Phosphorylation of Cardiac Troponin I at Tyrosine 26 Decreases Thin Filament Calcium Sensitivity

Abstract

The troponin complex is a critical molecular switch involved in transducing the calcium activating signal into contraction. Troponin I (TnI), the inhibitory subunit of the complex, is phosphorylated as a key regulatory mechanism to alter the calcium regulation of contraction. Altered cardiac contraction is a hallmark of heart failure with several studies demonstrating increased myofibrillar calcium sensitivity. Recent work has identified a novel phosphorylation of TnI at Tyr-26 that is decreased in heart failure with unknown functional effects. Similar to the location of the desensitizing TnI Ser-23/24 phosphorylation, TnI Tyr-26 is located in the unique cardiac TnI N-terminal extension. These data lead us to hypothesize that the N-terminal Tyr-26 phosphorylation of TnI decreases calcium sensitivity of the thin filament, the loss of which may contribute to the altered calcium sensitivity observed in heart failure. To assess the regulatory effects of Tyr-26 phosphorylation, we employed recombinant human cardiac TnI containing phosphate at Tyr-26 induced by treatment with a tyrosine kinase and TnI Tyr-26 phosphomimetic substitutions (Glu or Asp). The effect of TnI Tyr-26 phosphorylation on myofilament calcium sensitivity was assessed by measuring calcium binding to troponin C (TnC) in reconstituted thin filaments. Results demonstrate both Tyr-26 phosphorylation and phosphomimetics decrease calcium binding to TnC compared to filaments reconstituted with non-phosphorylated TnI. To further investigate the effects of TnI Tyr-26 phosphorylation on myofilament deactivation we measured the rate of calcium disassociation from TnC. Results demonstrate filaments containing either Tyr-26 phosphorylated TnI or phosphomimetics increase the rate of calcium dissociation from TnC. Our findings suggest that TnI Tyr-26 phosphorylation functions similarly to Ser-23/24 N-terminal phosphorylation to decrease myofilament calcium sensitivity and increase myofilament relaxation. The loss of TnI Tyr-26 phosphorylation may therefore contribute to altered cardiac contraction in heart failure.