Calsequestrin mutation and catecholaminergic polymorphic ventricular tachycardia: a simulation study of cellular mechanism

Abstract

Objectives: Patients with a missense mutation of the calsequestrin 2 gene (CASQ2) are at risk for catecholaminergic polymorphic ventricular tachycardia. This mutation (CASQ2(D307H)) results in decreased ability of CASQ2 to bind Ca2+ in the sarcoplasmic reticulum (SR). In this theoretical study, we investigate a potential mechanism by which CASQ2(D307H) manifests its pro-arrhythmic consequences in patients.

Methods: Using simulations in a model of the guinea pig ventricular myocyte, we investigate the mutation's effect on SR Ca2+ storage, the Ca2+ transient (CaT), and its indirect effect on ionic currents and membrane potential. We model the effects of isoproterenol (ISO) on Ca(V)1.2 (the L-type Ca2+ current, I(Ca(L))) and other targets of beta-adrenergic stimulation.

Results: ISO increases I(Ca(L)), prolonging action potential (AP) duration (Control: 172 ms, +ISO: 207 ms, at cycle length of 1500 ms) and increasing CaT (Control: 0.79 microM, +ISO: 1.61 microM). ISO increases I(Ca(L)) by reducing the fraction of channels which undergo voltage-dependent inactivation and increasing transitions from a non-conducting to conducting mode of channel gating. CASQ2(D307H) reduces SR storage capacity, thereby reducing the magnitude of CaT (Control: 0.79 microM, CASQ2(D307H): 0.52 microM, at cycle length of 1500 ms). The combined effect of CASQ2(D307H) and ISO elevates SR free Ca2+ at a rapid rate, leading to store-overload-induced Ca2+ release and delayed afterdepolarization (DAD). If resting membrane potential is sufficiently elevated, the Na+-Ca2+ exchange-driven DAD can trigger I(Na) and I(Ca(L)) activation, generating a triggered arrhythmogenic AP.

Conclusions: The CASQ2(D307H) mutation manifests its pro-arrhythmic consequences due to store-overload-induced Ca2+ release and DAD formation due to excess free SR Ca2+ following rapid pacing and beta-adrenergic stimulation.

FIGURE 1

(A) Schematic of the five compartment myocyte model (bulk myoplasm, JSR – junctional SR, NSR – network SR, mito – mitochondria, and t-tubular subspace). (B) State diagram of the L-type Ca²⁺ channel Markov model. The conducting mode, ModeV (grey) consists of four closed states (C₀, C₁, C₂, and C₃), a single conducting state (O), an inactivation state into which channels move rapidly (I_Vf) following depolarization, and an inactivation state into which channels enter more slowly (I_Vs). ModeCa (black) is a non-conducting mode representing channels which have inactivated due to Ca²⁺. (C) Modal transitions of the Ca_V1.2 Markov model. From any of the states in ModeV, channels may inactivate via CDI (shown as a transition from ModeV to ModeCa). Channels can also transition into Mode0, a non-conducting mode that serves as a reserve of channels that are activated in the presence of ISO. Details are in the reference [8] and Online Supplement.

FIGURE 2

(A) I_Ca(L) steady-state inactivation curves during control conditions. Simulations (line traces) are compared to experimental data from Findlay (symbols) [10] for conditions where Ca²⁺ is not the charge carrier (□) (VDI) and where Ca²⁺ is the charge carrier (■) (VDI + CDI). (B) Ca_V1.2 steady state inactivation curves following the application of 0.1 μM isoproternol. As in Panel A, the line traces are simulated curves and the symbols are experimental data [10] for conditions where Ca²⁺ is not the charge carrier (△) and where Ca²⁺ is the charge carrier (▲). The protocol for both Panels A and B is shown in the inset of Panel A. The experimental data were measured at room temperature and the simulations conducted at 37°C; times shown in the inset are the experimental times adjusted to 37°C utilizing a Q₁₀=2. In the experiments, the non-Ca²⁺ charge carrier was Mg²⁺, which we simulate by eliminating CDI in the model. The experimental data were recorded with ryanodine in the solution, simulated here by setting G_rel = 0. (C) Ca_V1.2 single channel traces generated using the model in the absence of CDI. The voltage is clamped to 20 mV from a holding potential of −100 mV for a duration of 150 ms every 600 ms. Left column: model results during control conditions and right column: model results following application of ISO. The ensemble (whole cell) current is shown below the single channel traces. (D) Experimentally measured single channel traces [12] generated utilizing the same protocol as Panel A. The ensemble current is shown below the single channel traces.

FIGURE 3

State residencies of the L-type Ca²⁺ channel during the time course of the action potential at CL=1500 ms in the absence of ISO (black curve) and in the presence of ISO (grey curve). For identification of the channel states, refer to Fig. 1B.

FIGURE 4

Top to bottom: Action potential (AP), calcium transient ([Ca²⁺]_i), L-type Ca²⁺ current (I_Ca(L)), Na⁺-Ca²⁺ exchange current (I_NaCa), and free JSR Ca²⁺ ([Ca²⁺]_JSR) for combinations of six different protocols: 1) slow pacing (CL=1500 ms) - Panels A and B. 2) fast pacing (CL=500 ms) – Panels C and D. 3) Wild type (WT) – non-shaded columns. 4) CASQ2^D307H – shaded columns. 5) Without Isoproterenol (−ISO) – black traces. 6) With Isoproterenol (+ISO) – grey traces. In all panels, the last paced beat is shown after pacing for 5 minutes at the indicated cycle length followed by a period where no stimulus is applied. Note that only the combination of fast rate, CASQ2^D307H mutation, and +ISO results in generation of delayed afterdepolarization (DAD) after cessation of pacing (arrow in Panel D). The DAD is generated when [Ca²⁺]_JSR reaches a threshold concentration, which results in opening of RyRs and spontaneous Ca²⁺ release. The resulting rise in [Ca²⁺]_i leads to increased depolarizing I_NaCa current as it removes the excess Ca²⁺.

FIGURE 5

DAD that triggers a spontaneous AP. Conditions are the same as Fig. 4D that resulted in DAD formation (CL=500 ms, +CASQ2^D307H mutation, +ISO) with additional reduction of I_K1. The arrow in Panel A indicates the last paced beat which is followed by a DAD that generates a spontaneous AP. Due to the elevated RMP and increased excitability (due to smaller I_K1), the depolarization is large enough to trigger activation of both I_Na (Panel E) and I_Ca(L) (Panel C) leading to the generation of an AP. A second spontaneous Ca²⁺ release event also occurs, but does not generate sufficient depolarizing current to generate a triggered AP.

FIGURE 6

Pause induced early afterdepolarization (EAD). The myocyte is paced for 40 beats at CL=500 ms (S1) followed by a 1000 ms pause before the next stimulus (S2). Panel A: Last paced and the post-pause APs; arrows indicate time of stimuli. Thin black trace shows control AP (pre-pause APD₉₀=151 ms, post-pause APD₉₀=168 ms); thick grey trace shows AP for 55% reduction of I_Kr and I_Ks (pre-pause APD₉₀=243 ms, post-pause AP develops EAD and APD₉₀=424 ms); dashed trace shows that the EAD is abolished by resetting I_Ks to its value at S1, thus preventing additional I_Ks deactivation during the long pause (pre-pause APD₉₀=243 ms, post-pause APD₉₀=229 ms). Panel B: Corresponding [Ca²⁺]_i. Panels C, D, and E: I_Ca(L), I_Kr, and I_Ks, respectively.