(A) Ventricular cardiac myocytes isolated from nontransgenic (NTG) or conditional Nix-overexpressing (Nix OE) mouse hearts were loaded with Fura-2 AM and analyzed for caffeine-stimulated [Ca²⁺]i by monitoring the 510 nm emission during rapidly alternating excitation at 340 and 380 nm. Data are reported as the 340:380 nm emission ratio. A representative pair of tracings is shown (left). Group data (right) represent mean ± SEM of 24 NTG and 42 Nix OE cardiac myocytes from 3 pairs of hearts. Caf, caffeine. (B) Ventricular cardiac myocytes isolated from WT or Nix-knockout (Nix^–/–) mouse hearts were loaded with Fura-2 AM and analyzed for caffeine-stimulated [Ca²⁺]i as above. A representative pair of tracings is shown (left). Group data (right) represent mean ± SEM of 25 WT and 50 Nix^–/– cardiac myocytes from 5 pairs of hearts. (C) Crude cardiac extracts from Nix-null (Nix^–/–) and WT hearts were subjected to immunoblotting for RYR, SERCA, NCX, PLN, and CSQN (50 μg protein/lane). (D) Representative peak I_Ca traces recorded from a holding potential of –50 mV to the indicated test potentials in patch-clamped isolated Nix-null and WT cardiac myocytes. (E) Representative traces of Na⁺/Ca²⁺ exchange current induced by a rapid solution change from 150 mM Na⁺ to 150 mM Li⁺ (indicated above) at a holding potential of –40 mV, recorded from Nix-null and WT cardiac myocytes.