Regulatory light chain mutants linked to heart disease modify the cardiac myosin lever arm

Abstract

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

Figure 1

The human β-cardiac myosin S1 motor binds the actin filament and generates tension with the lever-arm swing. In a muscle fiber the lever-arm links to the myosin filament where Load and Tension vectors are shown. The regulatory light chain (HCRLC in bold red) indicates positions 20, 134, and 162 with mutations implicated in HCM. The PAGFP is linked to the light chain (dashed yellow linker near G162) and reports lever-arm orientation. The ELC is shown in cyan. In the motor, black regions are the actin binding site, the blue α-helix is the switch 2 helix and the green β-sheet is the active site with entrance at Loop 1.

Figure 2

TIRF observation characterizes single myosin lever-arms in the muscle fiber using exchanged HCRLC-PAGFP. TIRF excitation uses 488 nm laser light focused on the back focal plane (BFP) to excite photoactivated PAGFP that emits with peak intensity at 520 nm. Infrared (800 nm) light focused on the fiber causes myosin SHG to image the periodic myosin structure and monitor sarcomere length. The microfluidic chamber was constructed from a brass master etched into the pattern shown in the insert. Three channels were constructed with depths of 20 (shown) to 40 μm to accommodate different fiber bundle sizes and to apply mild pressure on the contracting fiber to keep it stationary during observation. The channel is also the conduit to exchange solutions. TIRF and SHG experiments are performed sequentially.

Figure 3

Simplified cross-bridge cycle in contraction detailing myosin’s discrete sub-states (A-E) and their correspondence to lever-arm orientation. The rigor cross-bridge binds actin (Ac) in the A-state (M_A), exerts force F_rt, and maintains the PAGFP (green cylinder with arrow) tagged HCRLC (red) in the A-state orientation. ELC is shown in blue. ATP addition relaxes the fiber by binding to myosin and detaching actin. The relaxed cross-bridge assumes the E-state (M_E) with the lever-arm re-primed for a new power stroke. Ca²⁺ addition activates the fiber producing force F_at. In isometric contraction single lever-arms assume one of their intermediates (A-E) depicted here as M_B which is a high force producing intermediate having a characteristic orientation indicated by Φ.

Figure 4

SDS-PAGE of the fiber protein extract and purified proteins. Lane 1 is the untreated fiber control, lane 2 the E134A-PAGFP exchanged fiber, and lane 3 purified proteins identified on the right of the gel except for ELC and porcine RLC. Quantitative evaluation of the untreated and exchanged fibers was done with ImageJ and using the ELC content as standard. Error is ~10%. Stoichiometric replacement of the porcine RLC is indicated by the sum of Lane 2 bands at HCRLC-PAGFP and porcine RLC equaling 1 to within error. HC is heavy chain and TnC is troponin C.

Figure 5

Dipole moment spherical polar coordinates (β,α) scatter plot for fibers in rigor (blue solid square), isometric contraction (red solid square), and relaxation (open blue square) and for the WT and M20L species. Coordinates are defined relative to a lab frame z-axis parallel to the fiber symmetry axis, x-axis in the plane of the coverslip, and y-axis normal to the coverslip plane pointing into the aqueous medium. Right panels show enlargements of the rectangular regions in the scatter plots. They depict the arrow clusters associating various high free-energy active isometric coordinates in sub-states A-E (Figure 3) at the base of the arrow with their unique minimum free-energy rigor A-state at the pointy ends. Blue or green arrows designate a positive or negative projection scalar (eq. 3). Arrow clusters like those in the enlargements populate the entire scatter plot but were left out here for clarity. The full set of clustered arrows for WT and all mutant species is shown in Supporting Information (Figure S3). Blue and green arrows arranged like the hands of a clock are the average orientation of all the same colored clustered arrows (including those not shown here outside of the rectangules). They are referred to in the text as $\overrightarrow{\Delta }g$(±) and have amplitudes proportional to the average free-energy separating the isometric active cross-bridge from the minimum free-energy rigor A-state cross-bridge computed using eq. 2.

Figure 6

Φ-permutations for the active-rigor transitions in WT and mutant HCRLC-PAGFP exchanged permeabilized papillary muscle fibers. The histogram is discretized corresponding to A- to E-state intermediates. ∑ indicates total instances.

Figure 7

Φ-permutations for the active-rigor (Active) and relax-rigor (Relax) transitions in WT HCRLC-PAGFP exchanged permeabilized papillary muscle fibers. ∑ indicates total instances.

Figure 8

Φ-densities for single myosins in an isometric active fiber. The WT and mutant species maintain different Φ-densities in isometric contraction. The altered intermediate densities reflect lowered lever-arm stiffness due to mutation in HCRLC. The E134A mutant also has lower isometric tension (Table 1). ∑ indicates total instances.