Role of Troponin I Phosphorylation in Protein Kinase C-mediated Enhanced Contractile Performance of Rat Myocytes

Abstract

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...

Designing Heart Performance by Gene Transfer

“…The increased peak amplitude of shortening observed in response to ET-1 was also significantly blunted in myocytes expressing ssTnI. These findings demonstrated that PKC directly phosphorylates cTnI and cTnI phosphorylation during ET-1 activation of PKC accelerates relaxation and contributes to the increased amplitude of contraction (958).…”

“…In experiments with the neonatal cardiac isoform slow skeletal TnI (ssTnI), ET-1 activation of PKC did not significantly phosphorylate the ssTnI isoform (958). All indices of relaxation were prolonged in response to ET-1 in myocytes expressing the nonphosphorylatable ssTnI.…”

“…PKC-dependent cTnI phosphorylation decreases myofilament Ca 2ϩ sensitivity (92,653) and correlates with peak PKC-dependent contractile responses using multiple agonists (152, 426,598,688). However, the role cTnI phosphorylation plays in the contractile response has been difficult to define, in part because PKC phosphorylation of cTnI is linked to both accelerated and decreased relaxation rates (689,782,958,962). Gene transfer studies have established that PKC-mediated cTnI phosphorylation accelerates relaxation rate in response to the neurohormone ET-1.…”

“…A much larger body of literature has focused on direct and indirect modulation of cardiac function by PKC using gene transfer approaches (556,958). Divergent contractile responses are observed in response to PKC activation, with reports of both increases and decreases in contractile performance.…”

Mitochondrial Dysfunction and Apoptosis Underlie the Pathogenic Process in α-B-Crystallin Desmin-Related Cardiomyopathy

“…The cells tolerated significant levels of CryAB expression, with both the overall morphology and integrity of the contractile apparatus preserved ( Figure 1B), consistent with previous data that confirmed cell integrity and the lack of nonspecific structural or functional changes on adenomediated gene transfer. 10,11 Cells transfected with CryAB…”