2.1. Cell isolation and culture

All animal procedures were approved by the Teikyo University Chancellor's Animal Research Committee (Permit Number 12–031). Atrioventricular node and sinoatrial node were dissected out [15] from adult male guinea pig hearts (6–8 weeks old, 450–500 g body weight, SANKYO Labo Service Inc. Japan) and digested with collagenase and elastase in 0 Ca²⁺ Tyrode's solution (135 mM NaCl, 5.4 mM KCl, 0.33 mM NaH₂PO₄, 5.0 mM HEPES, 0.5 mM MgCl₂, and 5.5 mM glucose) as previously described [9,16]. Thereafter, the digested tissues were resuspended in high-glucose Dulbecco's Modified Eagle's Medium (0.45% glucose) supplemented with 10% fetal bovine serum (MP Biomedicals, Inc.) and triturated to obtain single cells. The cell suspension contained atrioventricular node cells as well as other types of cells naturally existing in the dissected tissue. 2 mL of the cell-containing medium was placed in a 35 mm culture dish (Greiner Bio-one) at a density of 2–5 × 10⁴ cells/cm². Isolated AVNCs and SANCs were cultured at 37 °C in a CO₂ (5 %) incubator. The culture medium was replaced every 3 days.