Estimating the Functional Capabilities of Sarcoplasmic Reticulum in Cardiac Muscle

A method was developed to estimate the amount of calcium which can be bound to the sarcoplasmic reticulum of the dog heart. Incubation conditions that permitted calcium oxalate uptake (steady-state filling or uptake rate) to be used as a marker for sarcoplasmic reticulum vesicles in homogenates and microsomal fractions were developed. By dividing the values for peak steady-state filling of a cardiac homogenate by those for peak steady-state filling of a sarcotubular-enriched fraction, we obtained a sarcoplasmic reticulum-homogenate ratio with units of mg sarcoplasmic reticulum × P/g wet heart, where P is the unknown fractional purity of the microsomal fraction. The mean value for the sarcoplasmic reticulum-homogenate ratio obtained from the steady-state filling studies was 6.9 mg sarcoplasmic reticulum × P/g wet heart. Similar values, 6.5 and 7.1 mg sarcoplasmic reticulum × P/g wet heart, were obtained when the rate of calcium oxalate uptake was used as the functional parameter for calculation of the sarcoplasmic reticulum-homogenate ratio. Evidence that sarcoplasmic reticulum vesicles in the homogenate are functionally the same as those in the isolated fraction was obtained. Calcium binding by the sarcotubule fraction was measured by either a spectrophotometric (murexide) or a Millipore filtration technique. Multiplication of the sarcoplasmic reticulum homogenate ratio by the amounts of calcium bound by sarcoplasmic reticulum vesicles (nmoles calcium/[mg sarcoplasmic reticulum × P]) provided an estimate of the ability of the cardiac sarcoplasmic reticulum to bind calcium. Application of this method indicated that the sarcoplasmic reticulum could bind 300–400 nmoles calcium/g wet heart at 10−5M free calcium and 170 nmoles calcium/g wet heart at 10−6M free calcium.