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 10-20 mg VH protein/ml (from 0.35 to 0.92 mu M for Indo-1 and from 0.20 to 0.76 mu M for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5-10 nmoles) yielded delta[Ca2+](B)/delta[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 mu M, 0.69 +/- 0.13 mu M and 0.53 +/- 0.10 mu M, 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 similar to 300 and 213 mu mol/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 similar to 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.