Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Monday Speaker Abstracts

WALKING A THIN (FILAMENT) LINE: INTEGRATING TIME-RESOLVED FRET AND COMPUTATION TO IDENTIFY NOVEL PATHOGENIC MECHANISMS IN HYPERTROPHIC CARDIOMYOPATHY Jil C. Tardiff ; 1 University of Arizona, Biomedical Engineering, Tucson, AZ, USA One of the most common and earliest clinical manifestations of hypertrophic cardiomyopathy (HCM) is a failure of the heart to efficiently relax in the context of an increase in hemodynamic demand. While in later stages of disease progression the mechanism for this impaired diastolic performance is multifactorial, the primary role of the cardiac thin filament (cTF) in the molecular initiation of relaxation via alterations in calcium exchange kinetics suggests a potential primary “trigger” for early onset disease. The physical interface between the N-lobe of TnC (Site II - calcium binding/release) and the N-terminus of cTnI (Ser23/24 PKA-substrate site) is a complex and dynamic domain that is intricately involved in the acceleration of calcium dissociation rate, tuning diastolic performance to meet hemodynamic demand. The structural mechanism for this observation not been studied in reconstituted cTF in-vitro systems, limiting our understanding of biologic and pathogenic HCM mechanisms. We hypothesize that N-cTnI contributes to stabilizing the calcium atom in Site II via direct interactions with cTnC and that phosphorylation of Ser 23/24 alters these interactions allowing for an increase in calcium dissociation rate. To address this, we performed TR-FRET experiments between cTnI-A28C to cTnC-84C, cTnI A17C to cTnC-84C, and cTnI-A9C to cTnC-84C in cTF to map N-cTnI with respect to cTnC under different biochemical conditions and in the presence of cTnT HCM mutations. We found that N-cTnI is more disordered in the absence of calcium and become more ordered and closer to cTnC upon the addition of calcium. Phosphorylation of Serine 23/24 caused an increase in distance and FWHM for the A28C and A17C sites, supporting an increase in calcium dissociation rate. MD simulations were employed to determine both average structures and predicted interactions. We predict that this central mechanism is disrupted in a subset of HCM mutations, representing a potential novel therapeutic target.

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