Final Report Summary - HEART-IPS (Congenital heart disease-associated arrhythmia:<br/>deciphering Hamamy syndrome, novel rare disease, using iPS cells)
In the growing population of patients with congenital heart defects (CHD), while arrhythmias are not a major issue for children, they are a leading complication during adulthood. My hypothesis is that the origin of some life-threatening arrhythmias in adult, lies in cardiac structural and electrophysiological development. Thus, studying causes of CHDs may reveal key steps, mis-regulated in adult patients with CHD, leading to arrhythmias.
Cardiac development and adult electrical function are finely regulated by transcription factors (TFs). Mutations in TFs have previously been linked to patients with CHD who are developing arrhythmias in adulthood. Hamamy syndrome is a newly described rare disease with CHD and rhythm disorders, caused by a mutation in IRX5 TF. In adult mice, Irx5 is known to regulate cardiac electrical function.
I hypothesize that IRX5 mutation in Hamamy-affected patients, leads to misregulation of heart development, promoting arrhythmic events during adulthood. The general objective of the two years HEART-iPS project was to understand role of Iroquois transcription factors in the control of generation and propagation of cardiac electrical impulse, to characterize the cardiac function altered by IRX5 mutation, and to understand the syndrome’s molecular mechanism.
To achieve these goals, we designed a multidisciplinary experimental plan that combines newly generated patient-derived induced pluripotent stem cells and transgenic mouse lines. First, HEART-iPS project provided the opportunity to fully develop at the host institution, the iPS cell technology with their cardiac differentiation followed by their analysis. This work resulted in a publication in the Journal of American Heart Association (Jouni et al., 2015). Using those technics, we then investigated cardiomyocytes differentiated from iPS cells generated from two Hamamy patients. We found that the Hamamy mutations in IRX5 did not alter IRX5 expression, however, they resulted in a loss of functionality. Based on the patient’s phenotype and previously published data in mouse models, we investigated (1) the role of IRX5 on the cardiac electrical activity, and (2) Hamamy syndrome’s pathophysiological mechanism.
(1) We recorded and compared action potentials from cardiomyocytes derived from control and Hamamy iPS cells and observed that cells bearing IRX5 mutated protein have a reduction in their action potential duration. We thus analyzed the expression of ion channels in Hamamy derived cardiomyocytes. As compared to control cells, the expression of KChiP2, the regulatory subunit of Ito current is increased, while the α-subunit, KCND3, is unaltered. With further experiments, we showed that IRX5 seem to directly inhibit KChiP2 expression, modulating the corresponding Ito current, and leading to the reduction of cardiac action potential duration. Furthermore, combining experiments on gene and protein expression analysis, immunofluorescence, and chromatin immunoprecipitation we found that IRX5 directly regulates the expression of Connexin 43, an actor of the electrical conduction, in human cardiac cells.
(2) Using Co-immunoprecipitation assays, we also identified new cardiac co-factors of IRX5: IRX3 and GATA4. Directly of interest here, these transcription factors have been shown in animal models to have a role in the conduction of cardiac electrical influx. Our data show that in control cardiomyocytes, IRX5 binds to IRX3 and GATA4 to regulate electrical conduction and that IRX5 proteins carrying Hamamy-specific mutations, sequesters these co-factors in non-functional heterodimers. This suggests that the IRX5 mutant protein alters Hamamy patient’s cardiac electrical conduction through a dominant-negative effect on other transcription factors. In parallel, analyses of a mouse model for Hamamy syndrome are currently in progress. Finally, we are working on assembling in a manuscript the results generated during the HEART-iPS contract, for publication.
With the conduction of this project, we have illustrated the ability of human iPS cell technology to model the abnormal functional phenotype of an inherited cardiac disorder. Overall, this study will provide immediate insight into understanding fundamental processes by which some arrhythmias in the adult originate in cardiac development. As such, it represents a promising model to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.
Being a recipient for the Marie Curie fellowship greatly supported my application for a tenure track position that I obtained at the first attempt at the end of the first year HEART-iPS contract. Conducting the Hamamy study allowed me to strongly establish my research domain at the host laboratory and to generate new collaborations.
Cardiac development and adult electrical function are finely regulated by transcription factors (TFs). Mutations in TFs have previously been linked to patients with CHD who are developing arrhythmias in adulthood. Hamamy syndrome is a newly described rare disease with CHD and rhythm disorders, caused by a mutation in IRX5 TF. In adult mice, Irx5 is known to regulate cardiac electrical function.
I hypothesize that IRX5 mutation in Hamamy-affected patients, leads to misregulation of heart development, promoting arrhythmic events during adulthood. The general objective of the two years HEART-iPS project was to understand role of Iroquois transcription factors in the control of generation and propagation of cardiac electrical impulse, to characterize the cardiac function altered by IRX5 mutation, and to understand the syndrome’s molecular mechanism.
To achieve these goals, we designed a multidisciplinary experimental plan that combines newly generated patient-derived induced pluripotent stem cells and transgenic mouse lines. First, HEART-iPS project provided the opportunity to fully develop at the host institution, the iPS cell technology with their cardiac differentiation followed by their analysis. This work resulted in a publication in the Journal of American Heart Association (Jouni et al., 2015). Using those technics, we then investigated cardiomyocytes differentiated from iPS cells generated from two Hamamy patients. We found that the Hamamy mutations in IRX5 did not alter IRX5 expression, however, they resulted in a loss of functionality. Based on the patient’s phenotype and previously published data in mouse models, we investigated (1) the role of IRX5 on the cardiac electrical activity, and (2) Hamamy syndrome’s pathophysiological mechanism.
(1) We recorded and compared action potentials from cardiomyocytes derived from control and Hamamy iPS cells and observed that cells bearing IRX5 mutated protein have a reduction in their action potential duration. We thus analyzed the expression of ion channels in Hamamy derived cardiomyocytes. As compared to control cells, the expression of KChiP2, the regulatory subunit of Ito current is increased, while the α-subunit, KCND3, is unaltered. With further experiments, we showed that IRX5 seem to directly inhibit KChiP2 expression, modulating the corresponding Ito current, and leading to the reduction of cardiac action potential duration. Furthermore, combining experiments on gene and protein expression analysis, immunofluorescence, and chromatin immunoprecipitation we found that IRX5 directly regulates the expression of Connexin 43, an actor of the electrical conduction, in human cardiac cells.
(2) Using Co-immunoprecipitation assays, we also identified new cardiac co-factors of IRX5: IRX3 and GATA4. Directly of interest here, these transcription factors have been shown in animal models to have a role in the conduction of cardiac electrical influx. Our data show that in control cardiomyocytes, IRX5 binds to IRX3 and GATA4 to regulate electrical conduction and that IRX5 proteins carrying Hamamy-specific mutations, sequesters these co-factors in non-functional heterodimers. This suggests that the IRX5 mutant protein alters Hamamy patient’s cardiac electrical conduction through a dominant-negative effect on other transcription factors. In parallel, analyses of a mouse model for Hamamy syndrome are currently in progress. Finally, we are working on assembling in a manuscript the results generated during the HEART-iPS contract, for publication.
With the conduction of this project, we have illustrated the ability of human iPS cell technology to model the abnormal functional phenotype of an inherited cardiac disorder. Overall, this study will provide immediate insight into understanding fundamental processes by which some arrhythmias in the adult originate in cardiac development. As such, it represents a promising model to study disease mechanisms, optimize patient care (personalized medicine), and aid in the development of new therapies.
Being a recipient for the Marie Curie fellowship greatly supported my application for a tenure track position that I obtained at the first attempt at the end of the first year HEART-iPS contract. Conducting the Hamamy study allowed me to strongly establish my research domain at the host laboratory and to generate new collaborations.