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.

Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo.

OBJECTIVE: Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). CONCLUSIONS: CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Common pathogenic mechanism in patients with dropped head syndrome caused by different mutations in the MYH7 gene.

Mutations in the MYH7 gene are the source of an allelic series of diseases, including various cardiomyopathies and skeletal myopathies that usually manifest in adulthood. We observed a 1.5 y.o. male patient with congenital weaknesses of the axial muscles, "dropped head" syndrome, and dilated cardiomyopathy. The clinical evaluation included medical history, an echocardiogram, electromyography, and a histopathological study. The genetic evaluation included whole exome sequencing. Muscle biopsy samples from the proband were used for mRNA extraction. We revealed a novel genetic variant c.5655 + 5G > C in the MYH7 gene. The analysis of the cDNA showed an in-frame skipping of exon 38 (p.1854_1885del). This variant and two previously published mutations (c.5655G > A and c.5655 + 1G > A), also presumably leading to exon 38 skipping, were studied by expression analysis in the HEK293T cell line transfected with 4 plasmids containing the MYH7 minigene (wt, c.5655G > C, c.5655 + 1G > A and c.5655 + 5G > A). A quantitative difference in expression was shown for cell lines with each of the three mutant plasmids. All mutation carriers had a similar phenotype and included congenital axial myopathy and variable cardiac involvement. Prominent dropped head syndrome was mentioned in all patients. Early-onset axial myopathy with a dropped head syndrome is a distinct clinical entity within MYH7-related disorders. We suggest that mutations in the MYH7 gene affecting the C-terminal domain of beta-myosin heavy chain should also be considered as a possible cause in cases of early-onset myopathy with "dropped head" syndrome.

Electrostatic interactions in the force-generating region of the human cardiac myosin modulate ADP dissociation from actomyosin.

Human cardiac myosin has two isoforms, alpha and beta, sharing significant sequence similarity, but different in kinetics: ADP release from actomyosin is an order of magnitude faster in the alpha myosin isoform. Apparently, small differences in the sequence are responsible for distinct local inter-residue interactions within alpha and beta isoforms, leading to such a dramatic difference in the rate of ADP release. Our analysis of structural kinetics of alpha and beta isoforms using molecular dynamics simulations revealed distinct dynamics of SH1:SH2 helix within the force-generation region of myosin head. The simulations showed that the residue R694 of the helix forms two permanent salt bridges in the beta isoform, which are not present in the alpha isoform. We hypothesized that the isoform-specific electrostatic interactions play a role in the difference of kinetic properties of myosin isoforms. We prepared R694N mutant in the beta isoform background to destabilize electrostatic interactions in the force-generating region of the myosin head. Our experimental data confirm faster ADP release from R694N actomyosin mutant, but is not as dramatic as the difference of kinetics of ADP release in the alpha and beta isoforms.

Recovery of left ventricular function following in vivo reexpression of cardiac myosin binding protein C.

The loss of cardiac myosin binding protein C (cMyBP-C) results in left ventricular dilation, cardiac hypertrophy, and impaired ventricular function in both constitutive and conditional cMyBP-C knockout (MYBPC3 null) mice. It remains unclear whether the structural and functional phenotypes expressed in the MYBPC3 null mouse are reversible, which is an important question, since reduced expression of cMyBP-C is an important cause of hypertrophic cardiomyopathy in humans. To investigate this question, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of WT cMyBP-C on the MYBPC3 null background. Functional Tet-Off mice expressing WT cMyBP-C (FT-WT) were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary doxycycline administration, cMyBP-C was expressed at normal levels in FT-WT myocardium, which exhibited similar levels of steady-state force and in vivo left ventricular function as WT mice. Introduction of dietary doxycycline for four weeks resulted in a partial knockdown of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching those observed in MYBPC 3 null mice. Subsequent withdrawal of doxycycline from the diet resulted in the reexpression of cMyBP-C to levels comparable to those observed in WT mice, along with near-complete recovery of in vivo ventricular function. These results show that the cardiac phenotypes associated with MYBPC3 null mice are reversible. Our work also validates the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain.

Myosin light chain 2 ( MYL2) gene encodes the myosin regulatory light chain (RLC) simultaneously in heart ventricles and in slow-twitch skeletal muscle. Using transgenic mice with cardiac-specific expression of the human R58Q-RLC mutant, we sought to determine whether the hypertrophic cardiomyopathy phenotype observed in papillary muscles (PMs) of R58Q mice is also manifested in slow-twitch soleus (SOL) muscles. Skinned SOL muscles and ventricular PMs of R58Q animals exhibited lower contractile force that was not observed in the fast-twitch extensor digitorum longus muscles of R58Q vs. wild-type-RLC mice, but mutant animals did not display gross muscle weakness in vivo. Consistent with SOL muscle abnormalities in R58Q vs. wild-type mice, myosin ATPase staining revealed a decreased proportion of fiber type I/type II only in SOL muscles but not in the extensor digitorum longus muscles. The similarities between SOL muscles and PMs of R58Q mice were further supported by quantitative proteomics. Differential regulation of proteins involved in energy metabolism, cell-cell interactions, and protein-protein signaling was concurrently observed in the hearts and SOL muscles of R58Q mice. In summary, even though R58Q expression was restricted to the heart of mice, functional similarities were clearly observed between the hearts and slow-twitch skeletal muscle, suggesting that MYL2 mutated models of hypertrophic cardiomyopathy may be useful research tools to study the molecular, structural, and energetic mechanisms of cardioskeletal myopathy associated with myosin RLC.-Kazmierczak, K., Liang, J., Yuan, C.-C., Yadav, S., Sitbon, Y. H., Walz, K., Ma, W., Irving, T. C., Cheah, J. X., Gomes, A. V., Szczesna-Cordary, D. Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain.

Single cardiac ventricular myosins are autonomous motors.

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

KEY POINTS: Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle. Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament. Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto-myosin ATPase cycle. Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto-myosin ATPase. Thick filament regulation is a promising target for novel therapeutics in heart disease. ABSTRACT: Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin-containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules - omecamtiv mecarbil (OM) and blebbistatin (BS) - that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small-molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin-myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin-myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co-operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Auxotonic to isometric contraction transitioning in a beating heart causes myosin step-size to down shift.

Myosin motors in cardiac ventriculum convert ATP free energy to the work of moving blood volume under pressure. The actin bound motor cyclically rotates its lever-arm/light-chain complex linking motor generated torque to the myosin filament backbone and translating actin against resisting force. Previous research showed that the unloaded in vitro motor is described with high precision by single molecule mechanical characteristics including unitary step-sizes of approximately 3, 5, and 8 nm and their relative step-frequencies of approximately 13, 50, and 37%. The 3 and 8 nm unitary step-sizes are dependent on myosin essential light chain (ELC) N-terminus actin binding. Step-size and step-frequency quantitation specifies in vitro motor function including duty-ratio, power, and strain sensitivity metrics. In vivo, motors integrated into the muscle sarcomere form the more complex and hierarchically functioning muscle machine. The goal of the research reported here is to measure single myosin step-size and step-frequency in vivo to assess how tissue integration impacts motor function. A photoactivatable GFP tags the ventriculum myosin lever-arm/light-chain complex in the beating heart of a live zebrafish embryo. Detected single GFP emission reports time-resolved myosin lever-arm orientation interpreted as step-size and step-frequency providing single myosin mechanical characteristics over the active cycle. Following step-frequency of cardiac ventriculum myosin transitioning from low to high force in relaxed to auxotonic to isometric contraction phases indicates that the imposition of resisting force during contraction causes the motor to down-shift to the 3 nm step-size accounting for >80% of all the steps in the near-isometric phase. At peak force, the ATP initiated actomyosin dissociation is the predominant strain inhibited transition in the native myosin contraction cycle. The proposed model for motor down-shifting and strain sensing involves ELC N-terminus actin binding. Overall, the approach is a unique bottom-up single molecule mechanical characterization of a hierarchically functional native muscle myosin.

Size and speed of the working stroke of cardiac myosin in situ.

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 µm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.

Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction.

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

Mutations in MYH7 reduce the force generating capacity of sarcomeres in human familial hypertrophic cardiomyopathy.

AIMS: Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling. METHODS AND RESULTS: Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick (MYBPC3, MYH7) and thin (TPM1, TNNI3, TNNT2) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m²) and HCMsmn (26 ± 3 kN/m²) compared with donor (36 ± 2 kN/m²). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 µm²). Low Fmax in MYBPC3mut, TNNI3mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7mut, TPM1mut, and TNNT2mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7mut, TPM1mut, and TNNT2mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m²). In addition, force was lower in MYH7mut cardiomyocytes compared with MYBPC3mut, HCMsmn, and donor at submaximal [Ca²âº]. CONCLUSION: Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca²âº]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

Prostanoid-mediated inotropic responses are attenuated in failing human and rat ventricular myocardium.

Prostanoid-modulatory approaches in heart failure patients have displayed effects which may seem to be mutually incompatible. Both treatment with prostanoids and inhibition of prostanoid synthesis have resulted in increased mortality in heart failure patients. Currently, it is unknown if prostanoids mediate contractile effects in failing human heart and if this can explain some of the clinical effects seen after prostanoid modulatory treatments. Therefore, the objectives of this study were to determine if prostanoids could elicit direct inotropic responses in human ventricle, and if so to determine if they are modified in failing ventricle. Contractile force was measured in left ventricular strips from non-failing or failing human and rat hearts. The ratio of phosphorylated to non-phosphorylated myosin light chain 2 (MLC-2) was measured by Western blotting in myocardial strips, and the levels of prostanoid FP receptor mRNA and protein were measured in rat by real-time RT-PCR and receptor binding assays. In non-failing human hearts, prostanoids evoked a positive inotropic effect and an increase of MLC-2 phosphorylation which was absent in failing human hearts. In failing rat heart, the prostanoid FP receptor-mediated inotropic response and prostanoid FP receptor-density was reduced by ~40-50% compared to non-failing rat heart. Prostanoids mediate a sustained positive inotropic response in non-failing heart, which appears to be down regulated in failing heart. The pathophysiological significance of changes in prostanoid-mediated inotropic support in the failing heart remains to be determined.

Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease.

Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure.

[Disease progression and systolic dysfunction in patients with hypertrophic cardiomyopathy: genetic basis, pathophysiology and clinical presentation]. / Progressione di malattia e disfunzione sistolica nei pazienti con cardiomiopatia ipertrofica: basi genetiche, fisiopatologia e presentazione clinica.

Progressive heart failure associated with left ventricular remodeling and systo-diastolic dysfunction is one of the most severe complications of hypertrophic cardiomyopathy (HCM). Such condition, for the lack of a better term, is referred to as end-stage (ES) HCM. During the last decade, we have begun to understand the mechanisms underlying progression from a hyperdynamic left ventricle to the striking patterns of ES. To date, different aspects of HCM progression remain obscure, including potential strategies for management and prevention. On the basis of recent evidence, it is appropriate to emphasize these aspects, which may be difficult to identify, particularly in the early stages when systolic function appears relatively preserved. Nevertheless, it is at these early stages that treatment may potentially interfere with the clinical evolution of HCM toward ES and heart failure. The possibility of early identification of patients at risk of ES progression may ultimately impact on the natural history of the disease in this challenging patient subgroup.

Regulation of synapse structure and function by distinct myosin II motors.

Ongoing synaptic function and rapid, bidirectional plasticity are both controlled by regulatory mechanisms within dendritic spines. Spine actin dynamics maintain synapse structure and function, and cytoskeletal rearrangements in these structures trigger structural and functional plasticity. Therefore, proteins that interact with actin filaments are attractive candidates to regulate synaptic actin dynamics and, thus, synapse structure and function. Here, we have cloned the rat isoform of class II myosin heavy chain MyH7B in brain. Unexpectedly, this isoform resembles muscle-type myosin II rather than the ubiquitously expressed nonmuscle myosin II isoforms, suggesting that a rich functional diversity of myosin II motors may exist in neurons. Indeed, reducing the expression of MyH7B in mature neurons caused profound alterations to dendritic spine structure and excitatory synaptic strength. Structurally, dendritic spines had large, irregularly shaped heads that contained many filopodia-like protrusions. Neurons with reduced MyH7B expression also had impaired miniature EPSC amplitudes accompanied by a decrease in synaptic AMPA receptors, which was linked to alterations of the actin cytoskeleton. MyH7B-mediated control over spine morphology and synaptic strength was distinct from that of a nonmuscle myosin, myosin IIb. Interestingly, when myosin IIb expression and MyH7B expression were simultaneously knocked-down in neurons, a third, more pronounced phenotype emerged. Together, our data provide evidence that distinct myosin II isoforms work together to regulate synapse structure and function in cultured hippocampal neurons. Thus, myosin II motor activity is emerging as a broad regulatory mechanism for control over complex actin networks within dendritic spines.

Heart failure in 2010.

The Heart Failure Congress 2010 in Berlin presented the latest trials and trends in the medical and mechanical therapy of heart failure in the presence of impaired or preserved left ventricular ejection fraction. It covered all aspects of heart failure from epidemiology through basic and translational science to prevention. The congress highlighted new drugs, novel biomarkers, updated trials, the role of imaging in risk stratification and the importance of telecare in the reduction of heart failure readmission.

Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.

Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.

Cutting edge: cardiac myosin activates innate immune responses through TLRs.

Autoimmune attack on the heart is linked to host immune responses against cardiac myosin, the most abundant protein in the heart. Although adaptive immunity is required for disease, little is known about innate immune mechanisms. In this study we report that human cardiac myosin (HCM) acted as an endogenous ligand to directly stimulate human TLRs 2 and 8 and to activate human monocytes to release proinflammatory cytokines. In addition, pathogenic epitopes of human cardiac myosin, the S2 fragment peptides S2-16 and S2-28, stimulated TLRs directly and activated human monocytes. Our data suggest that cardiac myosin and its pathogenic T cell epitopes may link innate and adaptive immunity in a novel mechanism that could promote chronic inflammation in the myocardium.

A novel approach to improve cardiac performance: cardiac myosin activators.

Decreased systolic function is a central factor in the pathogenesis of heart failure, yet there are no safe medical therapies to improve cardiac function in patients. Currently available inotropes, such as dobutamine and milrinone, increase cardiac contractility at the expense of increased intracellular concentrations of calcium and cAMP, contributing to increased heart rate, hypotension, arrhythmias, and mortality. These adverse effects are inextricably linked to their inotropic mechanism of action. A new class of pharmacologic agents, cardiac myosin activators, directly targets the kinetics of the myosin head. In vitro studies have demonstrated that these agents increase the rate of effective myosin cross-bridge formation, increasing the duration and amount of myocyte contraction, and inhibit non-productive consumption of ATP, potentially improving myocyte energy utilization, with no effect on intracellular calcium or cAMP. Animal models have shown that this novel mechanism increases the systolic ejection time, resulting in improved stroke volume, fractional shortening, and hemodynamics with no effect on myocardial oxygen demand, culminating in significant increases in cardiac efficiency. A first-in-human study in healthy volunteers with the lead cardiac myosin activator, CK-1827452, as well as preliminary results from a study in patients with stable chronic heart failure, have extended these findings to humans, demonstrating significant increases in systolic ejection time, fractional shortening, stroke volume, and cardiac output. These studies suggest that cardiac myosin activators offer the promise of a safe and effective treatment for heart failure. A program of clinical studies are being planned to test whether CK-1827452 will fulfill that promise.