Abstract 5404: Perturbation of Tropomyosin Amino Acid Residues Binding to Actin at the “Off” State of Tropomyosin Increase Cardiac Muscle Dynamics

Abstract

Tropomyosin (TM), an essential thin filament protein, binds along F-actin in association with troponin and plays a central role in regulating cardiac muscle dynamics. Previous structural studies have shown that TM molecule consists of 14 negatively charged, alternating α- and β-bands along its length, responsible for binding to actin. The 7 α-bands of TM are implicated in the binding to actin at the “off” state where actomyosin crossbridging is blocked. The 7 β-bands of TM bind to actin at the “on” state that permits actomyosin interaction and results in force production. In this study, we hypothesize that if the α-bands are perturbed by changing the charged residues at that region, it will cause TM molecule “slipping” at the “off” state that will allow TM to be in an extended “on” state leading to enhanced cardiac muscle dynamics. To test this hypothesis we have generated TG mouse lines expressing α-TM with mutations in the α-bands located in the carboxy terminus (α-TM CTM). More specifically, codons encoding negatively charged amino acids (aspartate or glutamate) at positions 212, 219, 223, 255 and 259 were changed to lysine by site-directed mutagenesis. Molecular analyses revealed one of the TG lines α-TM CTM-6 expressed 100% of mutant protein. Echocardiogram data analyses showed that α-TM CTM-6 hearts exhibited significant decrease in heart rate (10%; p<0.05, n=6)) in the absence or presence of dobutamine. Furthermore, the percentage fractional shortening and ejection fraction were significantly increased (13% and 15%, respectively, p<0.05, n=6) in TG hearts when compared to non-transgenic (NTG) controls. To investigate the mechanisms for the increase in cardiac muscle dynamics in the TG hearts, we measured force and calcium levels in the papillary muscle fiber. Force- [Ca2+]i data analyses showed that the α-TM CTM TG hearts produced more force per given [Ca2+]i when compared to NTG hearts, indicating that α-TM CTM myofilaments exhibit an increase in Ca2+ sensitivity. Protein modeling analyses show surface charges of α-TM CTM molecule is altered. We propose that the changes in charged residues in the α-bands located in the C-terminus of TM molecule perturb the “off” state, which augment the crossbridge kinetics and lead to enhanced cardiac muscle performance.