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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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Interaction between Cx43 and N-Cad proteins in human LA.A. Representative double immunostaining image of Cx43 (green) and N-Cad (red). B-E. Enlarged images from a cropped area of image A including the Cx43/N-Cad overlay image (B), N-Cad (red; C), N-Cad pixel-by-pixel matched Cx43 signals (D), and non-N-Cad co-localized Cx43 (E). Arrows indicate stellate Cx43 pixels are in close proximity with the N-Cad pixel but do not pixel-by-pixel co-localize with N-Cad. F. Immunoblotting images of co-immunoprecipitated N-Cad protein with immunoprecipitated Cx43 proteins. Right column is the immunoprecipitated negative control without primary Cx43 antibody.
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pone-0104357-g002: Interaction between Cx43 and N-Cad proteins in human LA.A. Representative double immunostaining image of Cx43 (green) and N-Cad (red). B-E. Enlarged images from a cropped area of image A including the Cx43/N-Cad overlay image (B), N-Cad (red; C), N-Cad pixel-by-pixel matched Cx43 signals (D), and non-N-Cad co-localized Cx43 (E). Arrows indicate stellate Cx43 pixels are in close proximity with the N-Cad pixel but do not pixel-by-pixel co-localize with N-Cad. F. Immunoblotting images of co-immunoprecipitated N-Cad protein with immunoprecipitated Cx43 proteins. Right column is the immunoprecipitated negative control without primary Cx43 antibody.

Mentions: Initially, we used a pixel-by-pixel determination approach to define the relationship between double immuno-stained Cx43 and N-Cad fluorescent signals on atrial tissue sections (Fig. 1). Pixels were considered as N-Cad co-localized Cx43 (Fig. 1E) if they were positively stained pixels in both Cx43 and N-Cad channels. Any positively stained Cx43 pixels that were negatively stained in the N-Cad channel were considered as N-Cad non-colocalized Cx43 pixels (Fig. 1F). Integrated intensity of all positively stained Cx43 pixels was defined as total Cx43 (Cx43T). However, we discovered that ID associated Cx43 did not always co-localize with N-Cad in a pixel-by-pixel fashion in the rabbit LA tissue sections (Figs. 1E & 1F). Figs.1D-1F shows a typical example of Cx43 clusters that distributed closely with the N-Cad plaques in general, but some Cx43 pixels (red arrows of Figs. 1D,1F) did not overlap with the N-Cad pixels, despite being in close proximity. Moreover, this phenomenon found in rabbit atrium was also discovered in human atrial tissues (Figs. 2A-2E). Since we were concerned that such pixel-by-pixel determination of N-Cad matched Cx43 could lead to an underestimation of Cx43E-E and overestimation of Cx43S-S, we next sought to consider whether stellate Cx43 signals were free-standing Cx43 proteins or were closely associated with N-Cad on IDs. To define the relationship between Cx43 and N-Cad, we performed immunoprecipitation experiments using human atrial tissue homogenates. Cx43 proteins were immunoprecipitated (IPed) with a specific anti-Cx43 antibody. We then performed immunoblotting to detect co-immunoprecipitated (co-IPed) N-Cad with Cx43 proteins as previously described [26], [28]. We found a greater amount of IPed Cx43 that was associated with a lesser abundance of co-IPed N-Cad (Fig. 2F). While it may not be an optimal approach to compare protein abundance detected from using two different antibodies in the setting of immunoblotting, our co-IP data further confirmed the relationship between Cx43 and N-Cad found in immunostaining images. Both results from using either IHC or co-IP techniques indicate that although N-Cad is part of the Cx43 molecular complex, the two molecules may not interact in a one-to-one matched abundance. This mismatched abundance of colocalized Cx43 and N-Cad and interior localization of N-Cad to Cx43 plaque is consistent with the findings from several other cellular systems [29]–[31].


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)

Interaction between Cx43 and N-Cad proteins in human LA.A. Representative double immunostaining image of Cx43 (green) and N-Cad (red). B-E. Enlarged images from a cropped area of image A including the Cx43/N-Cad overlay image (B), N-Cad (red; C), N-Cad pixel-by-pixel matched Cx43 signals (D), and non-N-Cad co-localized Cx43 (E). Arrows indicate stellate Cx43 pixels are in close proximity with the N-Cad pixel but do not pixel-by-pixel co-localize with N-Cad. F. Immunoblotting images of co-immunoprecipitated N-Cad protein with immunoprecipitated Cx43 proteins. Right column is the immunoprecipitated negative control without primary Cx43 antibody.
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Related In: Results  -  Collection

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

pone-0104357-g002: Interaction between Cx43 and N-Cad proteins in human LA.A. Representative double immunostaining image of Cx43 (green) and N-Cad (red). B-E. Enlarged images from a cropped area of image A including the Cx43/N-Cad overlay image (B), N-Cad (red; C), N-Cad pixel-by-pixel matched Cx43 signals (D), and non-N-Cad co-localized Cx43 (E). Arrows indicate stellate Cx43 pixels are in close proximity with the N-Cad pixel but do not pixel-by-pixel co-localize with N-Cad. F. Immunoblotting images of co-immunoprecipitated N-Cad protein with immunoprecipitated Cx43 proteins. Right column is the immunoprecipitated negative control without primary Cx43 antibody.
Mentions: Initially, we used a pixel-by-pixel determination approach to define the relationship between double immuno-stained Cx43 and N-Cad fluorescent signals on atrial tissue sections (Fig. 1). Pixels were considered as N-Cad co-localized Cx43 (Fig. 1E) if they were positively stained pixels in both Cx43 and N-Cad channels. Any positively stained Cx43 pixels that were negatively stained in the N-Cad channel were considered as N-Cad non-colocalized Cx43 pixels (Fig. 1F). Integrated intensity of all positively stained Cx43 pixels was defined as total Cx43 (Cx43T). However, we discovered that ID associated Cx43 did not always co-localize with N-Cad in a pixel-by-pixel fashion in the rabbit LA tissue sections (Figs. 1E & 1F). Figs.1D-1F shows a typical example of Cx43 clusters that distributed closely with the N-Cad plaques in general, but some Cx43 pixels (red arrows of Figs. 1D,1F) did not overlap with the N-Cad pixels, despite being in close proximity. Moreover, this phenomenon found in rabbit atrium was also discovered in human atrial tissues (Figs. 2A-2E). Since we were concerned that such pixel-by-pixel determination of N-Cad matched Cx43 could lead to an underestimation of Cx43E-E and overestimation of Cx43S-S, we next sought to consider whether stellate Cx43 signals were free-standing Cx43 proteins or were closely associated with N-Cad on IDs. To define the relationship between Cx43 and N-Cad, we performed immunoprecipitation experiments using human atrial tissue homogenates. Cx43 proteins were immunoprecipitated (IPed) with a specific anti-Cx43 antibody. We then performed immunoblotting to detect co-immunoprecipitated (co-IPed) N-Cad with Cx43 proteins as previously described [26], [28]. We found a greater amount of IPed Cx43 that was associated with a lesser abundance of co-IPed N-Cad (Fig. 2F). While it may not be an optimal approach to compare protein abundance detected from using two different antibodies in the setting of immunoblotting, our co-IP data further confirmed the relationship between Cx43 and N-Cad found in immunostaining images. Both results from using either IHC or co-IP techniques indicate that although N-Cad is part of the Cx43 molecular complex, the two molecules may not interact in a one-to-one matched abundance. This mismatched abundance of colocalized Cx43 and N-Cad and interior localization of N-Cad to Cx43 plaque is consistent with the findings from several other cellular systems [29]–[31].

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