Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling

Figures

• 

  Figure 1. CaT alternans is enhanced in heart failure. CaT traces recorded in AP-clamped atrial myocytes isolated from normal (Control) and HF hearts paced with N-W ((Aa), paced at 1.19 Hz), W-W ((Ba) paced at 1.19 Hz) and N-N ((Ca) paced at 1.56 Hz) pacing protocols. Bottom traces in (a) panels show pacing protocols. Mean ± SEM of ARs observed in control and HF atrial myocytes paced at different frequencies with AP-clamp N-W (Ab), W-W (Bb) N-N (Cb) pacing protocols. Data was collected from cells isolated from 19 control and 7 HF rabbits, number of cells for every data point is shown in parentheses. Statistics calculated using Šidàk’s mixed-effects multigroup comparison test.

• 

  Figure 2. AP morphology and ICa in normal and HF atrial myocytes. (A): (a) Atrial APs recorded in normal and HF current clamped myocytes at 1 Hz. (b) Mean ± SEM and individual AP durations at 50% and 90% of repolarization in control (N/n = 23/28) and HF (N/n = 5/7) myocytes recorded at 1 Hz pacing. Statistical analysis performed using Welch t-test. (B): (a) Representative ICa traces in normal and HF atrial myocytes recorded during step stimulation protocol (bottom). (b) I-V curves of ICa recorded in normal (N/n = 4/10) and HF (N/n = 7/16) atrial myocytes. No statistical differences between ICa amplitude in control and HF myocytes (Šidàk’s test).

• 

  Figure 3. Inhibition of CaMKII activity decreases risk of atrial alternans. (A): (a) CaT traces recorded in control and after application of CaMKII blocker KN-93 (1 µM; top) and its inactive analogue KN-92 (1 µM; bottom). (b) Mean ± SEM and individual CaT ARs recorded in control and after the application of KN-93 and KN-92 (paired t-test). (B): (a) CaT traces recorded in control cells and in cells preincubated with AIP (1 µM) at 1.6 Hz pacing. (b) Mean ± SEM CaT ARs observed at various pacing frequencies in control cells and in cells preincubated for ≥30 min with AIP (1 µM). (c) Mean ± SEM CaT ARs at different pacing rates in control and AIP-treated cells exhibiting alternans (in this data set non-alternating cells were removed from the analysis. (d) Fraction of cells exhibiting alternans at given frequencies in control cells (N/n = 8/35) and cells preincubated with AIP (N/n = 8/24).

• 

  Figure 4. Stimulation of IP3 signaling by ET-1 and Ang II has a dual effect on CaT alternans. (A): (a) CaT ARs recorded over time in control and in the presence of 100 nM ET-1. Top: CaT traces observed at the times marked by the corresponding letter in the bottom graph. (b) Mean ± SEM and individual CaT ARs observed in control, after 0.5–2 min of application of ET-1 at the point of maximal effect of ET-1 (acute effect), after 10 min of ET-1 application and following ≥5 min washout of ET-1 (N/n = 3/8; Tukey’s test). (B): (a) CaT ARs recorded over time in control and in the presence of 500 nM Ang II. Top: CaT traces observed at the times marked by the corresponding letter in the bottom graph. (b) Mean ± SEM and individual CaT ARs observed in control, after acute application of 500 nM of Ang II, after 10 min of Ang II application and after ≥5 min washout (N/n = 4/9 Tukey’s test).

• 

  Figure 5. Effect of endothelin-1 and angiotensin II on CaT amplitude. (A): (a) CaT amplitude recorded over time in control and in the presence of 100 nM ET-1 in atrial cell paced at 0.5 Hz. Inset: Overlay of CaTs recorded in control, after acute and 10 min application of ET-1. (b) Mean ± SEM and individual CaT amplitudes in control, maximal transient effect of ET-1 (ET-1 acute) and after 10 min application of 100 nM ET-1 (N/n = 4/13, Tukey’s test). (B): (a) CaT amplitude recorded over time in control and in the presence of 500 nM Ang II. Inset: Overlay of CaTs recorded in control, after acute and 10 min application of Ang II. (b) Mean ± SEM and individual CaT amplitudes in control, after acute and 10 min application of 100 nM Ang II (N/n = 6/9, Tukey’s test).

• 

  Figure 6. Effect of endothelin-1 on SR Ca2+ load. (A) [Ca2+]SR measured with the low affinity Ca2+ indicator Cal520N in control and in 100 nM ET-1 in cells paced at 0.5 Hz. Bottom: depletions of [Ca2+]SR recorded at times indicated by the corresponding letter in the top graph. (B,C): Mean ± SEM and individual depletions of [Ca2+]SR (B) and diastolic [Ca2+]SR (C) measured in control, during transient ET-1 effect and after 10 min application of ET-1 (N/n = 2/7). Statistics: Tukey’s test.

• 

  Figure 7. IP3 receptor blocker 2-APB enhances CaT alternans. (A): Traces of CaTs (top) and CaT ARs recorded over time in control and in presence of 2-APB (5 µM). (B): Mean ± SEM and individual CaT ARs observed in control, in 2-APB and after washout of 2-APB (N/n = 4/10, Tukey’s test). (C): Mean ± SEM and individual CaT amplitudes (a) and diastolic [Ca2+]i (b) observed in control and in the presence of 2-APB recorded at 0.5 Hz pacing in the absence of alternans (N/n = 3/12, paired t-test). (D): Application of 2-APB in the presence of Ang II further increases CaT AR (N/n = 2/7; Tukey’s test). (E): Mean ± SEM and individual CaT ARs recorded in control and after application of TRPC6/3 channel blocker GSK2833503A (GSK; 200 nM) and simultaneous application of GSK and 2-APB (N/n = 4/7; Tukey’s test).

• 

  Figure 8. IP3R block prevents transient effect of Ang II on CaT alternans and CaT amplitude. (A): (a) CaTs (top) and CaT ARs (bottom) recorded over time in control, in 5 µM 2-APB and during simultaneous application of 2-APB and 500 nM Ang II. (b) Mean ± SEM and individual CaT ARs recorded in control, in 2-APB and in 2-APB together with Ang II after 1 min (time point when typically the transient effect of Ang II is observed under control conditions) and after 10 min of Ang II application (N/n = 3/8, Tukey’s test). (B): Application of IP3 receptor blocker 2-APB (5 µM) prevented the acute effect of Ang II on CaT amplitude (0.5 Hz pacing, N/n = 3/12, Tukey’s test). (C): TRPC6/3 channel blocker GSK2833503A (200 nM) had no effect on CaT amplitude and did not prevent transient decrease of CaT amplitude during ET-1 application (pacing at 0.5 Hz; N/n = 3/8, Tukey’s test).