In Situ Time-Resolved FRET Reveals Effects of Sarcomere Length on Cardiac Thin-Filament Activation

Abstract

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-μm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.

Functional significance of C-terminal mobile domain of cardiac troponin I

“…Cardiac troponin complex containing wild-type cTnC, cTnI(1–167) or cTnI(1–193) and wild-type cTnT was reconstituted at a molar ratio of 1:1:1 cTnC:cTnI:cTnT using a protocol published previously [40, 60]. Endogenous troponin in the detergent skinned rat papillary muscle fibers were exchanged by overnight incubation of fibers in exchange solution containing 25µM of reconstituted cTn complex followed by another overnight incubation of fibers in exchange solution containing 10µM of only cTnC.…”

“…The incubation was followed by a 20-minute sonication of samples in a water bath at 4°C and centrifugation at 10k rpm. Equal amounts of supernatants fractions were loaded to 4–20% SDS-PAGE gels for Western blotting analysis following the protocol established previously [40, 61, 62]. Loading amounts were standardized by estimating the total protein concentration using a Protein Assay Kit (Biorad #5000121).…”

“…Different characteristics of the myofilaments contribute to LDA, such as the inter-filament lattice spacing [24–26], the distinctive structural changes in thick and thin filaments [27, 28], the orientation and number of cross-bridges [29–31], titin-based passive tension [32–37], and the increase in positive feedback on myocardial contractile regulation [38–40]. Our previous studies have shown that Ca 2+ -induced opening of the N-domain of cTnC [40], and Ca 2+ -induced movement of C-terminus of cTnI [41] are both SL- and cross-bridge-dependent. These results suggest an interplay between the thin filament- and thick filament-based regulatory mechanisms of LDA [27].…”

Thin Filament Structure and the Steric Blocking Model

“…A view emerged that Ca 2+ binding to troponin, thereby releasing tropomyosin inhibition, is not sufficient to activate the thin filament fully. Indeed, if a multiple step coupled reaction is involved in the myofilament activation process, as shown years later, any form of myosin-induced tropomyosin movement linked to Ca 2+ activation may promote the binding of more myosin to actin by "potentiating" local tropomyosin movement as part of a cooperative dynamic (61,130,133).…”

“…Tropomyosin is often described as fundamental to the cooperative allosteric regulation of muscle contraction (47,129,130,133,145). The term allosteric when conjoined with descriptions of steric effects can be confusing.…”