Modeling the Atrioventricular Conduction Axis Based on Human Pluripotent Stem Cell-Derived Cardiac Assembloids

Research Background

The atrioventricular (AV) conduction axis is responsible for electrical conduction between the atrium and the ventricle and is a core component of the cardiac electrophysiological system. The delay function of AV conduction ensures coordinated contraction between the atrium and ventricle to maintain normal blood flow. Myocardial cells in the AV node region have slow impulse conduction characteristics; this delay is crucial for blood filling. Dysfunction in the AV conduction system can lead to severe arrhythmias and contraction abnormalities, such as AV conduction block. However, existing research models, like mouse and zebrafish models, have limitations in simulating key features of the human AV conduction system, highlighting the urgent need for a more physiologically relevant human model to study the pathology of the AV node region.

Research Objectives and Methods

To this end, scientists, including Jiuru Li, used human induced pluripotent stem cell (hiPSC)-derived cardiac assembloids to simulate the AV conduction axis, thus deeply exploring the functional mechanisms and related pathology of the AV conduction system. The research team is from the Amsterdam UMC and the Amsterdam Cardiovascular Sciences Institute, and their findings were published in the journal “Cell Stem Cell.” The focus of the study was to induce cardiac mesoderm cells to differentiate into AV canal myocardial cells (AVCMs) using Wnt2 and retinoic acid (RA), thereby reconstructing the AV conduction axis through a multi-component tissue system and verifying its effectiveness in simulating complex arrhythmias such as AV conduction block.

Research Process

This study employed a monolayer guided differentiation method to generate AV canal myocardial cells, treating hiPSCs with Wnt2 and RA on day 4 to guide their differentiation into AV canal myocardial cells. On approximately day 10, all myocardial cell subtypes (including AV node, atrial, and ventricular) exhibited visible contractile activity. Flow cytometry at 18-20 days showed that 70%-90% of the terminally differentiated myocardial cells expressed the TNNT2 protein, marking successful differentiation.

To further verify the identity of these cells, the research team analyzed the transcriptome of differentiated cells using single-cell RNA sequencing (scRNA-seq). They found that the gene expression of myocardial cells treated with Wnt2 and RA closely resembled in vivo AV canal myocardial cells, especially with higher expression levels of specific genes such as TBX2 and TBX3, displaying AVCM characteristics. Moreover, researchers repeated the experiments using different hiPSC lines to ensure the reproducibility of the differentiation process.

Research Results

The study demonstrated that Wnt2 and RA treatment successfully differentiated hiPSCs into myocardial cells with AV canal characteristics, whose gene expression and electrophysiological properties are similar to in vivo AV node cells. Evaluating the electrophysiological properties of AVCMs through single-cell patch clamp experiments revealed significant characteristics in depolarization speed, action potential duration, and calcium homeostasis, further verifying their identity as myocardial cells in the AV node region.

To more authentically simulate the AV conduction axis, the research team cultured different myocardial cell subtypes (including atrial, AV canal, and ventricular cells) into three-dimensional spheroids, forming the so-called “cardiac assembloids.” In these assembloids, electrical impulses transmitted from the atrial to the ventricular end, exhibiting a fast-slow-fast conduction pattern consistent with in vivo. Further immunofluorescence staining confirmed the specific expression patterns of cells in different regions, demonstrating the authenticity of the AV conduction axis model.

Pathological Study of LMNA Gene Mutation

The study also explored the pathological mechanism of AV conduction block caused by LMNA gene mutation. LMNA gene mutations are known to be associated with AV conduction block and other arrhythmias. Researchers isolated hiPSCs from patients carrying LMNA mutations to generate AVCMs and construct assembloids. They found that these mutant assembloids exhibited impulse conduction block phenomena at higher frequencies (2Hz and 3Hz). Further analysis revealed calcium-handling abnormalities in mutant AVCMs, mainly characterized by excessive calcium release and delayed calcium transient decay, leading to slow myocardial cell depolarization, which may be fundamental to AV conduction block.

To address this issue, the research team attempted to use the chemical molecule S107 to reduce calcium leakage. They found it could effectively reduce abnormal depolarization and alleviate AV conduction block, validating its potential therapeutic value.

Conclusion and Significance

The study demonstrated that AVCMs obtained through Wnt2 and RA guided differentiation exhibit the characteristics of in vivo AV canal myocardial cells and successfully reconstructed the AV conduction axis in three-dimensional assembloids. The method of generating AVCMs is simple and efficient, applicable to multiple hiPSC lines, and holds broad application potential. This model can be used not only to simulate complex arrhythmias but also to provide new research tools for understanding the fundamental biological mechanisms of the AV node region.

Research Highlights

1. AV Conduction Axis Model Construction: Using pluripotent stem cell-derived AVCM assembloids to authentically simulate the fast-slow-fast conduction pattern of the AV conduction axis, making it a powerful tool for studying AV conduction pathology.
2. Genetic Mutation Research: Assembloids based on LMNA gene mutation revealed conduction block caused by calcium homeostasis abnormalities, providing new insights into the etiology of complex arrhythmias.
3. Potential Therapeutic Target: The study showed that calcium homeostasis modulator S107 alleviates AV conduction block, offering new intervention strategies for AV node pathology.

Research Limitations

Although this study constructed an AV conduction axis model similar to in vivo, the model lacked non-myocardial cells in the AV node region (such as fibroblasts and endothelial cells). In the future, methods like three-dimensional bioprinting could be considered to further enhance model complexity. Additionally, hiPSC-derived myocardial cells are not yet mature and differ from adult in vivo myocardial cells, which is an important direction for future research.

Summary

This study innovatively constructed a functional AV conduction axis model using human pluripotent stem cells, providing a powerful tool for studying the physiological and pathological processes of the cardiac conduction system. By simulating arrhythmia pathology associated with genetic mutations and testing potential therapeutic strategies, this platform demonstrates its great potential in researching complex heart diseases.