Sarcomere Lengthdependent Effects on the Ca2+-Troponin Regulation in Skinned Myocardial Fiber from Titin RBM20 Deletion Mice

Abstract

The length dependent activation of the myofilaments has been considered the cellular basis underlying the Frank-Starling law of the heart trough dynamic and complex interplays between a multitude of thick- and thin-filament components. Among these components, both troponin and giant protein titin are likely key players, but the mechanism, by which the length dependent myofilament Ca2+ regulation is linked functionally to titin, is elusive. In this study we investigate the link using in situ FRET technique to monitor how the length dependent Ca2+-induced conformational change of the N-domain of cTnC within myocardium is modulated by alteration of titin compliance that directly determines the passive tension of myocardium. To achieve the objective, FRET donor and acceptor modified cTnC(13C/51C)AEDANS-DDPM was reconstituted into chemically-skinned myocardial fibers from wild-type mice and RBM20 deficient mice that expresses more compliant N2BA titin. The Ca2+-induced conformational change in cTnC under different conditions were quantitatively characterized using time-resolved FRET measurements. Our results from wild type myocardium showed that sarcomere length dependent enhancement of Ca2+-troponin regulation was found to be directly associated with strong crossbridge and it was diminished when the strong crossbridge was inhibited. When measurements were performed with skinned myocardial fibers from RBM20 deletion animals, the length dependent change was blunted. These results support that titin based passive tension development of myocardium is indeed involved in sarcomere length modulated Ca2+-troponin regulation and that the strong crossbridge plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation.