Abstract 161: TRPC6 Activator Hyperforin Facilitates Arrhythmias Via Activating Store-operated Calcium Entry

Abstract

Hyperforin, a major antidepressant constituent of St. John’s wort ( Hypericum perforatum ), exhibits high selectivity in activating the canonical transient potential channel 6 (TRPC6). Our previous studies have shown that TRPC6 may modulate Ca 2+ handling via store-operated Ca 2+ entry (SOCE) in adult ventricular myocytes. In the present study, we aim to test whether hyperforin is arrhythmogenic by facilitating Ca 2+ waves. Intracellular Ca 2+ fluorescence (F/F 0 ) was imaged in Fluo-4-AM loaded ventricular myocytes isolated from adult mice (2-4 month old). SOCE was evaluated by increasing the external Ca 2+ concentration ([Ca 2+ ] o ) from 0 to 1 mM after caffeine (10 mM) and thapsigargin (10 µM) were used to completely deplete SR Ca 2+ . Hyperforin (0.1-10 µM) perfusion increased SOCE up to 3.7 folds (F/F 0 = 3.73 ± 0.42, n = 7, compared to the control F/F 0 = 1.69 ± 0.37, n = 9, p < 0.05), in a concentration dependent fashion. Whole-cell currents were recorded using ramp pulses from -110 mV to +50 mV, while K + , Na + , L-type Ca 2+ , and Na + -Ca 2+ exchange currents were pre-blocked. Hyperforin at a lower concentration (0.1 µM) induced a significant increase of inward current (from -1.44 ± 0.23 pA/pF to -2.56 ± 0.36 pA/pF, n = 8, p < 0.05), which was then inhibited by the SOCE blocker Gd 3+ (1 mM) (-0.43 ± 0.22 pA/pF, n = 8, p < 0.05). Hyperforin promotes spontaneous CaWs ([Ca 2+ ] o 4 mM) by increasing the frequency to 180± 14 % (n = 11. p < 0.05), which can be attenuated by Gd 3+ (1 mM), or SKF-96265 (10 μM). In addition, both the amplitudes of Ca 2+ transients (F/F 0 =1.92 ± 0.09 to 2.09 ± 0.10, n = 12, p < 0.05) and calcium content (control: F/F 0 = 2.03 ± 0.11, n=16 to 2.12 ± 0.13, n = 11, p < 0.05) were enhanced by acute hyperforin perfusion (0.1 µM), when the cells were paced at a pacing cycle length of 2 seconds. The proarrhythmic effect of hyperforin was also confirmed in Langendorff-perfused hearts, from which optical membrane voltage mapping and EKG were simultaneously recorded. In conclusion, the TRPC6 activator hyperforin exhibits an arrhythmogenic effect in the heart. Its action is likely to be mediated by generating an inward current and increasing the calcium load in cardiac myocytes through the SOCE pathway. Caution should be taken when prescribing this drug to patients with heart disease.