Ph.D Thesis


Ph.D StudentGruber Amit
SubjectOptogenetics Control of Human Induced Pluripotent Stem Cell
Derived Cardiomyocytes
DepartmentDepartment of Medicine
Supervisor PROF. Lior Gepstein
Full Thesis textFull thesis text - English Version


Abstract

Background: Optogenetics, a technology based on light-sensitive proteins, has emerged as an experimental paradigm to modulate neuronal and cardiomyocyte excitability. In this thesis, we aimed to develop high-resolution optogenetic approaches to modulate excitability in single cells and engineered heart tissue models derived from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). With the combination of optogenetics and hiPSC technologies we aim to model inherited arrhythmogenic syndromes and re-entrant cardiac arrhythmias, treat the underlying arrhythmogenic causes, and terminate arrhythmia when it emerges.

 

Methods: For the different aims in this thesis, we used healthy control and patient-specific hiPSC-CMs. Patient-specific cells carried mutations for either the long or short QT syndromes (LQTS or SQTS, respectively). To allow optogenetic control we delivered different optogenetic genes using either direct viral transduction of target cardiomyocytes or indirect sensitization to light via engineered opsin-donor cells. The direct and the indirect approaches were used for single cell and tissue models, respectively. The tissue models were comprised from a mixture of genetically engineered HEK293 cells expressing a light-sensitive cationic channel (i.e. CoChR or CheRiff) and hiPSC-CMs. These well-calibrated co-cultures were generated as either two-dimensional hiPSC-derived cardiac cell-sheet (hiPSC-CCSs) or three-dimensional engineered heart tissue (EHT) models. Optical stimulations were delivered in high temporal resolution, and when necessary, could also be delivered in complex spatial illumination patterns thanks to a high-resolution digital micromirror device (DMD).  Whole-cell patch clamp, optical monitoring and force measurements techniques were then performed to evaluate the electrophysiological and mechanical consequences of the optical perturbations in different experimental models.

 

Results: We divide the results into two parts. In part one, we revealed the ability to optogenetically pace and shape conduction patterns in hiPSC-CCSs and EHTs using complex geometrical illumination patterns. This allowed to establish in-vitro models for optogenetic-based cardiac resynchronization therapy, where electrical activation could be synchronized in both the hiPSC-CCS and EHT models, resulting in improved contractile activity in the EHT model. Next, we were able to establish reentrant activity (rotors) in our hiPSC-derived tissue models through an optogenetic cross-field optical stimulation protocol. We then studied the mechanism by which application of diffuse illumination can terminate arrhythmias, and we characterized the potency of reduced exposure to light in terminating rotors, identifying a critical mass for cardioversion.

In part two of the results, we demonstrate how precise timing of optical stimulation during an action potential (AP) can modulate the cardiomyocyte's AP properties, correct the repolarization abnormalities associated with the arrhythmogenic LQTS and SQTS, and even eliminate the arrhythmogenic early after depolarizations (EADs) in LQTS cells.

 

Conclusions: Our results demonstrate the unique potential in combining optogenetics and hiPSC technologies to derive light-controllable human cardiomyocytes and cardiac tissue models. This strategy can be used to manipulate excitability in a functional, reversible, and localized manner. This approach may bring a unique value for physiological/pathophysiological studies, for disease modeling, and for development of optogenetics-based treatments: cardiac pacing, resynchronization, defibrillation, and treatment of repolarization abnormalities.