Whole-cell patch-clamp recording from cardiomyocytes

Ventricular myocytes were isolated from mice of either sex, as previously described (Alliance for Cellular Signaling Procedure Protocol PP00000125) (103), maintained at 37°C, and aerated with 98% O₂ and 2% CO₂. Calcium currents (I_Ca) were recorded in whole-cell mode at room temperature from calcium-tolerant myocytes within 1 to 24 hours of isolation. The extracellular solution contained 137 mM NaCl, 1.8 mM CaCl₂, 25 mM CsCl, 0.5 mM MgCl₂, 10 mM Hepes, and 10 mM glucose (pH 7.4). Patch pipettes (1 to 1.5 megohms) were filled with 120 mM CsCl, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1 mM EGTA, 1 mM MgCl₂, 1 mM Na₂GTP, 5 mM phosphocreatine, and 10 mM Hepes (pH 7.2). I_Ca was elicited by depolarizing steps. A prepulse from −80 to −40 mV was used to inactivate fast Na⁺ currents and then stepped to voltages between −40 and +60 mV (10-mV increments). I_Ca was recorded by an Axopatch 200B amplifier (Axon Instruments) and stored on a computer through an analog-digital converter (DIGIDATA 1332A; Axon Instruments). Protocols were controlled by pClamp 9 software (Axon Instruments). I_Ca was measured as the difference between the peak inward current and the current after the test pulse ended. After establishing a stable baseline, the effect of 1 μM ISO on I_Ca was examined.