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Combining induced pluripotent stem cells, tissue engineering, optogenetic and chemogenetic concepts for the study and treatment of atrial fibrillation

Periodic Reporting for period 2 - iPS-ChOp-AF (Combining induced pluripotent stem cells, tissue engineering, optogenetic and chemogenetic concepts for the study and treatment of atrial fibrillation)

Reporting period: 2019-09-01 to 2021-02-28

Cardiac arrhythmias are defined as any deviation from the normal pattern or rate of the cardiac electrical excitation and result in significant morbidity and mortality. For example, atrial fibrillation (AF), the most common clinical arrhythmia, is associated with a doubling in all-cause mortality, a five-fold increase in stroke (being responsible for 30% of all stroke cases), and often with the development of progressive heart failure.

Major hurdles in studying and developing better treatments for cardiac rhythm disorders, such as AF, have been the lack of suitable human cardiac tissue models and specifically those reflecting patient/disease-specific abnormalities; the paucity of methods for long-term repeated analysis of the tissue's electrophysiological (EP) properties; and the inability to perform targeted, high-resolution, functional, reversible perturbations of the system.

To address the aforementioned challenges, we propose to combine advances in genetics, human induced pluripotent stem cells (hiPSC) and genome-editing (CRISPR) technologies, development biology-inspired differentiating systems that yield specific heart cell subtypes, novel tissue engineering strategies, state-of-the-art electrophysiological mapping techniques, and emerging concepts from the fields of optogenetics and chemogenetics.

The end-result should go beyond the state-of-the-art and provide novel experimental platforms to study cardiac electrophysiology at the single-cell and tissue levels and eventually lead to a paradigm shift in the way we can study and treat human cardiac arrhythmias.

To achieve the overall goal of the current project, our first aim was to establish efficient differentiation protocols from hiPSCs to derive specific cardiac cell subtypes, primarily focusing on the ability to generate atrial cells. We next aimed to combine advances in clinical genetics, hiPSC, and genome editing (CRISPR) technologies to identify genetic causes of AF, to develop patient/disease-specific hiPSC models of familial AF, and subsequently to perform detailed cellular phenotyping of the generated hiPSC-derived atrial cells. Since arrhythmias in AF are re-entrant and therefore cannot be modelled at the cellular level, our next aim focused on establishing novel tissue models. Initially, we aimed to establish 2D large-scale hiPSC-derived cardiac cellular-sheets, which can be used to model atrial re-entrant arrhythmic activity ("rotors"). Next, we aimed to introduce tissue-engineering concepts to establish 3D atrial tissue models of AF.

Our next aim involved integrating optogenetics tools into our models. Specifically, we aimed to use light sensitive proteins (channels and pumps) coupled with targeted illumination to perform controllable functional perturbations to the system. Our new approach aims to functionally modulate (augment or suppress) the tissue's electrical properties at a high spatial and temporal resolutions and in a controlled and reversible manner. Beyond serving as a novel approach to study arrhythmia mechanisms, the results of this stage can lead to development of novel optogenetics-based treatments of AF and other arrhythmias. The final methodology ("chemogenetics") involves introduction of engineered proteins (G-protein coupled receptors or ion channels) into the targeted cells, whose function can be controlled externally by specific pharmacology. Chemogenetics does not allow the same-level of spatiotemporal control to modulate excitability as optogenetics, but may be more practical clinically. Our final aim will focus on evaluating the potential optogenetics and chemogenetics based therapeutic concepts in animal models of AF.
The overall objective of this project was to develop novel experimental models and tools to gain mechanistic insights and to develop new treatments for cardiac arrhythmias in general, and atrial fibrillation (AF) specifically. To achieve this goal, in our first Aim, we successfully created patient/disease-specific human induced pluripotent stem cell (hiPSC) models from patients inflicted with a variety of inherited arrhythmogenic disorders that could give rise to atrial fibrillation (AF). We next developed and optimized differentiation protocols to derive purified cell-populations of atrial cells from the different hiPSC lines. Detailed molecular, structural, and functional studies confirmed the presence of an atrial-specific phenotypes of the differentiated cells. These studies have also raised important insights into the abnormal cellular electrophysiological properties associated with this genetic rhythm disorders involving abnormal action potential or calcium-handling properties.

Since arrhythmias, like AF, are usually reentrant in nature they can only be induced and studied at the tissue level. To tackle this challenge, in the second aim, we focused on establishing tissue models from the hiPSC-derived atrial or ventricular cells. Initially, we were able to establish a unique two-dimensional tissue model, whose electrical properties could be studied in detail using a unique optical mapping system. Using this model we were able to recapitulate and provide mechanistic insights into the clinical phenotype of a familial arrhythmogenic syndrome termed the short QT syndrome (SQTS). Our results demonstrated the ability to induce reentrant arrhythmias in the SQTS-hiPSC derived tissue models, to study the mechanisms involved in their generation and perpetuation, and to study the potential therapeutic effects of different anti-arrhythmic drugs. The development of the aforementioned hiPSCs-based "disease-in-a-dish" model represents a paradigm shift in the way we study inherited cardiac disorders, could lead to better understanding of such diseases, to development of novel therapies, and to the ability to individualize patient-specific therapies.

Moving from two-dimensions to the three-dimensional level, we were able to use advanced tissue engineering strategies (hydrogel and decellularization/recellularization approaches) coupled with the use of hiPSC-derive chamber-specific cardiomyocytes to generate clinically-relevant tissue-constructs. The generated atrial tissues demonstrated atrial-specific functional activity, could be used to model atrial fibrillation through the induction of reentrant activity, and was used to study the effects of different drugs for their potential anti-arrhythmic activity.

Since drug therapy for AF has been hampered by its limited efficacy, global activity and significant side effects, in the third Aim, we introduced an out-of-the-box approach (optogenetics) as an alternative therapeutic strategy. The optogenetic approach utilizes light-sensitive channels and pumps, which are expressed in the targeted tissues, coupled with targeted illumination to modulate the electrical properties of excitable tissues. Using this strategy, we were able augment (using ChR2, a light-sensitive cationic channel) or to suppress (using ACR-2, a light-sensitive anionic channel) the electrical activity in our 2D/3D hiPSC-derived cardiac tissue engineered models. Congenitally, we were able to provide proof-of-concept evidence for the ability to use optogenetic tools to achieve optogenetic "biological pacing", to synchronized electrical activity and improve the tissue's mechanical performance ("optogenetics cardiac resynchronization therapy"), and to induce and terminate reentrant arrhythmias ("optogenetics defibrillation").
Our current results so far in the project has already introduced several concepts, which are beyond the current state of the art. These include:
(1) The concepts of hiPSC-based modelling of inherited and acquired arrhythmias focused mainly on the cellular levels before the initiation of this project. Work resulting from this project, significantly advanced the field by providing means to study such arrhythmias also at the tissue level. This is crucial since the major mechanism underlying most clinical cardiac arrhythmias is reentry, which can only be formed at the tissue level.
(2) One of the limitations of hiPSC-modeling of cardiac disorders was that the differentiated cells contained a mixed population of different heart cell types (ventricular, atrial and nodal/pacemaker cells). In the current project, by using development biology guided differentiation protocols we were able to derive purified populations of either ventricular or atrial cells, with the latter cells being used to model atrial disorders like AF.
(3) Previous tissue engineering strategies could not really recapitulate the human heart either because they used non-human cells or scaffolds that did not reflect the cardiac anatomy. By combing human-specific cardiac cell subtypes (using the aforementioned hiPSC differentiation protocols) and chamber-specific anatomically-accurate scaffolds (using organ decellularization/recellularization processes) we could generate clinically-relevant tissue engineered chambers such as atrial structures.
(4) Traditional anti-arrhythmic therapies are aimed at modifying the abnormal electrophysiological substrate either by focal injury (surgery or radiofrequency catheter ablation), implantable devices, or by specific pharmacology. The latter approach may be significantly hampered by its global cardiac action, low efficacy, and significant pro-arrhythmic. Here we aimed to tackle these challenges by introducing the concept of optogenetics, which allows targeted, high-resolution, functional, and reversible perturbations of the system; potentially serving as a novel anti-arrhythmic concept that can bypass many of these shortcomings.

In the second half of the project we aim to continue and expand the work on each of these aspects. In addition, we plan to also introduce the concept of chemogenetics. Chemogenetics is a parallel discipline that involves the use of modified genetically encoded actuator-coupled receptors that are selectively activated by specific pharmacologically. This approach holds great therapeutic promise, since the used ligands can be administered orally with high specificity to the target cells thereby limiting potential adverse effects.
We next plan to implement such optogenetic and chemogenetic tools in our hiPSC based models at multiple levels with the goals of: (1) inducing targeted perturbation of the system to gain insights into basic arrhythmia mechanisms and (2) to develop novel therapeutic strategies for the treatment of AF. Finally, we will use the lessons learned from our in vitro models to try to derive similar optogenetics and chemogenetic therapeutic strategies also using in vivo animal models of cardiac arrhythmias and atrial fibrillation.