Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin

Abstract

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure–function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM–D166V) and dilated (DCM–D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM–D94A mice, HCM–D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM–D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM–D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM–D166V, the DCM–D94A model favored the energy-conserving SRX state, but the structure/function–pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM–D166V and DCM–D94A mutations.

Variants identify sarcomere inter-protein contacts distinguishing inheritable cardiac muscle diseases

“…The ST-RLC co-domain interactions (characteristic to a destabilized SRX in FHC) are not detected at the highest significance level in DCM or LVN species again implying their C-terminus SNVs are SRX detectors but not disruptors such that the MYBPC3 C-terminus segment SNVs do not influence SRX stability. The DCM result agrees with a previous analysis of myosin variants [34, 35]. The 2D-CG map for FHC SNVs appears to eliminate the free head (Level 1)-RLC (Level 3) contact destabilizing the SRX conformation.…”

Small Angle X-ray Diffraction as a Tool for Structural Characterization of Muscle Disease

“…Small-angle X-ray diffraction patterns in D94A papillary muscle fibers at submaximal Ca 2+ concentrations showed reduced I 1,1 / I 1,0 compared to controls, supporting the hypocontractile state of the D94A myosin motors including a reduced calcium sensitivity of force. In a follow up study [ 50 ], X-ray diffraction patterns were collected as a function of calcium activated force to generate full I 1,1 /I 1,0 -pCa curves, but the structure/function-pCa data were similar to WT indicating that the D94A mutant does not result in a shift of the equilibrium of relaxed heads from the OFF to ON states, consistent with the observed increased SRX population relative to wild type at rest. Myosin Binding protein C and DCM …”

“…These differences in sarcomere structure were partially or fully reversed by pseudo-phosphorylation of D166V fibers from homozygous and heterozygous S15D-D166V mice, respectively, indicating that pseudo-phosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the HCM phenotype. In a follow up study [ 50 ], the investigators showed that both of force/pCa (Ca 2+ -sensitivity) and lI 1,1 /I 1,0 -pCa were left shifted in HCM-D166V myocardium so that over most of the submaximal activation range, cross-bridges were closer to actin-containing thin filaments facilitating activation. These results accorded with biochemical assays where the population of myosin heads in SRX were reduced indicating that SRX-to-DRX transitions are promoted in HCM-D166V mutant myocardium consistent with the phenotype of hypercontractility characteristic of HCM.…”

“…The thick filaments (thick dots) form a 2D hexagonal lattice and the thin filaments (thin dots) interdigitate into the trigonal points between the thick filaments. ( B ) Equatorial reflections (up panel) and their one-dimensional intensity profiles (bottom panel) from permeabilized murine myocardium under resting (pCa 8) and sub-maximally activated state (pCa5.8) (based on reference [ 50 ]).…”

Effects of omecamtiv mecarbil on the contractile properties of skinned porcine left atrial and ventricular muscles

“…This unique relaxed state has come to be widely known as the “super-relaxed state” (SRX) (see reviews and references therein: e.g., Cooke, 2011 ; McNamara et al, 2015 ; Irving, 2017 ; Spudich, 2019 ; Craig and Padrón, 2022 ). SRX is reportedly in equilibrium with the disordered-relaxed state (DRX) in which myosin heads are in closer proximity to thin filaments (e.g., Anderson et al, 2018 ; Gollapudi et al, 2021 ; Yuan et al, 2022 ), and ready to produce force. A decrease in the number of myosin molecules in SRX would increase the proportion of the heads available to form force-generating cross-bridges (e.g., Schmid and Toepfer, 2021 ).…”