4.7. Patch–Clamp Recordings in Cardiomyocytes

Isolated myocytes were transferred to a shallow chamber and allowed to settle for at least 30 min before being superfused with the external bathing solution (Tyrode’s solution with nicardipine 10 µM for I_to or without nicardipine for I_Ca-L). Only Ca²⁺-tolerant rod-shaped cells, with clear cross-striations and lacking any visible blebs on their surfaces were used. All experiments were performed at room temperature (23–25 °C). Ionic currents were recorded using the whole-cell configuration of the patch–clamp technique with an Axopatch-1D patch–clamp amplifier (Axon Instruments, Inc., USA). Recording pipettes were obtained from borosilicate tubes (Sutter Instruments, Novato, CA, USA) and had a tip resistance of 2–5 MΩ when filled with the internal solution (in mmol/L): CsCl 110; TEACl 30; Mg-ATP 5; EGTA 10; HEPES 10 and Na-GTP 0.1 to record the L-type Ca²⁺ current, I_Ca-L or KCl 125; NaCl 10; MgCl₂ 5; HEPES 10 and EGTA 5 to record the transient outward K⁺ current, I_to.

Following the patch rupture, whole cell membrane capacitances were measured from integration of the capacitive transients elicited by voltage steps from −70 mV to −75, −65 and −60 mV, which did not activate any voltage-dependent membrane current. For both I_to and I_Ca,L recording, voltage pulses ranging from −50 to +60 mV, starting from a holding potential of −60 mV preceded by pre-pulse to −40 mV were applied and current amplitudes were normalized to cell capacitance and expressed as pA/pF. Steady-state activation curve of I_to or I_ca-L was obtained from the I–V ratio, calculating the conductance (G) to the K⁺ or Ca²⁺ ion, respectively.

To evaluate the voltage dependence of the I_to stationary inactivation (Steady-state inactivation), a double-pulse protocol was used ranging from −90 to +30 mV, starting from a holding potential of −80 mV followed by pulse to +60 mV. Recovery from inactivation of I_to was assessed by a double-pulse protocol from −80 to +60 mV, where the time between two consecutive pulses was variable.

To evaluate the voltage dependence of the I_Ca-L steady-state inactivation, a double-pulse protocol was used ranging from −90 to +40 mV, starting from a holding potential of −80 followed by a pulse to 0 mV.