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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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Using N-Cad as an internal housekeeping reference for imaging quantification.A-B. Confocal images of Cx43 (green) and N-Cad (red) double immunostaining on two LA tissue sections obtained from two young rabbits (Rabbit-1 and Rabbit-2). C. Difference of quantitative total Cx43 raw data between the two confocal images obtained from Rabbit-1 and Rabbit-2 LA. D. A comparable amount of total Cx43 between the two rabbit LA samples when Cx43 raw data was normalized to N-Cad. E. Immunoblotting image of total Cx43 and GAPDH protein expression in Rabbit-1 and Rabbit-2 LA tissue homogenates. F. Comparison of quantitative immuno-stained Cx43 signal intensity with or without N-Cad normalization in aged rabbit LA.
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pone-0104357-g005: Using N-Cad as an internal housekeeping reference for imaging quantification.A-B. Confocal images of Cx43 (green) and N-Cad (red) double immunostaining on two LA tissue sections obtained from two young rabbits (Rabbit-1 and Rabbit-2). C. Difference of quantitative total Cx43 raw data between the two confocal images obtained from Rabbit-1 and Rabbit-2 LA. D. A comparable amount of total Cx43 between the two rabbit LA samples when Cx43 raw data was normalized to N-Cad. E. Immunoblotting image of total Cx43 and GAPDH protein expression in Rabbit-1 and Rabbit-2 LA tissue homogenates. F. Comparison of quantitative immuno-stained Cx43 signal intensity with or without N-Cad normalization in aged rabbit LA.

Mentions: Tissue sectioning and histological processing variability can impact Cx43 quantification. Fig. 5 shows an example of different quantitative results of Cx43 from two healthy young rabbit hearts. One image of a Cx43/N-Cad double immunostained LA section from young healthy rabbit 1 (Rabbit-1_LA) apparently presented a greater amount of Cx43 than that of another LA section from health young rabbit 2 (rabbit-2_LA; Figs. 5A-5C). However, after normalizing quantified Cx43 raw data to the co-stained N-Cad quantitative values, abundances of total Cx43 on the two tissue sections were comparable (Fig. 5D). To further confirm the protein expression levels of Cx43 in the two healthy young rabbit LA tissue samples, an immunoblotting assay was performed. Fig. 5E reveals that the Cx43 protein expression levels were similar in the two young rabbit LA samples (Fig. 5D). Immunoblotting results were therefore consistent with the quantitative Cx43 result normalized with N-Cad. Our data suggest that the difference of quantitative IHC Cx43 values between the two young rabbit atrial sections were not due to Cx43 expression variation. To further validate the effect of N-Cad normalization in reducing quantification errors, we quantified Cx43/N-Cad double stained images from four aged rabbit atrial sections. Fig. 5F shows N-Cad normalization significantly reduced variations of quantified immunostained Cx43 signals. Thus, our results suggest that N-Cad could be an effective internal reference for eliminating quantification errors due to tissue sectioning and tissue fiber orientation variables.


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)

Using N-Cad as an internal housekeeping reference for imaging quantification.A-B. Confocal images of Cx43 (green) and N-Cad (red) double immunostaining on two LA tissue sections obtained from two young rabbits (Rabbit-1 and Rabbit-2). C. Difference of quantitative total Cx43 raw data between the two confocal images obtained from Rabbit-1 and Rabbit-2 LA. D. A comparable amount of total Cx43 between the two rabbit LA samples when Cx43 raw data was normalized to N-Cad. E. Immunoblotting image of total Cx43 and GAPDH protein expression in Rabbit-1 and Rabbit-2 LA tissue homogenates. F. Comparison of quantitative immuno-stained Cx43 signal intensity with or without N-Cad normalization in aged rabbit LA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104357-g005: Using N-Cad as an internal housekeeping reference for imaging quantification.A-B. Confocal images of Cx43 (green) and N-Cad (red) double immunostaining on two LA tissue sections obtained from two young rabbits (Rabbit-1 and Rabbit-2). C. Difference of quantitative total Cx43 raw data between the two confocal images obtained from Rabbit-1 and Rabbit-2 LA. D. A comparable amount of total Cx43 between the two rabbit LA samples when Cx43 raw data was normalized to N-Cad. E. Immunoblotting image of total Cx43 and GAPDH protein expression in Rabbit-1 and Rabbit-2 LA tissue homogenates. F. Comparison of quantitative immuno-stained Cx43 signal intensity with or without N-Cad normalization in aged rabbit LA.
Mentions: Tissue sectioning and histological processing variability can impact Cx43 quantification. Fig. 5 shows an example of different quantitative results of Cx43 from two healthy young rabbit hearts. One image of a Cx43/N-Cad double immunostained LA section from young healthy rabbit 1 (Rabbit-1_LA) apparently presented a greater amount of Cx43 than that of another LA section from health young rabbit 2 (rabbit-2_LA; Figs. 5A-5C). However, after normalizing quantified Cx43 raw data to the co-stained N-Cad quantitative values, abundances of total Cx43 on the two tissue sections were comparable (Fig. 5D). To further confirm the protein expression levels of Cx43 in the two healthy young rabbit LA tissue samples, an immunoblotting assay was performed. Fig. 5E reveals that the Cx43 protein expression levels were similar in the two young rabbit LA samples (Fig. 5D). Immunoblotting results were therefore consistent with the quantitative Cx43 result normalized with N-Cad. Our data suggest that the difference of quantitative IHC Cx43 values between the two young rabbit atrial sections were not due to Cx43 expression variation. To further validate the effect of N-Cad normalization in reducing quantification errors, we quantified Cx43/N-Cad double stained images from four aged rabbit atrial sections. Fig. 5F shows N-Cad normalization significantly reduced variations of quantified immunostained Cx43 signals. Thus, our results suggest that N-Cad could be an effective internal reference for eliminating quantification errors due to tissue sectioning and tissue fiber orientation variables.

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