Myosin-actin crossbridge independent sarcomere length induced Ca2+ 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 Ca²⁺ sensitivity of myocardial contraction at a longer sarcomere length, leading to an enhanced maximal force at the same level of Ca²⁺. 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 Ca²⁺sensitivity, 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 Ca²⁺ 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)_AEDANS-DDPM, 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 Ca²⁺ 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 Ca²⁺ sensitivity in either the presence or the absence of strong XBs, suggesting that the SL-induced Ca²⁺ 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 Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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.