Single molecule mechanics resolves the earliest events in force generation by cardiac myosin.

Key steps of cardiac mechanochemistry, including the force-generating working stroke and the release of phosphate (Pi), occur rapidly after myosin-actin attachment. An ultra-high-speed optical trap enabled direct observation of the timing and amplitude of the working stroke, which can occur within <200 µs of actin binding by ß-cardiac myosin. The initial actomyosin state can sustain loads of at least 4.5 pN and proceeds directly to the stroke or detaches before releasing ATP hydrolysis products. The rates of these processes depend on the force. The time between binding and stroke is unaffected by 10 mM Pi which, along with other findings, indicates the stroke precedes phosphate release. After Pi release, Pi can rebind enabling reversal of the working stroke. Detecting these rapid events under physiological loads provides definitive indication of the dynamics by which actomyosin converts biochemical energy into mechanical work.

Positive cardiac inotrope omecamtiv mecarbil activates muscle despite suppressing the myosin working stroke.

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.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine ß-cardiac myosin.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

Unc45b forms a cytosolic complex with Hsp90 and targets the unfolded myosin motor domain.

Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45b(Flag)) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of approximately 19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45b(Flag) fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45b(Flag) re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45b(Flag)/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45b(Flag) does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45b(Flag) enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding.

Unc45 activates Hsp90-dependent folding of the myosin motor domain.

Myosin folding and assembly in striated muscle are mediated by the general chaperones Hsc70 and Hsp90 and involve a myosin-specific co-chaperone related to the Caenorhabditis elegans gene unc-45. Two unc-45 genes are found in vertebrates, a general cell isoform, unc45a, and a striated muscle-specific isoform, unc45b. We have investigated the role of both isoforms of mouse Unc45 in myosin folding using an in vitro synthesis and folding assay. A smooth muscle myosin motor domain (MD) fused to green fluorescent protein (GFP) (MD::GFP) was used as substrate, and folding was measured by native gel electrophoresis and functional assays. In the absence of Unc45, the MD::GFP chimera folds poorly. Addition of either Unc45a or Unc45b dramatically enhances the folding in a reaction that is dependent on Hsp90 ATPase activity. Unc45a is more effective than Unc45b with a higher apparent affinity and greater extent of folding. The Unc45-Hsp90 chaperone complex acts late in the folding pathway and promotes motor domain maturation after release from the ribosome. Unc45a behaves kinetically as an activator of the folding reaction by stimulating the rate of the Hsp90-dependent folding by >20-fold with an apparent K(act) of 33 nm. This analysis of vertebrate Unc45 isoforms clearly demonstrates a direct role for Unc45 in Hsp90-mediated myosin motor domain folding and highlights major differences between the isoforms in substrate specificity and mechanism.

Chaperone-mediated folding and assembly of myosin in striated muscle.

De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.

Mutations in the motor domain modulate myosin activity and myofibril organization.

We have investigated the functional impact on cardiac myofibril organization and myosin motor activity of point mutations associated with familial hypertrophic cardiomyopathies (FHC). Embryonic chicken cardiomyocytes were transfected with vectors encoding green fluorescent protein (GFP) fused to a striated muscle myosin heavy chain (GFP-myosin). Within 24 hours of transfection, the GFP-myosin is found co-assembled with the endogenous myosin in striated myofibrils. The wild-type GFP-myosin had no effect on the organization of the contractile cytoskeleton of the cardiomyocytes. However, expression of myosin with the R403Q FHC mutation resulted in a small but significant decrease in myofibril organization, and the R453C and G584R mutations caused a more dramatic increase in myofibril disarray. The embryonic cardiomyocytes beat spontaneously in culture and this was not affected by expression of the wild-type or mutant GFP-myosin. For the biochemical analysis of myosin motor activity, replication defective adenovirus was used to express the wild-type and mutant GFP-myosin in C2C12 myotubes. The R403Q mutation enhanced actin filament velocity but had no effect on the myosin duty ratio. The R453C and G584R mutations impaired actin filament movement and both increased the duty ratio. The effects of these mutations on myosin motor activity correlate with changes in myofibril organization of live cardiomyocytes. Thus, mutations associated with hypertrophic cardiomyopathies that alter myosin motor activity can also impair myofibril organization.

Folding of the striated muscle myosin motor domain.

We have investigated the folding of the myosin motor domain using a chimera of an embryonic striated muscle myosin II motor domain fused on its COOH terminus to a thermal stable, fast folding variant of green fluorescent protein (GFP). In in vitro expression assays, the GFP domain of the chimeric protein, S1(795)GFP, folds rapidly enabling us to monitor the folding of the motor domain using fluorescence. The myosin motor domain folds very slowly and transits through multiple intermediates that are detectable by gel filtration chromatography. The distribution of the nascent protein among these intermediates is strongly dependent upon temperature. At 25 degrees C and above the predominant product is an aggregate of S1(795)GFP or a complex with other lysate proteins. At 0 degrees C, the motor domain folds slowly via an energy independent pathway. The unusual temperature dependence and slow rate suggests that folding of the myosin motor is highly susceptible to off-pathway interactions and aggregation. Expression of the S1(795)GFP in the C2C12 muscle cell line yields a folded and functionally active protein that exhibits Mg(2+)ATP-sensitive actin-binding and myosin motor activity. In contrast, expression of S1(795)GFP in kidney epithelial cell lines (human 293 and COS 7 cells) results in an inactive and aggregated protein. The results of the in vitro folding assay suggest that the myosin motor domain does not fold spontaneously under physiological conditions and probably requires cytosolic chaperones. The expression studies support this conclusion and demonstrate that these factors are optimized in muscle cells.