Abstract
The Ca2+ binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca2+ binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca2+ affinity by 27% in the steady-state measurement and increased the Ca2+ dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca2+ sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca2+ sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca2+ sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca2+ sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent.
Highlights
The cardiac troponin trimeric complex plays an essential role in regulating cardiac contractility
The complexes were found to be stable in the absence of free cardiac troponin C (cTnC), and free cardiac troponin (cTn) complexes were not detected in the reconstituted thin filament preparations (TFP)
A measurable Ca2+-dependent IAANS fluorescence decrease was observed in all four cTn complexes and no significant differences in the steady-state Ca2+ binding or dissociation parameters were observed (Table 2)
Summary
The cardiac troponin (cTn) trimeric complex plays an essential role in regulating cardiac contractility. It consists of a highly conserved Ca2+ binding subunit (cTnC) [1], an inhibitory subunit (cTnI), and a tropomyosin binding subunit (cTnT). Ca2+ binding to cTnC triggers conformational changes in the cTnI-actin interaction and other thin filament proteins. These changes produce strong, forcegenerating interactions between actin and myosin. The Ca2+induced disruption of the cTnI-actin interaction is modulated by the PKA phosphorylation of cTnI, feedback from strong actinmyosin interaction and is is highly dependent on the sarcomere length [2]
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