Abstract 4351517: Desensitization of the Cardiac Troponin Complex by TnI Phosphorylation and Epigallocatechin-3-gallate

Introduction: The objective of this research is to experimentally demonstrate desensitization of thin filaments to Ca 2+ due to troponin I (TnI) phosphorylation and (-)-epigallocatechin-3-gallate (EGCG) binding and to explain the molecular mechanisms via computational modeling. While TnI inhibitory function regulates myocardial contraction via systolic Ca 2+ -dependent activation of thin filaments, its function can be modulated via phosphorylation of Ser23/24 at the N-terminal region of TnI, leading to an apparent Ca 2+ - desensitization. Similarly, EGCG found to be abundant in green tea desensitizes the troponin complex via mechanisms to be determined. The hypothesis is that TnI phosphorylation and EGCG binding desensitize thin filaments via enhanced stabilization of protein-protein interactions within the troponin complex. Methods: In vitro motility assays quantified Ca 2+ -dependent sliding velocities in phosphorylated TnI bound to thin filaments. AlphaFold3 recreated de novo structural models of phosphorylated TnI bound to the troponin (Tn) complex. Molecular docking simulations employed PyRx to model EGCG binding to Tn. Results: TnI phosphorylation and 20 µM ECGC treatments significantly reduced the maximum sliding velocity of thin filaments (-49±7%, p = 7x10 -6 ; and -58±8%, p =5x10 -5 respectively) and decreased the pCa 50 sensitivity (-3±2%, p = 1x10 -3 ; and -8±4%, p =4x10 -3 respectively) relative to controls. AlphaFold3 revealed that TnI phosphorylation led to the formation of an α-helix at the Ser23/24 phosphorylation sites across Tn complex isoforms four mammalian species including human. Molecular docking simulations localized the ECGC binding site onto the Tn complex that resulted from hydrogen-bonds between EGCG and the troponin C (TnC) C-lobe (residues 120-161) as well at the beginning of the TnI IT arm directly downstream from the TnI Ser 23/24 phosphorylation sites (residues 34-71). Conclusions: The results suggest that EGCG and TnI Ser23/24 phosphorylation desensitization mechanisms are allosteric, with phosphorylation modifying interactions between TnI and TnC N-terminal domains and EGCG between TnI and TnC C-terminal domain. The results of this wok could inform the development of more targeted therapies to treat diastolic heart diseases such as HFpEF and cardiomyopathies derived from over-sensitization of thin filaments to Ca 2+ .