Monitoring Cardiac Troponin Structural Changes using In-Situ Time-Resolved FRET: Implications on the Regulatory Roles of Cross-Bridges and Sarcomere Length

Abstract

During cardiac thin filament activation, the N-domain of Ca2+-binding cardiac troponin C (N-cTnC) interacts with the actomyosin inhibitory troponin I (cTnI) subunit, which concomitantly opens the cTnC N-domain and leads to force generation. Recently, we used in situ steady state FRET measurements based on N-cTnC opening to determine that strongly bound cross-bridges (XBs) stabilize this Ca2+-sensitizing N-cTnC-cTnI interaction. However, the method was unable to determine how N-cTnC opening is affected by sarcomere length (SL). In this study, we used time-resolved in situ FRET to monitor the effects of Ca2+-occupancy, XB state, and SL on N-cTnC opening in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM) modified double-cysteine mutant cTnC(13C/51C)AEDANS-DDPM was reconstituted into skinned muscle fibers to examine the N domain of cTnC (N-cTnC) opening. To study the effect of SL on structural transitions of cTnC, we monitored the protein structural transitions at low and high [Ca2+] and SL 1.8 and 2.2 μm. Mg2+-ADP and sodium orthovanadate (Vi) were used to examine the effects of non-cyclng strong and weak XBs, respectively. We found that strongly bound XBs alter structural transitions of cardiac troponin only at 2.2 μm. On the other hand, Vi blunted the SL dependent opening of N-cTnC such that weak XBs have no effect on N-cTnC at either [Ca2+] or SL. In addition, distance distribution analysis indicated that N-cTnC adopts four unique conformations associated with the four states of thin filament regulation, and that N-cTnC conformational equilibria are caused by cycling XBs. Based on our findings, we conclude that the observed dependence of myosin positive feedback regulation on SL is an important determinant of the Frank-Starling law of the heart.