Cryo-EM structure of the human cardiac myosin filament.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly1. Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years2. Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin's motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state3; how titin and cMyBP-C may contribute to length-dependent activation4; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease5,6. The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

Cryo-EM structure of the human cardiac myosin filament.

Pumping of the heart is powered by filaments of the motor protein myosin, which pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here, we have solved the structure of the main (cMyBP-C-containing) region of the human cardiac filament to 6 Å resolution by cryo-EM. The reconstruction reveals the architecture of titin and cMyBP-C for the first time, and shows how myosin's motor domains (heads) form 3 different types of motif (providing functional flexibility), which interact with each other and with specific domains of titin and cMyBP-C to dictate filament architecture and regulate function. A novel packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps generate the cardiac super-relaxed state 3 , how titin and cMyBP-C may contribute to length-dependent activation 4 , and how mutations in myosin and cMyBP-C might disrupt interactions, causing disease 5, 6 . A similar structure is likely in vertebrate skeletal myosin filaments. The reconstruction resolves past uncertainties, and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs.

Variants of the myosin interacting-heads motif.

Under relaxing conditions, the two heads of myosin II interact with each other and with the proximal part (S2) of the myosin tail, establishing the interacting-heads motif (IHM), found in myosin molecules and thick filaments of muscle and nonmuscle cells. The IHM is normally thought of as a single, unique structure, but there are several variants. In the simplest ("canonical") IHM, occurring in most relaxed thick filaments and in heavy meromyosin, the interacting heads bend back and interact with S2, and the motif lies parallel to the filament surface. In one variant, occurring in insect indirect flight muscle, there is no S2-head interaction and the motif is perpendicular to the filament. In a second variant, found in smooth and nonmuscle single myosin molecules in their inhibited (10S) conformation, S2 is shifted ∼20 Šfrom the canonical form and the tail folds twice and wraps around the interacting heads. These molecule and filament IHM variants have important energetic and pathophysiological consequences. (1) The canonical motif, with S2-head interaction, correlates with the super-relaxed (SRX) state of myosin. The absence of S2-head interaction in insects may account for the lower stability of this IHM and apparent absence of SRX in indirect flight muscle, contributing to the quick initiation of flight in insects. (2) The ∼20 Šshift of S2 in 10S myosin molecules means that S2-head interactions are different from those in the canonical IHM. This variant therefore cannot be used to analyze the impact of myosin mutations on S2-head interactions that occur in filaments, as has been proposed. It can be used, instead, to analyze the structural impact of mutations in smooth and nonmuscle myosin.

Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle.

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This "interacting-heads motif" (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3-4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3-4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.

Structural basis of the super- and hyper-relaxed states of myosin II.

Super-relaxation is a state of muscle thick filaments in which ATP turnover by myosin is much slower than that of myosin II in solution. This inhibited state, in equilibrium with a faster (relaxed) state, is ubiquitous and thought to be fundamental to muscle function, acting as a mechanism for switching off energy-consuming myosin motors when they are not being used. The structural basis of super-relaxation is usually taken to be a motif formed by myosin in which the two heads interact with each other and with the proximal tail forming an interacting-heads motif, which switches the heads off. However, recent studies show that even isolated myosin heads can exhibit this slow rate. Here, we review the role of head interactions in creating the super-relaxed state and show how increased numbers of interactions in thick filaments underlie the high levels of super-relaxation found in intact muscle. We suggest how a third, even more inhibited, state of myosin (a hyper-relaxed state) seen in certain species results from additional interactions involving the heads. We speculate on the relationship between animal lifestyle and level of super-relaxation in different species and on the mechanism of formation of the super-relaxed state. We also review how super-relaxed thick filaments are activated and how the super-relaxed state is modulated in healthy and diseased muscles.

Caracterización de pacientes operados por cáncer pulmonar en el Hospital Universitario "Manuel Ascunce Domenech" / Characterization of Patients Operated on for Pulmonary Cancer at Manuel Ascunce Domenech Pulmonary Cancer

Introducción: La cirugía es el tratamiento de elección que ofrece la curación a un grupo de pacientes afectados de cáncer pulmonar, aun así, la supervivencia global para todos los estadios a 5 años se mantiene baja. Objetivo: Caracterizar los pacientes operados de cáncer pulmonar en el período comprendido desde el 1ro de marzo del 2015 hasta el 1ro de agosto del 2020. Métodos: Se realizó un estudio, observacional, retrospectivo de corte transversal, para describir el comportamiento de la morbimortalidad quirúrgica de los pacientes portadores de cáncer pulmonar en el Hospital Provincial Universitario "Manuel Ascunce Domenech" de la ciudad de Camagüey, desde marzo del 2015 y agosto del 2020. El universo de estudio se conformó por 211 pacientes operados con diagnóstico de cáncer pulmonar. Resultados: La edad que predominó fue entre 61 a 70 años y el sexo masculino. El adenocarcinoma y el carcinoma epidermoide fueron los diagnósticos histológicos más frecuentes. La lobectomía superior derecha fue la resección que con más frecuencia se realizó, así como algún tipo de linfadenectomía mediastinal fue realizada en más de la mitad de los casos. Las complicaciones más frecuentes fueron las relacionadas con la fuga de aire. El índice de fallecidos fue bajo encontrándose las complicaciones cardiovasculares y el tromboembolismo pulmonar, las causas de mayor frecuencia. Conclusiones: La cirugía en el cáncer pulmonar es el tratamiento de elección en etapas tempranas con una baja morbilidad y mortalidad en pacientes que reúnan las condiciones de operabilidad(AU)

Introduction: Surgery is the treatment of choice that offers cure to a group of patients affected by pulmonary cancer; yet five-year overall survival for all stages remains low. Objective: To characterize the patients operated on for pulmonary cancer in the period from March 1, 2015 to August 1, 2020. Methods: An observational, retrospective and cross-sectional study was carried out to describe the behavior of surgical morbidity and mortality since March 2015 and August 2020 in patients with pulmonary cancer at Manuel Ascunce Domenech University Provincial Hospital in Camagüey City. The study universe was made up of 211 patients operated on with a diagnosis of pulmonary cancer. Results: There was a predominance of the age 61-70 years and the male sex. Adenocarcinoma and squamous cell carcinoma were the most frequent histological diagnoses. Right upper lobectomy was the most frequently performed resection, as well as some type of mediastinal lymphadenectomy, performed in more than half of the cases. The most frequent complications were those related to air leakage. The death rate was low, with cardiovascular complications and pulmonary thromboembolism being the most frequent causes. Conclusions: Surgery is the treatment of choice for pulmonary cancer in early stages, with low morbidity and mortality in patients who meet operability conditions(AU)

Evaluación de técnicas mínimamente invasivas en el diagnóstico de enfermedades benignas y malignas del tórax / An Assessment of Minimally Invasive Techniques for the Diagnosis of Benign and Malignant Thoracic Diseases

Introducción: Los cambios en la tecnología médica abarcan todas las especialidades y dentro de ellas, en la cirugía general. Las técnicas mínimamente invasivas han ocupado un papel cimero en el diagnóstico y tratamiento de enfermedades en la cavidad torácica. Objetivo: Describir el uso de las técnicas mínimamente invasivas en el diagnóstico de enfermedades intratorácicas. Métodos: Se realizó un estudio observacional, retrospectivo de corte transversal para evaluar el uso de técnicas mínimamente invasivas en el diagnóstico de las enfermedades intratorácicas en el Hospital Universitario "Manuel Ascunce Domenech" desde enero de 2017 hasta mayo de 2020. El universo estuvo compuesto por 104 pacientes con sospecha diagnóstica de enfermedades benignas o malignas del tórax. Resultados: La mayor parte de los pacientes con enfermedades benignas y malignas del tórax tenían una edad de 49 (±7,05) años, sin significación intersexo. La hipertensión arterial predominó como principal comorbilidad. La videotoracoscopia fue la técnica mínimamente invasiva más empleada y la sospecha de cáncer pulmonar y enfermedad pleural constituyeron las indicaciones más frecuentes que motivaron el proceder. El diagnóstico posoperatorio que predominó en la serie fue el cáncer pulmonar. Las técnicas mínimamente invasivas empleadas mostraron una alta validez. La mayoría de los pacientes egresaron vivos y sin complicaciones. Conclusiones: El uso de técnicas mínimamente invasivas en varias enfermedades intratorácicas son de vital importancia para definir diagnóstico y tratamiento(AU)

Introduction: Changes in medical technology cover all specialties and, within them, general surgery. Minimally invasive techniques have played a paramount role in the diagnosis and treatment of thoracic cavity diseases. Objective: To describe the use of minimally invasive techniques in the diagnosis of intrathoracic diseases. Methods: An observational, retrospective and cross-sectional study was carried out, from January 2017 to May 2020, in order to assess the use of minimally invasive techniques in the diagnosis of intrathoracic diseases at Manuel Ascunce Domenech University Hospital. The universe was made up of 104 patients with suspected benign or malignant diseases of the chest. Results: Most of the patients with benign and malignant chest diseases were 49 (± 7.05) years old, without intersex significance. Arterial hypertension predominated as the main comorbidity. Videothoracoscopy was the most widely used minimally invasive technique, while suspicion of lung cancer and pleural disease were the most frequent indications that motivated the procedure. The postoperative diagnosis that predominated in the series was lung cancer. The minimally invasive techniques used showed high validity. Most of the patients were discharged alive and without complications. Conclusions: The use of minimally invasive techniques in various intrathoracic diseases are of vital importance to define diagnosis and treatment(AU)

Maniobras de movilización visceral en el trauma vascular abdominal / Visceral mobilization maneuvers in abdominal vascular trauma

RESUMEN Fundamento: el trauma vascular abdominal constituye un reto para el cirujano. El control vascular es una condición sine qua non en los pacientes que reciben intervención quirúrgica frente a esta letal enfermedad, donde las maniobras de movilización visceral constituyen una herramienta clave en el tratamiento. Objetivo: explicar las maniobras de movilización visceral que pueden auxiliar al cirujano frente a un trauma vascular abdominal. Métodos: se realizó una búsqueda en las bases de datos Scopus, Medline, Cochrane, Hinari y Redalyc se utilizaron los descriptores: trauma vascular, abdomen, maniobras, movilización visceral. Resultados: se abordaron las maniobras de movilización visceral a tener en cuenta en el trauma vascular abdominal con una explicación de los principales pasos técnicos en cada una de ellas. Conclusiones: el conocimiento y aplicación de las maniobras de movilización visceral constituyen una herramienta útil en el tratamiento de pacientes con trauma vascular abdominal.

ABSTRACT Background: abdominal vascular trauma is a challenge for the surgeon. Vascular control is a sine qua non condition in patients who receive surgical intervention against this lethal pathology, where visceral mobilization maneuvers are a key tool in treatment. Objective: to explain the visceral mobilization maneuvers that can help the surgeon against abdominal vascular trauma. Methods: a search of the Scopus, Medline, Cochrane, Hinari and Redalyc databases was performed using the descriptors: vascular trauma, abdomen, maneuvers, visceral mobilization. Results: visceral mobilization maneuvers to be taken into account in abdominal vascular trauma were addressed with an explanation of the main technical steps in each of them. Conclusions: the knowledge and application of visceral mobilization maneuvers constitute a useful tool in the treatment of patients with abdominal vascular trauma.

Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms.

Myosin molecules in the relaxed thick filaments of striated muscle have a helical arrangement in which the heads of each molecule interact with each other, forming the interacting-heads motif (IHM). In relaxed mammalian skeletal muscle, this helical ordering occurs only at temperatures >20°C and is disrupted when temperature is decreased. Recent x-ray diffraction studies of live tarantula skeletal muscle have suggested that the two myosin heads of the IHM (blocked heads [BHs] and free heads [FHs]) have very different roles and dynamics during contraction. Here, we explore temperature-induced changes in the BHs and FHs in relaxed tarantula skeletal muscle. We find a change with decreasing temperature that is similar to that in mammals, while increasing temperature induces a different behavior in the heads. At 22.5°C, the BHs and FHs containing ADP.Pi are fully helically organized, but they become progressively disordered as temperature is lowered or raised. Our interpretation suggests that at low temperature, while the BHs remain ordered the FHs become disordered due to transition of the heads to a straight conformation containing Mg.ATP. Above 27.5°C, the nucleotide remains as ADP.Pi, but while BHs remain ordered, half of the FHs become progressively disordered, released semipermanently at a midway distance to the thin filaments while the remaining FHs are docked as swaying heads. We propose a thermosensing mechanism for tarantula skeletal muscle to explain these changes. Our results suggest that tarantula skeletal muscle thick filaments, in addition to having a superrelaxation-based ATP energy-saving mechanism in the range of 8.5-40°C, also exhibit energy saving at lower temperatures (<22.5°C), similar to the proposed refractory state in mammals.

Cryo-EM structure of the inhibited (10S) form of myosin II.

Myosin II is the motor protein that enables muscle cells to contract and nonmuscle cells to move and change shape1. The molecule has two identical heads attached to an elongated tail, and can exist in two conformations: 10S and 6S, named for their sedimentation coefficients2,3. The 6S conformation has an extended tail and assembles into polymeric filaments, which pull on actin filaments to generate force and motion. In 10S myosin, the tail is folded into three segments and the heads bend back and interact with each other and the tail3-7, creating a compact conformation in which ATPase activity, actin activation and filament assembly are all highly inhibited7,8. This switched-off structure appears to function as a key energy-conserving storage molecule in muscle and nonmuscle cells9-12, which can be activated to form functional filaments as needed13-but the mechanism of its inhibition is not understood. Here we have solved the structure of smooth muscle 10S myosin by cryo-electron microscopy with sufficient resolution to enable improved understanding of the function of the head and tail regions of the molecule and of the key intramolecular contacts that cause inhibition. Our results suggest an atomic model for the off state of myosin II, for its activation and unfolding by phosphorylation, and for understanding the clustering of disease-causing mutations near sites of intramolecular interaction.

Valor de la linfadenectomía mediastinal en la cirugía del cáncer pulmonar / The Value of Mediastinal Lymphadenectomy in Lung Cancer Surgery

RESUMEN Introducción: La evaluación del mediastino en los pacientes que reciben tratamiento quirúrgico por cáncer del pulmón tiene sus orígenes en la necesidad de definir la extensión anatómica de cada estación nodal, lo cual es indispensable para la categorización patológica de los nódulos linfáticos. Objetivo: Evaluar la utilidad de la linfadenectomía mediastinal en el tratamiento quirúrgico del cáncer pulmonar. Métodos: Se realizó un estudio analítico de carácter prospectivo en el período comprendido entre enero de 2015 al 31 de agosto de 2018. El universo estuvo conformado por 96 pacientes con algún tipo de linfadenectomía mediastinal en el tratamiento quirúrgico del cáncer pulmonar dentro del período señalado. Se utilizaron métodos estadísticos, teóricos y empíricos. Resultados: La edad promedio fue 58,69 ± 9,343, el sexo masculino fue el más afectado y el adenocarcinoma el tipo histológico que predominó. La etapa clínica y patológica mayoritaria fue la III A y la lobectomía superior derecha la intervención más realizada. Se efectuaron un total de 76 disecciones ganglionares sistemáticas, seguidas por 18 muestreos ganglionares y 2 biopsias. Las linfadenectomías realizadas provocaron cambios de estadios en 46 pacientes. El análisis del valor predictivo positivo y negativo, así como de la sensibilidad y la especificidad fueron altos, así como las complicaciones escasas. Conclusiones: La linfadenectomía mediastinal como parte del tratamiento quirúrgico en el cáncer pulmonar constituye un pilar fundamental en la estadificación patológica del TNM, al mostrar una exactitud diagnóstica alta(AU)

ABSTRACT Introduction: Evaluation of the mediastinum in patients receiving surgical treatment for lung cancer has its origins in the need to define the anatomical extension of each node station, which is essential for the pathological categorization of lymph nodes. Objective: To evaluate the usefulness of mediastinal lymphadenectomy in the surgical treatment of lung cancer. Methods: A prospective and analytical study was carried out in the period from January 2015 to August 31, 2018. The universe consisted of 96 patients with some type of mediastinal lymphadenectomy in the surgical treatment of lung cancer within the indicated period. Statistical, theoretical and empirical methods were used. Results: The average age was 58.69 ± 9.343. The male sex was the most affected. Adenocarcinoma was the histological type that predominated. The most manifested clinical and pathological stage was III A. Right upper lobectomy was the most performed intervention. A total of 76 systematic lymph node dissections were performed, followed by 18 lymph node samples and two biopsies. The lymphadenectomies performed caused stage changes in 46 patients. The analysis of positive and negative predictive value, as well as sensitivity and specificity, were high. There were few complications. Conclusions: Mediastinal lymphadenectomy as part of the surgical treatment in lung cancer constitutes a fundamental procedure for the pathological staging of TNM, as it shows high diagnostic accuracy(AU)

Evaluación de la linfoadenectomía transcervical en la sistemática diagnóstica del cáncer pulmonar / Evaluation of transcervical lymphadenectomy in the diagnostic system of lung cancer

RESUMEN Fundamento: el estudio ganglionar del mediastino en los pacientes afectos de cáncer pulmonar es de vital importancia, al permitir trazar la estrategia terapéutica. Objetivo: evaluar la técnica de linfoadenectomía transcervical como parte de la sistemática diagnóstica en el cáncer pulmonar. Métodos: se realizó una investigación analítica, longitudinal prospectiva en pacientes, se usó la linfoadenectomía transcervical como parte de la sistemática diagnóstica del cáncer pulmonar en el Hospital Universitario Manuel Ascunce Domenech, de la provincia Camagüey. El universo estuvo compuesto por 346 pacientes y la muestra, de tipo probabilístico aleatorio simple, quedó conformada por 65 pacientes. Resultados: predominaron los pacientes masculinos y mayores de 60 años. El tipo histológico más frecuente fue el adenocarcinoma y los lóbulos superiores fueron los más afectados. Se extirparon un total de 205 ganglios y la mayoría fueron positivos, la negatividad, en la histología, predominó en los ganglios menores de 1 cm diagnosticados en el estudio tomográfico. Existió migración de estadios en la correlación clínico patológica, así como la sensibilidad y especificidad fueron de un 100 % en la muestra estudiada. Las complicaciones fueron escasas. Conclusiones: la linfoadenectomía transcervical constituye una herramienta diagnóstica en los pacientes con cáncer pulmonar y afectación ganglionar mediastinal.

ABSTRACT Background: the lymph node study of the mediastinum in patients with lung cancer is of vital importance, as it allows the therapeutic strategy to be outlined. Objective: to evaluate the transcervical lymphadenectomy technique as part of the diagnostic system in lung cancer. Methods: a prospective longitudinal analytical investigation was carried out in patients in whom transcervical lymphadenectomy was used as part of the diagnostic system for lung cancer at the Hospital Universitario Manuel Ascunce Domenech, in the province of Camagüey. The universe was made up of 346 patients and the sample, of a simple random probability type, consisted of 65 patients. Results: male patients over 60 years prevailed. The most frequent histological type was adenocarcinoma and the upper lobes were the most affected. A total of 205 nodes were removed and the majority were positive, negativity, in histology, predominated in nodes less than 1 cm diagnosed in the tomographic study. There was migration of stages in the clinical pathological correlation, as well as the sensitivity and specificity were 100% in the studied sample. Complications were rare. Conclusions: transcervical lymphadenectomy constitutes a diagnostic tool in patients with lung cancer and mediastinal lymph node involvement.

Pacientes con infección intrabdominal en la unidad de cuidados intensivos / Patients with Intrabdominal Infection in the Intensive Care Unit

RESUMEN Los pacientes con infección intrabdominal en unidad de cuidados intensivos representan un reto diagnóstico y terapéutico, tanto en el mundo como en Cuba y es responsable del aumento de un periodo de invalidez transitoria y de mortalidad de una parte de los pacientes. El objetivo del artículo fue profundizar en los conocimientos actuales sobre el tratamiento de la infección intrabdominal en la unidad de cuidados intensivos. Se realizó revisión tipo exploratorio-descriptivo para profundizar el tema y se consultaron fuentes primarias y secundarias de información de las bases de datos Scopus, Medline, Cochrane, Hinary y Redalyc bajo los criterios de sus revisores. La infección intrabdominal en los pacientes bajo cuidados intensivos suscita un mal pronóstico, por lo que requiere de un diagnóstico oportuno y un tratamiento eficaz. Los índices de mortalidad en estos enfermos apenas han decrecido a pesar de los avances tecnológicos(AU)

ABSTRACT Patients with intraabdominal infection in the intensive care unit represent a diagnostic and therapeutic challenge, both in Cuba and worldwide. Such condition is responsible for the increase in the period of temporary disability and mortality in part of the patients. The aim of the article was to deepen the current knowledge on treatment of intraabdominal infection in the intensive care unit. An exploratory-descriptive review was carried out to deepen the subject and primary and secondary sources of information were consulted from the Scopus, Medline, Cochrane, Hinary and Redalyc databases, following the criteria of their reviewers. Intraabdominal infection in patients under intensive care presents poor prognosis, which requires prompt diagnosis and effective treatment. Mortality rates in these patients have barely decreased despite technological advances(AU)

The myosin interacting-heads motif present in live tarantula muscle explains tetanic and posttetanic phosphorylation mechanisms.

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing-proposed in vertebrate muscle-is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca2+-binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. METHODS: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. RESULTS: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. CONCLUSIONS: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

18O labeling on Ser45 but not on Ser35 supports the cooperative phosphorylation mechanism on tarantula thick filament activation.

Thick filaments from some striated muscles are regulated by phosphorylation of myosin regulatory light chains (RLCs). A tarantula thick filament quasi-atomic model achieved by cryo-electron microscopy has advanced our understanding on how this regulation occurs. In native thick filaments, an asymmetric intramolecular interaction between the actin-binding region of one myosin head ("blocked") and the converter region of the other head ("free") switches both heads off, establishing the myosin interacting-heads motif (IHM). This structural finding, together with motility assays, sequence analysis, and mass spectrometry (MS) observations have suggested a cooperative phosphorylation activation (CPA) mechanism for thick filament activation. In the CPA mechanism, some myosin free heads are phosphorylated constitutively in Ser35 by protein kinase C (PKC) and -under Ca2+ control - others (free or blocked) heads temporally on Ser45 by myosin light chain kinase (MLCK), in a way that explains both force development and post-tetanic potentiation in tarantula striated muscle. We tested this model using MS to verify if Ca2+-activation phosphorylates de novo un-phosphorylated Ser35 heads. For this purpose, we standardized an approach based on 18O isotopic ATP labeling to accurately detect by MS-MS the RLC phosphorylation under Ca2+-activation. MS spectra showed de novo18O incorporation only on Ser45 but not on Ser35. As the constitutive Ser35 phosphorylation cannot be dephosphorylated, this result suggests that the number of RLCs on free heads with constitutively phosphorylated Ser35 does remain constant on Ca2+-activation supporting that the myosin has a basal activation and force modulation or potentiation is controlled by MLCK Ser45 phosphorylation.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.

Lessons from a tarantula: new insights into myosin interacting-heads motif evolution and its implications on disease.

Tarantula's leg muscle thick filament is the ideal model for the study of the structure and function of skeletal muscle thick filaments. Its analysis has given rise to a series of structural and functional studies, leading, among other things, to the discovery of the myosin interacting-heads motif (IHM). Further electron microscopy (EM) studies have shown the presence of IHM in frozen-hydrated and negatively stained thick filaments of striated, cardiac, and smooth muscle of bilaterians, most showing the IHM parallel to the filament axis. EM studies on negatively stained heavy meromyosin of different species have shown the presence of IHM on sponges, animals that lack muscle, extending the presence of IHM to metazoans. The IHM evolved about 800 MY ago in the ancestor of Metazoa, and independently with functional differences in the lineage leading to the slime mold Dictyostelium discoideum (Mycetozoa). This motif conveys important functional advantages, such as Ca2+ regulation and ATP energy-saving mechanisms. Recent interest has focused on human IHM structure in order to understand the structural basis underlying various conditions and situations of scientific and medical interest: the hypertrophic and dilated cardiomyopathies, overfeeding control, aging and hormone deprival muscle weakness, drug design for schistosomiasis control, and conditioning exercise physiology for the training of power athletes.

Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca2+-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes.

Cardiac ß-myosin variants cause hypertrophic (HCM) or dilated (DCM) cardiomyopathy by disrupting sarcomere contraction and relaxation. The locations of variants on isolated myosin head structures predict contractility effects but not the prominent relaxation and energetic deficits that characterize HCM. During relaxation, pairs of myosins form interacting-heads motif (IHM) structures that with other sarcomere proteins establish an energy-saving, super-relaxed (SRX) state. Using a human ß-cardiac myosin IHM quasi-atomic model, we defined interactions sites between adjacent myosin heads and associated protein partners, and then analyzed rare variants from 6112 HCM and 1315 DCM patients and 33,370 ExAC controls. HCM variants, 72% that changed electrostatic charges, disproportionately altered IHM interaction residues (expected 23%; HCM 54%, p=2.6×10-19; DCM 26%, p=0.66; controls 20%, p=0.23). HCM variant locations predict impaired IHM formation and stability, and attenuation of the SRX state - accounting for altered contractility, reduced diastolic relaxation, and increased energy consumption, that fully characterizes HCM pathogenesis.