Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images

Antoni Oliver-Gelabert,Manuel Vázquez-Sancho,Laura García-Mendívil,Marta Matamala-Adell,Emiliano Raúl Diez,Fernando Sorribas-Berjón,José María Vallejo-Gil,Narcisa Tribulova,Esther Pueyo,Katarína Andelová,Pedro Carlos Fresneda-Roldán,Laura Ordovás,Javier Fañanás-Mastral,Carlos Ballester-Cuenca

doi:10.3390/biom10091334

Abstract

Cardiomyocytes’ geometry and connexin 43 (CX43) amount and distribution are structural features that play a pivotal role in electrical conduction. Their quantitative assessment is of high interest in the study of arrhythmias, but it is usually hampered by the lack of automatic tools. In this work, we propose a software algorithm (Myocyte Automatic Retrieval and Tissue Analyzer, MARTA) to automatically detect myocytes from fluorescent microscopy images of cardiac tissue, measure their morphological features and evaluate the expression of CX43 and its degree of lateralization. The proposed software is based on the generation of cell masks, contouring of individual cells, enclosing of cells in minimum area rectangles and splitting of these rectangles into end-to-end and middle compartments to estimate CX43 lateral-to-total ratio. Application to human ventricular tissue images shows that mean differences between automatic and manual methods in terms of cardiomyocyte length and width are below 4 m. The percentage of lateral CX43 also agrees between automatic and manual evaluation, with the interquartile range approximately covering from 3% to 30% in both cases. MARTA is not limited by fiber orientation and has an optimized speed by using contour filtering, which makes it run hundreds of times faster than a trained expert. Developed for CX43 studies in the left ventricle, MARTA is a flexible tool applicable to morphometric and lateralization studies of other markers in any heart chamber or even skeletal muscle. This open-access software is available online.

Highlights

The size and shape of cardiomyocytes (CMs) are major determinants of the electrical propagation in the heart [1,2,3,4]
Human and rat ventricular tissue images obtained by using various combinations of antibodies and fluorophores and imaged with different microscopes were analyzed to show the versatility of our proposed software
Input images d1 and d2 correspond to the same image d but in the first case using one channel (F-actin) and in the second case using two channels (F-actin and connexin 43 (CX43))

Summary

Introduction

The size and shape of cardiomyocytes (CMs) are major determinants of the electrical propagation in the heart [1,2,3,4]. Conduction through extracellular spaces depends on tissue structure. The arrangement of myocytes, non-myocyte cells and connective tissue influence the properties of the extracellular conductor [5]. Electrical conduction in the heart is highly determined by the expression and distribution of connexins. Connexins are the proteins that form intercellular channels in the gap junctions of the myocardial tissue [6]. Gap junctions allow electrical and metabolic connection between CMs, giving the myocardial tissue a syncytium behavior. Gap junctions are concentrated at the intercalated discs between myocytes, located at their longitudinal ends. This lateralization process can occur in a relatively short time period or remain stable [7]

Methods

Results

Conclusion