Coppini HCM AIVPA Ancona 6 Maggio 2017.pptx

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1. STATE-OF-THE-ART OF HMC, HUMAN AND VETERINARY REALITY (PART 2) Raffaele Coppini University of Florence Department NeuroFarBa , division of Pharmacology Human HCM: state of the art and focus on drug therapy

2. Hypertrophic cardiomyopathy (HCM) is a primary disorder of the myocardium, characterized by the presence of increased left ventricular (LV) wall thickness* that is not solely explained by abnormal loading conditions. DEFINITION European Society of Cardiology Guidelines Eur . H. Journal 2015 * >15mm or >12mm in patients with pathogenic gene mutations

3. Mayo clinic website: “http://www.mayoclinic.com/health/medical/IM00586” CARDIOMIOPATIA IPERTROFICA (HCM) Prevalenza : 1/500 Prima causa di morte improvvisa cardiaca <40aa Importante causa di scompenso cardiaco a tutte le età Cuore Normale HCM No ostruzione Ostruzione OSTRUZIONE DINAMICA

4. MORTE CARDIACA IMPROVVISA NEI GIOVANI (<40aa) 1000 casi /anno in Italia HCM è la prima causa di MI negli atleti Generalmente avviene a causa di una aritmia letale ( fibrillazione ventricolare )

5. SCOMPENSO CARDIACO IN HCM 5-10% dei casi di HCM evolve verso lo scompenso Lo scompenso è più frequente se le mutazioni sono in specifici geni ( filamento sottile ) FORMA RESTRITTIVA FORMA DILATATIVA Da Coppini et al. JACC 2014

6. Malattia del sarcomero Mutazioni sarcomeriche in >65% dei pazienti Anomalie funzionali dei miofilamenti

7. MYH7 (36%) MYL-2 TPM1 Complex TNNT2 TNNI2 MYBPC3 (57%) Myofilament Positive (62%) Myofilament Negative (38%) Florence Cohort (>1000 pts .)

8. GENE TROPONINA T TNNT2 GENE A-ACTINA ACTC MIOSINA Catene leggere regolatrici MYL2 GENE A-TROPOMIOSINA TPM1 GENE PROTEINA C MYBPC3 GENE Beta-MIOSINA Catene pesanti MYH7 MIOSINA Catene leggere essenziali MYL3 GENE TROPONINA I TNNI3 TEST GENETICO TRADIZIONALE: 8 geni sarcomerici “Richard, Circulation 2003”

9. THE ERA OF NEXT GENERATION SEQUENCING (>90 genes ) 10 HCM MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1, LAMP2, CSRP3, PRKAG2, ACTA1,TCAP, TNNC1, ACTN2, JPH2, TTN, ANKRD1, MYH6, MYOZ2, GLA, ACTC1,CALM3, MYO6, OBSCN, NEXN, FKTN, SMC1A, CALR3, COX15, MYLK2, CASQ2, CAV3, DES, FXN, NDUFV2, PLN, RAF1, SRI, VCL DCM MYBPC3, MYH7, TNNI3, TNNT2, TPM1, CSRP3, TCAP, TNNC1, ACTN2, TTN, ANKRD1, MYH6, MYOZ2, ACTC1, DES, PLN, VCL, LMNA, TAZ, MYOZ1, FHL2, LDB3/ZASP, SCN5A, EYA4, SGCD, BAG3, TMPO, DMD, ABCC9, SCO2,XIRP2, CMYA5, PDE4DIP, XIRP1, UNC45B, MYO6, EMD, PLEC, MLYCD, DNAJC19, SLC25A3 ARVC DSG2, DSP, PKP2, DSC2, TMEM43, JUP, RYR2, TGFB3,TTN RCM MYL2,MYL3, PRKAG2,GLA, TNNI3 LVNC CASQ2, MYBPC3, MYH7,TNNI3,TNNT2,TPM1,ACTC1,PLN,LMNA,TAZ,LDB3/ZASP,SCN5A,DTNA, DMPK FLNA AKAP9, ANK2, CACNA1C, CACNB2, CASQ2, CAV3, GPD1L, KCNE1,KCNE2,KCNE3, KCNJ2,KCNQ1,RYR2, SCN1B,SCN4B,SCN5A, SNTA1 CHANNELLOPATHIES Courtesy of Dr. Francesca Girolami, Careggi, Florence

10. Yacoub, M. H. (2014) Cardiomyopathy on the move Nat. Rev. Cardiol . doi:10.1038/nrcardio.2014.157 GENES AND CARDIOMYOPATHIES

11. MYH7 (36%) MYL-2 TPM1 Complex TNNT2 TNNI2 MYBPC3 (57%) Myofilament Positive (62%) Myofilament Negative (38%) Florence Cohort (>1000 pts .)

12. MYH7 (36%) MYL-2 TPM1 Complex TNNT2 TNNI2 MYBPC3 (57%) Myofilament Positive (62%) Myofilament Negative (38%) Florence Cohort (>1000 pts .) GENOTYPE-PHENOTYPE CORRELATIONS

13. GENOTYPE-PHENOTYPE CORRELATIONS Thin-filament HCM: Increased risk of sudden death Increased risk of heart failure (systolic or diastolic) Coppini et al. JACC 2014

14. Onde Q, ipertrofia e sovraccarico del VS (QRS ampi) DIAGNOSI: ECG

15. Il Cuore Normale DIAGNOSI: ECOCARDIOGRAFIA ASSE LUNGO

16. Il Cuore Normale DIAGNOSI: ECOCARDIOGRAFIA APICALE 4-CAMERE

17. Cardiomiopatia Ipertrofica DIAGNOSI: ECOCARDIOGRAFIA ASSE LUNGO: Ipertrofia settale

18. Cardiomiopatia Ipertrofica DIAGNOSI: ECOCARDIOGRAFIA ASSE CORTO: Ipertrofia spesso anche della parete libera

19. Cardiomiopatia Ipertrofica DIAGNOSI: ECOCARDIOGRAFIA APICALE 4-CAMERE: Funzione sistolica supernormale Disfunzione diastolica

20. DIAGNOSI: ECOCARDIOGRAFIA Doppler Trans mitrale Doppler Vene polm Tissue - doppler

21. RISONANZA MAGNETICA

22. IMAGING AVANZATO Contrast MRI: LGE (fibrosis) PET (Ammonia): Local blood flow (microvascualar dysfunction)

23. Hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 Obstructive STAGES OF HCM

24. Hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 Obstructive STAGES OF HCM 30-35%

25. Ostruzione al TEVS: SAM ECOCARDIOGRAFIA Insuff . Mitr

26. Ostruzione al TEVS: Gradiente ECOCARDIOGRAFIA AO LV LA

27. Ostruzione: Terapia chirurgica ECOCARDIOGRAFIA Miectomia settale transaortica PRE POST Scomparsa del SAM Abbattimento del gradiente

28. Hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 Obstructive STAGES OF HCM

29. Ipocinetico-dilatativa

30. Restrittiva

31. Hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 Obstructive STAGES OF HCM

32. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - β blockers - Diuretics - Verapamil - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines Obstructive - β blockers TREATMENT OF HCM-RELATED SYMPTOMS

33. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - β blockers - Diuretics - Verapamil - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines Obstructive - β blockers Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) TREATMENT OF HCM-RELATED SYMPTOMS

34. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers (I B) - Verapamil / Diltiazem (I B) - Disopyramide (I B) - β blockers (II C) - Diuretics (II C) - ACEi – ARBs (II C) -MRA (II C) -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines (CLASS OF RECOMMENDATION) Obstructive - β blockers (II C) Arrhythmias - β blockers (I C) - Amiodarone (I C) - Sotalol (II C) -Anti- coagulants (AF) (I B) - β blockers (II C) - Diuretics (II C) - Verapamil (II C) CLASS I B = Benefit greatly exceeds the risk; limited evidence from a single randomized trial or multiple nonrandomized studies . CLASS I C = Treatment should be administered; evidence only from expert opinions/ case studies. CLASS II C = Benefit exceeds the risk and it is reasonable to treat; evidence only from expert opinions/ case studies.

35. Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Preventing phenotype development in HCM mutation carriers DRUG THERAPY IN HCM: OUTLINE

36. Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Preventing phenotype development in HCM mutation carriers OUTLINE

37. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - β blockers - Diuretics - Verapamil - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines Obstructive - β blockers TREATMENT OF HCM-RELATED SYMPTOMS

38. ROLE OF DISOPYRAMIDE First-line drug for LVOT obstruction (Class I B indication ), alone or in addition to β - blockers or verapamil . Consistently reduces LVOT gradient ( negative inotropic effect ? ) Improves survival in obstructive patients Mechanisms poorly studied Disopyramide + β blocker Disopyramide β blocker Disopyramide in the Florence cohort of obstructive patients (>250)

39. Diso DISOPYRAMIDE IMPROVES SURVIVAL

40. HCM patients with Obstruction Septal myectomy Surgical Sample 1 cm Diastole Left Atrium Left Ventricle LVOT Aorta CONTROLS: non-failing surgical patients: septal thickness < 14mm, ejection fraction >55%, aortic valve disease Basal inter-ventricular septum Turbulent blood flow in the LVOT STUDY COHORT : HCM patients with symptomatic obstruction Late Sodium Current in Hypertrophic Cardiomyopathy Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy . Circulation 2013 HCM Control Calibration bar = 10 µm Cardiomyocytes

41. EC50: 5.29±1.55 µM Therapeutic conc .: 5 µM Normalized 1 mN /mm 2 # p<0.05, paired Coppini et al. Unpublished Data # # DISOPYRAMIDE IN HUMAN HCM MYOCARDIUM

42. DISOPYRAMIDE IN HUMAN HCM CARDIOMYOCYTES Means±SEM from 13 cells/ 4 patients Disopyramide shortens APD Disopyramide reduces diastolic Ca and the amplitude of Ca transients Effects mediated by I NaL block ?

43. Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Preventing phenotype development in HCM mutation carriers OUTLINE

44. BETA BLOCKERS IN HCM First-line drug for LVOT obstruction (Class I B indication ) . To be used in patients with ICD and recurrent arrhythmias . Primary arrhythmia prevention ? No apparent effects on survival in adult patients Improve survival in children with HCM (single study ). High-dose beta- blockers Low-dose beta- blockers No beta- blockers

45. BETA BLOCKERS IN HCM Hypertrophic cardiomyopathy (HCM) presenting in childhood has higher mortality (annual rate 4.6% to 5.8%) than in adult life (1% to 4%) HCM-related deaths during childhood are: Sudden (arrhythmic) 48% Heart failure 36% Surgical complications 16% Hypertrophic cardiomyopathy during childhood  is associated with increased activity of cardiac sympathetic nerves (extreme sympathetic over-activity)

46. High-dose beta- blockers Standard or no Therapy -Standard Dose βB, n = 18 patients; 0.8 to 4 mg/kg/day of propranolol) -High-dose therapy (HDβB), n = 26 patients; 5 to 23 mg/kg/day of propranolol, or equivalent doses of metoprolol or atenolol) -No specific therapy, n=20 BETA BLOCKERS IN HCM

47. Likely beneficial for symptomatic patients with no obstruction (Class II B indication ). Nadolol (80-120 mg/d) to be preferred in obstructive pts . over propranolol or metoprolol (Class II C). Bisoprolol (5-10 mg/d) to be preferred in end-stage (II C). All studies with β - blockers in HCM are 20+ years old Beta- blockers reduce obstruction and improve diastolic function , however the mechanisms are unclear No studies on human cardiomyocytes or samples BETA BLOCKERS IN HCM: CONTROVERSIES

48. Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Preventing phenotype development in HCM mutation carriers OUTLINE

49. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - β blockers - Diuretics - Verapamil - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines Obstructive - β blockers Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) TREATMENT OF HCM-RELATED SYMPTOMS

50. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - I NaL blockers - β blockers - Diuretics - Verapamil - I NaL blockers - Perhexiline - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines ----------------- Promising Future Options Obstructive - β blockers - I NaL blockers - Perhexiline Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) - I NaL blockers DISEASE-MODIFYING THERAPIES? -Anti- oxydants - ARBs - Statins

51. Polakit Teekakirikul et al. J Cell Biol 2012;199:417-421 TARGETING CELLULAR AND EXTRACELLULAR REMODELLING

52. EXPERIMENTAL THERAPIES: STATINS Atorvastatin reduced hypertrophy in HCM rabbits …. but failed to reduce hypertrophy in a pilot study on 32 patients

53. Reduced fibrosis EXPERIMENTAL THERAPIES: SARTANS Baseline 1 y treatment Reduced TGF- β Disarray persists Change in LGE% Change in LV Mass Losartan reduced fibrosis in transgenic rabbits and reduced LGE and LV mass in patients after 1 year of treatment ( pilot study on 20 patients )

54. EXPERIMENTAL THERAPIES: SARTANS Single center, randomized, placebo-controlled trial with losartan in HCM Primary endpoint : change in LV mass at MR 133 total patients (50% per branch ) No significant change in LV mass after 12 months of treatment when compared with placebo INHERIT TRIAL

55. Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Preventing phenotype development in HCM mutation carriers OUTLINE

56. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 - β blockers - Verapamil / Diltiazem - Disopyramide - I NaL blockers - β blockers - Diuretics - Verapamil - I NaL blockers - Perhexiline - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines ----------------- Promising Future Options Obstructive - β blockers - I NaL blockers - Perhexiline Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) - I NaL blockers DISEASE-MODIFYING THERAPIES? -Anti- oxyd . - ARBs - Statins

57. ** Increased I NaL Results Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy . Circulation 2013 Control HCM Prolonged APD Late Sodium Current in Hypertrophic Cardiomyopathy Stimuli = Early After Depolarization , EAD Cellular arrhythmias Delayed relaxation

58. ATP ase NCX I NaL PLB Sarcoplasmic reticulum Ca Na Ca Na K Na Myofilaments Ca RyR SERCA Sarcolemma T-tubule I CaL Ca Mitochondria mNCX Ca Na ATP produced MCU K Na H NHE I K Na Na I NaT H Na ETC H P Ca NHE P AP Ca Transient Force From: Coppini R et al. Regulation of intracellular Na(+) in health and disease : pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract . 2013 NKA Consequences of Increased I NaL in cardiomyocytes CONTROL CARDIOMYOCYTE

59. ATP ase NCX ( forward ) I NaL PLB Ca Na Ca Na K Myofilaments Ca RyR SERCA Sarcolemma T-tubule I CaL Ca Mitochondria ↓Ca Na ATP produced MCU K Na H NKA NHE I K Na Na I NaT H Na ETC H P NHE AP Ca 2+ Force Ca LEAK P P P Sarcoplasmic reticulum NCX (reverse) Na mNCX ↑ CaMKII Na + + ↑ROS + + ↓ pH From: Coppini R et al. Regulation of intracellular Na(+) in health and disease : pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract . 2013 Consequences of Increased I NaL in HCM cardiomyocytes HCM CARDIOMYOCYTE

60. Effects of Late Sodium Current Inhibition. Direct effects ( decreased net inward current during AP plateau) Shortening of AP duration Suppression of Early after-depolarizations ( EADs ) Increased repolarization stability Mediated by the reduction of [Na + ] i ↓ [Ca 2+ ] i (NCX) ↓ Delayed after- depolarizations (DADs) Ameliorated Diastolic function and reduction of myocardial ischemia Effects on long term remodelling Beneficial effects of I NaL block Na + Na + Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late  I Na  in Development of Cardiac Arrhythmias. Handb Exp Pharmacol . 2014

61. Antiarrhythmic Drugs that inhibit I NaL Modified from: Antzelevitch C. et al. The Role of Late I Na in Development of Cardiac Arrhythmias Handb Exp Pharmacol.  2015. DRUG I NaL / I NaT I NaL / I Kr Amiodarone 13 ≤ 1.5 Flecainide 2.9–5 < 0.1–2 Ranolazine 9–38 1.5–2 GS-967 50-100 >100 Selectivity for I NaL ( uM ) Late I Na Peak I Na Modified from: Belardinelli L. et al. Cardiac late Na + current : Proarrhythmic effects , roles in long QT syndromes , and pathological relationship to CaMKII and oxidative stress. Heart Rhythm  2015

62. Ranolazine : Key Clinical Trials ROLE N=746 Chronic angina Chaitman BR et al. J Am Coll Cardiol 2004;43:1375-82 Chaitman BR, et al. JAMA. 2004;291:309-316 Stone PH, et al. J Am Coll Cardiol . 2006;48:566-575 Morrow DA, et al. JAMA. 2007;297:1775-1783 Kosiborod M et al. J Am Coll Cardiol 2013;61:2038–45 Koren MJ et al. J AM Coll Cardiol 2007;49:1027-34 CARISA N=823 Chronic angina Ranolazine vs placebo on top of standard therapy ERICA N=565 Chronic angina Ranolazine vs placebo on top of amlodipine 10mg MERLIN TIMI-36 N=6560 Non-STE ACS Ranolazine vs placebo on top of standard care MARISA N=191 Chronic angina Ranolazine vs placebo Total patients enrolled = 9,088 TERISA N=949 Chronic Angina/ DM2 Ranolazine vs placebo on top of 1 to 2 antianginals Approved for Chronic Stable Angina treatment Studies in pts with Diabetes , Neuropathic Pain , Heart Failure , HFpEF

63. Effects of Late Sodium Current Inhibition. Direct effects ( decreased net inward current during AP plateau) Shortening of AP duration Suppression of Early after-depolarizations ( EADs ) Increased repolarization stability Mediated by the reduction of [Na + ] i ↓ [Ca 2+ ] i (NCX) ↓ Delayed after- depolarizations (DADs) Ameliorated Diastolic function and reduction of myocardial ischemia Effects on long term remodelling Beneficial effects of I NaL block Na + Na + Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late  I Na  in Development of Cardiac Arrhythmias. Handb Exp Pharmacol . 2014

64. HCM Basal HCM + Ran HCM Basal HCM + Ran HCM + TTX Transmembrane Current I NaL Inhibition shortens APD and abolish EADs in human HCM Ranolazine 10µM Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy . Circulation 2013

65. HCM Basal HCM + Ran HCM + TTX Transmembrane Current I NaL Inhibition shortens APD and abolish EADs in human HCM = Early After Depolarization (EAD) Stimuli Ranolazine 10µM Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy . Circulation 2013

66. 0.2 Hz ## ## ## ## p<0.01, paired Means±SEM from 25 cells/ 9 patients I NaL Inhibition shortens APD and abolish EADs in human HCM GS-967 1µM Coppini et al. Unpublished Data

67. Effects of Late Sodium Current Inhibition. Direct effects ( decreased net inward current during AP plateau) Shortening of AP duration Suppression of Early after-depolarizations ( EADs ) Increased repolarization stability Mediated by the reduction of [Na + ] i ↓ [Ca 2+ ] i (NCX) ↓ Delayed after- depolarizations (DADs) Ameliorated Diastolic function and reduction of myocardial ischemia Effects on long term remodelling Beneficial effects of I NaL block Na + Na + Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late  I Na  in Development of Cardiac Arrhythmias. Handb Exp Pharmacol . 2014

68. ATP ase NCX ( forward ) I NaL PLB Ca Na Ca Na K Myofilaments Ca RyR SERCA Sarcolemma T-tubule I CaL Ca Mitochondria ↓Ca Na ATP produced MCU K Na H NKA NHE I K Na Na I NaT H Na ETC H P NHE AP Ca 2+ Force Ca LEAK P P P Sarcoplasmic reticulum NCX (reverse) Na mNCX ↑ CaMKII Na + + ↑ROS + + ↓ pH From: Coppini R et al. Regulation of intracellular Na(+) in health and disease : pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract . 2013 HCM CARDIOMYOCYTE Consequences of Increased I NaL in cardiomyocytes

69. ATP ase NCX ( forward ) I NaL PLB Ca Na Ca Na K Na Myofilaments Ca RyR SERCA Sarcolemma T-tubule I CaL Ca Mitochondria Ca Na ATP produced MCU K Na H NHE I K Na Na I NaT H Na ETC H P NHE AP Ca 2+ Force Ca LEAK P P P Sarcoplasmic reticulum NCX (reverse) Na Ranolazine mNCX NKA ↓ CaMKII ↑ROS - - From: Coppini R et al. Regulation of intracellular Na(+) in health and disease : pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract . 2013 HCM CARDIOMYOCYTE GS-967 Consequences of I NaL inhibition in HCM cardiomyocytes

70. HCM Basal HCM + Ran * * ** Ranolazine decreases [Na + ] i ** * ** Ranolazine hastens Ca transient kinetics Modified from: Coppini C., Mugelli A. et al. Late Sodium Current Inhibition Reverses Electromechanical Dysfunction in Hypertrophic Cardiomyopathy . Circulation 2013 I NaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes

71. ** ** ** HCM Basal HCM + Ran Control ** =p<0.01 Ranolazine reduces intracellular diastolic [Ca] I NaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes

72. 0.2 Hz 0.5 Hz 1 Hz ## ## # ## ## ## # p<0.05, paired ## p<0.01, paired Means±SEM from 15 cells/ 6 patients I NaL inhibition ameliorates Ca homeostasis in HCM cardiomyocytes GS-967

73. Effects of Late Sodium Current Inhibition. Direct effects ( decreased net inward current during AP plateau) Shortening of AP duration Suppression of Early after-depolarizations ( EADs ) Increased repolarization stability and reduced spatial APD dispersion Mediated by the reduction of [Na + ] i ↓ [Ca 2+ ] i (NCX) ↓ Delayed after- depolarizations (DADs) Ameliorated Diastolic function and reduction of myocardial ischemia Effects on long term remodelling Beneficial effects of I NaL block Na + Na + Modified from: Antzelevitch C, Belardinelli L et al.: The Role of Late  I Na  in Development of Cardiac Arrhythmias. Handb Exp Pharmacol . 2014

74. Ranolazine lowers diastolic tension and accelerates twitch relaxation Basal Ran Wash out 3mN/mm 2 HCM Basal HCM+ Ran 75 I NaL inhibition ameliorates diastolic function in HCM myocardium 10 mN /mm 2 # # # # # p<0.05, paired Means±SEM from 8 trabeculae / 7 patients GS-967 Ranolazine

75. 1 mN /mm 2 # # # n.s. # n.s. # p<0.05 RANOLAZINE CAN BE A DRUG FOR OBSTRUCTION

76. 1 mN /mm 2 # # # n.s. # n.s. # p<0.05 RANOLAZINE CAN BE A DRUG FOR OBSTRUCTION # Basal Contraction Diso Ran

77. Clinical Trials with Late Na Current Blockers in HCM RESTYLE-HCM Recruitment : 80 non- obtructive symptomatic patients with reduced exercise capacity (VO 2 max <75% of predicted ) Double- blind , randomized , placebo- controlled multicenter study (11 centers across europe ) Primary Objective : improved exercise capacity at CPET Secondary Objectives : improved diastolic function , ameliorated symptomatic status, lowered proBNP , reduced arrhythmic burden

78. Clinical Trials with Late Na Current Blockers in HCM RESTYLE-HCM Objective : demonstrate the efficacy of ranolazine in improving exercise capacity and reducing symptoms in HCM patients Ranolazine or placebo added on top of standard treatment Primary : objective not met (p≈0.10) Secondary : significant reduction of proBNP , reduced arrhythmic burden at Holter Placebo V3 Treatment phase Ranolazine Time 0 V4 V6 3 ° month 5 ° month V6 V4 3 ° month V5 V5 1 ° month 1 ° month V3 Placebo Time 0 Treatment phase V1 Ranolazine 500 mg bid Time -14 d V2 Ranolazine 750 mg bid Time -7 d Titration phase Titration phase Placebo Time -14d V1 Placebo Time -7d V2 5 ° month Ranolazine 1000 mg bid screening Symptomatic standard therapy RESULTS ( preliminary : final statistics analysis ongoing ) Problems : low number of pts ., difficult recruitment

79. 40 specialized centers in USA and Europe (Italy, Germany, UK) to enrol 180 symptomatic patients (including obstructive) . PRIMARY: improvement of exercise capacity ( VO 2 max at CPET ) SECONDARY: improvement of: symptomatic status (questionnaire), arrhythmias, diastolic function, degree of obstruction (gradient) Level 2/3, randomized , double- blind , placebo- controlled , with GS-6615 Objectives TREATMENT: 30mg single dose, 3 mg/die for 12 weeks, then 6 mg/die for 12 weeks ( total 24 weeks) Clinical Trials with Late Na Current Blockers in HCM

80. Exercise Capacity Diastolic Function QoL Arrhythmias Zio Patch Echo MLHFQ CPET (VO 2 ) Obstruction (GS-6615)

81. Clinical Trials with Late Na Current Blockers in HCM The first multicentric randomized trial in HCM with a novel drug ( eleclazine ) Aimed at registration with specific HCM indication Results expected beginning of 2017 Based on solid preclinical data on human samples

82. Late sodium current inhibition in symptomatic HCM: from basic science to randomized trials Disopyramide : new roles for an old drug β - blockers : new mechanisms and perspectives Other experimental therapies for HCM Preventing phenotype development in HCM mutation carriers OUTLINE

83. Current therapies Stages of hypertrophic cardiomyopathy (HCM) Modified from Iacopo Olivotto et al. Circ Heart Fail. 2012;5:535-546 Non-Hypertrophic Development of LV Hypertropy No current therapies are able to prevent cardiac phenotype development in young carriers of mutations associated with HCM Non-Hypertrophic Non-Hypertrophic Healthy mutation carrier Prevention of HCM phenotype

84. Pharmacological Therapy of hypertrophic cardiomyopathy at different Stages Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 No disease expression - Diltiazem -Ranolazine/ new I NaL blockers -Gene therapy - β blockers - Verapamil / Diltiazem - Disopyramide - I NaL blockers - β blockers - Diuretics - Verapamil - I NaL blockers - Perhexiline - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines ----------------- Promising Future Options Obstructive - β blockers - I NaL blockers - Perhexiline Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) - I NaL blockers PHENOTYPE PREVENTION -Anti- oxydants - ARBs - Statins

85. Prevention of HCM phenotype

86. Means ± S.E. from 38 patients: 18 Diltiazem and 20 Placebo Age, yrs : 14,1 ± 1,7 Diltiazem and 17,3 ± 2,1 Placebo During treatment After treatment Prevention of HCM phenotype

87. ATP ase NCX ( forward ) I NaL PLB Ca Na Ca Na K Na Myofilaments Ca RyR SERCA Sarcolemma T-tubule I CaL Ca Mitochondria Ca Na ATP produced MCU K Na H NHE I K Na Na I NaT H Na ETC H P NHE AP Ca 2+ Force Ca LEAK P P P Sarcoplasmic reticulum NCX (reverse) Na Ranolazine mNCX NKA ↓ CaMKII ↑ROS - - From: Coppini R et al. Regulation of intracellular Na(+) in health and disease : pathophysiological mechanisms and implications for treatment. Glob Cardiol Sci Pract . 2013 HCM CARDIOMYOCYTE I NaL inhibition may exert long-term effects on myocardial remodeling

88. Genotyping 45 ranolazine- treated mice 90 newborn (R92Q or WT) siblings 45 vehicle-treated mice 1 month 11-12 months 22 mice   R92Q (R92Q-RAN) WT (WT-RAN) R92Q (R92Q-KET) WT (WT-KET) Chow 0.5% Ran . + 0.03% Ketoc . 6 couples of progenitors ( ♀WT+♂R92Q) 22 mice   22 mice   22 mice   Chow 0.03% Ketoc . (vehicle) -In vivo experiments ( Echo , MRI) -Ex vivo experiments (cells, trabeculae,hystology , PCR) WT R92Q -KET R92Q -RAN LV LA LAA LV LA LAA LV LA LAA WT R92Q-KET E A E A R92Q-RAN E A ** * * ** NS ** WT R92Q -KET R92Q -RAN I NaL inhibition exerts beneficial effects on myocardial remodeling in HCM R92Q-TnT transgenic mice were treated with Ran lifelong Ran lifelong treatment prevented the development of LV hypertrophy and LA dilatation in mutants Ran lifelong treatment prevented the development of diastolic dysfunction in mutants ** NS ** Bar=1mm Bar=1mm * = p<0.05 ** =p<0.01 Coppini et al. Unpublished

89. 1 minute after Gd injection 30 minutes after Gd injection WT R92Q-KET R92Q-RAN RV LV RV LV RV LV END-DIASTOLE END-SYSTOLE ** NS ** ** NS * ** NS * ** NS * Cardiac Magnetic Resonance ** ** ** WT R92Q-KET R92Q-RAN ** * * R92Q-KET WT R92Q-RAN Histology I NaL inhibition prevents structural myocardial remodeling in HCM

90. Administration of ranolazine in HCM mice prevents LV hypertrophy and hypercontractility , ameliorates diastolic function and reduces extracellular fibrosis Ranolazine-treatment normalizes cellular dysfunction Ranolazine or novel late Na current blockers may be employed in young carriers of HCM-mutations to prevent phenotype onset and disease progression Prevention of HCM phenotype

91. GENE THERAPY In homozygous Mybpc3 mutants , reintroduction of WT Mybpc3 gene via AAV- mediated cardiac-specific gene-transfer prevents hypertrophy and functional deterioration (single injection at 1 week of age ).

92. THE BRIGHT FUTURE: HCM AS A TREATABLE DISEASE Modified from: Iacopo Olivotto et al. Circ Heart Fail. 2012 No disease expression - Diltiazem -Ranolazine/ new I NaL blockers -Gene therapy - β blockers - Verapamil / Diltiazem - Disopyramide - I NaL blockers - β blockers - Diuretics - Verapamil - I NaL blockers - Perhexiline - β blockers - Diuretics - ACEi – ARBs -MRA -------------- Current therapies according to AHA 2013 and ESC 2014 HCM guidelines ----------------- Promising Future Options Obstructive - β blockers - I NaL blockers - Perhexiline Arrhythmias - β blockers - Amiodarone - Sotalol -Anti- coagulants (AF) - I NaL blockers PHENOTYPE PREVENTION -Anti- oxydants - ARBs - Statins

93. Dept. Physiology University of Florence N. Piroddi B. Scellini J.M. Pioner F. Gentile B. Tosi C. Tesi C. Ferrantini C. Poggesi Referral Center for Cardiomyopathies AOU Careggi – Firenze I. Olivotto F. Cecchi B. Tomberli K. Baldini Dept. Pharmacology University of Florence L. Sartiani V. Spinelli M. del Lungo L. Diolaiuti L. Dini S. Blescia L. Mazzoni E. Cerbai Genetics AOU Careggi F. Girolami S. Bardi F. Torricelli Cardiac Surgery Units AOU Careggi: M.Yacoub, A.Rossi, S.Stefano Villa Beatrice: G. Popov LENS : C. Crocini M. Scardigli L. Sacconi F. Pavone Thanks T ucson : J. Tardiff Gilead Sciences L. Belardinelli S. Rajamani Fundings :