Calculation of total cytosolic Ca²⁺ buffers in RVCM and LVCM. (A and B) Ca²⁺ binding ratio values from five times of experiments for LVCM (A) and RVCM (B) were plotted as a function of [Ca²⁺]_i. Total buffer was the sum of fura-2 and hypothetical Ca²⁺ binding species S. The variables reflecting chemical properties of buffers were calculated by fitting curves using Eq. 2 (total buffer) and Eq. 1 (intrinsic buffer). (C and D) The amount of total buffer (B_tot) is larger in LVCM (C), while that of fura-2 (Fura_tot) is not different (D). (E)S_tot, total amount of intrinsic buffer, is also larger in LVCM. (F) The dissociation constants of intrinsic buffer (K_d,S) were acquired using Eq. 3. (G and H) Ca²⁺ bound to total buffer, fura-2, and intrinsic buffer was calculated (Calc.) as a function of [Ca²⁺]_i with the differential equation and buffer parameters. The dotted lines indicate the Ca²⁺ bound to the intrinsic buffer. All statistical tests performed using Student’s t test (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.