Actin-activated Myosin ATPase Activity Research Articles

A Novel Variant in TPM3 Causing Muscle Weakness and Concomitant Hypercontractile Phenotype.

A novel variant of unknown significance c.8A > G (p.Glu3Gly) in TPM3 was detected in two unrelated families. TPM3 encodes the transcript variant Tpm3.12 (NM_152263.4), the tropomyosin isoform specifically expressed in slow skeletal muscle fibers. The patients presented with slowly progressive muscle weakness associated with Achilles tendon contractures of early childhood onset. Histopathology revealed features consistent with a nemaline rod myopathy. Biochemical in vitro assays performed with reconstituted thin filaments revealed defects in the assembly of the thin filament and regulation of actin-myosin interactions. The substitution p.Glu3Gly increased polymerization of Tpm3.12, but did not significantly change its affinity to actin alone. Affinity of Tpm3.12 to actin in the presence of troponin ± Ca2+ was decreased by the mutation, which was due to reduced interactions with troponin. Altered molecular interactions affected Ca2+-dependent regulation of the thin filament interactions with myosin, resulting in increased Ca2+ sensitivity and decreased relaxation of the actin-activated myosin ATPase activity. The hypercontractile molecular phenotype probably explains the distal joint contractions observed in the patients, but additional research is needed to explain the relatively mild severity of the contractures. The slowly progressive muscle weakness is most likely caused by the lack of relaxation and prolonged contractions which cause muscle wasting. This work provides evidence for the pathogenicity of the TPM3 c.8A > G variant, which allows for its classification as (likely) pathogenic.

Functional comparison of phosphomimetic S15D and T160D mutants of myosin regulatory light chain exchanged in cardiac muscle preparations of HCM and WT mice.

In this study, we investigated the rescue potential of two phosphomimetic mutants of the myosin regulatory light chain (RLC, MYL2 gene), S15D, and T160D RLCs. S15D-RLC mimics phosphorylation of the established serine-15 site of the human cardiac RLC. T160D-RLC mimics the phosphorylation of threonine-160, identified by computational analysis as a high-score phosphorylation site of myosin RLC. Cardiac myosin and left ventricular papillary muscle (LVPM) fibers were isolated from a previously generated model of hypertrophic cardiomyopathy (HCM), Tg-R58Q, and Tg-wild-type (WT) mice. Muscle specimens were first depleted of endogenous RLC and then reconstituted with recombinant human cardiac S15D and T160D phosphomimetic RLCs. Preparations reconstituted with recombinant human cardiac WT-RLC and R58Q-RLC served as controls. Mouse myosins were then tested for the actin-activated myosin ATPase activity and LVPM fibers for the steady-state force development and Ca2+-sensitivity of force. The data showed that S15D-RLC significantly increased myosin ATPase activity compared with T160D-RLC or WT-RLC reconstituted preparations. The two S15D and T160D phosphomimetic RLCs were able to rescue Vmax of Tg-R58Q myosin reconstituted with recombinant R58Q-RLC, but the effect of S15D-RLC was more pronounced than T160D-RLC. Low tension observed for R58Q-RLC reconstituted LVPM from Tg-R58Q mice was equally rescued by both phosphomimetic RLCs. In the HCM Tg-R58Q myocardium, the S15D-RLC caused a shift from the super-relaxed (SRX) state to the disordered relaxed (DRX) state, and the number of heads readily available to interact with actin and produce force was increased. At the same time, T160D-RLC stabilized the SRX state at a level similar to R58Q-RLC reconstituted fibers. We report here on the functional superiority of the established S15 phospho-site of the human cardiac RLC vs. C-terminus T160-RLC, with S15D-RLC showing therapeutic potential in mitigating a non-canonical HCM behavior underlined by hypocontractile behavior of Tg-R58Q myocardium.

Role of calponin 2 in cell migration and mechanoregulation

Calponin is a family of actin filament-associated regulatory proteins and functions via inhibiting actin-activated myosin ATPase activity. Among three calponin isoforms in vertebrates, calponin 2 encoded by Cnn2 gene expresses in smooth muscles and non-muscle cells such as monocytes/macrophages, endothelial cells, epithelial cells, fibroblasts, myoblasts, and lung alveolar cells. Our group previously reported that calponin 2 regulates the rate of cell proliferation and migration by stabilizing the actin cytoskeleton.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega‐7,9 trans‐fat rumenic acid

Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the β-cardiac myosin-2 motor domain with an EC50 of 200nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine β-cardiac myosin, the EC50 for rumenic acid varies from 10.5μM in the absence of actin to 200nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of β-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5μM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin–actin interaction

Actin–myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin–myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin–myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Synthesis and Evaluation of 4-Hydroxycoumarin Imines as Inhibitors of Class II Myosins.

Inhibitors of muscle myosin ATPases are needed to treat conditions that could be improved by promoting muscle relaxation. The lead compound for this study ((3-(N-butylethanimidoyl)ethyl)-4-hydroxy-2H-chromen-2-one; BHC) was previously discovered to inhibit skeletal myosin II. BHC and 34 analogues were synthesized to explore structure-activity relationships. The properties of analogues, including solubility, stability, and toxicity, suggest that the BHC scaffold may be useful for developing therapeutics. Inhibition of actin-activated ATPase activity of fast skeletal and cardiac muscle myosin II, inhibition of skeletal muscle contractility ex vivo, and slowing of in vitro actin-sliding velocity were measured. Several analogues with aromatic side arms showed improved potency (half-maximal inhibitory concentration (IC50) <1 μM) and selectivity (≥12-fold) for skeletal myosin versus cardiac myosin compared to BHC. Several analogues blocked neurotransmission, suggesting that they are selective for nonmuscle myosin II over skeletal myosin. Competition and molecular docking studies suggest that BHC and blebbistatin bind to the same site on myosin.

Phosphomimetic-mediated invitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain.

Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+ -troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic (Tg) R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where Ser-15 at the phosphorylation site was substituted with aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities invitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in Tg mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium toward the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients.

Fropofol decreases force development in cardiac muscle.

Supranormal contractile properties are frequently associated with cardiac diseases. Anesthetic agents, including propofol, can depress myocardial contraction. We tested the hypothesis that fropofol, a propofol derivative, reduces force development in cardiac muscles via inhibition of cross-bridge cycling and may therefore have therapeutic potential. Force and intracellular Ca2+ concentration ([Ca2+]i) transients of rat trabecular muscles were determined. Myofilament ATPase, actin-activated myosin ATPase, and velocity of actin filaments propelled by myosin were also measured. Fropofol dose dependently decreased force without altering [Ca2+]i in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca2+-activated force ( Fmax) and increased the [Ca2+]i required for 50% of Fmax (Ca50) at steady state without affecting the Hill coefficient in both intact and skinned cardiac fibers. The drug also depressed cardiac myofibrillar and actin-activated myosin ATPase activity. In vitro actin sliding velocity was significantly reduced when fropofol was introduced during rigor binding of cross-bridges. The data suggest that the depressing effects of fropofol on cardiac contractility are likely to be related to direct targeting of actomyosin interactions. From a clinical standpoint, these findings are particularly significant, given that fropofol is a nonanesthetic small molecule that decreases myocardial contractility specifically and thus may be useful in the treatment of hypercontractile cardiac disorders.-Ren, X., Schmidt, W., Huang, Y., Lu, H., Liu, W., Bu, W., Eckenhoff, R., Cammarato, A., Gao, W. D. Fropofol decreases force development in cardiac muscle.

Sarcomeric perturbations of myosin motors lead to dilated cardiomyopathy in genetically modified MYL2 mice

Dilated cardiomyopathy (DCM) is a devastating heart disease that affects about 1 million people in the United States, but the underlying mechanisms remain poorly understood. In this study, we aimed to determine the biomechanical and structural causes of DCM in transgenic mice carrying a novel mutation in the MYL2 gene, encoding the cardiac myosin regulatory light chain. Transgenic D94A (aspartic acid-to-alanine) mice were created and investigated by echocardiography and invasive hemodynamic and molecular structural and functional assessments. Consistent with the DCM phenotype, a significant reduction of the ejection fraction (EF) was observed in ∼5- and ∼12-mo-old male and female D94A lines compared with respective WT controls. Younger male D94A mice showed a more pronounced left ventricular (LV) chamber dilation compared with female counterparts, but both sexes of D94A lines developed DCM by 12 mo of age. The hypocontractile activity of D94A myosin motors resulted in the rightward shift of the force-pCa dependence and decreased actin-activated myosin ATPase activity. Consistent with a decreased Ca2+ sensitivity of contractile force, a small-angle X-ray diffraction study, performed in D94A fibers at submaximal Ca2+ concentrations, revealed repositioning of the D94A cross-bridge mass toward the thick-filament backbone supporting the hypocontractile state of D94A myosin motors. Our data suggest that structural perturbations at the level of sarcomeres result in aberrant cardiomyocyte cytoarchitecture and lead to LV chamber dilation and decreased EF, manifesting in systolic dysfunction of D94A hearts. The D94A-induced development of DCM in mice closely follows the clinical phenotype and suggests that MYL2 may serve as a new therapeutic target for dilated cardiomyopathy.

Interactions and Functions of Myosin 16 Domains

The unconventional myosin 16b (Myo16b), which may have a role in the neuronal development and the regulation of cell cycle, can be found both in the nucleus and in the cytoplasm. Myo16b contains a motor domain, a short neck region, an itrinsically disordered tail (My16Tail) and an ankyrin-repeat containing N-terminal domain, called the ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g. in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c) and My16Ank shows a sequence similarity to MYPT1. We characterized the Myo16b domains: the My16Ank, the motor domain, and the My16Tail in vitro using recombinant protein fragments. We tested the effects of My16Ank on the myosin motor function using skeletal muscle myosin as a model system. The results showed that My16Ank bound to skeletal muscle myosin (KD ∼ 2.4 µM) and the actin-activated ATPase activity of the skeletal myosin was increased in the presence of My16Ank. Besides, we showed, that My16Ank strongly bound to PP1cα (KD ∼ 540 nM) and also to PP1cδ (KD ∼ 600 nM) isoforms. The prolin-rich My16Tail is supposed to bind profilin, but we found no direct interaction between My16Tail and fluorescent profilin-1 using anisotropy measurements. Meanwhile, we also tested the interaction between My16Tail and My16Ank domains, which showed a moderate affinity (KD ∼ 13 µM). Our results suggest that one function My16Ank is probably to regulate the motor function of Myo16. It may influence the motor activity and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, yet unidentified, intracellular proteins. My16Tail can bind My16Ank assuming a regulatory function by backfolding of the tail to the N-terminal of the Myo16.

Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).

Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-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 β-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 β-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 mayfurther increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

Molecular and Functional Effects of a Splice Site Mutation in the MYL2 Gene Associated with Cardioskeletal Myopathy and Early Cardiac Death in Infants.

The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.

Arachidonic Acid Directly Binds and Activates Beta-Cardiac Myosin in the Regulated Cardiac Actomyosin Complex

Mutation-induced dysfunction or misfolding of heart muscle sarcomere components have been linked to dilated and hypertrophic cardiomyopathies. We have previously shown that the small molecule EMD57033 increases β-cardiac myosin activity and acts as a pharmacological chaperone that stabilizes and refolds the motor domain. Following up on this finding, we investigated whether metabolites such as fatty acids can have a similar effect on β-cardiac myosin. Arachidonic acid (AA) was previously reported as an activator of smooth muscle myosin. We found that among all fatty acids tested, AA induced the largest effect on actin-activated ATPase activity of β-cardiac myosin. Closer characterization revealed a 13-fold increase in β-cardiac myosins affinity for actin in the presence of ATP and a 7-fold increase in the rate of phosphate release. We further aimed to elucidate the effect of AA-induced myosin activation in the context of a reconstituted human cardiac actin-troponin-tropomyosin complex. Both, assays monitoring ATPase and in vitro motility showed a significant increase in activity over a wide range of Ca2+ concentrations. Similar to the effect of EMD57033, the addition of AA increases the stability of myosin and reverses the loss of myosin activity caused by temperature stress. The refolding reaction can be monitored by the recovery of the intrinsic protein fluorescence signal that is associated with ATP binding and active turnover. Alternatively, we followed the clear reduction in the number of immobile filaments following the addition of AA.

Effects of IVS6-1 Mutation in MYl2 Associated with Cardioskeletal Myopathy and Early Cardiac Death of Infants

The homozygous appearance of intronic mutation (IVS6-1) in MYL2 encoding myosin ventricular/slow skeletal myosin regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy. The affected infants died between 4 and 6 months of age due to dilated or hypertrophic cardiomyopathy. In this report we have investigated, the molecular mechanism and functional consequences associated with IVS6-1 using recombinant human cardiac IVS6-1-RLC reconstituted in RLC-depleted porcine cardiac muscle preparations. The results were compared with those obtained for recombinant human cardiac WT-RLC. Despite the ability of the mutant to become phosphorylated, IVS6-1 displayed a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT (0.18±0.2 s-1 vs. 0.25±0.02 s-1; n=3), indicating a slower myosin cross-bridge turnover rate of the mutant. Likewise, stopped flow kinetics of the ATP induced dissociation of the acto-myosin complex showed a significantly reduced slope of the kobs-[MgATP] relationship for IVS6-1 reconstituted myosin (4.3±0.02×105 M-1 s-1, n=5), depicting slower second-order MgATP binding rates compared with WT (5.3±0.02×105 M-1 s-1, n=5). Steady-state fluorescence binding experiments of mutant vs. WT reconstituted myosin to pyrene labeled F-actin under rigor conditions demonstrated significantly reduced binding affinity of IVS6-1 (Kd=16.7±2.8 nM, n=3) compared with WT (Kd=4.6±1.3 nM, n=3). In skinned porcine cardiac muscles, a significant decrease in maximal force was observed for IVS6-1 reconstituted fibers compared with WT (27.1±1.0 kN/m2 vs. 34.9±2.4 kN/m2, n=6). The Ca2+ sensitivity and cooperativity were significantly different in IVS6-1 (pCa50=5.66±0.01, nH=2.9±0.15) vs. WT (pCa50=5.48±0.01, nH=2.2±0.14). In conclusion, we demonstrate that IVS6-1 may lead to cardiac dysfunction by disrupting the acto-myosin interaction (steady-state and kinetics) and compromising the ability of the mutant myosin to develop contractile force.

Novel familial dilated cardiomyopathy mutation in MYL2 affects the structure and function of myosin regulatory light chain.

Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild-type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC-mutant or WT-reconstituted porcine cardiac preparations. The mutation induced a reduction in the α-helical content of the RLC, and imposed intra-molecular rearrangements. The phosphorylation of RLC by Ca²⁺/calmodulin-activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild-type. No changes in the myofibrillar ATPase-pCa relationship were observed in wild-type- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle, with no change in the calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with wild-type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype.

Disease Causing Troponin C Mutations Have Varied Effects on Actin Regulatory States

Cardiomyopathy associated mutations in troponin often affect the actin-activated ATPase activity of myosin and the response of force and ATPase activity to Ca2+. Three hypertrophic cardiomyopathy TnC mutants A8V, C84Y, and D145E were examined for their effects on the distribution of states of regulated actin in vitro. In the absence of activating Ca2+, regulated actin filaments containing the C84Y mutant produced a 2.5x elevation in ATPase activity of skeletal myosin subfragment 1 compared to wild type; the other mutants had normal activity. We examined acrylodan-tropomyosin fluorescence changes that occur following rapid detachment of S1 from regulated actin in the absence of Ca2+. All troponin mutants gave a fluorescence increase indicative of a normal transition into the inactive state. The A8V mutant differed from wild type and other TnC mutants at saturating Ca2+. Actin filaments containing the A8V TnC mutant produced an ATPase rate in Ca2+ that was 1.3x wild type. For comparison, the Δ14TnT mutant that destabilizes the inactive state was 1.7x wild type. Interestingly, the effects of the A8V-TnC and Δ14-TnT mutants were additive leading to a Ca2+-activated rate 2.3x wild type. The A8V TnC mutant appeared to favor the active state relative to wild type but only in the presence of Ca2+. This behavior differed from the Δ14 mutation of TnT where the distribution was also altered at low Ca2+ conditions.

Actin-Activated Atpase and Actin Sliding Velocities are Similarly Influenced by Actin-Myosin Binding Kinetics

Detachment-limited models of muscle shortening assume that with increasing myosin densities maximal velocities are achieved when at least one myosin head is bound to a given actin filament. However, numerous studies suggest that actin sliding velocities, V, are influenced by actin-myosin attachment kinetics, suggesting that V should saturate - along with actin-activated myosin ATPase activity, v - when myosin heads saturate binding sites on actin. Here we used an in vitro motility assay to measure both V and v in the same flow cell. We observed that both V and v exhibited similar saturation kinetics with increasing myosin densities and that increasing ionic strength similarly decreased the Km for both activation curves. Using the same in vitro assay, we measured the calcium-dependence of V for regulated thin filaments (actin filaments reconstituted with tropomyosin and troponin) and observed that the myosin-dependence of calcium sensitivity (pCa50) exhibited saturation kinetics similar to that of V and v. These results suggest that saturation of V, v, and pCa50 all occur when myosin saturates binding sites on actin. This is consistent with estimates showing that saturating myosin densities in a motility assay correspond to a linear spacing of myosin along an actin filament that is similar to the repeat of myosin binding sites along one side of the filament.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60Å2 to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (Vmax) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller Kapp) than that of the wild-type actin, with the Vmax being almost unchanged. The Kapp and Vmax of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/Kapp reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

Caldesmon Regulates Axon Extension through Interaction with Myosin II

To begin the process of forming neural circuits, new neurons first establish their polarity and extend their axon. Axon extension is guided and regulated by highly coordinated cytoskeletal dynamics. Here we demonstrate that in hippocampal neurons, the actin-binding protein caldesmon accumulates in distal axons, and its N-terminal interaction with myosin II enhances axon extension. In cortical neural progenitor cells, caldesmon knockdown suppresses axon extension and neuronal polarity. These results indicate that caldesmon is an important regulator of axon development.