Benjamin Templeton – Biomedical Science

Project Title: Regulation of troponin-I calpain mediated proteolysis by Tyr-26 phosphorylation

Project Mentor: Brandon Biesiadecki – Physiology and Cell Biology

Abstract:

Introduction:  Cardiac muscle contraction and relaxation is regulated by the interaction of myosin with actin to produce force.  Myosin’s interaction to actin is dependent upon the binding of the signaling molecule Ca²⁺ to myofilament regulatory proteins troponin and tropomyosin.  At low intracellular Ca²⁺, tropomyosin and troponin I (TnI) of the troponin complex sterically inhibit myosin binding to actin.  Upon increased concentration of Ca²⁺intracellularly, troponin and tropomyosin undergo structural rearrangement on the actin filament, removing the steric inhibition of TnI and tropomyosin, thus allowing myosin and actin interaction.  The inhibitory subunit of TnI can be post-translationally modified to alter the force-per-unit Ca²⁺ created by myosin binding to actin, thus modulating cardiac contraction and relaxation.  Myocardial ischemia (MI) and subsequent cardiac myocyte death is a significant contributor to development of heart failure.  During MI, elevated intracellular Ca²⁺ results in activation of the protease calpain which cleaves TnI at its amino terminus, negatively affecting cardiac function.  Previous research has found that phosphorylation of TnI at amino acid residues Ser-23/24 can reduce cleavage by calpain.  In addition to Ser-23/24, TnI is also phosphorylated at Tyr-26 during ischemia with unknown functions.  We hypothesized that phosphorylation at the Tyr-26 site will reduce cleavage by calpain similarly to phosphorylation at Ser-23/24.

Methods: Calpain cleavage assays of purified TnI and troponin complex containing unphosphorylated (wild-type), Ser-23/24 pseudo-phosphorylated, and Tyr-26 pseudo-phosphorylated TnI were employed to evaluate TnI susceptibility to calpain.  Western blot was used to quantify TnI cleavage of each protein at varied time points.  Changes in cleavage levels were assessed.

Results: The presence of TnI Tyr-26 pseudo-phosphorylation decreased calpain-mediated TnI cleavage compared to wild-type non-phosphorylated TnI.  The cleavage levels were found to be like that of TnI Ser-23/24 pseudo-phosphorylation in both purified TnI and the troponin complex.

Conclusion: Calpain-mediated cleavage reduction caused by TnI Tyr26 pseudo-phosphorylation is significant to maintain myofibril force production, potentially improving the outcome of MI by limiting cardiac myocyte death.  Future directions will look at the effect of TnI Tyr26 pseudo-phosphorylation on calpain-mediated cleavage of TnI in myofilaments isolated from cardiac myocytes to better simulate physiological conditions.