Phosphorylation of troponin I and phospholamban during catecholamine stimulation of rabbit heart

Abstract

During adrenergic stimulation heart cells develop increased force and there is an increase in the rate of rise and fall of force. These changes are thought to be triggered by the binding of catecholamine to the β-receptor, which in turn signals alterations in membranes and the contracto-regulatory protein complex by multiple, coordinated mechanisms involving Ca²⁺, cyclic nucleotides and protein phosphorylation^1,2. This view is supported by results of in vitro studies with cardiac myofibrils^3,4 and vesicles of sarcoplasmic reticulum (SR)^5–7 showing that these organelles have sites of protein phosphorylation that may act as effectors of the adrenergic signal giving rise to the altered twitch dynamics. Phosphorylation of troponin I (TnI), a regulatory protein of cardiac myofibrils, results in a decreased steady-state affinity of troponin (TnC) for Ca²⁺, an increase in the off rate for Ca²⁺ exchange with TnC⁸ and a rightward shift of the relationship between free Ca²⁺ and myofibrillar force⁹ or ATPase^3,4. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, increases the velocity of Ca²⁺ transport by SR vesicles, the affinity of the transport protein for Ca²⁺ and the turnover of elementary steps of the ATPase reaction^5–7,10,11. Taken together, these findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Yet, although it is known that TnI is phosphorylated during adrenergic stimulation of beating heart^12–14 there has been no clear evidence that phospholamban is phosphorylated at the same time. We now report that during the peak of the inotropic response of the rabbit heart to perfusion with isoproterenol, there is simultaneous phosphorylation of TnI and an 11,000-molecular weight (M_r) protein associated with SR, which is probably monomeric phospholamban.