Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells

Bundles of cells from rat right ventricular myocardium were made "hyperpermeable" by an overnight soak in 10 mM EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764). In this preparation the cytoplasmic concentration of Ca++ and ATP could be controlled while sarcolemmal receptors and enzymes were retained. The Ca sensitivity of the tissues (as indicated by the pCa for 50% maximum activation) was altered to different extents in the presence of [32Pgamma]ATP by treatment with cyclic nucleotides, catecholamines, or a low concentration of nonionic detergent. The proteins of the tissue were then isolated by SDS-polyacrylamide gel electrophoresis, and the identity of 32P-labeled proteins was determined. The Ca sensitivity is inversely related to the relative amount of 32P incorporated into the inhibitory subunit of troponin (TNI). Extrapolation of the relation to the lowest Ca sensitivity observed gives a stoichiometry of about 0.8 mol PO4 per mol TNI. These results support the hypothesis that Ca sensitivity of cardiac myofibrils is regulated by a phosphrylation of TNI that is stimulated by cyclic AMP (cAMP) and inhibited by cGMP.