Passive Ca²⁺ binding in ventricular myocardium of neonatal and adult rats

In this study, passive Ca²⁺ binding was determined in ventricular homogenates (VH) from neonatal (4-6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca²⁺ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca²⁺ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca²⁺] ([Ca²⁺](F)) was measured with Indo-1 and bound Ca²⁺ ([Ca²⁺](B)) was the difference between [Ca²⁺](F) and total Ca²⁺. Apparent Ca²⁺ dissociation constants (K(d)) for BAPTA and Indo-1 were increased by 10-20mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl₂ additions (2.5-10 nmoles) yielded δ[Ca²⁺](B)/δ[Ca²⁺](F) for the sum of [Ca²⁺](B) at all three classes of binding sites. From this function, the apparent number of endogenous sites (B(en)) and their K(d) (K(en)) were determined. Similar K(en) values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, B(en) was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ~300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that B(en) determined in VH underestimates cellular B(en) by 29%. Although B(en) values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing B(en) per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca²⁺ binding capacity at high-affinity sites is ~300 and 176 mmol/l cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca²⁺ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.