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

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

3. 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.

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

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

6. Cardiac Sodium Channelopathies: One Gene, Many Diseases
Brugada
Syndrome
Cardiac Conduction Defect
Sick Sinus Syndrome
SCN5A (Gene)
Nav1.5 (Protein)
Atrial Fibrillation
Still Birth
Considering the importance of intact cardiac sodium channels for proper cardiac function, mutations in the SCN5A gene underlie a variety of different heritable heart diseases either due to a gain-of-function or a loss-of-function of cardiac sodium channels.
My projects are specifically focused on two major sodium channel related arrhythmias; one is a LQT3 syndrome which caused by gain-of-function of Na channel and another is BrS which caused by loss-of-fuction of Na channel.
Red arrow signifies gain of function and blue arrow loss of function of the Nav1.5 channel.
Sudden Infant Death Syndrome
Long QT 3 Syndrome
Dilated Cardiomyopathy
Gain-of-function
Loss-of-function

7. Same Genetic Mutation, Different Genetic Disease Phenotype???1 2 3
A very interesting and not well-understood aspect aspect of most heritable diseases, including LQTS and BrS, is that not every individual of a given genotype shows the expected phenotype.
Say we are studying three individual who carry the same disease-causing mutation; 2 of these individuals suffer from the disease but exhibit different symptoms, while the third person is completely unaffected.
These findings are the basis for the concepts of penetrance and expressivity.
In LQT3 and BrS, they frequently display incomplete penetrance, a phenomenon where an individual who carries a disease causing mutation, but is asymptomatic…!!!
Penetrance: the proportion of individuals with a specific genotype who manifest that genotype at the phenotype level.
Incomplete penetrance: not every individual of a given genotype shows the expected phenotype; that is, the phenotypic effects of the allele are not always seen in the individual.
Expressivity: the degree to which a particular genotype is expressed in the phenotype
Variable expressivity: a variable phenotype is seen among individuals of the same genotype (with respect to the trait in question)
Variable Expressivity
Incomplete Penetrance

8. Why do phenotypes show differences in penetrance and expressivity???
Unresolved Question:
Why do phenotypes show differences in penetrance and expressivity???
This observation raised an interesting question that why do phenotypes show differences in penetrance and expressivity within family members harboring the same disease-causing mutation?
Answering these questions is not an easy task. However, understanding the mechanisms underlying the genotype-phenotype discordance in heritable cardiac diseases is important for understanding the disease process, patient counseling, and select a proper therapeutic approach for affected patients, especially for asymptomatic family members.

9. Pedigree of an Asymptomatic Family Carrying a Gain-of-Function Mutation of Sodium Channels
H558R
H558R+P2006A 1 2 I II
H558R+P2006
Going back to this family that I mentioned earlier, further genotyping revealed that all of the asymptomatic carriers of a defective P2006A mutation also carry the H558R polymorphism: one is hetrozygous for the H558R polymorphism and 3 are homozygous for the H558R polymorphism.
P2006A
H558R
Shinlapawittayatorn et al., Heart Rhythm 2011;8(3):455-62

10. 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.
Based on the observation that this asymptomatic individual all carry the H558R polymorphism in addition to the disease causing mutation.
Therefore, this led to the over all hypothesis of my projects that sodium channel H558R polymorphism may contribute to the genotype-phenotype discordance observed in heritable arrhythmias by acting as diseases modifying gene.

11. 2 Peer Reviewed Articles From PhD Project
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): (IF = 4.043)
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): (IF = 4.246)

12. 4 Other Peer Reviewed Articles
Shinlapawittayatorn K, Deschênes I. Sodium Channel Polymorphisms and Arrhythmogenic Events: Pro-Arrhythmic or Anti-Arrhythmic? (in preparation)
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)
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)
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): (IF = 4.218)

13. 1 Editorial Comments
Shinlapawittayatorn K, Deschênes I. Alteration of Tyrosine Kinase Signaling: Another Player in the Arrhythmogenesis of Atrial Fibrillation? Heart Rhythm 2010;7(9): (IF = 4.246)

14. 10 Peer Reviewed Abstracts
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.
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.
Kuri B, Nassal DM, Shinlapawittayatorn K, Ficker E, Deschênes I. Identification of KChIP2 in Guinea Pig Heart. Biophys J 2011.
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.
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.
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.
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.
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.
Shinlapawittayatorn K, Du X, Liu H, Kaufman ES, Deschênes I. A Common SCN5A Polymorphism Restores the Biophysical Defects of LQT3 Mutations. Biophys J 2009.
Shinlapawittayatorn K, Kaufman ES, Deschênes I. SCN5A Polymorphism Decreases Arrhythmogenic Events in a Family Carrying a LQT3 Mutation. Biophys J 2008;94:3087.

15. 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

16. 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

17. Acknowledgements Ph.D. Thesis Guidance Committee Thomas M. Nosek, PhD
George R. Dubyak, PhD
Stephen W. Jones, PhD
Kevin J. Donahue, MD
Kenneth R. Laurita, PhD
Robert D. Harvey, PhD
Isabelle Deschênes, PhD
Case Western Reserve University
MetroHealth Medical Center
Finally, I would like to acknowledge the families affected by these devastating diseases and trust that the knowledge derived herein will not only advance the science but also ultimately translate into refined care for our affected patients.
Chiang Mai Medical School
CERT Center
Nipon Chattipakorn, MD, PhD

18. 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)

19. Cardiac Voltage-Gated Sodium Channel (Gene: SCN5A, Protein: Nav1.5)
α (260 kD) NH 2
COOH + DI
DII
DIII
DIV β2 β1
β (36 kD) S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
which is a relative large glycosylated integral membrane protein consisting of 2,016 amino acids with a molecular weight of about kilo Dalton.
A functional cardiac sodium channel constitute 4 homologous domains (I to IV) each consisting of 6 transmembrane segments (S1 to S6). The S4 segments in each of the four homologous domains contain positively charged lysine (K) and Arginine (R) residues that detect changes in the transmembrane potential. The bold S5-6 linker sequences are “P-segments” that line the outer pore as shown here.
The III-IV linker plays a major role in fast inactivation…!!!
The cardiac sodium channel can be regulated by auxiliary beta-subunits. However, the exact stoichiometry of Nav1.5 and NavB subunits is not clear.
S4 segments:Voltage sensors
S5-S6 loops: Pore of the channel
Domains III-IV linker: Inactivation gate

20. Heterologous Expression of Nav1.5
HEK
cells
Patch clamp
Fluorescence
Nav1.5
GFP-IRES
Vector
SCN5A-WT
SCN5A-P2006A
SCN5A-H558R-P2006A
HEK293 cells
For functional analysis, sodium channels were expressed in HEK293 cells and then analyzed their function with whole-cell patch clamp recording.
1 day 20

21. FRETc = (IDA – aIAA – dIDD)/IDD
Using Fluorescence Resonance Energy Transfer (FRET) to Examine Sodium Channel Folding
Emission
FRETc = (IDA – aIAA – dIDD)/IDD
FRET
Excitation
CFP
YFP
Acceptor
Fluorescence Resonance Energy Transfer (FRET) based approach has been shown to be a powerful technique for probing protein interaction either intermolecular and intramolecular interaction and the principle is this:
If the 2 fluorophores come within a 100 Å (10 nm) of each other there is transfer of energy.
The changing in FRET signal indicated that the 3-D conformation of the channel has changed so that the distances between YFP and CFP were changed.
Because of this property FRET based approach is particular useful and allow us to probe protein folding.
Therefore, we employed a FRET based approach to study folding of sodium channel in the presence or absence of the peptide fragments.
CFP = Donor YFP= Acceptor
Three cubes to isolate the signal of CFP and YFP and FRET
CFP (EX:440/20m EM:480/30m)
Excites both but measures signal where only CFP emits
YFP (EX:500 EM:530)
Measures near exclusive YFP EM by preferential EX of YFP
FRET (EX:440 EM:535)
EX CFP at 440 and measures EM at 535
Donor C N N C
FRET

22. Model of Rescued a Gain-of-Function Mutation by the H558R Polymorphism
Ventricular myocytes action potential
Inward
Outward
INa
ICa IK 1 30
-30
-60
-90 2
Membrane potential (mV) 3 4 4
Na+
Stabilized Inactivation
Na+
Defective Inactivation
In the presence of the H558R polymorphism, the persistent currents were reduced back to WT level suggesting that H558R polymorphism might play an important role in the stabilization of sodium channels’ fast inactivation leading to the restoration of APD toward the WT level.
This observation may contribute to the absence of a typical LQT3 phenotype in those individuals that carry both the mutant and the polymorphism.
0 nA
Persistent sodium current
Peak sodium current

23. Fragment Design Nav1.5 R558-40aa R558-20aa H558-20aa DI DII DIII DIV
282 + + + +
558
NH2
COOH
Therefore, as a proof-of-concept we also created recombinant DNA encoding for peptide fragments centered on amino acid 558, the site of the polymorphism.
Here is the 40 amino acid fragment and the 20 amino acid fragment containing the R558. Moreover, we also created the 20 amino acid fragment containing the H558.
For the sake of simplicity, I will call R558 as “polymorphic fragment” and H558 as “WT-fragment”.
G538
S577
R558
R558-40aa
N548
L567
R558
R558-20aa
N548
L567
H558
H558-20aa 23