Model of Timothy Syndrome to Screen Drugs with Induced Pluripotent Stem Cells


Principal Investigator: Masayuki Yazawa
Abstract: Abstract: Prolonged QT interval, the electrical manifestation of repolarization in ventricular myocytes, is a major cause of cardiac arrhythmia and sudden death. Long QT syndrome (LQTS) can have a genetic basis or be induced by drug exposure or physiological stress. Drug-induced LQTS is a side effect of many drugs that have approved and is a common cause of drug failure in clinical trials. Though many of the genes are reported to cause LQTS, the mechanisms underlying the disease in humans are incompletely understood. My career goal is to develop novel systems to uncover molecular and cellular mechanisms underlying human cardiac arrhythmia and to find lead compounds for pharmaceutical applications to treat arrhythmia. My personal motivation for this study is that I have a grandmother who had suffered severe arrhythmia and then died last year. As a professional scientist I'd like to contribute to cardiovascular fields to help as many patients suffering arrhythmia as possible. Key elements of my career goal are 1) to develop human models of cardiac arrhythmia to examine how cardiac arrhythmia occurs in human hearts;2) to develop screen methods using human cells to find new lead compounds that have better effects but less side effects than present ones. To accomplish this goal, I have focused calcium signaling in heart function and development since undergraduate studies. This is because depletion of calcium related molecules in mice induced lethal cardiac dysfunction in most cases and many mutations in the molecules are reported to be associated with human cardiac diseases including LQTS. Here I propose to study a missense mutation in the L-type Ca2+ channel, CaV1.2, which causes LQTS and lethal arrhythmia in patients with Timothy syndrome (TS) in order to explore the effect of the TS mutation on the electrical activity and contraction of human cardiomyocytes (CMs). While TS is a rare disorder, CaV1.2 channels play important roles in generation of action potential and in excitation- contraction coupling for heart muscles. Therefore, human model of TS would be a useful platform to study mechanisms of arrhythmia and to test drugs for future treatment of cardiac arrhythmia. In preliminary studies, to develop human models of TS, I reprogrammed human skin cells from two TS patients to generate induced pluripotent stem cells (iPSCs) and differentiated these cells into CMs. Electrophysiological recording and Ca2+ imaging studies of these cells revealed irregular contraction, excess Ca2+ influx, prolonged action potentials, delayed afterdepolarizations and irregular Ca2+ signaling. Using these cells I found that roscovitine restored the electrical and Ca2+ signaling properties of TS CMs. The approach using iPSC-derived CMs provides new opportunities for studying the molecular and cellular mechanisms of cardiac arrhythmias in humans and for developing new drugs to treat these diseases. However, it is still difficult to screen a library of chemical compounds to treat lethal arrhythmia using human iPSC-derived CMs because electrophysiological recordings are not easily used for developing medium- throughput screen to find lead compounds to treat cardiac disease. Therefore, the goal of this project is to develop and validate an iPSC-based screening method that can be used to identify therapies for cardiac arrhythmia. This goal encompasses the approaches as follow: 1) Further characterization of phenotypes in TS cardiomyocytes: Using a variety of assays I will ask how TS mutation induce lethal ventricular tachycardia and whether TS mutation alters proliferation, differentiation, gene expression, contractility and ultra-structures in human CMs to uncover further molecular and cellular mechanisms that underlie cardiac arrhythmia of TS. 2) Direct screen of drugs to rescue TS phenotypes: Several families of ion channel blockers are used clinically as well as [unreadable]-blockers to prevent lethal cardiac arrhythmia. However, it is not clear that these blockers can rescue the cardiac phenotypes observed in TS CMs. I will test these blockers for their ability to restore normal Ca2+ responses and reduce irregular contraction in TS CMs. In addition, I will also test derivates of roscovitine, which are tested to rescue the cellular phenotypes of TS. 3) Development of screen methods to find lead compounds: To develop medium throughput screen systems for a library of chemical compounds to rescue the cardiac phenotypes of TS, I will test two different methods based on relative motion and calcium response in TS CMs using automated fluorescent microscopes. To validate the systems, I will used [unreadable]-agonists and roscovitine, which have been tested on TS CMs, to optimize experimental conditions for the methods to assess the reproducibility as determined by Z' value. Finally, I will conduct a pilot screen in TS CMs using LOPAC 1280 compounds that have been used in human, which is available through Stanford high-throughput screening facility. These approaches using human cardiac model of TS would be very unique and innovative to understand the mechanisms underlying human cardiac arrhythmia. The proposed systems to screen a library of compounds to rescue TS phenotypes will provide a platform to find novel lead compounds that would be clinically useful for the treatment of not only TS but also other cardiac arrhythmias.
Funding Period: 2012-01-01 - 2016-02-29
more information: NIH RePORT

Detail Information

Research Grants30

  1. Model of Timothy Syndrome to Screen Drugs with Induced Pluripotent Stem Cells
    Masayuki Yazawa; Fiscal Year: 2013
    ..The proposed systems to screen a library of compounds to rescue TS phenotypes will provide a platform to find novel lead compounds that would be clinically useful for the treatment of not only TS but also other cardiac arrhythmias. ..
  2. CaMKII and InsP3-Mediated Signaling in Cardiac Myocytes
    Donald M Bers; Fiscal Year: 2013
    ..Results will greatly increase our understanding of the roles of CaMKll and InsP3R in cardiac myocytes during ECC, arrhythmogenesis &nuclear signaling in normal, Hyp and HF cardiac myocytes. ..
  3. Fibroblast Growth Factor Homologous Factors: Modulation of L-type Calcium Channel
    ..This will demonstrate that FGF13 is a prominent player in the cardiac action potential through its modulation of the LTCC and define FGF13 dysfunction as a mediator of acquired and inherited arrhythmias. ..
  4. Diverse Roles of Reactive Oxygen Species and Inflammation in Vascular Disease
    Kathy K Griendling; Fiscal Year: 2013
    ..Ultimately, this research may establish new unifying concepts linking conditions that alter vascular oxidant stress and inflammation to the molecular processes underlying vasculopathies. (End of Abstract) ..
  5. Caveolae and Cardiac Repolarization
    TIMOTHY KAMP; Fiscal Year: 2013
    ..These studies will provide mechanistic new insights into the control of cardiac repolarization and abnormalities in disease which can result in arrhythmias. ..
  6. Ca2+ Regulation in Newly Formed Ventricular Myocytes
    Steven R Houser; Fiscal Year: 2013
    ..Our preclinical studies in animal models will apply these strategies to determine if they might be useful for treating patients with many different forms of cardiac injury. ..
  7. Mechanisms of PMN and Endothelial-Mediated Lung Inflammation and Injury
    Asrar B Malik; Fiscal Year: 2013
    ..abstract_text> ..
  8. Endophenotypes of Sleep Apnea and Role of Obesity
    ALLAN IAN PACK; Fiscal Year: 2013
    ..It will lead to a new molecular signature of OSA that could transform the practice of medicine in this area in a new, cost-effective way. ..
    John V Fahy; Fiscal Year: 2013
    ..Including studies in human biospecimens in a PPG that promises to advance understanding of airway TH2 inflammation in ways that are highly relevant to patients with asthma. ..
  10. Regulation of the Cardiac Sodium Channel by SIRTUIN1
    Barry London; Fiscal Year: 2013
    ..In doing so, it will open the door for pharmacologic SIRT1 activators as potential therapeutic agents in patients at risk for cardiac arrhythmias dues to inherited and acquired disturbances in cardiac INa. ..
    Carmen W Dessauer; Fiscal Year: 2013
    ..Specific Aims will 1) examine the enzymatic regulation of AC9 and real-time production of cAMP in cardiac myocytes, 2) identify and characterize AC9 complexes in cardiac myocytes, and 3) examine the functional roles of AC9 in heart. ..
  12. Cell-type Specificity of Heritable Channelopathies: Role of the Purkinje Fiber
    Vivek Iyer; Fiscal Year: 2013
    ..3. To utilize multidimensional simulation to determine the impact of mutations in the Purkinje fiber system and interplay with the ventricle, establishing a link between genetic lesion and arrhythmia on the whole organ level. ..
  13. Human Induced Pluripotent Cell Models of Pediatric Cardiac Disorders
    Ihor R Lemischka; Fiscal Year: 2013
    ..These studies could have important impact on the development of novel therapeutic strategies for these myocardial diseases, for which our current approaches are not curative. ..
  14. Phosphorylation of cMyBP-C Modulates Cardiac Arrhythmias
    Ramzi J Khairallah; Fiscal Year: 2013
    ..For the PI, the investigation nicely supports his long-term plan of conducting interdisciplinary research related to cardiac function, with the prospect of broadening our understanding of the pathogenesis of heart failure. ..
  15. Multi-scale Modeling of Calcium Mediated Triggered Activity in the Heart
    Yohannes Shiferaw; Fiscal Year: 2013
    ..Our computer models will shed light on the underlying mechanisms by bridging the gap between ion channels, cell electrophysiology, and tissue scale electrical activity. ..
  16. From human keratinocytes to biological pacemakers
    Ira S Cohen; Fiscal Year: 2013
    ..If the HFKT-pacemaker functions well in the canine heart, a future goal would be to advance this novel autologous, cellular approach towards clinical deployment. ..
  17. Immune-Based Interventions Against Infectious Diseases
    Alan L Rothman; Fiscal Year: 2013
    ..3. Recruit promising junior investigators and provide mentoring by established NIH-funded researchers. 4. Support a multidisciplinary research program led by junior investigators in translational infectious diseases immunology. ..
  18. The Role of the Transient Outward Current in Genetically Elusive Sudden Death
    John R Giudicessi; Fiscal Year: 2013
    ..Further, it offers the possibility of unveiling novel biology through the exploration of still enigmatic pathology. ..
  19. Optogenetic Multiparametric Assay for HT Cardiotoxicity Testing
    Fabio Cerignoli; Fiscal Year: 2013
    ..Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing. ..
    Jeanne M Nerbonne; Fiscal Year: 2013
  21. Molecular Physiology of HERG (KCNH2) Pottasium Channels
    Matthew C Trudeau; Fiscal Year: 2013
    ..Our long-term objectives are to determine the fundamental molecular basis of gating and modulation in cardiac IKr channels, in an effort to better treat inherited LQTS and prevent acquired LQTS. ..
  22. Advancing Experimental Models to Study Intercellular Crosstalk of Cardiac Cells
    Ulrike Mende; Fiscal Year: 2013
    ..Furthermore, we anticipate that the models that will be developed and the insights gained in this project will facilitate research on intercellular crosstalk between other cell types in the cardiac field and beyond. ..
    Sandor Gyorke; Fiscal Year: 2013
    ..S. This proposal will study how improper regulation of calcium by the muscle cells of the heart contributes to arrhythmias. Information gained from this study may help design better therapies for arrhythmias. ..
  24. Molecular basis of Ca2+ leak in heart
    GEORGE BLAIR WILLIAMS; Fiscal Year: 2013
    ..For the PI, the investigation nicely supports his long-term plan to combine theoretical investigations with practical and informative tests with the prospect of broadening our understanding of cardiac cellular function. ..
  25. Function and Integration of Stem Cell-derived Cardiac Tissue Patch
    Nenad Bursac; Fiscal Year: 2013
    ..The knowledge obtained in this project will allow us to pursue in the future engineering of a functional cardiac tissue patch made of human stem cells for potential clinical applications. ..
  26. Heart and Muscle K+ Channels: Assembly and Regulation
    Gideon Koren; Fiscal Year: 2013
  27. Discovery and Development of Therapeutic Genes for CHF
    H Kirk Hammond; Fiscal Year: 2013
    ..Four Cores will support the Program: Digital Imaging (Dr. Farquhar);Vector Production (Dr. Miyanohara);Translational Systems (Dr. Hammond) and Clinical &Administrative (Dr. Hammond). ..
  28. PPG - Mechanisms of Cardiovascular Protection and Disease
    Donald D Heistad; Fiscal Year: 2013
    ..abstract_text> ..