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Comparative Study
. 2010 Jun;298(6):C1603-9.
doi: 10.1152/ajpcell.00435.2009. Epub 2010 Mar 24.

Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart

Keng-Leong Ang  1 , Lincoln T Shenje, Sean Reuter, Mark H Soonpaa, Michael Rubart, Loren J Field, Manuel Galiñanes
Affiliations
Comparative Study

Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart

Keng-Leong Ang et al. Am J Physiol Cell Physiol. 2010 Jun.

Abstract

Accurate nuclear identification is crucial for distinguishing the role of cardiac myocytes in intrinsic and experimentally induced regenerative growth of the myocardium. Conventional histologic analysis of myocyte nuclei relies on the optical sectioning capabilities of confocal microscopy in conjunction with immunofluorescent labeling of cytoplasmic proteins such as troponin T, and dyes that bind to double-strand DNA to identify nuclei. Using heart sections from transgenic mice in which the cardiomyocyte-restricted alpha-cardiac myosin heavy chain promoter targeted the expression of nuclear localized beta-galactosidase reporter in >99% of myocytes, we systematically compared the fidelity of conventional myocyte nuclear identification using confocal microscopy, with and without the aid of a membrane marker. The values obtained with these assays were then compared with those obtained with anti-beta-galactosidase immune reactivity in the same samples. In addition, we also studied the accuracy of anti-GATA4 immunoreactivity for myocyte nuclear identification. Our results demonstrate that, although these strategies are capable of identifying myocyte nuclei, the level of interobserver agreement and margin of error can compromise accurate identification of rare events, such as cardiomyocyte apoptosis and proliferation. Thus these data indicate that morphometric approaches based on segmentation are justified only if the margin of error for measuring the event in question has been predetermined and deemed to be small and uniform. We also illustrate the value of a transgene-based approach to overcome these intrinsic limitations of identifying myocyte nuclei. This latter approach should prove quite useful when measuring rare events.

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Figures

Fig. 1.
Fig. 1.
Representative maximal projection images used in the study. A–C: images of myocytes predominantly in the transverse orientation. D–F: myocytes orientated predominantly in the longitudinal orientation. Pseudocoloring: red = troponin T, blue = 4′,6-diamidino-2-phenylindole (DAPI), white = wheat germ agglutinin (WGA), and green = β-galactosidase (β-GAL) myocyte nucleus.
Fig. 2.
Fig. 2.
Typical problems encountered in nuclear identification. Yellow arrow shows a nonmyocyte nucleus surrounded by the myocyte cytoplasm, which was thought to be of myocyte origin. Light blue arrow points to a misidentified myocyte nucleus that was located in the periphery of a myocyte (see text for detailed explanation of arrows). Pseudocoloring: white = cell membrane delineated by WGA, red = troponin T, blue = DAPI nuclear counterstain, and green = β-GAL myocyte nucleus.
Fig. 3.
Fig. 3.
Relationship between GATA4 and β-GAL immune reactivity. A: nuclei with positive β-GAL immune reactivity (pseudocolored green). B and C: GATA4 positive nuclei (red) and DAPI (blue) nuclei staining, respectively, within the same field. The merged image is represented in D. The yellow arrows point to nuclei that had positive GATA4, and β-GAL immune reactivities. The white arrow points to a GATA4 positive nucleus, which lacked β-GAL signal.
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