The Spatiotemporal Development of Intercalated Disk in Three-Dimensional Engineered Heart Tissues Based on Collagen/Matrigel Matrix

Jin Zhou,Yan Wang,Shuang-Hong Lü,Qiu-Xia Lin,Xia Li,Jun-Jie Li,Yao Shu,Cui-Mi Duan,Yong-Chao Mou,Hong-Yu Sun,Chang-Yong Wang,Rong-Yu Tang,Abhay Pandit

doi:10.1371/journal.pone.0081420

Abstract

Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.

Highlights

The last decade has witnessed great improvement in cardiac tissue engineering, and a variety of biomaterials, including naturally polymers, synthetic polymers and decellularized extracellular matrix (ECM), have been developed to construct cardiac tissues with rhythmic contraction in vitro [1,2,3,4]
Via intercalated disk (ID), cardiomyocytes are electromechanically coupled into a functional syncitium, and it is essential for normal morphology and function of myocardium [7]
Remodeling of cardiomyocytes in the engineered heart tissues (EHTs) The neonatal rat cardiomyocytes lost their classic morphology in collagen/Matrigel matrix at first, and most cells shrank to spheroids

Summary

Introduction

The last decade has witnessed great improvement in cardiac tissue engineering, and a variety of biomaterials, including naturally polymers, synthetic polymers and decellularized extracellular matrix (ECM), have been developed to construct cardiac tissues with rhythmic contraction in vitro [1,2,3,4]. Highly organized sarcomeres and specialized intercellular junctions, i.e., intercalated disk (ID) can be observed in engineered heart tissues (EHTs), suggesting that some ultra-structural features of EHTs resemble that of native myocardium [5,6]. ID, composed of adherens junctions, gap junctions and desmosomes, is a complex and highly orchestrated structure. Via ID, cardiomyocytes are electromechanically coupled into a functional syncitium, and it is essential for normal morphology and function of myocardium [7]. The formation of specialized ID may be closely associated with the quality and function of EHTs. And current research has demonstrated that adherens junctions and/or gap junctions can be detected by the expression of their hallmark proteins in the EHTs [3,8]

Methods

Results

Conclusion