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C. Frank Starmer Medical University of South Carolina

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  1. Making Complex Arrhythmias from Simple Mechanisms:Exploring Anti- and Proarrhythmic Effects of Na Channel Blockade with the Guarded Receptor Paradigm C. Frank Starmer Medical University of South Carolina

  2. LA RA RV LV 15.5 mm Mechanism of defibrillation failure (monophasic cathodal truncated exponential shock, -100 V, 8 ms) Dynamics of transmembrane potential tachycardia fibrillation shock

  3. How To Initiate Reentry or Fibrillation:The cardiac vulnerable period conduction refractory Partial Conduction (arrhythmia) Refractory: s1s2 = 2.1 Excitable: s1s2 = 2.3 Vulnerable: s1s2 = 2.2

  4. Ion Channel Blockade Reduces Excitability (Anti- effect) and Slows Conduction (Pro- effect)Historical observations that provided a foundation for a model of ion channel blockade: Johnson and McKinnon (1957) (memory) West and Amory (1960) (use-dependence) Armstrong (1967) (open channel block) Heistracher (1971) (frequency-dependence) Carmeliet (1988) (trapping)

  5. Steady-state Frequency-dependent AP Alterations: Quinidine dV/dt(max) decreases with increased stim rate AP amplitude decreases with increased stim rate Johnson and McKinnon JPET 460-468, 1957

  6. Freq-dependent Quinidine Block:Alteration of AP Duration Increased stim rate slows repolarization West and Amory: JPET 130:183-193,1960

  7. An Early Model of Use-dependent Blockade West and Amory: JPET 130:183-193,1960

  8. Frequency- as well as Use-dependence: Detailed Characterization of Ajmaline Blockade dV/dt(max) reduced with repeated stimulation: note approx exponential decrease with stimulation number Steady-state dV/dt(max) Reduced with faster stimulation Heistracher. Naunyn-Schmeideberg’s Archiv Fur Pharmakologie 269:199-213, 1971

  9. Voltage and Time-dependent TEA Block of K+ Channels Control: no “inactivation” + TEA: Apparent “inactivation” +90 mV IK CP -46 mV Armstrong. J. Gen Physiol 54:553-575, 1969

  10. Once a Drug Molecule Blocks the Channel, Can it Escape? i.e. is it possible to trap it in the channel

  11. Is use-dependent channel blockade a “special” process or is it simply a variant of ordinary ligand-receptor interactions?If it’s a variant - what variant? From These Observations, One Wonders:

  12. Ordinary (not use-dependent) Chemistry:Reacting with a Continuously Accessible SiteNo possibility of use- or frequency dependence a Ligand + Receptor LR-Complex b b = a/(a + b) l = a + b b(t) = b + (b0 - b) e-l t (b- b0)/2 = Kd = b/a

  13. How to Build a Model that Displaysuse- and frequency dependence? a(V) Unblocked + Drug Blocked b(V) A necessary condition: Either a Real or Apparent Voltage-dependent Equilibrium Dissociation Constant: Kd = b(V) / a(V)

  14. Modeling Apparent Voltage Dependence Of the Equilibrium Dissociation Constant Voltage-dependent Access to the Binding Site (a+b)/b kD Inaccessible Blocked l

  15. Hypothesis: Control of Binding Site Access by Channel Conformation accessible inaccessible

  16. Blockade During Accessible and Inaccessible Intervals: Accessible Conformation Inaccessible Conformation a Channel + D Blocked Channel + D Blocked b b

  17. Characterization of Access Control:Guarded Receptor Model (when channel transition time << drug binding time) G*k Unblocked Channel + Drug Blocked Channel where G and T act as “switches” that control binding site accessibility T*l G = “guard function” controls drug ingress: e.g. h, m, m3h, d, n, n4 T = “trap function” controls drug egress: e.g. m3h, h In reality, the guard and trap functions are hypothesized to reflect specific channel protein conformations, and not arbitrary model parameters Starmer, Grant, Strauss. Biophys J 46:15-27, 1984 Starmer and Grant. Mol Pharm 28:348-356,1985 Starmer. Biometry 44:549-559, 1989

  18. Combining Gated Access with Repetitive Stimulation makes Use-dependent Blockade:Switched Accessibility to a Binding Site Starmer and Grant. Mol. Pharm 28:348-356, 1985 ta tr b(t) = b - (b0 - b) e-(k + l)*t bactivated = ass - (a0 - ass) e-l*n brecov = rss - (b0 - rss) e-l*n la l = la ta + lr tr lr U B U B

  19. Dissecting the Mechanism of Use-Dependent Blockade: Using Voltage Clamp Protocols to Amplify or Attenuate Blockade

  20. Continuous Access Associated with Channel Inactivation (shift in “apparent” h) (1-h)a Unblocked + Drug Blocked b V(cond) block Starmer et. al. Amer. J Physiol 259:H626-H634, 1990

  21. Transient Access Associated with Channel OpeningPulse duration: 2 ms 2 ms 150 350 ms 550 Gilliam et al Circ Res 65:723-739, 1989

  22. Shift in Apparent Activation:Evidence of Open (?) Channel Access Control 10 ms Starmer et. Al. J. Mol Cell Cardiol 23:73-83, 1991

  23. Exploring a Model of Use-Dependent Blockade Analytical Description: block associated with the nth pulse: bn = bss + (b0 - bss) e -(la ta + lr tr)n Are the Analytical Predictions Testable? Use-dependent rate: l = la ta + lr tr Steady-state block: bss = a + g (r + a) Steady-state slope: g = (1 - e-lr tr) / (1 - e-l )

  24. Testing the Model • Pulse-train stimulation evokes an exponential pattern of use-dependent block • There is a linear relation between exponential rate and stimulus recovery interval • There is a linear relation between steady-state block and a function of the recovery interval (g) • There is a shift in the midpoint of channelavailability and /or activation (depending on the access control mechanism)

  25. Test 1. Frequency-dependent Lidocaine Uptake:Exponential Pulse-to-pulse Blockade (50 ms) .65 .35 .15 Gilliam et al Circ Res 65:723-739, 1989

  26. Test 2: Linear Uptake Rate, Linear Steady State Block ta constant and tr variable Linear Uptake Rate l = la ta + lr tr Linear Steady-State Block bss = a + g(r- a)

  27. Test 3: Shifting Apparent Inactivation (channel availability) (1-h)a Unblocked + Drug Blocked b K = 3940 /M/s l = .678 /s KD = 18.8 mM = 10.76 mV DV = s ln(1 + D/KD) Obs DV = 9 mV

  28. Test 4: Shifting Apparent Channel ActivationNimodipine Blockade of Ca++ Channels da Unblocked + Drug Blocked b KD = .38 nM DV = 40.1 mV DV = k (1 + D/KD) = 43.4 mV

  29. Exploiting the “Therapeutic” Potential of Use-dependent BlockadeCellular Antiarrhythmic ResponseMulticellular Proarrhythmic Response

  30. Therapeutic Potential: Cellular Effects of Blockade (Antiarrhythmic)Prolonging Recovery of Excitability:Control and with Use-dependent Blockade

  31. Therapeutic Potential: Multicellular Effects of Blockade(Proarrhythmic)Slowed Conduction, Increased Vulnerable Period Why? Propagation: Responses to Excitation 1) no response 2) front propagates away from stimulation site 3) front propagates in some directions and fails to propagate in other direction (proarrhythmic)

  32. Premature Excitation:The Vulnerable Period • Normal excitation: cells are in the rest state • Premature excitation: Following a propagating wave is a refractory region that recovers to the resting state. Stimulation in the transition region can be proarrhythmic

  33. The Dynamics of Vulnerability Using a simple 2 current model (Na: inward; K: outward) we can demonstrate role of introducing a stimulus within and outside the interval of vulnerability: We demonstrate the paradox of channel blockade: block extends the refractory period, slows conduction and increases the VP Here, we switch to Matlab, to demonstrate the dynamic events defining the Vulnerable Period

  34. Demonstrating the Vulnerable Period: ControlRefractory Period = 352 ms VP = 3 ms

  35. Demonstrating Extension of the VP: DrugRefractory Period = 668 ms VP = 59 ms

  36. Use-dependent Extension of the VP

  37. 2-D Responses to Premature Excitation:Note geometric distance between 1st and 2nd fronts(refractory, unidirectional conduction, bidirectional conduction) conduction refractory unidirectional conduction Refractory: s1s2 = 2.1 Excitable: s1s2 = 2.3 Vulnerable: s1s2 = 2.2

  38. Extending the VP with Na Channel Block:Fact or Fantasy? Starmer et. al. Amer. J. Physiol 262:H1305-1310, 1992

  39. More Apparent Complexity: Monomorphic and Polymorphic Reentry and ECG Monomorphic Polymorphic gna = 2.3 Polymorphic gNa = 2.25 gNa = 4.5

  40. Major Lessons Learned FromIdeas Originating in Studies of Johnson, Heistracher and Carmaliet

  41. Antiarrhythmic Extended refractory interval and reduced excitability leading to PVC suppression Proarrhythmic Extends the vulnerable period (increases the probability of a PVC initiating reentry) Slowed conduction increase the probability of sustained reentry Increases probability of wavefront fractionation Use caution when “repairing” channels that aren’t broken:Blockade of normal Na Channels

  42. Repairing Channels that are Broken (e.g. SCN5A) may have Clinical Utility:Blockade of “defective” channels diminishes EADs in LQT Syndrome, Heart Failure, Epilepsy

  43. Long QT Syndrome:Links to Mutant Na and K Channels Q T

  44. Stable and Unstable Action Potentials Human Ventricular Cells Beeler-Reuter Model

  45. Yet Another Variant: Epilepsy

  46. Summary • Use- and Frequency Na channel block are consistent with “ordinary” binding to a periodically accessible site • Tonic block is compatible with block of inactivated channels at the rest potential. • Tests are available to validate the applicability of the guarded-receptor paradigm to observations of drug-channel interactions

  47. For individual cells: use-dependent Na channel block reduces excitability (prolongs the refractory period (antiarrhythmic effect) • For connected cells (tissue): reduced excitability ALSO slows propagation which extends the vulnerable period (proarrhythmic effect) • The guarded receptor paradigm is a tool for “in numero” exploration of channel blockade in both cellular and multicellular preparations and direct characterization of anti- and proarrhythmic effects

  48. Apparent Trapping of Quinidine and Disopyramide 100 uM Diso 5 uM Quinidine Zilberter et. Al. Amer. J. Physiol 266:H2007-H2017, 1994