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The action potential and the underlying ionic currents Norbert Jost, PhD The propagation of the stimulation in the heart Left atria Sinus node His Bundle Left ventricle Conduction velocity in m/s AV node Left Bundle branch Time to arrive from AV to the respective place (ms) Right atria Right ventricle Right Bundle branch Purkinje fibres Bovine Purkinje fibres system marked with ink The action potential in a ventricular myocyte 1. EXCITABILITY Small triggering stimulus large response „Action Potential” Action potential Threshold and autogenerative excitation Ca- channel / Ca- current to intracellular space K- channel / K- current from intracellular space to extracellullar space 4. Re-establish of the diastolic (resting) membrane potential ION AT 3. REPOLARIZATION IZ AR from extracellullar space L PO RE Na-channel / Na-current DEPOLARIZATION 2. DEPOLARIZATION The ECG and the action potential I. The ECG and the action potential II. REFRACTORINESS ERP = Effective Refractory Period The shortest time needed for reactivation of the heart muscle Depends on ? 1. Repolarization of the myocytes (K-channels) 2. The actual size of the depolarizing currents (Na- and Ca - channels) Ventricle/atria Na-channel SA/AV node Ca-channel Multi-cellular Organization Functional syntitium = The ability for cardiomuscles to contract all at once = Gap Junction Channel (small resistance) IMPULSE CONDUCTION Direction of the impulse propagation 1. The speed of depolarization (Vmax) - depends on fast sodium current Velocity at which each domino falls 2. Action potential amplitude - depends on fast sodium current Height of the domino 3. Treshold of activation - depends on fast sodium current The energy needed to push the domino 4. The cells internal resistance / the resistance between the cells (ri) - depends on the gap junctions What is the medium resistance (water, air, vacuum) Sodium channels (atria, ventricle) or calcium channels (sinus and AV- node) Outline of membrane currents of sinus node cells: current profiles (drawn by hand) are time aligned with the action potential. Purkinje fibre Diastolic depolarization The main potassium currents in the ventricular and atrial muscle And many other currents and mechanisms !!! Species differences in APD and ERP Action potential and currents in sinus node and Purkinje fibre Diastolés depolarizáció Action potential and currents in sinus node A more positive resting potential ! The pacemaker current (If) To induce the spontanous diastolic depolarization If Action potential and fast sodium current (INa) in atria and ventricle The fast sodium channel (INa) 50 mV Wu et al, Heart Rhythm, 2008, 5(12):1726-34 INa 100 ms Resting state Na + Active state Na + Na + Na outside m h inside - h inside Na + h - m m inside + Na + outside outside Inactive state + Blockers of the fast sodium channel (Ina) Na-channel Tetrodotoxin (TTX) binds specifically to sodium channels by mimicking the hydrated Na+ ion, denying entry to Na+ ions. TTX binding site Na channel The slow (late) sodium channel INaL 50 mV ATX 100 ms Wu et al, Heart Rhythm, 2008, 5(12):1726-34 Action potential and the L type calcium current (ICaL) in atria and ventricle Atria L type calcium current (ICa) 50 mV ICaL 100 ms Varro et al, Br. J. Pharmacol, (2001) 133, 625 – 634. Activation kinetics Resting potential + Ca Ca + outside Inactivation kinetics Actíve Ca outside h Re-activation kinetics m - inside Ca + h h inside + Ca outside m - + Ca + m inside Inactíve + L type calcium current (ICa) S4 – voltage sensor Loop S5-S6 – ion conductance and selectivity 2 subunit complex Intracellular -subunit Action potential and the transient outward potassium current (Ito) in atria and ventricle Csatorna fehérje Transient outward potassium current (Ito) 50 mV Virag et al, unpublished 100 ms Resting potential Activation Inactivation „Notch” Effect of selective Ito blockade on the action potential Repolarization lengthens The notch disappears Virag et al, unpublished Action potential and the rapid and slow componets of the delayed rectifier potassium currents (IKr and IKs) in atria and ventricle The fast and slow delayed rectifier potassium currents (IKr and IKs) 1000 ms 5000 ms 30 mV 30 mV -40 mV -40 mV 100 pA 50 pA There is a fast inactivation also ! 2500 ms Activation 25 pA Resting potential 2500ms Deactivation 500 ms The fast and slow delayed rectifier potassium currents (IKr and IKs) Controll 1 µM E-4031 +30 mV -40 mV -80 mV 250 ms Difference current 50 pA E-4031 sensitive (IKr) Controll 100 nM L-735,821 0 pA L-735,821 sensitive (IKs) 50 mV 200 ms Varro et al, J.Physiol. 2000; 523.1: 67-81 200 ms Action potential and the inward rectifier potassium current (IK1) in atria and ventricle The inward rectifier potassium currents (IKr and IKs) The ” inward” rectification is regulated (inhibited) by intracellular cations (Mg2+, Ca*, polyaminok) under depolarization 10 M BaCl2 Control 0 mV Control 0 pA 60 mV -90 mV 36 s - 50 mV 1000 pA 10 M BaCl2 -120 mV cycle length = 1000 ms -120 -80 -40 0 40 (mV) 200 ms Biliczki et al, Br. J. Pharmacol, 2002,137(3):361-368. Resting potential Activation Deactivation Summary – the four main repolarizing current under the action potential Atria specific currents: The ultrarapid delayed rectifier potassium current (IKur) Kamra Pitvar ? Ionáram Csatorna fehérje Ionáram Atria specific currents: The ultrarapid delayed rectifier potassium current (IKur) IKur Gao et al, Br. J. Pharmacol, 2005; 144, 595-604 Resting potential Activation Inactivation IKur szelektív gátlásának hatása az akciós potenciálra Wang et al. Circ. Res. 1993, 73: 1061 Wettwer et al. Circulation 2004;110:2299-2306 Pitvarszelektív áramok – az acetilkolin függő káliumáram (IK,Ach) Dobrev et al. Circulation 2005;112:3697-3706 Az IK,ACh gátlása lehetséges kezelési mód a krónikus PF esetében ?!? Other ligand dependent current: the ATP sensitive potassium current (IKATP) SARCOLEMMAL CHLORIDE CHANNELS Cl- channels Activated by PKA (ICl.PKA) and PKC (ICl.PKA) Cl - Channels Regulated by Cell Volume (ICl.vol) Other Cl - Channels (activated by Cytoplasmic Ca2+ (Ito2), purinergic Receptors (ICl,ATP), etc) ECl under normal physiological conditions in the range of -65 to -45 mV thus membrane Cl- channels have the unique ability, compared with cation channels, to contribute both inward as well as outward current during the cardiac AP Cl- channels activated by PK (ICl.PK) ! 21 % of the studied atrial myocytes James et al, Circ Res, 79, 201-207, 1996 Cl- channels activated by PK (ICl.PK) Levesque et al, Pflug Archiv, 424, 54-62, 1993 Molecular background, tissue and species distribution of ICl.PK Gene encoding: (CFTR) (cystic fibrosis transmembrane conductance regulator) Hume et al, Physiol Rev, 80, 31-81, 2000 Present in: - adult ventricular, but not in atrial (?) or sinoatrial nodal cells in guinea pig, rabbit, and cat and human (?!) Cl - Channels Regulated by Cell Volume (ICl.vol) Volume-regulated anion channels are now known to be ubiquitously expressed in mammalian cells and play an important role in cell volume homeostasis. An increase in cell volume activates outwardly rectifying chloride channel, which inactivate at positive membrane potentials. Exposure to hypotonic solutions is the most common technique used to swell cells and activate ICl.vol Current sensitive to SIDS and 9-AC. Exists also a basally active current Cl - Channels Regulated by Cell Volume (ICl.vol) Sorota, Circ res, 70, 679-687, 1992 Main anion channels and transporters Hume et al, Physiol Rev, 80, 31-81, 2000 -Receptors and KV Channels permeability for K+ agonist K+ Adenylate cyclase Out Gs PKA + ATP In + cAMP IKS Channel (KCNQ1+KCNE1) Muscarinic Receptors and Pacemaker Channels ACh Adenylate cyclase Out M2 Gi cAMP ATP cAMP In Pacemaker Channel (HCN4) -Receptors and Ca2+ Channels agonist Adenylate cyclase Out Gs + + + ATP cAMP PKA In Ca2+ L-type Ca2+ Channel Correspondence between cardiac ionic currents and channel proteins Computer modelling V (mV) 50 epi epi 8 0 % I to1 block endo endo 8 0 % I to1 block • • • 0 • -50 • -100 0 100 200 T im e (m s) 300 400 Decker 2009 canine model 1000 beats, 1 Hz "epi" is the standard model (which was built to represent epi data) "endo" has Gto reductions according to Liu, Gintant and Antzelevitch Circ Res 1992 (1/5th as much Ito and epi) "endo" has Gks reductions according to Liu & Antzelevitch Circ Res 1995 (11/35 as much Iks and epi) Control human and dog APs BCL = 1000 ms Human model is based on the dog model by scaling: factor_IK1 = 0.3779; %0.65 pA/pF vs 1.72pA/pF factor_Ito = 0.9*0.77; % dog: 0.9 x normal HR model factor_ICaL = 1.3; factor_IKr = 1; factor_IKs = 0.22; % dog = 4.5 x human human IK1 block (0.3xgK1) dog IKr block (0.25xgKr) human dog human IKs block (0.5 x gKs) dog human dog IKr + IKs human IKr + IK1 dog THANK YOU FOR YOUR ATTENTION !