Curriculum vitae Balázs Ördög

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2 Curriculum vitae Balázs Ördög : Szent István Univ. M.Sc., Engineer of agriculture (biotechnology) : Montreal Heart Institue, postdoc : Univ. Szeged, Dept. Medical Biology, Ph.D :

3 Origin of phenotypic variance Genetic factors Environmental factors Signs of genetic determination of disease Family aggregation Unusually young age of patient

4 Why genetics? Don`t you want to know the future? (genetic risk factors of disease) Personalized medicine Humans are the best model of human disease Why deal with extremely rare genetic diseases?

5 Ion channel structure and function: ion selectivity, the KCSA model

6 Ion channel structure and function: tertiary structure

7 The cardiac action potential Phase 0: depolarization Na + influx Phase 1: initial repolarization K + outflux Phase 2: plateau K + outflux Ca 2+ influx 0 mv Phase 3: repolarization K + outflux Phase 4: resting potential

8 Inward rectifier K + channels 1 2 I K1 Subunits: Kir2.1 (KCNJ2) Kir2.2 (KCNJ12) Kir2.3 (KCNJ4) Kir2.4 (KCNJ14) 0 AP Function: AP phase 4, resting potential I K1 Dysfunction: Long QT Syndrome Short QT Syndrome Andersen-Tawil Syndrome Brugada Syndrome

9 Transient outward rectifying K + channels I + to : transient outward K current Pore-forming subunits: Kv1.4 (KCNA4), Kv4.2 (KCND2), Kv4.3 (KCND3) Homo- or heteromeric structure Auxiliary subunits: KChIP2, DPP6, KCNE1-5? Function AP Phase 1 Region-specific AP configurations Disfunction Brugada Syndrome Idiopathic Ventricular Fibrillation 50 mv -35 mv -80 mv

10 Na + channels Voltage-gated, Na + selective Pore-forming subunit: Nav1.5, SCN5A Auxiliary subunits: Navβ1, 2, 3 (SCNB1, 2, 3) Function: AP phase 0 Excitability and Conduction velocity Dysfunction: Long QT Syndrome Brugada Syndrome AP I NA

11 Ca 2+ channels AP I Ca Subunits: Pore-forming: α 1C (Cav1.2, CACNA1C) Auxiliary: α 2 /δ, β 2 Function: AP plateau, excitation-contraction coupling Disfunction: Atrial Fibrillation, Heart Failure, Thymothy syndrome...

12 Ca 2+ channel function: excitationcontraction coupling

13 Delayed rectifier K + channels AP Delayed outward rectifiers I Kr Fast activating component: I Kr, herg (KCNH2) Slowly activating component: I Ks, KvLQT1 (KCNQ1) Auxiliary subunits: KCNE1 5 Function: AP phase 2 and 3 Disfunction: long QT Syndrome I Ks

14 Role of auxiliary subunits: IK s channels, an illustrative example KvLQT1 or mink produce no current when expressed alone. Inject mink RNA into Xenopus oocytes big current. Why? Auxiliary subunits can modify: Gating Protein folding, trafficking Subcellular localization Sensitivity to blockers Receptor- and/or liganddependent effects

15 Hereditary ion channel diseases Sudden cardiac death cases per year USA: 300 k UK: 70 k HU: k Coronary heart disease is the underlying condition in the majority of SCD cases In 40% of cases SCD occurs in the absance of structural heart disease

16 Congenital Long QT Syndrome Frequency: approx. 1 in 10 k 15% of carriers show no QT elongation: Genetic testing of family members warranted Romano-Ward Syndrome: Autosomal dominant QT elongation and torsade de pointes Jervell and Lange-Nielsen Syndrome: Autosomal recessive Sensorineural deafness QT elongation and torsade de pointes Adrenerg stimuli promote cardiac events Torsades de pointes

17 Congenital Long QT Syndrome

18 AP configuration heterogeneity in the heart Penetrance of an ion channel mutation varies from region to region or from cell to cell, causing increased heterogeneity.

19 Acquired Long QT Syndrome Drugs with arrhytmogenic side-effects (mostly I Kr blockers) Generic name Brand name Class / Clinical use Albuterol Ventolin Bronchodilator / Asthma Albuterol Proventil Bronchodilator / Asthma Amitriptyline Elavil Antidepressant / depression Amoxapine Asendin Antidepressant / depression Ampicillin Omnipen Antibiotic / infection Arsenic tiroxide Trisenox Anti-cancer / Leukemia Chloral hydrate Noctec Sedative / sedation/ insomnia Citalopram Celexa Anti-depressant / depression Clarithromycin Biaxin Antibiotic / bacterial infection Cocaine Cocaine Local anesthetic Ephedrine Broncholate Bronchodilator decongestant / Allergies sinusitis Ephedrine Rynatuss Bronchodilator decongestant / Allergies sinusitis Fenfluramine Pondimin Appetite suppressant / dieting, weight loss Fluconazole Diflucan Anti-fungal / fungal infection Halofantrine Halfan Anti-malarial / malaria infection Ketoconazole Nizoral Anti-fungal / fungal infection Levalbuterol Xopenex Bronchodilator / asthma Moxifloxacin Avelox Antibiotic / bacterial infection Nicardipine Cardene Anti-hypertensive / high blood pressure Norepinephrine Levophed Vasconstrictor Inotrope / shock, low blood pressure Phentermine Fastin Appetite suppressant / dieting, weight loss Tacrolimus Prograf Immunosuppressant / Immune suppression Tamoxifen Nolvadex Anti-cancer / breast cancer Venlafaxine Effexor Anti-depressant / depression Amino acids with aromatic side chains in the herg channel (Tyr-652, Phe-656) Prmosuous target of blockers

20 Repolarization Reserve Repolarization is maintained by the concerted action of several differeint ionic currents The blockade of one particular repolarizing current does not necessarily lead to QT elongation, because of the compensatory effect of other repol. currents Blocking of a second repolarizing current has a synergistic effect Consequences: Patients with normal QTc may be at risk of arrhythmia (e.g. LQTS mutation carriers) Blocking two or more repolarizing currents is a bad idea

21 Repolarization reserve Genotype (e.g. asymptomatic LQT mutation carrier) + Environment (drug side-effect) Depletion of repolarization reserve increased APD, QT variability, afterdepolarisations TdP, VF, SCD

22 Brugada Syndrome SCD from ventricular tachyarrhytmia ECG signature: ST segment elavation Brugada mutations SCN5A I Na CACNA1C I Ca CACNAB2 I Ca KCNE3 I to Autosomal dominant Particularly frequent in South-Asia (30 / 100 k cases per year) Responsible for 20% of unexplained SCD cases Annual mortality of diagnosed patients: 8.2% Mean age of death of carriers: 41y

23 Brugada szindróma Depolarizing currents Brugada mutations SCN5A I Na CACNA1C I Ca CACNAB2 I Ca KCNE3 I to Repolarizing currents Depolarization-repolarization balance is disrupted (which is particularly fragile in the epicardial layer of the right ventricle) Right ventricular epicardium APD Epi-endo voltage gradient Increased transmural and epicardial repolarization dispresion ventricular tachyarrhytmia

24 Hereditary Ca 2+ channel disfunction: Timothy syndrome Average age of death: 2.5 years Symptoms elongated QT interval and syndactyly heart malformations (ASD, VSD, PFO, Tetralogy of Fallot) arrhythmias (VT, TdP, VF) autistic spectrum disorder: impaired communication and socialization, delayed development of speech and language Genetic cause: gain-of-function mutation in CACNA1C (Cav1.2)

25 Hereditary RYR disfunction: Catecolaminergic polymorphic ventricular tachycardia Prevalence 1 in 10K est. 15% of SCD cases in the young 60% of cases experience cardiac events by age of 20 mortality: 50% by age of 30 Symptoms syncope during excersize or in response to emotional stress disease progression: ventrciular extra beats and bigeminy, bidirectional or polymorphic tachycardia, VF Genetic causes: Ryanodine receptor, RYR (60% of CPVT cases) Calsequestrin-2, CASQ2 (1-2% of CPVT cases)

26 Ca 2+ overload results in SOICR, which causes triggered activity SOICR: store overload-induced calcium release

27 Unifying theory for CPVT

28 Idiopathic ventricular fibrillation (IVF) In 10% of SCD cases Survival rate at ag of 58y: ~50% VF recurrence: 30% 20% of IVF cases show family aggregation Normal ECG SCD is the first symptom

29 Idiopathic ventricular fibrillation (IVF) Pedigree of 3 Dutch families with IVF

30 Idiopathic ventricular fibrillation (IVF) Linkage analysis pointed to 7q36

31 Idiopathic ventricular fibrillation (IVF) Increase DPP6 expresszion in right septal biopsies

32 DPP6: an auxiliary subunit of I to channels in Purkinje cells AV-GFP-DPP6 mrna protein P DPP6 IRES GFP pa DPP6 overexpression VM PC

33 DPP6: an auxiliary subunit of I to channels in Purkinje cells DPP6 gene silencing AdV-DPP6-KD Ω CMV GFP pa VM PC

34 IVF (putative) pathomechanism Increased DPP6 expression I to gain of function in Purkinje cells Increased spatial repol. dispersion between Purkinje cells and cardiomyocytes Arrhytmia substrate

35 Viral gene transfer Lentivirus In vitro applications for proliferative cell types stable cell lines ease of use Adenovirus In vitro and in vivo ( atiral painting ) broad host range tranzient transduction laborous to produce Adeno-asszociated virus (AAV9) transduces the heart in vivo produced easily limited size of payload The payload can be: Overexpression of proteins Gene silencing MicroRNA manipulations (overexpression or knockdown)

36 Andersen-Tawil Syndrome: Complex clinical manifestation prominent U wave Frequent ventricular extrasystolae QT elongation (50% of ATS cases, LQT7) Bidirectional ventricular tachycardia (32% of ATS cases) Periodic paralysis Anatomic features: broad forehead, micrognathia, low set ears, clinodactyly, syndactyly

37 Andersen-Tawil szindróma: Klinikai manifesztáció

38 Andersen-Tawil szindróma: Klinikai manifesztáció

39 Andersen-Tawil szindróma: KCNJ2 (Kir2.1, I K1 ) diszfunkció AP I K1

40 Andersen-Tawil szindróma: KCNJ2 302 funkcionális karatkerizálása Heterológ expresszió KCNJ2 WT KCNJ2 WT + KCNJ2 Δ302 1:1 Voltage clamp protokoll +50 mv KCNJ2 WT + KCNJ2 Δ302 3:1-80 mv -140 mv 300 ms KCNJ2 Δ302

41 Andersen-Tawil szindróma: KCNJ2 302 funkcionális karatkerizálása

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