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1.
Nat Commun ; 15(1): 4885, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849353

RESUMO

Inherited cardiomyopathies are common cardiac diseases worldwide, leading in the late stage to heart failure and death. The most promising treatments against these diseases are small molecules directly modulating the force produced by ß-cardiac myosin, the molecular motor driving heart contraction. Omecamtiv mecarbil and Mavacamten are two such molecules that completed phase 3 clinical trials, and the inhibitor Mavacamten is now approved by the FDA. In contrast to Mavacamten, Omecamtiv mecarbil acts as an activator of cardiac contractility. Here, we reveal by X-ray crystallography that both drugs target the same pocket and stabilize a pre-stroke structural state, with only few local differences. All-atom molecular dynamics simulations reveal how these molecules produce distinct effects in motor allostery thus impacting force production in opposite way. Altogether, our results provide the framework for rational drug development for the purpose of personalized medicine.


Assuntos
Simulação de Dinâmica Molecular , Contração Miocárdica , Ureia , Contração Miocárdica/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/química , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Miosinas Ventriculares/metabolismo , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Animais , Benzilaminas , Uracila/análogos & derivados
2.
J Gen Physiol ; 156(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38709176

RESUMO

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in ß-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human ß-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.


Assuntos
Miosinas Cardíacas , Cardiomiopatia Dilatada , Miosinas Ventriculares , Animais , Humanos , Actinas/metabolismo , Actinas/genética , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Mutação , Contração Miocárdica/fisiologia , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo
3.
Arch Biochem Biophys ; 754: 109961, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38492659

RESUMO

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.


Assuntos
Atrofia Muscular , Miosinas Ventriculares , Ratos , Animais , Miosinas Ventriculares/metabolismo , Atrofia Muscular/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo
4.
J Phys Chem B ; 128(13): 3113-3120, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38516963

RESUMO

Human ß-cardiac myosin plays a critical role in generating the mechanical forces necessary for cardiac muscle contraction. This process relies on a delicate dynamic equilibrium between the disordered relaxed state (DRX) and the super-relaxed state (SRX) of myosin. Disruptions in this equilibrium due to mutations can lead to heart diseases. However, the structural characteristics of SRX and the molecular mechanisms underlying pathogenic mutations have remained elusive. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the conformational changes in myosin. Our findings indicate that the size of the phosphate-binding pocket can serve as a valuable metric for characterizing the transition from the DRX to SRX state. Importantly, we established a global dynamic coupling network within the myosin motor head at the residue level, elucidating how the pathogenic mutation E483K impacts the equilibrium between SRX and DRX through allosteric effects. Our work illuminates molecular details of SRX and offers valuable insights into disease treatment through the regulation of SRX.


Assuntos
Simulação de Dinâmica Molecular , Miosinas Ventriculares , Humanos , Miosinas , Coração , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo
5.
Biochemistry (Mosc) ; 89(1): 116-129, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38467549

RESUMO

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.


Assuntos
Actinas , Proteína C , Actinas/metabolismo , Proteína C/metabolismo , Proteínas de Transporte/metabolismo , Cálcio/metabolismo , Miosinas Atriais , Miosinas Ventriculares/metabolismo , Miosinas/metabolismo , Miocárdio/metabolismo , Trifosfato de Adenosina/metabolismo , Isoformas de Proteínas/metabolismo , Ligação Proteica
6.
Sci Rep ; 14(1): 3915, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365813

RESUMO

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo , Miosinas Ventriculares/farmacologia , Edição de Genes , Miocárdio , Miócitos Cardíacos/metabolismo , Diferenciação Celular , Miosinas/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo
7.
Proc Natl Acad Sci U S A ; 121(9): e2315472121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38377203

RESUMO

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in ß-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human ß, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in ß myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in ß myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not ß, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.


Assuntos
Miosinas , Miosinas Ventriculares , Humanos , Miosinas Ventriculares/genética , Miosinas/metabolismo , Adenosina Trifosfatases/metabolismo , Mutação , Actinas/metabolismo , Músculo Esquelético/metabolismo
8.
PLoS Comput Biol ; 19(5): e1011099, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37200380

RESUMO

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.


Assuntos
Miosinas Cardíacas , Miosinas Ventriculares , Humanos , Miosinas Cardíacas/química , Miosinas Cardíacas/metabolismo , Miosinas Ventriculares/química , Miosinas Ventriculares/metabolismo , Coração , Miocárdio/metabolismo , Miosinas/química , Ureia/metabolismo
9.
Nat Commun ; 14(1): 3166, 2023 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-37258552

RESUMO

To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human ß-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the 'off' state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the 'off' state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability.


Assuntos
Miosinas Cardíacas , Cardiomiopatia Hipertrófica , Humanos , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Microscopia Crioeletrônica , Coração , Cardiomiopatia Hipertrófica/genética
11.
Nature ; 611(7937): 818-826, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36385524

RESUMO

Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.


Assuntos
Linfócitos T CD8-Positivos , Imunoterapia , Miocardite , Miosinas Ventriculares , Animais , Camundongos , Autoantígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/deficiência , Antígeno CTLA-4/genética , Imunoterapia/efeitos adversos , Miocardite/induzido quimicamente , Miocardite/etiologia , Miocardite/mortalidade , Miocardite/patologia , Miosinas Ventriculares/imunologia
12.
Elife ; 112022 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-36422472

RESUMO

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.


Assuntos
Cardiomiopatia Dilatada , Miosinas Ventriculares , Humanos , Miosinas Ventriculares/genética , Miosinas Cardíacas , Cardiomiopatia Dilatada/genética , Miosinas/genética , Mutação , Miocárdio , Trifosfato de Adenosina
13.
J Gen Physiol ; 154(10)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36053243

RESUMO

The ß-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal Musculus soleus (M. soleus) type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between M. soleus and ventricular myosin, which could influence functional parameters, such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules, we made use of in vitro motility assays. We found that ventricular myosin moved actin filaments with ∼0.9 µm/s significantly faster than M. soleus myosin (0.3 µm/s). Filaments prepared from isolated actin are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from M. soleus or ventricular tissue, the gliding velocity of M. soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and M. soleus ß-myosin, a difference became evident. M. soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding. We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between M. soleus and ventricular myosin persisted when native thin filaments were used.


Assuntos
Actinas , Cadeias Leves de Miosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosinas Ventriculares
14.
Genes (Basel) ; 13(9)2022 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-36140722

RESUMO

Cardiac dysfunction accelerates the risk of heart failure, and its pathogenesis involves a complex interaction between genetic and environmental factors. Variations in myosin affect contractile abilities of cardiomyocytes and cause structural and functional abnormalities in myocardium. The study aims to find the association of MYH7 rs121913642 (c.1594 T>C) and rs121913645 (c.667G>A) variants with cardiac dysfunction in the Punjabi Pakistani population. Patients with heart failure (n = 232) and healthy controls (n = 205) were enrolled in this study. MYH7 variant genotyping was performed using tetra ARMS-PCR. MYH7 rs121913642 TC genotype was significantly more prevalent in the patient group (p < 0.001). However, MYH7 rs121913645 genotype frequencies were not significantly different between the patient and control groups (p < 0.666). Regression analysis also revealed that the rs121913642 C allele increases the risk of cardiac failure by ~2 [OR:1.98, CI: 1.31−2.98, p < 0.001] in comparison to the T allele. High levels of the cardiac enzymes cardiac troponin I (cTnI) and CK-MB were observed in patients. There was also an increase in total cholesterol, LDL cholesterol, and uric acid in patients compared to the healthy control group (p < 0.001). In conclusion, the MYH7 gene variant rs121913642 is genetically associated with cardiac dysfunction and involved in the pathogenesis of HF.


Assuntos
Cardiopatias , Insuficiência Cardíaca , Cadeias Pesadas de Miosina/genética , Miosinas Cardíacas/genética , LDL-Colesterol/genética , Insuficiência Cardíaca/genética , Humanos , Mutação , Fenótipo , Troponina I/genética , Ácido Úrico , Miosinas Ventriculares/genética
15.
Elife ; 112022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35767336

RESUMO

Mutations in the lever arm of ß-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of ß-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.


Assuntos
Cardiomiopatia Hipertrófica , Miosinas Ventriculares , Actinas/genética , Actinas/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Mutação , Relação Estrutura-Atividade , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
16.
J Biol Chem ; 298(7): 102070, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35623390

RESUMO

The myosin II motors are ATP-powered force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta (ß-MyHC) is primarily expressed in the ventricular myocardium and in slow-twitch muscle fibers, such as M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular ß-cardiac myosin (ßM-II); however, the direct assessment of biochemical and mechanical features of the native myosins is limited. By employing optical trapping, we examined the mechanochemical properties of native myosins isolated from the rabbit heart ventricle and soleus muscles at the single-molecule level. We found purified motors from the two tissue sources, despite expressing the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. We demonstrate ßM-II was approximately threefold faster in the actin filament-gliding assay than SolM-II. The maximum actomyosin (AM) detachment rate derived in single-molecule assays was also approximately threefold higher in ßM-II, while the power stroke size and stiffness of the "AM rigor" crossbridge for both myosins were comparable. Our analysis revealed a higher AM detachment rate for ßM-II, corresponding to the enhanced ADP release rates from the crossbridge, likely responsible for the observed differences in the motility driven by these myosins. Finally, we observed a distinct myosin light chain 1 isoform (MLC1sa) that associates with SolM-II, which might contribute to the observed kinetics differences between ßM-II and SolM-II. These results have important implications for the choice of tissue sources and justify prerequisites for the correct myosin heavy and light chains to study cardiomyopathies.


Assuntos
Miosinas Cardíacas , Cadeias Pesadas de Miosina , Animais , Ventrículos do Coração , Miosina Tipo II , Miosinas , Isoformas de Proteínas , Coelhos , Miosinas Ventriculares
17.
Biophys J ; 121(12): 2449-2460, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35591788

RESUMO

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a "nanosurfer" assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human ß-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0-C2 or C1-C2) on nanotubes every 14 nm. Both C0-C2 and C1-C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0-C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0-C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of ß-cardiac myosin contractility.


Assuntos
Miosinas Cardíacas , Miosinas Ventriculares , Miosinas Cardíacas/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Miocárdio/metabolismo , Miosinas/metabolismo , Fosforilação , Miosinas Ventriculares/análise , Miosinas Ventriculares/metabolismo
18.
Cell Mol Life Sci ; 78(23): 7309-7337, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34704115

RESUMO

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.


Assuntos
Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Miosinas Ventriculares/metabolismo , Sequência de Aminoácidos , Função Atrial/fisiologia , Pressão Sanguínea/fisiologia , Humanos , Miócitos Cardíacos/metabolismo , Miofibrilas/fisiologia , Domínios Proteicos , Isoformas de Proteínas , Função Ventricular/fisiologia
19.
J Mol Biol ; 433(23): 167295, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34627791

RESUMO

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.


Assuntos
Benzilaminas/química , Modelos Moleculares , Conformação Proteica , Uracila/análogos & derivados , Miosinas Ventriculares/química , Benzilaminas/farmacologia , Miosinas/antagonistas & inibidores , Miosinas/química , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Análise Espectral , Relação Estrutura-Atividade , Uracila/química , Uracila/farmacologia , Miosinas Ventriculares/antagonistas & inibidores
20.
Cardiovasc Ther ; 2021: 5554569, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34257705

RESUMO

Ginkgolide B (GB) is an active ingredient extracted from Ginkgo biloba leaves. However, the effects of GB on cardiac hypertrophy remain unclear. The study is aimed at determining whether GB could alleviate cardiac hypertrophy and exploring its underlying molecular mechanism. Rat cardiomyocyte cell line H9c2 cells were pretreated with GB and incubated with angiotensin II (Ang II) to simulate an in vitro cardiac hypertrophy model. Cell viability, cell size, hypertrophy markers, and autophagy were determined in H9c2 cells after Ang II treatment. Proteins involved in autophagy and the SIRT1 pathway were determined by western blot. Our data demonstrated that GB attenuated Ang II-induced cardiac hypertrophy and reduced the mRNA expressions of hypertrophy marker, atrial natriuretic peptide (ANP), and ß-myosin heavy chain (ß-MHC). GB further increased Ang II-induced autophagy in H9c2 cells and modulated expressions of autophagy-related proteins Beclin1 and P62. Modulation of autophagy using autophagy inhibitor 3-methyladenine (3-MA) could abrogate GB-downregulated transcription of NPPA. We then showed that GB attenuated Ang II-induced oxidative stress and reduction in SIRT1 and FoxO1 protein expression. Finally, the effect of GB on autophagy and cardiac hypertrophy could be reversed by SIRT1 inhibitor EX-527. GB inhibits Ang II-induced cardiac hypertrophy by enhancing autophagy via the SIRT1-FoxO1 signaling pathway and might be a potential agent in treating pathological cardiac hypertrophy.


Assuntos
Angiotensina II/toxicidade , Autofagia/efeitos dos fármacos , Ginkgolídeos/farmacologia , Lactonas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Sirtuína 1/metabolismo , Animais , Fator Natriurético Atrial/genética , Cardiomegalia/tratamento farmacológico , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Miócitos Cardíacos/patologia , Substâncias Protetoras/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Miosinas Ventriculares/genética
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