Cardiomyocyte? March 14 th 2013

Transcription

1 What Makes a Cardiomyocyte a Cardiomyocyte? Kyle Kolaja March 14 th 2013

2 What Makes a Cardiomyocyte a Cardiomyocyte? Shouldn t proliferate Should beat Should be fairly pure Should look like and express genes like a heart cell Should respond to cardioactive drugs 2

3 Cardiomyocyte a Cardiomyocyte Rationale from a toxicologist perspective Two schools of thought Bits and pieces look right Cell-based system responds like a heart Bits and Pieces Characterization Gene expression Channel activity/function Purity and impact on endpoints Cell-based systems Arrhythmia Hypertrophy Toxicity 3

4 icell Cardiomyocytes are more similar to Adult Human Heart Samples than Primary Cultures CDI cells d28 and d120 Primary Heart cultures Troponin I = at background = ES cell Babiarz et al Stem Cells Dev. Jan

5 icell Cardiomyocytes are more similar to Adult Human Heart Samples than Primary Cultures CDI cells d28 and d120 Primary Heart cultures Troponin I = at background = ES cell Babiarz et al Stem Cells Dev. Jan

6 Cardiomyocyte Manufacturing Process CREATE & MAINTAIN EXPAND DIFFERENTIATE FINSH & CRYOPRESERVE Cardiomyocyte y Production Quality Quantity Purity Purity and Morphology Differentiation with expression-based selection can yield >98% purity Cryopreservation makes easier for everyone Morphology with sarcomeres & striated myofibers

7 Confirmation of Cardiomyocyte Nature icell Cardiomyocytes Express major cardiac proteins Exhibit cardiac action potential wave forms Functionally active cardiac channels Respond as expected to channel blockers Ma et. al Am J Physiol Heart Circ Physiol. (2011) High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents 7

8 Impedance Detection of Cardiomyocyte Beating 96-well E-eplate Seconds 10 log) Cell Index ( Hours 8

9 Drug-induced changes in beat rate Indication of functional cardiac ion channels xcelligence: Impedance MEA: Electrical Field Control Na+ Channel Blocker Pace-maker Current Blocker Agonist of β- Adrenergic Receptor 9

10 Does impedance detect the physical beating or the electrical changes? Pre-drug Blebbistatin: Blocks myosin II in an actin-detached state 15 min MEA 15 min 0µM

11 In vitro Detection of Arrhythmias with Human icell Cardiomyocytes Pre-drug Post-drug 11

12 In vitro Detection of Arrhythmias using Human icell Cardiomyocytes E-4031 causes arrhythmia DMSO E-4031 Nifedipine rescues E-4031-induced arrhythmia DMSO + Nif E Nif Misner et al Br J Pharmacol Apr;165(8): Functional Rescue of Drug-induced Arrhythmia 12

13 Human Cardiomyocytes (hcar) Assay ips cell-derived human cardiomyocytes IB20= lowest tested concentration resulting in 20% irregular beats 13

14 hcar Assay Validation: 23 compounds with known in vivo effect 12 Pro-arrhythmic 11 Non-arrhythmic IB20 30 um One False Positive No False Negatives IB20= lowest tested concentration resulting in 20% i l b Drug IB20 Clinical herg QT (µm) arrhythmia Dofetilide (+) (+) (+) Ouabain 0.03 ( ) ( ) (+) Aconitine 0.03 ( ) ( ) (+) Cisapride 0.03 (+) (+) (+) E (+) (+) (+) Astemizole 0.03 (+) (+) (+) Terfenadine 0.3 (+) (+) (+) Flecainide 1 (+) (+) (+) Alfuzosin 1 ( ) (+) ( ) Thioridazine 3 (+) (+) (+) Quinidine 10 (+) (+) (+) Erythromycin 30 (+) (+) (+) Sotalol 30 (+) (+) (+) Fluoxetine >30 (+) (+) ( ) Verapamil >30 (+) (±) ( ) Moxifloxacin >100 (+) (+) (+) Amiodarone >100 (+) (+) (+) Ranolazine >100 (+) (+) ( ) Captopril >100 ( ) ( ) ( ) Rofecoxib >100 ( ) ( ) ( ) Amoxicillin >1000 ( ) ( ) ( ) irregular beats Aspirin >1000 ( ) ( ) ( ) Nifedipine >3 ( ) ( ) ( ) 14 Guo et al. Toxicol Sci Sep;123(1):281-9.

15 Human Cardiomyocytes (hcar) Assay Next Round of Validation 83 Compounds ~82% -- arrhy. prediction >90% -- QT prediction 30 Internal Compounds 80% -- arrhy. prediction 95% -- QT prediction 15

16 Hallmarks of Cardiac Hypertrophy Increased cell size Enhanced protein synthesis Higher organization of the sarcomere Re-activation of the fetal gene program Expression of B-type natriuretic protein (BNP) Normal Untreated + PE Kuwahara K et al. J Pharmacol Sci 2012;119: Cell Size Protein Synthesis BNP Expression Ac ctin Reorg Diseased Lister K et al. Cardiovasc Res 2006;70: Glenn D et al. Hypertension 2009;53:

17 Hypertrophy Application Suite BNP qpcr Measure activation of BNP gene (NPPB) ELISA Quantify levels of secreted BNP Flow Cytometry Detect BNP expression High Content Analysis Image BNP expression, Cell Size, Cell Thaw and Plate icell Cardiomyocytes Induce with ET-1 Detect BNP Coat Plate with Fibronectin Replace Medium

18 NHLBI Family Blood Pressure Program GWAS now icell Cardiomyocytes GWAS identification of associated SNPs with left Ventricular Hypertrophy Phenotyping through cardiovascular phenotypes and risk factors Family-based ascertainment NHLBI, Next Generation Genetic Association Studies, RFA-HL CDI will make pure ventricular cardiomyocytes from 250 different patients 100 most informative families derived from HyperGEN cohort Induce hypertrophy phenotype in vitro, perform molecular analyses Attempt to correlate GWAS findings with in vitro phenotype

19 CDI Grant Milestones You are here Grant Year Calendar Year Grant Phases Research Development Sample identification and Optimize parallel ips and CM production Cell Production Production Plan 25 donors completed or in progress donors donors donors

20 Example Pre and Post Selection Purity and Yield Sample ID O1144 Start of Selection (day 12) End of Selection (day 16) ctnt-33.0% ctnt-88.9% T CM yield (start of selection): 256M CM yield (end of selection): 280M QC Criteria at cryo (day 30) >90% purity (ctnt+) > 4.3 million viable cells per cryovial >50 million total yield (200M target) Mycoplasma negative Identity screen (STR) match Observed beating rate This will drift up to ~97% by day 30

21 Studying the mechanism of Sunitinib (Sutent) mediated cardiotoxicity Sutent multi-targeted (RTK) inhibitor for oncology indication Cardiac dysfunction and toxicity congestive heart failure and left ventricular systolic dysfunction Inhibits herg and causes arrhythmia Previous work done mostly with NRVMs Binds 150 kinases Inhibits proliferation/survival/angiogenesis VEGFR1-3, PDGFR(α and β), c-kit, FLT3, CSF1R, and RET AMP Kinase cellular gas gauge RSK ribosomal s6 kinase -downstream of ERK Aurora Kinase - cell prolif/, Chromatid seg., mitosis

22 Advancing the Understanding of Sutent Cardiotoxicity Sutent potently cytotoxic in icell CMs Confirmed the inhibition of AMPK by sutent but minimal protective effect seen by AMPK agonists Inhibit herg, Ca++ cycling and NaV1.5 -> arrhythmia RSK inhibitors not cardiotoxic icell CMs ideal RSK inhibitor inhibits herg and causes arrhythmia model to assess cardiotoxicity, Aurora Kinases electrophysiology and contractility tilit effects in parallel Multi-factorial combination of cytotoxicity, cardiac conduction abnormalities, hypoxia, pan- kinase inhibition leading to suppressed response/accommodation mechanisms 22

23 Jaspamide Cardiotoxicity Jaspamide (a cyclodepsipeptide isolated from the marine sponge Jaspis johnstoni ) a potential cancer therapeutic agent Pulmonary edema and cardiac hemorrhage and congestion in preclincal tox species Jaspamide (10 lm) inhibited Kv1.5 activity by 98.5%. And also inhibited Cav1.2, Cav3.2, and HCN2; but not herg icell CMs ideal model to assess cardiotoxicity, electrophysiology and contractility effects in parallel Affects actin binding to cytoskeleton 23

24 Opportunity for ips-derived Cardiomyocytes in Cardiac Research Functional aspects of PSC-derived cardiomyocytes highlight an improved in vitro tool for studying cardiotoxicity Gene expression Channel activity/function Purity and impact on endpoints Rhythmic beating and response to treatment Doesn t proliferate Maintain in culture for prolonged periods Example cell-based systems just beginning Arrhythmia and hypertrophy Mechanistic toxicity regulatory filings Continued validation through empirical testing 24