Myosin-actin crossbridge independent sarcomere length induced Ca sensitivity changes in skinned myocardial fibers: Role of myosin heads.

Sarcomere length-dependent activation (LDA) is essential to engaging the Frank-Starling mechanism in the beat-to-beat regulation of cardiac output. Through LDA, the heart increases the Ca sensitivity of myocardial contraction at a longer sarcomere length, leading to an enhanced maximal force at the same level of Ca. Despite its importance in both normal and pathological states, the molecular mechanism underlying LDA, especially the origin of the sarcomere length (SL) induced increase in myofilament Casensitivity, remains elusive. The aim of this study is to interrogate the role of changes in the state of myosin heads during diastole as well as effects of strong force-generating cross-bridges (XB) as determinants of SL-induced Ca sensitivity of troponin in membrane-free (skinned) rat myocardial fibers. Skinned myocardial fibers were reconstituted with troponin complex containing a fluorophore-modified cardiac troponin C, cTnC(13C/51C), and recombinant cardiac troponin I (cTnI) mutant, ΔSP-cTnI, in which the switch peptide (Sp) of cTnI was replaced by a non-functional peptide link to partially block the force-generating reaction of myosin with actin. We used the reconstituted myocardial fibers as a platform to investigate how Ca sensitivity of troponin within skinned myocardial fibers responds to sarcomere stretch with variations in the status of myosin-actin XBs. Muscle mechanics and fluorescence measurements clearly showed similar SL-induced increases in troponin Ca sensitivity in either the presence or the absence of strong XBs, suggesting that the SL-induced Ca sensitivity change is independent of reactions of force generating XB with the thin filament. The presence of mavacamten, a selective myosin-motor inhibitor known to promote transition of myosin heads from the weakly actin-bound state (ON or disordered relaxed (DRX) state) to the ordered off state (OFF or super-relaxed (SRX) state), blunted the observed SL-induced increases in Ca sensitivity of troponin regardless of the presence of XBs, suggesting that the presence of the myosin heads in the weakly actin bound state, is essential for Ca-troponin to sense the sarcomere stretch. Results from skinned myocardial fibers reconstituted with troponin containing engineered TEV digestible mutant cTnI and cTnT suggest that the observed SL effect on Ca sensitivity may involve potential interactions of weakly bound myosin heads with troponin in the actin/Tm cluster region interacting with cTnT-T1 and residues 182-229 of cTnT-T2. The mechanical stretch effects may then be subsequently transmitted to the N-cTnC via the IT arm of troponin and the N-terminus of cTnI. Our findings strongly indicate that SL-induced potential myosin-troponin interaction in diastole, rather than strong myosin-actin XBs, may be an essential molecular mechanism underlying LDA of myofilament.