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. 2018 Apr:117:72-81.
doi: 10.1016/j.yjmcc.2018.02.009. Epub 2018 Feb 13.

Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle

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Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle

Thomas Kampourakis et al. J Mol Cell Cardiol. 2018 Apr.

Abstract

Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics.

Keywords: Cardiac muscle regulation; Hypertrophic cardiomyopathy; Myosin; Myosin regulatory light chain; Polarized fluorescence.

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Figures

Fig. 1
Fig. 1
R58Q stabilizes the OFF conformation of the myosin heads. (A) Left: Structure of the RLC-region of myosin with RLC and myosin heavy chain (MHC) shown in green and yellow, respectively. Arginine 58 is shown in van-der-Waals representation, and the BSR probe attached to the E-helix and putative Ca2+/Mg2+ binding site are shown in purple and grey, respectively. Right: OFF and ON orientations of the myosin heads with respect to the filament axis (FA; black arrow), reported by parallel (high value of the order parameter <P2>) and perpendicular (low <P2>) orientations of the E-helix probe, respectively. (B) <P2 > measured from WT- (green) and R58Q-BSR-cRLC-E (blue) exchanged ventricular trabeculae in relaxing conditions (REL, pCa 9) and full calcium activation (ACT, pCa 4.3) at ~1.9 μm sarcomere length. (C) Decrease in <P2 > (Δ < P2>) on calcium activation for WT- and R58Q-BSR-cRLC-E exchanged trabeculae. (D) ATPase activity of cardiac myofibrils in relaxing conditions and full calcium activation exchanged with either WT- (green) or R58Q-RLC (blue). (E) Maximum isometric force and (F) rate of force re-development of WT- and R58Q-BSR-cRLC-E exchanged ventricular trabeculae. Means ± SEM (n = 4–16). Statistical significance of difference between WT and R58Q groups was assessed with a two-tailed unpaired Student's t-test: *p < .05, **p < .01, ***p < .001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Effects of R58Q mutation on isolated cardiac myosin and RLC. (A) SDS-PAGE of RLC-depleted porcine β-cardiac myosin, and myosin reconstituted with either WT- and/or R58Q-RLC. The endogenous (eRLC) and recombinant RLC (rRLC) are labelled accordingly. (B) F-actin dependent ATPase activity of RLC-exchanged β-cardiac myosins from (A) (for details see Materials and Methods). Data points fitted to the Michaelis-Menten equation (solid lines). (C) Protein stability of WT- and R58Q-RLCs were assessed by Micro-scale Thermophoresis (MST) against increasing concentrations of Guanidine Hydrochloride (GdnHCl). Data points between 0 and 3 mol/L GdnHCl were fitted to a Hill equation (solid lines). (D) Binding of WT- and R58Q-RLC to N-terminal (C0C2) domains of cMyBP-C assessed by MST. Means ± SEM (n = 4–6 for C0C2; n = 1 for C3C5).
Fig. 3
Fig. 3
R58Q perturbs the effect of sarcomere length on maximum force but not that on calcium sensitivity of cardiac trabeculae. Normalized (A and B) and absolute (C and D) force-pCa relations of WT- (A and C) and R58Q-BSR-cRLC-E (B and D) exchanged trabeculae at short (~1.9 μm) and long (~2.2 μm) sarcomere length. Force is normalized to maximum force at 1.9 μm sarcomere length. <P2 > -pCa relations for WT- and R58Q-BSR-cRLC-E exchanged trabeculae measured in parallel with force are shown in (E) and (F), respectively. Dashed and dotted lines in (B), (D) and (F) denote Hill fits for WT-BSR-cRLC-E exchanged trabeculae at short and long sarcomere length, respectively. Means ± SEM (n = 4–7).
Fig. 4
Fig. 4
RLC phosphorylation restores myofilament function in the presence of R58Q-RLC. (A) In vitro kinase assay of WT- and R58Q-RLC incubated without (−) and with cMLCK (+) and analyzed by urea-glycerol PAGE. (B) In situ phosphorylation of WT- and R58Q-RLC exchanged into cardiac myofibrils (CMF) analyzed by Phostag™-SDS-PAGE and Western blot against RLC. (C) Time-course of phosphorylation of native, and WT- and R58Q-RLC exchanged CMFs. (D) Active isometric force (FActive) and (E) Δ < P2 > of R58Q-BSR-cRLC-E exchanged trabeculae before (−) and after RLC phosphorylation (+) to ~0.3 mol Pi/mol RLC (Fig. S6) at short sarcomere length (~1.9 μm), and after RLC phosphorylation at long sarcomere length (~2.2 μm). The values obtained for WT-BSR-cRLC-E at each sarcomere length are indicated by dotted lines and labelled accordingly. (F) Rate of force re–development of R58Q-BSR-cRLC-E exchanged trabeculae before (blue) and after RLC phosphorylation (purple). Means ± SEM (n = 4–7). Statistical significance of differences of values were assessed with a two-tailed paired Student's t-test: *p < .05, **p < .01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Model for the effect of R58Q on myofilament activation in the heart. Cardiac contraction is controlled by regulatory structural transitions in both actin-containing thin and myosin-containing thick filaments. During diastole (left) both thin and thick filament are in the OFF state. Tropomyosin (brown lines) blocks myosin-binding sites on actin (white and grey circles), and myosin head domains (space filling representation, green) are sequestered on the surface of the thick filament backbone. During systole (right) calcium (black circle) binds to troponin C (red) and activates the thin filaments by removing inhibitory interactions of troponin I (yellow) and troponin T (purple) with actin, followed by tropomyosin moving azimuthally away from myosin-binding sites on actin. Calcium activation of the thin filament activates myosin heads through so far unknown mechanisms (indicated by vertical arrows), resulting in release of the heads from the surface of the thick filament to become available for actin binding and force-generation. The R58Q mutation in the myosin RLC (blue) reduces cardiac contractility by stabilizing the thick filament OFF state and preventing myosin heads from leaving the thick filament surface. RLC phosphorylation (+P) restores thick filament activation in the presence of the R58Q mutation by destabilizing the OFF state and promoting the myosin head ON state. Homology models for human β-cardiac myosin were created by the Spudich laboratory (http://spudlab.stanford.edu/homology-models/). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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