Cardiac function and phosphorylation of contractile proteins

Abstract The primary regulation of cardiac contractility is probably through changes in the level of cytoplasmic free Ca2+. In the stimulation of contraction by catecholamines, secondary controls may be present at the level of the contractile proteins. Troponin-I, a subunit of the troponin complex of the thin filament, and C-protein, a thick filament component, are both phosphorylated in perfused hearts in response to catecholamines over time courses similar to that for the increase in contraction. Both proteins are also phosphorylated rapidly in vitro by cyclic-AMP-dependent protein kinase. Phosphorylation of troponin-I causes a decrease in the sensitivity of both cardiac myofibrillar ATPase and tension development of skinned fibre preparations to Ca2+, and also an increase in the rate of dissociation of Ca2+ from isolated troponin. These results support the hypothesis that the role of phosphorylation of cardiac troponin-I is to contribute to the increased rate of relaxation of the heart that is observed with catecholamines. C-protein is phosphorylated to a maximum of 4-5 mol phosphate per mole protein both in vivo and in vitro. At present, however, the functions of both C-protein itself and its phosphorylation are unknown. Dephosphorylation of these contractile proteins after catecholamine stimulation is slow in perfused heart, although the rate can be increased by cholinergic agents. Phosphorylase, in contrast, is rapidly dephosphorylated under these circumstances. Phosphoprotein phosphatases relatively specific for phosphorylase have been identified in rat heart, whereas troponin-I appears to be dephosphorylated by general phosphatases. These observations account for the different rates of dephosphorylation of phosphorylase and the contractile proteins, but do not explain the slow dephosphorylation of the latter. In control perfused hearts myosin P-light chain was 50 % phosphorylated, and this was not changed by perfusion with positive inotropic agents or by short-term ischaemia. It was also unchanged during long-term hormonal modifications. Perfusions with 32P1 in rat heart give a half-time for the turnover of phosphate bound to the P-light chain of 2-4 min, showing that the myosin light chain kinase and phosphatase are active in the heart. It is hypothesized that under control conditions the kinase is already fully active, and that an increase in cytoplasmic Ca2+ cannot therefore cause further activation of the enzyme.