Journal article
In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation
Biophysical journal, Vol.107(3), pp.682-693
08/05/2014
Handle:
https://hdl.handle.net/2376/107139
PMCID: PMC4129473
PMID: 25099807
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.
Metrics
10 Record Views
Details
- Title
- In situ time-resolved FRET reveals effects of sarcomere length on cardiac thin-filament activation
- Creators
- King-Lun Li - Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WashingtonDaniel Rieck - Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WashingtonR John Solaro - The Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IllinoisWenji Dong - 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
- Publication Details
- Biophysical journal, Vol.107(3), pp.682-693
- Academic Unit
- Chemical Engineering and Bioengineering, School of
- Publisher
- United States
- Grant note
- R01 HL80186-5S1 / NHLBI NIH HHS R21 HL109693 / NHLBI NIH HHS T32GM008336 / NIGMS NIH HHS R01 HL064035 / NHLBI NIH HHS T32 GM008336 / NIGMS NIH HHS P01 HL062426 / NHLBI NIH HHS R01 HL64035 / NHLBI NIH HHS R01 HL080186 / NHLBI NIH HHS
- Identifiers
- 99900546985601842
- Language
- English
- Resource Type
- Journal article