Multicellular In vitro Models of Cardiac Arrhythmias: Focus on Atrial Fibrillation

Abstract

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.

Keywords: (conditionally) immortalized cardiomyocyte; (induced) pluripotent stem cell-derived cardiomyocyte; arrhythmia research; atrial fibrillation; disease modeling; in vitro model; primary cardiomyocyte.

Figure 1

Overview of the different 2D multicellular in vitro models of AF. The figure is divided in 4 top panels describing the different techniques to generate 2D multicellular in vitro atrial arrhythmia models and a bottom panel depicting the various applications of these models (i.e., fundamental research, drug screening, disease modeling and regenerative medicine). The 1st panel from the left describes the generation of the HL-1 cell line. This cell line was derived from a transgenic mice in which expression of SV40 Tag is driven by the atrium-specific human NPPA promoter (HsNPPA-Tag). This resulted in mice carrying right atrial tumors, which were isolated and enzymatically dissociated yielding cells that could be kept in culture as the HL-1 cell line. This cell line can proliferate while retaining an immature AM phenotype. The 2nd panel from the left shows how AMs and non-CMs (e.g., fibroblasts) can be derived from atrial tissue samples of animals (e.g., neonatal rats) by enzymatic digestion and cultured in vitro. The resulting mixed atrial cell preparations also served as starting material for the conditional immortalization procedure depicted in the 3rd panel from the left, which led to the generation of iAM cell lines including iAM-1. The conditional immortalization was achieved by transducing primary AMs with a repressor-based lentiviral Tet-on system (represented by the lentiviral particle), in which the muscle-specific promoter MHCK7 drives the expression of a temperature-sensitive mutant of SV40 Tag designated tsA58 (iMHCK7.Tag-tsA58). The addition of doxycycline results in dedifferentiation and proliferation of the cells while its removal causes redifferentiation toward fully functional AMs. The 4th panel from the left describes the use of (i)PSCs, which are generated either by the disruption of embryos (ESCs) or by reprogramming somatic cells (iPSCs) using viral (e.g., Sendai virus) or non-viral vector-based methods. The (i)PSCs can proliferate virtually indefinitely and be differentiated in a mixed population of immature CM-like cells by a multistep procedure recapitulating the different stages of cardiac development and involving the use of specific inducers. After cell sorting/enrichment, these cells as well as the cells generated by the other techniques, can be used for different applications as depicted in the lower panel. The lower right picture merely serves to indicate that AMs/atrial tissue constructs might be applied in the future for reparative purposes (like the repair of atrial septal defects) and does not want to suggest their suitability for the regeneration of ventricular tissue.