Carotid Plaque Score is Associated with 10-year Major Adverse Cardiovascular Events in Low-Intermediate Risk Patients Referred to a General Cardiology Community Clinic.

AIMS: Atherosclerotic carotid plaque assessments have not been integrated into routine clinical practice due to the time-consuming nature of both imaging and measurements. Plaque score, Rotterdam method, is simple, quick, and only requires 4-6 B-mode ultrasound images. The aim was to assess the benefit of plaque score in a community cardiology clinic to identify patients at risk for major adverse cardiovascular events (MACE). METHODS AND RESULTS: Patients ≥40 years presenting for risk assessment were given a carotid ultrasound. Exclusions included a history of vascular disease or MACE and being >75 years. Kaplan-Meier curves and hazard ratios were performed. The left and right common carotid artery (CCA), bulb, and internal carotid artery (ICA) were given 1 point per segment if plaque present (plaque score 0 to 6). Administrative data holdings at ICES were used for 10-year event follow-up. Of 8,472 patients, 60% were females (n = 5,121). Plaque was more prevalent in males (64% vs 53.9%; P <0.0001). The 10-year MACE cumulative incidence estimate was 6.37% with 276 events (males 6.9 % vs females 6.0%; P = 0.004). Having both maximal CCA IMT <1.00 mm and plaque score = 0, was associated with less events. A plaque score <2 was associated with a low 10-year event rate (4.1%) compared to 2-4 (8.7%) and 5-6 (20%). CONCLUSION: A plaque score ≥2 can re-stratify low-intermediate risk patients to a higher risk for events. Plaque score may be used as a quick assessment in a cardiology office to guide treatment management of patients.

Four phases of a force transient emerge from a binary mechanical system.

Accurate models of muscle contraction are important for understanding both muscle performance and the therapeutics that enhance physiological function. However, models are only accurate and meaningful if they are consistent with physical laws. A single muscle fiber contains billions of randomly fluctuating atoms that on the spatial scale of a muscle fiber generate unidirectional force and power output. This thermal system is formally constrained by the laws of thermodynamics, and a recently developed thermodynamic model of muscle force generation provides qualitative descriptions of the muscle force-velocity relationship, muscle force generation, muscle force transients, and the thermodynamic work loop of muscle with a thermodynamic (not molecular) power stroke mechanism. To demonstrate the accuracy of this model requires that its outputs be quantitatively compared with experimentally observed muscle function. Here I show that a two-state thermodynamic model accurately describes the experimentally observed four-phase force transient response to both mechanical and chemical perturbations. This is the simplest possible model of one of the most complex characteristic signatures of muscle mechanics.

The Kinetic and Energetic Pull of Chemical Entropy.

Single molecule mechanics studies clearly show that the molecular mechanism of muscle contraction is a force-generating myosin motor switch. However, muscle mechanics and energetics cannot be accounted for by summing up the force-generating chemical steps of independent myosin motors - the energetic contribution of the gradient of myosin motors across the force-generating chemical step is required. Chemistry (i.e., the Gibbs free energy equation) describes this energetic contribution as a chemical activity, whereas statistical mechanics describes it as entropic. Here, I show that while mathematically these two energetic terms are similar, physically they are fundamentally different. The entropic interpretation implies a novel thermodynamic model of chemical kinetics in which a chemical reaction is pulled down the entropic energy landscape of an ensemble of molecules rather than being pushed through mass action. I show that the transition from chemical activity to entropy is physical and occurs when thermal fluctuations isolated within N independent molecules become distributed among those molecules. With this transition chemical activity is lost when the N degrees of freedom of independent molecules physically and irreversibly collapse into one ensemble system within which heat is delocalized among the N molecules in the form of entropy.

Cells solved the Gibbs paradox by learning to contain entropic forces.

As Nature's version of machine learning, evolution has solved many extraordinarily complex problems, none perhaps more remarkable than learning to harness an increase in chemical entropy (disorder) to generate directed chemical forces (order). Using muscle as a model system, here I describe the basic mechanism by which life creates order from disorder. In short, evolution tuned the physical properties of certain proteins to contain changes in chemical entropy. As it happens these are the "sensible" properties Gibbs postulated were needed to solve a paradox that has intrigued and challenged scientists and philosophers for over 100 years.

Four Phases of a Force Transient Emerge from a Binary Mechanical System.

Models of muscle contraction are important for guiding drug discovery, drug validation, and clinical decision-making with the goal of improving human health. Models of muscle contraction are also key to discovering clean energy technologies from one of the most efficient and clean-burning machines on the planet. However, these important goals can only be met through muscle models that are based on science. Most every model and mechanism (e.g., a molecular power stroke) of muscle contraction described in the literature to date is based on a corpuscular mechanic philosophy that has been challenged by science for over two decades. A thermodynamic model and mechanisms (e.g., a molecular switch) of muscle contraction is supported by science but has not yet been tested against experimental data. Here, I show that following a rapid perturbation to the free energy of a thermodynamic muscle system, a transient force response emerges with four phases, each corresponding to a different clearly-defined thermodynamic (not molecular) process. I compare these four phases to those observed in two classic muscle transient experiments. The observed consistency between model and data implies that the simplest possible model of muscle contraction (a binary mechanical system) accurately describes muscle contraction.

The Problem with Inventing Molecular Mechanisms to Fit Thermodynamic Equations of Muscle.

Almost every model of muscle contraction in the literature to date is a molecular power stroke model, even though this corpuscular mechanism is opposed by centuries of science, by 85 years of unrefuted evidence that muscle is a thermodynamic system, and by a quarter century of direct observations that the molecular mechanism of muscle contraction is a molecular switch, not a molecular power stroke. An ensemble of molecular switches is a binary mechanical thermodynamic system from which A.V. Hill's muscle force-velocity relationship is directly derived, where Hill's parameter a is the internal force against which unloaded muscle shortens, and Hill's parameter b is the product of the switch displacement, d, and the actin-myosin ATPase rate. Ignoring this model and the centuries of thermodynamics that preceded it, corpuscularians continue to develop molecular power stroke models, adding to their 65-year jumble of "new", "innovative", and "unconventional" molecular mechanisms for Hill's a and b parameters, none of which resemble the underlying physical chemistry. Remarkably, the corpuscularian community holds the thermodynamicist to account for these discrepancies, which, as outlined here, I have done for 25 years. It is long past time for corpuscularians to be held accountable for their mechanisms, which by all accounts have no foundation in science. The stakes are high. Molecular power stroke models are widely used in research and in clinical decision-making and have, for over half a century, muddied our understanding of the inner workings of one of the most efficient and clean-burning machines on the planet. It is problematic that corpuscularians present these models to stakeholders as science when in fact corpuscularians have been actively defending these models against science for decades. The path forward for scientists is to stop baseless rejections of muscle thermodynamics and to begin testing corpuscular and thermodynamic mechanisms with the goal of disproving one or the other of these hypotheses.

Cells Solved the Gibbs Paradox by Learning to Contain Entropic Forces.

As Nature's version of machine learning, evolution has solved many extraordinarily complex problems, none perhaps more remarkable than learning to harness an increase in chemical entropy (disorder) to generate directed chemical forces (order). Using muscle as a model system, here I unpack the basic mechanism by which life creates order from disorder. In short, evolution tuned the physical properties of certain proteins to contain changes in chemical entropy. As it happens, these are the "sensible" properties Gibbs postulated were needed to solve his paradox.

Thermodynamics and Kinetics of a Binary Mechanical System: Mechanisms of Muscle Contraction.

Biological motors function at the interface of biology, physics, and chemistry, and it remains unsettled what rules from which disciplines account for how these motors work. Myosin motors are enzymes that catalyze the hydrolysis of ATP through a mechanism involving a switch-like myosin structural change (a lever arm rotation) induced by actin binding that generates a small displacement of an actin filament. In muscle, individual myosin motors are widely assumed to function as molecular machines having mechanical properties that resemble those of muscle. In a fundamental departure from this perspective, here, I show that muscle more closely resembles a heat engine with mechanical properties that emerge from the thermodynamics of a myosin motor ensemble. The transformative impact of thermodynamics on our understanding of how a heat engine works guides a parallel transformation in our understanding of how muscle works. I consider the simplest possible model of force generation: a binary mechanical system. I develop the mechanics, energetics, and kinetics of this system and show that a single binding reaction generates force when muscle is held at a fixed length and performs work when muscle is allowed to shorten. This creates a network of thermodynamic binding pathways that resembles many of the characteristic mechanical and energetic behaviors of muscle including the muscle force-velocity relationship, heat output by shortening muscle, four phases of a muscle tension transient, spontaneous oscillatory contractions, and force redevelopment. Analogous to the thermodynamic (Carnot) cycle for a heat engine, isothermal and adiabatic binding and detachment reactions create a thermodynamic cycle for muscle that resembles cardiac pressure-volume loops (i.e., how the heart works). This paper provides an outline for how to re-interpret muscle mechanic data using thermodynamics - an ongoing effort that will continue providing novel insights into how muscle and molecular motors work.

Evaluation of Sublethal Toxicity of Nitrite to a Suite of Aquatic Organisms in Support of the Derivation of a Chronic Environmental Water Quality Benchmark.

Nitrite is a naturally-occurring inorganic compound that occurs in aquatic environments as an intermediary between nitrate and ammonia in the nitrogen cycle. It is a contaminant of potential concern resulting from anthropogenic activities in some cases. While the acute toxicity of nitrite has been characterized in previous studies, its sublethal toxicity is less understood. To determine the sublethal toxicity of nitrite on freshwater organisms, a suite of organisms was tested including: two salmonids (Oncorhynchus mykiss and O. kisutch), an alga (Pseudokirchneriella subcapitata), an aquatic macrophyte (Lemna minor), and three invertebrates (Ceriodaphnia dubia, Chironomus dilutus, and Neocloeon triangulifer). Test organisms were exposed to nitrite concentrations ranging between 0.02 and 1.28 mg/L nitrite (NO2-N). The toxicity tests were conducted according to procedures specified in standardized methods, allowing for the estimation of multiple endpoints for each test species. Species sensitivity distributions (SSDs) were generated using endpoints from the toxicity testing results, as well as data from previous studies, from which water chemistry approximated that used in the tests (i.e., < 5 mg/L chloride, an important toxicity-modifying factor for nitrite). The mayfly, N. triangulifer, was the most sensitive species, followed by the two salmonids (which represented the second and third most sensitive species), although they were not as sensitive to nitrite exposure as reported in previous studies. The fifth percentile hazard concentration (HC5) generated from the SSD could be used for derivation of regulatory benchmarks and threshold values for site-specific aquatic risk assessments.

A chemical thermodynamic model of motor enzymes unifies chemical-Fx and powerstroke models.

Molecular motors play a central role in many biological processes, ranging from pumping blood and breathing to growth and wound healing. Through motor-catalyzed chemical reactions, these nanomachines convert the chemical free energy from ATP hydrolysis into two different forms of mechanical work. Motor enzymes perform reversible work, wrev, through an intermediate step in their catalyzed reaction cycle referred to as a working step, and they perform Fx work when they move a distance, x, against a force, F. In a powerstroke model, wrev is performed when the working step stretches a spring within a given motor enzyme. In a chemical-Fx model, wrev is performed in generating a conserved Fx potential defined external to the motor enzyme. It is difficult to find any common ground between these models even though both have been shown to account for mechanochemical measurements of motor enzymes with reasonable accuracy. Here, I show that, by changing one simple assumption in each model, the powerstroke and chemical-Fx model can be reconciled through a chemical thermodynamic model. The formal and experimental justifications for changing these assumptions are presented. The result is a unifying model for mechanochemical coupling in motor enzymes first presented by A.V. Hill in 1938 that is consistent with single-molecule structural and mechanical data.

Velocity of myosin-based actin sliding depends on attachment and detachment kinetics and reaches a maximum when myosin-binding sites on actin saturate.

Molecular motors such as kinesin and myosin often work in groups to generate the directed movements and forces critical for many biological processes. Although much is known about how individual motors generate force and movement, surprisingly, little is known about the mechanisms underlying the macroscopic mechanics generated by multiple motors. For example, the observation that a saturating number, N, of myosin heads move an actin filament at a rate that is influenced by actin-myosin attachment and detachment kinetics is accounted for neither experimentally nor theoretically. To better understand the emergent mechanics of actin-myosin mechanochemistry, we use an in vitro motility assay to measure and correlate the N-dependence of actin sliding velocities, actin-activated ATPase activity, force generation against a mechanical load, and the calcium sensitivity of thin filament velocities. Our results show that both velocity and ATPase activity are strain dependent and that velocity becomes maximized with the saturation of myosin-binding sites on actin at a value that is 40% dependent on attachment kinetics and 60% dependent on detachment kinetics. These results support a chemical thermodynamic model for ensemble motor mechanochemistry and imply molecularly explicit mechanisms within this framework, challenging the assumption of independent force generation.

Effects of Maternally Transferred Egg Selenium on Embryo-Larval Survival, Growth, and Development in Arctic Grayling (Thymallus arcticus).

Selenium (Se) toxicity to fish is primarily manifested via maternal transfer to the eggs, which may result in adverse effects on larval survival and development. The present study assessed the effects of egg Se concentrations derived via maternal transfer on early life-stage development, survival, and growth of Arctic grayling (Thymallus arcticus), a salmonid species not previously assessed for Se sensitivity. Fish gametes were collected from 4 streams in Alaska known to exhibit a range of egg Se concentrations. Eggs were fertilized and reared in the laboratory from hatch through post-swim-up. Larvae were assessed for survival, length, and weight, as well as deformities (skeletal, craniofacial, fin-fold) and edema based on a graduated severity index. Eggs from a total of 47 females were collected, with egg Se concentrations ranging from 3.3 to 33.9 mg kg-1 dry weight. No relationships were observed between larval endpoints evaluated and parent females' egg, muscle, or whole-body Se concentrations. Therefore, Se 10% effective concentrations (EC10s) were defined as the maximum measured Se concentrations: >33.9, >17.6, and >19.7 mg kg-1 dry weight for eggs, muscle, and whole-body tissue, respectively. Collectively, these data indicate that Arctic grayling are relatively insensitive to maternally transferred Se compared to other fish species. Environ Toxicol Chem 2021;40:380-389. © 2020 SETAC.

S-Nitrosoglutathione Reductase Underlies the Dysfunctional Relaxation to Nitric Oxide in Preterm Labor.

Tocolytics show limited efficacy to prevent preterm delivery. In uterine smooth muscle cGMP accumulation following addition of nitric oxide (NO) has little effect on relaxation suggesting a role for protein S-nitrosation. In human myometrial tissues from women in labor at term (TL), or spontaneously in labor preterm (sPTL), direct stimulation of soluble guanylyl cyclase (sGC) fails to relax myometrium, while the same treatment relaxes vascular smooth muscle completely. Unlike term myometrium, effects of NO are not only blunted in sPTL, but global protein S-nitrosation is also diminished, suggesting a dysfunctional response to NO-mediated protein S-nitrosation. Examination of the enzymatic regulator of endogenous S-nitrosoglutathione availability, S-nitrosoglutathione reductase, reveals increased expression of the reductase in preterm myometrium associated with decreased total protein S-nitrosation. Blockade of S-nitrosoglutathione reductase relaxes sPTL tissue. Addition of NO donor to the actin motility assay attenuates force. Failure of sGC activation to mediate relaxation in sPTL tissues, together with the ability of NO to relax TL, but not sPTL myometrium, suggests a unique pathway for NO-mediated relaxation in myometrium. Our results suggest that examining the action of S-nitrosation on critical contraction associated proteins central to the regulation of uterine smooth muscle contraction can reveal new tocolytic targets.

A mixed-kinetic model describes unloaded velocities of smooth, skeletal, and cardiac muscle myosin filaments in vitro.

In vitro motility assays, where purified myosin and actin move relative to one another, are used to better understand the mechanochemistry of the actomyosin adenosine triphosphatase (ATPase) cycle. We examined the relationship between the relative velocity (V) of actin and myosin and the number of available myosin heads (N) or [ATP] for smooth (SMM), skeletal (SKM), and cardiac (CMM) muscle myosin filaments moving over actin as well as V from actin filaments moving over a bed of monomeric SKM. These data do not fit well to a widely accepted model that predicts that V is limited by myosin detachment from actin (d/ton), where d equals step size and ton equals time a myosin head remains attached to actin. To account for these data, we have developed a mixed-kinetic model where V is influenced by both attachment and detachment kinetics. The relative contributions at a given V vary with the probability that a head will remain attached to actin long enough to reach the end of its flexible S2 tether. Detachment kinetics are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 ± 3 nm, 22 ± 9 nm, and 22 ± 2 nm, respectively). In contrast, L is shorter (8 ± 3 nm) when myosin monomers are attached to a surface. This suggests that the behavior of the S2 domain may be an important mechanical feature of myosin filaments that influences unloaded shortening velocities of muscle.

Using ultrahigh-resolution mass spectrometry and toxicity identification techniques to characterize the toxicity of oil sands process-affected water: The case for classical naphthenic acids.

Previous assessments of oil sands process-affected water (OSPW) toxicity were hampered by lack of high-resolution analytical analysis, use of nonstandard toxicity methods, and variability between OSPW samples. We integrated ultrahigh-resolution mass spectrometry with a toxicity identification evaluation (TIE) approach to quantitatively identify the primary cause of acute toxicity of OSPW to rainbow trout (Oncorhynchus mykiss). The initial characterization of OSPW toxicity indicated that toxicity was associated with nonpolar organic compounds, and toxicant(s) were further isolated within a range of discrete methanol fractions that were then subjected to Orbitrap mass spectrometry to evaluate the contribution of naphthenic acid fraction compounds to toxicity. The results showed that toxicity was attributable to classical naphthenic acids, with the potency of individual compounds increasing as a function of carbon number. Notably, the mass of classical naphthenic acids present in OSPW was dominated by carbon numbers ≤16; however, toxicity was largely a function of classical naphthenic acids with ≥17 carbons. Additional experiments found that acute toxicity of the organic fraction was similar when tested at conductivities of 400 and 1800 µmhos/cm and that rainbow trout fry were more sensitive to the organic fraction than larval fathead minnows (Pimephales promelas). Collectively, the results will aid in developing treatment goals and targets for removal of OSPW toxicity in water return scenarios both during operations and on mine closure. Environ Toxicol Chem 2017;36:3148-3157. © 2017 SETAC.

Evaluation of the effect of water type on the toxicity of nitrate to aquatic organisms.

A suite of acute and chronic toxicity tests were conducted to evaluate the sensitivity of freshwater organisms to nitrate (as sodium nitrate). Acute exposures with rainbow trout (Onchorhynchus mykiss) and amphipods (Hyalella azteca), as well as chronic exposures with H. azteca (14-d survival and growth), midges (Chironomus dilutus; 10-d survival and growth), daphnids (Ceriodaphnia dubia; 7-d survival and reproduction), and fathead minnows (Pimephales promelas; 7-d survival and growth) were used to determine sublethal and lethal effect concentrations. Modification of nitrate toxicity was investigated across a range of ionic strengths, created through the use of very soft water, and standard preparations of synthetic soft, moderately-hard and hard dilution waters. The most sensitive species tested were C. dubia and H. azteca, in soft water, with reproduction and growth IC25 values of 13.8 and 12.2 mg/L NO3-N, respectively. All of the organisms exposed to nitrate demonstrated significantly reduced effects with increasing ionic strength associated with changes in water type. Possible mechanisms responsible for the modifying effect of increasing major ion concentrations on nitrate toxicity are discussed.

The role of phosphorus in the metabolism of arsenate by a freshwater green alga, Chlorella vulgaris.

A freshwater microalga, Chlorella vulgaris, was grown in the presence of varying phosphate concentrations (<10-500µg/L P) and environmentally realistic concentrations of arsenate (As(V)) (5-50µg/L As). Arsenic speciation in the culture medium and total cellular arsenic were measured using AEC-ICP-MS and ICP-DRC-MS, respectively, to determine arsenic biotransformation and uptake in the various phosphorus scenarios. At high phosphate concentration in the culture medium, >100µg/L P, the uptake and biotransformation of As(V) was minimal and dimethylarsonate (DMAs(V)) was the dominant metabolite excreted by C. vulgaris, albeit at relatively low concentrations. At common environmental P concentrations, 0-50µg/L P, the uptake and biotransformation of As(V) increased. At these higher As-uptake levels, arsenite (As(III)) was the predominant metabolite excreted from the cell. The concentrations of As(III) in these low P conditions were much higher than the concentrations of methylated arsenicals observed at the various P concentrations studied. The switchover threshold between the (small) methylation and (large) reduction of As(V) occurred around a cellular As concentration of 1fg/cell. The observed nearly quantitative conversion of As(V) to As(III) under low phosphate conditions indicates the importance of As(V) bio-reduction at common freshwater P concentrations. These findings on the influence of phosphorus on arsenic uptake, accumulation and excretion are discussed in relation to previously published research. The impact that the two scenarios of As(V) metabolism, As(III) excretion at high As(V)-uptake and methylarsenical excretion at low As(V)-uptake, have on freshwater arsenic speciation is discussed.

Dietary selenium disrupts hepatic triglyceride stores and transcriptional networks associated with growth and Notch signaling in juvenile rainbow trout.

Dietary Se has been shown to adversely affect adult fish by altering growth rates and metabolism. To determine the underlying mechanisms associated with these observations, we measured biochemical and transcriptomic endpoints in rainbow trout following dietary Se exposures. Treatment groups of juvenile rainbow trout were fed either control Lumbriculus variegatus worms or worms cultured on selenized yeast. Selenized yeast was cultured at four nominal doses of 5, 10, 20 or 40mg/kg Se dry weight (measured dose in the worms of 7.1, 10.7, 19.5, and 31.8mg/kgSedw respectively) and fish were fed for 60days. At 60 d, hepatic triglycerides, glycogen, total glutathione, 8-isoprostane and the transcriptome response in the liver (n=8/group) were measured. Fish fed the nominal dose of 20 and 40mg/kg Se dry weight had lower body weight and a shorter length, as well as lower triglyceride in the liver compared to controls. Evidence was lacking for an oxidative stress response and there was no change in total glutathione, 8-isoprostane levels, nor relative mRNA levels for glutathione peroxidase isoforms among groups. Microarray analysis revealed that molecular networks for long-chain fatty acid transport, lipid transport, and low density lipid oxidation were increased in the liver of fish fed 40mg/kg, and this is hypothesized to be associated with the lower triglyceride levels in these fish. In addition, up-regulated gene networks in the liver of 40mg/kg Se treated fish included epidermal growth factor receptor signaling, growth hormone receptor, and insulin growth factor receptor 1 signaling pathways. These molecular changes are hypothesized to be compensatory and related to impaired growth. A gene network related to Notch signaling, which is involved in cell-cell communication and gene transcription regulation, was also increased in the liver following dietary treatments with both 20 and 40mg/kg Se. Transcriptomic data support the hypothesis that dietary Se increases the expression of networks for growth-related signaling cascades in addition to those related to fatty acid synthesis and metabolism. We propose that the disruption of metabolites related to triglyceride processing and storage, as well as gene networks for epidermal growth factor and Notch signaling in the liver, represent key molecular initiating events for adverse outcomes related to growth and Se toxicity in fish.

Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.

Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcat for both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Km values were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat /Km ) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions. SIGNIFICANCE OF THE STUDY: Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.