Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers

Abstract

Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 μm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca²⁺ sensitivity for 2.5 vs. 2.0 μm sarcomere length fibers (pCa₅₀ = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca²⁺ sensitivity of force was attenuated, though the Ca²⁺ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by ∼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s^-1). In the presence of dextran, cross-bridge detachment was ∼20% slower at 2.5 vs. 2.0 μm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s^-1). However, osmotic compression of fibers at either 2.5 or 2.0 μm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.

Figure 1

Dextran and longer sarcomere length both increased tension according to the tension-pCa relationship. (A) Ca²⁺-activated tension-pCa relationship at 2.0 μm and 2.5 μm sarcomere lengths in solution containing 0% dextran. (B) Ca²⁺-activated tension-pCa relationship at 2.0 μm and 2.5 μm sarcomere length in solution containing 4% dextran. The dashed line is for comparison with 2.0 μm fibers in 0% dextran solution. Fits are to the three-parameter Hill equation. ^∗p < 0.05 between sarcomere lengths.

Figure 2

Dextran and sarcomere length did not influence viscoelasticity under relaxed conditions. Elastic (top) and viscous (bottom) moduli were plotted against frequency for 2.0 μm and 2.5 μm sarcomere length at pCa 8 and 5 mM [MgATP]. (A and C) Elastic and viscous moduli for fibers in dextran-free solution. (B and D) Elastic and viscous moduli for fibers in solution containing 4% dextran. Gray shading represents single-sided error bars (upward for 2.0 μm sarcomere length and downward for 2.5 μm sarcomere length) associated with the mean data points at each frequency.

Figure 3

Sarcomere length influenced viscoelasticity under activated conditions. Elastic (top) and viscous (bottom) moduli were plotted against frequency for 2.0 μm and 2.5 μm sarcomere length at pCa 4.8 and 5 mM [MgATP]. (A and C) Elastic and viscous moduli for fibers in dextran-free solution. (B and D) Elastic and viscous moduli for fibers in solution containing 4% dextran. Leftward shifts in the C-process of the viscous modulus indicate slower myosin detachment rates. Gray shading represents single-sided error bars (upward for 2.0 μm sarcomere length and downward for 2.5 μm sarcomere length) associated with the mean data points at each frequency.

Figure 4

Sarcomere length and dextran both influenced cross-bridge kinetics as [MgATP] was varied. Parameter fits to Eq. 2 are plotted against [MgATP] for fibers of 2.0 μm and 2.5 μm sarcomere lengths at pCa 4.8 in 0% dextran (left column) and 4% dextran solutions (right column). Dashed lines are for comparison with 2.0 μm fibers in 0% dextran solution. (A–D) Increases in A and decreases in k as [MgATP] decreases are representative of increased viscoelastic stiffness and fiber elasticity. (E–H) Increases in the magnitude parameters B and C at lower [MgATP] indicate increases in cross-bridge binding. (I–L) Decreases in 2πb and 2πc as [MgATP] decreases toward rigor represent slower rates of myosin attachment and detachment rates, respectively. ^∗p < 0.05 between sarcomere lengths.

Figure 5

Sarcomere length and dextran both influenced cross-bridge kinetics as pCa is varied. Parameter fits to Eq. 2 are plotted against pCa for fibers of 2.0 μm and 2.5 μm sarcomere lengths in 5mM [MgATP] solution containing 0% dextran (left column) or 4% dextran (right column). Dashed lines are for comparison with 2.0 μm fibers in 0% dextran solution. (A–D) Increases in A and decreases in k as [Ca²⁺] decreases are representative of increased viscoelastic stiffness and fiber elasticity. (E–H) Increases in the magnitude parameters B and C at lower [Ca²⁺] indicate increases in cross-bridge binding. (I–L) Decreases in 2πb and 2πc as [MgATP] decreases from 4.8 pCa represent slower rates of myosin attachment and detachment rates, respectively. ^∗p < 0.05 between sarcomere lengths.