Ph.D Thesis


Ph.D StudentShiti Assad
SubjectAtrial Cardiomycytes Derived from Human Induced
Pluripotent Stem Cells for Studing Atrial
Fibrillation
DepartmentDepartment of Medicine
Supervisor PROF. Lior Gepstein


Abstract

Atrial fibrillation (AF) is the most common sustained arrhythmia and is responsible for ~30% of all strokes. One of the limitations in studying the mechanisms underlying AF and in developing better treatment has been the lack of human atrial tissue models. The human induced pluripotent stem cells (hiPSCs) technology has been widely utilized in the field of cardiac disease modeling and drug screening. However, most functional studies using hiPSC-cardiomyocytes (CMs), to date, have been primarily focused on the single-cell level and utilized a mixture of differentiated hiPSC-CMs subtypes. We aimed to tackle these challenges by establishing differentiation schemes to generate atrial cells on one hand and functional tissue models on the other, eventually generating atrial multicellular tissue model that was utilized to study arrhythmia initiation and maintenance in inherited cases of AF.

Initially, we established a hiPSC-based two-dimensional - cardiac cell sheet model, that coupled with an optical mapping system, allowed to track the tissue’s electrical activity and study conduction, repolarization, and arrhythmogenesis. The resulting platform enabled pharmacological testing and monitoring of drug-induced arrhythmogenesis (manifested as early-after-depolarizations, triggered beats, or sustained spiral-waves) as well as its prevention using pharmacological and electrical means. By deploying phase mapping, we could track spiral-wave biophysical properties and study its initiation, perpetuation, and termination.

Next, we established both an EB-based and monolayer-based chamber-specific hiPSC differentiation schemes. The resulting atrial CMs displayed unique molecular (connexin40 upregulation and lack of MLC2v expression) and electrical characteristics (presence of IKACh, and short action potential with triangular morphology).

After establishment of the atrial differentiation protocol and tissue model, we continued to generate hiPSC-derived atrial cell sheets. We then utilized these models for studying two AF linked genetic conditions, at both the cellular and tissue levels. We first studied the atrial phenotype of the short QT syndrome (SQTS). When compared to healthy unrelated control and a CRISPR/CAS9-corrected isogenic control, SQTS-hiPSC atrial cells displayed an augmented IKr current, shortened action potential (AP) and abbreviated refractory period. Similarly, the SQTS hiPSC-atrial cell sheets displayed shortened AP as well as accelerated and extra curved spiral-wave arrhythmias. Studying potential drug treatments, we observed that both the abnormal electrical substrate and the enhanced arrhythmia stability parameters were reversed by the application of quinidine and vernakalant, but not by sotalol.

We next utilized the CRISPR/CAS9 system to introduce an AF-linked connexin40 mutation into healthy hiPSCs. The latter were differentiated to subtype-specific atrial and ventricular CMs and studied at the cellular and tissue level. Interestingly, the presence of the mutation did not alter AP properties in patch-clamp recordings of isolated atrial cells nor did it affect conduction properties of hiPSC-derived ventricular tissues. Only the combination of connexin40 mutant hiPSC-atrial CMs in a 2D cell sheet model revealed an abnormal phenotype manifested by atrial-restricted conduction slowing as well as increased arrhythmia inducibility and stability.

The work in the current project presents novel in-vitro platforms for modeling AF, which can elucidate the mechanisms underlying AF on the cellular and tissue level and eventually allow testing existing and innovative anti-arrhythmic interventions.