After 3 minutes of stirring for 3 different periods, the ECLl suspensions were centrifuged, and 1 mmol / L CaCl2. After all, the isolated cells were in KB-L Solution suspended with 0.5 mg / ml BSA, and at room temperature for 30 min to 1 h before the experiments. The sticks with clear cross strips expertised without automatic BI6727 Volasertib contraction Gt were used in this study. Voltage clamp Aufzeichnungsstr Me the L-type calcium channels Le under voltage clamp in the whole cell configuration of the patch clamp technique has been registered. Cardiomyocytes were placed in a tray on the plate of an inverted microscope, and were continuously at a constant speed with a peroxide-L Solution containing NaCl 137, CaCl2 1.8, MgCl2 1, 5.4 CsCl TTX 0 perfused, 02, 4 AP 4, HEPES 10 and glucose 10th Individual cells were clamped with a patch clamp amplifier Stronger.
Physiological signals recorded pCLAMP 6.0. Pipettes Registration whole cell patch-clamp  and have resistors Walls. 1-3 mV The pipette L Solution contained the CsCl 130, MgCl2 1, Na2ATP 5, Na2GTP 0.5, EGTA 11 and 10 HEPES. I Ca L current was measured under the conditions described above. K-Str Me were sung by internal Cs and 4 in the AP infusion, And free external K L Suppressed solution. Na current was suppressed by TTX. Na, K pump current in L solutions Of K and Na Complimentary toiletries L Inactivated solutions without pipette. Membrane Str me With Na Ca2 exchange was assigned by Na and low Ca2 pipette L Eliminated solutions.
Administration of nifedipine of Badl Solution could completely Constantly inhibit peak I Ca, L 1 min, the best Firmed that the measured current due I Ca, L IV relationship I Ca, L was in by plotting the peak amplitude of the current response to voltage pulses reaches to potentials 240-70 mV from a holding potential of 240 mV. The activation state of equilibrium of I Ca was determined by applying L 200 ms depolarizing pulses between 270 mV and 30 mV from a holding potential of 270 mV. The inactivation of the station Ren I Ca L is through the application of a pulse protocol with two pulses, before 1 s 270-30 mV and a sequence of 200 ms test pulses 0 mV from a holding potential determined by 270 mV. Recovering I Ca, L from the inactivation was measured with a dual-pulse protocol, consisting of a 200 ms pulse to 0 mV by conditioning ms test pulse to 0 mV from 200 followed examined from a holding potential of 270 mV in steps of 20 ms interval increased from 20 500 ms.
normalize membrane beaches me in Cm, the gegenw rtige capacity t of fa transient in response to a 5 mV hyperpolarizing pulse has been integrated, and to give the predetermined voltage divided Cm measured total for each cell. Different concentrations of NaHS were applied by a fast fan. All experiments were carried out at an ambient temperature of 21 23uC. Cell culture and identification of proteins containing sulfhydryl groups H9c2 cells were cultured in 100 mm plates with Dulbecco’s modified Eagle’s medium supplemented with 10% f Fetal K Calf serum administered, 2 mmol / l glutamine, 100 incubated U / ml penicillin and 100 mg / ml streptomycin.