The Action Potential Clamp Technique as a Tool for Risk Stratification of Sinus Bradycardia Due to Loss-of-Function Mutations in HCN4: An In Silico Exploration Based on In Vitro and In Vivo Data

Abstract

These days, in vitro functional analysis of gene variants is becoming increasingly important for risk stratification of cardiac ion channelopathies. So far, such risk stratification has been applied to SCN5A, KCNQ1, and KCNH2 gene variants associated with Brugada syndrome and long QT syndrome types 1 and 2, respectively, but risk stratification of HCN4 gene variants related to sick sinus syndrome has not yet been performed. HCN4 is the gene responsible for the hyperpolarization-activated 'funny' current I_f, which is an important modulator of the spontaneous diastolic depolarization underlying the sinus node pacemaker activity. In the present study, we carried out a risk classification assay on those loss-of-function mutations in HCN4 for which in vivo as well as in vitro data have been published. We used the in vitro data to compute the charge carried by I_f (Q_f) during the diastolic depolarization phase of a prerecorded human sinus node action potential waveform and assessed the extent to which this Q_f predicts (1) the beating rate of the comprehensive Fabbri-Severi model of a human sinus node cell with mutation-induced changes in I_f and (2) the heart rate observed in patients carrying the associated mutation in HCN4. The beating rate of the model cell showed a very strong correlation with Q_f from the simulated action potential clamp experiments (R² = 0.95 under vagal tone). The clinically observed minimum or resting heart rates showed a strong correlation with Q_f (R² = 0.73 and R² = 0.71, respectively). While a translational perspective remains to be seen, we conclude that action potential clamp on transfected cells, without the need for further voltage clamp experiments and data analysis to determine individual biophysical parameters of I_f, is a promising tool for risk stratification of sinus bradycardia due to loss-of-function mutations in HCN4. In combination with an I_f blocker, this tool may also prove useful when applied to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from mutation carriers and non-carriers.

Keywords: HCN4 channels; action potential; cellular electrophysiology; computer simulations; human; hyperpolarization-activated current; pacemaker activity; patch clamp; sinoatrial node.

Figure 1

Schematic topology of the HCN4 protein and the set of loss-of-function mutations in HCN4 associated with familial sinus bradycardia for which both clinical and in vitro data were available, requiring that these clinical data include quantitative heart rate data from at least two mutation carriers. Tetramers of HCN4 α-subunits form the cardiac ion channels that conduct the hyperpolarization-activated ‘funny’ current (I_f). The HCN4 protein has six transmembrane segments (S1–S6), a pore-forming loop (P), and intracellular N- and C-termini. The voltage sensor of the channel is formed by the positively charged S4 helix. The C-terminus contains the C-linker (dotted line) and the cyclic nucleotide-binding domain (CNBD), which is known to mediate cyclic AMP (cAMP)-dependent changes in HCN channel gating. Colored dots indicate the location of the loss-of-function mutations in the HCN4 protein of the present study. This set of mutations includes eleven substitutions (R375C, R378C, A414G, G480R, Y481H, G482R, A485V, K530N, R550C, R666Q, and S672R) and one truncation (695X).

Figure 2

Voltage dependence of wild type (WT; solid blue lines) and heteromeric R375C mutant (WT + R375C; orange dotted lines) HCN4 current (de)activation. (A) Steady-state activation (y_∞). Horizontal arrow: the mutation-induced hyperpolarizing shift in half-maximum activation voltage. Vertical arrow: the mutation-induced decrease in maximally available HCN4 current. (B) Time constant of (de)activation (τ_y). Horizontal arrow: the mutation-induced hyperpolarizing shift in the bell-shaped curve. Upward vertical arrow: the mutation-induced decrease in the rate of (de)activation at highly negative membrane potentials. Downward vertical arrow: the mutation-induced increase in the rate of (de)activation at less negative membrane potentials.

Figure 3

Charge carried by I_f during diastolic depolarization. (A) Prerecorded AP waveform of an isolated human sinus node pacemaker cell. During the diastolic depolarization from the maximum diastolic potential (MDP) to the take-off potential (TOP), which takes 538 ms, the membrane potential (V_m) depolarizes by 23 mV. (B) Associated reconstructed WT I_f, which carries a charge of 1.00 pC (filled area) as an inward current during diastolic depolarization. (C) Associated reconstructed WT + R375C I_f, which carries a charge of 0.20 pC during diastolic depolarization. The AP waveform of panel A is a typical waveform obtained from a set of single isolated human SAN pacemaker cells [61], and the I_f curve of panel B is reconstructed from this typical AP waveform and the I_f equations of the Fabbri–Severi model [62], which are based on the I_f data obtained in voltage clamp experiments on the same set of single-isolated human SAN pacemaker cells [61,63].

Figure 4

Electrical activity of the Fabbri–Severi model of a human SAN pacemaker cell with its default ‘wild-type’ I_f (WT; solid blue lines) and heteromeric R375C mutant I_f (WT + R375C; orange dotted lines) at different levels of autonomic tone. (A) Vagal tone (simulated ACh concentration of 20 nmol/L). (B) No rate modulation (default model). (C) β-Adrenergic tone (‘High Iso’ settings of the model).

Figure 5

The beating rate of the Fabbri–Severi model of a human SAN pacemaker cell with its default ‘wild-type’ I_f (WT) and heteromeric mutant I_f, simulated with the settings presented in Section 3.2 as a function of Q_f (Section 3.3) at different levels of autonomic tone. ^a I_f parameters are based on Milano et al. [71]. ^b I_f parameters are based on Schweizer et al. [74]. Dashed lines are linear fits.

Figure 6

Minimum, average, and maximum heart rates obtained during 24 h Holter recordings from heterozygous carriers of the mutations in HCN4 as indicated or from non-carriers of the same family (Table 1) as a function of Q_f (Section 3.3). ^a I_f parameters are based on Milano et al. [71]. ^b I_f parameters are based on Schweizer et al. [74]. Dashed lines are linear fits.

Figure 7

(A) Resting heart rates and (B) maximum heart rates during exercise testing from heterozygous carriers of the mutations in HCN4 as indicated or from non-carriers of the same family (Table 2) as a function of Q_f (Section 3.3). Dashed lines are linear fits.