Pursuing a Ph D My Journey of Discovery

Pursuing a Ph. D: My Journey of Discovery and Future Research Directions Krekwit Shinlapawittayatorn, MD, Ph. D Cardiac Electrophysiology Research & Training Center (CERT) Department of Physiology, Chiang Mai University November 21 st, 2011

Introduction: Education • 2004 Doctor of Medicine (M. D. ) Chiang Mai University Chiang Mai, Thailand • 2006 -2011 Ph. D. (Physiology & Biophysics) Case Western Reserve University Cleveland, OH, USA

Case Western Reserve University (CWRU) and Department of Physiology and Biophysics • CWRU was founded in 1826. • CWRU is a private research university located in Cleveland, OH, USA. • The University is associated with 16 Nobel Laureates. • The department is currently ranked #9 based on NIH funding.

Dissertation Modulations of Sodium Channel Long QT and Brugada Syndrome Mutations by a Common Sodium Channel Polymorphism

Genetic Defects of Cardiac Ion Channels: From the Bench to Bedside

Cardiac Sodium Channelopathies: One Gene, Many Diseases Brugada Cardiac Conduction Defect Syndrome SCN 5 A (Gene) Still Birth Nav 1. 5 (Protein) Sudden Infant Death Syndrome Gain-of-function Long QT 3 Syndrome Sick Sinus Syndrome Atrial Fibrillation Dilated Cardiomyopathy Loss-of-function

Same Genetic Mutation, Different Genetic Disease Phenotype? ? ? 1 2 Variable Expressivity 3 Incomplete Penetrance

Unresolved Question: Why do phenotypes show differences in penetrance and expressivity? ? ?

Pedigree of an Asymptomatic Family Carrying a Gain-of. Function Mutation of Sodium Channels I 1 H 558 R 2 H 558 R+P 2006 A II 1 H 558 R 2 H 558 R+P 2006 A H 558 R+P 2006 H 558 R+P 2006 A Shinlapawittayatorn et al. , Heart Rhythm 2011; 8(3): 455 -62

General hypothesis This led us to hypothesize that sodium channel H 558 R polymorphism may contribute to the genotype-phenotype discordance observed in heritable arrhythmias by acting as diseases modifying gene.

2 Peer Reviewed Articles From Ph. D Project 1. Shinlapawittayatorn K, Dudash L, Poelzing S, Ficker E, and Deschênes I. Cardiac Sodium Channel Fragments Spanning H 558 R Polymorphism Rescue Defective Trafficking of a Brugada Syndrome Mutation. Circ Cardiovasc Genet 2011; 4(5): 500 -9. (IF = 4. 043) 2. Shinlapawittayatorn K, Du X, Liu H, Ficker E, Kaufman ES, Deschênes I. A Common SCN 5 A Polymorphism Restores the Biophysical Defects of SCN 5 A Mutations. Heart Rhythm 2011; 8(3): 455 -62. (IF = 4. 246)

4 Other Peer Reviewed Articles 1. Shinlapawittayatorn K, Deschênes I. Sodium Channel Polymorphisms and Arrhythmogenic Events: Pro-Arrhythmic or Anti-Arrhythmic? (in preparation) 2. Shinlapawittayatorn K, Sorrentino S, Forleo C, Anaclerio M, Iacoviello M, Guida P, Favale S, Ficker E, Santis DD, Nalin I, Deschênes I. Evidence for a Novel Gene (KCNQ 1) Underlying Brugada Syndrome. (in preparation) 3. Abu Jawdeh BG, Khan S, Deschênes I, Hoshi M, Goel M, Lock JT, Shinlapawittayatorn K, Babcock G, Lakhe-Reddy S, De. Caro G, Yadav SP, Mohan ML, Naga Prasad SV, Schilling WP, Ficker E, and Schelling JR. Phosphoinositide Binding Differentially Regulates NHE 1 Na+/H+ Exchanger-Dependent Proximal Tubule Cell Survival. J Biol Chem 2011 (in press, IF = 5. 328) 4. Hsu K, Han J, Shinlapawittayatorn K, Deschênes I, Marbán E. Membrane Potential Depolarization As a Triggering Mechanism for Vpu-Mediated HIV-1 Release. Biophysical Journal 2010; 99(6): 1718 -25. (IF = 4. 218)

1 Editorial Comments 1. Shinlapawittayatorn K, Deschênes I. Alteration of Tyrosine Kinase Signaling: Another Player in the Arrhythmogenesis of Atrial Fibrillation? Heart Rhythm 2010; 7(9): 1253 -4. (IF = 4. 246)

10 Peer Reviewed Abstracts 1. Shinlapawittayatorn K, Du X, Liu H, Nassal DM, Liu H, Enweane P, Deschênes I. A Novel Lossof-Function Mechanism for Brugada Syndrome Sodium Channel Mutations. Heart Rhythm 2011. 2. Shinlapawittayatorn K, Nassal DM, Liu H, Ficker E, Deschênes I. Dominant-Negative Suppression of Sodium Channel Activity By a Brugada Syndrome Mutation Observed in Cardiomyocytes. Biophys J 2011. 3. Kuri B, Nassal DM, Shinlapawittayatorn K, Ficker E, Deschênes I. Identification of KCh. IP 2 in Guinea Pig Heart. Biophys J 2011. 4. Du X, Enweana P, Shinlapawittayatorn K, Liu H, Deschênes I. A Novel Mechanism of Action for Sodium Channel Brugada Syndrome Mutations. Heart Rhythm 2010; 7(11): 1716. 5. Shinlapawittayatorn K, Du X, Liu H, Ficker E, Deschênes I. Do Sodium Channel α-α. Interactions Contribute to Loss-of-Function Observed in Brugada Syndrome? Biophys J 2010. 6. Sorrentino S, Shinlapawittayatorn K, Forleo C, Anaclerio M, Iacoviello M, Nalin I, De Santis D, Zaccaria M, Ficker E, Guida P, Deschênes I, Favale S. Evidence for a Novel Gene (KCNQ 1) Underlying Brugada Syndrome. Societa Italiana di Cardiologia-70○ Congresso Nazionale 2009. 7. Sorrentino S, Shinlapawittayatorn K, Forleo C, Anaclerio M, Iacoviello M, De Santis D, Nalin I, Ficker E, Favale S, Deschênes I. A Novel Gene (KCNQ 1) Is Involved in Brugada Syndrome. ESC Congress 2009. 8. Shinlapawittayatorn K, Sorrentino S, Anaclerio M, Guida P, Iacoviello M, Favale S, Ficker E, Forleo C, Deschênes I. Evidence for a Novel Gene (KCNQ 1) Underlying Brugada Syndrome. Heart Rhythm 2009. Shinlapawittayatorn K, Du X, Liu H, Kaufman ES, Deschênes I. A Common SCN 5 A Polymorphism

Honors and Awards • 2011 Baltimore, • 2009 Reserve • 2009 Finalist of Student Research Achievement Award (Category: Membrane Biophysics), 55 nd Annual Meeting of the Biophysical Society, Maryland, USA First Place Graduate Student Poster Presentation (Department Annual Retreat), Department of Physiology and Biophysics, Case Western University, Cleveland, Ohio, USA First Place of the Trainee’s Poster Presentation Competition (Research Festival), Metro. Health Medical Center, Case Western Reserve University, • 2008 American Heart Association Pre-doctorol Fellowship Award (Percentile Rank: 0. 93), American Heart Association, Great Rivers Affiliate, USA • 2008 Long Beach, • Cleveland, Ohio, USA Finalist of Student Research Achievement Award (Category: Membrane Biophysics), 52 nd Annual Meeting of the Biophysical Society, California, USA Basis of First Place of the Trainee’s Oral Presentation Competition (Genetic Cardiovascular Disease), Metro. Health Medical Center, Case 2007 Recknagel Graduate Student Best Academic Record, Department of 2007 Western Reserve • Physiology University, Cleveland, Ohio, USA and Biophysics, Case Western Reserve University,

Future Directions • Patch clamp recording facility (research and training) - Isolated cardiomyocytes - Drug screening - Molecular autopsy - Personalized medicine: patient-specific i. PSC • TRF research grant for new scholar

Acknowledgements Ph. D. Thesis Guidance Committee Thomas M. Nosek, Ph. D George R. Dubyak, Ph. D Stephen W. Jones, Ph. D Kevin J. Donahue, MD Kenneth R. Laurita, Ph. D Robert D. Harvey, Ph. D Isabelle Deschênes, Ph. D Case Western Reserve University Metro. Health Medical Center Chiang Mai Medical School CERT Center Nipon Chattipakorn, MD, Ph. D

Thank You For Your Attention “nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual form of nature, by careful investigation of the rarer forms of disease” William Harvey (1657)

Cardiac Voltage-Gated Sodium Channel (Gene: SCN 5 A, Protein: Nav 1. 5) α (260 k. D) NH 2 DI + + S 1 S 2 S 3 S 4 S 5 β 2 β (36 k. D) DII S 6 + S 5 S 1 S 2 S 3 S 4 + DIII S 6 + + S 1 S 2 S 3 S 4 S 5 COOH NH 2 S 4 segments: Voltage sensors S 5 -S 6 loops: Pore of the channel Domains III-IV linker: Inactivation gate NH 2 DIV S 6 + + S 1 S 2 S 3 S 4 S 5 S 6 β 1 COOH

Heterologous Expression of Nav 1. 5 Patch clamp Nav 1. 5 GFP-IRES Vector SCN 5 A-WT SCN 5 A-P 2006 A SCN 5 A-H 558 R-P 2006 A HEK cells 1 day HEK 293 cells Fluorescence HEK cells

Using Fluorescence Resonance Energy Transfer (FRET) to Examine Sodium Channel Folding FRETc = (IDA – a. IAA – d. IDD)/IDD Emission FRET Excitation N CFP YFP Donor Acceptor C N FRET C

Na+ Membrane potential (m. V) Model of Rescued a Gain-of-Function Mutation by the H 558 R Polymorphism Ventricular myocytes action potential 1 30 INa 2 ICa 0 Outw 0 -30 3 d Inwar -60 4 4 -90 Na+ 0 n. A Stabilized Inactivation Defective Inactivation IK Persistent sodium current Peak sodium current ard

Fragment Design Nav 1. 5 DI DIII DIV + + 282 + + 558 NH 2 COOH R 558 -40 aa G 538 R 558 -20 aa H 558 -20 aa N 548 R 558 S 577 L 567 H 558 L 567