RESUMO
In skeletal muscle, nebulin stabilizes and regulates the length of thin filaments, but the underlying mechanism remains nebulous. In this work, we used cryo-electron tomography and subtomogram averaging to reveal structures of native nebulin bound to thin filaments within intact sarcomeres. This in situ reconstruction provided high-resolution details of the interaction between nebulin and actin, demonstrating the stabilizing role of nebulin. Myosin bound to the thin filaments exhibited different conformations of the neck domain, highlighting its inherent structural variability in muscle. Unexpectedly, nebulin did not interact with myosin or tropomyosin, but it did interact with a troponin T linker through two potential binding motifs on nebulin, explaining its regulatory role. Our structures support the role of nebulin as a thin filament "molecular ruler" and provide a molecular basis for studying nemaline myopathies.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miofibrilas/ultraestrutura , Actinas/química , Actinas/metabolismo , Animais , Tomografia com Microscopia Eletrônica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Musculares/genética , Mutação , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miofibrilas/química , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Miopatias da Nemalina/metabolismo , Miosinas/química , Miosinas/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Músculos Psoas/química , Músculos Psoas/metabolismo , Músculos Psoas/ultraestrutura , Sarcômeros/química , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
Time-resolved X-ray diffraction of isolated fast-twitch muscles of mice was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by a complete recovery of the folded motor conformation on the filament backbone but by incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscles.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético , Miosinas , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/química , Músculo Esquelético/fisiologia , Miosinas/química , Miosinas/metabolismo , Miosinas/fisiologia , Sarcômeros/química , Sarcômeros/fisiologiaRESUMO
Estrogen deficiency has a significant influence on the excitation-contraction coupling in the ventricular myocardium but its impact on the atrial contractile function has not been studied. We have compared the effects of estrogen deficiency on the contractility and cytosolic Ca2+ transient of single cardiomyocytes isolated from the left atrium (LA) and the left ventricle (LV) of rats subjected to ovariectomy (OVX) or sham surgery (Sham). The characteristics of actin-myosin interaction were studied in an in vitro motility assay. We found that OVX decreased the contractility of LV single cardiomyocytes but increased that of LA myocytes. The disturbance of ventricular mechanical function may be explained by the acceleration of Ca2+ transient and reduced Ca2+ sensitivity of the actin-myosin interaction. The augmentation of LA contractility may be explained by accelerated cross-bridge kinetics and increased end-diastolic sarcomere length, which may lead to elevated tension in atrial cells due to the Frank-Starling mechanism.
Assuntos
Estrogênios/deficiência , Ventrículos do Coração/citologia , Miocárdio/metabolismo , Função Ventricular , Actinas/metabolismo , Animais , Cálcio/metabolismo , Feminino , Insuficiência Cardíaca , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Miosinas/metabolismo , Ovariectomia , Fosforilação , Ratos , Sarcômeros/química , Sarcômeros/metabolismoRESUMO
Vertebrate striated muscle thin filaments are thought to be thermodynamically activated in response to an increase in Ca2+ concentration. We tested this hypothesis by measuring time intervals for gliding runs and pauses of individual skeletal muscle thin filaments in cycling myosin motility assays. A classic thermodynamic mechanism predicts that if chemical potential is constant, transitions between runs and pauses of gliding thin filaments will occur at constant rate as given by a Poisson distribution. In this scenario, rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distribution that slopes negatively for all observable run times. However, we determined that relative density of observed run times fits an exponential only at low Ca2+ levels that activate filament gliding. Further titration with Ca2+, or adding excess regulatory proteins tropomyosin and troponin, shifted the relative density of short run times to fit the positive slope of a gamma distribution, which derives from waiting times between Poisson events. Events that arise during a run and prevent the chance of ending a run for a random interval of time account for the observed run time distributions, suggesting that the events originate with cycling myosin. We propose that regulatory proteins of the thin filament require the mechanical force of cycling myosin to achieve the transition state for activation. During activation, combinations of cycling myosin that contribute insufficient activation energy delay deactivation.
Assuntos
Citoesqueleto de Actina/química , Cálcio/química , Contração Muscular , Miosinas/química , Sarcômeros/química , Citoesqueleto de Actina/metabolismo , Animais , Cálcio/metabolismo , Miosinas/metabolismo , Coelhos , Sarcômeros/metabolismoRESUMO
High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Subfragmentos de Miosina/ultraestrutura , Tropomiosina/ultraestrutura , Troponina/ultraestrutura , Citoesqueleto de Actina/química , Actinas/química , Animais , Cálcio/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Imagem Molecular , Contração Muscular/genética , Músculo Esquelético/química , Músculo Esquelético/ultraestrutura , Miocárdio/química , Miocárdio/ultraestrutura , Subfragmentos de Miosina/química , Miosinas/química , Ligação Proteica , Coelhos , Sarcômeros/química , Sarcômeros/ultraestrutura , Tropomiosina/química , Troponina/químicaRESUMO
Sarcomeric signaling complexes are important to sustain proper sarcomere structure and function, however, the mechanisms underlying these processes are not fully elucidated. In a gene trap experiment, we found that vascular cell adhesion protein 1 isoform X2 (VCAP1X2) mutant embryos displayed a dilated cardiomyopathy phenotype, including reduced cardiac contractility, enlarged ventricular chamber and thinned ventricular compact layer. Cardiomyocyte and epicardial cell proliferation was decreased in the mutant heart ventricle, as was the expression of pAKT and pERK. Contractile dysfunction in the mutant was caused by sarcomeric disorganization, including sparse myofilament, blurred Z-disc, and decreased gene expression for sarcomere modulators (smyd1b, mypn and fhl2a), sarcomeric proteins (myh6, myh7, vmhcl and tnnt2a) and calcium regulators (ryr2b and slc8a1a). Treatment of PI3K activator restored Z-disc alignment while injection of smyd1b mRNA restored Z-disc alignment, contractile function and cardiomyocyte proliferation in ventricles of VCAP1X2 mutant embryos. Furthermore, injection of VCAP1X2 variant mRNA rescued all phenotypes, so long as two cytosolic tyrosines were left intact. Our results reveal two tyrosine residues located in the VCAP1X2 cytoplasmic domain are essential to regulate cardiac contractility and the proliferation of ventricular cardiomyocytes and epicardial cells through modulating pAKT and pERK expression levels.
Assuntos
Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ventrículos do Coração/metabolismo , Mutagênese , Contração Miocárdica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sarcômeros/química , Sarcômeros/fisiologia , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Molécula 1 de Adesão de Célula Vascular/química , Molécula 1 de Adesão de Célula Vascular/genética , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
Over 20 mutations in ß-cardiac myosin heavy chain (ß-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase-light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged ß-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged ß-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.
Assuntos
Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Miopatias Distais/genética , Fibras Musculares Esqueléticas/citologia , Mutação , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Animais , Miosinas Cardíacas/metabolismo , Linhagem Celular , Técnicas In Vitro , Camundongos , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Estrutura Secundária de Proteína , Ratos , Sarcômeros/química , Sarcômeros/metabolismoRESUMO
The response of titin to mechanical forces is a major determinant of the function of the heart. When placed under a pulling force, the unstructured regions of titin uncoil while its immunoglobulin (Ig) domains unfold and extend. Using single-molecule atomic force microscopy, we show that disulfide isomerization reactions within Ig domains enable a third mechanism of titin elasticity. Oxidation of Ig domains leads to non-canonical disulfide bonds that stiffen titin while enabling force-triggered isomerization reactions to more extended states of the domains. Using sequence and structural analyses, we show that 21% of titin's I-band Ig domains contain a conserved cysteine triad that can engage in disulfide isomerization reactions. We propose that imbalance of the redox status of myocytes can have immediate consequences for the mechanical properties of the sarcomere via alterations of the oxidation state of titin domains.
Assuntos
Conectina/química , Dissulfetos/química , Elasticidade , Domínios de Imunoglobulina , Animais , Conectina/metabolismo , Cisteína/química , Cisteína/metabolismo , Isomerismo , Microscopia de Força Atômica , Modelos Moleculares , Oxirredução , Dobramento de Proteína , Desdobramento de Proteína , Coelhos , Sarcômeros/química , Sarcômeros/metabolismoRESUMO
Telethonin anchors the N-terminal region of titin in the Z-disk of the sarcomere by binding to two immunoglobulin-like (Ig) domains (Z1 and Z2) of titin (Z1Z2). Thereby telethonin plays an important role in myofibril assembly and in muscle development and functional regulation. The expression and purification of recombinant telethonin is very challenging. In previous studies, recombinant telethonin expressed from E. coli was refolded in the presence of Z1Z2. Here, we report various strategies to establish a reliable and efficient protocol for the preparation of telethonin and titin Z1Z2 protein. First, a co-expression strategy was designed to obtain soluble Z1Z2/telethonin complexes. The concentration of antibiotics and the type of expression vector were found to be important for achieving high yields of purified complex. Second, the five cysteine residues of telethonin were mutated to serine to avoid severe problems with cysteine oxidation. Third, a short version of telethonin (telethonin1-90) was designed to avoid the proteolytic degradation observed for longer constructs of the protein. The short telethonin formed a highly stable complex with Z1Z2 with no degradation being observed for 30 days at 4 °C. Fourth, an improved refolding protocol was developed to achieve high yields of Z1Z2/telethonin complex. Finally, based on the crystal structure in which Z1Z2 and telethonin1-90 assemble into a 2:1 complex, a single chain fusion protein was designed, comprising two Z1Z2 modules that are connected by flexible linkers N- and C-terminally of the telethonin1-90. Expression of this fusion protein, named ZTZ, affords high yields of soluble expressed and purified protein.
Assuntos
Conectina/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Conectina/biossíntese , Conectina/química , Conectina/genética , Escherichia coli/genética , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Musculares/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sarcômeros/química , Sarcômeros/genéticaRESUMO
Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of ß-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from ß-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Benzilaminas/química , Miosinas Cardíacas/química , Subfragmentos de Miosina/química , Sarcômeros/química , Uracila/análogos & derivados , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Miosinas Cardíacas/metabolismo , Cardiomegalia/metabolismo , Bovinos , Subfragmentos de Miosina/metabolismo , Sarcômeros/metabolismo , Uracila/químicaRESUMO
The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Hipertrófica Familiar/genética , Modelos Moleculares , Mutação Puntual , Sarcômeros/metabolismo , Tropomiosina/metabolismo , Troponina T/metabolismo , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Hipertrófica Familiar/metabolismo , Biologia Computacional , Humanos , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sarcômeros/química , Tropomiosina/química , Tropomiosina/genética , Troponina/química , Troponina/genética , Troponina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Troponina I/química , Troponina I/genética , Troponina I/metabolismo , Troponina T/química , Troponina T/genéticaRESUMO
Microscopy in combination with contrast-increasing dyes allows the visualization and analysis of organs, tissues, and various cells. Because of their better resolution, the development of confocal and laser microscopes enables the investigations of cell components, which are labeled with fluorescent dyes. The imaging of living cells on subcellular level (also in vivo) needs a labeling by gene transfection of GFP or similar labeled proteins. We present a method for visualization of cell structure in skeletal and heart muscle by label-free Second Harmonic Generation (SHG) microscopy and describe analytic methods for quantitative measurements of morphology and dynamics in skeletal muscle fibers.
Assuntos
Fibras Musculares Esqueléticas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Microscopia de Geração do Segundo Harmônico/métodos , Animais , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Imageamento Tridimensional , Microscopia Confocal , Fibras Musculares Esqueléticas/química , Miócitos Cardíacos/química , Miosinas/química , Fótons , Sarcômeros/químicaRESUMO
In the last decade, improvements in electron microscopy and image processing have permitted significantly higher resolutions to be achieved (sometimes <1 nm) when studying isolated actin and myosin filaments. In the case of actin filaments the changing structure when troponin binds calcium ions can be followed using electron microscopy and single particle analysis to reveal what happens on each of the seven non-equivalent pseudo-repeats of the tropomyosin α-helical coiled-coil. In the case of the known family of myosin filaments not only are the myosin head arrangements under relaxing conditions being defined, but the latest analysis, also using single particle methods, is starting to reveal the way that the α-helical coiled-coil myosin rods are packed to give the filament backbones.
Assuntos
Citoesqueleto de Actina/química , Miosinas/química , Citoesqueleto de Actina/ultraestrutura , Animais , Humanos , Microscopia Eletrônica , Miosinas/ultraestrutura , Sarcômeros/química , Sarcômeros/ultraestrutura , Difração de Raios XRESUMO
Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.
Assuntos
Actinina/metabolismo , Conectina/metabolismo , Actinina/química , Sequência de Aminoácidos , Animais , Conectina/química , Cisteína/química , Cistina/química , Humanos , Mutagênese Sítio-Dirigida , Pinças Ópticas , Domínios Proteicos , Mapeamento de Interação de Proteínas , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Sarcômeros/química , Sarcômeros/ultraestrutura , Estresse MecânicoRESUMO
The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.
Assuntos
Cardiomiopatia Restritiva , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Sarcômeros , Troponina I , Substituição de Aminoácidos , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Proteína Quinase C/química , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Sarcômeros/química , Sarcômeros/genética , Sarcômeros/metabolismo , Relação Estrutura-Atividade , Troponina I/química , Troponina I/genética , Troponina I/metabolismoRESUMO
The aim of this experiment was to use metabolomic techniques to investigate the energy metabolism in lamb M. longissimus thoracis et lumborum subjected to very fast chilling (VFC) post-mortem. The tissue was prepared by 2 different operators and subjected to very fast chilling (less than 0°C within 1.5h of slaughter) or typical chilling regimes (Control; 0°C within 22h of slaughter). Non-targeted metabolomic analysis ((1)H NMR) and targeted analysis ((31)P NMR, HPLC-PDA and HPLC-MS/MS) were used to examine the change in muscle metabolites post-mortem. One VFC treatment, which resulted in a colder core temperature and more tender meat, had higher levels of glycolytic intermediate metabolites pre-rigor as well as more of the end-products of adenosine and nicotine nucleotide metabolism pre-rigor, relative to conventionally chilled treatments. In conclusion, VFC to less than 0°C within 1.5h of slaughter causes considerable changes in metabolism and rigor onset, which are associated with tender meat.
Assuntos
Temperatura Baixa , Carne/análise , Metabolômica , Músculo Esquelético/química , Mudanças Depois da Morte , Adenosina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Manipulação de Alimentos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Fosfocreatina/metabolismo , Sarcômeros/química , Carneiro Doméstico , Espectrometria de Massas em TandemRESUMO
Molecular chaperones are commonly identified by their ability to suppress heat-induced protein aggregation. The muscle-specific molecular chaperone UNC-45B is known to be involved in myosin folding and is trafficked to the sarcomeres A-band during thermal stress. Here, we identify temperature-dependent structural changes in the UCS chaperone domain of UNC-45B that occur within a physiologically relevant heat-shock range. We show that distinct changes to the armadillo repeat protein topology result in exposure of hydrophobic patches, and increased flexibility of the molecule. These rearrangements suggest the existence of a novel thermosensor within the chaperone domain of UNC-45B. We propose that these changes may function to suppress aggregation under stress by allowing binding to a wide variety of aggregation prone loops on its client.
Assuntos
Proteínas do Domínio Armadillo/química , Resposta ao Choque Térmico , Chaperonas Moleculares/química , Dobramento de Proteína , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Temperatura Alta , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/genética , Sarcômeros/química , Sarcômeros/genética , Sarcômeros/metabolismoRESUMO
During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca(2+) and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca(2+)-induced opening of N-cTnC and is presumed to also destabilize cTnI-actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca(2+)-sensitizing N-cTnC-cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca(2+)-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca(2+) and 1.80 and 2.2-µm sarcomere length. Mg(2+)-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca(2+)] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca(2+) sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.