Figure 3. Conditioning of the beating frequency by electrical stimulation is mediated by KCNH2.

(a,b) Immunostains of troponin (green) and hERG (red), counterstained with 4,6-diamidino-2-phenylindole (DAPI, blue); scale bar, 50 μm. Frequency-dependent increases in Troponin and hERG (n=3, P<0.05, one-way ANOVA with post hoc Tukey test). (c) qPCR of KCNH2 confirms the upregulation with stimulation (average±s.e.m. of fold change). *Statistically significant differences between the 2-Hz group and the 1-, 0.5-Hz and control groups (n=3, P<0.05, one-way ANOVA with post hoc Tukey test). (d) Representative action potentials at baseline (solid) and after addition of the KCNH2 inhibitor E-4031 (dashed). The dashed black line represents 0 mV. (e,f) Action potential duration (APD90) and maximum diastolic potential at baseline and when exposed to E-4031. *Significant difference between experimental electrically stimulated groups and control unstimulated cells at baseline. **Significant difference for an experimental group before and after treatment with E-4031 (control: n=13, 0.5 Hz: n=5, 1 Hz: n=9, 2 Hz: n=15, P<0.05, two-way ANOVA with post hoc Tukey test). (g) The differential beating frequency after 7 days of stimulation was abolished with the addition of the E-4031 and was regained after washing out the drug. *Statistically significant differences between the 2-Hz group and the 1-, 0.5-Hz and control groups (n=8, P<0.05). NS denotes no statistical difference between control, 0.5-, 1- and 2-Hz groups when exposed to E-4031. **Statistically significant difference for the 2-Hz group between the baseline, when exposed to E-4031, and following the washout of E-4031 (n=8, P<0.05, two-way ANOVA with post hoc Tukey test). (h) Frequency of cardiomyocytes throughout the stimulation period in the presence of E-4031, and following the washout of E-4031 after the stimulation period (n=8).