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 X 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 X P/g wet heart. Similar values, 6.5 and 7.1 mg sarcoplasmic reticulum x 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 x 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 10~5Mfree calcium and 170 nmoles calcium/g wet heart at lO^Mfree calcium. KEY WORDSheart calcium uptake rate excitation-contraction coupling dog homogenate of heart oxalate• Estimating the amount of sarcoplasmic reticulum in cardiac muscle is difficult because of the lack of a specific marker and because a procedure for the quantitative isolation of a pure fraction has not been developed. Estimates made from electron micrographs (1, 2) provide information only on the volume of muscle occupied by the sarcoplasmic reticulum. Before functional information can be derived from such data, it is necessary to know the mass that such volumes represent and the specific activity of the function under consideration, e.g., the rate of calcium uptake per mass of sarcoplasmic reticulum.
The amounts of calcium required to achieve various levels of myofibrillar activation in the dog heart were determined by measuring the dependence of myofibrillar calcium binding, myofibrillar adenosinetriphosphatase (ATPase), and isometric tension on free calcium concentration. Myofibrillar ATPase was half-maximal at 2.4 x 10 -6 M free calcium, and tension development was half-maximal at 2.0 x 10 -6 M free calcium. No simple relation between calcium binding and activation was found. For example, between 10 -8 M and 10 -6 M free calcium, an appreciable amount of calcium was bound to the myofibrils, but there was little activation of isometric tension. On the other hand, myofibrillar calcium binding was not saturated at levels of free calcium at which both tension and ATPase were maximal; therefore, it appears that only a portion of the total myofibrillar calcium binding sites control ATPase and tension. Using the information derived from the binding and activation studies together with our determination of the myofibrillar content of the dog heart, 47.5 mg myofibrillar protein/g wet heart, we calculated the calcium required to achieve various levels of myofibrillar activation in the intact ventricle. By this calculation method, development of half-maximal tension required 22.4 µmoles calcium/kg wet heart, and development of maximal isometric tension required 92.8 µmoles/kg wet heart.
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