Binding pocket dynamics along the recovery stroke of human ß-cardiac myosin.

The druggability of small-molecule binding sites can be significantly affected by protein motions and conformational changes. Ligand binding, protein dynamics and protein function have been shown to be closely interconnected in myosins. The breakthrough discovery of omecamtiv mecarbil (OM) has led to an increased interest in small molecules that can target myosin and modulate its function for therapeutic purposes (myosin modulators). In this work, we use a combination of computational methods, including steered molecular dynamics, umbrella sampling and binding pocket tracking tools, to follow the evolution of the OM binding site during the recovery stroke transition of human ß-cardiac myosin. We found that steering two internal coordinates of the motor domain can recapture the main features of the transition and in particular the rearrangements of the binding site, which shows significant changes in size, shape and composition. Possible intermediate conformations were also identified, in remarkable agreement with experimental findings. The differences in the binding site properties observed along the transition can be exploited for the future development of conformation-selective myosin modulators.

Two Classes of Myosin Inhibitors, Para-nitroblebbistatin and Mavacamten, Stabilize ß-Cardiac Myosin in Different Structural and Functional States.

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure-function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, 'ultra-relaxed' state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human ß-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil.

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human ß-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human ß-cardiac myosin subfragment 1 (M2ß-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Biophysical properties of human ß-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the ß-cardiac myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human ß-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human ß-cardiac myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.

Omecamtiv Mecarbil Enhances the Duty Ratio of Human ß-Cardiac Myosin Resulting in Increased Calcium Sensitivity and Slowed Force Development in Cardiac Muscle.

The small molecule drug omecamtiv mecarbil (OM) specifically targets cardiac muscle myosin and is known to enhance cardiac muscle performance, yet its impact on human cardiac myosin motor function is unclear. We expressed and purified human ß-cardiac myosin subfragment 1 (M2ß-S1) containing a C-terminal Avi tag. We demonstrate that the maximum actin-activated ATPase activity of M2ß-S1 is slowed more than 4-fold in the presence of OM, whereas the actin concentration required for half-maximal ATPase was reduced dramatically (30-fold). We find OM does not change the overall actin affinity. Transient kinetic experiments suggest that there are two kinetic pathways in the presence of OM. The dominant pathway results in a slow transition between actomyosin·ADP states and increases the time myosin is strongly bound to actin. However, OM also traps a population of myosin heads in a weak actin affinity state with slow product release. We demonstrate that OM can reduce the actin sliding velocity more than 100-fold in the in vitro motility assay. The ionic strength dependence of in vitro motility suggests the inhibition may be at least partially due to drag forces from weakly attached myosin heads. OM causes an increase in duty ratio examined in the motility assay. Experiments with permeabilized human myocardium demonstrate that OM increases calcium sensitivity and slows force development (ktr) in a concentration-dependent manner, whereas the maximally activated force is unchanged. We propose that OM increases the myosin duty ratio, which results in enhanced calcium sensitivity but slower force development in human myocardium.

Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human ß-Cardiac Myosin.

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in ß-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human ß-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human ß-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

Analytical comparison of natural and pharmaceutical ventricular myosin activators.

Ventricular myosin (ßMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive myosins for which persistent actin association is the priority. The only pharmaceutical ßMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro ßMys step-size, motility velocity, and actin-activated myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize ßMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting ßMys to a near processive myosin. The OM conversion dramatically increases force and modestly increases power over the native ßMys. Contrasting motility modification due to OM with that from the natural myosin activator, specific ßMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical ßMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.

Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules.

Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.

Human essential myosin light chain isoforms revealed distinct myosin binding, sarcomeric sorting, and inotropic activity.

AIMS: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity. METHODS AND RESULTS: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac ß-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rß-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or ß-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rß-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged. CONCLUSION: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Enhancement of the recycling and activation of beta-adrenergic receptor by Rab4 GTPase in cardiac myocytes.

We investigate the role of Rab4, a Ras-like small GTPase coordinating protein transport from the endosome to the plasma membrane, on the recycling and activation of endogenous beta-adrenergic receptor (beta-AR) in HL-1 cardiac myocytes in vitro and transgenic mouse hearts in vivo. Beta1-AR, the predominant subtype of beta-AR in HL-1 cardiac myocytes, was internalized after stimulation with isoproterenol (ISO) and fully recycled at 4 h upon ISO removal. Transient expression of Rab4 markedly facilitated recycling of internalized beta-AR to the cell surface and enhanced beta-AR signaling as measured by ISO-stimulated cAMP production. Transgenic overexpression of Rab4 in the mouse myocardium significantly increased the number of beta-AR in the plasma membrane and augmented cAMP production at the basal level and in response to ISO stimulation. Rab4 overexpression induced concentric cardiac hypertrophy with a moderate increase in ventricle/body weight ratio and posterior wall thickness and a selective up-regulation of the beta-myosin heavy chain gene. These data provide the first evidence indicating that Rab4 is a rate-limiting factor for the recycling of endogenous beta-AR and augmentation of Rab4-mediated traffic enhances beta-AR function in cardiac myocytes.

The regulatory effects of tropomyosin and troponin-I on the interaction of myosin loop regions with F-actin.

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088-5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac beta-myosin, presumed to be sites of interaction with F-actin (residues 554-584, 622-646, and 633-660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134-147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin.

Impact of beta-myosin heavy chain isoform expression on cross-bridge cycling kinetics.

Myosin heavy chain (MHC) isoforms alpha and beta have intrinsically different ATP hydrolysis activities (ATPase) and therefore cross-bridge cycling rates in solution. There is considerable evidence of altered MHC expression in rodent cardiac disease models; however, the effect of incremental beta-MHC expression over a wide range on the rate of high-strain, isometric cross-bridge cycling is yet to be ascertained. We treated male rats with 6-propyl-2-thiouracil (PTU; 0.8 g/l in drinking water) for short intervals (6, 11, 16, and 21 days) to generate cardiac MHC patterns in transition from predominantly alpha-MHC to predominantly beta-MHC. Steady-state calcium-dependent tension development and tension-dependent ATP consumption (tension cost; proportional to cross-bridge cycling) were measured in chemically permeabilized (skinned) right ventricular muscles at 20 degrees C. To assess dynamic cross-bridge cycling kinetics, the rate of force redevelopment (ktr) was determined after rapid release-restretch of fully activated muscles. MHC isoform content in each experimental muscle was measured by SDS-PAGE and densitometry. alpha-MHC content decreased significantly and progressively with length of PTU treatment [68 +/- 5%, 58 +/- 4%, 37 +/- 4%, and 27 +/- 6% for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Tension cost decreased, linearly, with decreased alpha-MHC content [6.7 +/- 0.4, 5.6 +/- 0.5, 4.0 +/- 0.4, and 3.9 +/- 0.3 ATPase/tension for 6, 11, 16, and 21 days, respectively; P < 0.001 (ANOVA)]. Likewise, ktr was significantly and progressively depressed with length of PTU treatment [11.1 +/- 0.6, 9.1 +/- 0.5, 8.2 +/- 0.7, and 6.2 +/- 0.3 s(-1) for 6, 11, 16, and 21 days, respectively; P < 0.05 (ANOVA)] Thus cross-bridge cycling, under high strain, for alpha-MHC is three times higher than for beta-MHC. Furthermore, under isometric conditions, alpha-MHC and beta-MHC cross bridges hydrolyze ATP independently of one another.

Cardiac myosin isoforms from different species have unique enzymatic and mechanical properties.

The mammalian heart contains two cardiac myosin isoforms: beta-myosin heavy chain (MHC) is found predominantly in the ventricles of large mammals, and alpha-MHC is expressed in the atria. The sequence identity between these isoforms is approximately 93%, with nonidentical residues clustered in discrete, functionally important domains associated with actin binding and ATPase activity. It is well-established that rabbit alpha-cardiac myosin has a 2-fold greater unloaded shortening velocity than beta-cardiac myosin but a 2-fold lower average isometric force. Here, we test the generality of these relationships for another large mammal, the pig, as well as for a small rodent, the mouse, which expresses alpha-MHC in its ventricles throughout adulthood. Hydrophobic interaction chromatography (HIC) was used to purify myosin from mouse, rabbit, and pig hearts. The superior resolving power of HIC made it possible to prepare highly homogeneous, enzymatically active myosin from small amounts of tissue. The movement of actin filaments by myosin was measured in an in vitro motility assay. The same assay could be used to determine average isometric force by loading the actin filaments with increasing concentrations of alpha-actinin to stop filament motion. We conclude that myosin from the mouse has significantly higher velocities for both alpha and beta isoforms than myosin from rabbits and pigs, even though the 2-fold difference in velocity between isoforms is maintained. Unlike the larger mammals, however, the small rodent generates the same high isometric force for both alpha and beta isoforms. Thus, nature has adapted the function of cardiac myosin isoforms to optimize power output for hearts of a given species.

Simultaneous quantification of human cardiac alpha- and beta-myosin heavy chain proteins by MALDI-TOF mass spectrometry.

We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the alpha and beta protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. Alpha-MyHC (726-741) and beta-MyHC (724-739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was synthesized for use as an internal standard (IS). The measured ion current ratios of synthetic alpha-MyHC (726-741), beta-MyHC (724-739), and IS peptides were used to generate standard curves. A known quantity of the IS was added to the MyHC digests. The measured ion current ratios were converted to the actual quantities of the isoform-specific peptides and hence the actual quantity of each protein isoform by reference to the standard curves. This method is of general applicability, especially when isoform quantification is required.

Molecular mechanics of mouse cardiac myosin isoforms.

Two myosin isoforms are expressed in myocardium, alphaalpha-homodimers (V(1)) and betabeta-homodimers (V(3)). V(1) exhibits higher velocities and myofibrillar ATPase activities compared with V(3). We also observed this for cardiac myosin from normal (V(1)) and propylthiouracil-treated (V(3)) mice. Actin velocity in a motility assay (V(actin)) over V(1) myosin was twice that of V(3) as was the myofibrillar ATPase. Myosin's average force (F(avg)) was similar for V(1) and V(3). Comparing V(actin) and F(avg) across species for both V(1) and V(3), our laboratory showed previously (VanBuren P, Harris DE, Alpert NR, and Warshaw DM. Circ Res 77: 439-444, 1995) that mouse V(1) has greater V(actin) and F(avg) compared with rabbit V(1). Mouse V(3) V(actin) was twice that of rabbit V(actin). To understand myosin's molecular structure and function, we compared alpha- and beta-cardiac myosin sequences from rodents and rabbits. The rabbit alpha- and beta-cardiac myosin differed by eight and four amino acids, respectively, compared with rodents. These residues are localized to both the motor domain and the rod. These differences in sequence and mechanical performance may be an evolutionary attempt to match a myosin's mechanical behavior to the heart's power requirements.