Titin-Based Modulation of Sarcomere Structure as Revealed by Equatorial X-Ray Diffraction

Abstract

Myofilament Length Dependent Activation (LDA) is the cellular mechanism underlying the Frank-Starling Law of the Heart. LDA is defined as an increase in force for a given amount of calcium when the muscle is stretched to a longer sarcomere length. Despite its physiological importance, the sensor that underlies LDA is not known. To address this issue we use X-ray diffraction of intact twitching rat papillary muscle where muscle initially at maximum sarcomere length (Lmax) is quickly released to slack length (Slack). X-ray patterns are taken is a 10 ms window just prior to force generation.Previous analyses of the data did not support the hypothesis that there was a radially outward movement of myosin heads at Lmax requiring some other explanation. The equatorial portion of our X-ray patterns show 5 pairs of well-resolved diffraction spots from the 1,0 to the 3, 0 equatorial reflection. This allowed calculation of two dimensional electron density maps (ED maps) comparing Lmax and Slack from both wild type rats and mutant rats with exceptionally long titin (J Mol Cell Cardiol. 44:983-91 2008) where the passive tension generated at Lmax, as well as LDA, is much reduced. Difference ED maps (Lmax-Slack) from WT rats indicate a better localization of both thick and thin filaments at Lmax plus the existence of bridging density joining the thick and thin filaments at Lmax and not at slack. In difference ED maps from the titin mutant rats, the bridging structures are absent and only the thick filaments appeared better localized. These results support a model where a small number of crossbridges transmit titin-based strain from the thick filament to the thin filament as part of the mechanism of LDA. Supported by NIH R01HL075494 and 9 P41 GM103622.