Julien S. Davis

The Overall Pattern of Cardiac Contraction Depends on a Spatial Gradient of Myosin Regulatory Light Chain Phosphorylation

Evolution of the human heart has incorporated a variety of successful strategies for motion used throughout the animal kingdom. One such strategy is to add the efficiency of torsion to compression so that blood is wrung, as well as pumped, out of the heart. Models of cardiac torsion have assumed uniform contractile properties of muscle fibers throughout the heart. Here, we show how a spatial gradient of myosin light chain phosphorylation across the heart facilitates torsion by inversely altering tension production and the stretch activation response. To demonstrate the importance of cardiac light chain phosphorylation, we cloned a myosin light chain kinase from a human heart and have identified a gain-in-function mutation in two individuals with cardiac hypertrophy.

Sensing Stretch Is Fundamental

Stretch induces changes in cardiomyocyte biology that are implicated in heart failure, but the mechanism by which stretch is sensed and signals are transduced is unknown. New understanding of the Z disc elements of contractile units are beginning to elucidate the mechanism of stretch sensing and its relation to cardiac adaptation and disease.

Kinetic Effects of Myosin Regulatory Light Chain Phosphorylation on Skeletal Muscle Contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).

Mechanistic role of movement and strain sensitivity in muscle contraction.

Tension generation can be studied by applying step perturbations to contracting muscle fibers and subdividing the mechanical response into exponential phases. The de novo tension-generating isomerization is associated with one of these phases. Earlier work has shown that a temperature jump perturbs the equilibrium constant directly to increase tension. Here, we show that a length jump functions quite differently. A step release (relative movement of thick and thin filaments) appears to release a steric constraint on an ensemble of noncompetent postphosphate release actomyosin cross-bridges, enabling them to generate tension, a concentration jump in effect. Structural studies [Taylor KA, et al. (1999) Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle. Cell 99:421–431] that map to these kinetics indicate that both catalytic and lever arm domains of noncompetent myosin heads change angle on actin, whereas lever arm movement alone mediates the power stroke. Together, these kinetic and structural observations show a 13-nm overall interaction distance of myosin with actin, including a final 4- to 6-nm power stroke when the catalytic domain is fixed on actin. Raising fiber temperature with both perturbation techniques accelerates the forward, but slows the reverse rate constant of tension generation, kinetics akin to the unfolding/folding of small proteins. Decreasing strain, however, causes both forward and reverse rate constants to increase. Despite these changes in rate, the equilibrium constant is strain-insensitive. Activation enthalpy and entropy data show this invariance to be the result of enthalpy–entropy compensation. Reaction amplitudes confirm a strain-invariant equilibrium constant and thus a strain-insensitive ratio of pretension- to tension-generating states as work is done.

Force Generation Simplified

Raising the temperature of a maximally Ca2+-activated muscle fiber causes a sigmoidal increase in tension. The kinetics that govern this process can be explored by step-heating the fiber a few degrees with a laser temperature-jump. A biexponential increase in tension results; a third exponential phase that opposes this biphasic rise in tension is only observed when phosphate, a reaction product normally at low concentration, is added to the fiber. This chapter explains how the temperature dependencies of isometric tension and the temperature jump kinetics interrelate, and how these insights have modified and simplified our understanding of current mechanisms of force generation. The fast kinetic phase of the tension rise appears associated with single-step force generation or a power stroke, a process largely isolated from adjacent steps in the crossbridge cycle. The amplitude of the slow phase of the tension rise exhibits a remarkable ∼1:1 ratio to the amplitude of the fast, tension generating phase above 10°C. The similarity of these two amplitudes, that combine to give the complete rise in isometric tension with temperature, appear to fit a model in which one of a pair of myosin heads generates force while the second head is poised to function after the power stroke of the first has occurred. The phase with the negative amplitude seen with added phosphate points to a mechanism in which phosphate release is indirectly linked to the tension generation by forward flow through the cross-bridge cycle to tension generation.

Kinetic Effects of Fiber Type on the Two Subcomponents of the Huxley-Simmons Phase 2 in Muscle

The Huxley-Simmons phase 2 controls the kinetics of the first stages of tension recovery after a step-change in fiber length and is considered intimately associated with tension generation. It had been shown that phase 2 is comprised of two distinct unrelated phases. This is confirmed here by showing that the properties of phase 2(fast) are independent of fiber type, whereas those of phase 2(slow) are fiber type dependent. Phase 2(fast) has a rate of 1000-2000 s(-1) and is temperature insensitive (Q(10) approximately 1.16) in fast, medium, and slow speed fibers. Regardless of fiber type and temperature, the amplitude of phase 2(fast) is half (approximately 0.46) that of phase 1 (fiber instantaneous stiffness). Consequently, fiber compliance (cross-bridge and thick/thin filament) appears to be the common source of both phase 1 elasticity and phase 2(fast) viscoelasticity. In fast fibers, stiffness increases in direct proportion to tension from an extrapolated positive origin at zero tension. The simplest explanation is that tension generation can be approximated by two-state transition from attached preforce generating (moderate stiffness) to attached force generating (high stiffness) states. Phase 2(slow) is quite different, progressively slowing in concert with fiber type. An interesting interpretation of the amplitude and rate data is that reverse coupling of phase 2(slow) back to P(i) release and ATP hydrolysis appears absent in fast fibers, detectable in medium speed fibers, and marked in slow fibers contracting isometrically. Contracting slow and heart muscles stretched under load could employ this enhanced reversibility of the cross-bridge cycle as a mechanism to conserve energy.

Cross-Bridge Populations and the Biphasic Time Course of Muscle Relaxation

A biphasic time course of relaxation follows Ca2+ removal from a contracting muscle fiber--a small linear decline is followed by a faster large exponential drop in tension to the relaxed state. Our aim is to see if recent insights into tension generation and the cross-bridge cycle can shed light on these kinetics (Davis and Epstein, 2009). In fully activated isometric rabbit psoas fibers two distinct and roughly equal populations of myosin heads are attached to actin. Half are termed “competent” and are capable of generating tension, while the other half are termed “noncompetent” and serve to “buffer” tension. For example, a step-decrease in fiber length releases a steric constraint on noncompetent cross-bridges thereby triggering tension recovery.We investigate whether the “buffer” of noncompetent AMD bridges might support the linear phase, with the exponential decline (kREL FAST) mediated by the decay of the remaining competent cross-bridge population. To do this, the linear phase was treated as a steady-state reaction with duration and not slope used to determine kREL SS. We found kREL SS normalized to kCAT (fiber ATPase) if a reasonable ∼21% of myosin heads constitute the noncompetent buffer. This leaves the final exponential decline to the relaxed state governed by the discharge of tension generating competent cross-bridges on exhaustion of the noncompetent buffer population. Additional support comes from the observations that added Pi decreases the duration and increases the slope of the linear phase with virtually no effect on kREL FAST (Tesi et al, 2002), and that a slow Tn Ca2+ off switch prolongs the linear phase (Kreutziger et al, 2008). Arrhenius plots of kCAT, kREL SS and kREL FAST are similar in slope, suggesting a common rate-limiting step of strain-sensitive ADP cross-bridge dissociation for all processes.