Calcium Dependence of Myosin Filament Structure in Demembranated Trabeculae from Rat Heart

Abstract

The contraction of the heart is modulated by regulatory structural changes in the myosin filament, in addition to the classical calcium-dependent regulation of the actin filament. Dual-filament regulation of cardiac muscle is best studied in intact force-generating sarcomeres, because it involves inter-filament signalling and mechano-sensing via signalling pathways that are still poorly understood. We have recently characterised by X-ray diffraction the structural dynamics of the thick filament associated with contraction and relaxation of electrically-paced intact trabeculae from rat hearts (Brunello et al., 2020 PNAS 117:8177). Here we complemented that approach using demembranated trabeculae to measure the structural changes in the myosin filament associated with the steady-state force-pCa relation. Trabeculae were activated at different [Ca2+] by temperature-jump from 2°C to 27°C in the presence of 3% Dextran to recover the physiological inter-filament lattice spacing. The calcium-dependence of the spacing of the M6 reflection, associated with the axial periodicity of the thick filament backbone, was similar to that of force, consistent with the idea of a stress-sensing mechanism in the filament backbone. Increasing [Ca2+] from the diastolic value (pCa 7.0) to pCa 6.0 induced a decrease in the intensities of the first myosin layer line and of the forbidden myosin-based reflections, that are signatures of the diastolic structure of the thick filament, by ∼50% and ∼80% respectively, with respect to the change observed at maximal [Ca2+] (pCa 4.7). In contrast, active force at pCa 6 was only ∼20% of that at maximal [Ca2+]. These results indicate that the regulatory transition in the myosin filament from the diastolic to the ON state is more sensitive to calcium than force generation. Supported by British Heart Foundation, Wellcome Trust, UK and ESRF, France.