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. 2015 Aug 15:580:14-21.
doi: 10.1016/j.abb.2015.06.014. Epub 2015 Jun 25.

Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

Anastasia Karabina  1 , Katarzyna Kazmierczak  2 , Danuta Szczesna-Cordary  2 , Jeffrey R Moore  3
Affiliations

Myosin regulatory light chain phosphorylation enhances cardiac β-myosin in vitro motility under load

Anastasia Karabina et al. Arch Biochem Biophys. .

Abstract

Familial hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy and myofibrillar disarray, and often results in sudden cardiac death. Two HCM mutations, N47K and R58Q, are located in the myosin regulatory light chain (RLC). The RLC mechanically stabilizes the myosin lever arm, which is crucial to myosin's ability to transmit contractile force. The N47K and R58Q mutations have previously been shown to reduce actin filament velocity under load, stemming from a more compliant lever arm (Greenberg, 2010). In contrast, RLC phosphorylation was shown to impart stiffness to the myosin lever arm (Greenberg, 2009). We hypothesized that phosphorylation of the mutant HCM-RLC may mitigate distinct mutation-induced structural and functional abnormalities. In vitro motility assays were utilized to investigate the effects of RLC phosphorylation on the HCM-RLC mutant phenotype in the presence of an α-actinin frictional load. Porcine cardiac β-myosin was depleted of its native RLC and reconstituted with mutant or wild-type human RLC in phosphorylated or non-phosphorylated form. Consistent with previous findings, in the presence of load, myosin bearing the HCM mutations reduced actin sliding velocity compared to WT resulting in 31-41% reductions in force production. Myosin containing phosphorylated RLC (WT or mutant) increased sliding velocity and also restored mutant myosin force production to near WT unphosphorylated values. These results point to RLC phosphorylation as a general mechanism to increase force production of the individual myosin motor and as a potential target to ameliorate the HCM-induced phenotype at the molecular level.

Keywords: Cardiac ventricular myosin; Hypertrophic cardiomyopathy; Load dependence; Regulatory light chain phosphorylation.

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Figures

Fig. 1
Fig. 1
Gel electrophoresis of RLC phosphorylation and exchange reactions. (A) Charge separation of dephosphorylated and phosphorylated bacterially expressed human ventricular RLCs is shown on an 8 M urea 10% polyacrylamide gel. Due to a more acidic isoelectric point phosphorylated RLCs migrate further than their respective dephosphorylated counterparts. WT RLC (lane 1), WT Phos RLC (lane 2), N47K RLC (lane 3), N47K Phos RLC (lane 4), R58Q RLC (lane 5), R58Q Phos RLC (lane 6). (B) SDS–PAGE of PC myosin RLC exchange reaction shows ELC and RLC bands from PC myosin (lane 1), PC RLC depleted myosin (lane 2; 85% depletion), and PC RLC depleted myosin fully reconstituted with hvRLCs (lanes 3–8), same order as panel A.
Fig. 2
Fig. 2
Frictional loading assays measure the force production of a bed of myosins. Average actin filament sliding velocity and SEs of the average sliding velocity are plotted as a function of α-actinin added to the motility assay surface. Average actin sliding velocity is determined from a minimum of 3 experiments. Plots are shown for myosins bearing RLCs in the dephosphorylated (circles, solid line) and phosphorylated (triangles, dashed line) states for (A) N47K, (B) R58Q, and (C) WT RLCs.
Fig. 3
Fig. 3
Average force values of cardiac β-myosins. Force values and SE are determined from the fit of the average data to Eq. (1). N47K and R58Q mutant myosins display significantly reduced force production (p < 0.05, 0.02, respectively) compared to WT myosin force values (dotted line). Phosphorylation of WT, N47K and R58Q myosins results in significantly increased force production (p < 0.03, 0.05, 0.03, respectively). When compared to WT phosphorylated myosin, N47K phosphorylated myosin exhibited borderline significance (p = 0.0502) and R58Q phosphorylated myosin was significantly different (p < 0.02).
Fig. 4
Fig. 4
Power output of cardiac β-myosins. (Top panel) Power is derived by converting α-actinin concentration in the frictional loading assays to a frictional force using Eq. (2), and then taking the product of force and velocity. Power curves are generated by fitting the force-power data to the empirically derived Hill equation (Eq. (4)). Shown is a representative fit of the Hill Power Equation to transformed data for WT phosphorylated myosin. (Bottom panel) Power output curves are shown in the absence of transformed data for better visualization and comparison of power trends. Isometric force values (±SE) estimated from frictional loading motility assays are used to constrain the fit and are shown on the x-axis. Cardiac myosins bearing hvRLCs in the phosphorylated state (dashed lines) show increased power output compared to hvRLCs in the dephosphorylated states (solid lines).
Fig. 5
Fig. 5
Regulatory light chain binds the myosin heavy chain (MHC) at hinge region. (Left) During the powerstroke actin (tan filament) is displaced by the myosin crossbridge which is tethered to the thick filament (blue horizontal rectangle). The myosin lever arm is stabilized by the essential light chain (ELC) and regulatory light chain (RLC), and undergoes a ~70° angle swing from the pre-powerstroke state to the post-powerstroke state. The ability of myosin generate the powerstroke against load will depend on the stiffness of the myosin light chain binding domain. (Right) A three-dimensional representation of the RLC (magenta) wrapping around the MHC (blue) at a lever arm hinge region illustrates positions of HCM mutations (black) and the cation binding site (green) within the RLC. The first 18 residues of the RLC N-terminus are missing from the structure and therefore the RLC phosphorylation site at Ser15 is not shown. The RLC structure is modified from the crystal structure of Rayment et al., 1993 (PDB: 2MYS). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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