Abstract 312: Troponin I Phosphorylation Crosstalk
The troponin (Tn) complex is a critical regulatory and integrative hub for myofilament post-translational modifications that regulate cardiac contraction. Beta-adrenergic-induced protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI) at Ser-23/24 is a major myofilament cardiac contractile modulator. In addition to this PKA-induced phosphorylation, cTnI can be simultaneously phosphorylated at a number of other residues. To fully understand the molecular mechanisms that underlie cardiac muscle regulation at the myofilament level, cTnI phosphorylations must be studied as integrated events. As an initial step towards understanding the integration of these events, we employed a phosphomimetic approach to investigate the effect of cTnI Ser-150 pseudo-phosphorylation (TnI-S150D) on cTnI PKA pseudo-phosphorylation (TnI-S23/24D) function within the thin filament. The combinatorial effect of these phosphorylation events were determined by measuring the binding of calcium to troponin C in thin filaments reconstituted with human Tn. The steady-state calcium sensitivity of TnC was increased ∼2.3 fold in thin filaments reconstituted with TnI-S150D containing Tn, while TnI-S23/24D containing filaments exhibited an ∼3.9 fold decrease of calcium sensitivity. Importantly, in the presence of both cTnI pseudo-phosphorylations (TnI-S23/24/150D) calcium binding was not different from that of wild-type. Likewise, calcium dissociation from thin filaments reconstituted with TnI-S23/24D was increased by ∼3.5 fold, while TnI-S150D containing filaments demonstrated a decrease in calcium dissociation of ∼2.3 fold compared to wild-type filaments. Interestingly the combined pseudo-phosphorylations of TnI-S23/24/150D only blunted TnI-23/24D calcium dissociation kinetics to ∼1.2 fold of the TnI-S23/24D filaments alone. These findings demonstrate the integrated effects of cTnI site-specific phosphorylation crosstalk to affect the function of cTnI PKA-induced phosphorylation. These data further highlight the importance of understanding the integrated role of Tn phosphorylation crosstalk on cardiac contractile regulation.
- © 2012 by American Heart Association, Inc.