Basal myosin light chain phosphorylation is a determinant of Ca2+ sensitivity of force and activation dependence of the kinetics of myocardial force development

It is generally recognized that ventricular myosin regulatory light chains (RLC) are ∼40% phosphorylated under basal conditions, and there is little change in RLC phosphorylation with agonist stimulation of myocardium or altered stimulation frequency. To establish the functional consequences of basal RLC phosphorylation in the heart, we measured mechanical properties of rat skinned trabeculae in which ∼7% or ∼58% of total RLC was phosphorylated. The protocol for achieving ∼7% phosphorylation of RLC involved isolating trabeculae in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC from its baseline level. Subsequent phosphorylation to ∼58% of total was achieved by incubating BDM-treated trabeculae in solution containing smooth muscle myosin light chain kinase, calmodulin, and Ca2+ (i.e., MLCK treatment). After MLCK treatment, Ca2+ sensitivity of force increased by 0.06 pCa units and maximum force increased by 5%. The rate constant of force development ( ktr) increased as a function of Ca2+ concentration in the range between pCa 5.8 and pCa 4.5. When expressed versus pCa, the activation dependence of ktr appeared to be unaffected by MLCK treatment; however, when activation was expressed in terms of isometric force-generating capability (as a fraction of maximum), MLCK treatment slowed ktr at submaximal activations. These results suggest that basal phosphorylation of RLC plays a role in setting the kinetics of force development and Ca2+ sensitivity of force in cardiac muscle. Our results also argue that changes in RLC phosphorylation in the range examined here influence actin-myosin interaction kinetics differently in heart muscle than was previously reported for skeletal muscle.

Cooperative Mechanisms in the Activation Dependence of the Rate of Force Development in Rabbit Skinned Skeletal Muscle Fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca2+ binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca2+ dependence of the rate constant of force redevelopment (ktr) in skinned single fibers in which cross-bridge and Ca2+ binding were also perturbed. Ca2+ sensitivity of tension, the steepness of the force-pCa relationship, and Ca2+ dependence of ktr were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non–force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca2+, 3 μM NEM-S1 increased ktr to maximal values, and higher concentrations of NEM-S1 (6 or 10 μM) increased ktr to greater than maximal values. NEM-S1 also accelerated ktr at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 μM NEM-S1 increased ktr to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of ktr. These results show that ktr is not maximal in control fibers, even at saturating [Ca2+], and suggest that activation dependence of ktr is due to the combined activating effects of Ca2+ binding to TnC and cross-bridge binding to actin.