the first and second plateaus indicates the amount of lateralized Cx43S-S.
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pone-0104357-g004: Determining an optimal radius of DNCU for Cx43E-E and Cx43S-S quantification. A-B & L-M.Representative Cx43 (green) and N-Cad (red) double immunostaining confocal images (A, L) and grayscale images (B, M) of enlarged single myocytes containing Cx43E-E only (A) or both Cx43E-E and Cx43S-S (L). C-J & N-U. Grayscale images of the enlarged single myocytes with an incrementally increased DNCU radius (1, 5, 7, 30 IPD, respectively) for both N-Cad (left column) and Cx43 (right column). Arrows (U) indicate the inclusion of lateralized Cx43 by an extensively enlarged radius of the DNCU. K & V. Summarized data of quantitative Cx43 values corresponding to a continuously increased radii of DNCU when myocytes present Cx43E-E only (K) or present both Cx43E-E and Cx43S-S (V). The Y-axis value of the first plateau reflects quantitative amount of ID located Cx43E-E (red label) and the difference of the Y-axis value between the first and second plateaus indicates the amount of lateralized Cx43S-S.

Mentions: We quantified all the Cx43 pixels that were covered by a group of DNCUs and analyzed the relationship between the Cx43 value and an incrementally extended DNCU radius (from 0 up to 40 IPDs; Fig 4). When all the Cx43 located at the ID areas and Cx43S-S pixels were absent (Fig. 4A), all positive-stained Cx43 pixels were defined as the Cx43E-E pixels. The quantitative value of Cx43 rapidly increased when the radius of DNCU was extended to 5 IPDs of the DNCU radius (Figs. 4C-4F). When the DNCU radius exceeded 5 IPDs, the quantitative value of Cx43 reached a peak and then remained at the plateau. At this plateau phase, the quantitative Cx43 peak value was not influenced by continuously increased DNCU radius (Figs. 4G-4J, 4K; red arrow). However, when both Cx43E-E and Cx43S-S were present (Fig. 4L), two peak plateaus of quantitative Cx43 values were found along with a continuously increased DNCU radius (up to 30 IPDs; Figs. 4N-4V). Fig. 4V shows that the value of Cx43 increased rapidly when IPD <5. When the DNCU radius increased up to 5 IPDs, the quantitative Cx43 value remained at a plateau. After the first plateau, continuously increasing the DNCU radius led to a second fast rising phase of quantitative Cx43 followed by a second plateau phase (Fig. 4V; blue arrow). While the first plateau phase reflects the amount of N-Cad associated Cx43E-E (Fig. 4V, red arrow), the second plateau phase was caused by the inclusion of N-Cad non-co-localized Cx43S-S signals (DNCU-uncovered Cx43; Fig. 4V, blue arrow). Thus, the Y-axis value of the first plateau phase reflects the quantitative amount of Cx43E-E, while the difference between the first and second plateaus at the Y-axis represents the quantitative amount of 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)

Determining an optimal radius of DNCU for Cx43E-E and Cx43S-S quantification. A-B & L-M.Representative Cx43 (green) and N-Cad (red) double immunostaining confocal images (A, L) and grayscale images (B, M) of enlarged single myocytes containing Cx43E-E only (A) or both Cx43E-E and Cx43S-S (L). C-J & N-U. Grayscale images of the enlarged single myocytes with an incrementally increased DNCU radius (1, 5, 7, 30 IPD, respectively) for both N-Cad (left column) and Cx43 (right column). Arrows (U) indicate the inclusion of lateralized Cx43 by an extensively enlarged radius of the DNCU. K & V. Summarized data of quantitative Cx43 values corresponding to a continuously increased radii of DNCU when myocytes present Cx43E-E only (K) or present both Cx43E-E and Cx43S-S (V). The Y-axis value of the first plateau reflects quantitative amount of ID located Cx43E-E (red label) and the difference of the Y-axis value between the first and second plateaus indicates the amount of lateralized Cx43S-S.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126721&req=5

pone-0104357-g004: Determining an optimal radius of DNCU for Cx43E-E and Cx43S-S quantification. A-B & L-M.Representative Cx43 (green) and N-Cad (red) double immunostaining confocal images (A, L) and grayscale images (B, M) of enlarged single myocytes containing Cx43E-E only (A) or both Cx43E-E and Cx43S-S (L). C-J & N-U. Grayscale images of the enlarged single myocytes with an incrementally increased DNCU radius (1, 5, 7, 30 IPD, respectively) for both N-Cad (left column) and Cx43 (right column). Arrows (U) indicate the inclusion of lateralized Cx43 by an extensively enlarged radius of the DNCU. K & V. Summarized data of quantitative Cx43 values corresponding to a continuously increased radii of DNCU when myocytes present Cx43E-E only (K) or present both Cx43E-E and Cx43S-S (V). The Y-axis value of the first plateau reflects quantitative amount of ID located Cx43E-E (red label) and the difference of the Y-axis value between the first and second plateaus indicates the amount of lateralized Cx43S-S.
Mentions: We quantified all the Cx43 pixels that were covered by a group of DNCUs and analyzed the relationship between the Cx43 value and an incrementally extended DNCU radius (from 0 up to 40 IPDs; Fig 4). When all the Cx43 located at the ID areas and Cx43S-S pixels were absent (Fig. 4A), all positive-stained Cx43 pixels were defined as the Cx43E-E pixels. The quantitative value of Cx43 rapidly increased when the radius of DNCU was extended to 5 IPDs of the DNCU radius (Figs. 4C-4F). When the DNCU radius exceeded 5 IPDs, the quantitative value of Cx43 reached a peak and then remained at the plateau. At this plateau phase, the quantitative Cx43 peak value was not influenced by continuously increased DNCU radius (Figs. 4G-4J, 4K; red arrow). However, when both Cx43E-E and Cx43S-S were present (Fig. 4L), two peak plateaus of quantitative Cx43 values were found along with a continuously increased DNCU radius (up to 30 IPDs; Figs. 4N-4V). Fig. 4V shows that the value of Cx43 increased rapidly when IPD <5. When the DNCU radius increased up to 5 IPDs, the quantitative Cx43 value remained at a plateau. After the first plateau, continuously increasing the DNCU radius led to a second fast rising phase of quantitative Cx43 followed by a second plateau phase (Fig. 4V; blue arrow). While the first plateau phase reflects the amount of N-Cad associated Cx43E-E (Fig. 4V, red arrow), the second plateau phase was caused by the inclusion of N-Cad non-co-localized Cx43S-S signals (DNCU-uncovered Cx43; Fig. 4V, blue arrow). Thus, the Y-axis value of the first plateau phase reflects the quantitative amount of Cx43E-E, while the difference between the first and second plateaus at the Y-axis represents the quantitative amount of 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