Pursuing a PhD: My Journey of Discovery and Future Research Directions

Krekwit Shinlapawittayatorn, MD, PhDCardiac Electrophysiology Research & Training Center (CERT)

Department of Physiology, Chiang Mai University

November 21st, 2011

Introduction: Education

• 2004 Doctor of Medicine (M.D.)Chiang Mai UniversityChiang Mai, Thailand

• 2006-2011 Ph.D. (Physiology & Biophysics)Case Western Reserve UniversityCleveland, 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

SCN5A (Gene)

Nav1.5 (Protein)

Sudden Infant Death

Syndrome

Still Birth

Brugada

Syndrome

Cardiac Conduction Defect

Sick Sinus Syndrome

Dilated Cardiomyopath

y

Atrial Fibrillation

Gain-of-function

Loss-of-function

Long QT 3 Syndrome

Same Genetic Mutation, Different Genetic Disease Phenotype???

1 2 3

Variable Expressivity

Incomplete Penetrance

Why do phenotypes show differences in penetrance and expressivity???

Unresolved Question:

Pedigree of an Asymptomatic Family Carrying a Gain-of-Function Mutation of Sodium Channels

Shinlapawittayatorn et al., Heart Rhythm 2011;8(3):455-62

H558R H558R

H558R H558R+P2006A H558R H558R+P2006A

H558R+P2006A

1 2

1 2

I

II

H558R H558R

H558R H558R+P2006 H558R H558R+P2006A

H558R+P2006A

1 2

1 2

I

II

P2006AH558R

General hypothesis

This led us to hypothesize that sodium channel H558R polymorphism may contribute to the genotype-phenotype discordance observed in

heritable arrhythmias by acting as diseases modifying gene.

2 Peer Reviewed Articles From PhD Project

1. Shinlapawittayatorn K, Dudash L, Poelzing S, Ficker E, and Deschênes I. Cardiac Sodium Channel Fragments Spanning H558R 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 SCN5A Polymorphism Restores the Biophysical Defects of SCN5A 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 (KCNQ1) 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, DeCaro G, Yadav SP, Mohan ML, Naga Prasad SV, Schilling WP, Ficker E, and Schelling JR. Phosphoinositide Binding Differentially Regulates NHE1 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 Loss-of-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 KChIP2 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 (KCNQ1) 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 (KCNQ1) 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 (KCNQ1) Underlying Brugada Syndrome. Heart Rhythm 2009.

9. Shinlapawittayatorn K, Du X, Liu H, Kaufman ES, Deschênes I. A Common SCN5A Polymorphism Restores the Biophysical Defects of LQT3 Mutations. Biophys J 2009.

10. Shinlapawittayatorn K, Kaufman ES, Deschênes I. SCN5A Polymorphism Decreases Arrhythmogenic Events in a Family Carrying a LQT3 Mutation. Biophys J 2008;94:3087.

Honors and Awards

• 2011 Finalist of Student Research Achievement Award (Category: Membrane Biophysics), 55nd Annual Meeting of the Biophysical Society, Baltimore, Maryland, USA

• 2009 First Place Graduate Student Poster Presentation (Department Annual Retreat), Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA

• 2009 First Place of the Trainee’s Poster Presentation Competition (Research Festival), MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio, USA

• 2008 American Heart Association Pre-doctorol Fellowship Award (Percentile Rank: 0.93), American Heart Association, Great Rivers Affiliate, USA

• 2008 Finalist of Student Research Achievement Award (Category: Membrane Biophysics), 52nd Annual Meeting of the Biophysical Society, Long Beach, California, USA

• 2007 First Place of the Trainee’s Oral Presentation Competition (Genetic Basis of Cardiovascular Disease), MetroHealth Medical Center, Case Western Reserve University, Cleveland, Ohio, USA

• 2007 Recknagel Graduate Student Best Academic Record, Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA

Future Directions

• Patch clamp recording facility (research and training)

- Isolated cardiomyocytes

- Drug screening

- Molecular autopsy

- Personalized medicine: patient-specific iPSC

• TRF research grant for new scholar

Acknowledgements

MetroHealth Medical Center

Case Western Reserve University

Ph.D. Thesis Guidance CommitteeThomas M. Nosek, PhDGeorge R. Dubyak, PhDStephen W. Jones, PhDKevin J. Donahue, MDKenneth R. Laurita, PhDRobert D. Harvey, PhDIsabelle Deschênes, PhD

Chiang Mai Medical School

CERT CenterNipon Chattipakorn, MD, PhD

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: SCN5A, Protein: Nav1.5)

α (260 kD)

NH2COOH

++

++

++

++

COOH COOH

NH2NH2DI DII DIII DIV

β2 β1

β (36 kD)

S1 S2S3 S4S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6

S4 segments:Voltage sensors

S5-S6 loops: Pore of the channel

Domains III-IV linker: Inactivation gate

Heterologous Expression of Nav1.5

HEKcells

Patch clamp

Fluorescence

HEKcells

HEKcells

1 day

Nav1.5

GFP-IRES

Vector

SCN5A-WT

SCN5A-P2006A

SCN5A-H558R-P2006A

HEK293 cells

Using Fluorescence Resonance Energy Transfer (FRET) to Examine Sodium Channel Folding

CFP

Donor

YFP

Acceptor

Excitation

FRET

EmissionFRETc = (IDA – aIAA –

dIDD)/IDD

CNC

N

FRET

Persistent sodium current

Model of Rescued a Gain-of-Function Mutation by the H558R Polymorphism

Ventricular myocytes action potential

0 nA

30

0

-30

-60

-90

Mem

bra

ne

po

ten

tial

(m

V)

Peak sodium current

4

0

1 2

4

3

Na+

Defective Inactivation

Na+

Stabilized Inactivation

Inward

Outward

INa

ICa

IK

Fragment Design

COOH

NH2

++

++

++

++

282

558

N548

L567R558

R558-20aaR558-20aaG538

S577

R558

R558-40aaR558-40aa

DI DII DIII DIV

Nav1.5

N548

L567H558

H558-20aaH558-20aa