Cardiomyocyte-Specific STIM1 (Stromal Interaction Molecule 1) Depletion in the Adult Heart Promotes the Development of Arrhythmogenic Discordant Alternans

Figures

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  Figure 1.. Inducible, cardiomyocyte-specific STIM1 (stromal interaction molecule 1) knockdown model.Representative Western blot and quantification of STIM1 protein expression on whole heart tissue (A) and isolated cardiomyocytes (B) on day 8 after initial tamoxifen injection. Statistical test: Mann-Whitney unpaired t test shows NS (P=0.4667) on heart lysates (n=6/group) and ***P<0.0001 on isolated cardiomyocytes (n=6 and n=7 respectively for STIM1-Ctl [control] and STIM1-KD [knock-down]). C, Survival curve of STIM1-Ctl (blue line) and STIM1-KD mice (red line), from day 1 to day 8. Tamoxifen injection was performed from day 1 to day 5 (green rectangle). n=22 and 23 respectively for STIM1-Ctl and STIM1-KD mice.

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  Figure 2.. Cardiac remodeling of adult STIM1-KD (stromal interaction molecule 1 knockdown) mice.Echocardiographic (echo) measurements at baseline (day 1), 3, 5, and 8 days after the first tamoxifen injection in STIM1-Ctl (control), STIM1-KD, and Cre-Ctl mice. A, Representative echo images of STIM1-Ctl and STIM1-KD mice at baseline and days 5 and 8 after the first tamoxifen injection. Different parameters were measured by echo: (B) heart rate, (C) percentage of cardiac fractional shortening (%FS), (D) left ventricular internal diameter during systole (LVIDs) and diastole (LVIDd), (E) interventricular septum thickness during systole (IVSs) and diastole (IVSd), and (F) left ventricular posterior wall thickness during systole (LVPWs) and diastole (LVPWd). The number of animal is embedded in the bar graphs for each time point (n). Statistics for echo parameters are summarized in Table 1.

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  Figure 3.. Arrhythmia susceptibility of STIM1-KD (stromal interaction molecule 1 knockdown) hearts. A, Representative action potential traces recorded from STIM-KD hearts after challenge with rapid stimulation. Action potentials document a wide range of arrhythmic activity from frequent after depolarizations to nonsustained and sustained ventricular tachycardia/ventricular fibrillation (VT/VF). B, Left, Representative action potential traces from Cre-Ctl (Cre-recombinase–control), STIM1-Ctl, and STIM1-KD hearts recorded during and immediately after the cessation of the rapid stimulus train (marked by black arrow). Note presence of action potential alternans before onset of VT/VF in STIM1-KD heart. Right, Incidence of VT/VF in all groups.

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  Figure 4.. Electrophysiological remodeling in STIM1-KD (stromal interaction molecule 1 knockdown) hearts.Langendorff-perfused hearts from STIM1-KD and STIM1-Ctl (control) mice were loaded with the voltage–sensitive dye, di-4-ANEPPS and paced from the left ventricular at pacing cycle lengths (PCLs) from 140 to 90 ms in 10 ms decrements. A, Conduction velocity (CV) was plotted against PCL. Statistics: STIM1-KD (n=7) vs STIM1-Ctl (n=9), 2-way ANOVA followed by Sidak’s multiple comparison test. B, Representative depolarization isochrone maps recorded at PCL 140 ms for STIM1-Ctl and STIM1-KD hearts. C, Comparison of average CV in ventricular tachycardia/ventricular fibrillation (VT/VF) (+) vs VT/VF (−) STIM1-KD hearts. D, Representative western blot of Cx43 and phospho-Cx43 (pSer368) protein expression in STIM1-Ctl (n=6) and STIM1-KD hearts (n=7) and quantification of the protein expression ratio. E, Representative western blot of Nav1.5 protein in STIM1-Ctl (n=6) and STIM1-KD hearts (n=7) and quantification of the protein expression. F, Representative western blot of phospho-CAMKII (pThr286) protein in STIM1-Ctl (n=6) an STIM1-KD hearts (n=7) and quantification of the protein expression.…

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  Figure 5.. Expression of molecular determinants of conduction in STIM1-Ctl (stromal interaction molecule 1 control) and STIM1-KD (knockdown) hearts. A, Average steady-state action potential duration (APD75) values measured over a range of pacing cycle length (PCL). Analysis is based on 7 STIM1-KD and 9 STIM1-Ctl hearts. B, Range of APD75 values measured across the mapping field as an index of APD heterogeneity. Analysis is based on 6 STIM1-KD and 7 STIM1-Ctl mice. C, Comparison of APD75 heterogeneity in ventricular tachycardia/ventricular fibrillation (VT/VF) (+) vs VT/VF (−) STIM1-KD hearts. D, mRNA expression of K-channel subunits: Kv1.4, Kv4.2, Kv4.3, and KCHIP2 in STIM1-Ctl and STIM1-KD hearts (n=4/group). Statistical analysis using the unpaired t test. Data presented as mean±SEM.

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  Figure 6.. Pacing-induced action potential alternans in STIM1-KD (stromal interaction molecule 1 knockdown) hearts. A, Representative action potential (AP) traces documenting the emergence of AP alternans before the onset of ventricular tachycardia/ventricular fibrillation (VT/VF) in STIM1-KD hearts. B, Representative AP traces during progressive elevation in pacing rate in STIM1-Ctl (control; left) and STIM1-KD (right) hearts illustrating presence of substantial pacing-induced AP alternans (red frame) in the latter but not former. C, Average ΔAPD75 measured at pacing cycle length 140 ms and 70 ms in STIM1-Ctl (n=7) andSTIM1-KD (n=6) hearts. Statistical analysis detailed in Table 2. D, Average ΔAPD75 as a measure of alternans magnitude in VT/VF(+) and VT/VF(−) STIM1-KD hearts. Statistical analysis: t test (P=0.0297). Data represent mean±SEM.

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  Figure 7.. Spatially discordant action potential alternans and their relevance to functional reentry in STIM1-KD (stromal interaction molecule 1 knockdown) hearts. A, Contour maps depicting the spatial distribution of ΔAPD75 in STIM1-Ctl (control; left) and STIM1-KD (right) hearts at basal pacing cycle length (PCL 140 ms) and elevated (PCL 70 ms) pacing rates. STIM1-KD but not STIM1-Ctl hearts exhibited a spatial discordant alternans profile marked by adjacent zones of opposite alternans polarity. Also shown are representative action potential tracings from the zones of opposite polarity illustrating the long (L)-short (S) (red) and short-long (blue) sequences. B, Examples of actions potential traces displaying discordant alternans in 4 different STIM1-KD hearts. C, Representative phase maps recorded during the early onset of ventricular tachycardia/ventricular fibrillation (VT/VF) showing stable rotors that are localized along the nodal line separating the out-of-phase alternans regions.