Engineered heart tissue: a novel tool to study the ischemic changes of the heart in vitro

Abstract

Background: Understanding the basic mechanisms and prevention of any disease pattern lies mainly on development of a successful experimental model. Recently, engineered heart tissue (EHT) has been demonstrated to be a useful tool in experimental transplantation. Here, we demonstrate a novel function for the spontaneously contracting EHT as an experimental model in studying the acute ischemia-induced changes in vitro.

Methodology/principal findings: EHT was constructed by mixing cardiomyocytes isolated from the neonatal rats and cultured in a ring-shaped scaffold for five days. This was followed by mechanical stretching of the EHT for another one week under incubation. Fully developed EHT was subjected to hypoxia with 1% O(2) for 6 hours after treating them with cell protective agents such as cyclosporine A (CsA) and acetylcholine (ACh). During culture, EHT started to show spontaneous contractions that became more synchronous following mechanical stretching. This was confirmed by the increased expression of gap junctional protein connexin 43 and improved action potential recordings using an optical mapping system after mechanical stretching. When subjected to hypoxia, EHT demonstrated conduction defects, dephosphorylation of connexin-43, and down-regulation of cell survival proteins identical to the adult heart. These effects were inhibited by treating the EHT with cell protective agents.

Conclusions/significance: Under hypoxic conditions, the EHT responds similarly to the adult myocardium, thus making EHT a promising material for the study of cardiac functions in vitro.

Figure 1. Experimental Protocol.

Experimental protocol of the study.

Figure 2. Characterization of the fully developed EHT.

Samples were collected for electron microscopy (A) and western blotting (B1) after mechanical stretching. Tubulin was used as a loading control. Densitometry analysis was perfomed as explained in the methods. **P<0.001 versus before stretch. For optical mapping (B2), The EHT was loaded with voltage sensitive dye and images were captured with a CMOS-based high speed and high resolution optical mapping system.

Figure 3. Response of EHT to hypoxic stress.

A. Representative Immunoblotting analysis of connexin 43 in EHTs subjected to hypoxic stresses. Arrows indicate positions of phosphorylated isoform of connexin 43 (43 kDa) and nonphosphorylated isoform of connexin 43 (41 kDa) bands, respectively. Quantitative densitometric analysis represents the phosphorylated isoform of connexin 43. Values are mean ± SD ^#P<0.05 versus normal group and ^*P<0.05 versus hypoxic group. N = 5 in each group. B. Representative images showing the conduction defect evaluated by optical mapping, following exposure of EHTs to hypoxia, which was reverted by treatment with ACh. The synchronous conduction was lost in the EHT subjected to hypoxia. However, treatment with ACh prevented the conduction defect.

Figure 4. Cell survival Cascade analysis.

Representative immunoblot and quantitative analysis of Akt (A) and Bcl-2 (B) in EHTs exposed to hypoxia. Values are mean ± SD ^#P<0.05 versus normal group and ^*P<0.05 versus hypoxic group. N = 5 in each group.