THE AUSTRALIAN NATIONAL UNIVERSITY

1. THE AUSTRALIAN NATIONAL UNIVERSITY The Cardiac ActionpotentialChristian StrickerAssociate Professor for Systems PhysiologyANUMS/JCSMR - ANUChristian.Stricker@anu.edu.auhttp://stricker.jcsmr.anu.edu.au/Cardiac_Actionpotential.pptx

3. Cardiovascular Part in Block 2 • Week 1 • Cardiac Action Potential • Modulation of the Action Potential • Basis of the ECG • Week 2 • Pressures, Flows and Volumes during the Cardiac Cycle • Practical: ECG • CO as HR·SV and Introduction to Starling's Law • Cardiac Work and Coupling of Venous Return and CO • Regulation of Blood Pressure. • Week 3 • Week 4 • Neural and Humoral Regulation of Blood Pressure • PV Loops and Principles of Left and Right Heart Failure • Vessels of the Systemic Circulation • Week 5 • Special Circulations (coronary, etc.) • Practical: Cardiovascular Simulations (in week 6)

4. Aims The students should • know the cellular specialisations supporting AP spread; • distinguish the AP phases in cardiac myocytes and ICS; • be aware of important in- and outward currents, how they contribute to cardiac AP and what their properties are; • be cognisant of determinants of the refractory period; • recognise determinants of AP propagation in the tissue; • be able to argue why there is a hierarchy of pacemakers; • appreciate the different AP shapes and repolarisations; and • be cognisant of drugs that target ion channels involved in AP.

5. Contents • Two types of cells in heart • Relevant anatomy incl. histological specialisations • Myocytes – coupled via gap junctions forming a functional syncytium • Cardiac impulse conduction system (ICS) • Cardiac myocyte • RMP, AP and currents involved 5 phases and determinants, relationship to contraction, refractory periods, determinants of action potential propagation • Cardiac ICS • RMP, AP and currents involved in ICS 3 phases and determinants, pacemaker currents incl. If and ICaTdeterminants of action potential propagation • AP and drug targets

6. Cardiac Specialisations • Cardiac myocytes • Intercalated discs • Desmosomes • complexes of cell adhesion proteins and linking proteins • Gap junctions: connexins • “electrical syncytium” • Impulse conduction system (ICS) • Specialised cells expressing different ion channels and amounts of contractile protein • Sinoatrial node (SA) • Round cells: pacemakers • Elongated cells: conductors • Atrioventricular node (AV) • Purkinje fibres

7. I. AP in the Cardiac Myocyte

8. RMP and AP of Cardiac Myocyte Draper & Weidmann (1951), J Physiol 115:74-94 • Ventricular myocyte (~80 BPM) • RMP: ~ -90 mV, ± constant! • AP peak: 20 – 30 mV; amplit.: ~120 mV. • Duration: 300 – 450 ms → 200 x longer than neuronal AP! • Shape depends on location in heart. • Net inward currents depolarise. • On: Na, Ca channels (voltage-gated). • Off: Some K channels • Net outward currents hyperpolarise. • On: various different K channels (voltage-dep., G protein coupled and ATP gated). • Off: Na, Ca channels • 5 Phases of AP (0 – 3 ± fixed dur.) 0. Fast depolarisation: Na+ “overshoot” • Early (partial) repolarisation (fast) • Plateau (Ca2+ shoulder) • Final repolarisation (slow) • Restoration of RMP: “diastole”, variable in duration

9. Currents in Different Phases • Phase 0 • Threshold @ -60 – 70 mV (negative!) • Activation of fast INa; deactivation of IK1 • Overshoot determined by [Na+] • Phase 1 • Inactivation of fast INa (NaV 1.5) • Activation of K channels: Ito,f&s • INCX(Na-Ca exchange – early hyperpolarising) • Phase 2 • ICaL(L-type, blocked by nifedipine) • INCX(Na-Ca exchange – late depolarising) • Phase 3 • Deactivation of ICaL • Activation of IK (del. rectifier (DR), IKs, IKr) • Re-activation of INaand ICaL • Phase 4 • Activation of fast IK1(inward rectifier, IR) • Re-establishes RMP Modified from Rhoades & Bell (2009), 3rd Ed.

10. Relationship to Contraction • AP peaks before Ca2+rise. • Peak of the Ca2+ signal during phase 2. • ICaL. • INCX(aids in Ca2+ influx early – reversal as Na+↑, but later Ca2+efflux - lowers). • Twitch peaks when Ca2+ is already decaying. • Highly efficient Ca2+extrusion mechanisms. • Excitation-contraction coupling. Spurgeon et al. (1990), Am J Physiol 258:H574

11. Refractory Periods in Myocyte • Absolute refractory period (ARP): Very few Na+ channels can be reactivated above Vm > -50 mV. • Effective refractory period (ERP): only after this, myocytes nearby can be activated as earlier current spread is too small for activation. • Relative refractory period (RRP): follows ARP; during this time no full AP can be generated (smaller amplitude and slower rise). • Full AP generated again after Vm hyperpolarised to ~ <-70 mV. Berne & Levy, 2008

12. Action Potential Propagation Modified from Raff & Levitzky (2011) • Passive spread of AP: “slow” – about 1 m/s • Depolarisation spreads passively, accelerated by gap junctions between cells (intercellularcurrent ahead). • Extracellular current spread (extracell. current back). • Propagation speed dependent on • Gap junction conductance: the larger – the faster. • Fibre diameter: the larger – the faster (Ri↓).

13. II. AP in the Cardiac ICS

14. RMP and AP in SAN and AVN • Atrial ICS • (Rabbit heart; ~ 96 BPM) • RMP: variable, and much more positive than in myocyte; when all conductances blocked, ~ -35 mV • Slow rising phase: Not INa • Peak amplitude: ~20 mV (depends on location within node; less positive in centre) • Duration: 200 – 250 ms • Threshold for AP: -50 – -40 mV • 3 Phases of AP (0 – 3 ± fixed; 4 var.) 0. Depolarisation (ICaLmuch slower) • Repolarisation (slow) • Variable RMP (linear increase in time to threshold).

15. Currents in Pacemaker Cells Kohnhardt et al. (1976), Basic Res Cardiol 71:17 • Phase 0 • Mostly Ca2+spike. • Amplitude is [Ca2+]-dependent. • ICaL(L-type) blocked by nifedipine; RMP). • Not much INa involved (blocked by TTX). • INa remains inactivated as hyperpolarisation not sufficiently big and long – it is there… • Phase 3 • Inactivation of ICa. • Activation of IK (delayed rectifier, DR). • Phase 4 (“Pacemaker potential”) • Early de-activation of fast IK (del. rectifier). • Turning off an outward is seen as net inward current. • Followed by opening of a set of channels that generate inward currents – predominantly carried by Na+(see next).

16. Pacemaker Currents/Potential • = decay of “RMP” until Ca2+spike. • Arises from a mixture of at least the following currents: • If - Current has “funny” properties – largest. • Hyperpolarisation-activated cation current (activated during phase 3). • Mixed cation current: largely Na+ inward current → depolarisation; slow activation / deactivation. • Erev = -20 – -10 mV. • Tetramericchannel, gated via cyclic nucleotides (cAMP/cGMP bind to intracellular tail). • cA/GMP↑: channel blocked and vice versa. • Current modulated fast withcA/GMP↓↑ (see next lecture). • 4 genes (HCN1 - 4). • In humans, mostly HCN4. • HCN4 viral transfection in dogs can restore “sick sinus” to normal pacing. • Blocked by ivabradine – but PMP not fully blocked. • ICaT – T-type Ca2+ current > -55 mV (late 4). • There is small amount of L-type current present, too. • INCX – in reverse mode (3 Na+ against 1 Ca2+) as Na+ enters via HCN channels. • Likely other Na+ conductance(s) involved. • IbNa (leak?, TRPM4, NaV1.5; see next lecture). Berne & Levy, 2008

17. Hierarchy of Pacemakers • Normally, 3-tiered system for pacing • SA node: 60 – 100 BPM • Natural rate around 100 BPM • Slower due to ANS (parasympathetic innervation). • AV node: 40 – 60 BPM • Smaller If → slower depolarisation → HR↓. • Is typically subservient to SA node; takes over if not paced in time (supraventricular pacing). • Delay of ~160 ms: gap junction coupling↓ and small cells (delay line): impulse propagationslow. • Purkinje cells: 20 – 40 BPM • Even slower Ifthan AV node; plus • RMP much more hyperpolarised (-90 mV). • Cells have Na+ current in them (reactivated…). • Cells with highest AP rate set heart rate as all other ICS cells are depolarised by them and functionally rendered “inactive”.

18. Spread of Excitation Rhoades & Bell (2009), 3rd Ed. • Atria excited within 80 – 90 ms (right earlier than left). • First excitation seen with 140 ms delay in top septal areas: AV delay. • Ventricles fully excited within 50 – 60 ms; right slightly earlier than left. • Compared to atria, speedup due to larger and better coupled Purkinje fibres generating bigger currents → faster depolarisation.

19. AP Time-Courses in Heart Modified from Barrett et al. (2010), 23rd Ed. • Purkinje cells have longest AP: prevents ventric. arrhythmia. • Longest APs are subendocardial, shortest subepicardial. • Last ventricular cells to depolarise are the first to repolarise. • Mechanism: ? – gradients of channel expression

20. III. Drug targets in AP

21. Currents as Therapeutic Targets • Several currents involved in AP generation are targeted via drugs used in clinical settings. • Some drugs used for cardioversion may target several currents unspecifically.

22. Take-Home Messages • Gap junctions are instrumental in spreading APs. • Cardiac myocyte and ICS have different ionic currents. • AP in myocytes has 5 phases during which specific currents are activated/inactivated. • AP propagation speeds up in larger fibres and when gap junction conductance is large. • Refractory period is determined by extent of repolari-sation (re-activation of Na+ channels). • Pacemaker current is largely carried by HCN channels with no NaV current. • There is a 3-tiered hierarchy of pacemakers running at different frequencies: SAN > AVN > Purkinje fibres. • A multitude of drugs is used that affect AP.

23. MCQ Joe Parsons, a 23 year-old very fit medical student participates in a study by a drug company, which aims to block HCN channels. Which of the following statements best describes the expected effect of an HCN block on the heart? • Drop in heart rate (bradycardia). • Lengthening of the actionpotential in Purkinje cells. • Decreased depolarisation rate in atrio-ventricular cells. • Early delayed rectifier activation in sino-atrial node cells. • Increased T-type calcium current in sino-atrial node cells.

24. That’s it folks…

25. MCQ Joe Parsons, a 23 year-old very fit medical student participates in a study by a drug company, which aims to block HCN channels. Which of the following statements best describes the expected effect of an HCN block on the heart? • Drop in heart rate (bradycardia). • Lengthening of the actionpotential in Purkinje cells. • Decreased depolarisation rate in atrio-ventricular cells. • Early delayed rectifier activation in sino-atrial node cells. • Increased T-type calcium current in sino-atrial node cells.