Troponin I Tyrosine Phosphorylation Decreases Myofilament Ca2+ Sensitivity and Accelerates Deactivation

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The Ohio State University

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The post-translational modification of troponin by phosphorylation is a central mechanism to modulate calcium activated cardiac contraction. Over 95% of phosphorylation in the heart occurs on serine or threonine residues. It has been recently shown that phosphorylation of the troponin inhibitory subunit (TnI) at Tyr-26 is decreased in human heart failure with functional effects such as decreasing thin filament calcium sensitivity and accelerating calcium dissociation. This discovery has lead us to hypothesize that the phosphorylation of the other tyrosine residues in TnI, including TnI Tyr-29, Tyr-112 (and TnI Tyr-134 found mouse cardiac muscle), will also exhibit functional effects. Kinase prediction calculation suggests that these additional tyrosine residues are even more likely to be phosphorylated than Tyr-26. To test the regulatory effects of TnI Tyr-phosphorylation, we generated, expressed and purified cardiac TnI containing phosphomimetic substitutions (Tyr to Glu or Tyr to Asp). Purified proteins were used to form the troponin complex and functional effects determined by measuring calcium binding to Troponin C (TnC) in reconstituted thin filaments. Our results confirm Tyr-26 pseudo-phosphorylation decrease calcium binding to TnC on the thin filament and demonstrate a similar decrease in calcium binding to TnC with the pseudo-phosphorylation of Tyr-29 in thin filaments containing human cardiac TnI and thin filaments containing mouse cardiac Tyr- 134 TnI. To investigate the effects of TnI Tyr phosphorylation on myofilament deactivation, we measured the rate of calcium dissociation from TnC. Results demonstrate filaments containing Tyr-26 and Tyr- 29 pseudo-phosphorylated TnI accelerate the rate of calcium dissociation from TnC. Based upon these results we conclude that the pseudo-phosphorylation of TnI at these additional Tyr-residues play a functional role in modulating force production of the heart.



Troponin Tyrosine Phosphorylation