RESUMEN
The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-ß chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-ß chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.
Asunto(s)
Miosinas Cardíacas , Miocardio , Sarcómeros , Conectina/química , Conectina/metabolismo , Conectina/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Miocardio/química , Miocardio/citología , Miocardio/ultraestructura , Sarcómeros/química , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Miosinas Cardíacas/química , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/ultraestructuraRESUMEN
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.
Asunto(s)
Miosinas Cardíacas , Microscopía por Crioelectrón , Miocardio , Humanos , Miosinas Cardíacas/química , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , Conectina/química , Conectina/metabolismo , Conectina/ultraestructura , Miocardio/química , Miocardio/ultraestructuraRESUMEN
The fundamental basis of muscle contraction 'the sliding filament model' (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954) and the 'swinging, tilting crossbridge-sliding filament mechanism' (Huxley, 1969; Huxley and Brown, 1967) nucleated a field of research that has unearthed the complex and fascinating role of myosin structure in the regulation of contraction. A recently discovered energy conserving state of myosin termed the super relaxed state (SRX) has been observed in filamentous myosins and is central to modulating force production and energy use within the sarcomere. Modulation of myosin function through SRX is a rapidly developing theme in therapeutic development for both cardiovascular disease and infectious disease. Some 70â years after the first discoveries concerning muscular function, modulation of myosin SRX may bring the first myosin targeted small molecule to the clinic, for treating hypertrophic cardiomyopathy (Olivotto et al., 2020). An often monogenic disease HCM afflicts 1 in 500 individuals, and can cause heart failure and sudden cardiac death. Even as we near therapeutic translation, there remain many questions about the governance of muscle function in human health and disease. With this review, we provide a broad overview of contemporary understanding of myosin SRX, and explore the complexities of targeting this myosin state in human disease.This article has an associated Future Leaders to Watch interview with the authors of the paper.
Asunto(s)
Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Contracción Miocárdica/fisiología , Biomarcadores , Miosinas Cardíacas/ultraestructura , Cardiomiopatía Hipertrófica/tratamiento farmacológico , Cardiomiopatía Hipertrófica/etiología , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Modelos Biológicos , Terapia Molecular Dirigida , Mutación , FosforilaciónRESUMEN
A number of cardiac myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) are linked to mutations in cardiac muscle myosin filament proteins, including myosin and myosin binding protein C (MyBP-C). To understand the myopathies it is necessary to know the normal 3D structure of these filaments. We have carried out 3D single particle analysis of electron micrograph images of negatively stained isolated myosin filaments from rabbit cardiac muscle. Single filament images were aligned and divided into segments about 2x430A long, each of which was treated as an independent 'particle'. The resulting 40A resolution 3D reconstruction showed both axial and azimuthal (no radial) myosin head perturbations within the 430A repeat, with successive crown rotations of approximately 60 degrees , 60 degrees and 0 degrees , rather than the regular 40 degrees for an unperturbed helix. However, it is shown that the projecting density peaks appear to start at low radius from origins closer to those expected for an unperturbed helical filament, and that the azimuthal perturbation especially increases with radius. The head arrangements in rabbit cardiac myosin filaments are very similar to those in fish skeletal muscle myosin filaments, suggesting a possible general structural theme for myosin filaments in all vertebrate striated muscles (skeletal and cardiac).
Asunto(s)
Miosinas Cardíacas/ultraestructura , Miocardio/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Animales , Miosinas Cardíacas/química , Imagenología Tridimensional , Microscopía Electrónica , Conformación Proteica , ConejosRESUMEN
Contraction of the heart results from interaction of the myosin and actin filaments. Cardiac myosin filaments consist of the molecular motor myosin II, the sarcomeric template protein, titin, and the cardiac modulatory protein, myosin binding protein C (MyBP-C). Inherited hypertrophic cardiomyopathy (HCM) is a disease caused mainly by mutations in these proteins. The structure of cardiac myosin filaments and the alterations caused by HCM mutations are unknown. We have used electron microscopy and image analysis to determine the three-dimensional structure of myosin filaments from wild-type mouse cardiac muscle and from a MyBP-C knockout model for HCM. Three-dimensional reconstruction of the wild-type filament reveals the conformation of the myosin heads and the organization of titin and MyBP-C at 4 nm resolution. Myosin heads appear to interact with each other intramolecularly, as in off-state smooth muscle myosin [Wendt T, Taylor D, Trybus KM, Taylor K (2001) Proc Natl Acad Sci USA 98:4361-4366], suggesting that all relaxed muscle myosin IIs may adopt this conformation. Titin domains run in an elongated strand along the filament surface, where they appear to interact with part of MyBP-C and with the myosin backbone. In the knockout filament, some of the myosin head interactions are disrupted, suggesting that MyBP-C is important for normal relaxation of the filament. These observations provide key insights into the role of the myosin filament in cardiac contraction, assembly, and disease. The techniques we have developed should be useful in studying the structural basis of other myosin-related HCM diseases.