Removal of the cardiac myosin regulatory light chain increases isometric force production

Abstract

The myosin neck, which is supported by the interactions between light chains and the underlying alpha-helical heavy chain, is thought to act as a lever arm to amplify movements originating in the globular motor domain. Here, we studied the role of the cardiac myosin regulatory light chains (RLCs) in the capacity of myosin to produce force using a novel optical-trap-based isometric force in vitro motility assay. We measured the isometric force and actin filament velocity for native porcine cardiac (PC) myosin, RLC-depleted PC (PC(depl)) myosin, and PC myosin reconstituted with recombinant bacterially expressed human cardiac RLC (PC(recon)). RLC depletion reduced unloaded actin filament velocity by 58% and enhanced the myosin-based isometric force approximately 2-fold. No significant change between PC and PC(depl) preparations was observed in the maximal rate of actin-activated myosin ATPase activity. Reconstitution of PC(depl) myosin with human RLC partially restored the velocity and force levels to near untreated values. The reduction in unloaded velocity after RLC extraction is consistent with the myosin neck acting as a lever, while the enhancement in isometric force can be directly related to enhancement of unitary force. The force data are consistent with a model in which the neck region behaves as a cantilevered beam.

Figure 1.

A) Schematic representation of RLC deletion from PC myosin: native PC myosin (a) and PC_depl myosin (b). B) SDS-PAGE of myosin: 4–20% Bio-Rad precast gel of native PC myosin (lane 1), PC_depl myosin (9.7±2% endogenous RLC; lane 2), and PC_recon myosin (138±28%; lane 3; 7.5 μg/ml sample loaded per well).

Figure 2.

Actin-activated myosin ATPase activity of PC, PC_depl, and PC_recon myosin. Steady-state actin-activated myosin ATPase activity was measured as a function of actin concentration. Error bars = average ± se values of 10–12 experiments. Solid lines represent the best fit to the equation y = m₁ + m₂ν, where m₁ is the myosin ATPase activity at 0 μM actin, m₂ is the maximal ATPase activity, and ν is the fractional saturation of binding sites and is a root of the quadratic equation: naν² − (K_d+na+b) ν + b = 0, where a and b are total concentration of myosin (a=1.9 μM) and actin, respectively; n is the apparent stoichiometry; and K_d is the apparent equilibrium dissociation constant. The following K_d (μM) values were obtained for PC, PC_depl, and PC_recon myosin: 3.9, 1.0, and 5.7, respectively. Apparent stoichiometry n values were 2.0, 4.3, and 1.0 for PC, PC_depl, and PC_recon myosin.

Figure 3.

Rate of actin movement in in vitro motility assays. Rates are shown for native PC myosin (solid bar), PC_depl myosin (open bar), and PC_recon myosin (hatched bar). Bar graph indicates mean ± se velocity (μm/s). There was a significant decrease in velocity (P=2.63×10⁻³) for PC_depl myosin (N=2) compared with PC myosin (N=2), where N is the number of myosin preparations used. There was also a significant decrease in velocity (P=4.14×10⁻⁴) for PC_recon myosin (N=4) compared with PC myosin (N=2).

Figure 4.

Duty cycle measurements for myosins. Actin filament velocity (mean±se) was plotted as a function of myosin motor surface density for PC myosin (shaded squares), PC_recon myosin (solid circles), and PC_depl myosin (open squares). Note dependence of actin filament velocity on number of interacting heads. Myosin motility data were best fit by theoretical curves (Eq. 1) representing duty cycles of f = 0.024 ± 0.004, f = 0.018 ± 0.004, and f = 0.022 ± 0.004 for PC, PC_recon, and PC_depl myosin, respectively. Data were normalized to V_max for each myosin tested by fitting to the relation V_obs = V_max × [1−(1−f^N)]. Normalization allows easy visual comparison of the rate of rise in velocity at low myosin densities. Normalized data were fit to Eq. 1 to obtain estimates of f; 25 filaments from 2–4 movies were analyzed for each concentration of myosin (each point on plot).

Figure 5.

Effects of α-actinin on actin filament velocity in the frictional loading assay. Effect of immobilized α-actinin on actin filament velocity for PC myosin (solid squares), PC_depl myosin (open squares), and PC_recon myosin (solid circles). Lines are least-squares regression fits.

Figure 6.

A) Schematic diagram of the laser trap used in this study. L, lenses; M, mirror; QD, quadrant detector; CCD, charge-coupled device. B) Isometric force is shown with native PC myosin (solid bar) and PC_depl myosin (open bar). Note a significant 115% increase in isometric force (P=1.33×10⁻⁸) for PC_depl myosin (N=13) compared with PC myosin (N=14). Isometric force data are means ±sd. Inset: raw displacement time series showing an individual force measurement plotted as a function of time for a PC myosin-coated trapped bead. Distance between bead position at 0 force and the extent of bead displacement (plateau) was used to calculate force.

Figure 7.

F_uni models. A) Linear spring located in S2 hinge. Force F is proportional to l (model I). B) Torsional spring located at head-neck junction. F is inversely proportional to length of neck l (model II). C) LC-binding domain acts as a cantilevered beam. In this model, F is inversely proportional to l² (model III).