Cardiomyocyte Culture and Electrophysiology

Adult (8–12 weeks) sinoatrial nodal (SAN) cells and ventricular myocytes were isolated as described (Anderson et al., 2018) and used within 8 h of isolation. Coverslips containing SAN cells or ventricular myocytes were transferred to a perfusion chamber and electrophysiological recordings were conducted as described (Anderson et al., 2018). In brief, whole-cell access was obtained in a bath consisting of (in mM): 130 NaCl, 5.4 KCl, 1 CaCl₂, 1 MgCl₂, 5.5 glucose, 5 HEPES/NaOH (pH 7.4). CCh-currents (holding potential of -70 mV) were measured in a high-K⁺ bath solution consisting of (in mM): 120 NaCl, 25 KCl, 1 CaCl₂, 1 MgCl₂, 5.5 glucose, 5 HEPES/NaOH (pH 7.4). CCh was applied via ValveLink 8.2 rapid perfusion system (AutoMate Scientific, Berkeley, CA, United States). BaCl₂ (5 μM) was included in the bath solution for ventricular myocyte recordings to block I_K1, which has been shown to mask GIRK-dependent currents (Beckmann et al., 2008). Studies using these recording conditions, and Girk1^-/- and/or Girk4^-/- mice, have shown definitively that the inward currents evoked by cholinergic agonists are mediated entirely by GIRK channel activation (Bettahi et al., 2002; Posokhova et al., 2013; Anderson et al., 2018). Steady-state CCh-induced currents (pA) were normalized to cell capacitance (pF), and experiments that did not have stable, low access resistances (<20 MΩ) were not included.