Contents

📒 Yang 2016

In silico prediction of drug therapy in catecholaminergic polymorphic ventricular tachycardia1

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Introduction

catecholaminergic polymorphic ventricular tachycardia (CPVT)

  • Mutation of RyR2 (inherited) and / or calsequestrin (CASQ2)
  • arrhythmic episodes during excitement (catecholaminergic surge)
  • 1st line treatment: beta-blockers
  • 30% of patients with limited improvements (still arrhythmic), requiring ICD implantation

Methods

Animal Experiment

  • six R176Q/+ and two wild‐type (WT) mice with ECG telemetry

Molecular dynamics and free energy simulations

  • Force‐field parameters for flecainide: general automated atomic model parameterization (GAAMP)

Computational ventricular myocyte models

  • mouse and rabbit in silico models, ‘knocking out’ the SR Ca2+ buffer calsequestrin
  • Morotti–Grandi mouse cardiac cell model2 and Soltis–Saucerman rabbit cardiac cell model
  • cardiac Na+ channel and I Kr and their interaction with flecainide
  • with cellular electrophysiology, Ca2+ handling, PKA, CaMKII

Wild‐type Na+ channel models and inclusion of drug binding

  • flecainide, ranolazine and lidocaine
  • Rates were also constrained by experimental data and microscopic reversibility Table1
Flecainide
  • drug binding to red states https://wol-prod-cdn.literatumonline.com/cms/attachment/bdcee2fb-c358-4499-ba9d-a5990e01d28e/tjp6929-gra-0001.png
  • Table2
Ranolazine
Lidocaine

RyR2–flecainide drug‐channel interaction model development

  • Shannon–Bers Markov model formulation of the RyR2 with drug bound state DO (open-state block) https://wol-prod-cdn.literatumonline.com/cms/attachment/02cf70fd-0a96-4150-9945-f90369e4fac9/tjp6929-gra-0002.png

  • Testing IC50 of flecainide https://wol-prod-cdn.literatumonline.com/cms/attachment/2bed5f04-0365-4f25-a0d7-836a1ddb9bca/tjp6929-fig-0001-m.jpg

  • equimolar concentrations of flecainide inside and outside of the cell

  • reduction to 20% maximal conductance of the channel

  • IC50 of 16 μM in single channel experiment, but in the model and reality, 2 μM IC50 is used in the simulation since multiple RyRs are coupled and Ca transient are different.

  • IC50 for wave inhibition in the isoproterenol‐stimulated Ca2+ waves in CASQ2(−/−) mice (IC50 = 2.0 ± 0.2 μM; Hwang et al. 2011), was used

https://user-images.githubusercontent.com/40054455/86726451-27c5fe00-c05d-11ea-8cee-cc7eac709fd5.PNG Table5

Simulation of I Kr blockade

$$ G_{\mathrm{IKr}}=G_{\mathrm{IKr}, \max } \times\left(\frac{1}{1+\left([\operatorname{Drug}] / \mathrm{IC}_{50}\right)^{n}}\right) $$

Where n = 1, flecainide 1.5 μM, ranolazine 12 μM

Simulation of effect of metoprolol on β‐adrenergic activity and CaMKII blocking effects of KN‐62

https://user-images.githubusercontent.com/40054455/86726458-298fc180-c05d-11ea-8696-e7110796031d.PNG

  • metoprolol (IC50 = 105 nM); KN‐62 (IC50 = 468 nM)

https://wol-prod-cdn.literatumonline.com/cms/attachment/76a93ca7-ae1c-459f-9046-75b9e75e6cd8/tjp6929-fig-0002-m.jpg

CPVT CASQ2(−/−) model development

  • set the calsequestrin buffer concentration and the time derivative of the calsequestrin buffer concentration to 0
  • regulation of Na+/K+‐ATPase activity by PKA as mediated by phospholemman were incorporated from the Yang–Saucerman model
  • previously validated Na+ channel (Moreno et al. 2011) and modified RyR2 channel were inserted into the cellular model (Soltis & Saucerman, 2010).

Stochastic single channel RyR2 simulations

  • modelled stochastically in Monte Carlo simulations using Gillespie’s algorithm
  • Parameters were constrained by fixed concentrations of flecainide (0 and 50 μM), luminal Ca2+ (1 mM), and junctional Ca2+ (0.1 μM), mimicking experimental conditions
  • approaching the expected mean open time (T o), mean closed time (T c), and open probability (P o)

Cellular and tissue simulations

Mouse ventricular myocyte model
  • INa channel was replaced by a new Markov model and adjusted three transition rates for the mouse model
  • −9.5 pA pF−1 current stimulus for 5 ms in single cells for 150 s at 2 Hz pacing frequency
Rabbit ventricular myocyte model
  • Cells were virtually paced (using a −80 pA pF−1 current stimulus for 0.5 ms in single cells and −500 pA pF−1 stimulus for 2.0 ms in tissues) after ‘resting’ for 10 mins
1D tissue sims
  • a fibre of 165 cells (1.65 cm) with reflective boundary conditions
  • Transmural heterogeneity was incorporated into the tissue by a linear decrease to 25% maximal I to conductance
  • The diffusion coefficient Dx was set to 0.002 cm2 ms−1 to establish a conduction velocity of 61–73 cm s−1

Results

https://wol-prod-cdn.literatumonline.com/cms/attachment/e5311ad8-3d35-444d-a61e-a798ea9762c2/tjp6929-fig-0003-m.jpg
Response single ryanodine receptors to flecainide
A: EXP vs sim B: open probability (P o, left), mean open time (middle, T o), and mean closed time (T c)

https://wol-prod-cdn.literatumonline.com/cms/attachment/6e399f2d-b1bb-4591-9c5e-1117867c9df7/tjp6929-fig-0004-m.jpg
An in silico calsequestrin (CASQ2(−/−)) knockout model recapitulates the CPVT phenotype

  • Upper half: mouse; lower half rabbit
  • virtual calsequestrin rabbit knockout for cross species testing (small animals vs large animals)
  • application of 1 μM ISO induces spontaneous Ca2+ release events, triggering APs

https://wol-prod-cdn.literatumonline.com/cms/attachment/20ecb486-9e06-475c-9b89-c9eb35e19492/tjp6929-fig-0005-m.jpg
Atomic scale prediction of flecainide transport across the membrane

  • With a pK a value of 9.8, 97% of flecainide will exist in the protonated form at physiological pH
  • hydrophobic environment of the lipid bilayers promotes stabilization of the neutral form, allowing for flecainide entry upon deprotonation
  • potentials of mean force (ΔPMF (Z)) and free energies for charged and neutral flecainide (ΔΔG A)

https://wol-prod-cdn.literatumonline.com/cms/attachment/402d8de0-a01a-4cd4-8ec9-1d21eab2a83a/tjp6929-fig-0006-m.jpg
Model prediction of flecainide mechanism

https://wol-prod-cdn.literatumonline.com/cms/attachment/0cb623c4-304a-49f0-ba8e-3bbbb1efff35/tjp6929-fig-0007-m.jpg
Full parameter space of flecainide efficacy for INa, RyR2 and both targets in combination

https://wol-prod-cdn.literatumonline.com/cms/attachment/d135d5b7-c8b2-4c25-b999-f9a732bad0ae/tjp6929-fig-0008-m.jpg
Simulation of the physiological effects of isoproterenol on heart rate

https://wol-prod-cdn.literatumonline.com/cms/attachment/0d147dc0-f7c5-46ba-8bc3-199b1844da5f/tjp6929-fig-0009-m.jpg
Space–time representations of one‐dimensional simulations of flecainide effects on CPVT at BCL 500ms

  • 2 μM flecainide acting on the RyR2 alone was sufficient for therapeutic suppression of DADs and triggered APs
  • 2 μM flecainide acting on I Na (e.g. Fig. 9 B and C) slowed tissue conduction velocity from 66 to 44 cm s−1 as compared with 2 μM flecainide acting on RyR2 alone

https://wol-prod-cdn.literatumonline.com/cms/attachment/a8f7727d-b3e6-4c58-b17a-7038cb68af63/tjp6929-fig-0010-m.jpg
Simulated effects of low clinical dose flecainide effect on RyR only in CPVT one‐dimensional tissue

https://wol-prod-cdn.literatumonline.com/cms/attachment/458016f9-04fd-4156-82b0-a59e638f25d5/tjp6929-fig-0011-m.jpg
Simulated high clinically relevant doses of lidocaine and ranolazine in CPVT myocytes

https://wol-prod-cdn.literatumonline.com/cms/attachment/554d8c15-8559-4647-a268-09baa0cd46e2/tjp6929-fig-0012-m.jpg
Predicted low‐dose polytherapy for CPVT

  • multidrug combination of flecainide, β‐blockade, and CaMKII inhibition would demonstrate efficacy in CPVT

https://user-images.githubusercontent.com/40054455/86726465-2a285800-c05d-11ea-832e-a7c7325d86ce.PNG
simulated our drug combination of 0.5 μM flecainide, 70 nM metoprolol and 312 nM KN‐62 in CASQ2(−/−) in 1D tissue

  • Better conduction velocity for multidrug regime (less flecainide required)

https://wol-prod-cdn.literatumonline.com/cms/attachment/9b3368bb-a35b-4413-b3c6-0a6529f93b14/tjp6929-fig-0013-m.jpg
Simulated drug effects in mouse ventricular myocytes

  • drug combination of 0.5 μM flecainide, 70 nM metoprolol and 312 nM KN‐62 in the mouse CASQ2(−/−) virtual myocyte completely normalized the cellular phenotype.

https://wol-prod-cdn.literatumonline.com/cms/attachment/43a29d93-0ecf-4995-8b3e-d119771d44e8/tjp6929-fig-0014-m.jpg
Experimental test of predicted polytherapy (in vivo mouse model)
https://user-images.githubusercontent.com/40054455/86726469-2ac0ee80-c05d-11ea-9811-f2d03d8ed5f1.PNG https://user-images.githubusercontent.com/40054455/86726473-2b598500-c05d-11ea-9094-f9dcefcd1da8.PNG

Discussion

  • Optimal pharmacological management of CPVT remains a clinical challenge (high failuer rates for current treatment)
  • Physics‐based molecular dynamics approach to simulate the partitioning of flecainide by computing free energy profiles for flecainide transport in the extracellular, membrane and intracellular compartments. This process is extremely rapid, occurring with a diffusion coefficient of ∼10−7 cm s−1, only an order of magnitude lower than in the bulk water.
  • Virtual mouse and rabbit CASQ2(−/−) cells with β‐agonist reproduced the CVPT phenotype
  • The absence of calsequestrin as a buffer speeds up the dynamics of SR refilling, providing more interaction time for SR Ca2+ to activate the RyR2, and results in functionally upregulated RyR2 activity
  • Diastolic intervals in the rabbit are longer than in the mouse, and the model predicted that they could allow sufficient time to promote spontaneous diastolic release and triggered activity
  • The model predicted that Na+ channel blockade – even at the high clinical doses – was not sufficient to explain the antiarrhythmic effects seen clinically with flecainide
  • Simulations with lidocaine (Moreno et al. 2011) as an additional control of pure Na+ channel blockade resulted in good concordance with experimental results. Na+ channel blockade is insufficient to resolve CPVT arrhythmia triggers
  • All three drugs achieved better efficacy when used in combination with either or both of the other drugs tested. efficacy at lower drug concentrations potentially limits the danger of off‐target and drug‐related side‐effects and are consistent with clinical reports

Limitations

  • Different CPVT mutations in vivo: ultrastructure, protein expression, RyR2, Ca2+ dynamics
  • No explicitly subcellular Ca2+ dynamics (subcellular Ca2+ waves)

Reference


  1. Yang P-C, Moreno JD, Miyake CY, et al. In silico prediction of drug therapy in catecholaminergic polymorphic ventricular tachycardia. J Physiol (Lond) 2016;594(3):567-593. doi:10.1113/JP271282. PMC4784170 ↩︎

  2. Morotti S, Edwards AG, McCulloch AD, Bers DM, Grandi E. A novel computational model of mouse myocyte electrophysiology to assess the synergy between Na+ loading and CaMKII. J Physiol. 2014;592(6):1181-97. PMC3961080 ↩︎