title: 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.
authors.affiliation: Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington.
authors.forename: King-Lun
authors.initials: KL
authors.lastname: Li
authors.affiliation: Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rieck
authors.affiliation: The Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, Illinois.
authors.forename: R John
authors.initials: RJ
authors.lastname: Solaro
authors.affiliation: Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington; Integrative Neuroscience and Physiology, Washington State University, Pullman, Washington. Electronic address: wdong@vetmed.wsu.edu.
authors.forename: Wenji
authors.initials: W
authors.lastname: Dong
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology; D051381:Rats; D012518:Sarcomeres / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000199:Actins; D019209:Troponin C; D002118:Calcium
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