The Effects of SEA0400 on Ca2+ Transient Amplitude and Proarrhythmia Depend on the Na+/Ca2+ Exchanger Expression Level in Murine Models

Abstract

Background/Objective: The cardiac Na⁺/Ca²⁺ exchanger (NCX) has been identified as a promising target to counter arrhythmia in previous studies investigating the benefit of NCX inhibition. However, the consequences of NCX inhibition have not been investigated in the setting of altered NCX expression and function, which is essential, since major cardiac diseases (heart failure/atrial fibrillation) exhibit NCX upregulation. Thus, we here investigated the effects of the NCX inhibitor SEA0400 on the Ca²⁺ transient amplitude and on proarrhythmia in homozygous NCX overexpressor (OE) and heterozygous NCX knockout (hetKO) mice compared to corresponding wild-types (WT_OE/WT_hetKO). Methods/Results: Ca²⁺ transients of field-stimulated isolated ventricular cardiomyocytes were recorded with fluo-4-AM or indo-1-AM. SEA0400 (1 μM) significantly reduced NCX forward mode function in all mouse lines. SEA0400 (1 μM) significantly increased the amplitude of field-stimulated Ca²⁺ transients in WT_OE, WT_hetKO, and hetKO, but not in OE (% of basal; OE = 98.7 ± 5.0; WT_OE = 137.8 ± 5.2^*; WT_hetKO = 126.3 ± 6.0^*; hetKO = 140.6 ± 12.8^*; ^*p < 0.05 vs. basal). SEA0400 (1 μM) significantly reduced the number of proarrhythmic spontaneous Ca²⁺ transients (sCR) in OE, but increased it in WT_OE, WT_hetKO and hetKO (sCR per cell; basal/+SEA0400; OE = 12.5/3.7; WT_OE = 0.2/2.4; WT_hetKO = 1.3/8.8; hetKO = 0.2/5.5) and induced Ca²⁺ overload with subsequent cell death in hetKO. Conclusion: The effects of SEA0400 on Ca²⁺ transient amplitude and the occurrence of spontaneous Ca²⁺ transients as a proxy measure for inotropy and cellular proarrhythmia depend on the NCX expression level. The antiarrhythmic effect of SEA0400 in conditions of increased NCX expression promotes the therapeutic concept of NCX inhibition in heart failure/atrial fibrillation. Conversely, in conditions of reduced NCX expression, SEA0400 suppressed the NCX function below a critical level leading to adverse Ca²⁺ accumulation as reflected by an increase in Ca²⁺ transient amplitude, proarrhythmia and cell death. Thus, the remaining NCX function under inhibition may be a critical factor determining the inotropic and antiarrhythmic efficacy of SEA0400.

Keywords: Ca2+ cycling; Na+/Ca2+ exchanger; arrhythmia; heart failure; novel antiarrhythmic strategies; transgenic mice.

Figure 1

SEA0400 mediated NCX forward mode inhibition in caffeine application experiments. Representative tracings of caffeine-induced Ca²⁺ transients illustrate the different decay rates in OE (A), hetKO (C) and respective WTs as a direct measure of NCX-mediated Ca²⁺ removal under basal conditions and during NCX inhibition by SEA0400. Quantification of time to 50% decay (T₅₀) of caffeine-induced Ca²⁺ transients confirmed increased NCX forward mode function in OE (B) and decreased in hetKO (D), and verified NCX forward mode inhibition by SEA0400 in all investigated groups but to different levels. ^*p < 0.05 vs. basal; ⁺p < 0.05 OE/hetKO vs. WT_OE/WT_hetKO basal; ANOVA on ranks test.

Figure 2

Relative effect of increasing SEA0400 concentrations on the amplitude of field stimulated Ca²⁺ transients as a measure for inotropy. SEA0400 mediated a significant relative increase of the Ca²⁺ transient amplitude vs. basal in WT_OE (A), WT_hetKO and hetKO (B), but not in OE (A). Thus, the effect of SEA0400 on the Ca²⁺ transient amplitude seems to depend on the respective NCX expression level. ^*p < 0.05 vs. basal; two-way repeated measures ANOVA test.

Figure 3

Quantification of spontaneous Ca²⁺ releases (sCR) as a measure for proarrhythmia. (A) Representative tracings illustrate the occurrence of numerous sCR (arrows below respective tracings) between field-stimulated Ca²⁺ transients (stim, arrows above respective tracings) in OE but not in hetKO in absence of SEA0400. (B) Quantification of sCR/cell under basal conditions and during exposure with 1 μM SEA0400. (C) Number of sCR-positive cardiomyocytes under basal conditions and during exposure with 1 μM SEA0400. SEA0400 significantly reduced the occurrence of sCR/cell in OE. Conversely, in hetKO SEA0400 increased the occurrence of sCR. Hence, the effect of SEA0400 on the occurrence of proarrhythmic spontaneous Ca²⁺ transients again depends on the NCX expression level. ^*p < 0.05 vs. basal; ⁺p < 0.05 vs. corresponding WT basal; two-way repeated measures ANOVA test; z-test.

Figure 4

Quantification of Ca²⁺ hypercontractions as a measure for proarrhythmia. In a fraction of cardiomyocytes application of 1 μM SEA0400 caused a sudden increase of the diastolic Ca²⁺ level with highly frequent contractions and subsequent cell death (“Ca²⁺ hypercontraction”) (B) as illustrated by the representative tracing in (A). The proportion of cells with Ca²⁺ hypercontractions was significantly increased only in the hetKO group. ^*p < 0.05 vs. basal; ⁺p < 0.05 vs. WT; Fisher's exact test with R.