Diastolic transient inward current in long QT syndrome type 3 is caused by Ca2+ overload and inhibited by ranolazine

Abstract

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na(+) channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca(2+))-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized Delta KPQ LQT-3 mutation but the link to altered Ca(2+) cycling related to mutant Na(+) channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca(2+) load and induction of spontaneous diastolic inward current (I(TI)) in myocytes expressing Delta KPQ Na(+) channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca(2+), and measurement of SR Ca(2+) content using a caffeine dump methodology. We compared the Ca(2+) content of Delta KPQ(+/-) myocytes displaying I(TI) to those without spontaneous diastolic activity and found that I(TI) induction correlates with higher sarcoplasmic reticulum (SR) Ca(2+). Both spontaneous diastolic I(TI) and underlying Ca(2+) waves are inhibited by ranolazine at concentrations that preferentially target I(NaL) during prolonged depolarization. Furthermore, ranolazine I(TI) inhibition is accompanied by a small but significant decrease in SR Ca(2+) content. Our results provide the first direct evidence that induction of diastolic transient inward current (I(TI)) in Delta KPQ(+/-) myocytes occurs under conditions of elevated SR Ca(2+) load.

Figures

Figure 1. ITI induction in ΔKPQ+/- myocytes correlates with elevated [Ca2+]SR

A. Schematic of conditioning voltage pulse train used to induce ITI (Methods). ITI and [Ca2+]SR were measured at the holding potential (-75 mV) as indicated by the arrow in the diagram. B,C. Shown are high gain current recordings during the 3 last pulses of the conditioning train followed by 10 s at the holding potential in a cell which lacked ITI (B, left) and a cell in which ITI was induced (C, left). In the same cells, [Ca2+]SR was estimated by applying caffeine (10 mM, grey bars) and integrating the caffeine-induced INCX (B,C right, arrows) triggered after the conditioning train of voltage pulses. D. Bar graph summarizes mean [Ca2+]SR in cells without (open, n = 9) and with (closed, n = 11) ITI (**: p < 0.05) (D). [Ca2+]SR of single experiments is plotted to reveal threshold for ITI (E). Vertical dashed line: mean [Ca2+]SR estimated by others in cells that generate ITI [18].

Figure 2. Ranolazine (20 μM) inhibits ITI and related diastolic Ca2+ wave

Representative currents (upper rows), line scans (middle rows), and averaged Ca2+ signals (lower rows) recorded during the last pulse of the conditioning train protocol (Methods) and followed by 10 s at the return holding potential (-75 mV). A. Control conditions reveal ITI induction (A, upper panel, arrow) that was associated with a Ca2+ wave (A, middle and lower panels, arrows). B. Exposure to ranolazine (20 μM) inhibited ITI (B, upper panel) and the accompanying Ca2+ wave (B, middle and lower panels).

Figure 3. Summary data: ranolazine inhibits ITI, diastolic Ca2+ wave, but not systolic Ca2+ transient

Both systolic and diastolic Ca2+ events were determined by integrating normalized fluo-4 transient signals as described in Methods. The bar graphs summarize (A) mean ITI density per cell (n = 18); (B) mean Ca2+ wave density (n = 18); and (C) mean systolic Ca2+ density as measured during the final pulse of the conditioning train (n = 18) in control (open) and after exposure to ranolazine (20 μM) (closed). **: p < 0.05, paired.

Figure 4. Ranolazine inhibits ITI in a concentration-dependent manner

Representative high gain recordings from three different myocytes show currents in response to the 3 last pulses of conditioning trains followed by 10 s return to the holding potential before (left) and after (right) exposure to the indicated ranolazine concentrations: 10 μM (upper row), 50 μM (middle row) and 100 μM (lower row). Arrows indicate ITI in relevant panels. Zero current is indicated (0) in each panel.

Figure 5. Ranolazine inhibition of ITI and INaL share similar concentration-dependence

A. Bar graphs show mean ITI density per cell in control conditions (open) and after exposure to 10 μM (left; n = 5), 50 μM (middle; n = 8) and 1000 μM (right; n = 4) ranolazine (closed). **: p<0.05, paired. B. Summary plot of the concentration-dependence ranolazine inhibition of INa peak (●), INaL (○) and ITIs (). Smooth curves are best fits of concentration-response relationships to INaL, INaP and ITI data (see Methods).

Figure 6. Ranolazine decreases [Ca2+]SR

Representative high gain recordings during the 3 last pulses of the conditioning train and 10 s at the holding potential (-75mV) after the train in the absence (A) and presence of 50 μM ranolazine (B). Induction (A, left, arrow) and ranolazine-dependent inhibition of ITI (B, left) was verified before estimating [Ca2+]SR in both conditions using caffeine-induced INCX (A and B, right, arrows). Zero current is indicated (0) in each panel. C. Bar graph summarizes normalized mean [Ca2+]SR before and after exposure to ranolazine, n = 5, p < 0.05, paired (C).