Periodic Reporting for period 4 - iPS-ChOp-AF (Combining induced pluripotent stem cells, tissue engineering, optogenetic and chemogenetic concepts for the study and treatment of atrial fibrillation)
Periodo di rendicontazione: 2022-09-01 al 2023-08-31
Cardiac arrhythmias lead to abnormalities in the rate or rhythm of the heartbeats, which result in a beating rate that is too quick, too slow, or irregular. The different cardiac arrhythmias are responsible for substantial worldwide morbidity and mortality and consequentially in significant burden on the health care systems. For example, atrial fibrillation (AF), the most common arrhythmia type, 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 arrhythmias, such as AF, have been the lack of suitable human cardiac tissue models and specifically those reflecting patient/disease-specific functional and structural abnormalities and the inability to perform targeted, high-resolution, functional, and reversible perturbations of the system.
To address these challenges, we aimed to combine advances in genetics, human induced pluripotent stem cells (hiPSCs) and genome-editing (CRISPR) technologies, development biology-inspired differentiating systems yielding specific heart cell subtypes, novel tissue engineering strategies, state-of-the-art electrophysiology methodologies, and emerging concepts from the fields of optogenetics and chemogenetics.
To achieve the overall goal of the project, our first aim was to establish efficient differentiation protocols from hiPSCs to derive specific cardiac cell subtypes, primarily focusing on atrial cells. We next aimed to combine advances in clinical genetics, hiPSC, and genome editing (CRISPR) technologies to develop and study patient/disease-specific hiPSC models of familial AF. Since arrhythmias in AF are re-entrant and therefore cannot be modelled at the cellular level, the second aim focused on establishing novel cardiac tissue models to study arrhythmia mechanisms and therapies, initially simple 2D models and later by using advanced tissue-engineering concepts to establish complex 3D atrial tissue models of AF.
Our third and fourth aims involved integrating optogenetic and chemogenetic tools into these tissue models and eventually into animal models. Optogenetics involves the expression, by gene therapy means, of light sensitive proteins (ion channels) in different electrically-active tissues, while chemogenetics involves introduction of engineered proteins (ligand-specific engineered receptors) into the targeted cells, whose function can be controlled externally by specific pharmacology. Using these approaches, we aimed to provide mechanistic insights and to develop novel anti-arrhythmic therapies for arrhythmias like AF.
By completing the aforementioned goals of the project, the deliverables went beyond the state-of-the-art by providing novel experimental platforms to study cardiac electrophysiology at the single-cell and tissue levels. This can eventually to lead to a paradigm shift in the way human cardiac arrhythmias can studied and treated.
Major hurdles in studying and developing better treatments for cardiac arrhythmias, such as AF, have been the lack of suitable human cardiac tissue models and specifically those reflecting patient/disease-specific functional and structural abnormalities and the inability to perform targeted, high-resolution, functional, and reversible perturbations of the system.
To address these challenges, we aimed to combine advances in genetics, human induced pluripotent stem cells (hiPSCs) and genome-editing (CRISPR) technologies, development biology-inspired differentiating systems yielding specific heart cell subtypes, novel tissue engineering strategies, state-of-the-art electrophysiology methodologies, and emerging concepts from the fields of optogenetics and chemogenetics.
To achieve the overall goal of the project, our first aim was to establish efficient differentiation protocols from hiPSCs to derive specific cardiac cell subtypes, primarily focusing on atrial cells. We next aimed to combine advances in clinical genetics, hiPSC, and genome editing (CRISPR) technologies to develop and study patient/disease-specific hiPSC models of familial AF. Since arrhythmias in AF are re-entrant and therefore cannot be modelled at the cellular level, the second aim focused on establishing novel cardiac tissue models to study arrhythmia mechanisms and therapies, initially simple 2D models and later by using advanced tissue-engineering concepts to establish complex 3D atrial tissue models of AF.
Our third and fourth aims involved integrating optogenetic and chemogenetic tools into these tissue models and eventually into animal models. Optogenetics involves the expression, by gene therapy means, of light sensitive proteins (ion channels) in different electrically-active tissues, while chemogenetics involves introduction of engineered proteins (ligand-specific engineered receptors) into the targeted cells, whose function can be controlled externally by specific pharmacology. Using these approaches, we aimed to provide mechanistic insights and to develop novel anti-arrhythmic therapies for arrhythmias like AF.
By completing the aforementioned goals of the project, the deliverables went beyond the state-of-the-art by providing novel experimental platforms to study cardiac electrophysiology at the single-cell and tissue levels. This can eventually to lead to a paradigm shift in the way human cardiac arrhythmias can studied and treated.
We succeeded in meeting the overall objective of this project, which was to develop novel hiPSC-based experimental models and tools in order to gain mechanistic insights and to develop new treatments for cardiac arrhythmias, and specifically AF. In the first Aim, we created disease-specific hiPSC models of AF either directly from patients with genetic AF or by introducing the relevant mutation to control hiPSCs by gene editing. We next derived atrial cells from healthy and disease hiPSC lines and confirmed their atrial-specific molecular and functional properties. These studies raised important insights into the abnormal cellular properties associated with the different genetic causes of AF including abnormal action potential, conduction, or calcium-handling properties.
Since AF is reentrant in nature and cannot be modeled at the cellular level, in Aim 2 we established novel cardiac tissue models. Initially, we generated 2D hiPSC-derived cardiac cell-sheets, which were used to model and study atrial reentrant arrhythmic activity. Next, we introduced tissue-engineering concepts to establish 3D atrial tissue models of AF. These included engineered heart tissues (EHTs), in which the atrial or ventricular cells were embedded in a collagen or cardiac extracellular matrix (ECM) hydrogel to generate ring-shaped tissues, and decellularization/recellularization approaches, in which acellular heart-derived atrial or ventricular scaffolds were seeded with the appropriate hiPSC-derived cardiac cells. Using these models we were able to recapitulate and provide mechanistic insights into the clinical phenotype of different acquired and inherited AF syndromes, to evaluate atrial-specific anti-arrhythmic drugs, and develop novel molecular therapies.
In the third and fourth aims, we integrated optogenetic and chemogenetic tools into the hiPSC-based and animal cardiac arrhythmia models. By using gene therapy to express light-sensitive or ligand-activated ion channels, we were able to achieve remote controllable functional perturbations of the cardiac tissue models. Our new approach allowed to functionally modulate (augment-pace or suppress-silence electrical activity) the tissue's electrical properties at a high spatial and temporal resolutions by light or specific pharmacology in a reversible manner. Consequentially, we were able to establish a novel approach to study arrhythmia mechanisms as well as to provide proof-of-concept evidence for the ability to use optogenetic/chemogenetic tools as future novel anti-arrhythmic therapies (optogenetic “pacing”, “cardioversion” and “molecular ablation”).
The development of the aforementioned hiPSCs-based "disease-in-a-dish" models coupled with the use of opto-/chemo-genetic tools represents a paradigm shift in the way we study cardiac rhythm disorders, to development of novel therapies, and to individualize patient-specific therapies.
Since AF is reentrant in nature and cannot be modeled at the cellular level, in Aim 2 we established novel cardiac tissue models. Initially, we generated 2D hiPSC-derived cardiac cell-sheets, which were used to model and study atrial reentrant arrhythmic activity. Next, we introduced tissue-engineering concepts to establish 3D atrial tissue models of AF. These included engineered heart tissues (EHTs), in which the atrial or ventricular cells were embedded in a collagen or cardiac extracellular matrix (ECM) hydrogel to generate ring-shaped tissues, and decellularization/recellularization approaches, in which acellular heart-derived atrial or ventricular scaffolds were seeded with the appropriate hiPSC-derived cardiac cells. Using these models we were able to recapitulate and provide mechanistic insights into the clinical phenotype of different acquired and inherited AF syndromes, to evaluate atrial-specific anti-arrhythmic drugs, and develop novel molecular therapies.
In the third and fourth aims, we integrated optogenetic and chemogenetic tools into the hiPSC-based and animal cardiac arrhythmia models. By using gene therapy to express light-sensitive or ligand-activated ion channels, we were able to achieve remote controllable functional perturbations of the cardiac tissue models. Our new approach allowed to functionally modulate (augment-pace or suppress-silence electrical activity) the tissue's electrical properties at a high spatial and temporal resolutions by light or specific pharmacology in a reversible manner. Consequentially, we were able to establish a novel approach to study arrhythmia mechanisms as well as to provide proof-of-concept evidence for the ability to use optogenetic/chemogenetic tools as future novel anti-arrhythmic therapies (optogenetic “pacing”, “cardioversion” and “molecular ablation”).
The development of the aforementioned hiPSCs-based "disease-in-a-dish" models coupled with the use of opto-/chemo-genetic tools represents a paradigm shift in the way we study cardiac rhythm disorders, to development of novel therapies, and to individualize patient-specific therapies.
The achievements made in the project introduced several concepts that are beyond the state of the art.
(1) Development of advanced, patient-specific tissue models of AF. This was made possible by combing patient/disease-specific hiPSCs; CRISPR-based genome-editing; hiPSC differentiation strategies to derive atrial cells; and advanced tissue engineering strategies.
(2) Use of advanced tissue engineering techniques. These 2D/3D cardiac tissue models allowed to assess complex electrophysiological phenomena such as conduction and reentrant arrhythmias, and were especially useful for studying AF. By using decellularization/recellularization and 3D bioprinting we were also able to introduce anatomical and microarchitecture properties to the models further enhancing their clinical relevance.
(3) Introducing optogenetic and chemogenetic concepts that coupled with the hiPSC-based tissue models introduced a new paradigm for arrhythmia mechanistic studies and treatment. Traditional research tools and anti-arrhythmic therapies are hampered by their global cardiac action, low efficacy, and pro-arrhythmia (drugs), irreversible and destructive nature (ablation), and pain (defibrillation). Here we overcame these shortcomings by introducing the concept of optogenetics and chemogenetics, which allow targeted, high-resolution, functional, and reversible perturbations of the system; serving as a unique tool to study arrhythmia mechanism and potentially also as novel anti-arrhythmic strategies.
(1) Development of advanced, patient-specific tissue models of AF. This was made possible by combing patient/disease-specific hiPSCs; CRISPR-based genome-editing; hiPSC differentiation strategies to derive atrial cells; and advanced tissue engineering strategies.
(2) Use of advanced tissue engineering techniques. These 2D/3D cardiac tissue models allowed to assess complex electrophysiological phenomena such as conduction and reentrant arrhythmias, and were especially useful for studying AF. By using decellularization/recellularization and 3D bioprinting we were also able to introduce anatomical and microarchitecture properties to the models further enhancing their clinical relevance.
(3) Introducing optogenetic and chemogenetic concepts that coupled with the hiPSC-based tissue models introduced a new paradigm for arrhythmia mechanistic studies and treatment. Traditional research tools and anti-arrhythmic therapies are hampered by their global cardiac action, low efficacy, and pro-arrhythmia (drugs), irreversible and destructive nature (ablation), and pain (defibrillation). Here we overcame these shortcomings by introducing the concept of optogenetics and chemogenetics, which allow targeted, high-resolution, functional, and reversible perturbations of the system; serving as a unique tool to study arrhythmia mechanism and potentially also as novel anti-arrhythmic strategies.