Molecular Mechanisms Underlying Human Cardiac Cell Junction Maturation and Disease Using Human iPSC

Heart disease is the number one cause of death and disability in California and in the United States. Especially devastating is Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), an inherited form of heart disease associated with a high frequency of arrhythmias and sudden cardiac death in young people, including young athletes, who despite their appearance of health are struck down by this type of heart disease. Even though it is inherited, early detection is hindered because people carrying the genetic code have highly variable clinical symptoms, making ARVC and catastrophic cardiac events very hard to predict and avoid. Evidence suggests that this heart disease is caused by mistakes in the genetic code essential for holding the mechanical integrity of heart muscle cells together or cell junctions. What is missing is an understanding of the basic biology of these heart muscle cell junctions in humans and appropriate human model systems to study their dynamics in heart disease, which is important since other heart diseases also share some of these same heart cell defects. Our goal is to understand the basic biology of how human heart muscle cell junctions mature and what happens in disease, by studying ARVC. Human iPS cells are a unique population of stem cells from our own tissues, such as skin, that have the same genetic information as the rest of our bodies. Thus, hiPS from people who carry the ARVC heart disease mistakes can be used in our laboratory to provide a true human model of that disease. During the first year of our grant, we have enrolled sufficient numbers of normal and ARVC donors into our study. We have collected skin biopsy tissues from donors as means to generate hiPS cells. Our results show that hiPS cell lines can be efficiently generated from both normal and ARVC donors and we have extensively characterized their profiles, such that we know they are bona fide stem cell lines and can be used as a model system to dissect defects in cardiac cell junction biology between these various different hiPS lines. We have also developed efficient and robust methodologies to generate heart muscle cells from hiPS from normal and ARVC donors that carry mistakes in the genetic code for cell junction components and are now in the midst of characterizing their molecular, genetic, biochemical and functional profiles to identify features in these cells that are unique for ARVC. Through our previous studies, we identified new pathways that may be important causes of ARVC, thus we will also use our hiPS lines, to confirm whether these new pathways are truly important in human ARVC disease progression and if our approaches reverse disease progression. Towards this goal, we have generated novel tools to increase and decrease a component of this pathway in order to test these approaches and have preliminary data to show that these tools are efficient in altering levels of this component in heart muscle cells, which we are now applying towards understanding these pathways in hiPS derived heart muscle cells and reversing defects in heart muscle cells from ARVC hiPS derived lines. Based on our progress, we have met all of the milestones stated in our grant proposal and in some cases, surpassed some milestones. We believe progress over the next year, will allow us to define some of the key cellular defects in ARVC and advance our understanding of how cell-cell junctions mature in hiPS and highlight tools that influence the microenvironment of the hiPS in a dish, to accelerate this process.

Overall, we have been able to achieve the milestones proposed for Year 2 of the grant. We have generated a panel of control and ARVC hiPSC lines using integration-free based methods. We provide evidence of our method to generate robust numbers of hiPSC-derived cardiac cells that express desmosomal cell-cell junction proteins. We show ARVC lines that display disease symptom-specific features (adipogenic or arrhythmic), which phenocopy the striking and differential symptoms found in respective individual ARVC-patients as tools to study human ARVC. We also uncover desmosomal defects in hiPSC-derived cardiac muscle cells that underlie the disease features found in ARVC cells. We have also published two reviews in the field of cell-cell junctional remodeling and stem cell approaches that helps to further our understanding of this field in cardiomyocytes, that is relevant to human disease and our research using hiPS.

Overall, we have been able to complete the milestones proposed for our grant. We have generated a unique panel of control and ARVC hiPSC lines using integration-free methods. We provide evidence of our method to generate robust numbers of hiPSC derived cardiac cells that express key components of the cardiac muscle cell-cell junction include mechanical junctions and electrical junctions. We show that our ARVC hiPSC lines display disease symptom-specific features (adipogenic and arrhythmic), which phenocopy the striking and differential diagnosis observed in our ARVC donor hearts and provide a platform to study the varying disease features underlying ARVC. We uncover novel and classic molecular and ultrastructural defects underlying the arrhythmogenic defects in our ARVC hiPSC lines that mimic the gradation in disease severity observed in ARVC donor hearts. We exploit conventional ARVC drugs to determine their impact on arrhythmogenic behavior and reversibility of phenotypes in our cells. We have published 4 articles in the field of cell-cell junction remodeling, protein turnover and stem cell approaches that further our understanding of this field in cardiac muscle cells as well as filed a provisional patent application on the use of a novel drug discovery system for fat arrhythmogenic disorders that exploit the genetic diversity and clinical features observed in our ARVC lines.