A cardioexcitatory substance from ganglia of the clam Macrocallista nimbosa, formerly designated peak C, is the tetrapeptide amide Phe-Met-Arg-Phe-NH2. Its structure was determined by the combined use of Edman dansyl degradation and tryptic digestion. The structure was confirmed by synthesis. This neuropeptide is active at about 10(-8)M when assayed on molluscan muscle.

Omecamtiv mecarbil (OM) is a positive cardiac inotrope in phase-3 clinical trials for treatment of heart failure. Although initially described as a direct myosin activator, subsequent studies are at odds with this description and do not explain OM-mediated increases in cardiac performance. Here we show, via single-molecule, biophysical experiments on cardiac myosin, that OM suppresses myosin’s working stroke and prolongs actomyosin attachment 5-fold, which explains inhibitory actions of the drug observed in vitro. OM also causes the actin-detachment rate to become independent of both applied load and ATP concentration. Surprisingly, increased myocardial force output in the presence of OM can be explained by cooperative thin-filament activation by OM-inhibited myosin molecules. Selective suppression of myosin is an unanticipated route to muscle activation that may guide future development of therapeutic drugs.

Visual Abstract

Response to induction chemotherapy as studied in this trial was not useful as a predictive marker for ultimate outcome or organ conservation. Overall, however, this regimen offers good disease control and survival for patients with locally advanced oropharyngeal carcinoma, comparable with other concurrent chemoradiation programs. Further study of similar protocols is indicated.

Myosin IC (myo1c), a widely expressed motor protein that links the actin cytoskeleton to cell membranes, has been associated with numerous cellular processes, including insulin-stimulated transport of GLUT4, mechanosensation in sensory hair cells, endocytosis, transcription of DNA in the nucleus, exocytosis, and membrane trafficking. The molecular role of myo1c in these processes has not been defined, so to better understand myo1c function, we utilized ensemble kinetic and single-molecule techniques to probe myo1c's biochemical and mechanical properties. Utilizing a myo1c construct containing the motor and regulatory domains, we found the force dependence of the actin-attachment lifetime to have two distinct regimes: a force-independent regime at forces <1 pN, and a highly force-dependent regime at higher loads. In this force-dependent regime, forces that resist the working stroke increase the actinattachment lifetime. Unexpectedly, the primary force-sensitive transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins. This force-sensing behavior is unique amongst characterized myosins and clearly demonstrates mechanochemical diversity within the myosin family. Based on these results, we propose that myo1c functions as a slow transporter rather than a tension-sensitive anchor.is a widely expressed myosin-I isoform that has been associated with several important cellular processes, including endocytosis (1), exocytosis (2) (including insulin-stimulated GLUT4 translocation to the cell membrane; refs. 3-5), membrane ruffling (6), transcription of DNA in the nucleus (7,8), and mechanosensing in sensory hair cells (9-13). Although it is known that myo1c links cell membranes to the actin cytoskeleton (14, 15), its molecular role in these cellular processes has not been determined. For example, in its proposed role in exocytosis, it is not known if myo1c acts as a motor for transport, moving vesicles into position for plasma membrane fusion and/or as a tension-sensitive anchor that docks exocytic vesicles to the actin cytoskeleton and plasma membrane.Most members of the myosin family share the same kinetic pathway for ATP hydrolysis, in which force-generating structural changes are linked to release of inorganic phosphate and ADP, but different myosin isoforms have evolved different biochemical reaction rates and force-dependent kinetics to suit their cellular functions. For example, in myosins that are thought to act as tension-sensitive anchors, the kinetic steps that limit actomyosin detachment are highly sensitive to load. In contrast, myosins that are thought to act as transporters have actin-detachment kinetics that are less sensitive to load, allowing work to be performed over a range of forces. Thus, insight into the molecular role of myosin in the cell can be gained from evaluating the kinetic and mechanical properties of the motor.Previous biochemical analyses have shown that myo1c is a lowduty ratio motor (i.e., it spends most of its biochemical cycle detached from act...

The heart adjusts its power output to meet specific physiological needs through the coordination of several mechanisms, including force-induced changes in contractility of the molecular motor, the β-cardiac myosin (βCM). Despite its importance in driving and regulating cardiac power output, the effect of force on the contractility of a single βCM has not been measured. Using single molecule optical-trapping techniques, we found that βCM has a two-step working stroke. Forces that resist the power stroke slow the myosin-driven contraction by slowing the rate of ADP release, which is the kinetic step that limits fiber shortening. The kinetic properties of βCM are affected by load, suggesting that the properties of myosin contribute to the force-velocity relationship in intact muscle and play an important role in the regulation of cardiac power output.

Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a major indicator for heart transplant. The disease is frequently caused by mutations of sarcomeric proteins; however, it is not well understood how these molecular mutations lead to alterations in cellular organization and contractility. To address this critical gap in our knowledge, we studied the molecular and cellular consequences of a DCM mutation in troponin-T, ΔK210. We determined the molecular mechanism of ΔK210 and used computational modeling to predict that the mutation should reduce the force per sarcomere. In mutant cardiomyocytes, we found that ΔK210 not only reduces contractility but also causes cellular hypertrophy and impairs cardiomyocytes’ ability to adapt to changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and disease). These results help link the molecular and cellular phenotypes and implicate alterations in mechanosensing as an important factor in the development of DCM.

Molecular motors convert chemical energy into mechanical movement, generating forces necessary to accomplish an array of cellular functions. Since molecular motors generate force, they typically work under loaded conditions where the motor mechanochemistry is altered by the presence of a load. Several biophysical techniques have been developed to study the loaded behavior and force generating capabilities of molecular motors yet most of these techniques require specialized equipment. The frictional loading assay is a modification to the in vitro motility assay that can be performed on a standard epifluorescence microscope, permitting the high-throughput measurement of the loaded mechanochemistry of molecular motors. Here, we describe a model for the molecular basis of the frictional loading assay by modeling the load as a series of either elastic or viscoelastic elements. The model, which calculates the frictional loads imposed by different binding proteins, permits the measurement of isotonic kinetics, force-velocity relationships, and power curves in the motility assay. We show computationally and experimentally that the frictional load imposed by alpha-actinin, the most widely employed actin binding protein in frictional loading experiments, behaves as a viscoelastic rather than purely elastic load. As a test of the model, we examined the frictional loading behavior of rabbit skeletal muscle myosin under normal and fatigue-like conditions using alpha-actinin as a load. We found that, consistent with fiber studies, fatigue-like conditions cause reductions in myosin isometric force, unloaded sliding velocity, maximal power output, and shift the load at which peak power output occurs. V C 2010 Wiley-Liss, Inc.Key Words: alpha-actinin, isotonic, force-velocity relationship, power, skeletal muscle fatigue Introduction T he myosin family of molecular motors is a diverse superfamily of actin binding proteins (ABP) that convert the energy from ATP hydrolysis into motion and force in order to accomplish an array of cellular functions [for review, see Sellers, 2000;O'Connell et al., 2007]. The in vitro motility assay is a useful tool for studying the molecular basis of myosin-based movement under a variety of well controlled experimental conditions Tyska and Warshaw, 2002]. This technique permits the study of myosin mechanics while at the same time, retaining many of the features of solution biochemistry. In the in vitro motility assay, myosin is bound to a cover glass surface and a solution containing fluorescently labeled actin filaments is applied to the myosin surface in the presence of ATP, allowing for the measurement of actin filament movement via fluorescence microscopy [Kron and Spudich, 1986]. The typical motility assay measures the sliding velocity of myosin in the absence of an exogenously added load, providing important information on the actomyosin contractile mechanism. Under physiological conditions, however, myosins typically operate against a load, making it desirable to study myosin force generation...