Potential Arrhythmogenic Role of TRPC Channels and Store-Operated Calcium Entry Mechanism in Mouse Ventricular Myocytes

Figures

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  FIGURE 1. Store-operated Ca2+ entry measured in adult mouse ventricular myocytes. (A) A representative recording of SOCE from an adult mouse ventricular myocyte. Ca2+ fluorescence intensity (F/F0) in normal Tyrode’s solution (1 mM Ca2+) and Ca2+-free (Ca2+) Tyrode’s solution was measured. SOCE was detected under partial SR Ca depletion by 10 μM CPA (an inhibitor of SERCA) alone or complete depletion by 10 μM CPA and 10 mM caffeine (CPA + Caff). (B) Summary of F/F0 responses (SOCE) recorded in the presence of 10 μM CPA or 1 μM thapsigargin (Tha, another SERCA blocker) (CPA/Tha) alone (1.7 ± 0.4) or together with caffeine (2.7 ± 0.7, ∗p < 0.05), suggesting the complete depletion of SR Ca is required for maximal SOCE activation. (C,D) Representative traces of SOCE and its inhibition by TRPC or SOCE blockers (i.e., Gd3+ and ML-9). (E) Summary data demonstrating the putative SOCE was inhibited by SOCE/TRPC channel blockers (39.8 ± 4.5% inhibition by 1 mM Gd3+ and 31.8 ± 6.3% inhibition by 10 μM ML-9. ∗p < 0.05 compared to control, Student’s t-Test) but not SEA0400 (4.9 ± 2.3%).

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  FIGURE 2. Store-operated Ca2+ entry inhibition by functional antibodies or inhibitors of TRPCs. (A–C) Representative traces of Ca2+ fluorescence (F/F0) recorded from myocytes pre-treated with TRPC channel antibodies for 30 min before recording. SOCEs were evaluated using a standard protocol (i.e., 0 Ca + Tha + Caff). (D) A representative trace of SOCE after pretreatment with Gd3+ 1 mM for 100 s. (E) A bar graph summarizing the effects of the TRPC antibodies and Gd3+ on SOCE (Ctl: 2.4 ± 0.2, TRPC1 Ab: 1.4 ± 0.2, TRPC3 Ab:1.2 ± 0.2, TRPC6 Ab: 1.1 ± 0.1 and Gd3+:1.2 ± 0.1, Pyr3: 1.2 ± 0.1, Student’s t-Test), suggesting TRPC channels account for SOCE.

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  FIGURE 3. TRPC6 activator hyperforin potentiates the SOCE in adult mouse cardiac myocytes. (A–C) Representative recordings of SOCE from adult mouse myocytes. Ca2+ fluorescence intensity (F/F0) in normal Tyrode’s solution (1 mM Ca2+) and Ca2+-free (0 Ca) Tyrode’s solution was measured. SOCE was detected under complete depletion by 0.1 μM thapsigargin and 1 mM caffeine (Tha + Caff), and was further enhanced by hyperforin (HF) at different concentrations. (D) Summary of F/F0 responses (SOCE) in the presence of 0.1, 1, and 10 μM hyperforin, which potentiated SOCE in a concentration-dependent fashion. (∗p < 0.05 compared to control, Student’s t-Test).

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  FIGURE 4. Hyperforin enhances CaT amplitude. (A) A representative trace of CaTs facilitated by hyperforin (0.1 and 1 μM) when the cell was paced at a pacing cycle length (PCL) of 2 s. (B) Summary of the amplitudes of CaTs potentiated by hyperforin. F/F0 increased from 1.9 ± 0.1 to 2.1 ± 0.1, n = 10 at 0.1 μM and from 2.0 ± 0.2 to 2.4 ± 0.1, n = 8 at 1 μM hyperforin, respectively (∗p < 0.05). (C) Representative traces of calcium transients were measured before and after a 90s-perfusion of hyperforin (0.1 – 1 μM) while the cells were kept quiescent. (D) Summary of calcium transient amplitude by hyperforin perfusion. (E) A representative trace of Ca2+ waves (with [Ca2+] 4 mM) facilitated by hyperforin (0.1 μM). (F) Summary of the changes in Ca2+ wave frequencies caused by hyperforin, which promotes the Ca2+ waves by increasing the frequency to 180.5 ± 13.5% (n = 11, ∗p < 0.05 vs. control).

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  FIGURE 5. SR depletion and the presence of hyperforin induced significant inward current. (A) A representative plot of currents measured at –110 mV over time. The same treatment protocol to deplete SR Ca2+ (Tha + Caff) was used. (B) I–V curves at time points a–d (control, calcium free, after SR depletion, and in the presence of Gd3+, respectively) indicated in (A). (C) Summary of the inward current densities at –110 mV measured under control and after SR depletion (∗p < 0.05, Student’s t-Test). (D) A representative plot of currents measured at –110 mV over time, and the presence of hyperforin induced significant inward current entry. (E) Summary of the inward current densities at –110 mV measured under control and after SR depletion. Hyperforin (0.1 μM) induced a significant increase of inward current (from –1.4 ± 0.2 pA/pF to –2.6 ± 0.4 pA/pF, n = 8, ∗p < 0.05), which was then inhibited by the SOCE blocker Gd3+ (1 mM) (–0.4 ± 0.2 pA/pF, n = 8, ∗p < 0.05, Student’s t-Test).

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  FIGURE 6. Store-operated calcium entry contributes to cellular arrhythmogenesis. (A) Top panel: A Ca2+ fluorescence (F/F0) recording. (a,b): Simultaneous recordings of linescan image, whole-cell calcium fluorescence (F/F0) and action potentials (AP) at the time windows a and b indicated in the top panel. The cell was paced at a pacing cycle length (PCL) of 6 s (indicated by the arrows underneath the AP trace). EADs, DADs and triggered APs appeared after SOCE was activated, indicating SOCE contributes to the generation of arrhythmias. (B) In another cell, the same protocol as in A was used to activate SOCE. The SOCE-induced EADs (indicated by the asterisks), DADs, and triggered APs (c) were eliminated by Gd3+ (1 mM) (d). The 0 mV level is indicated by the dotted line in each panel. (C) Expanded region within the red box as indicated in (Ab). (D) Expanded region within the red box as indicated in (Bc). Note initiation (indicated by the dotted vertical lines) of the spontaneous depolarization phase (DADs triggering action potential) and occurrence of EADs (indicated by ∗) preceded the elevation of intracellular Ca2+. TA, triggered action potential; St, stimulation.

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  FIGURE 7. Proarrhythmic effect of hyperforin in ex vivo hearts. (A,B) Pseudo-Lead II ECG signals recorded from control (ctl) and 20 min after perfusion with 100 nM hyperforin (HF). Programmed S1–S2 stimulations were applied to evaluate the vulnerability to arrhythmias in each case. Sustained VT was observed in HF-treated heart. (C) Summarized arrhythmia scores for control and hyperforin groups, ∗p < 0.05, compared to control.