Control wells containing iPS cells were cultured in mTeSR1 medium without UVC irradiation

digitized at 10 kHz, and stored on a computer hard disk for further analysis. Resveratrol Attenuates H2O2-Increased INaL Intracellular Ca2+ Fluorescence Measurement Myocytes were loaded with fura-2-AM for 10 min at 25uC, and fluorescence measurements were recorded with a dual excitation fluorescence photomultiplier system. Myocytes were imaged through an Olympus IX-70 Fluor 40 6 oil objective. The cells were field stimulated with a suprathreshold voltage and at a frequency of 0.5 Hz, 3-ms duration, using a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator. The polarity of the stimulatory electrodes was reversed frequently to avoid possible build up of electrolyte by-products. Cells were exposed to light emitted by a 75-W lamp and passed through either a 340- or 380-nm filter while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 340 nm for 0.5 s then at 380 nm for the duration of the recording protocol. The 360 excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca2+ level were inferred from the ratio of the fura-fluorescence intensity at both wavelengths. Intracellular Ca2+ fluorescence measurements were assessed using the following indices: diastolic intracellular Ca2+ level , electrically stimulated rise in intracellular Ca2+ , maximal velocity of Ca2+ rise and Ca2+ decay. Data Analysis Whole-cell LOXO 101 web recordings were analyzed using clampfit 9.0 and PulseFit. Results Effects of Resveratrol and TTX on INa.L Under Normal Condition To identify INa.L, the current was recorded first in the absence and then in the presence of 4 mM TTX with 300 ms voltage steps from a holding potential of 2120 to 220 mV. The values of current recorded before and after application of TTX were 20.40060.050 and 20.15460.038 pA pF21, respectively, indicating that this TTXsensitive current recorded was INa.L. When INa.L was recorded under normal condition using depolarizing pulses with a duration of 300 ms applied at 0.25 Hz from a HP of 2120 mV in 10 mV increments between 270 and 220 mV, administration of 10, 20, 40 and 80 mM resveratrol resulted in decreased amplitudes of INa.L 4 Resveratrol Attenuates H2O2-Increased INaL in a concentration dependent manner. Effects of Resveratrol, Ranolazine and TTX on the Increased INa.L by H2O2 Currents were recorded using depolarizing pulses with a duration of 300 ms at a rate of 0.25 Hz from a HP of 2120 mV, in 10 mV increments between 270 and 220 mV. Administration of resveratrol at concentrations of 10, 20, 40 and 80 mM resulted in a decrease in the amplitudes of INa.L in a concentration dependent manner in myocytes exposed to H2O2. H2O2 increased the amplitudes of INa.L but 10, 20, 40 and 80 mM resveratrol decreased the amplitudes of INa.L in the continued presence of H2O2. Shown in figure 2B are the I-V relationships of INa.L after the sequential application of 300 mM H2O2, 10, 20, 40 and 80 mM resveratrol respectively, without a shift of the voltage at which the INa.L amplitude was maximal. Resveratrol Attenuates H2O2-Increased INaL 6 Resveratrol Attenuates H2O2-Increased INaL resveratrol on the gINa.L induced by 300 mM H2O2 with an IC50 of 26.192 mM. Ranolazine attenuated the increased INa.L in the presence of 300 mM H2O2 in a concentration dependent manner. Shown in figure 3B are the I-V relationships of INa.L after th