Investigation of the Effects of the Short QT Syndrome D172N Kir2.1 Mutation on Ventricular Action Potential Profile Using Dynamic Clamp

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K(+) current, I(K1). The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of I(K1) using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic I(K1) was inhibited with a low concentration (50 µM) of Ba(2+) ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic I(K1) through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) I(K1) with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD(90)) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD(90) and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant I(K1) to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations

Markov models of use-dependence and reverse use-dependence during the mouse cardiac action potential.

The fast component of the cardiac transient outward current, I(Ktof), is blocked by a number of drugs. The major molecular bases of I(Ktof) are Kv4.2/Kv4.3 voltage-gated potassium channels. Drugs with similar potencies but different blocking mechanisms have differing effects on action potential duration (APD). We used in silico analysis to determine the effect of I(Ktof)-blocking drugs with different blocking mechanisms on mouse ventricular myocytes. We used our existing mouse model of the action potential, and developed 4 new Markov formulations for I(Ktof), I(Ktos), I(Kur), I(Ks). We compared effects of theoretical I(Ktof)-specific channel blockers: (1) a closed state, and (2) an open channel blocker. At concentrations lower or close to IC(50), the drug which bound to the open state always had a much greater effect on APD than the drug which bound to the closed state. At concentrations much higher than IC(50), both mechanisms had similar effects at very low pacing rates. However, an open state binding drug had a greater effect on APD at faster pacing rates, particularly around 10 Hz. In summary, our data indicate that drug effects on APD are strongly dependent not only on IC(50), but also on the drug binding state

Dose dependent blockade of I_Ktof.

<p>The effect of various concentrations of drug on the action potential for <b>A:</b> open state block, and <b>B:</b> closed state block. The degree of block was determined by holding at −70 mV, the applying a test pulse to +50 mV for 500 ms. <b>C:</b> Open state binding of Drug O. <b>D:</b> Closed state binding of Drug C. Change in peak (▪), change in total current flow (○). Solid lines are Boltzmann fits to the data .</p

Effect of drugs O and C on APD prolongation.

<p><b>A:</b> simulated action potentials of the mouse ventricular model for the epicardial and endocardial cells. Pacing rate was 1 Hz. Relative APD prolongation normalized to the maximum prolongation with I_Ktof completely blocked was determined at various drug concentrations for drug O (○) and drug C (▪) on <b>B:</b> endocardium and <b>C:</b> Epicardium. Pacing rate was 1 Hz.</p

Restitution curves for epicardial cells.

<p><b>A:</b> 1 Hz pacing; <b>B:</b> 2 Hz pacing; <b>C:</b> 10 Hz pacing. Top: APD30; Middle: APD75; Bottom: APD30. Control in the absence of drug (▪), 0.1 mM drug C (•), 1 mM drug C (▴), 0.1 mM drug O (○), 1 mM drug O (Δ).</p

Example of APs recorded with different S1–S2 intervals.

<p>The first AP (control) represents the last beat of the pacing train at a cycle length of 1s (S1) on endocardial cells. APs are shown for S1–S2 intervals of 60, 100, 200, and 300 ms. Peak amplitudes are shown above the AP. APD30 is 4.89 ms, 8.17 ms, 6.11 ms, 5.58 ms, and 5.56 ms; APD75 is 18.11 ms, 22.07 ms, 18.65 ms, 17.81 ms, and 17.81 ms; and APD90 is 29.82 ms, 32.73 ms, 30.13 ms, 29.54 ms, and 29.47 ms for control, 60, 100, 200, and 300 ms S1–S2 intervals respectively.</p