Our goal was to define the role of phosphorylated cardiac troponin-I in the adult myocyte contractile performance response to activated protein kinase C. In agreement with earlier work, endothelin enhanced both adult rat myocyte contractile performance and cardiac troponin-I phosphorylation. Protein kinase C participated in both responses. The role of cardiac troponin-I phosphorylation in the contractile function response to protein kinase C was further investigated using gene transfer into myocytes of troponin-I isoforms/mutants lacking one or more phosphorylation sites previously identified in purified cardiac troponin-I. Sarcomeric replacement with slow skeletal troponin-I-abrogated protein kinase C-mediated troponin-I phosphorylation. In functional studies, endothelin slowed relaxation in myocytes expressing slow skeletal troponin-I, while the relaxation rate increased in myocytes expressing cardiac troponin-I. Based on these results, acceleration of myocyte relaxation during protein kinase C activation largely depended on cardiac troponin-I phosphorylation. Experiments with troponin-I isoform chimeras provided evidence that phosphorylation sites in the amino portion of cardiac troponin I-mediated the protein kinase C acceleration of relaxation. The cardiac troponin-I Thr-144 phosphorylation site identified in earlier biochemical studies was not significantly phosphorylated during the acute contractile response. Thus, aminoterminal protein kinase C-dependent phosphorylation sites in cardiac troponin-I are likely responsible for the accelerated relaxation observed in adult myocytes.
Troponin I (TnI)1 is a key regulatory protein within the thin filament of the contractile apparatus. Different isoforms of TnI influence myofilament Ca 2ϩ sensitivity (1) and contribute to developmental changes in myofilament function. In particular, the transition from slow skeletal TnI (ssTnI) expression in embryonic/fetal hearts to the cardiac isoform expressed exclusively in adult hearts appears to "fine tune" myofilament regulatory function (1, 2). The Ca 2ϩ sensitivity properties of the myofilaments also can be modified by TnI phosphorylation (3). For example, -adrenergic activation of protein kinase A (PKA) phosphorylates cardiac TnI (cTnI), which reduces myofilament Ca 2ϩ sensitivity (3) and contributes to accelerated relaxation in intact adult myocardium (4). TnI also is phosphorylated by activated protein kinase C (PKC) (5, 6), and while an association exists between PKC-mediated cTnI phosphorylation and contractile function (7), the specific role of TnI in the contractile response remains unclear.The activation and expression pattern of multiple PKC isoforms in the heart changes under pathophysiological conditions, which may influence the role of PKC-mediated TnI phosphorylation in the contractile function response. In adult myocardium, the classical ␣ isoform, as well as the novel ␦ and ⑀ isoforms are normally expressed in both rats and humans (8 -10), while expression of the classical  isoform is absent from adult m...